# Patent

## Title
Corynebacterium glutamicum genes encoding metabolic pathway proteins

## Abstract

Isolated nucleic acid molecules, designated MP nucleic acid molecules, which encode novel MP proteins from Corynebacterium glutamicum are described. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing MP nucleic acid molecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated MP proteins, mutated MP proteins, fusion proteins, antigenic peptides and methods for the improvement of production of a desired compound from C. glutamicum based on genetic engineering of MP genes in this organism.

## Background
RELATED APPLICATIONS
The present application is an continuation in part of U.S. patent application Ser. No. 09/606,740, filed Jun. 23, 2000. This application is also a continuation in part of U.S. patent application Ser. No. 09/603,124, filed Jun. 23, 2000. The present application claims priority to prior filed U.S. Provisional Patent Application Ser. No. 60/141,031, filed Jun. 25, 1999, U.S. Provisional Patent Application Ser. No. 60/142,101, filed Jul. 2, 1999, U.S. Provisional Patent Application Ser. No. 60/148,613, filed Aug. 12, 1999, U.S. Provisional Patent Application Ser. No. 60/187,970, filed Mar. 9, 2000, and also to German Patent Application No. 19931420.9, filed Jul. 8, 1999. The entire contents of all of the aforementioned applications are hereby expressly incorporated herein by this reference.


BACKGROUND OF THE INVENTION
Certain products and by-products of naturally-occurring metabolic processes in cells have utility in a wide array of industries, including the food, feed, cosmetics, and pharmaceutical industries. These molecules, collectively termed ‘fine chemicals’, include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, and enzymes. Their production is most conveniently performed through large-scale culture of bacteria developed to produce and secrete large quantities of a particular desired molecule. One particularly useful organism for this purpose is Corynebacterium glutamicum, a gram positive, nonpathogenic bacterium. Through strain selection, a number of mutant strains have been developed which produce an array of desirable compounds. However, selection of strains improved for the production of a particular molecule is a time-consuming and difficult process.
SUMMARY OF THE INVENTION
The invention provides novel bacterial nucleic acid molecules which have a variety of uses. These uses include the identification of microorganisms which can be used to produce fine chemicals (e.g., amino acids, such as, for example, lysine and methionine), the modulation of fine chemical production in C. glutamicum or related bacteria, the typing or identification of C. glutamicum or related bacteria, as reference points for mapping the C. glutamicum genome, and as markers for transformation. These novel nucleic acid molecules encode proteins, referred to herein as metabolic pathway (MP) proteins.
C. glutamicum is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids. The MP nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, e.g., by fermentation processes. Modulation of the expression of the MP nucleic acids of the invention, or modification of the sequence of the MP nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism (e.g., to improve the yield or production of one or more fine chemicals from a Corynebacterium or Brevibacterium species). In a preferred embodiment, the MP genes of the invention are combined with one or more genes involved in the same or different metabolic pathway to modulate the production of one or more fine chemicals from a microorganism.
The MP nucleic acids of the invention may also be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof, or to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present. Although Corynebacterium glutamicum itself is nonpathogenic, it is related to species pathogenic in humans, such as Corynebacterium diphtheriae (the causative agent of diphtheria); the detection of such organisms is of significant clinical relevance.
The MP nucleic acid molecules of the invention may also serve as reference points for mapping of the C. glutamicum genome, or of genomes of related organisms. Similarly, these molecules, or variants or portions thereof, may serve as markers for genetically engineered Corynebacterium or Brevibacterium species.
The MP proteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, performing an enzymatic step involved in the metabolism of certain fine chemicals, including amino acids, e.g., lysine and methionine, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides, and trehalose. Given the availability of cloning vectors for use in Corynebacterium glutamicum, such as those disclosed in Sinskey et al., U.S. Pat. No. 4,649,119, and techniques for genetic manipulation of C. glutamicum and the related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 (1984)), the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of one or more fine chemicals.
This improved production or efficiency of production of a fine chemical may be due to a direct effect of manipulation of a gene of the invention, or it may be due to an indirect effect of such manipulation. Specifically, alterations in C. glutamicum metabolic pathways for amino acids, e.g., lysine and methionine, vitamins, cofactors, nucleotides, and trehalose may have a direct impact on the overall production of one or more of these desired compounds from this organism. For example, optimizing the activity of a lysine or a methionine biosynthetic pathway protein or decreasing the activity of a lysine or methionine degradative pathway protein may result in an increase in the yield or efficiency of production of lysine or methionine from such an engineered organism. Alterations in the proteins involved in these metabolic pathways may also have an indirect impact on the production or efficiency of production of a desired fine chemical. For example, a reaction which is in competition for an intermediate necessary for the production of a desired molecule may be eliminated, or a pathway necessary for the production of a particular intermediate for a desired compound may be optimized. Further, modulations in the biosynthesis or degradation of, for example, an amino acid, e.g., lysine or methionine, a vitamin, or a nucleotide may increase the overall ability of the microorganism to rapidly grow and divide, thus increasing the number and/or production capacities of the microorganism in culture and thereby increasing the possible yield of the desired fine chemical.
The nucleic acid and protein molecules of the invention, alone or in combination with one or more nucleic acid and protein molecules of the same or different metabolic pathway, may be utilized to directly improve the production or efficiency of production of one or more desired fine chemicals from Corynebacterium glutamicum (e.g., methionine or lysine). Using recombinant genetic techniques well known in the art, one or more of the biosynthetic or degradative enzymes of the invention for amino acids, e.g., lysine and methionine, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides, or trehalose may be manipulated such that its function is modulated. For example, a biosynthetic enzyme may be improved in efficiency, or its allosteric control region destroyed such that feedback inhibition of production of the compound is prevented. Similarly, a degradative enzyme may be deleted or modified by substitution, deletion, or addition such that its degradative activity is lessened for the desired compound without impairing the viability of the cell. In each case, the overall yield or rate of production of the desired fine chemical may be increased.
It is also possible that such alterations in the protein and nucleotide molecules of the invention may improve the production of other fine chemicals besides the amino acids, e.g., lysine and methionine, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides, and trehalose through indirect mechanisms. Metabolism of any one compound is necessarily intertwined with other biosynthetic and degradative pathways within the cell, and necessary cofactors, intermediates, or substrates in one pathway are likely supplied or limited by another such pathway. Therefore, by modulating the activity of one or more of the proteins of the invention, the production or efficiency of activity of another fine chemical biosynthetic or degradative pathway may be impacted. For example, amino acids serve as the structural units of all proteins, yet may be present intracellularly in levels which are limiting for protein synthesis; therefore, by increasing the efficiency of production or the yields of one or more amino acids within the cell, proteins, such as biosynthetic or degradative proteins, may be more readily synthesized. Likewise, an alteration in a metabolic pathway enzyme such that a particular side reaction becomes more or less favored may result in the over- or under-production of one or more compounds which are utilized as intermediates or substrates for the production of a desired fine chemical.
This invention provides novel nucleic acid molecules which encode proteins, referred to herein as metabolic pathway (“MP”) proteins, which are capable of, for example, performing an enzymatic step involved in the metabolism of molecules important for the normal functioning of cells, such as amino acids, e.g., lysine and methionine, vitamins, cofactors, nucleotides and nucleosides, or trehalose. Nucleic acid molecules encoding an MP protein are referred to herein as MP nucleic acid molecules. In a preferred embodiment, an MP protein, alone or in combination with one or more proteins of the same or different metabolic pathway, performs an enzymatic step related to the metabolism of one or more of the following: amino acids, e.g., lysine and methionine, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides, and trehalose. Examples of such proteins include those encoded by the genes set forth in Table 1.
Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding an MP protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of MP-encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth as the odd-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5), or the coding region or a complement thereof of one of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more homologous to a nucleotide sequence set forth as an odd-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5), or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences set forth as an even-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6). The preferred MP proteins of the present invention also preferably possess at least one of the MP activities described herein.
In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence having an even-numbered SEQ ID NO in the Sequence Listing, such as SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6), e.g., sufficiently homologous to an amino acid sequence of the invention such that the protein or portion thereof maintains an MP activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to perform an enzymatic reaction in a amino acid, e.g., lysine or methionine, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathway. In one embodiment, the protein encoded by the nucleic acid molecule is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more homologous to an amino acid sequence of the invention (e.g., an entire amino acid sequence selected from those having an even-numbered SEQ ID NO in the Sequence Listing, such as SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6). In another preferred embodiment, the protein is a full length C. glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention (encoded by an open reading frame shown in the corresponding odd-numbered SEQ ID NO in the Sequence Listing (e.g., SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5).
In another preferred embodiment, the isolated nucleic acid molecule is derived from C. glutamicum and encodes a protein (e.g., an MP fusion protein) which includes a biologically active domain which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of one of the even-numbered SEQ ID NOs in the Sequence Listing, such as SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6) and is able to catalyze a reaction in a metabolic pathway for an amino acid, e.g., lysine or methionine, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose, or one or more of the activities set forth in Table 1, and which also includes heterologous nucleic acid sequences encoding a heterologous polypeptide or regulatory regions.
In another embodiment, the isolated nucleic acid molecule is at least 15 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO in the Sequence Listing, such as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5). Preferably, the isolated nucleic acid molecule corresponds to a naturally-occurring nucleic acid molecule. More preferably, the isolated nucleic acid encodes a naturally-occurring C. glutamicum MP protein, or a biologically active portion thereof.
Another aspect of the invention pertains to vectors, e.g., recombinant expression vectors, containing the nucleic acid molecules of the invention, alone or in combination with one or more nucleic acid molecules involved in the same or different pathway, and host cells into which such vectors have been introduced. In one embodiment, such a host cell is used to produce an MP protein by culturing the host cell in a suitable medium. The MP protein can be then isolated from the medium or the host cell.
Yet another aspect of the invention pertains to a genetically altered microorganism in which one or more MP genes, alone or in combination with one or more genes involved in the same or different metabolic pathway, have been introduced or altered. In one embodiment, the genome of the microorganism has been altered by introduction of a nucleic acid molecule of the invention encoding one or more wild-type or mutated MP sequences as transgenes alone or in combination with one or more nucleic acid molecules involved in the same or different metabolic pathway. In another embodiment, one or more endogenous MP genes within the genome of the microorganism have been altered, e.g., functionally disrupted, by homologous recombination with one or more altered MP genes. In another embodiment, one or more endogenous or introduced MP genes, alone or in combination with one or more genes of the same or different metabolic pathway in a microorganism have been altered by one or more point mutations, deletions, or inversions, but still encode functional MP proteins. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of one or more MP genes in a microorganism, alone or in combination with one or more MP genes or in combination with one or more genes of the same or different metabolic pathway, has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of one or more MP genes is modulated. In a preferred embodiment, the microorganism belongs to the genus Corynebacterium or Brevibacterium, with Corynebacterium glutamicum being particularly preferred. In a preferred embodiment, the microorganism is also utilized for the production of a desired compound, such as an amino acid, with lysine and methionine being particularly preferred. In a particularly preferred embodiment, the MP gene is the metZ gene (SEQ ID NO:1), metC gene (SEQ ID NO:3), or the RXA00657 gene (SEQ ID NO:5), alone or in combination with one or more MP genes of the invention or in combination with one or more genes involved in methionine and/or lysine metabolism.
In another aspect, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth in Table 1 and in the Sequence Listing as SEQ ID NOs 1 through 122) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject.
Still another aspect of the invention pertains to an isolated MP protein or portion, e.g., biologically active portion, thereof. In a preferred embodiment, the isolated MP protein or portion thereof, alone or in combination with one or more MP proteins of the invention or in combination with one or more proteins of the same or different metabolic pathway, can catalyze an enzymatic reaction involved in one or more pathways for the metabolism of an amino acid, e.g., lysine or methionine, a vitamin, a cofactor, a nutraceutical, a nucleotide, a nucleoside, or trehalose. In another preferred embodiment, the isolated MP protein or portion thereof, is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: in the Sequence Listing, such as SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6) such that the protein or portion thereof maintains the ability to catalyze an enzymatic reaction involved in one or more pathways for the metabolism of an amino acid, a vitamin, a cofactor, a nutraceutical, a nucleotide, a nucleoside, or trehalose.
The invention also provides an isolated preparation of an MP protein. In preferred embodiments, the MP protein comprises an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing such as SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6). In another preferred embodiment, the invention pertains to an isolated full length protein which is substantially homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO of the Sequence Listing such as SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6) (encoded by an open reading frame set forth in a corresponding odd-numbered SEQ ID NO: of the Sequence Listing such as SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5). In yet another embodiment, the protein is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing such as SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6). In other embodiments, the isolated MP protein comprises an amino acid sequence which is at least about 50% or more homologous to one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing such as SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6) and is able to catalyze an enzymatic reaction in an amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathway either alone or in combination one or more MP proteins of the invention or any protein of the same or different metabolic pathway, or has one or more of the activities set forth in Table 1.
Alternatively, the isolated MP protein can comprise an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, or is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more homologous to a nucleotide sequence of one of the even-numbered SEQ ID NOs set forth in the Sequence Listing. It is also preferred that the preferred forms of MP proteins also have one or more of the MP bioactivities described herein.
The MP polypeptide, or a biologically active portion thereof, can be operatively linked to a non-MP polypeptide to form a fusion protein. In preferred embodiments, this fusion protein has an activity which differs from that of the MP protein alone. In other preferred embodiments, this fusion protein, when introduced into a C. glutamicum pathway for the metabolism of an amino acid, vitamin, cofactor, nutraceutical, results in increased yields and/or efficiency of production of a desired fine chemical from C. glutamicum. In particularly preferred embodiments, integration of this fusion protein into an amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathway of a host cell modulates production of a desired compound from the cell.
In another aspect, the invention provides methods for screening molecules which modulate the activity of an MP protein, either by interacting with the protein itself or a substrate or binding partner of the MP protein, or by modulating the transcription or translation of an MP nucleic acid molecule of the invention.
Another aspect of the invention pertains to a method for producing a fine chemical. This method involves the culturing of a cell containing one or more vectors directing the expression of one or more MP nucleic acid molecules of the either alone or in combination one or more MP nucleic acid molecules of the invention or any nucleic acid molecule of the same or different metabolic pathway, such that a fine chemical is produced. In a preferred embodiment, this method further includes the step of obtaining a cell containing such a vector, in which &cell is transfected with a vector directing the expression of an MP nucleic acid. In another preferred embodiment, this method further includes the step of recovering the fine chemical from the culture. In a particularly preferred embodiment, the cell is from the genus Corynebacterium or Brevibacterium, or is selected from those strains set forth in Table 3. In another preferred embodiment, the MP genes is the metZ gene (SEQ ID NO:1), metC gene (SEQ ID NO:3), or the gene designated as RXA00657 (SEQ ID NO:5) (see Table 1), alone or in combination with one or more MP nucleic acid molecules of the invention or with one or more genes involved in methionine and/or lysine metabolism. In yet another preferred embodiment, the fine chemical is an amino acid, e.g., L-lysine and L-methionine.
Another aspect of the invention pertains to methods for modulating production of a molecule from a microorganism. Such methods include contacting the cell with an agent which modulates MP protein activity or MP nucleic acid expression such that a cell associated activity is altered relative to this same activity in the absence of the agent. In a preferred embodiment, the cell is modulated for one or more C. glutamicum amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathways, such that the yields or rate of production of a desired fine chemical by this microorganism is improved. The agent which modulates MP protein activity can be an agent which stimulates MP protein activity or MP nucleic acid expression. Examples of agents which stimulate MP protein activity or MP nucleic acid expression include small molecules, active MP proteins, and nucleic acids encoding MP proteins that have been introduced into the cell. Examples of agents which inhibit MP activity or expression include small molecules and antisense MP nucleic acid molecules.
Another aspect of the invention pertains to methods for modulating yields of a desired compound from a cell, involving the introduction of a wild-type or mutant MP gene into a cell, either alone or in combination one or more MP nucleic acid molecules of the invention or any nucleic acid molecule of the same or different metabolic pathway, either maintained on a separate plasmid or integrated into the genome of the host cell. If integrated into the genome, such integration can be random, or it can take place by homologous recombination such that the native gene is replaced by the introduced copy, causing the production of the desired compound from the cell to be modulated. In a preferred embodiment, said yields are increased. In another preferred embodiment, said chemical is a fine chemical. In a particularly preferred embodiment, said fine chemical is an amino acid. In especially preferred embodiments, said amino acid are L-lysine and L-methionine. In another preferred embodiment, said gene is the metZ gene (SEQ ID NO:1), metC gene (SEQ ID NO:3), or the RXA00657 gene (SEQ ID NO:5), alone or in combination with one or more MP nucleic acid molecules of the invention or with one or more genes involved in methionine and/or lysine metabolism.


DETAILED DESCRIPTION OF THE INVENTION
The present invention provides MP nucleic acid and protein molecules which are involved in the metabolism of certain fine chemicals in Corynebacterium glutamicum, including amino acids, e.g., lysine and methionine, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides, and trehalose. The molecules of the invention may be utilized in the modulation of production of fine chemicals from microorganisms, such as C. glutamicum, either directly (e.g., where modulation of the activity of a lysine or methionine biosynthesis protein has a direct impact on the production or efficiency of production of lysine or methionine from that organism), or may have an indirect impact which nonetheless results in an increase of yield or efficiency of production of the desired compound (e.g., where modulation of the activity of a nucleotide biosynthesis protein has an impact on the production of an organic acid or a fatty acid from the bacterium, perhaps due to improved growth or an increased supply of necessary co-factors, energy compounds, or precursor molecules). The MP molecules may be utilized alone or in combination with other MP molecules of the invention, or in combination with other molecules involved in the same or a different metabolic pathway (e.g., lysine or methione metabolism). In a preferred embodiment, the MP molecules are the metZ (SEQ ID NO:1), metC (SEQ ID NO:3), or RXA00657 (SEQ ID NO:5) nucleic acid molecules and the proteins encoded by these nucleic acid molecules (SEQ ID NO:2, SEQ ID NO.:4 and SEQ ID NO.:6, respectively). Aspects of the invention are further explicated below.
I. Fine Chemicals
The term ‘fine chemical’ is art-recognized and includes molecules produced by an organism which have applications in various industries, such as, but not limited to, the pharmaceutical, agriculture, and cosmetics industries. Such compounds include organic acids, such as tartaric acid, itaconic acid, and diaminopimelic acid, both proteinogenic and non-proteinogenic amino acids, purine and pyrimidine bases, nucleosides, and nucleotides (as described e.g. in Kuninaka, A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, and references contained therein), lipids, both saturated and unsaturated fatty acids (e.g., arachidonic acid), diols (e.g., propane diol, and butane diol), carbohydrates (e.g., hyaluronic acid and trehalose), aromatic compounds (e.g., aromatic amines, vanillin, and indigo), vitamins and cofactors (as described in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27, “Vitamins”, p. 443-613 (1996) VCH: Weinheim and references therein; and Ong, A. S., Niki, E. & Packer, L. (1995) “Nutrition, Lipids, Health, and Disease” Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research—Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, polyketides (Cane et al. (1998) Science 282: 63-68), and all other chemicals described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and references therein. The metabolism and uses of certain of these fine chemicals are further explicated below.
A. Amino Acid Metabolism and Uses
Amino acids comprise the basic structural units of all proteins, and as such are essential for normal cellular functioning in all organisms. The term “amino acid” is art-recognized. The proteinogenic amino acids, of which there are 20 species, serve as structural units for proteins, in which they are linked by peptide bonds, while the nonproteinogenic amino acids (hundreds of which are known) are not normally found in proteins (see Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97 VCH: Weinheim (1985)). Amino acids may be in the D- or L-optical configuration, though L-amino acids are generally the only type found in naturally-occurring proteins. Biosynthetic and degradative pathways of each of the 20 proteinogenic amino acids have been well characterized in both prokaryotic and eukaryotic cells (see, for example, Stryer, L. Biochemistry, 3rd edition, pages 578-590 (1988)). The ‘essential’ amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine), so named because they are generally a nutritional requirement due to the complexity of their biosyntheses, are readily converted by simple biosynthetic pathways to the remaining 11 ‘nonessential’ amino acids (alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, and tyrosine). Higher animals do retain the ability to synthesize some of these amino acids, but the essential amino acids must be supplied from the diet in order for normal protein synthesis to occur.
Aside from their function in protein biosynthesis, these amino acids are interesting chemicals in their own right, and many have been found to have various applications in the food, feed, chemical, cosmetics, agriculture, and pharmaceutical industries. Lysine is an important amino acid in the nutrition not only of humans, but also of monogastric animals such as poultry and swine. Glutamate is most commonly used as a flavor additive (mono-sodium glutamate, MSG) and is widely used throughout the food industry, as are aspartate, phenylalanine, glycine, and cysteine. Glycine, L-methionine and tryptophan are all utilized in the pharmaceutical industry. Glutamine, valine, leucine, isoleucine, histidine, arginine, proline, serine and alanine are of use in both the pharmaceutical and cosmetics industries. Threonine, tryptophan, and D/L-methionine are common feed additives. (Leuchtenberger, W. (1996) Amino aids—technical production and use, p. 466-502 in Rehm et al. (eds.) Biotechnology vol. 6, chapter 14a, VCH: Weinheim). Additionally, these amino acids have been found to be useful as precursors for the synthesis of synthetic amino acids and proteins, such as N-acetylcysteine, S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan, and others described in Ulmann's Encyclopedia of Industrial Chemistry, vol. A2, p. 57-97, VCH: Weinheim, 1985.
The biosynthesis of these natural amino acids in organisms capable of producing them, such as bacteria, has been well characterized (for review of bacterial amino acid biosynthesis and regulation thereof, see Umbarger, H. E. (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate is synthesized by the reductive amination of α-ketoglutarate, an intermediate in the citric acid cycle. Glutamine, proline, and arginine are each subsequently produced from glutamate. The biosynthesis of serine is a three-step process beginning with 3-phosphoglycerate (an intermediate in glycolysis), and resulting in this amino acid after oxidation, transamination, and hydrolysis steps. Both cysteine and glycine are produced from serine; the former by the condensation of homocysteine with serine, and the latter by the transferal of the side-chain β-carbon atom to tetrahydrofolate, in a reaction catalyzed by serine transhydroxymethylase. Phenylalanine and tyrosine are synthesized from the glycolytic and pentose phosphate pathway precursors erythrose 4-phosphate and phosphoenolpyruvate in a 9-step biosynthetic pathway that differ only at the final two steps after synthesis of prephenate. Tryptophan is also produced from these two initial molecules, but its synthesis is an 11-step pathway. Tyrosine may also be synthesized from phenylalanine, in a reaction catalyzed by phenylalanine hydroxylase. Alanine, valine, and leucine are all biosynthetic products of pyruvate, the final product of glycolysis. Aspartate is formed from oxaloacetate, an intermediate of the citric acid cycle. Asparagine, methionine, threonine, and lysine are each produced by the conversion of aspartate. Isoleucine is formed from threonine.
The biosynthetic pathways leading to methionine have been studied in diverse organisms. The first step, acylation of homoserine, is common to all of the organisms, even though the source of the transferred acyl groups is different. Escherichia coli and the related species use succinyl-CoA (Michaeli, S. and Ron, E. Z. (1981) Mol. Gen. Genet. 182, 349-354), while Saccharomyces cerevisiae (Langin, T., et al. (1986) Gene 49, 283-293), Brevibacterium flavum (Miyajima, R. and Shiio, I. (1973) J. Biochem. 73, 1061-1068; Ozaki, H. and Shiio, I. (1982) J. Biochem. 91, 1163-1171), C. glutamicum (Park, S.-D., et al. (1998) Mol. Cells 8, 286-294), and Leptospira meyeri (Belfaiza, J. et al. (1998) 180, 250-255; Bourhy, P., et al. (1997) J. Bacteriol. 179, 4396-4398) use acetyl-CoA as the acyl donor; Formation of homocysteine from acylhomoserine can occur in two different ways. E. coli uses the transsulfuration pathway which is catalyzed by cystathionine γ-synthase (the product of metB) and cystathionine β-lyase (the product of metC). S. cerevisiae (Cherest, H. and Surdin-Kerjan, Y. (1992) Genetics 130, 51-58), B. flavum (Ozaki, H. and Shiio, I. (1982) J. Biochem. 91, 1163-1171), Pseudomonas aeruginosa (Foglino, M., et al. (1995) Microbiology 141, 431-439), and L. meyeri (Belfaiza, J., et al. (1998) J. Bacteriol. 180, 250-255) utilize the direct sulfhydrylation pathway which is catalyzed by acylhomoserine sulfhydrylase. Unlike closely related B. flavum which uses only the direct sulfhydrylation pathway, enzyme activities of the transsulfuration pathway have been detected in the extracts of the C. glutamicum cells and the pathway has been proposed to be the route for methionine biosynthesis in the organism (Hwang, B-J., et al. (1999) Mol. Cells 9, 300-308; Kase, H. and Nakayama, K. (1974) Agr. Biol. Chem. 38, 2021-2030; Park, S.-D., et al. 1998) Mol. Cells 8, 286-294).
Although some genes involved in methionine biosynthesis in C. glutamicum have been isolated, information on the biosynthesis of methionine in C. glutamicum is still very limited. No genes other than metA and metB have been isolated from the organism. To understand the biosynthetic pathways leading to methionine in C. glutamicum, we have isolated and characterized the metC gene (SEQ ID NO:3) and the metZ (also called metY) gene (SEQ ID NO:1) of C. glutamicum (see Table 1).
Amino acids in excess of the protein synthesis needs of the cell cannot be stored, and are instead degraded to provide intermediates for the major metabolic pathways of the cell (for review see Stryer, L. Biochemistry 3rd ed. Ch. 21 “Amino Acid Degradation and the Urea Cycle” p. 495-516 (1988)). Although the cell is able to convert unwanted amino acids into useful metabolic intermediates, amino acid production is costly in terms of energy, precursor molecules, and the enzymes necessary to synthesize them. Thus it is not surprising that amino acid biosynthesis is regulated by feedback inhibition, in which the presence of a particular amino acid serves to slow or entirely stop its own production (for overview of feedback mechanisms in amino acid biosynthetic pathways, see Stryer, L. Biochemistry, 3rd ed. Ch. 24: “Biosynthesis of Amino Acids and Heme” p. 575-600 (1988)). Thus, the output of any particular amino acid is limited by the amount of that amino acid present in the cell.
B. Vitamin, Cofactor, and Nutraceutical Metabolism and Uses
Vitamins, cofactors, and nutraceuticals comprise another group of molecules which the higher animals have lost the ability to synthesize and so must ingest, although they are readily synthesized by other organisms, such as bacteria. These molecules are either bioactive substances themselves, or are precursors of biologically active substances which may serve as electron carriers or intermediates in a variety of metabolic pathways. Aside from their nutritive value, these compounds also have significant industrial value as coloring agents, antioxidants, and catalysts or other processing aids. (For an overview of the structure, activity, and industrial applications of these compounds, see, for example, Ullman's Encyclopedia of Industrial Chemistry, “Vitamins” vol. A27, p. 443-613, VCH: Weinheim, 1996.) The term “vitamin” is art-recognized, and includes nutrients which are required by an organism for normal functioning, but which that organism cannot synthesize by itself. The group of vitamins may encompass cofactors and nutraceutical compounds. The language “cofactor” includes nonproteinaceous compounds required for a normal enzymatic activity to occur. Such compounds may be organic or inorganic; the cofactor molecules of the invention are preferably organic. The term “nutraceutical” includes dietary supplements having health benefits in plants and animals, particularly humans. Examples of such molecules are vitamins, antioxidants, and also certain lipids (e.g., polyunsaturated fatty acids).
The biosynthesis of these molecules in organisms capable of producing them, such as bacteria, has been largely characterized (Ullman's Encyclopedia of Industrial Chemistry, “Vitamins” vol. A27, p. 443-613, VCH: Weinheim, 1996; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley & Sons; Ong, A. S., Niki, E. & Packer, L. (1995) “Nutrition, Lipids, Health, and Disease” Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research—Asia, held Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press: Champaign, Ill. X, 374 S).
Thiamin (vitamin B1) is produced by the chemical coupling of pyrimidine and thiazole moieties. Riboflavin (vitamin B2) is synthesized from guanosine-5′-triphosphate (GTP) and ribose-5′-phosphate. Riboflavin, in turn, is utilized for the synthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The family of compounds collectively termed ‘vitamin B6’ (e.g., pyridoxine, pyridoxamine, pyridoxa-5′-phosphate, and the commercially used pyridoxin hydrochloride) are all derivatives of the common structural unit, 5-hydroxy-6-methylpyridine. Pantothenate (pantothenic acid, (R)-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-β-alanine) can be produced either by chemical synthesis or by fermentation. The final steps in pantothenate biosynthesis consist of the ATP-driven condensation of β-alanine and pantoic acid. The enzymes responsible for the biosynthesis steps for the conversion to pantoic acid, to β-alanine and for the condensation to panthotenic acid are known. The metabolically active form of pantothenate is Coenzyme A, for which the biosynthesis proceeds in 5 enzymatic steps. Pantothenate, pyridoxal-5′-phosphate, cysteine and ATP are the precursors of Coenzyme A. These enzymes not only catalyze the formation of panthothante, but also the production of (R)-pantoic acid, (R)-pantolacton, (R)-panthenol (provitamin B5), pantetheine (and its derivatives) and coenzyme A.
Biotin biosynthesis from the precursor molecule pimeloyl-CoA in microorganisms has been studied in detail and several of the genes involved have been identified. Many of the corresponding proteins have been found to also be involved in Fe-cluster synthesis and are members of the nifS class of proteins. Lipoic acid is derived from octanoic acid, and serves as a coenzyme in energy metabolism, where it becomes part of the pyruvate dehydrogenase complex and the α-ketoglutarate dehydrogenase complex. The folates are a group of substances which are all derivatives of folic acid, which is turn is derived from L-glutamic acid, p-amino-benzoic acid and 6-methylpterin. The biosynthesis of folic acid and its derivatives, starting from the metabolism intermediates guanosine-5′-triphosphate (GTP), L-glutamic acid and p-amino-benzoic acid has been studied in detail in certain microorganisms.
Corrinoids (such as the cobalamines and particularly vitamin B12) and porphyrines belong to a group of chemicals characterized by a tetrapyrole ring system. The biosynthesis of vitamin B12 is sufficiently complex that it has not yet been completely characterized, but many of the enzymes and substrates involved are now known. Nicotinic acid (nicotinate), and nicotinamide are pyridine derivatives which are also termed ‘niacin’. Niacin is the precursor of the important coenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) and their reduced forms.
The large-scale production of these compounds has largely relied on cell-free chemical syntheses, though some of these chemicals have also been produced by large-scale culture of microorganisms, such as riboflavin, Vitamin B6, pantothenate, and biotin. Only Vitamin B12 is produced solely by fermentation, due to the complexity of its synthesis. In vitro methodologies require significant inputs of materials and time, often at great cost.
C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses
Purine and pyrimidine metabolism genes and their corresponding proteins are important targets for the therapy of tumor diseases and viral infections. The language “purine” or “pyrimidine” includes the nitrogenous bases which are constituents of nucleic acids, co-enzymes, and nucleotides. The term “nucleotide” includes the basic structural units of nucleic acid molecules, which are comprised of a nitrogenous base, a pentose sugar (in the case of RNA, the sugar is ribose; in the case of DNA, the sugar is D-deoxyribose), and phosphoric acid. The language “nucleoside” includes molecules which serve as precursors to nucleotides, but which are lacking the phosphoric acid moiety that nucleotides possess. By inhibiting the biosynthesis of these molecules, or their mobilization to form nucleic acid molecules, it is possible to inhibit RNA and DNA synthesis; by inhibiting this activity in a fashion targeted to cancerous cells, the ability of tumor cells to divide and replicate may be inhibited. Additionally, there are nucleotides which do not form nucleic acid molecules, but rather serve as energy stores (i.e., AMP) or as coenzymes (i.e., FAD and NAD).
Several publications have described the use of these chemicals for these medical indications, by influencing purine and/or pyrimidine metabolism (e.g. Christopherson, R. I. and Lyons, S. D. (1990) “Potent inhibitors of de novo pyrimidine and purine biosynthesis as chemotherapeutic agents.” Med. Res. Reviews 10: 505-548). Studies of enzymes involved in purine and pyrimidine metabolism have been focused on the development of new drugs which can be used, for example, as immunosuppressants or anti-proliferants (Smith, J. L., (1995) “Enzymes in nucleotide synthesis.” Curr. Opin. Struct. Biol. 5: 752-757; (1995) Biochem Soc. Transact. 23: 877-902). However, purine and pyrimidine bases, nucleosides and nucleotides have other utilities: as intermediates in the biosynthesis of several fine chemicals (e.g., thiamine, S-adenosyl-methionine, folates, or riboflavin), as energy carriers for the cell (e.g., ATP or GTP), and for chemicals themselves, commonly used as flavor enhancers (e.g., IMP or GMP) or for several medicinal applications (see, for example, Kuninaka, A. (1996) Nucleotides and Related Compounds in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, p. 561-612). Also, enzymes involved in purine, pyrimidine, nucleoside, or nucleotide metabolism are increasingly serving as targets against which chemicals for crop protection, including fungicides, herbicides and insecticides, are developed.
The metabolism of these compounds in bacteria has been characterized (for reviews see, for example, Zalkin, H. and Dixon, J. E. (1992) “de novo purine nucleotide biosynthesis”, in: Progress in Nucleic Acid Research and Molecular Biology, vol. 42, Academic Press:, p. 259-287; and Michal, G. (1999) “Nucleotides and Nucleosides”, Chapter 8 in: Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, Wiley: New York). Purine metabolism has been the subject of intensive research, and is essential to the normal functioning of the cell. Impaired purine metabolism in higher animals can cause severe disease, such as gout. Purine nucleotides are synthesized from ribose-5-phosphate, in a series of steps through the intermediate compound inosine-5′-phosphate (IMP), resulting in the production of guanosine-5′-monophosphate (GMP) or adenosine-5′-monophosphate (AMP), from which the triphosphate forms utilized as nucleotides are readily formed. These compounds are also utilized as energy stores, so their degradation provides energy for many different biochemical processes in the cell. Pyrimidine biosynthesis proceeds by the formation of uridine-5′-monophosphate (UMP) from ribose-5-phosphate. UMP, in turn, is converted to cytidine-5′-triphosphate (CTP). The deoxy-forms of all of these nucleotides are produced in a one step reduction reaction from the diphosphate ribose form of the nucleotide to the diphosphate deoxyribose form of the nucleotide. Upon phosphorylation, these molecules are able to participate in DNA synthesis.
D. Trehalose Metabolism and Uses
Trehalose consists of two glucose molecules, bound in α, α-1,1 linkage. It is commonly used in the food industry as a sweetener, an additive for dried or frozen foods, and in beverages. However, it also has applications in the pharmaceutical, cosmetics and biotechnology industries (see, for example, Nishimoto et al., (1998) U.S. Pat. No. 5,759,610; Singer, M. A. and Lindquist, S. (1998) Trends Biotech. 16: 460-467; Paiva, C. L. A. and Panek, A. D. (1996) Biotech. Ann. Rev. 2: 293-314; and Shiosaka, M. (1997) J. Japan 172: 97-102). Trehalose is produced by enzymes from many microorganisms and is naturally released into the surrounding medium, from which it can be collected using methods known in the art.
II. Elements and Methods of the Invention
The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as MP nucleic acid and protein molecules (see Table 1), which play a role in or function in one or more cellular metabolic pathways. In one embodiment, the MP molecules catalyze an enzymatic reaction involving one or more amino acid, e.g., lysine or methionine, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathways. In a preferred embodiment, the activity of one or more MP molecules of the present invention, alone or in combination with molecules involved in the same or different metabolic pathway (e.g., methionine or lysine metabolism), in one or more C. glutamicum metabolic pathways for amino acids, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides or trehalose has an impact on the production of a desired fine chemical by this organism. In a particularly preferred embodiment, the MP molecules of the invention are modulated in activity, such that the C. glutamicum metabolic pathways in which the MP proteins of the invention are involved are modulated in efficiency or output, which either directly or indirectly modulates the production or efficiency of production of a desired fine chemical by C. glutamicum. In a preferred embodiment, the fine chemical is an amino acid, e.g., lysine or methionine. In another preferred embodiment, the MP molecules are metZ, metY, and/or RXA00657 (see Table 1).
The language, “MP protein” or “MP polypeptide” includes proteins which play a role in, e.g., catalyze an enzymatic reaction, in one or more amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside or trehalose metabolic pathways. Examples of MP proteins include those encoded by the MP genes set forth in Table 1 and by the odd-numbered SEQ ID NOs. The terms “MP gene” or “MP nucleic acid sequence” include nucleic acid sequences encoding an MP protein, which consist of a coding region and also corresponding untranslated 5′ and 3′ sequence regions. Examples of MP genes include those set forth in Table 1. The terms “production” or “productivity” are art-recognized and include the concentration of the fermentation product (for example, the desired fine chemical) formed within a given time and a given fermentation volume (e.g., kg product per hour per liter). The term “efficiency of production” includes the time required for a particular level of production to be achieved (for example, how long it takes for the cell to attain a particular rate of output of a fine chemical). The term “yield” or “product/carbon yield” is art-recognized and includes the efficiency of the conversion of the carbon source into the product (i.e., fine chemical). This is generally written as, for example, kg product per kg carbon source. By increasing the yield or production of the compound, the quantity of recovered molecules, or of useful recovered molecules of that compound in a given amount of culture over a given amount of time is increased. The terms “biosynthesis” or a “biosynthetic pathway” are art-recognized and include the synthesis of a compound, preferably an organic compound, by a cell from intermediate compounds in what may be a multistep and highly regulated process. The terms “degradation” or a “degradation pathway” are art-recognized and include the breakdown of a compound, preferably an organic compound, by a cell to degradation products (generally speaking, smaller or less complex molecules) in what may be a multistep and highly regulated process. The language “metabolism” is art-recognized and includes the totality of the biochemical reactions that take place in an organism. The metabolism of a particular compound, then, (e.g., the metabolism of an amino acid such as glycine) comprises the overall biosynthetic, modification, and degradation pathways in the cell related to this compound.
The MP molecules of the present invention may be combined with one or more MP molecules of the invention or one or more molecules of the same or different metabolic pathway to increase the yield of a desired fine chemical. In a preferred embodiment, the fine chemical is an amino acid, e.g., lysine or methionine. Alternatively, or in addition, a byproduct which is not desired may be reduced by combination or disruption of MP molecules or other metabolic molecules (e.g., molecules involved in lysine or methionine metabolism). MP molecules combined with other molecules of the same or a different metabolic pathway may be altered in their nucleotide sequence and in the corresponding amino acid sequence to alter their activity under physiological conditions, which leads to an increase in productivity and/or yield of a desired fine chemical. In a further embodiment, an MP molecule in its original or in its above-described altered form may be combined with other molecules of the same or a different metabolic pathway which are altered in their nucleotide sequence in such a way that their activity is altered under physiological conditions which leads to an increase in productivity and/or yield of a desired fine chemical, e.g., an amino acid such as methionine or lysine.
In another embodiment, the MP molecules of the invention, alone or in combination with one or more molecules of the same or different metabolic pathway, are capable of modulating the production of a desired molecule, such as a fine chemical, in a microorganism such as C. glutamicum. Using recombinant genetic techniques, one or more of the biosynthetic or degradative enzymes of the invention for amino acids, e.g., lysine or methionine, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides, or trehalose may be manipulated such that its function is modulated. For example, a biosynthetic enzyme may be improved in efficiency, or its allosteric control region destroyed such that feedback inhibition of production of the compound is prevented. Similarly, a degradative enzyme may be deleted or modified by substitution, deletion, or addition such that its degradative activity is lessened for the desired compound without impairing the viability of the cell. In each case, the overall yield or rate of production of one of these desired fine chemicals may be increased.
It is also possible that such alterations in the protein and nucleotide molecules of the invention may improve the production of other fine chemicals besides the amino acids, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides, and trehalose. Metabolism of any one compound is necessarily intertwined with other biosynthetic and degradative pathways within the cell, and necessary cofactors, intermediates, or substrates in one pathway are likely supplied or limited by another such pathway. Therefore, by modulating the activity of one or more of the proteins of the invention, the production or efficiency of activity of another fine chemical biosynthetic or degradative pathway may be impacted. For example, amino acids serve as the structural units of all proteins, yet may be present intracellularly in levels which are limiting for protein synthesis; therefore, by increasing the efficiency of production or the yields of one or more amino acids within the cell, proteins, such as biosynthetic or degradative proteins, may be more readily synthesized. Likewise, an alteration in a metabolic pathway enzyme such that a particular side reaction becomes more or less favored may result in the over- or under-production of one or more compounds which are utilized as intermediates or substrates for the production of a desired fine chemical.
The isolated nucleic acid sequences of the invention are contained within the genome of a Corynebacterium glutamicum strain available through the American Type Culture Collection, given designation ATCC 13032. The nucleotide sequence of the isolated C. glutamicum MP DNAs and the predicted amino acid sequences of the C. glutamicum MP proteins are shown in the Sequence Listing as odd-numbered SEQ ID NOs and even-numbered SEQ ID NOs, respectively. Computational analyses were performed which classified and/or identified these nucleotide sequences as sequences which encode metabolic pathway proteins, e.g., proteins involved in the methionine or lysine metabolic pathways.
The present invention also pertains to proteins which have an amino acid sequence which is substantially homologous to an amino acid sequence of the invention (e.g., the sequence of an even-numbered SEQ ID NO of the Sequence Listing). As used herein, a protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence is least about 50% homologous to the selected amino acid sequence, e.g., the entire selected amino acid sequence. A protein which has an amino acid sequence which is substantially homologous to a selected amino acid sequence can also be least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more homologous to the selected amino acid sequence.
An MP protein of the invention, or a biologically active portion or fragment thereof, alone or in combination with one or more proteins of the same or different metabolic pathway, can catalyze an enzymatic reaction in one or more amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathways, or have one or more of the activities set forth in Table 1 (e.g., metabolism of methionine or lysine biosynthesis).
Various aspects of the invention are described in further detail in the following subsections:
A. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that encode MP polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes or primers for the identification or amplification of MP-encoding nucleic acid (e.g., MP DNA). As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. This term also encompasses untranslated sequence located at both the 3′ and 5′ ends of the coding region of the gene: at least about 100 nucleotides of sequence upstream from the 5′ end of the coding region and at least about 20 nucleotides of sequence downstream from the 3′end of the coding region of the gene. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated MP nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (e.g, a C. glutamicum cell). Moreover, an “isolated” nucleic acid molecule, such as a DNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having a nucleotide sequence of an odd-numbered SEQ ID NO of the Sequence Listing, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a C. glutamicum MP DNA can be isolated from a C. glutamicum library using all or portion of one of the odd-numbered SEQ ID NO sequences of the Sequence Listing as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO:) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence (e.g., a nucleic acid molecule encompassing all or a portion of one of the nucleic acid sequences of the invention (e.g., an odd-numbered SEQ ID NO of the Sequence Listing) can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this same sequence). For example, mRNA can be isolated from normal endothelial cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299) and DNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers for polymerase chain reaction amplification can be designed based upon one of the nucleotide sequences shown in the Sequence Listing. A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to an MP nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises one of the nucleotide sequences shown in the Sequence Listing. The nucleic acid sequences of the invention, as set forth in the Sequence Listing, correspond to the Corynebacterium glutamicum MP DNAs of the invention. This DNA comprises sequences encoding MP proteins (i.e., the “coding region”, indicated in each odd-numbered SEQ ID NO: sequence in the Sequence Listing), as well as 5′ untranslated sequences and 3′ untranslated sequences, also indicated in each odd-numbered SEQ ID NO: in the Sequence Listing. Alternatively, the nucleic acid molecule can comprise only the coding region of any of the nucleic acid sequences of the Sequence Listing.
For the purposes of this application, it will be understood that some of the MP nucleic acid and amino acid sequences set forth in the Sequence Listing have an identifying RXA, RXN, RXS, or RXC number having the designation “RXA”, “RXN”, “RXS”, or “RXC” followed by 5 digits (i.e., RXA, RXN, RXS, or RXC). Each of the nucleic acid sequences comprises up to three parts: a 5′ upstream region, a coding region, and a downstream region. Each of these three regions is identified by the same RXA, RXN, RXS, or RXC designation to eliminate confusion. The recitation “one of the odd-numbered sequences of the Sequence Listing”, then, refers to any of the nucleic acid sequences in the Sequence Listing, which may also be distinguished by their differing RXA, RXN, RXS, or RXC designations. The coding region of each of these sequences is translated into a corresponding amino acid sequence, which is also set forth in the Sequence Listing, as an even-numbered SEQ ID NO: immediately following the corresponding nucleic acid sequence. For example, the coding region for RXA00115 is set forth in SEQ ID NO:69, while the amino acid sequence which it encodes is set forth as SEQ ID NO:70. The sequences of the nucleic acid molecules of the invention are identified by the same RXA, RXN, RXS, or RXC designations as the amino acid molecules which they encode, such that they can be readily correlated. For example, the amino acid sequences designated RXA00115, RXN00403, and RXS03158 are translations of the coding regions of the nucleotide sequences of nucleic acid molecules RXA00115, RXN00403, and RXS03158, respectively. The correspondence between the RXA, RXN, RXS, and RXC nucleotide and amino acid sequences of the invention and their assigned SEQ ID NOs is set forth in Table 1.
Several of the genes of the invention are “F-designated genes”. An F-designated gene includes those genes set forth in Table 1 which have an ‘F’ in front of the RXA, RXN, RXS, or RXC designation. For example, SEQ ID NO:77, designated, as indicated on Table 1, as “F RXA00254”, is an F-designated gene.
Also listed on Table 1 are the metZ (or metY) and metC genes (designated as SEQ ID NO:1 and SEQ ID NO:3, respectively. The corresponding amino acid sequence encoded by the metZ and metC genes are designated as SEQ ID NO:2 and SEQ ID NO:5, respectively.
In one embodiment, the nucleic acid molecules of the present invention are not intended to include those compiled in Table 2.
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of one of the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof. A nucleic acid molecule which is complementary to one of the nucleotide sequences of the invention is one which is sufficiently complementary to one of the nucleotide sequences shown in the Sequence Listing (e.g., the sequence of an odd-numbered SEQ ID NO:) such that it can hybridize to one of the nucleotide sequences of the invention, thereby forming a stable duplex.
In still another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more homologous to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing), or a portion thereof. Ranges and identity values intermediate to the above-recited ranges, (e.g., 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. In an additional preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to one of the nucleotide sequences of the invention, or a portion thereof.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the coding region of the sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of an MP protein. The nucleotide sequences determined from the cloning of the MP genes from C. glutamicum allows for the generation of probes and primers designed for use in identifying and/or cloning MP homologues in other cell types and organisms, as well as MP homologues from other Corynebacteria or related species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense strand of one of the nucleotide sequences of the invention (e.g., a sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing), an anti-sense sequence of one of these sequences, or naturally occurring mutants thereof. Primers based on a nucleotide sequence of the invention can be used in PCR reactions to clone MP homologues. Probes based on the MP nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells which misexpress an MP protein, such as by measuring a level of an MP-encoding nucleic acid in a sample of cells from a subject e.g., detecting MP mRNA levels or determining whether a genomic MP gene has been mutated or deleted.
In one embodiment, the nucleic acid molecule of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO of the Sequence Listing) such that the protein or portion thereof maintains the ability to catalyze an enzymatic reaction in an amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathway. As used herein, the language “sufficiently homologous” refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain as an amino acid residue in a sequence of one of the even-numbered SEQ ID NOs of the Sequence Listing) amino acid residues to an amino acid sequence of the invention such that the protein or portion thereof is able to catalyze an enzymatic reaction in a C. glutamicum amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside or trehalose metabolic pathway. Protein members of such metabolic pathways, as described herein, function to catalyze the biosynthesis or degradation of one or more of: amino acids, vitamins, cofactors, nutraceuticals, nucleotides, nucleosides, or trehalose. Examples of such activities are also described herein. Thus, “the function of an MP protein” contributes to the overall functioning of one or more such metabolic pathway and contributes, either directly or indirectly, to the yield, production, and/or efficiency of production of one or more fine chemicals. Examples of MP protein activities are set forth in Table 1.
In another embodiment, the protein is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more homologous to an entire amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing).
Portions of proteins encoded by the MP nucleic acid molecules of the invention are preferably biologically active portions of one of the MP proteins. As used herein, the term “biologically active portion of an MP protein” is intended to include a portion, e.g., a domain/motif, of an MP protein that catalyzes an enzymatic reaction in one or more C. glutamicum amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathways, or has an activity as set forth in Table 1. To determine whether an MP protein or a biologically active portion thereof can catalyze an enzymatic reaction in an amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathway, an assay of enzymatic activity may be performed. Such assay methods are well known to those of ordinary skill in the art, as detailed in Example 8 of the Exemplification.
Additional nucleic acid fragments encoding biologically active portions of an MP protein can be prepared by isolating a portion of one of the amino acid sequences of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing), expressing the encoded portion of the MP protein or peptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the MP protein or peptide.
The invention further encompasses nucleic acid molecules that differ from one of the nucleotide sequences of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing) (and portions thereof) due to degeneracy of the genetic code and thus encode the same MP protein as that encoded by the nucleotide sequences of the invention. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in the Sequence Listing (e.g., an even-numbered SEQ ID NO:). In a still further embodiment, the nucleic acid molecule of the invention encodes a full length C. glutamicum protein which is substantially homologous to an amino acid sequence of the invention (encoded by an open reading frame shown in an odd-numbered SEQ ID NO: of the Sequence Listing).
It will be understood by one of ordinary skill in the art that in one embodiment the sequences of the invention are not meant to include the sequences of the prior art, such as those Genbank sequences set forth in Table 2, which was available prior to the present invention. In one embodiment, the invention includes nucleotide and amino acid sequences having a percent identity to a nucleotide or amino acid sequence of the invention which is greater than that of a sequence of the prior art (e.g., a Genbank sequence (or the protein encoded by such a sequence) set forth in Table 2). For example, the invention includes a nucleotide sequence which is greater than and/or at least 45% identical to the nucleotide sequence designated RXA00657 SEQ ID NO:5 One of ordinary skill in the art would be able to calculate the lower threshold of percent identity for any given sequence of the invention by examining the GAP-calculated percent identity scores set forth in Table 4 for each of the three top hits for the given sequence, and by subtracting the highest GAP-calculated percent identity from 100 percent. One of ordinary skill in the art will also appreciate that nucleic acid and amino acid sequences having percent identities greater than the lower threshold so calculated (e.g., at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%; 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more identical) are also encompassed by the invention.
In addition to the C. glutamicum MP nucleotide sequences set forth in the Sequence Listing as odd-numbered SEQ ID NOs, it will be appreciated by one of ordinary skill in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of MP proteins may exist within a population (e.g., the C. glutamicum population). Such genetic polymorphism in the MP gene may exist among individuals within a population due to natural variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding an MP protein, preferably a C. glutamicum MP protein. Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the MP gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in MP that are the result of natural variation and that do not alter the functional activity of MP proteins are intended to be within the scope of the invention.
Nucleic acid molecules corresponding to natural variants and non-C. glutamicum homologues of the C. glutamicum MP DNA of the invention can be isolated based on their homology to the C. glutamicum MP nucleic acid disclosed herein using the C. glutamicum DNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising a nucleotide sequence of an odd-numbered SEQ ID NO: of the Sequence Listing. In other embodiments, the nucleic acid is at least 30, 50, 100, 250 or more nucleotides in length. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 65%, more preferably at least about 70%, and even more preferably at least about 75% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to one of ordinary skill in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to a nucleotide sequence of the invention corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural C. glutamicum MP protein.
In addition to naturally-occurring variants of the MP sequence that may exist in the population, one of ordinary skill in the art will further appreciate that changes can be introduced by mutation into a nucleotide sequence of the invention, thereby leading to changes in the amino acid sequence of the encoded MP protein, without altering the functional ability of the MP protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in a nucleotide sequence of the invention. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of one of the MP proteins (e.g., an even-numbered SEQ ID NO: of the Sequence Listing) without altering the activity of said MP protein, whereas an “essential” amino-acid residue is required for MP protein activity. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved in the domain having MP activity) may not be essential for activity and thus are likely to be amenable to alteration without altering MP activity.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding MP proteins that contain changes in amino acid residues that are not essential for MP activity. Such MP proteins differ in amino acid sequence from a sequence of an even-numbered SEQ ID NO: of the Sequence Listing yet retain at least one of the MP activities described herein. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50% homologous to an amino acid sequence of the invention and is capable of catalyzing an enzymatic reaction in an amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathway, or has one or more activities set forth in Table 1. Preferably, the protein encoded by the nucleic acid molecule is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% homologous to one of the amino acid sequences of the invention.
To determine the percent homology of two amino acid sequences (e.g., one of the amino acid sequences of the invention and a mutant form thereof) or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence (e.g., one of the amino acid sequences of the invention) is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence (e.g., a mutant form of the amino acid sequence), then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid “homology” is equivalent to amino acid or nucleic acid “identity”). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100).
An isolated nucleic acid molecule encoding an MP protein homologous to a protein sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) can be created by introducing one or more nucleotide substitutions, additions or deletions into a nucleotide sequence of the invention such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into one of the nucleotide sequences of the invention by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an MP protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an MP coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an MP activity described herein to identify mutants that retain MP activity. Following mutagenesis of the nucleotide sequence of one of the odd-numbered SEQ ID NOs of the Sequence Listing, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using, for example, assays described herein (see Example 8 of the Exemplification).
In addition to the nucleic acid molecules encoding MP proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An “antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded DNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire MP coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding an MP protein. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the entire coding region of SEQ ID NO.:1 (metZ) comprises nucleotides 363 to 1673). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding MP. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).
Given the coding strand sequences encoding MP disclosed herein (e.g., the sequences set forth as odd-numbered SEQ ID NOs in the Sequence Listing), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of MP mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of MP mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of MP mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a cell or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an MP protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense molecule can be modified such that it specifically binds to a receptor or an antigen expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecule to a peptide or an antibody which binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong prokaryotic, viral, or eukaryotic promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave MP mRNA transcripts to thereby inhibit translation of MP mRNA. A ribozyme having specificity for an MP-encoding nucleic acid can be designed based upon the nucleotide sequence of an MP DNA disclosed herein (i.e., SEQ ID NO:1 (metZ). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an MP-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, MP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.
Alternatively, MP gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an MP nucleotide sequence (e.g., an MP promoter and/or enhancers) to form triple helical structures that prevent transcription of an MP gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.
Another aspect of the invention pertains to combinations of genes involved in methionine and/or lysine metabolism and the use of to combinations of genes involved in methionine and/or lysine metabolism in the methods of the invention. Preferred combinations are the combination of metZ with metC, metB (encoding Cystathionine-Synthase), metA (encoding homoserine-O-acetyltransferase), metE (encoding Methionine Synthase), metH (encoding Methionine Synthase), hom (encoding homoserine dehydrogenase), asd (encoding aspartatesemialdehyd dehydrogenase), lysC/ask (encoding aspartokinase) and rxa00657 (herein designated as SEQ ID NO.:5), dapA, (gene encoding DIHYDRODIPICOLINATE SYNTHASE), dapB (gene encoding DIHYDRODIPICOLINATE REDUCTASE), dapC (gene encoding 2,3,4,5-tetrahydropyridine-2-carboxylate N-succinyltransferase), dapD/argD (gene encoding acetylornithine transaminase), dapE (gene encoding succinyldiaminopimelate desuccinylase), dapF (gene encoding diaminopimelate epimerase), lysA (gene encoding diaminopimelate decarboxylase), ddh (gene encoding diaminopimelate dehydrogenase), lysE (gene encoding for the lysine exporter), lysG (gene encoding for the exporter regulator), hsk (gene encoding homoserine kinase) as well as genes involved in anaplerotic reaction such as ppc (gene encoding phosphoenolpyruvate carboxylase), ppcK (gene encoding phosphoenolpyruvate carboxykinase), pycA (gene encoding pyruvate carboxylase), accD, accA, accB, accC (genes encoding for subunits of acetyl-CoA-carboxylase), as well as genes of the pentose-phosphate pathway, gpdh genes encoding glucose-6-phophate-dehydrogenase, opcA, pgdh (gene encoding 6-phosphogluconate-dehydrogenase), ta (gene encoding transaldolase), tk (gene encoding gene encoding transketolase), pgl (gene encoding 6-PHOSPHOGLUCONO-LACTONASE), ripe (gene encoding RIBULOSE-PHOSPHATE 3-EPIMERASE) rpe (gene encoding RIBOSE 5-PHOSPHATE EPIMERASE) or combinations of the above-mentioned genes of the pentose-phosphate-pathways, or other MP genes of the invention.
The genes may be altered in their nucleotide sequence and in the corresponding amino acid sequence resulting in derivatives in such a way that their activity is altered under physiological conditions which leads to an increase in productivity and/or yield of a desired fine chemical, e.g., an amino acid such as methionine or lysine. One class of such alterations or derivatives is well known for the nucleotide sequence of the ask gene encoding aspartokinase. These alterations lead to removal of feed back inhibition by the amino acids lysine and threonine and subsequently to lysine overproduction. In a preferred embodiment the metZ gene or altered forms of the metZ gene are used in a Corynebacterium strain in combination with ask, hom, metA and metH or derivatives of these genes. In another preferred embodiment metZ or altered forms of the metZ gene are used in a Corynebacterium strain in combination with ask, hom, metA and metE or derivatives of these genes. In a more preferred embodiment, the gene combinations metZ or altered forms of the metZ gene are combined with ask, hom, metA and metH or derivatives of these genes, or metZ is combined with ask hom, metA and metE or derivatives of these genes in a Corynebacterium strain and sulfur sources such as sulfates, thiosulfates, sulfites and also more reduced sulfur sources such as H2S and sulfides and derivatives are used in the growth medium. Also, sulfur sources such as methyl mercaptan, methanesulfonic acid, thioglycolates, thiocyanates, thiourea, sulfur containing amino acids such as cysteine and other sulfur containing compounds can be used. Another aspect of the invention pertains to the use of the above mentioned gene combinations in a Corynebacterium strain which is, before or after introduction of the genes, mutagenized by radiation or by mutagenic chemicals well-known to one of ordinary skill in the art and selected for resistance against high concentrations of the fine chemical of interest, e.g. lysine or methionine or analogues of the desired fine chemical such as the methionine analogues ethionine, methyl methionine, or others. In another embodiment, the gene combinations mentioned above can be expressed in a Corynebacterium strain having particular gene disruptions. Preferred are gene disruptions that encode proteins that favor carbon flux to undesired metabolites. Where methionine is the desired fine chemical the formation of lysine may be unfavorable. In such a case the combination of the above mentioned genes should proceed in a Corynebacterium strain bearing a gene disruption of the lysA gene (encoding diaminopimelate decarboxylase) or the ddh gene (encoding the meso-diaminopimelate dehydrogenase catalysing the conversion of tetrahydropicolinate to meso-diaominopimelate). In a preferred embodiment, a favorable combination of the above-mentioned genes are all altered in such a way that their gene products are not feed back inhibited by end products or metabolites of the biosynthetic pathway leading to the desired fine chemical. In the case that the desired fine chemical is methionine, the gene combinations may be expressed in a strain previously treated with mutagenic agents or radiation and selected for the above-mentioned resistance. Additionally, the strain should be grown in a growth medium containing one or more of the above mentioned sulfur sources.
In another embodiment of the invention, a gene was identified from the genome of Corynebacterium glutamicum as a gene coding for a hypothetical transcriptional regulatory protein. This gene is described as RXA00657. The nucleotide sequence of RXA00657 corresponds to SEQ ID NO:5. The amino acid sequence of RXA00657 corresponds to SEQ ID NO:6. It was found that when the RXA00657 gene, as well as upstream and downstream regulatory regions described in the examples, was cloned into a vector capable of replicating in Corynebacterium glutamicum and transformed and expressed in a lysine producing strain such as ATCC13286, that this strain produced more lysine compared to the strain transformed with the same plasmid lacking the aforementioned nucleotide fragment RXA00657. In addition to the observation that the lysine titer was increased in the mentioned strain, the selectivity determined by the molar amount of lysine produced compared to the molar amount of sucrose consumed was increased (see Example 14). Overexpression of RXA00657 in combination with the overexpression of other genes either directly involved in the lysine specific pathway such as lysC, dapA, dapB, dapC, dapD, dapF, ddh, lysE, lysG, and lysR results in an increase in the production of lysine compared to RXA00657 alone.
B. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an MP protein (or a portion thereof) or combinations of genes wherein at least one gene encodes for an MP protein. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, repressor binding sites, activator binding sites, enhancers and other expression control elements (e.g., terminators, polyadenylation signals, or other elements of mRNA secondary structure). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells. Preferred regulatory sequences are, for example, promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-, lpp-lac-, lacIq, T7-, T5-, T3-, gal-, trc-, ara-, SP6-, arny, SPO2, λ-PR- or λ PL, which are used preferably in bacteria. Additional regulatory sequences are, for example, promoters from yeasts and fungi, such as ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH, promoters from plants such as CaMV/35S, SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin- or phaseolin-promoters. It is also possible to use artificial promoters. It will be appreciated by one of ordinary skill in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., MP proteins, mutant forms of MP proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of MP proteins in prokaryotic or eukaryotic cells. For example, MP genes can be expressed in bacterial cells such as C. glutamicum, insect cells (using baculovirus expression vectors), yeast and other fungal cells (see Romanos, M. A. et al. (1992) “Foreign gene expression in yeast: a review”, Yeast 8: 423-488; van den Hondel, C. A. M. J. J. et al. (1991) “Heterologous gene expression in filamentous fungi” in: More Gene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p. 396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J. F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae and multicellular plant cells-(see Schmidt, R. and Willmitzer, L. (1988) High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledon explants” Plant Cell Rep.: 583-586), or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein but also to the C-terminus or fused within suitable regions in the proteins. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. In one embodiment, the coding sequence of the MP protein is cloned into a pGEX expression vector to create a vector encoding a fusion protein comprising, from the N-terminus to the C-terminus, GST-thrombin cleavage site-X protein. The fusion protein can be purified by affinity chromatography using glutathione-agarose resin. Recombinant MP protein unfused to GST can be recovered by cleavage of the fusion protein with thrombin.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11, pBdCl, and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89; and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter. For transformation of other varieties of bacteria, appropriate vectors may be selected. For example, the plasmids pIJ101, pIJ364, pIJ702 and pIJ361 are known to be useful in transforming Streptomyces, while plasmids pUB110, pC194, or pBD214 are suited for transformation of Bacillus species. Several plasmids of use in the transfer of genetic information into Corynebacterium include pHM1519, pBL1, pSA77, or pAJ667 (Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
One strategy to maximize recombinant protein expression is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the bacterium chosen for expression, such as C. glutamicum (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the MP protein expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234), 2μ, pAG-1, Yep6, Yep13, pEMBLYe23, pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and methods for the construction of vectors appropriate for use in other fungi, such as the filamentous fungi, include those detailed in: van den Hondel, C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge University Press: Cambridge, and Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York (IBSN 0 444 904018):
Alternatively, the MP proteins of the invention can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In another embodiment, the MP proteins of the invention may be expressed in unicellular plant cells (such as algae) or in plant cells from higher plants (e.g., the spermatophytes, such as crop plants). Examples of plant expression vectors include those detailed in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) “New plant binary vectors with selectable markers located proximal to the left border”, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W. (1984) “Binary Agrobacterium vectors for plant transformation”, Nucl. Acid. Res. 12: 8711-8721, and include pLGV23, pGHlac+, pBIN19, pAK2004, and pDH51 (Pouwels et al., eds. (1985) Cloning Vectors. Elsevier: New York IBSN 0 444 904018).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to MP mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, an MP protein can be expressed in bacterial cells such as C. glutamicum, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those of ordinary skill in the art. Microorganisms related to Corynebacterium glutamicum which may be conveniently used as host cells for the nucleic acid and protein molecules of the invention are set forth in Table 3.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection”, “conjugation” and “transduction” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., linear DNA or RNA (e.g., a linearized vector or a gene construct alone without a vector) or nucleic acid in the form of a vector (e.g., a plasmid, phage, phasmid, phagemid, transposon or other DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an MP protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
To create a homologous recombinant microorganism, a vector is prepared which contains at least a portion of an MP gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the MP gene. Preferably, this MP gene is a Corynebacterium glutamicum MP gene, but it can be a homologue from a related bacterium or even from a mammalian, yeast, or insect source. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous MP gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous MP gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous MP protein). In the homologous recombination vector, the altered portion of the MP gene is flanked at its 5′ and 3′ ends by additional nucleic acid of the MP gene to allow for homologous recombination to occur between the exogenous MP gene carried by the vector and an endogenous MP gene in a microorganism. The additional flanking MP nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see e.g., Thomas, K. R., and Capecchi, M. R. (1987) Cell 51: 503 for a description of homologous recombination vectors). The vector is introduced into a microorganism (e.g., by electroporation) and cells in which the introduced MP gene has homologously recombined with the endogenous MP gene are selected, using art-known techniques.
In another embodiment, recombinant microorganisms can be produced which contain selected systems which allow for regulated expression of the introduced gene. For example, inclusion of an MP gene on a vector placing it under control of the lac operon permits expression of the MP gene only in the presence of IPTG. Such regulatory systems are well known in the art.
In another embodiment, an endogenous MP gene in a host cell is disrupted (e.g., by homologous recombination or other genetic means known in the art) such that expression of its protein product does not occur. In another embodiment, an endogenous or introduced MP gene in a host cell has been altered by one or more point mutations, deletions, or inversions, but still encodes a functional MP protein. In still another embodiment, one or more of the regulatory regions (e.g., a promoter, repressor, or inducer) of an MP gene in a microorganism has been altered (e.g., by deletion, truncation, inversion, or point mutation) such that the expression of the MP gene is modulated. One of ordinary skill in the art will appreciate that host cells containing more than one of the described MP gene and protein modifications may be readily produced using the methods of the invention, and are meant to be included in the present invention.
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an MP protein. Accordingly, the invention further provides methods for producing MP proteins using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an MP protein has been introduced, or into which genome has been introduced a gene encoding a wild-type or altered MP protein) in a suitable medium until MP protein is produced. In another embodiment, the method further comprises isolating MP proteins from the medium or the host cell.
C. Isolated MP Proteins
Another aspect of the invention pertains to isolated MP proteins, and biologically active portions thereof. An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of MP protein in which the protein is separated from cellular components of the cells in which it is naturally or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of MP protein having less than about 30% (by dry weight) of non-MP protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-MP protein, still more preferably less than about 10% of non-MP protein, and most preferably less than about 5% non-MP protein. When the MP protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. The language “substantially free of chemical precursors or other chemicals” includes preparations of MN protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of MP protein having less than about 30% (by dry weight) of chemical precursors or non-MP chemicals, more preferably less than about 20% chemical precursors or non-MP chemicals, still more preferably less than about 10% chemical precursors or non-MP chemicals, and most preferably less than about 5% chemical precursors or non-MP chemicals. In preferred embodiments, isolated proteins or biologically active portions thereof lack contaminating proteins from the same organism from which the MP protein is derived. Typically, such proteins are produced by recombinant expression of, for example, a C. glutamicum MP protein in a microorganism such as C. glutamicum. 
An isolated MP protein or a portion thereof of the invention can catalyze an enzymatic reaction in an amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathway, or has one or more of the activities set forth in Table 1. In preferred embodiments, the protein or portion thereof comprises an amino acid sequence which is sufficiently homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) such that the protein or portion thereof maintains the ability to catalyze an enzymatic reaction in an amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathway. The portion of the protein is preferably a biologically active portion as described herein. In another preferred embodiment, an MP protein of the invention has an amino acid sequence set forth as an even-numbered SEQ ID NO: of the Sequence Listing. In yet another preferred embodiment, the MP protein has an amino acid sequence which is encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention (e.g., a sequence of an odd-numbered SEQ ID NO: of the Sequence Listing). In still another preferred embodiment, the MP protein has an amino acid sequence which is encoded by a nucleotide sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more homologous to one of the nucleic acid sequences of the invention, or a portion thereof. Ranges and identity values intermediate to the above-recited values, (e.g., 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. The preferred MP proteins of the present invention also preferably possess at least one of the MP activities described herein. For example, a preferred MP protein of the present invention includes an amino acid sequence encoded by a nucleotide sequence which hybridizes, e.g., hybridizes under stringent conditions, to a nucleotide sequence of the invention, and which can catalyze an enzymatic reaction in an amino acid, vitamin, cofactor, nutraceutical, nucleotide, nucleoside, or trehalose metabolic pathway, or which has one or more of the activities set forth in Table 1.
In other embodiments, the MP protein is substantially homologous to an amino acid sequence of the invention (e.g., a sequence of an even-numbered SEQ ID NO: of the Sequence Listing) and retains the functional activity of the protein of one of the amino acid sequences of the invention yet differs in amino acid sequence due to natural variation or mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the MP protein is a protein which comprises an amino acid sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90%, or 91%, 92%, 93%, 94%, and even more preferably at least about 95%, 96%, 97%, 98%, 99%, 99.7% or more homologous to an entire amino acid sequence of the invention and which has at least one of the MP activities described herein. Ranges and identity values intermediate to the above-recited values, (e.g., 70-90% identical or 80-95% identical) are also intended to be encompassed by the present invention. For example, ranges of identity values using a combination of any of the above values recited as upper and/or lower limits are intended to be included. In another embodiment, the invention pertains to a full length C. glutamicum protein which is substantially homologous to an entire amino acid sequence of the invention.
Biologically active portions of an MP protein include peptides comprising amino acid sequences derived from the amino acid sequence of an MP protein, e.g., an amino acid sequence of an even-numbered SEQ ID NO: of the Sequence Listing or the amino acid sequence of a protein homologous to an MP protein, which include fewer amino acids than a full length MP protein or the full length protein which is homologous to an MP protein, and exhibit at least one activity of an MP protein. Typically, biologically active portions (peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise a domain or motif with at least one activity of an MP protein. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein. Preferably, the biologically active portions of an MP protein include one or more selected domains/motifs or portions thereof having biological activity.
MP proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the MP protein is expressed in the host cell. The MP protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, an MP protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native MP protein can be isolated from cells (e.g., endothelial cells), for example using an anti-MP antibody, which can be produced by standard techniques utilizing an MP protein or fragment thereof of this invention.
The invention also provides MP chimeric or fusion proteins. As used herein, an MP “chimeric protein” or “fusion protein” comprises an MP polypeptide operatively linked to a non-MP polypeptide. An “MP polypeptide” refers to a polypeptide having an amino acid sequence corresponding to MP, whereas a “non-MP polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the MP protein, e.g., a protein which is different from the MP protein and which is derived from the same or a different organism. Within the fusion protein, the term “operatively linked” is intended to indicate that the MP polypeptide and the non-MP polypeptide are fused in-frame to each other. The non-MP polypeptide can be fused to the N-terminus or C-terminus of the MP polypeptide. For example, in one embodiment the fusion protein is a GST-MP fusion protein in which the MP sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant MP proteins. In another embodiment, the fusion protein is an MP protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of an MP protein can be increased through use of a heterologous signal sequence.
Preferably, an MP chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques; For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An MP-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the MP protein.
Homologues of the MP protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the MP protein. As used herein, the term “homologue” refers to a variant form of the MP protein which acts as an agonist or antagonist of the activity of the MP protein. An agonist of the MP protein can retain substantially the same, or a subset, of the biological activities of the MP protein. An antagonist of the MP protein can inhibit one or more of the activities of the naturally occurring form of the MP protein, by, for example, competitively binding to a downstream or upstream member of the MP cascade which includes the MP protein. Thus, the C. glutamicum MP protein and homologues thereof of the present invention may modulate the activity of one or more metabolic pathways in which MP proteins play a role in this microorganism.
In an alternative embodiment, homologues of the MP protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the MP protein for MP protein agonist or antagonist activity. In one embodiment, a variegated library of MP variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of MP variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential MP sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of MP sequences therein. There are a variety of methods which can be used to produce libraries of potential MP homologues from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential MP sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of the MP protein coding can be used to generate a variegated population of MP fragments for screening and subsequent selection of homologues of an MP protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an MP coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the MP protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of MP homologues. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify MP homologues (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
In another embodiment, cell based assays can be exploited to analyze a variegated MP library, using methods well known in the art.
D. Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, fusion proteins, primers, vectors, and host cells described herein can be used in one or more of the following methods: identification of C. glutamicum and related organisms; mapping of genomes of organisms related to C. glutamicum; identification and localization of C. glutamicum sequences of interest; evolutionary studies; determination of MP protein regions required for function; modulation of an MP protein activity; modulation of the activity of an MP pathway; and modulation of cellular production of a desired compound, such as a fine chemical.
The MP nucleic acid molecules of the invention have a variety of uses. First, they may be used to identify an organism as being Corynebacterium glutamicum or a close relative thereof. Also, they may be used to identify the presence of C. glutamicum or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of C. glutamicum genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a C. glutamicum gene which is unique to this organism, one can ascertain whether this organism is present. Although Corynebacterium glutamicum itself is not pathogenic to humans, it is related to species which are human pathogens, such as Corynebacterium diphtheriae. Corynebacterium diphtheriae is the causative agent of diphtheria, a rapidly developing, acute, febrile infection which involves both local and systemic pathology. In this disease, a local lesion develops in the upper respiratory tract and involves necrotic injury to epithelial cells; the bacilli secrete toxin which is disseminated through this lesion to distal susceptible tissues of the body. Degenerative changes brought about by the inhibition of protein synthesis in these tissues, which include heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen, result in the systemic pathology of the disease. Diphtheria continues to have high incidence in many parts of the world, including Africa, Asia, Eastern Europe and the independent states of the former Soviet Union. An ongoing epidemic of diphtheria in the latter two regions has resulted in at least 5,000 deaths since 1990.
In one embodiment, the invention provides a method of identifying the presence or activity of Cornyebacterium diphtheriae in a subject. This method includes detection of one or more of the nucleic acid or amino acid sequences of the invention (e.g., the sequences set forth as odd-numbered or even-numbered SEQ ID NOs, respectively, in the Sequence Listing) in a subject, thereby detecting the presence or activity of Corynebacterium diphtheriae in the subject. C. glutamicum and C. diphtheriae are related bacteria, and many of the nucleic acid and protein molecules in C. glutamicum are homologous to C. diphtheriae nucleic acid and protein molecules, and can therefore be used to detect C. diphtheriae in a subject.
The nucleic acid and protein molecules of the invention may also serve as markers for specific regions of the genome. This has utility not only in the mapping of the genome, but also for functional studies of C. glutamicum proteins. For example, to identify the region of the genome to which a particular C. glutamicum DNA-binding protein binds, the C. glutamicum genome could be digested, and the fragments incubated with the DNA-binding protein. Those which bind the protein may be additionally probed with the nucleic acid molecules of the invention, preferably with readily detectable labels; binding of such a nucleic acid molecule to the genome fragment enables the localization of the fragment to the genome map of C. glutamicum, and, when performed multiple times with different enzymes, facilitates a rapid determination of the nucleic acid sequence to which the protein binds. Further, the nucleic acid molecules of the invention may be sufficiently homologous to the sequences of related species such that these nucleic acid molecules may serve as markers for the construction of a genomic map in related bacteria, such as Brevibacterium lactofermentum. 
The MP nucleic acid molecules of the invention are also useful for evolutionary and protein structural studies. The metabolic processes in which the molecules of the invention participate are utilized by a wide variety of prokaryotic and eukaryotic cells; by comparing the sequences of the nucleic acid molecules of the present invention to those encoding similar enzymes from other organisms, the evolutionary relatedness of the organisms can be assessed. Similarly, such a comparison permits an assessment of which regions of the sequence are conserved and which are not, which may aid in determining those regions of the protein which are essential for the functioning of the enzyme. This type of determination is of value for protein engineering studies and may give an indication of what the protein can tolerate in terms of mutagenesis without losing function.
Manipulation of the MP nucleic acid molecules of the invention may result in the production of MP proteins having functional differences from the wild-type MP proteins. These proteins may be improved in efficiency or activity, may be present in greater numbers in the cell than is usual, or may be decreased in efficiency or activity.
The invention also provides methods for screening molecules which modulate the activity of an MP protein, either by interacting with the protein itself or a substrate or binding partner of the MP protein, or by modulating the transcription or translation of an MP nucleic acid molecule of the invention. In such methods, a microorganism expressing one or more MP proteins of the invention is contacted with one or more test compounds, and the effect of each test compound on the activity or level of expression of the MP protein is assessed.
When the desired fine chemical to be isolated from large-scale fermentative culture of C. glutamicum is an amino acid, a vitamin, a cofactor, a nutraceutical, a nucleotide, a nucleoside, or trehalose, modulation of the activity or efficiency of activity of one or more of the proteins of the invention by recombinant genetic mechanisms may directly impact the production of one of these fine chemicals. For example, in the case of an enzyme in a biosynthetic pathway for a desired amino acid, improvement in efficiency or activity of the enzyme (including the presence of multiple copies of the gene) should lead to an increased production or efficiency of production of that desired amino acid. In the case of an enzyme in a biosynthetic pathway for an amino acid whose synthesis is in competition with the synthesis of a desired amino acid, any decrease in the efficiency or activity of this enzyme (including deletion of the gene) should result in an increase in production or efficiency of production of the desired amino acid, due to decreased competition for intermediate compounds and/or energy. In the case of an enzyme in a degradation pathway for a desired amino acid, any decrease in efficiency or activity of the enzyme should result in a greater yield or efficiency of production of the desired product due to a decrease in its degradation. Lastly, mutagenesis of an enzyme involved in the biosynthesis of a desired amino acid such that this enzyme is no longer is capable of feedback inhibition should result in increased yields or efficiency of production of the desired amino acid. The same should apply to the biosynthetic and degradative enzymes of the invention involved in the metabolism of vitamins, cofactors, nutraceuticals, nucleotides, nucleosides and trehalose.
Similarly, when the desired fine chemical is not one of the aforementioned compounds, the modulation of activity of one of the proteins of the invention may still impact the yield and/or efficiency of production of the compound from large-scale culture of C. glutamicum. The metabolic pathways of any organism are closely interconnected; the intermediate used by one pathway is often supplied by a different pathway. Enzyme expression and function may be regulated based on the cellular levels of a compound from a different metabolic process, and the cellular levels of molecules necessary for basic growth, such as amino acids and nucleotides, may critically affect the viability of the microorganism in large-scale culture. Thus, modulation of an amino acid biosynthesis enzyme, for example, such that it is no longer responsive to feedback inhibition or such that it is improved in efficiency or turnover may result in increased cellular levels of one or more amino acids. In turn, this increased pool of amino acids provides not only an increased supply of molecules necessary for protein synthesis, but also of molecules which are utilized as intermediates and precursors in a number of other biosynthetic pathways. If a particular amino acid had been limiting in the cell, its increased production might increase the ability of the cell to perform numerous other metabolic reactions, as well as enabling the cell to more efficiently produce proteins of all kinds, possibly increasing the overall growth rate or survival ability of the cell in large scale culture. Increased viability improves the number of cells capable of producing the desired fine chemical in fermentative culture, thereby increasing the yield of this compound. Similar processes are possible by the modulation of activity of a degradative enzyme of the invention such that the enzyme no longer catalyzes, or catalyzes less efficiently, the degradation of a cellular compound which is important for the biosynthesis of a desired compound, or which will enable the cell to grow and reproduce more efficiently in large-scale culture. It should be emphasized that optimizing the degradative activity or decreasing the biosynthetic activity of certain molecules of the invention may also have a beneficial effect on the production of certain fine chemicals from C. glutamicum. For example, by decreasing the efficiency of activity of a biosynthetic enzyme in a pathway which competes with the biosynthetic pathway of a desired compound for one or more intermediates, more of those intermediates should be available for conversion to the desired product. A similar situation may call for the improvement of degradative ability or efficiency of one or more proteins of the invention.
This aforementioned list of mutagenesis strategies for MP proteins to result in increased yields of a desired compound is not meant to be limiting; variations on these mutagenesis strategies will be readily apparent to one of ordinary skill in the art. By these mechanisms, the nucleic acid and protein molecules of the invention may be utilized to generate C. glutamicum or related strains of bacteria expressing mutated MP nucleic acid and protein molecules such that the yield, production, and/or efficiency of production of a desired compound is improved. This desired compound may be any natural product of C. glutamicum, which includes the final products of biosynthesis pathways and intermediates of naturally-occurring metabolic pathways, as well as molecules which do not naturally occur in the metabolism of C. glutamicum, but which are produced by a C. glutamicum strain of the invention. Preferred compounds to be produced by Corynebacterium glutamicum strains are the amino acids L-lysine and L-methionine.
In one embodiment, the metC gene encoding cystathionine β-lyase, the third enzyme in the methionine biosynthetic pathway, was isolated from Corynebacterium glutamicum. The translational product of the gene showed no significant homology with that of metC gene from other organisms. Introduction of the plasmid containing the metC gene into C. glutamicum resulted in a 5-fold increase in the activity of cystathionine β-lyase. The protein product, now designated MetC (corresponding to SEQ ID NO:4), which encodes a protein product of 35,574 Daltons and consists of 325 amino acids, is identical to the previously reported aecD gene (Rossol, I. and Puhler, A. (1992) J. Bacteriology 174, 2968-2977) except the existence of two different amino acids. Like aecD gene, when present in multiple copies, metC gene conferred resistance to S-(β-aminoethyl)-cysteine which is a toxic lysine analog. However, genetic and biochemical evidences suggest that the natural activity of metC gene product is to mediate methionine biosynthesis in C. glutamicum. Mutant strains of metC were constructed and the strains showed methionine prototrophy. The mutant strains completely lost their ability to show resistance to S-(γ-aminoethyl)-cysteine. These results show that, in addition to the transsulfuration, which is another biosynthetic pathway, the direct sulfhydrylation pathway is functional in C. glutamicum as a parallel biosynthetic route for methionine.
In yet another embodiment, it is also shown that the additional sulfhydrylation pathway is catalyzed by O-acetylhomoserine sulfhydrylase. The presence of the pathway is demonstrated by the isolation of the corresponding metZ (or metY) gene and enzyme (corresponding to SEQ ID NO:1 and SEQ ID NO:2, respectively). Among the eukaryotes, fungi and yeast species have been reported to have both the transsulfuration and direct sulfhydrylation pathway. Thus far, no prokaryotic organism which possesses both pathways has been found. Unlike E. coli which only possesses single biosynthetic route for lysine, C. glutamicum possesses two parallel biosynthetic pathways for the amino acid. The biosynthetic pathway for methionine in C. glutamicum is analogous to that of lysine in that aspect.
The gene metZ is located in the upstream region of metA, which is the gene encoding the enzyme catalysing the first step of methionine biosynthesis (Park, S.-D., et al. (1998) Mol. Cells 8, 286-294). Regions upstream and downstream of metA were sequenced to identify other met genes. It appears that metZ and metA form an operon. Expression of the genes encoding MetA and MetZ leads to overproduction of the corresponding polypeptides.
Surprisingly, metZ clones can complement methionine auxotrophic Escherichia coli metB mutant strains. This shows that the protein product of metZ catalyzes a step that can bypass the step catalyzed by the protein product of metB. MetZ was also disrupted and the mutant strain showed methionine prototrophy. Corynebacterium glutamicum metB and metZ double mutants were also constructed. The double mutant is auxotrophic for methionine. Thus, metZ encodes a protein catalysing the reaction from O-Acetyl-Homoserine to Homocysteine, which is one step in the sulfhydrylation pathway of methionine biosynthesis. Corynebacterium glutamicum contains both the transsulfuration and the sulfhydrylation pathway of methionine biosynthesis.
Introduction of metZ into C. glutamicum resulted in the expression of a 47,000 Dalton protein. Combined introduction of metZ and metA in C. glutamicum resulted in the appearance of metA and metZ proteins as shown by gel electrophoresis. If the Corynebacterium strain is a lysine overproducer, introduction of a plasmid containing metZ and metA resulted in a lower lysine titer but accumulation of homocysteine and methionine is detected.
In another embodiment metZ and metA were introduced into Corynebacterium glutamicum strains together with the hom gene, encoding the homoserine dehydrogenase, catalysing the conversion from aspartate semialdehyde to homoserine. Different hom genes from different organisms were chosen for this experiment. The Corynebacterium glutamicum hom gene can be used as well as hom genes from other procaryotes like Escherichia coli or Bacillus subtilis or the hom gene of eukaryotes such as Saccharomyces cerevisiae, Shizosaccharomyces pombe, Ashbya gossypii or algae, higher plants or animals. It may be that the hom gene is insensitive against feed back inhibition mediated by any metabolites that occur in the biosynthetic routes of the amino acids of the aspartate family, like aspatrate, lysine, threonine or methionine. Such metabolites are for example aspartate, lysine, methionine, threonine, aspartyl-phosphate, aspartate semialdehyd, homoserine, cystathionine, homocysteine or any other metabolite that occurs in this biosynthetic routes. In addition to the metabolites, the homoserine dehydrogenase may be insensitive against inhibition by analogues of all those metabolites or even against other compounds involved in this metabolism as there are other amino acids like cysteine or cofactors like vitamin B12 and all of its derivatives and S-adenosylmethionine and its metabolites and derivatives and analogues. The insensitivity of the homoserine dehydrogenase against all these, a part of these or only one of these compounds may either be its natural attitude or it may be the result from one or more mutations that resulted from classical mutation and selection using chemicals or irradiation or other mutagens. The mutations could also be introduced into the hom gene using gene technology, for example the introduction of site specific point mutations or by any method aforementioned for the MP or MP encoding DNA-sequences.
When a hom gene was combined with the metZ and metA genes and introduced into a Corynebacterium glutamicum strain that is a lysine overproducer, lysine accumulation was reduced and homocysteine and methionine accumulation was enhanced. A further enhancement of homocysteine and methionine concentrations can be achieved, if a lysine overproducing Corynebacterium glutamicum strain is used and a disruption of the ddh gene or the lysA gene was introduced prior to the transformation with DNA containing a hom gene and metZ and metA in combination. The overproduction of homocysteine and methionine was possible using different sulfur sources. Sulfates, thiosulfates, sulfites and also more reduced sulfur sources like H2S and sulfides and derivatives could be used. Also, organic sulfur sources like methyl mercaptan, thioglycolates, thiocyanates, thiourea, sulfur containing amino acids like cysteine and other sulfur containing compounds can be used to achieve homocysteine and methionine overproduction.
In another embodiment, the metC gene was introduced into a Corynebacterium glutamicum strain using aforementioned methods. The metC gene can be transformed into the strain in combination with other genes like metB, metA and metA. The hom gene can also be added. When the hom gene, the met C, metA and metB genes were combined on a vector and introduced into a Corynebacterium glutamicum strain, homocysteine and methionine overproduction was achieved. The overproduction of homocysteine and methionine was possible using different sulfur sources. Sulfates, thiosulfates, sulfites and also more reduced sulfur sources like H2S and sulfides and derivatives could be used. Also, organic sulfur sources like methyl mercaptan, thioglycolates, thiocyanates, thiourea, sulfur containing amino acids like cysteine and other sulfur containing compounds can be used to achieve homocysteine and methionine overproduction.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patent applications, patents, published patent applications, Tables, and the sequence listing cited throughout this application are hereby incorporated by reference.
EXEMPLIFICATION
Example 1
Preparation of Total Genomic DNA of Corynebacterium glutamicum ATCC13032
A culture of Corynebacterium glutamicum (ATCC 13032) was grown overnight at 30° C. with vigorous shaking in BHI medium (Difco). The cells were harvested by centrifugation, the supernatant was discarded and the cells were resuspended in 5 ml buffer-I (5% of the original volume of the culture—all indicated volumes have been calculated for 100 ml of culture volume). Composition of buffer-I: 140.34 g/l sucrose, 2.46 g/l MgSO4×7 H2O, 10 ml/l KH2PO4 solution (100 g/l, adjusted to pH 6.7 with KOH), 50 ml/l M12 concentrate (10 g/l (NH4)2SO4, 1 g/l NaCl, 2 g/l MgSO4×7 H2O, 0.2 g/l CaCl2, 0.5 g/l yeast extract (Difco), 10 ml/l trace-elements-mix (200 mg/l FeSO4×H2O, 10 mg/l ZnSO4×7 H2O, 3 mg/l MnCl2×4 H2O, 30 mg/l H3BO3 20 mg/l CoCl2×6 H2O, 1 mg/l NiCl2×6 H2O, 3 mg/l Na2MoO4×2 H2O, 500 mg/l complexing agent (EDTA or critic acid), 100 ml/l vitamins-mix (0.2 mg/l biotin, 0.2 mg/l folic acid, 20 mg/l p-amino benzoic acid, 20 mg/l riboflavin, 40 mg/l ca-panthothenate, 140 mg/l nicotinic acid, 40 mg/l pyridoxole hydrochloride, 200 mg/l myo-inositol). Lysozyme was added to the suspension to a final concentration of 2.5 mg/ml. After an approximately 4 h incubation at 37° C., the cell wall was degraded and the resulting protoplasts are harvested by centrifugation. The pellet was washed once with 5 ml buffer-I and once with 5 ml TE-buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The pellet was resuspended in 4 ml TE-buffer and 0.5 ml SDS solution (10%) and 0.5 ml NaCl solution (5 M) are added. After adding of proteinase K to a final concentration of 200 μg/ml, the suspension is incubated for ca. 18 h at 37° C. The DNA was purified by extraction with phenol, phenol-chloroform-isoamylalcohol and chloroform-isoamylalcohol using standard procedures. Then, the DNA was precipitated by adding 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol, followed by a 30 min incubation at −20° C. and a 30 min centrifugation at 12,000 rpm in a high speed centrifuge using a SS34 rotor (Sorvall). The DNA was dissolved in 1 ml TE-buffer containing 20 μg/ml RNaseA and dialysed at 4° C. against 1000 ml TE-buffer for at least 3 hours. During this time, the buffer was exchanged 3 times. To aliquots of 0.4 ml of the dialysed DNA solution, 0.4 ml of 2 M LiCl and 0.8 ml of ethanol are added. After a 30 min incubation at −20° C., the DNA was collected by centrifugation (13,000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany). The DNA pellet was dissolved in TE-buffer. DNA prepared by this procedure could be used for all purposes, including southern blotting or construction of genomic libraries.
Example 2
Construction of Genomic Libraries in Escherichia coli of Corynebacterium glutamicum ATCC13032
Using DNA prepared as described in Example 1, cosmid and plasmid libraries were constructed according to known and well established methods (see e.g., Sambrook, J. et al. (1989) “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, or Ausubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley & Sons.)
Any plasmid or cosmid could be used. Of particular use were the plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. Natl. Acad. Sci. USA, 75:3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol 134:1141-1156), plasmids of the pBS series (pBSSK+, pBSSK− and others; Stratagene, LaJolla, USA), or cosmids as SuperCos1 (Stratagene, LaJolla, USA) or Lorist6 (Gibson, T. J., Rosenthal A. and Waterson, R. H. (1987) Gene 53:283-286. Gene libraries specifically for use in C. glutamicum may be constructed using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
For the isolation of metC clones, E. coli JE6839 cells were transformed with the library DNA and plated onto the M9 minimal medium containing ampicillin and appropriate supplements. The plates were incubated at 37° C. for 5 days. Colonies were isolated and screened for the plasmid content. The complete nucleotide sequence of the isolated metC gene was determined by methods well-known to one of ordinary skill in the art.
Example 3
DNA Sequencing and Computational Functional Analysis
Genomic libraries as described in Example 2 were used for DNA sequencing according to standard methods, in particular by the chain termination method using ABI377 sequencing machines (see e.g., Fleischman, R. D. et al. (1995) “Whole-genome Random Sequencing and Assembly of Haemophilus Influenzae Rd., Science, 269:496-512). Sequencing primers with the following nucleotide sequences were used: 5′-GGAAACAGTATGACCATG-3′ (SEQ ID NO:123) or 5′-GTAAAACGACGGCCAGT-3′(SEQ ID NO.:124).
Example 4
In vivo Mutagenesis
In vivo mutagenesis of Corynebacterium glutamicum can be performed by passage of plasmid (or other vector) DNA through E. coli or other microorganisms (e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are impaired in their capabilities to maintain the integrity of their genetic information. Typical mutator strains have mutations in the genes for the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; for reference, see Rupp, W. D. (1996) DNA repair mechanisms, in: Escherichia coli and Salmonella, p. 2277-2294, ASM: Washington.) Such strains are well known to those of ordinary skill in the art. The use of such strains is illustrated, for example, in Greener, A. and Callahan, M. (1994) Strategies 7: 32-34.
Example 5
DNA Transfer Between Escherichia coli and Corynebacterium glutamicum 
Several Corynebacterium and Brevibacterium species contain endogenous plasmids (as e.g., pHM1519 or pBL1) which replicate autonomously (for review see, e.g., Martin, J. F. et al. (1987) Biotechnology, 5:137-146). Shuttle vectors for Escherichia coli and Corynebacterium glutamicum can be readily constructed by using standard vectors for E. coli (Sambrook, J. et al. (1989), “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley & Sons) to which a origin or replication for and a suitable marker from Corynebacterium glutamicum is added. Such origins of replication are preferably taken from endogenous plasmids isolated from Corynebacterium and Brevibacterium species. Of particular use as transformation markers for these species are genes for kanamycin resistance (such as those derived from the Tn5 or Tn903 transposons) or chloramphenicol (Winnacker, E. L. (1987) “From Genes to Clones—Introduction to Gene Technology, VCH, Weinheim). There are numerous examples in the literature of the construction of a wide variety of shuttle vectors which replicate in both E. coli and C. glutamicum, and which can be used for several purposes, including gene over-expression (for reference, see e.g., Yoshihama, M. et al. (1985) J. Bacteriol. 162:591-597, Martin J. F. et al. (1987) Biotechnology, 5:137-146 and Eikmanns, B. J. et al. (1991) Gene, 102:93-98).
Using standard methods, it is possible to clone a gene of interest into one of the shuttle vectors described above and to introduce such a hybrid vectors into strains of Corynebacterium glutamicum. Transformation of C. glutamicum can be achieved by protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53:399-303) and in cases where special vectors are used, also by conjugation (as described e.g. in Schäfer, A et al. (1990) J. Bacteriol. 172:1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using standard methods well-known in the art) and transforming it into E. coli. This transformation step can be performed using standard methods, but it is advantageous to use an Mcr-deficient E. coli strain, such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).
Genes may be overexpressed in C. glutamicum strains using plasmids which comprise pCG1 (U.S. Pat. No. 4,617,267) or fragments thereof, and optionally the gene for kanamycin resistance from TN903 (Grindley, N. D. and Joyce, C. M. (1980) Proc. Natl. Acad. Sci. USA 77(12): 7176-7180). In addition, genes may be overexpressed in C. glutamicum strains using plasmid pSL109 (Lee, H.-S. and A. J. Sinskey (1994) J. Microbiol. Biotechnol. 4: 256-263).
Aside from the use of replicative plasmids, gene overexpression can also be achieved by integration into the genome. Genomic integration in C. glutamicum or other Corynebacterium or Brevibacterium species may be accomplished by well-known methods, such as homologous recombination with genomic region(s), restriction endonuclease mediated integration (REMI) (see, e.g., DE Patent 19823834), or through the use of transposons. It is also possible to modulate the activity of a gene of interest by modifying the regulatory regions (e.g., a promoter, a repressor, and/or an enhancer) by sequence modification, insertion, or deletion using site-directed methods (such as homologous recombination) or methods based on random events (such as transposon mutagenesis or REMI). Nucleic acid sequences which function as transcriptional terminators may also be inserted 3′ to the coding region of one or more genes of the invention; such terminators are well-known in the art and are described, for example, in Winnacker, E. L. (1987) From Genes to Clones—Introduction to Gene Technology. VCH: Weinheim.
Example 6
Assessment of the Expression of the Mutant Protein
Observations of the activity of a mutated protein in a transformed host cell rely on the fact that the mutant protein is expressed in a similar fashion and in a similar quantity to that of the wild-type protein. A useful method to ascertain the level of transcription of the mutant gene (an indicator of the amount of mRNA available for translation to the gene product) is to perform a Northern blot (for reference see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York), in which a primer designed to bind to the gene of interest is labeled with a detectable tag (usually radioactive or chemiluminescent), such that when the total RNA of a culture of the organism is extracted, run on gel, transferred to a stable matrix and incubated with this probe, the binding and quantity of binding of the probe indicates the presence and also the quantity of mRNA for this gene. This information is evidence of the degree of transcription of the mutant gene. Total cellular RNA can be prepared from Corynebacterium glutamicum by several methods, all well-known in the art, such as that described in Bormann, E. R. et al. (1992) Mol. Microbiol. 6: 317-326.
To assess the presence or relative quantity of protein translated from this mRNA, standard techniques, such as SDS-acrylamide gel electrophoresis, were employed. The overproduction of metC and metZ in combination with metA in Corynebacterium glutamicum was demonstrated by this method. Western blot may also be employed (see, for example, Ausubel et al. (1988) Current Protocols in Molecular Biology, Wiley: New York). In this process, total cellular proteins are extracted, separated by gel electrophoresis, transferred to a matrix such as nitrocellulose, and incubated with a probe, such as an antibody, which specifically binds to the desired protein. This probe is generally tagged with a chemiluminescent or colorimetric label which may be readily detected. The presence and quantity of label observed indicates the presence and quantity of the desired mutant protein present in the cell.
Example 7
Growth of Escherichia coli and Genetically Modified Corynebacterium glutamicum—Media and Culture Conditions
E. coli strains are routinely grown in MB and LB broth, respectively (Follettie, M. T., et al. (1993) J. Bacteriol. 175, 4096-4103). Minimal media for E. coli is M9 and modified MCGC (Yoshihama, M., et al. (1985) J. Bacteriol. 162, 591-507). Glucose was added to a final concentration of 1%. Antibiotics were added in the following amounts (micrograms per milliliter): ampicillin, 50; kanamycin, 25; nalidixic acid, 25. Amino acids, vitamins, and other supplements were added in the following amounts: methionine, 9.3 mM; arginine, 9.3 mM; histidine, 9.3 mM; thiamine, 0.05 mM. E. coli cells were routinely grown at 37° C., respectively.
Genetically modified Corynebacteria are cultured in synthetic or natural growth media. A number of different growth media for Corynebacteria are both well-known and readily available (Lieb et al. (1989) Appl. Microbiol. Biotechnol., 32:205-210; von der Osten et al. (1998) Biotechnology Letters, 11:11-16; Patent DE 4,120,867; Liebl (1992) “The Genus Corynebacterium, in: The Procaryotes, Volume II, Balows, A. et al., eds. Springer-Verlag). These media consist of one or more carbon sources, nitrogen sources, inorganic salts, vitamins and trace elements. Preferred carbon sources are sugars, such as mono-, di-, or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose serve as very good carbon sources. It is also possible to supply sugar to the media via complex compounds such as molasses or other by-products from sugar refinement. It can also be advantageous to supply mixtures of different carbon sources. Other possible carbon sources are alcohols and organic acids, such as methanol, ethanol, acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds, or materials which contain these compounds. Exemplary nitrogen sources include ammonia gas or ammonia salts, such as NH4Cl or (NH4)2SO4, NH4OH, nitrates, urea, amino acids or complex nitrogen sources like corn steep liquor, soy bean flour, soy bean protein, yeast extract, meat extract and others.
The overproduction of sulfur containing amino acids like homocysteine and methionine was made possible using different sulfur sources. Sulfates, thiosulfates, sulfites and also more reduced sulfur sources like H2S and sulfides and derivatives can be used. Also, organic sulfur sources like methyl mercaptan, thioglycolates, thiocyanates, thiourea, sulfur containing amino acids like cysteine and other sulfur containing compounds can be used to achieve homocysteine and methionine overproduction
Inorganic salt compounds which may be included in the media include the chloride-, phosphorous- or sulfate-salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Chelating compounds can be added to the medium to keep the metal ions in solution. Particularly useful chelating compounds include dihydroxyphenols, like catechol or protocatechuate, or organic acids, such as citric acid. It is typical for the media to also contain other growth factors, such as vitamins or growth promoters, examples of which include biotin, riboflavin, thiamin, folic acid, nicotinic acid, pantothenate and pyridoxin. Growth factors and salts frequently originate from complex media components such as yeast extract, molasses, corn steep liquor and others. The exact composition of the media compounds depends strongly on the immediate experiment and is individually decided for each specific case. Information about media optimization is available in the textbook “Applied Microbiol. Physiology, A Practical Approach (eds. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). It is also possible to select growth media from commercial suppliers, like standard 1 (Merck) or BHI (grain heart infusion, DIFCO) or others.
All medium components are sterilized, either by heat (20 minutes at 1.5 bar and 121° C.) or by sterile filtration. The components can either be sterilized together or, if necessary, separately. All media components can be present at the beginning of growth, or they can optionally be added continuously or batchwise.
Culture conditions are defined separately for each experiment. The temperature should be in a range between 15° C. and 45° C. The temperature can be kept constant or can be altered during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0, and can be maintained by the addition of buffers to the media. An exemplary buffer for this purpose is a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES, ACES and others can alternatively or simultaneously be used. It is also possible to maintain a constant culture pH through the addition of NaOH or NH4OH during growth. If complex medium components such as yeast extract are utilized, the necessity for additional buffers may be reduced, due to the fact that many complex compounds have high buffer capacities. If a fermentor is utilized for culturing the micro-organisms, the pH can also be controlled using gaseous ammonia.
The incubation time is usually in a range from several hours to several days. This time is selected in order to permit the maximal amount of product to accumulate in the broth. The disclosed growth experiments can be carried out in a variety of vessels, such as microtiter plates, glass tubes, glass flasks or glass or metal fermentors of different sizes. For screening a large number of clones, the microorganisms should be cultured in microtiter plates, glass tubes or shake flasks, either with or without baffles. Preferably 100 ml shake flasks are used, filled with 10% (by volume) of the required growth medium. The flasks should be shaken on a rotary shaker (amplitude 25 mm) using a speed-range of 100-300 rpm. Evaporation losses can be diminished by the maintenance of a humid atmosphere; alternatively, a mathematical correction for evaporation losses should be performed.
If genetically modified clones are tested, an unmodified control clone or a control clone containing the basic plasmid without any insert should also be tested. The medium is inoculated to an OD600 of O.5-1.5 using cells grown on agar plates, such as CM plates (10 g/l glucose, 2.5 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast extract, 5 g/l meat extract, 22 g/l agar, pH 6.8 with 2M NaOH) that had been incubated at 30° C. Inoculation of the media is accomplished by either introduction of a saline suspension of C. glutamicum cells from CM plates or addition of a liquid preculture of this bacterium.
Example 8
In Vitro Analysis of the Function of Mutant Proteins
The determination of activities and kinetic parameters of enzymes is well established in the art. Experiments to determine the activity of any given altered enzyme must be tailored to the specific activity of the wild-type enzyme, which is well within the ability of one of ordinary skill in the art. Overviews about enzymes in general, as well as specific details concerning structure, kinetics, principles, methods, applications and examples for the determination of many enzyme activities may be found, for example, in the following references: Dixon, M., and Webb, E. C., (1979) Enzymes. Longmans: London; Fersht, (1985) Enzyme Structure and Mechanism. Freeman: New York; Walsh, (1979) Enzymatic Reaction Mechanisms. Freeman: San Francisco; Price, N. C., Stevens, L. (1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D., ed. (1983) The Enzymes, 3rd ed. Academic Press: New York; Bisswanger, H., (1994) Enzymkinetik, 2nd ed. VCH: Weinheim (ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., Graβ1, M., eds. (1983-1986) Methods of Enzymatic Analysis, 3rd ed., vol. I-XII, Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial Chemistry (1987) vol. A9, “Enzymes”. VCH: Weinheim, p. 352-363.
Cell extracts from Corynebacterium glutamicum were prepared as described previously (Park, S.-D., et al. (1998) Mol. Cells 8, 286-294). Cystathionine β-lyase was assayed as follows. The assay mixture contained 100 mM Tris-HCl (pH8.5), 0.1 mM NADH, 1 mM L-cystathionine, 5 units of L-lactate dehydrogenase, and appropriate amounts of crude extract. Optical changes were monitored at 340 nm. Assay for S-(□-aminoethyl)-cysteine (AEC) resistance was carried out as described in Rossol, I. and Pühler, A. (1992) J. Bacteriol. 174, 2968-77. The results of cystathionin β-lyase assays from extracts of different Corynebacterium glutamicum strains as well as results of AEC resistance assays of the same strain are summarized in Table 5, below.







TABLE 5







Expression of cystathionine β-lyasea













Activity
Growth





(nmol
on
Resistance


Strains
Properties
min−1 mg−1)
MMb
to AECc














C. glutamicum
—
146
+
+


ASO19E12


C. glutamicum
Empty vector
145
+
+


ASO19E12/pMT1


C. glutamicum
metC clone
797
+
++


ASO19E12/pSL173


C. glutamicum
metC mutantd
19
+
−


HL457


C. glutamicum
metC mutantd
23
+
−


HL459


E. coli JE6839
metC mutant
21
−
NDe





aThe enzyme was induced by growth to the stationary phase on the minimal medium containing 1% glucose. Cells were harvested, disrupted, and assayed for the activity as described in the Materials and Methods.


bMCGC minimal media was used. Growth was monitored on plates.


cCells were grown on plates containing 40 mM S-(β-aminoethyl)-cysteine (AEC) for 5 days.


dThe mutants were generated in this study.


eNot determined.






The ability of the metC clones to express cystathionine β-lyase was tested by enzymatic assay. Crude extracts prepared from the C. glutamicum ASO19E12 cells harboring plasmid pSL173 were assayed. Cells harboring the plasmid showed approximately a 5-fold increase in the activity of cystathionine β-lyase compared to those harboring the empty vector pMT1 (Table 5), apparently due to the gene-dose effect. SDS-PAGE analysis of crude extracts revealed a putative cystathionine β-lyase band with approximate Mr of 41,000. Intensity of each putative cystathionine β-lyase band agreed with the complementation and enzymatic assay data (Table 5). As described above, a region of metC appeared to be nearly identical to the previously reported aecD. Since the aecD gene was isolated on the basis of its ability to confer resistance to S-(β-aminoethyl)-cysteine (AEC), a toxic lysine analogue, we tested the protein product of metC for the presence of the activity. As shown in Table 5, cells overexpressing cystathionine β-lyase showed increased resistance to AEC. The strain carrying a mutation in metC gene (see below) completely lost its ability to show a resistant phenotype to AEC.
Assay for O-acetylhmoserine sulphydrylase was performed as follows (Belfaiza, J., et al. (1998) J. Bacteriol. 180, 250-255; Ravanel, S., M. Droux, and R. Douce (1995) Arch. Biochem. Biophys. 316, 572-584; Foglino, M. (1995) Microbiology 141, 431-439). Assay mixture of 0.1 ml contained 20 mM MOPS-NaOH (pH7.5), 10 mM O-acetylhomoserine, 2 mM Na2S in 50 mM NaOH, and an appropriate amount of enzyme. Immediately after the addition of Na2S which was added last, the reaction mixture was overlayed with 50 ul of mineral oil. After 30 minute incubation at 30° C., the reaction was stopped by boiling the mixture for 3 minutes. Homocysteine produced in the reaction was quantified as previously described (Yamagata, S. (1987) Method Enzymol. 143, 478-483.). Reaction mixture of 0.1 ml was taken and mixed with 0.1 ml of H2O, 0.6 ml of saturated NaCl, 0.1 ml of 1.5 M Na2CO3 containing 67 mM KCN, and 0.1 ml of 2% nitroprusside. After 1 minute incubation at room temperature, optical density was measured at 520 nm. Corynebacterium cells harboring additional copies of the metZ gene, e.g., a plasmid containing the metZ gene, exhibited significantly higher metZ enzyme activities than the same type of Corynebacterium cells without additional copies of the metZ gene.
The activity of proteins which bind to DNA can be measured by several well-established methods, such as DNA band-shift assays (also called gel retardation assays). The effect of such proteins on the expression of other molecules can be measured using reporter gene assays (such as that described in Kolmar, H. et al. (1995) EMBO J. 14: 3895-3904 and references cited therein). Reporter gene test systems are well known and established for applications in both pro- and eukaryotic cells, using enzymes such as beta-galactosidase, green fluorescent protein, and several others.
The determination of activity of membrane-transport proteins can be performed according to techniques such as those described in Gennis, R. B. (1989) “Pores, Channels and Transporters”, in Biomembranes, Molecular Structure and Function, Springer: Heidelberg, p. 85-137; 199-234; and 270-322.
Example 9
Analysis of Impact of Mutant Protein on the Production of the Desired Product
The effect of the genetic modification in C. glutamicum on production of a desired compound (such as an amino acid) can be assessed by growing the modified microorganism under suitable conditions (such as those described above) and analyzing the medium and/or the cellular component for increased production of the desired product (i.e., an amino acid). Such analysis techniques are well known to one of ordinary skill in the art, and include spectroscopy, thin layer chromatography, staining methods of various kinds, enzymatic and microbiological methods, and analytical chromatography such as high performance liquid chromatography (see, for example, Ullman, Encyclopedia of Industrial Chemistry, vol. A2, p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A. et al., (1987) “Applications of HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17; Rehm et al. (1993) Biotechnology, vol. 3, Chapter III: “Product recovery and purification”, page 469-714, VCH: Weinheim; Belter, P. A. et al. (1988) Bioseparations: downstream processing for biotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S. (1992) Recovery processes for biological materials, John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D. (1988) Biochemical separations, in: Ulmann's Encyclopedia of Industrial Chemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation and purification techniques in biotechnology, Noyes Publications.)
In addition to the measurement of the final product of fermentation, it is also possible to analyze other components of the metabolic pathways utilized for the production of the desired compound, such as intermediates and side-products, to determine the overall efficiency of production of the compound. Analysis methods include measurements of nutrient levels in the medium (e.g., sugars, hydrocarbons, nitrogen sources, phosphate, and other ions), measurements of biomass composition and growth, analysis of the production of common metabolites of biosynthetic pathways, and measurement of gasses produced during fermentation. Standard methods for these measurements are outlined in Applied Microbial Physiology, A Practical Approach, P. M. Rhodes and P. F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN: 0199635773) and references cited therein.
Example 10
Purification of the Desired Product from C. glutamicum Culture
Recovery of the desired product from the C. glutamicum cells or supernatant of the above-described culture can be performed by various methods well known in the art. If the desired product is not secreted from the cells, the cells can be harvested from the culture by low-speed centrifugation, the cells can be lysed by standard techniques, such as mechanical force or sonication. The cellular debris is removed by centrifugation, and the supernatant fraction containing the soluble proteins is retained for further purification of the desired compound. If the product is secreted from the C. glutamicum cells, then the cells are removed from the culture by low-speed centrifugation, and the supernate fraction is retained for further purification.
The supernatant fraction from either purification method is subjected to chromatography with a suitable resin, in which the desired molecule is either retained on a chromatography resin while many of the impurities in the sample are not, or where the impurities are retained by the resin while the sample is not. Such chromatography steps may be repeated as necessary, using the same or different chromatography resins. One of ordinary skill in the art would be well-versed in the selection of appropriate chromatography resins and in their most efficacious application for a particular molecule to be purified. The purified product may be concentrated by filtration or ultrafiltration, and stored at a temperature at which the stability of the product is maximized.
There are a wide array of purification methods known to the art and the preceding method of purification is not meant to be limiting. Such purification techniques are described, for example, in Bailey, J. E. & Ollis, D. F. Biochemical Engineering Fundamentals, McGraw-Hill: New York (1986).
The identity and purity of the isolated compounds may be assessed by techniques standard in the art. These include high-performance liquid chromatography (HPLC), spectroscopic methods, staining methods, thin layer chromatography, NIRS, enzymatic assay, or microbiologically. Such analysis methods are reviewed in: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry, (1996) vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566, 575-581 and p. 581-587; Michal, G. (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.
Example 11
Analysis of the Gene Sequences of the Invention
The comparison of sequences and determination of percent homology between two sequences are art-known techniques, and can be accomplished using a mathematical algorithm, such as the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to MP nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to MP protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, one of ordinary skill in the art will know how to optimize the parameters of the program (e.g., XBLAST and NBLAST) for the specific sequence being analyzed.
Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Meyers and Miller ((1988) Comput. Appl. Biosci. 4: 11-17). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, and include ADVANCE and ADAM. described in Torelli and Robotti (1994) Comput. Appl. Biosci 10:3-5; and FASTA, described in Pearson and Lipman (1988) P.N.A.S. 85:2444-8.
The percent homology between two amino acid sequences can also be accomplished using the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. The percent homology between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using standard parameters, such as a gap weight of 50 and a length weight of 3.
A comparative analysis of the gene sequences of the invention with those present in Genbank has been performed using techniques known in the art (see, e.g., Bexevanis and Ouellette, eds. (1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. John Wiley and Sons: New York). The gene sequences of the invention were compared to genes present in Genbank in a three-step process. In a first step, a BLASTN analysis (e.g., a local alignment analysis) was performed for each of the sequences of the invention against the nucleotide sequences present in Genbank, and the top 500 hits were retained for further analysis. A subsequent FASTA search (e.g., a combined local and global alignment analysis, in which limited regions of the sequences are aligned) was performed on these 500 hits. Each gene sequence of the invention was subsequently globally aligned to each of the top three FASTA hits, using the GAP program in the GCG software package (using standard parameters). In order to obtain correct results, the length of the sequences extracted from Genbank were adjusted to the length of the query sequences by methods well-known in the art. The results of this analysis are set forth in Table 4. The resulting data is identical to that which would have been obtained had a GAP (global) analysis alone been performed on each of the genes of the invention in comparison with each of the references in Genbank, but required significantly reduced computational time as compared to such a database-wide GAP (global) analysis. Sequences of the invention for which no alignments above the cutoff values were obtained are indicated on Table 4 by the absence of alignment information. It will further be understood by one of ordinary skill in the art that the GAP alignment homology percentages set forth in Table 4 under the heading “% homology (GAP)” are listed in the European numerical format, wherein a ‘,’ represents a decimal point. For example, a value of “40,345” in this column represents “40.345%”.
Example 12
Construction and Operation of DNA Microarrays
The sequences of the invention may additionally be used in the construction and application of DNA microarrays (the design, methodology, and uses of DNA arrays are well known in the art, and are described, for example, in Schena, M. et al. (1995) Science 270: 467470; Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367; DeSaizieu, A. et al. (1998) Nature Biotechnology 16: 45-48; and DeRisi, J. L. et al. (1997) Science 278: 680-686).
DNA microarrays are solid or flexible supports consisting of nitrocellulose, nylon, glass, silicone, or other materials. Nucleic acid molecules may be attached to the surface in an ordered manner. After appropriate labeling, other nucleic acids or nucleic acid mixtures can be hybridized to the immobilized nucleic acid molecules, and the label may be used to monitor and measure the individual signal intensities of the hybridized molecules at defined regions. This methodology allows the simultaneous quantification of the relative or absolute amount of all or selected nucleic acids in the applied nucleic acid sample or mixture. DNA microarrays, therefore, permit an analysis of the expression of multiple (as many as 6800 or more) nucleic acids in parallel (see, e.g., Schena, M. (1996) BioEssays 18(5): 427-431).
The sequences of the invention may be used to design oligonucleotide primers which are able to amplify defined regions of one or more C. glutamicum genes by a nucleic acid amplification reaction such as the polymerase chain reaction. The choice and design of the 5′ or 3′ oligonucleotide primers or of appropriate linkers allows the covalent attachment of the resulting PCR products to the surface of a support medium described above (and also described, for example, Schena, M. et al. (1995) Science 270: 467-470).
Nucleic acid microarrays may also be constructed by in situ oligonucleotide synthesis as described by Wodicka, L. et al. (1997) Nature Biotechnology 15: 1359-1367. By photolithographic methods, precisely defined regions of the matrix are exposed to light. Protective groups which are photolabile are thereby activated and undergo nucleotide addition, whereas regions that are masked from light do not undergo any modification. Subsequent cycles of protection and light activation permit the synthesis of different oligonucleotides at defined positions. Small, defined regions of the genes of the invention may be synthesized on microarrays by solid phase oligonucleotide synthesis.
The nucleic acid molecules of the invention present in a sample or mixture of nucleotides may be hybridized to the microarrays. These nucleic acid molecules can be labeled according to standard methods. In brief, nucleic acid molecules (e.g., mRNA molecules or DNA molecules) are labeled by the incorporation of isotopically or fluorescently labeled nucleotides, e.g., during reverse transcription or DNA synthesis. Hybridization of labeled nucleic acids to microarrays is described (e.g., in Schena, M. et al. (1995) supra; Wodicka, L. et al. (1997), supra; and DeSaizieu A. et al. (1998), supra). The detection and quantification of the hybridized molecule are tailored to the specific incorporated label. Radioactive labels can be detected, for example, as described in Schena, M. et al. (1995) supra) and fluorescent labels may be detected, for example, by the method of Shalon et al. (1996) Genome Research 6: 639-645).
The application of the sequences of the invention to DNA microarray technology, as described above, permits comparative analyses of different strains of C. glutamicum or other Corynebacteria. For example, studies of inter-strain variations based on individual transcript profiles and the identification of genes that are important for specific and/or desired strain properties such as pathogenicity, productivity and stress tolerance are facilitated by nucleic acid array methodologies. Also, comparisons of the profile of expression of genes of the invention during the course of a fermentation reaction are possible using nucleic acid array technology.
Example 13
Analysis of the Dynamics of Cellular Protein Populations (Proteomics)
The genes, compositions, and methods of the invention may be applied to study the interactions and dynamics of populations of proteins, termed ‘proteomics’. Protein populations of interest include, but are not limited to, the total protein population of C. glutamicum (e.g., in comparison with the protein populations of other organisms), those proteins which are active under specific environmental or metabolic conditions (e.g., during fermentation, at high or low temperature, or at high or low pH), or those proteins which are active during specific phases of growth and development.
Protein populations can be analyzed by various well-known techniques, such as gel electrophoresis. Cellular proteins may be obtained, for example, by lysis or extraction, and may be separated from one another using a variety of electrophoretic techniques. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins largely on the basis of their molecular weight. Isoelectric focusing polyacrylamide gel electrophoresis (IEF-PAGE) separates proteins by their isoelectric point (which reflects not only the amino acid sequence but also posttranslational modifications of the protein). Another, more preferred method of protein analysis is the consecutive combination of both IEF-PAGE and SDS-PAGE, known as 2-D-gel electrophoresis (described, for example, in Hermann et al. (1998) Electrophoresis 19: 3217-3221; Fountoulakis et al. (1998) Electrophoresis 19: 1193-1202; Langen et al. (1997) Electrophoresis 18: 1184-1192; Antelmann et al. (1997) Electrophoresis 18: 1451-1463). Other separation techniques may also be utilized for protein separation, such as capillary gel electrophoresis; such techniques are well known in the art.
Proteins separated by these methodologies can be visualized by standard techniques, such as by staining or labeling. Suitable stains are known in the art, and include Coomassie Brilliant Blue, silver stain, or fluorescent dyes such as Sypro Ruby (Molecular Probes). The inclusion of radioactively labeled amino acids or other protein precursors (e.g., 35S-methionine, 35S-cysteine, 14C-labelled amino acids, 15N-amino acids, 15NO3 or 15NH4+ or 13C-labelled amino acids) in the medium of C. glutamicum permits the labeling of proteins from these cells prior to their separation. Similarly, fluorescent labels may be employed. These labeled proteins can be extracted, isolated and separated according to the previously described techniques.
Proteins visualized by these techniques can be further analyzed by measuring the amount of dye or label used. The amount of a given protein can be determined quantitatively using, for example, optical methods and can be compared to the amount of other proteins in the same gel or in other gels. Comparisons of proteins on gels can be made, for example, by optical comparison, by spectroscopy, by image scanning and analysis of gels, or through the use of photographic films and screens. Such techniques are well-known in the art.
To determine the identity of any given protein, direct sequencing or other standard techniques may be employed. For example, N- and/or C-terminal amino acid sequencing (such as Edman degradation) may be used, as may mass spectrometry (in particular MALDI or ESI techniques (see, e.g., Langen et al. (1997) Electrophoresis 18: 1184-1192)). The protein sequences provided herein can be used for the identification of C. glutamicum proteins by these techniques.
The information obtained by these methods can be used to compare patterns of protein presence, activity, or modification between different samples from various biological conditions (e.g., different organisms, time points of fermentation, media conditions, or different biotopes, among others). Data obtained from such experiments alone, or in combination with other techniques, can be used for various applications, such as to compare the behavior of various organisms in a given (e.g., metabolic) situation, to increase the productivity of strains which produce fine chemicals or to increase the efficiency of the production of fine chemicals.
Example 14
Cloning of Genes by Application of the Polymerase Chain Reaction (PCR)
Genes can be amplified using specific oligonucleotides comprising either nucleotide sequences homologous to sequences of Corynebacterium glutamicum or other strains as well as recognition sites of restriction enzymes well known in the art (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Theses oligonucleotides can be used to amplify specific DNA-fragments containing parts of the chromosome of mentioned strains using DNA-polymerases such as T. aquaticus DNA-polymerase, P. furiosus DNA-polymerase, or P. woesei DNA-polymerase and dNTPs nucleotides in an appropriate buffer solution as described by the manufacturer.
Gene fragments such as coding sequences from RXA00657 including appropriate upstream and downstream regions not contained in the coding region of the mentioned gene can be amplified using the aforementioned technologies. Furthermore, these fragments can be purified from unincorporated oligonucleotides and nucleotides. DNA restriction enzymes can be used to produce protruding ends that can be used to ligate DNA fragments to vectors digested with complementary enzymes or compatible enzymes producing ends that can be used to ligate the DNA into the vectors mentioned in Sinskey et al., U.S. Pat. No. 4,649,119, and techniques for genetic manipulation of C. glutamicum and the related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al, J. Bacteriol. 162: 591-597 (1985); Katsumata et al., J. Bacteriol. 159: 306-311 (1984); and Santamaria et al., J. Gen. Microbiol. 130: 2237-2246 (1984). Oligonucleotides used as primers for the amplification of upstream DNA sequence, the coding region sequence and the downstream region of RXA00657 were as follows:











TCGGGTATCCGCGCTACACTTAGA;
(SEQ ID NO: 121)




 



GGAAACCGGGGCATCGAAACTTA.
(SEQ ID NO: 122)






Corynebacterium glutamicum chromosomal DNA with an amount of 200 ng was used as a template in a 100 μl reaction volume containing 2.5 U Pfu Turbo-PolymeraseTM (StratageneTM), and 200 μM dNTP-nucleotides The PCR was performed on a PCR-CyclerTM (Perkin Elmer 2400TM) using the following temperature/time protocol:

    1 cycle: 94° C.: 2 min.;
    20 cycle: 94° C.: 1 min.;
    52° C.: 1 min, 72° C.: 1.5 min.,
    1 cycle: 72° C.: 5 min.


Primers were removed from the resulting amplified DNA fragment and the resulting fragment was cloned into the blunt EcoRV site of pBS KS (StratageneTM). The fragment was excised by digestion with the restriction enzymes BamHI/XhoI and ligated into a BamHI SalI digested vector pB (SEQ ID NO.:125). The resulting vector is called pB RXA00657.
Resulting recombinant vectors can be analyzed using standard techniques described in e.g., Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and can be transferred into C. glutamicum using aforementioned techniques.
A Corynebacterium strain (ATCC 13286) was treated for a transformation as described. Transformation of C. glutamicum can be achieved by protoplast transformation (Kastsumata, R. et al. (1984) J. Bacteriol. 159306-311), electroporation (Liebl, E. et al. (1989) FEMS Microbiol. Letters, 53:399-303) and in cases where special vectors are used, also by conjugation (as described, e.g., in Schäfer, A. et al. (1990) J. Bacteriol. 172:1663-1666). It is also possible to transfer the shuttle vectors for C. glutamicum to E. coli by preparing plasmid DNA from C. glutamicum (using standard methods well-known in the art) and transforming it into E. coli. This transformation step can be performed using standard methods, but it is advantageous to use an Mcr-deficient E. coli strain, such as NM522 (Gough & Murray (1983) J. Mol. Biol. 166:1-19).
Transformation of a bacterial strain such as Corynebacterium glutamicum strain (ATCC 13286) was performed with a plasmid pB containing the aforementioned DNA regions of RXA00657 (SEQ ID NO.:6) and in another case with the vector pB (SEQ ID NO.: ) carrying no additional insertion of nucleic acids.
The resulting strains were plated on and isolated from CM-Medium (10 g/l Glucose 2.5 g/l NaCl, 2.0 g/l Urea, 10 g/l Bacto Peptone (Difco/Becton Dicinson/Sparks USATM), 5 g/l yeast extract (Difco/Becton Dicinson/Sparks USATM), 5 g/l meat extract (Difco/Becton Dicinson/Sparks USATM), 22 g/l Agar (Difco/Becton Dickinson/Sparks USATM) and 15 μg/ml kanamycin sulfate (Serva, Germany) with a adjusted with NaOH to pH of 6.8.
Strains isolated from the aforementioned agar medium were inoculated in 10 ml in a 100 ml shake flask containing no baffles in liquid medium containing 100 g/l sucrose 50 g/l (NH4)2SO4, 2.5 g/l NaCl, 2.0 g/l Urea, 10 g/l Bacto Peptone (Difco/Becton Dickinson/Sparks USA), 5 g/l yeast extract (Difco/Becton Dickinson/Sparks USA), 5 g/l meat extract (Difco/Becton Dickinson/Sparks USA), and 25 g/l CaCO3 (Riedel de Haen, Germany). Medium was a adjusted with NaOH to pH of 6.8.
Strains were incubated at 30° C. for 48 h. Supernatants of incubations were prepared by centrifugation 20′ at 12,000 rpm in an EppendorfTM microcentrifuge. Liquid supernatants were diluted and subjected to amino acid analysis (Standard methods for these measurements are outlined in Applied Microbial Physiology, A Practical Approach, P. M. Rhodes and P. F. Stanbury, eds., IRL Press, p. 103-129; 131-163; and 165-192 (ISBN: 0199635773) and references cited therein).
The results are shown in Table 6, below.







TABLE 6







Results:












Strain
Plasmid





ATCC 13286
contained
pB
pB RXA00657
















lysin produced

13.5
14.93



(g/l)



Selectivity

0.235
0.25



(mol lysine/



mol consumed



Saccharose)










Equivalents

Those of ordinary skill in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.







TABLE 1







Included Genes













Nucleic Acid
Amino Acid
Identification

NT
NT



SEQ ID NO
SEQ ID NO
Code
Contig.
Start
Stop
Function










Lysine biosynthesis













5
6
RXA00657



AMINOACID BIOSYNTHESIS REGULATOR


7
8
RXA02229
GR00653
2793
3617
DIAMINOPIMELATE EPIMERASE (EC 5.1.1.7)


9
10
RXS02970



ACETYLORNITHINE AMINOTRANSFERASE (EC 2.6.1.11)


11
12
F RXA01009
GR00287
4714
5943
ACETYLORNITHINE AMINOTRANSFERASE (EC 2.6.1.11)


13
14
RXC02390



MEMBRANE SPANNING PROTEIN INVOLVED IN LYSINE








METABOLISM


15
16
RXC01796



MEMBRANE ASSOCIATED PROTEIN INVOLVED IN LYSINE








METABOLISM


17
18
RXC01207



CYTOSOLIC PROTEIN INVOLVED IN METABOLISM OF LYSINE








AND THREONINE


19
20
RXC00657



TRANSCRIPTIONAL REGULATOR INVOLVED IN LYSINE








METABOLISM


21
22
RXC00552



CYTOSOLIC PROTEIN INVOLVED IN LYSINE METABOLISM


23
24
RXA00534
GR00137
4758
3496
ASPARTOKINASE ALPHA AND BETA SUBUNITS (EC 2.7.2.4)


25
26
RXA00533
GR00137
3469
2438
ASPARTATE-SEMIALDEHYDE DEHYDROGENASE (EC 1.2.1.11)


27
28
RXA02843
GR00842
543
4
2,3,4,5-TETRAHYDROPYRIDINE-2-CARBOXYLATE








N-SUCCINYLTRANSFERASE (EC 2.3.1.117)


29
30
RXA02022
GR00613
2063
3169
SUCCINYL-DIAMINOPIMELATE DESUCCINYLASE (EC 3.5.1.18)


31
32
RXA00044
GR00007
3458
4393
DIHYDRODIPICOLINATE SYNTHASE (EC 4.2.1.52)


33
34
RXA00863
GR00236
896
1639
DIHYDRODIPICOLINATE REDUCTASE (EC 1.3.1.26)


35
36
RXA00864
GR00236
1694
2443
probable 2,3-dihydrodipicolinate N-C6-lyase (cyclizing) (EC 4.3.3.-) -








Corynebacterium glutamicum


37
38
RXA02843
GR00842
543
4
2,3,4,5-TETRAHYDROPYRIDINE-2-CARBOXYLATE








N-SUCCINYLTRANSFERASE (EC 2.3.1.117)


39
40
RXN00355
VV0135
31980
30961
MESO-DIAMINOPIMELATE D-DEHYDROGENASE


41
42
F RXA00352
GR00068
861
4
MESO-DIAMINOPIMELATE D-DEHYDROGENASE (EC 1.4.1.16)


43
44
RXA00972
GR00274
3
1379
DIAMINOPIMELATE DECARBOXYLASE (EC 4.1.1.20)


45
46
RXA02653
GR00752
5237
7234
DIAMINOPIMELATE DECARBOXYLASE (EC 4.1.1.20)


47
48
RXA01393
GR00408
4249
3380
LYSINE EXPORT REGULATOR PROTEIN


49
50
RXA00241
GR00036
5443
6945
L-LYSINE TRANSPORT PROTEIN


51
52
RXA01394
GR00408
4320
5018
LYSINE EXPORTER PROTEIN


53
54
RXA00865
GR00236
2647
3549
DIHYDRODIPICOLINATE SYNTHASE (EC 4.2.1.52)


55
56
RXS02021



2,3,4,5-TRAHYDROPYRIDINE-2-CARBOXYLATE








N-SUCCINYLTRANSFERASE (EC 2.3.1.117)


57
58
RXS02157



ACETYLORNITHINE AMINOTRANSFERASE (EC 2.6.1.11)


59
60
RXC00733



ABC TRANSPORTER ATP-BINDING PROTEIN INVOLVED IN








LYSINE METABOLISM


61
62
RXC00861



PROTEIN INVOLVED IN LYSINE METABOLISM


63
64
RXC00866



ZN-DEPENDENT HYDROLASE INVOLVED IN LYSINE








METABOLISM


65
66
RXC02095



ABC TRANSPORTER ATP-BINDING PROTEIN INVOLVED IN








LYSINE METABOLISM


67
68
RXC03185



PROTEIN INVOLVED IN LYSINE METABOLISM







Metabolism of methionine and S-adenosyl methionine













1
2
metZ or met



O-ACETYLHOMOSERINE SULFHYDRYLASE (EC 4.2.99.10)


3
4
metC



Cystathionine-y-lyase


69
70
RXA00115
GR00017
5359
4313
HOMOSERINE O-ACETYLTRANSFERASE (EC 2.3.1.31)


71
72
RXN00403
VV0086
70041
68911
HOMOSERINE O-ACETYLTRANSFERASE


73
74
F RXA00403
GR00088
723
1832
HOMOSERINE O-ACETYLTRANSFERASE (EC 2.3.1.11)


75
76
RXS03158



CYSTATHIONINE GAMMA-SYNTHASE (EC 4.2.99.9)


77
78
F RXA00254
GR00038
2404
1811
CYSTATHIONINE GAMMA-SYNTHASE (EC 4.2.99.9)


79
80
RXA02532
GR00726
3085
2039
CYSTATHIONINE GAMMA-SYNTHASE (EC 4.2.99.9)


81
82
RXS03159



CYSTATHIONINE GAMMA-SYNTHASE (EC 4.2.99.9)


83
84
F RXA02768
GR00770
1919
2521
CYSTATHIONINE GAMMA-SYNTHASE (EC 4.2.99.9)


85
86
RXA00216
GR00032
16286
15297
5-methyltetrahydrofolate-homocysteine methyltransferase








(methionine synthetase)


87
94
RXN02197
GR00645
4552
4025
5-METHYLTETRAHYDROFOLATE-HOMOCYSTEINE








METHYLTRANSFERASE (EC 2.1.1.13)


89
90
RXN02198
VV0302
9228
11726
5-METHYLTETRAHYDROFOLATE-HOMOCYSTEINE








METHYLTRANSFERASE (EC 2.1.1.13)


91
91
F RXA02198
GR00646
2483
6
5-METHYLTETRAHYDROFOLATE-HOMOCYSTEINE








METHYLTRANSFERASE (EC 2.1.1.13)


93
94
RXN03074
VV0042
2238
1741
S-ADENOSYLMETHIONINE:2-DEMETHYLMENAQUINONE








METHYLTRANSFERASE (EC 2.1.-.-)


95
96
F RXA02906
GR10044
1142
645
S-ADENOSYLMETHIONINE:2-DEMETHYLMENAQUINONE








METHYLTRANSFERASE (EC 2.1.-.-)


97
98
RXN00132
VV0124
3612
5045
ADENOSYLHOMOCYSTEINASE (EC 3.3.1.1)


99
100
F RXA00132
GR00020
7728
7624
ADENOSYLHOMOCYSTEINASE (EC 3.3.1.1)


101
102
F RXA01371
GR00398
2339
3634
ADENOSYLHOMOCYSTEINASE (EC 3.3.1.1)


103
104
RXN02085



5-METHYLTETRAHYDROPTEROYLTRIGLUTAMATE--








HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.14)


105
106
F RXA02085
GR00629
3496
5295
5-METHYLTETRAHYDROPTEROYLTRIGLUTAMATE--








HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.14)


107
108
F RXA02086
GR00629
5252
5731
5-METHYLTETRAHYDROPTEROYLTRIGLUTAMATE--








HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.14)


109
110
RXN02648



5-METHYLTETRAHYDROPTEROYLTRIGLUTAMATE--








HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.14)


111
112
F RXA02648
GR00751
5254
4730
5-METHYLTETRAHYDROPTEROYLTRIGLUTAMATE--








HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.14)


113
114
F RXA02658
GR00752
14764
15447
5-METHYLTETRAHYDROPTEROYLTRIGLUTAMATE--








HOMOCYSTEINE METHYLTRANSFERASE (EC 2.1.1.14)


115
116
RXC02238



PROTEIN INVOLVED IN METABOLISM OF








S-ADENOSYLMETHIONINE, PURINES AND PANTOTHENATE


117
118
RXC00128



EXPORTED PROTEIN INVOLVED IN METABOLISM OF








PYRIDIMES AND ADENOSYLHOMOCYSTEINE







S-2adenosyl methionine (SAM) Biosynthesis













119
120
RXA02240
GR00654
7160
8380
S-ADENOSYLMETHIONINE SYNTHETASE (EC 2.5.1.6)
















TABLE 2







GENES IDENTIFIED FROM GENBANK










GenBank TM
Gene




Accession No.
Name
Gene Function
Reference





A09073
ppg
Phosphoenol pyruvate carboxylase
Bachmann, B. et al. “DNA fragment coding for phosphoenolpyruvat





corboxylase, recombinant DNA carrying said fragment, strains carrying the





recombinant DNA and method for producing L-aminino acids using said





strains,” Patent: EP 0358940-A 3 Mar. 21, 1990


A45579,

Threonine dehydratase
Moeckel, B. et al. “Production of L-isoleucine by means of recombinant


A45581,


micro-organisms with deregulated threonine dehydratase,” Patent: WO


A45583,


9519442-A 5 Jul. 20, 1995


A45585


A45587


AB003132
murC;

Kobayashi, M. et al. “Cloning, sequencing, and characterization of the ftsZ



ftsQ;

gene from coryneform bacteria,” Biochem. Biophys. Res. Commun.,



ftsZ

236(2): 383-388 (1997)


AB015023
murC;

Wachi, M. et al. “A murC gene from Coryneform bacteria,” Appl. Microbiol.



ftsQ

Biotechnol., 51(2): 223-228 (1999)


AB018530
dtsR

Kimura, E. et al. “Molecular cloning of a novel gene, dtsR, which rescues the





detergent sensitivity of a mutant derived from Brevibacterium





lactofermentum,” Biosci. Biotechnol. Biochem., 60(10): 1565-1570 (1996)


AB018531
dtsR1;



dtsR2


AB020624
murI
D-glutamate racemase


AB023377
tkt
transketolase


AB024708
gltB;
Glutamine 2-oxoglutarate



gltD
aminotransferase large and




small subunits


AB025424
acn
aconitase


AB027714
rep
Replication protein


AB027715
rep; 
Replication protein; aminoglycoside



aad
adenyltransferase


AF005242
argC
N-acetylglutamate-5-semialdehyde




dehydrogenase


AF005635
glnA
Glutamine synthetase


AF030405
hisF
cyclase


AF030520
argG
Argininosuccinate synthetase


AF031518
argF
Ornithine carbamolytransferase


AF036932
aroD
3-dehydroquinate dehydratase


AF038548
pyc
Pyruvate carboxylase


AF038651
dciAE;
Dipeptide-binding protein; adenine
Wehmeier, L. et al. “The role of the Corynebacterium glutamicum rel gene in



apt;
phosphoribosyltransferase; GTP
(p)ppGpp metabolism,” Microbiology, 144: 1853-1862 (1998)



rel
pyrophosphokinase


AF041436
argR
Arginine repressor


AF045998
impA
Inositol monophosphate phosphatase


AF048764
argH
Argininosuccinate lyase


AF049897
argC;
N-acetylglutamylphosphate reductase;



argJ;
ornithine acetyltransferase; N-



argB;
acetylglutamate kinase;



argD;
acetylornithine transminase;



argF;
ornithine carbamoyltransferase;



argR;
arginine repressor; argininosuccinate



argG;
synthase; argininosuccinate lyase



argH


AF050109
inhA
Enoyl-acyl carrier protein reductase


AF050166
hisG
ATP phosphoribosyltransferase


AF051846
hisA
Phosphoribosylformimino-5-amino-1-




phosphoribosyl-4-




imidazolecarboxamide isomerase


AF052652
metA
Homoserine O-acetyltransferase
Park, S. et al. “Isolation and analysis of metA, a methionine biosynthetic gene





encoding homoserine acetyltransferase in Corynebacterium glutamicum,” Mol.





Cells., 8(3): 286-294 (1998)


AF053071
aroB
Dehydroquinate synthetase


AF060558
hisH
Glutamine amidotransferase


AF086704
hisE
Phosphoribosyl-ATP-




pyrophosphohydrolase


AF114233
aroA
5-enolpyruvylshikimate 3-phosphate




synthase


AF116184
panD
L-aspartate-alpha-decarboxylase
Dusch, N. et al. “Expression of the Corynebacterium glutamicum panD gene




precursor
encoding L-aspartate-alpha-decarboxylase leads to pantothenate





overproduction in Escherichia coli,” Appl. Environ. Microbiol., 65(4)1530-1539





(1999)


AF124518
aroD;
3-dehydroquinase; shikimate



aroE
dehydrogenase


AF124600
aroC;
Chorismate synthase; shikimate



aroK;
kinase; 3-dehydroquinate synthase;



aroB;
putative cytoplasmic peptidase



pepQ


AF145897
inhA


AF145898
inhA


AJ001436
ectP
Transport of ectoine, glycine betaine,
Peter, H. et al. “Corynebacterium glutamicum is equipped with four secondary




proline
carriers for compatible solutes: Identification, sequencing, and characterization





of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine





betaine carrier, EctP,” J. Bacteriol., 180(22): 6005-6012 (1998)


AJ004934
dapD
Tetrahydrodipicolinate succinylase
Wehrmann, A. et al. “Different modes of diaminopimelate synthesis and their




(incompletei)
role in cell wall integrity: A study with Corynebacterium glutamicum,” J.





Bacteriol., 180(12): 3159-3165 (1998)


AJ007732
ppc;
Phosphoenolpyruvate-carboxylase; ?;



secG;
high affinity ammonium uptake



amt;
protein; putative ornithine-



ocd;
cyclodecarboxylase; sarcosine



soxA
oxidase


AJ010319
ftsY,
Involved in cell division; PII protein;
Jakoby, M. et al. “Nitrogen regulation in Corynebacterium glutamicum;



glnB,
uridylyltransferase (uridylyl-
Isolation of genes involved in biochemical characterization of corresponding



glnD;
removing enzmye); signal recognition
proteins,” FEMS Microbiol., 173(2): 303-310 (1999)



srp;
particle; low affinity ammonium



amtP
uptake protein


AJ132968
cat
Chloramphenicol aceteyl transferase


AJ224946
mqo
L-malate: quinone oxidoreductase
Molenaar, D. et al. “Biochemical and genetic characterization of the





membrane-associated malate dehydrogenase (acceptor) from Corynebacterium





glutamicum,” Eur. J. Biochem., 254(2): 395-403 (1998)


AJ238250
ndh
NADH dehydrogenase


AJ238703
porA
Porin
Lichtinger, T. et al. “Biochemical and biophysical characterization of the cell





wall porin of Corynebacterium glutamicum: The channel is formed by a low





molecular mass polypeptide,” Biochemistry, 37(43): 15024-15032 (1998)


D17429

Transposable element IS31831
Vertes et al. “Isolation and characterization of IS31831, a transposable element





from Corynebacterium glutamicum,” Mol. Microbiol., 11(4): 739-746 (1994)


D84102
odhA
2-oxoglutarate dehydrogenase
Usuda, Y. et al. “Molecular cloning of the Corynebacterium glutamicum





(Brevibacterium lactofermentum AJ12036) odhA gene encoding a novel type





of 2-oxoglutarate dehydrogenase,” Microbiology, 142: 3347-3354 (1996)


E01358
hdh;
Homoserine dehydrogenase;
Katsumata, R. et al. “Production of L-thereonine and L-isoleucine,” Patent: JP



hk
homoserine kinase
1987232392-A 1 Oct. 12, 1987


E01359

Upstream of the start codon of
Katsumata, R. et al. “Production of L-thereonine and L-isoleucine,” Patent: JP




homoserine kinase gene
1987232392-A 2 Oct. 12, 1987


E01375

Tryptophan operon


E01376
trpL;
Leader peptide; anthranilate synthase
Matsui, K. et al. “Tryptophan operon, peptide and protein coded thereby,



trpE

utilization of tryptophan operon gene expression and production of





tryptophan,” Patent: JP 1987244382-A 1 Oct. 24, 1987


E01377

Promoter and operator regions of
Matsui, K. et al. “Tryptophan operon, peptide and protein coded thereby,




tryptophan operon
utilization of tryptophan operon gene expression and production of





tryptophan,” Patent: JP 1987244382-A 1 Oct. 24, 1987


E03937

Biotin-synthase
Hatakeyama, K. et al. “DNA fragment containing gene capable of coding





biotin synthetase and its utilization,” Patent: JP 1992278088-A 1 Oct. 02, 1992


E04040

Diamino pelargonic acid
Kohama, K. et al. “Gene coding diaminopelargonic acid aminotransferase and




aminotransferase
desthiobiotin synthetase and its utilization,” Patent: JP 1992330284-A 1





Nov. 18, 1992


E04041

Desthiobiotinsynthetase
Kohama, K. et al. “Gene coding diaminopelargonic acid aminotransferase and





desthiobiotin synthetase and its utilization,” Patent: JP 1992330284-A 1





Nov. 18, 1992


E04307

Flavum aspartase
Kurusu, Y. et al. “Gene DNA coding aspartase and utilization thereof,” Patent:





JP 1993030977-A 1 Feb. 09, 1993


E04376

Isocitric acid lyase
Katsumata, R. et al. “Gene manifestation controlling DNA,” Patent: JP





1993056782-A 3 Mar. 09, 1993


E04377

Isocitric acid lyase N-terminal
Katsumata, R. et al. “Gene manifestation controlling DNA,” Patent: JP




fragment
1993056782-A 3 Mar. 09, 1993


E04484

Prephenate dehydratase
Sotouchi, N. et al. “Production of L-phenylalanine by fermentation,” Patent: JP





1993076352-A 2 Mar. 30, 1993


E05108

Aspartokinase
Fugono, N. et al. “Gene DNA coding Aspartokinase and its use,” Patent: JP





1993184366-A 1 Jul. 27, 1993


E05112

Dihydro-dipichorinate synthetase
Hatakeyama, K. et al. “Gene DNA coding dihydrodipicolinic acid synthetase





and its use,” Patent: JP 1993184371-A 1 Jul. 27, 1993


E05776

Diaminopimelic acid dehydrogenase
Kobayashi, M. et al. “Gene DNA coding Diaminopimelic acid dehydrogenase





and its use,” Patent: JP 1993284970-A 1 Nov. 02, 1993


E05779

Threonine synthase
Kohama, K. et al. “Gene DNA coding threonine synthase and its use,” Patent:





JP 1993284972-A 1 Nov. 02, 1993


E06110

Prephenate dehydratase
Kikuchi, T. et al. “Production of L-phenylalanine by fermentation method,”





Patent: JP 1993344881-A 1 Dec. 27, 1993


E06111

Mutated Prephenate dehydratase
Kikuchi, T. et al. “Production of L-phenylalanine by fermentation method,”





Patent: JP 1993344881-A 1 Dec. 27, 1993


E06146

Acetohydroxy acid synthetase
Inui, M. et al. “Gene capable of coding Acetohydroxy acid synthetase and its





use,” Patent: JP 1993344893-A 1 Dec. 27, 1993


E06825

Aspartokinase
Sugimoto, M. et al. “Mutant aspartokinase gene,” patent: JP 1994062866-A 1





Mar. 08, 1994


E06826

Mutated aspartokinase alpha subunit
Sugimoto, M. et al. “Mutant aspartokinase gene,” patent: JP 1994062866-A 1





Mar. 08, 1994


E06827

Mutated aspartokinase alpha subunit
Sugimoto, M. et al. “Mutant aspartokinase gene,” patent: JP 1994062866-A 1





Mar. 08, 1994


E07701
secY

Honno, N. et al. “Gene DNA participating in integration of membraneous





protein to membrane,” Patent: JP 1994169780-A 1 Jun. 21, 1994


E08177

Aspartokinase
Sato, Y. et al. “Genetic DNA capable of coding Aspartokinase released from





feedback inhibition and its utilization,” Patent: JP 1994261766-A 1 Sep. 20, 1994


E08178,

Feedback inhibition-released
Sato, Y. et al. “Genetic DNA capable of coding Aspartokinase released from


E08179,

Aspartokinase
feedback inhibition and its utilization,” Patent: JP 1994261766-A 1 Sep. 20, 1994


E08180,


E08181,


E08182


E08232

Acetohydroxy-acid isomeroreductase
Inui, M. et al. “Gene DNA coding acetohydroxy acid isomeroreductase,”





Patent: JP 1994277067-A 1 Oct. 04, 1994


E08234
secE

Asai, Y. et al. “Gene DNA coding for translocation machinery of protein,”





Patent: JP 1994277073-A 1 Oct. 04, 1994


E08643

FT aminotransferase and
Hatakeyama, K. et al. “DNA fragment having promoter function in




desthiobiotin synthetase promoter
coryneform bacterium,” Patent: JP 1995031476-A 1 Feb. 03, 1995




region


E08646

Biotin synthetase
Hatakeyama, K. et al. “DNA fragment having promoter function in





coryneform bacterium,” Patent: JP 1995031476-A 1 Feb. 03, 1995


E08649

Aspartase
Kohama, K. et al “DNA fragment having promoter function in coryneform





bacterium,” Patent: JP 1995031478-A 1 Feb. 03, 1995


E08900

Dihydrodipicolinate reductase
Madori, M. et al. “DNA fragment containing gene coding Dihydrodipicolinate





acid reductase and utilization thereof,” Patent: JP 1995075578-A 1 Mar. 20, 1995


E08901

Diaminopimelic acid decarboxylase
Madori, M. et al. “DNA fragment containing gene coding Diaminopimelic acid





decarboxylase and utilization thereof,” Patent: JP 1995075579-A 1 Mar. 20, 1995


E12594

Serine hydroxymethyltransferase
Hatakeyama, K. et al. “Production of L-trypophan,” Patent: JP 1997028391-A





1 Feb. 04, 1997


E12760,

transposase
Moriya, M. et al. “Amplification of gene using artificial transposon,” Patent:


E12759,


JP 1997070291-A Mar. 18, 1997


E12758


E12764

Arginyl-tRNA synthetase;
Moriya, M. et al. “Amplification of gene using artificial transposon,” Patent:




diaminopimelic acid decarboxylase
JP 1997070291-A Mar. 18, 1997


E12767

Dihydrodipicolinic acid synthetase
Moriya, M. et al. “Amplification of gene using artificial transposon,” Patent:





JP 1997070291-A Mar. 18, 1997


E12770

aspartokinase
Moriya, M. et al. “Amplification of gene using artificial transposon,” Patent:





JP 1997070291-A Mar. 18, 1997


E12773

Dihydrodipicolinic acid reductase
Moriya, M. et al. “Amplification of gene using artificial transposon,” Patent:





JP 1997070291-A Mar. 18, 1997


E13655

Glucose-6-phosphate dehydrogenase
Hatakeyama, K. et al. “Glucose-6-phosphate dehydrogenase and DNA capable





of coding the same,” Patent: JP 1997224661-A 1 Sep. 02, 1997


L01508
IlvA
Threonine dehydratase
Moeckel, B. et al. “Functional and structural analysis of the threonine





dehydratase of Corynebacterium glutamicum,” J. Bacteriol., 174: 8065-8072





(1992)


L07603
EC
3-deoxy-D-arabinoheptulosonate-7-
Chen, C. et al. “The cloning and nucleotide sequence of Corynebacterium



4.2.1.15
phosphate synthase
glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase gene,”





FEMS Microbiol. Lett., 107: 223-230 (1993)


L09232
IlvB;
Acetohydroxy acid synthase large
Keilhauer, C. et al. “Isoleucine synthesis in Corynebacterium glutamicum:



ilvN;
subunit; Acetohydroxy acid
molecular analysis of the ilvB-ilvN-ilvC operon,” J. Bacteriol., 175(17): 5595-5603



ilvC
synthase small subunit;
(1993)




Acetohydroxy acid isomeroreductase


L18874
PtsM
Phosphoenolpyruvate sugar
Fouet, A et al. “Bacillus subtilis sucrose-specific enzyme II of the




phosphotransferase
phosphotransferase system: expression in Escherichia coli and homology to





enzymes II from enteric bacteria,” PNAS USA, 84(24): 8773-8777 (1987); Lee, J. K.





et al. “Nucleotide sequence of the gene encoding the Corynebacterium





glutamicum mannose enzyme II and analyses of the deduced protein





sequence,” FEMS Microbiol. Lett., 119(1-2): 137-145 (1994)


L27123
aceB
Malate synthase
Lee, H-S. et al. “Molecular characterization of aceB, a gene encoding malate





synthase in Corynebacterium glutamicum,” J. Microbiol. Biotechnol.,





4(4): 256-263 (1994)


L27126

Pyruvate kinase
Jetten, M. S. et al. “Structural and functional analysis of pyruvate kinase from





Corynebacterium glutamicum,” Appl. Environ. Microbiol., 60(7): 2501-2507





(1994)


L28760
aceA
Isocitrate lyase


L35906
dtxr
Diphtheria toxin repressor
Oguiza, J. A. et al. “Molecular cloning, DNA sequence analysis, and





characterization of the Corynebacterium diphtheriae dtxR from Brevibacterium





lactofermentum,” J. Bacteriol., 177(2): 465-467 (1995)


M13774

Prephenate dehydratase
Follettie, M. T. et al. “Molecular cloning and nucleotide sequence of the





Corynebacterium glutamicum pheA gene,” J. Bacteriol., 167: 695-702 (1986)


M16175
5S

Park, Y-H. et al. “Phylogenetic analysis of the coryneform bacteria by 56



rRNA

rRNA sequences,” J. Bacteriol., 169: 1801-1806 (1987)


M16663
trpE
Anthranilate synthase, 5′ end
Sano, K. et al. “Structure and function of the trp operon control regions of





Brevibacterium lactofermentum, a glutamic-acid-producing bacterium,” Gene,





52: 191-200 (1987)


M16664
trpA
Tryptophan synthase, 3′end
Sano, K. et al. “Structure and function of the trp operon control regions of





Brevibacterium lactofermentum, a glutamic-acid-producing bacterium,” Gene,





52: 191-200 (1987)


M25819

Phosphoenolpyruvate carboxylase
O'Regan, M. et al. “Cloning and nucleotide sequence of the





Phosphoenolpyruvate carboxylase-coding gene of Corynebacterium





glutamicum ATCC13032,” Gene, 77(2): 237-251 (1989)


M85106

23S rRNA gene insertion sequence
Roller, C. et al. “Gram-positive bacteria with a high DNA G + C content are





characterized by a common insertion within their 23S rRNA genes,” J. Gen.





Microbiol., 138: 1167-1175 (1992)


M85107,

23S rRNA gene insertion sequence
Roller, C. et al. “Gram-positive bacteria with a high DNA G + C content are


M85108


characterized by a common insertion within their 23S rRNA genes,” J. Gen.





Microbiol., 138: 1167-1175 (1992)


M89931
aecD;
Beta C-S lyase; branched-chain
Rossol, I. et al. “The Corynebacterium glutamicum aecD gene encodes a C-S



brnQ;
amino acid uptake carrier;
lyase with alpha, beta-elimination activity that degrades aminoethylcysteine,”



yhbw
hypothetical protein yhbw
J. Bacteriol., 174(9): 2968-2977 (1992); Tauch, A. et al. “Isoleucine uptake in





Corynebacterium glutamicum ATCC 13032 is directed by the brnQ gene





product,” Arch. Microbiol., 169(4): 303-312 (1998)


S59299
trp
Leader gene (promoter)
Herry, D. M. et al. “Cloning of the trp gene cluster from a tryptophan-





hyperproducing strain of Corynebacterium glutamicum: identification of a





mutation in the trp leader sequence,” Appl. Environ. Microbiol., 59(3): 791-799





(1993)


U11545
trpD
Anthranilate
O'Gara, J. P. and Dunican, L. K. (1994) Complete nucleotide sequence of the




phosphoribosyltransferase
Corynebacterium glutamicum ATCC 21850 tpD gene.” Thesis, Microbiology





Department, University College Galway, Ireland.


U13922
cglIM;
Putative type II 5-cytosoine
Schafer, A. et al. “Cloning and characterization of a DNA region encoding a



cglIR;
methyltransferase; putative type II
stress-sensitive restriction system from Corynebacterium glutamicum ATCC



clgIIR
restriction endonuclease; putative
13032 and analysis of its role in intergeneric conjugation with Escherichia




type I or type III restriction
coli,” J. Bacteriol., 176(23): 7309-7319 (1994); Schafer, A. et al. “The




endonuclease
Corynebacterium glutamicum cglIM gene encoding a 5-cytosine in an McrBC-





deficient Escherichia coli strain,” Gene, 203(2): 95-101 (1997)


U14965
recA


U31224
ppx

Ankri, S. et al. “Mutations in the Corynebacterium glutamicumproline





biosynthetic pathway: A natural bypass of the proA step,” J. Bacteriol.,





178(15): 4412-4419 (1996)


U31225
proC
L-proline: NADP+ 5-oxidoreductase
Ankri, S. et al. “Mutations in the Corynebacterium glutamicumproline





biosynthetic pathway: A natural bypass of the proA step,” J. Bacteriol.,





178(15): 4412-4419 (1996)


U31230
obg;
?; gamma glutamyl kinase; similar to
Ankri, S. et al. “Mutations in the Corynebacterium glutamicumproline



proB;
D-isomer specific 2-hydroxyacid
biosynthetic pathway: A natural bypass of the proA step,” J. Bacteriol.,



unkdh
dehydrogenases
178(15): 4412-4419 (1996)


U31281
bioB
Biotin synthase
Serebriiskii, I. G., “Two new members of the bio B superfamily: Cloning,





sequencing and expression of bio B genes of Methylobacillus flagellatum and





Corynebacterium glutamicum,” Gene, 175: 15-22 (1996)


U35023
thtR;
Thiosulfate sulfurtransferase;
Jager, W. et al. “A Corynebacterium glutamicum gene encoding a two-domain



accBC
acyl CoA carboxylase
protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins,”





Arch. Microbiol., 166(2); 76-82 (1996)


U43535
cmr
Multidrug resistance protein
Jager, W. et al. “A Corynebacterium glutamicum gene conferring multidrug





resistance in the heterologous host Escherichia coli,” J. Bacteriol.,





179(7): 2449-2451 (1997)


U43536
clpB
Heat shock ATP-binding protein


U53587
aphA-3
3′5′′-aminoglycoside




phosphotransferase


U89648

Corynebacterium glutamicum




unidentified sequence involved in




histidine biosynthesis, partial




sequence


X04960
trpA;
Tryptophan operon
Matsui, K. et al. “Complete nucleotide and deduced amino acid sequences of



trpB;

the Brevibacterium lactofermentum tryptophan operon,” Nucleic Acids Res.,



trpC;

14(24): 10113-10114 (1986)



trpD;



trpE;



trpG;



trpL


X07563
lys A
DAP decarboxylase (meso-
Yeh, P. et al. “Nucleic sequence of the lysA gene of Corynebacterium




diaminopimelate decarboxylase,
glutamicum and possible mechanisms for modulation of its expression,” Mol.




EC 4.1.1.20)
Gen. Genet., 212(1): 112-119 (1988)


X14234
EC
Phosphoenolpyruvate carboxylase
Eikmanns, B. J. et al. “The Phosphoenolpyruvate carboxylase gene of



4.1.1.31

Corynebacterium glutamicum: Molecular cloning, nucleotide sequence, and





expression,” Mol. Gen. Genet., 218(2): 330-339 (1989); Lepiniec, L. et al.





“Sorghum Phosphoenolpyruvate carboxylase gene family: structure, function





and molecular evolution,” Plant. Mol. Biol., 21 (3): 487-502 (1993)


X17313
fda
Fructose-bisphosphate aldolase
Von der Osten, C. H. et al. “Molecular cloning, nucleotide sequence and fine-





structural analysis of the Corynebacterium glutamicum fda gene: structural





comparison of C. glutamicum fructose-1,6-biphosphate aldolase to class I and





class II aldolases,” Mol. Microbiol.,


X53993
dapA
L-2,3-dihydrodipicolinate
Bonnassie, S. et al. “Nucleic sequence of the dapA gene from




synthetase (EC 4.2.1.52)
Corynebacterium glutamicum,” Nucleic Acids Res., 18(21): 6421 (1990)


X54223

AttB-related site
Cianciotto, N. et al. “DNA sequence homology between att B-related sites of





Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium





glutamicum, and the attP site of lambdacorynephage,” FEMS. Microbiol,





Lett., 66: 299-302 (1990)


X54740
argS;
Arginyl-tRNA synthetase;
Marcel, T. et al. “Nucleotide sequence and organization of the upstream region



lysA
Diaminopimelate decarboxylase
of the Corynebacterium glutamicum lysA gene,” Mol. Microbiol., 4(11): 1819-1830





(1990)


X55994
trpL;
Putative leader peptide; anthranilate
Heery, D. M. et al. “Nucleotide sequence of the Corynebacterium glutamicum



trpE
synthase component 1
trpE gene,” Nucleic Acids Res., 18(23): 7138 (1990)


X56037
thrC
Threonine synthase
Han, K. S. et al. “The molecular structure of the Corynebacterium glutamicum





threonine synthase gene,” Mol. Microbiol., 4(10): 1693-1702 (1990)


X56075
attB-
Attachment site
Cianciotto, N. et al. “DNA sequence homology between att B-related sites of



related

Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium



site

glutamicum, and the attP site of lambdacorynephage,” FEMS. Microbiol,





Lett., 66: 299-302 (1990)


X57226
lysC-
Aspartokinase-alpha subunit;
Kalinowski, J. et al. “Genetic and biochemical analysis of the Aspartokinase



alpha;
Aspartokinase-beta subunit; aspartate
from Corynebacterium glutamicum,” Mol. Microbiol., 5(5): 1197-1204 (1991);



lysC-
beta semialdehyde dehydrogenase
Kalinowski, J. et al. “Aspartokinase genes lysC alpha and lysC beta overlap



beta;

and are adjacent to the aspertate beta-semialdehyde dehydrogenase gene asd in



asd

Corynebacterium glutamicum,” Mol. Gen. Genet., 224(3): 317-324 (1990)


X59403
gap;
Glyceraldehyde-3-phosphate;
Eikmanns, B. J. “Identification, sequence analysis, and expression of a



pgk;
phosphoglycerate kinase;
Corynebacterium glutamicum gene cluster encoding the three glycolytic



tpi
triosephosphate isomerase
enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate





kinase, and triosephosphate isomeras,” J. Bacteriol., 174(19): 6076-6086





(1992)


X59404
gdh
Glutamate dehydrogenase
Bormann, E. R. et al. “Molecular analysis of the Corynebacterium glutamicum





gdh gene encoding glutamate dehydrogenase,” Mol. Microbiol., 6(3): 317-326





(1992)


X60312
lysI
L-lysine permease
Seep-Feldhaus, A. H. et al. “Molecular analysis of the Corynebacterium





glutamicum lysI gene involved in lysine uptake,” Mol. Microbiol., 5(12): 2995-3005





(1991)


X66078
cop1
Ps1 protein
Joliff, G. et al. “Cloning and nucleotide sequence of the csp1 gene encoding





PS1, one of the two major secreted proteins of Corynebacterium glutamicum:





The deduced N-terminal region of PS1 is similar to the Mycobacterium antigen





85 complex,” Mol. Microbiol., 6(16): 2349-2362 (1992)


X66112
glt
Citrate synthase
Eikmanns, B. J. et al. “Cloning sequence, expression and transcriptional





analysis of the Corynebacterium glutamicum gltA gene encoding citrate





synthase,” Microbiol., 140: 1817-1828 (1994)


X67737
dapB
Dihydrodipicolinate reductase


X69103
csp2
Surface layer protein PS2
Peyret, J. L. et al. “Characterization of the cspB gene encoding PS2, an ordered





surface-layer protein in Corynebacterium glutamicum,” Mol. Microbiol.,





9(1): 97-109 (1993)


X69104

IS3 related insertion element
Bonamy, C. et al. “Identification of IS1206, a Corynebacterium glutamicum





IS3-related insertion sequence and phylogenetic analysis,” Mol. Microbiol.,





14(3): 571-581 (1994)


X70959
leuA
Isopropylmalate synthase
Patek, M. et al. “Leucine synthesis in Corynebacterium glutamicum: enzyme





activities, structure of leuA, and effect of leuA inactivation on lysine





synthesis,” Appl. Environ. Microbiol., 60(1): 133-140 (1994)


X71489
icd
Isocitrate dehydrogenase (NADP+)
Eikmanns, B. J. et al. “Cloning sequence analysis, expression, and inactivation





of the Corynebacterium glutamicum icd gene encoding isocitrate





dehydrogenase and biochemical characterization of the enzyme,” J. Bacteriol.,





177(3): 774-782 (1995)


X72855
GDHA
Glutamate dehydrogenase (NADP+)


X75083,
mtrA
5-methyltryptophan resistance
Heery, D. M. et al. “A sequence from a tryptophan-hyperproducing strain of


X70584


Corynebacterium glutamicum encoding resistance to 5-methyltryptophan,”





Biochem. Biophys. Res. Commun., 201(3): 1255-1262 (1994)


X75085
recA

Fitzpatrick, R. et al. “Construction and characterization of recA mutant strains





of Corynebacterium glutamicum and Brevibacterium lactofermentum,” Appl.





Microbiol. Biotechnol., 42(4): 575-580 (1994)


X75504
aceA;
Partial Isocitrate lyase; ?
Reinscheid, D. J. et al. “Characterization of the isocitrate lyase gene from



thiX

Corynebacterium glutamicum and biochemical analysis of the enzyme,” J.





Bacteriol., 176(12): 3474-3483 (1994)


X76875

ATPase beta-subunit
Ludwig, W. et al. “Phylogenetic relationships of bacteria based on comparative





sequence analysis, of elongation factor Tu and ATP-synthase beta-subunit





genes,” Antonie Van Leeuwenhoek, 64: 285-305 (1993)


X77034
tuf
Elongation factor Tu
Ludwig, W. et al. “Phylogenetic relationships of bacteria based on comparative





sequence analysis of elongation factor Tu and ATP-synthase beta-subunit





genes,” Antonie Van Leeuwenhoek, 64: 285-305 (1993)


X77384
recA

Billman-Jacobe, H. “Nucleotide sequence of a recA gene from





Corynebacterium glutamicum,” DNA Seq., 4(6): 403-404 (1994)


X78491
aceB
Malate synthase
Reinscheid, D. J. et al. “Malate synthase from Corynebacterium glutamicum





pta-ack operon encoding phosphotransacetylase: sequence analysis,”





Microbiology, 140: 3099-3108 (1994)


X80629
16S
16S ribosomal RNA
Rainey, F. A. et al. “Phylogenetic analysis of the genera Rhodococcus and



rDNA

Norcardia and evidence for the evolutionary origin of the genus Norcardia





from within the radiation of Rhodococcus species,” Microbiol., 141: 523-528





(1995)


X81191
gluA;
Glutamate uptake system
Kronemeyer, W. et al. “Structure of the gluABCD cluster encoding the



gluB;

glutamate uptake system of Corynebacterium glutamicum,” J. Bacteriol.,



gluC;

177(5): 1152-1158 (1995)



gluD


X81379
dapE
Succinyldiaminopimelate
Wehrmann, A. et al. “Analysis of different DNA fragments of




desuccinylase
Corynebacterium glutamicum complementing dapE of Escherichia coli,”





Microbiology, 40: 3349-56 (1994)


X82061
16S
16S ribosomal RNA
Ruimy, R. et al. “Phylogeny of the genus Corynebacterium deduced from



rDNA

analyses of small-subunit ribosomal DNA sequences,” Int. J. Syst. Bacteriol.,





45(4): 740-746 (1995)


X82928
asd;
Aspartate-semialdehyde
Serebrijski, I. et al. “Multicopy suppression by asd gene and osmotic stress-



lysC
dehydrogenase; ?
dependent complementation by heterologous proA in proA mutants,” J.





Bacteriol., 177(24): 7255-7260 (1995)


X82929
proA
Gamma-glutamyl phosphate
Serebrijski, I. et al. “Multicopy suppression by asd gene and osmotic stress-




reductase
dependent complementation by heterologous proA in proA mutants,” J.





Bacteriol., 177(24): 7255-7260 (1995)


X84257
16S
16S ribosomal RNA
Pascual, C. et al. “Phylogenetic analysis of the genus Corynebacterium based



rDNA

on 16S rRNA gene sequences,” Int. J. Syst. Bacteriol., 45(4): 724-728 (1995)


X85965
aroP;
Aromatic amino acid permease; ?
Wehrmann et al. “Functional analysis of sequences adjacent to dapE of C.



dapE

glutamicum proline reveals the presence of aroP, which encodes the aromatic





amino acid transporter,” J. Bacteriol., 177(20): 5991-5993 (1995)


X86157
argB;
Acetylglutamate kinase; N-acetyl-
Sakanyan, V. et al. “Genes and enzymes of the acetyl cycle of arginine



argC;
gamma-glutamyl-phosphate
biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early



argD;
reductase; acetylornithine
steps of the arginine pathway,” Microbiology, 142: 99-108 (1996)



argF;
aminotransferase; ornithine



argJ
carbamoyltransferase; glutamate




N-acetyltransferase


X89084
pta;
Phosphate acetyltransferase; acetate
Reinscheid, D. J. et al. “Cloning, sequence analysis, expression and inactivation



ackA
kinase
of the Corynebacterium glutamicum pta-ack operon encoding





phosphotransacetylase and acetate kinase,” Microbiology, 145: 503-513 (1999)


X89850
attB
Attachment site
Le Marrec, C. et al. “Genetic characterization of site-specific integration





functions of phi AAU2 infecting “Arthrobacter aureus C70,” J. Bacteriol.,





178(7): 1996-2004 (1996)


X90356

Promoter fragment F1
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90357

Promoter fragment F2
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90358

Promoter fragment F10
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90359

Promoter fragment F13
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90360

Promoter fragment F22
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90361

Promoter fragment F34
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90362

Promoter fragment F37
Patek, M. et al. “Promoters from C. glutamicum: cloning, molecular analysis





and search for a consensus motif,” Microbiology, 142: 1297-1309 (1996)


X90363

Promoter fragment F45
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90364

Promoter fragment F64
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90365

Promoter fragment F75
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90366

Promoter fragment PF101
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90367

Promoter fragment PF104
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X90368

Promoter fragment PF109
Patek, M. et al. “Promoters from Corynebacterium glutamicum: cloning,





molecular analysis and search for a consensus motif,” Microbiology,





142: 1297-1309 (1996)


X93513
amt
Ammonium transport system
Siewe, R. M. et al. “Functional and genetic characterization of the (methyl)





ammonium uptake carrier of Corynebacterium glutamicum,” J. Biol. Chem.,





271(10): 5398-5403 (1996)


X93514
betP
Glycine betaine transport system
Peter, H. et al. “Isolation, characterization, and expression of the





Corynebacterium glutamicum betP gene, encoding the transport system for the





compatible solute glycine betaine,” J. Bacteriol., 178(17): 5229-5234 (1996)


X95649
orf4

Patek, M. et al. “Identification and transcriptional analysis of the dapB-ORF2-





dapA-ORF4 operon of Corynebacterium glutamicum, encoding two enzymes





involved in L-lysine synthesis,” Biotechnol. Lett., 19: 1113-1117 (1997)


X96471
lysE;
Lysine exporter protein; Lysine
Vrljic, M. et al. “A new type of transporter with a new type of cellular



lysG
export regulator protein
function: L-lysine export from Corynebacterium glutamicum,” Mol.





Microbiol., 22(5): 815-826 (1996)


X96580
panB;
3-methyl-2-oxobutanoate
Sahm, H. et al. “D-pantothenate synthesis in Corynebacterium glutamicum and



panC;
hydroxymethyltransferase; pantoate-
use of panBC and genes encoding L-valine synthesis for D-pantothenate



xylB
beta-alanine ligase; xylulokinase
overproduction,” Appl. Environ. Microbiol., 65(5): 1973-1979 (1999)


X96962

Insertion sequence IS1207 and




transposase


X99289

Elongation factor P
Ramos, A. et al. “Cloning, sequencing and expression of the gene encoding





elongation factor P in the amino-acid producer Brevibacterium lactofermentum





(Corynebacterium glutamicum ATCC 13869),” Gene, 198: 217-222 (1997)


Y00140
thrB
Homoserine kinase
Mateos, L. M. et al. “Nucleotide sequence of the homoserine kinase (thrB) gene





of the Brevibacterium lactofermentum,” Nucleic Acids Res., 15(9): 3922 (1987)


Y00151
ddh
Meso-diaminopimelate D-
Ishino, S. et al. “Nucleotide sequence of the meso-diaminopimelate D-




dehydrogenase (EC 1.4.1.16)
dehydrogenase gene from Corynebacterium glutamicum,” Nucleic Acids Res.,





15(9): 3917 (1987)


Y00476
thrA
Homoserine dehydrogenase
Mateos, L. M. et al. “Nucleotide sequence of the homoserine dehydrogenase





(thrA) gene of the Brevibacterium lactofermentum,” Nucleic Acids Res.,





15(24): 10598 (1987)


Y00546
hom;
Homoserine dehydrogenase;
Peoples, O. P. et al. “Nucleotide sequence and fine structural analysis of the



thrB
homoserine kinase
Corynebacterium glutamicum hom-thrB operon,” Mol. Microbiol., 2(1): 63-72





(1988)


Y08964
murC;
UPD-N-acetylmuramate-alanine
Honrubia, M. P. et al. “Identification, characterization, and chromosomal



ftsQ/
ligase; division initiation protein or
organization of the ftsZ gene from Brevibacterium lactofermentum,” Mol. Gen.



divD;
cell division protein; cell division
Genet., 259(1): 97-104 (1998)



ftsZ
protein


Y09163
putP
High affinity proline transport system
Peter, H. et al. “Isolation of the putP gene of Corynebacterium





glutamicumproline and characterization of a low-affinity uptake system for





compatible solutes,” Arch. Microbiol., 168(2): 143-151 (1997)


Y09548
pyc
Pyruvate carboxylase
Peters-Wendisch, P. G. et al. “Pyruvate carboxylase from Corynebacterium





glutamicum: characterization, expression and inactivation of the pyc gene,”





Microbiology, 144: 915-927 (1998)


Y09578
leuB
3-isopropylmalate dehydrogenase
Patek, M. et al. “Analysis of the leuB gene from Corynebacterium





glutamicum,” Appl. Microbiol. Biotechnol., 50(1): 42-47 (1998)


Y12472

Attachment site bacteriophage Phi-16
Moreau, S. et al. “Site-specific integration of corynephage Phi-16: The





construction of an integration vector,” Microbiol., 145: 539-548 (1999)


Y12537
proP
Proline/ectoine uptake system protein
Peter, H. et al. “Corynebacterium glutamicum is equipped with four secondary





carriers for compatible solutes: Identification, sequencing, and characterization





of the proline/ectoine uptake system, ProP, and the ectoine/proline/glycine





betaine carrier, EctP,” J. Bacteriol., 180(22): 6005-6012 (1998)


Y13221
glnA
Glutamine synthetase I
Jakoby, M. et al. “Isolation of Corynebacterium glutamicum glnA gene





encoding glutamine synthetase I,” FEMS Microbiol. Lett., 154(1): 81-88 (1997)


Y16642
lpd
Dihydrolipoamide dehydrogenase


Y18059

Attachment site Corynephage 304L
Moreau, S. et al. “Analysis of the integration functions of φ304L: An





integrase module among corynephages,” Virology, 255(1): 150-159 (1999)


Z21501
argS;
Arginyl-tRNA synthetase;
Oguiza, J. A. et al. “A gene encoding arginyl-tRNA synthetase is located in the



lysA
diaminopimelate decarboxylase
upstream region of the lysA gene in Brevibacterium lactofermentum:




(partial)
Regulation of argS-lysA cluster expression by arginine,” J.





Bacteriol., 175(22): 7356-7362 (1993)


Z21502
dapA;
Dihydrodipicolinate synthase;
Pisabarro, A. et al. “A cluster of three genes (dapA, orf2, and dapB) of



dapB
dihydrodipicolinate reductase
Brevibacterium lactofermentum encodes dihydrodipicolinate reductase, and a





third polypeptide of unknown function,” J. Bacteriol., 175(9): 2743-2749





(1993)


Z29563
thrC
Threonine synthase
Malumbres, M. et al. “Analysis and expression of the thrC gene of the encoded





threonine synthase,” Appl. Environ. Microbiol., 60(7)2209-2219 (1994)


Z46753
16S
Gene for 16S ribosomal RNA



rDNA


Z49822
sigA
SigA sigma factor
Oguiza, J. A. et al “Multiple sigma factor genes in Brevibacterium





lactofermentum: Characterization of sigA and sigB,” J. Bacteriol., 178(2): 550-553





(1996)


Z49823
galE;
Catalytic activity UDP-galactose 4-
Oguiza, J. A. et al “The galE gene encoding the UDP-galactose 4-epimerase of



dtxR
epimerase; diphtheria toxin regulatory
Brevibacterium lactofermentum is coupled transcriptionally to the dmdR




protein
gene,” Gene, 177: 103-107 (1996)


Z49824
orf1;
?; SigB sigma factor
Oguiza, J. A. et al “Multiple sigma factor genes in Brevibacterium



sigB

lactofermentum: Characterization of sigA and sigB,” J. Bacteriol., 178(2): 550-553





(1996)


Z66534

Transposase
Correia, A. et al. “Cloning and characterization of an IS-like element present in





the genome of Brevibacterium lactofermentum ATCC 13869,” Gene,





170(1): 91-94 (1996)





1A sequence for this gene was published in the indicated reference. However, the sequence obtained by the inventors of the present application is significantly longer than the published version. It is believed that the published version relied on an incorrect start codon, and thus represents only a fragment of the actual coding region.













TABLE 3







Corynebacterium and Brevibacterium Strains Which May be Used in


the Practice of the Invention



























Other


Genus
species
ATCC
FERM
NRRI
CECT
NCIMB
CBS
NCTC
DSMZ
origin




















Brevibacterium
ammoniagenes
21054










Brevibacterium
ammoniagenes
19350


Brevibacterium
ammoniagenes
19351


Brevibacterium
ammoniagenes
19352


Brevibacterium
ammoniagenes
19353


Brevibacterium
ammoniagenes
19354


Brevibacterium
ammoniagenes
19355


Brevibacterium
ammoniagenes
19356


Brevibacterium
ammoniagenes
21055


Brevibacterium
ammoniagenes
21077


Brevibacterium
ammoniagenes
21553


Brevibacterium
ammoniagenes
21580


Brevibacterium
ammoniagenes
39101


Brevibacterium
butanicum
21196


Brevibacterium
divaricatum
21792
P928


Brevibacterium
flavum
21474


Brevibacterium
flavum
21129


Brevibacterium
flavum
21518


Brevibacterium
flavum


B11474


Brevibacterium
flavum


B11472


Brevibacterium
flavum
21127


Brevibacterium
flavum
21128


Brevibacterium
flavum
21427


Brevibacterium
flavum
21475


Brevibacterium
flavum
21517


Brevibacterium
flavum
21528


Brevibacterium
flavum
21529


Brevibacterium
flavum


B11477


Brevibacterium
flavum


B11478


Brevibacterium
flavum
21127


Brevibacterium
flavum


B11474


Brevibacterium
healii
15527


Brevibacterium
ketoglutamicum
21004


Brevibacterium
ketoglutamicum
21089


Brevibacterium
ketosoreductum
21914


Brevibacterium
lactofermentum



70


Brevibacterium
lactofermentum



74


Brevibacterium
lactofermentum



77


Brevibacterium
lactofermentum
21798


Brevibacterium
lactofermentum
21799


Brevibacterium
lactofermentum
21800


Brevibacterium
lactofermentum
21801


Brevibacterium
lactofermentum


B11470


Brevibacterium
lactofermentum


B11471


Brevibacterium
lactofermentum
21086


Brevibacterium
lactofermentum
21420


Brevibacterium
lactofermentum
21086


Brevibacterium
lactofermentum
31269


Brevibacterium
linens
9174


Brevibacterium
linens
19391


Brevibacterium
linens
8377


Brevibacterium
paraffinolyticum




11160


Brevibacterium
spec.





717.73


Brevibacterium
spec.





717.73


Brevibacterium
spec.
14604


Brevibacterium
spec.
21860


Brevibacterium
spec.
21864


Brevibacterium
spec.
21865


Brevibacterium
spec.
21866


Brevibacterium
spec.
19240


Corynebacterium
acetoacidophilum
21476


Corynebacterium
acetoacidophilum
13870


Corynebacterium
acetoglutamicum


B11473


Corynebacterium
acetoglutamicum


B11475


Corynebacterium
acetoglutamicum
15806


Corynebacterium
acetoglutamicum
21491


Corynebacterium
acetoglutamicum
31270


Corynebacterium
acetophilum


B3671


Corynebacterium
ammoniagenes
6872





2399


Corynebacterium
ammoniagenes
15511


Corynebacterium
fujiokense
21496


Corynebacterium
glutamicum
14067


Corynebacterium
glutamicum
39137


Corynebacterium
glutamicum
21254


Corynebacterium
glutamicum
21255


Corynebacterium
glutamicum
31830


Corynebacterium
glutamicum
13032


Corynebacterium
glutamicum
14305


Corynebacterium
glutamicum
15455


Corynebacterium
glutamicum
13058


Corynebacterium
glutamicum
13059


Corynebacterium
glutamicum
13060


Corynebacterium
glutamicum
21492


Corynebacterium
glutamicum
21513


Corynebacterium
glutamicum
21526


Corynebacterium
glutamicum
21543


Corynebacterium
glutamicum
13287


Corynebacterium
glutamicum
21851


Corynebacterium
glutamicum
21253


Corynebacterium
glutamicum
21514


Corynebacterium
glutamicum
21516


Corynebacterium
glutamicum
21299


Corynebacterium
glutamicum
21300


Corynebacterium
glutamicum
39684


Corynebacterium
glutamicum
21488


Corynebacterium
glutamicum
21649


Corynebacterium
glutamicum
21650


Corynebacterium
glutamicum
19223


Corynebacterium
glutamicum
13869


Corynebacterium
glutamicum
21157


Corynebacterium
glutamicum
21158


Corynebacterium
glutamicum
21159


Corynebacterium
glutamicum
21355


Corynebacterium
glutamicum
31808


Corynebacterium
glutamicum
21674


Corynebacterium
glutamicum
21562


Corynebacterium
glutamicum
21563


Corynebacterium
glutamicum
21564


Corynebacterium
glutamicum
21565


Corynebacterium
glutamicum
21566


Corynebacterium
glutamicum
21567


Corynebacterium
glutamicum
21568


Corynebacterium
glutamicum
21569


Corynebacterium
glutamicum
21570


Corynebacterium
glutamicum
21571


Corynebacterium
glutamicum
21572


Corynebacterium
glutamicum
21573


Corynebacterium
glutamicum
21579


Corynebacterium
glutamicum
19049


Corynebacterium
glutamicum
19050


Corynebacterium
glutamicum
19051


Corynebacterium
glutamicum
19052


Corynebacterium
glutamicum
19053


Corynebacterium
glutamicum
19054


Corynebacterium
glutamicum
19055


Corynebacterium
glutamicum
19056


Corynebacterium
glutamicum
19057


Corynebacterium
glutamicum
19058


Corynebacterium
glutamicum
19059


Corynebacterium
glutamicum
19060


Corynebacterium
glutamicum
19185


Corynebacterium
glutamicum
13286


Corynebacterium
glutamicum
21515


Corynebacterium
glutamicum
21527


Corynebacterium
glutamicum
21544


Corynebacterium
glutamicum
21492


Corynebacterium
glutamicum


B8183


Corynebacterium
glutamicum


B8182


Corynebacterium
glutamicum


B12416


Corynebacterium
glutamicum


B12417


Corynebacterium
glutamicum


B12418


Corynebacterium
glutamicum


B11476


Corynebacterium
glutamicum
21608


Corynebacterium
lilium

P973


Corynebacterium
nitrilophilus
21419



11594


Corynebacterium
spec.

P4445


Corynebacterium
spec.

P4446


Corynebacterium
spec.
31088


Corynebacterium
spec.
31089


Corynebacterium
spec.
31090


Corynebacterium
spec.
31090


Corynebacterium
spec.
31090


Corynebacterium
spec.
15954






20145


Corynebacterium
spec.
21857


Corynebacterium
spec.
21862


Corynebacterium
spec.
21863


Corynebacterium
Glutamicum*








ASO19


Corynebacterium
Glutamicum**








ASO19












E12


Corynebacterium
Glutamicum***








HL457


Corynebacterium
Glutamicum****








HL459





ATCC: American Type Culture Collection, Rockville, MD, USA


FERM: Fermentation Research Institute, Chiba, Japan


NRRL: ARS Culture Collection, Northern Regional Research Laboratory, Peoria, IL, USA


CECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain


NCIMB: National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK


CBS: Centraalbureau voor Schimmelcultures, Baam, NL


NCTC: National Collection of Type Cultures, London, UK


DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany


For reference see Sugawara, H. et al. (1993) World directory of collections of cultures of microorganisms: Bacteria, fungi and yeasts (4th edn), World federation for culture collections world data center on microorganisms, Saimata, Japen.


*Spontaneous rifampin-resistant mutant of C. glutamicum ATCC13059d Yoshihama et al., 1985


**Restriction-deficient variant of ASO19 Follettie et al., 1993


***metC-disrupted mutant of ASO19E12 This study


****metC-disrupted mutant of ASO19E12 This study













TABLE 4







ALIGNMENT RESULTS
















length





% homology
Date of


ID #
(NT)
Genbank Hit
Length
Accession
Name of Genbank Hit
Source of Genbank Hit
(GAP)
Deposit


















rxa00657
906
GB_BA1:AF064700
3481
AF064700
Rhodococous sp. NO1-1 CprS and CprR genes, complete cds.
Rhodococcus sp
40,265
15-Jul.-98


metz
1314
GB_BA2:MTV016
53662
AL021841
Mycobacterium tuberculosis H37Rv complete genome; segment 143/162.
Mycobacterium tuberculosis
61,278
23-Jun.-1999


metc
978
GB_BA2:CORCSLYS
2821
M89931
Corynebacterium glutamicum beta C-S lyase (aecD) and branched-chain amino acid upta
Corynebacterium glutamicum
99,591
04-Jun.-1998


rxa00023
3579
GB_EST33:A1776129
483
A1776129
EST257217 tomato resistant, Comell Lycopersicon esculentum cDNA clone
Lycopersicon esculentum
40,956
29-Jun.-1999







cLER17D3, mRNA sequence.




GB_EST33:A1776129
483
A1776129
EST257217 tomato resistant, Comell Lycopersicon esculentum cDNA clone
Lycopersicon esculentum
40,956
29-Jun.-1999







cLER17D3, mRNA sequence.


rxa00044
1059
EM_PAT:E11760
6911
E11760
Base sequence of sucrase gene.
Corynebacterium glutamicum
42,979
08-Oct.-1997










(Rel. 52,










Created)




GB_PAT:126124
6911
I26124
Sequence 4 from patent US 5556776.
Unknown.
42,979
07-Oct.-1996




GB_BA2:ECOUW89
176195
U00006
E. coli chromosomal region from 89.2 to 92.8 minutes.
Escherichia coli
39,097
17-Dec.-1993


rxa00064
1401
GB_PAT:E16763
2517
E16763
gDNA encoding aspartate transferase (AAT).
Corynebacterium glutamicum
95,429
28-Jul.-1999




GB_HTG2:AC007892
134257
AC007892
Drosophila melanogaster chromosome 3 clone BACR02O03 (D797) RPCI-98
Drosophila melanogaster
31,111
2-Aug.-1999







02.O.3 map 99B-99B strain y; cn bw sp, *** SEQUENCING IN PROGRESS







***, 113 unordered pieces.




GB_HTG2:AC007892
134257
AC007892
Drosophila melanogaster chromosome 3 clone BACRO2O03 (D797) RPCI-98
Drosophila melanogaster
31,111
2-Aug.-1999







02.O.3 map 99B-99B strain y; cn bw sp, *** SEQUENCING IN PROGRESS***,







113 unordered pieces.


rxa00072


rxa00105
798
GB_BA1:MTV002
56414
AL008967
Mycobacterium tuberculosis H37Rv complete genome; segment 122/162.
Mycobacterium tuberculosis
37,753
17-Jun.-1998




GB_BA1:ECU29581
71128
U29581
Escherichia coli K-12 genome; approximately 63 to 64 minutes.
Escherichia coli
35,669
14-Jan.-1997




GB_BA2:AE000366
10405
AE000366
Escherichia coil K-12 MG1655 section 256 of 400 of the complete genome.
Escherichia coli
35,669
12-Nov.-1998


rxa00106
579
GB_EST15:AA494237
367
AA494237
ng83f04.s1 NCI_CGAP_Pr6 Homo sapiens cDNA clone IMAGE:941407
Homo sapiens
42,896
20-Aug.-1997







similar to SW:DYR_LACCA P00381 DIHYDROFOLATE REDUCTASE;,







mRNA sequence.




GB_BA2:AF161327
2021
AF161327
Corynebacterium diphtheriae histidine kinase ChrS (chrS) and response
Corynebacterium diphtheriae
40,210
9-Sep.-1999







regulator ChrA (chrA) genes, complete cds.




GB_PAT:AR041189
654
AR041189
Sequence 4 from patent U.S. 5811286.
Unknown.
41,176
29-Sep.-1999


rxa00115
1170
GB_PR4:AC007110
148336
AC007110
Homo sapiens chromosome 17, clone hRPK.472_J_18, complete sequence.
Homo sapiens
36,783
30-Mar.-1999




GB_HTG3:AC008537
170030
AC008537
Homo sapiens chromosome 19 clone CIT-HSPC_490E21, *** SEQUENCING
Homo sapiens
40,296
2-Sep.-1999







IN PROGRESS ***, 93 unordered pieces.




GB_HTG3:AC008537
170030
AC008537
Homo sapiens chromosome 19 clone CIT-HSPC_490E21, *** SEQUENCING
Homo sapiens
40,296
2-Sep.-1999







IN PROGRESS ***, 93 unordered pieces.


rxa00116
1284
GB_BA2:AF062345
16458
AF062345
Caulobacter crescentus Sst1 (sst1), S-layer protein subunit (rsaA), ABC
Caulobacter crescentus
36,235
19-Oct.-1999







transporter (rsaD), membrane forming unit (rsaE), putative GDP-mannose-4,6-







dehydratase (lpaA), putative acetyltransferase (lpsB), putative perosamine







synthetase (lpsC), putative mannosyltransferase (lpsD), putative







mannosyltransferase (lpsE), outer membrane protein (rsaF), and putative







perosamine transferase (lpsE) genes, complete cds.




GB_PAT:I18647
3300
I18647
Sequence 6 from patent U.S. 5500353.
Unknown.
36,821
07-Oct.-1996




GB_GSS13:AQ446197
751
AQ446197
nbxb0062D16r CUGI Rice BAC Library Oryza sativa genomic clone
Oryza sativa
38,124
8-Apr.-1999







nbxb0062D16r, genomic survey sequence.


rxa00131
732
GB_BA1:MTY20B11
36330
Z95121
Mycobacterium tuberculosis H37Rv complete genome; segment 139/162.
Mycobacterium tuberculosis
43,571
17-Jun.-1998




GB_BA1:SAR7932
15176
AJ007932
Streptomyces argillaceus mithramycin biosynthetic genes.
Streptomyces argillaceus
41,116
15-Jun.-1999




GB_BA1:MTY20B11
36330
Z95121
Mycobacterium tuberculosis H37Rv complete genome; segment 139/162.
Mycobacterium tuberculosis
39,726
17-Jun.-1998


rxa00132
1557
GB_BA1:MTY20B11
36330
Z95121
Mycobacterium tuberculosis H37Rv complete genome; segment 139/162.
Mycobacterium tuberculosis
36,788
17-Jun.-1998




GB_IN2:TVU40872
1882
U40872
Trichomonas vaginalis S-adenosyl-L-homocysteine hydrolase gene, complete
Trichomonas vaginalis
61,914
31-Oct.-1996







cds.




GB_HTG6:AC010706
169265
AC010706
Drosophila melanogaster chromosome X clone BACR36D15 (D887) RPCI-98
Drosophila melanogaster
51,325
22-Nov.-1999







36.D.15 map 13C-13E strain y; cn bw sp, *** SEQUENCING IN PROGRESS







***, 74 unordered pieces.


rxa00145
1059
GB_BA1:MTCY2B12
20431
Z81011
Mycobacterium tuberculosis H37Rv complete genome; segment 61/162.
Mycobacterium tuberculosis
63,365
18-Jun.-1998




GB_BA1:PSEPYRBX
2273
L19649
Pseudomonas aeruginosa aspartate transcarbamoylase (pyrB) and
Pseudomonas aeruginosa
56,080
26-Jul.-1993







dihydroorotase-like (pyrX) genes, complete cds's.




GB_BA1:LLPYRBDNA
1468
X84262
L.leichmannil pyrB gene.
Lactobacillus leichmannii
47,514
29-Apr.-1997


rxa00146
1464
GB_BA1:MTCY2B12
20431
Z81011
Mycobacterium tuberculosis H37Rv complete genome; segment 61/162.
Mycobacterium tuberculosis
60,714
18-Jun.-1998




GB_BA1:MTCY154
13935
Z98209
Mycobacterium tuberculosis H37Rv complete genome; segment 121/162.
Mycobacterium tuberculosis
39,229
17-Jun.-1998




GB_BA1:MSGY154
40221
AD000002
Mycobacterium tuberculosis sequence from clone y154.
Mycobacterium tuberculosis
36,618
03-Dec.-1996


rxa00147
1302
GB_BA1:MTCY2B12
20431
Z81011
Mycobacterium tuberculosis H37Rv complete genome; segment 61/162.
Mycobacterium tuberculosis
61,527
18-Jun.-1998




GB_BA1:MSGB937CS
38914
L78820
Mycobacterium leprae cosmid B937 DNA sequence.
Mycobacterium leprae
59,538
15-Jun.-1996




GB_BA1:PAU81259
7285
U81259
Pseudomonas aeruginosa dihydrodipicolinate reductase (dapB) gene, partial
Pseudomonas aeruginosa
55,396
23-Dec.-1996







cds, carbamoylphosphate synthetase small subunit (carA) and







carbamoylphosphate synthetase large subunit (carB) genes, complete cds,







and FtsJ homolog (ftsJ) gene, partial cds.


rxa00156
1233
GB_BA1:SC9B10
33320
AL009204
Streptomyces coelicolor cosmid 9B10.
Streptomyces coelicolor
52,666
10-Feb.-1999




GB_BA2:AF002133
15437
AF002133
Mycobacterium avium strain GIR10 transcriptional regulator (mav81) gene,
Mycobacterium avium
54,191
26-Mar.-1998







partial cds, aconitase (acn), invasin 1 (inv1), invasin 2 (inv2), transcriptional







regulator (moxR), ketoacyl-reductase (fabG), enoyl-reductase (inhA) and







ferrochelatase (mav272) genes, complete cds.




GB_BA1:D85417
7984
D85417
Propionibacterium freudenreichii hemY, hemH, hemB, hemX, hemR and hemL
Propionibacterium
46,667
6-Feb.-1999







genes, complete cds.
freudenreichii


rxa00166
783
GB_HTG3:AC008167
174223
AC008167
Homo sapiensclone NH0172O13, *** SEQUENCING IN PROGRESS ***, 7
Homo sapiens
37,451
21-Aug.-1999







unordered pieces.




GB_HTG3:AC008167
174223
AC008167
Homo sapiensclone NH0172O13, *** SEQUENCING IN PROGRESS ***, 7
Homo sapiens
37,451
21-Aug.-1999







unordered pieces.




GB_HTG4:AC010118
80605
AC01118
Drosophila melanogaster chromosome 3L/62B1 clone RPC198-10D15, ***
Drosophila melanogaster
38,627
16-Oct.-1999







SEQUENCING IN PROGRESS ***, 51 unordered pieces.


rxa00198
672
GB_BA1:AB024708
8734
AB024708
Corynebacterium glutamicum gltB and gltD genes for glutamine 2-oxoglutarate
Corynebacterium glutamicum
92,113
13-Mar.-1999







aminotransferase large and small subunits, complete cds.




GB_BA1:AB024708
8734
AB024708
Corynebacterium glutamicum gltB and gltD genes for glutamine 2-oxoglutarate
Corynebacterium glutamicum
93,702
13-Mar.-1999







aminotransferase large and small subunits, complete cds.




GB_EST24:AI232702
528
AI232702
EST229390 Normalized rat kidney, Bento Soares Rattus sp. cDNA clone
Rattus sp.
34,221
31-Jan.-1999







RKICF35 3′ end, mRNA sequence.


rxa00216
1113
GB_HTG2:HSDJ850E9
117353
AL121758
Homo sapiens chromosome 20 clone RP5-850E9, *** SEQUENCING IN
Homo sapiens
37,965
03-Dec.-1999







PROGRESS ***, in unordered pieces.




GB_HTG2:HSDJ850E9
117353
AL121758
Homo sapiens chromosome 20 clone RP5-850E9, *** SEQUENCING IN
Homo sapiens
37,965
03-Dec.-1999







PROGRESS ***, in unordered pieces.




GB_PR2:CNS01DSA
159400
AL121766
Human chromosome 14 DNA sequence *** IN PROGRESS *** BAC R-412H8
Homo sapiens
38,796
11-Nov.-1999







of RPCI-11 library from chromosome 14 of Homo sapiens (Human), complete







sequence.


rxa00219
1065
GB_HTG2:AC005079_0
110000
AC005079
Homo sapiens clone RG252P22, *** SEQUENCING IN PROGRESS ***, 3
Homo sapiens
38,227
22-Nov.-1998







unordered pieces.




GB_HTG2:AC005079_1
110000
AC005079
Homo sapiens clone RG252P22, *** SEQUENCING IN PROGRESS ***, 3
Homo sapiens
38,227
22-Nov.-1998







unordered pieces.




GB_HTG2:AC005079_1
110000
AC005079
Homo sapiens clone RG252P22, *** SEQUENCING IN PROGRESS ***, 3
Homo sapiens
38,227
22-Nov.-1998







unordered pieces.


rxa00223
1212
GB_BA1:PPEA3NIF
19771
X99694
Plasmid pEA3 nitrogen fixation genes.
Enterobacter agglomerans
48,826
2-Aug.-1996




GB_BA2:AF128444
2477
AF128444
Rhodobacter capsulatus molybdenum cofactor biosynthetic gene cluster,
Rhodobacter capsulatus
40,135
22-Mar.-1999







partial sequence.




GB_HTG4:AC010111
138938
AC010111
Drosophila melanogaster chromosome 3L/70C1 clone RPCI98-9B18, ***
Drosophila melanogaster
39,527
16-Oct.-1999







SEQUENCING IN PROGRESS ***, 64 unordered pieces.


rxa00229
803
GB_BA2:AF124518
1758
AF124518
Corynebacterium glutamicum 3-dehydroquinase (aroD) and shikimate
Corynebacterium glutamicum
98,237
18-May-1999







dehydrogenase (aroE) genes, complete cds.




GB_PR3:AC004593
150221
AC004593
Homo sapiens PAC clone DJ0964C11 from 7p14-p15, complete sequence.
Homo sapiens
36,616
18-Apr.-1998




GB_HTG2:AC006907
188972
AC006907
Caenorhabditis elegans clone Y76B12, *** SEQUENCING IN PROGRESS ***,
Caenorhabditis elegans
37,095
26-Feb.-1999







25 unordered pieces.


rxa00241
1626
GB_BA1:CGLYSI
4232
X60312
C.glutamicum lysl gene for L-lysine permease.
Corynebacterium glutamicum
100,000
30-Jan.-1992




GB_HTG1:PFMAL13P1
192581
AL049180
Plasmodium falciparum chromosome 13 strain 3D7, *** SEQUENClNG IN
Plasmodium falciparum
34,947
11-Aug.-1999







PROGRESS ***, in unordered pieces.




GB_HTGI:PFMAL13P1
192581
AL049180
Plasmodium falciparum chromosome 13 strain 3D7, *** SEQUENCING IN
Plasmodium falciparum
34,947
11-Aug.-1999







PROGRESS ***, in unordered pieces.


rxa00262
1197
GB_IN2:EHU89655
3219
U89655
Entamoeba histolytica unconventional myosin IB mRNA, complete cds.
Entamoeba histolytica
36,496
23-May-1997




GB_IN2:EHU89655
3219
U89655
Entamoeba histolytica unconventional myosin IB mRNA, complete cds.
Entamoeba histolytica
37,544
23-May-1997


rxa00266
531
GB_RO:AF016190
2939
AF016190
Mus musculus connexin-36 (Cx36) gene, complete cds.
Mus musculus 
41,856
9-Feb.-1999




EM_PAT:E09719
3505
E09719
DNA encoding precursor protein of alkaline cellulase.
Bacillus sp.
34,741
08-Oct.-1997










(Rel. 52,










Created)




GB_PAT:E02133
3494
E02133
gDNA encoding alkaline cellulase.
Bacillus sp.
34,741
29-Sep.-1997


rxa00278
1155
GB_IN1:CELK05F6
36912
AF040653
Caenorhabditis elegans cosmid K05F6.
Caenorhabditis elegans
36,943
6-Jan.-1998




GB_BA1:CGU43535
2531
U43535
Corynebacterium glutamicum multidrug resistance protein (cmr) gene,
Corynebacterium glutamicum
36,658
9-Apr.-1997







complete cds.




GB_RO:RNU30789
3510
U30789
Rattus norvegicus clone N27 mRNA.
Rattus norvegicus
38,190
20-Aug.-1996


rxa00295
1125
GB_BA2:CGU31281
1614
U31281
Corynebacterium glutamicum biotin synthase (bioB) gene, complete cds.
Corynebacterium glutamicum
99,111
21-Nov.-1996




GB_BA1:BRLBIOBA
1647
D14084
Brevibacterium flavum gene for biotin synthetase, complete cds.
Corynebacterium glutamicum
98,489
3-Feb.-1999




GB_PAT:E03937
1005
E03937
DNA sequence encoding Brevibacterium flavum biotin-synthase.
Corynebacterium glutamicum
98,207
29-Sep.-1997


rxa00323
1461
GB_BA1:MTCY427
38110
Z70692
Mycobacterium tuberculosis H37Rv complete genome; segment 99/162.
Mycobacterium tuberculosis
35,615
24-Jun.-1999




GB_BA1:MSGB32CS
36404
L78818
Mycobacterium leprae cosmid B32 DNA sequence.
Mycobacterium leprae
60,917
15-Jun.-1996




GB_BA1:MTCY427
38110
Z70692
Mycobacterium tuberculosis H37Rv complete genome; segment 99/162.
Mycobacterium tuberculosis
44,606
24-Jun.-1999


rxa00324
3258
GB_BA1:MSGB32CS
36404
L78818
Mycobacterium leprae cosmid B32 DNA sequence.
Mycobacterium leprae
52,516
15-Jun.-1996




GB_BA1:MTCY427
38110
Z70692
Mycobacterium tuberculosis H37Rv complete genome; segment 99/162.
Mycobacterium tuberculosis
38,079
24-Jun.-1999




GB_OM:BOVELA
3242
J02717
Bovine elastin a mRNA, complete cds.
Bos taurus
39,351
27-Apr.-1993


rxa00330
1566
GB_BA1:CGTHRC
3120
X56037
Corynebacterium glutamicum thrC gene for threonine synthase (EC 4.2.99.2).
Corynebacterium glutamicum
99,808
17-Jun.-1997




GB_PAT:I09078
3146
I09078
Sequence 4 from Patent WO 8809819.
Unknown.
99,617
02-Dec.-1994




GB_BA1:BLTHRESYN
1892
Z29563
Brevibacterium lactofermentum; ATCC 13869;; DNA (genomic);.
Corynebacterium glutamicum
99,170
20-Sep.-1995


rxa00335
1554
GB_BA1:CGGLNA
3686
Y13221
Corynebacterium glutamicum glnA gene.
Corynebacterium glutamicum
100,000
28-Aug.-1997




GB_BA2:AF005635
1690
AF005635
Corynebacterium glutamicum glutamine synthetase (glnA) gene, complete cds.
Corynebacterium glutamicum
98,906
14-Jun.-1999




GB_BA1:MSGB27CS
38793
L78817
Mycobacterium leprae cosmid B27 DNA sequence.
Mycobacterium leprae
66,345
15-Jun.-1996


rxa00347
891
GB_EST27:AI455217
624
AI455217
LD21828.3prime LD Drosophila melanogaster embryo pOT2 Drosophila
Drosophila melanogaster
34,510
09-Mar.-1999







melanogaster cDNA clone LD21828 3prime, mRNA sequence.




GB_BA2:SSU30252
2891
U30252
Synechococcus PCC7942 nucleoside diphosphate kinase and ORF2 protein
Synechococcus PCC7942
37,084
29-Oct.-1999







genes, complete cds, ORF1 protein gene, partial cds, and neutral site I for







vector use.




GB_EST21:AA911262
581
AA911262
oe75a02.s1 NCI_CGAP_Lu5 Homo sapiens cDNA clone IMAGE:1417418 3′
Homo sapiens
37,500
21-Apr.-1998







similar to gb:AI8757 UROKINASE PLASMINOGEN ACTIVATOR SURFACE







RECEPTOR, GPI-ANCHORED (HUMAN);, mRNA sequence.


rxa00351
1578
GB_BA1:MLU15187
36138
U15187
Mycobacterium leprae cosmid L296.
Mycobacterium leprae
52,972
09-Mar.-1995




GB_IN2:AC004373
72722
AC004373
Drosophila melanogaster DNA sequence (P1 DS05273 (D80)), complete
Drosophila melanogaster
46,341
17-Jul-1998







sequence.




GB_IN2:AF145653
3197
AF145653
Drosophila melanogaster clone GH08860 BcDNA.GH08860
Drosophila melanogaster
49,471
14-Jun.-1999







(BcDNA.GHO8a6O) mRNA, complete cds.


rxa00365
727
GB_BA1:AB024708
8734
AB024708
Corynebacterium glutamicum gltB and gltD genes for glutamine 2-oxoglutarate
Corynebacterium glutamicum
96,556
13-Mar.-1999







aminotransferase large and small subunits, complete cds.




GB_BA1:MTCY1A6
37751
Z83864
Mycobacterium tuberculosis H37Rv complete genome; segment 159/162.
Mycobacterium tuberculosis
39,496
17-Jun.-1998




GB_BA1:SC3A3
15901
AL109849
Streptomyces coelicolor cosmid 3A3.
Streptomyces coelicolor A3(2)
37,946
16-Aug.-1999


rxa00366
480
GB_BA1:AB024708
8734
AB024708
Corynebacterium glutamicum gltB and gltD genes for glutamine 2-oxoglutarate
Corynebacterium glutamicum
99,374
13-Mar.-1999







aminotransferase large and small subunits, complete cds.




GB_BA1:MTCY1A6
37751
Z83864
Mycobacterium tuberculosis H37Rv complete genome: segment 159/162.
Mycobacterium tuberculosis
41,333
17-Jun.-1998




GB_BA1:SC3A3
15901
AL109849
Streptomyces coelicolor cosmid 3A3.
Streptomyces coelicolor A3(2)
37,554
16-Aug.-1999


rxa00367
4653
GB_BA1:AB024708
8734
AB024708
Corynebacterium glutamicum gltB and gltD genes for glutamine 2-
Corynebacterium glutamicum
99,312
13-Mar.-1999







oxoglutarate aminotransferase large and small subunits, complete cds.




GB_BA1:MTCY1A6
37751
Z83864
Mycobacterium tuberculosis H37Rv complete genome; segment 159/162.
Mycobacterium tuberculosis
36,971
17-Jun.-1998




GB_BA1:SC3A3
15901
AL109849
Streptomyces coelicolor cosmid 3A3.
Streptomyces coelicolor A3(2)
37,905
16-Aug.-1999


rxa00371
1917
GB_VI:SBVORFS
7568
M89923
Sugarcane bacilliform virus ORF 1, 2, and 3 DNA, complete cds.
Sugarcane bacilliform virus
35,843
12-Jun.-1993




GB_E5T37:AI96750
380
AI967505
Ljimpest03.215-c10 Ljimp Lambda HybriZap two-hybrid library Lotus japonicus
Lotus japonicus
42,593
24-Aug.-1999







cDNA clone LP215-03-c10 5′ similar to 60S ribosomal protein L39, mRNA







sequence.




GB_IN1:CELKO9H9
37881
AF043700
Caenorhabditls elegans cosmid K09H9.
Caenorhabditis elegans
34,295
22-Jan.-1998


rxa00377
1245
GB_BA1:CCU13664
1678
U13664
Caulobacter crescentus uroporphyrinogen decarboxylase homolog (hemE)
Caulobacter crescentus
36,832
24-Mar.-1995







gene, partial cds.




GB_PL1:ANSDGENE
1299
Y08866
A.nidulans sD gene.
Emericella nidulans
39,603
17-Oct.-1996




GB_GSS4:AQ730303
483
AQ730303
HS_5505_B1_C04_T7A RPCI-11 Human Male BAC Library Homo sapiens
Homo sapiens
36,728
15-Jul.-1999







genomic clone Plate=1081 Col=7 Row=F, genomic survey sequence.


rxa00382
1425
GB_BA1:PAHEML
4444
X82072
P.aeruginosa hemL gene.
Pseudomonas aeruginosa
54,175
18-Dec.-1995




GB_BA1:MTY25D10
40838
Z95558
Mycobacterium tuberculosis H37Rv complete genome; segment 28/162.
Mycobacterium tuberculosis
61,143
17-Jun.-1998




GB_BA1:MSGy224
40051
AD000004
Mycobacterium tuberculosis sequence from clone y224.
Mycobacterium tuberculosis
61,143
03-Dec.-1996


rxa00383
1467
GB_BA1:MLCB1222
34714
AL049491
Mycobacterium leprae cosmid B1222.
Mycobacterium leprae
43,981
27-Aug.-1999




GB_HTG2:AC006269
167171
AC006269
Homo sapiens chromosome 17 clone hRPK.515_E_23 map 17, ***
Homo sapiens
35,444
10-Jun.-1999







SEQUENCING IN PROGRESS ***, 2 ordered pieces.




GB_HTG2:AC007638
178053
AC007638
Homo sapiens chromosome 17 clone hRPK.515_O_17 map 17, ***
Homo sapiens
34,821
22-May-1999







SEQUENCING IN PROGRESS ***, 8 unordered pieces.


rxa00391
843
GB_E5T38:AW017053
613
AW017053
EST272398 Schistosoma mansoni male, Phil LoVerde/Joe Merrick
Schistosoma mansoni
40,472
10-Sep.-1999







Schistosoma mansoni cDNA done SMMAS14 5′ end, mRNA sequence.




GB_PAT:AR065852
32207
AR065852
Sequence 20 from patent U.S. 5849564.
Unknown.
38,586
29-Sep.-1999




GB_VI:AF148805
28559
AF148805
Kaposi's sarcoma-associated herpesvirus ORF 68 gene, partial cds; and ORF
Kaposi's sarcoma-associated
38,509
2-Aug.-1999







69, kaposin, v-FLIP, v-cyclin, latent nuclear antigen, ORF K14, v-GPCR,
herpesvirus







putative phosphoribosylformylglycinamldlne synthase, and LAMP







(LAMP) genes, complete cds.


rxa00393
1017
GB_BA1:MTY25D10
40838
Z95558
Mycobacterium tuberculosis H37Rv complete genome; segment 28/162.
Mycobacterium tuberculosis
36,308
17-Jun.-1998




GB_BA1:MSGY224
40051
AD000004
Mycobacterium tuberculosis sequence from clone y224.
Mycobacterium tuberculosis
39,282
03-Dec.-1996




GB_BA1:MLB1306
7762
Y13603
Mycobacterium leprae cosmid B1306 DNA.
Mycobacterium leprae
39,228
24-Jun.-1997


rxa00402
623
GB_BA2:AF052652
2096
AF052652
Corynebacterium glutamicum homoserine O-acetyltransferase (metA) gene,
Corynebacterium glutamicum
99,672
19-Mar.-1998







complete cds.




GB_BA2:AF109162
4514
AF109162
Corynebacterium diphtheriae heme uptake locus, complete sequence.
Corynebacterium diphtheriae
40,830
8-Jun.-1999




GB_BA2:AF092918
20758
AF092918
Pseudomonas alcaligenes outer membrane Xcp-secretion system gene
Pseudomonas alcaligenes
50,161
06-Dec.-1998







cluster.


rxa00403
1254
GB_BA2:AF052652
2096
AF052652
Corynebacterium glutamicum homoserine O-acetyltransferase (metA) gene,
Corynebacterium glutamicum
99,920
19-Mar.-1998







complete cds.




GB_BA1:MTV016
53662
AL021841
Mycobacterium tuberculosis H37Rv complete genome; segment 143/162.
Mycobacterium tuberculosis
52,898
23-Jun.-1999




GB_EST23:AI111288
750
A1111288
SWOvAMCAQO2AOSSK Onchocerca volvulus adult male cDNA (SAW98MLW-
Onchocerca volvulus
37,565
31-Aug.-1998







OvAM) Onchocerca volvulus cDNA clone SWOvAMCAQ02A05 5′, mRNA







sequence.


rxa00405
813
GB_BA1:MTV016
53662
AL021841
Mycobacterium tuberculosis H37Rv complete genome; segment 143/162.
Mycobacterium tuberculosis
57,259
23-Jun.-1999




GB_PR4:AC005145
143678
AC005145
Homo sapiens Xp22-166-169 GSHB-523A23 (Genome Systems Human BAC
Homo sapiens
34,179
08-Dec.-1998







library) complete sequence.




GB_BA1:MTV016
53662
AL021841
Mycobacterium tuberculosis H37Rv complete gename; segment 143/162.
Mycobacterium tuberculosis
40,169
23-Jun.-1999


rxa00420
1587
GB_BA1:MTY13D12
37085
Z80343
Mycobacterium tuberculosis H37Rv complete genome; segment 156/162.
Mycobacterium tuberculosis
62,031
17-Jun.-1998




GB_BA1:MSGY126
37164
AD000012
Mycobacterium tuberculosis sequence from clone y126.
Mycobacterium tuberculosis
61,902
10-Dec.-1996




GB_BA1:MSGB971CS
37566
L78821
Mycobacterium leprae cosmid B971 DNA sequence.
Mycobacterium leprae
39,651
15-Jun.-1996


rxa00435
1296
GB_BA1:AFACBBTZ
2760
M68904
Alcaligenes eutrophus chromsomal transketolase (cbbTc) and
Ralstonia eutropha
38,677
27-Jul.-1994







phosphoglycolate phosphatase (cbbZc) genes, complete cds.




GB_HTG4:AC009541
169583
AC009541
Homo sapiens chromosome 7, *** SEQUENCING IN PROGRESS ***, 25
Homo sapiens
36,335
12-Oct.-1999







unordered pieces.




GB_HTG4:AC009541
169583
AC009541
Homo sapiens chromosome 7, *** SEQUENCING IN PROGRESS ***, 25
Homo sapiens
36,335
12-Oct.-1999







unordered pieces.


rxa00437
579
GB_PR4:AC005951
155450
AC005951
Homo sapiens chromosome 17, clone hRPK.372_K_20, complete sequence.
Homo sapiens
31,738
18-Nov.-1998




GB_BA1:SC2A11
22789
AL031184
Streptomyces coelicolor cosmid 2A11.
Streptomyces coelicolor
43,262
5-Aug.-1998




GB_PR4:AC005951
155450
AC005951
Homo sapiens chromosome 17, done hRPK.372_K_20, complete sequence.
Homo sapiens
37,647
18-Nov.-1998


rxa00439
591
GB_BA1:MTV016
53662
AL021841
Mycobacterium tuberculosis H37Rv complete genome; segment 143/162.
Mycobacterium tuberculosis
37,088
23-Jun.-1999




GB_PL2:AF167358
1022
AF167358
Rumex acetosa expansin (EXP3) gene, partial cds.
Rumex acetosa
46,538
17-Aug.-1999




GB_HTG3:AC009120
269445
AC009120
Homo sapiens chromosome 16 clone RPCI-11_484E3, *** SEQUENCING IN
Homo sapiens
43,276
3-Aug.-1999







PROGRESS ***, 34 unordered pieces.


rxa00440
582
GB_BA2:SKZ86111
7860
Z86111
Streptomyces lividans rpsP, trmD, rplS, sipW, sipX, sipY, sipZ, mutT genes
Streptomyces lividans
43,080
27-Oct.-1999







and 4 open reading frames.




GB_BA1:SC2E1
38962
AL023797
Streptomyces coelicolor cosmid 2E1.
Streptomyces coelicolor
42,931
4-Jun.-1998




GB_BA1:SC2E1
38962
AL023797
Streptomyces coelicolor cosmid 2E1.
Streptomyces coelicolor
36,702
4-Jun.-1998


nca00441
1287
GB_PR2:HS173D1
117338
AL031984
Human DNA sequence from clone 173D1 on chromosome 1p36.21-
Homo sapiens
38,027
23-Nov.-1999







36.33.Contains ESTs, STSs and GSSs, complete sequence.




GB_HTG2:HSDJ719K3
267114
AL109931
Homo sapiens chromosome X clone RP4-719K3 map q21.1-21.31, ***
Homo sapiens
34,521
03-Dec.-1999







SEQUENCING IN PROGRESS ***, in unordered pieces.




GB_HTG2:H5DJ719K3
267114
AL109931
Homo sapiens chromosome X clone RP4-719K3 map q21.1-21.31, ***
Homo sapiens
34,521
03-Dec.-1999







SEQUENCING IN PROGRESS ***, in unordered pieces.


rxa00446
987
GB_BA1:SCD78
36224
AL034355
Streptomyces coelicolor cosmid D78.
Streptomyces coelicolor
56,410
26-Nov.-1998




GB_HTG4:AC009367
226055
AC009367
Drosophila melanogaster chromosome 3L/76A2 clone RPC198-48B15, ***
Drosophila melanogaster
34,959
16-Oct.-1999







SEQUENCING IN PROGRESS ***, 44 unordered pieces.




GB_HTG4:AC009367
226055
AC009367
Drosophila melanogaster chromosome 3L/76A2 clone RPC198-48B15, ***
Drosophila melanogaster
34,959
16-Oct.-1999







SEQUENCING IN PROGRESS ***, 44 unordered pieces.


rxa00448
1143
GB_PR3:AC003670
88945
AC003670
Homo sapiens 12q13.1 PAC RPCI1-130F5 (Roswell Park Cancer Institute
Homo sapiens
35,682
9-Jun.-1998







Human PAC library) complete sequence.




GB_HTG2:AF029367
148676
AF029367
Homo sapiens chromosome 12 clone RPCI-1 130F5 map 12q13.1, ***
Homo sapiens
31,373
18-Oct.-1997







SEQUENCING IN PROGRESS ***, 156 unordered pieces.




GB_HTG2:AF029367
148676
AF029367
Homo sapiens chromosome 12 clone RPCI-1 1 30F5 map 12q13.1, ***
Homo sapiens
31,373
18-Oct.-1997







SEQUENCING IN PROGRESS ***, 156 unordered pieces.


rxa00450
424
GB_HTG2:AC007824
133361
AC007824
Drosophila melanogaster chromosome 3 clone BACR02L16 (D715) RPCI-1998
Drosophila metanogaster
40,000
2-Aug.-1999







02.L16 map 89E-90A strain y; cn bw sp, SEQUENCING IN PROGRESS







***, 91 unordered pieces.




GB_HTG2:AC007824
133361
AC007824
Drosophila melanogaster chromosome 3 clone BACR02L16 (D715) RPCI-1998
Drosophila melanogaster
40,000
2-Aug.-1999







02.L.16 map 89E-90A strain y; cn bw sp, SEQUENCING IN PROGRESS







***′, 91 unordered pieces.




GB_EST35:AI816057
412
AI818057
wk14a08.x1 NCI_CGAP_Lym12 Homo sapiens cDNA clone IMAGE:2412278
Homo sapiens
35,714
24-Aug.-1999







3′ similar to gb:Y00764 UBIQUINOL-CYTOCHROME C REDUCTASE 11 KD







PROTEIN (HUMAN);, mRNA sequence.


rxa00461
975
GB_BA1:MLCB1779
43254
Z98271
Mycobacterium leprae cosmid B1779.
Mycobacterium leprae
39,308
8-Aug.-1997




GB_IN1:DMC86E4
29352
AL021086
Drosophila melanogaster cosmid clone 86E4.
Drosophila melanogaster
37,487
27-Apr.-1999




GB_GSS15:AQ640325
467
AQ640325
927P1-2H3.TP 927P1 Trypanosoma brucei genomic clone 927P1-2H3,
Trypanosoma brucei
38,116
8-Jul.-1999







genomic survey sequence.


rxa00465


rxa00487
1692
GB_BA1:BAGUAA
3866
Y10499
B.ammoniagenes guaA gene.
Corynebacterium
74,259
8-Jan.-1998








ammoniagenes




GB_BA2:U00015
42325
U00015
Mycobacterium leprae cosmid B1620.
Mycobacterium leprae
37,248
01-Mar.-1994




GB_BA1:MTCY78
33818
Z77165
Mycobacterium tuberculosis H37Rv complete genome; segment 145/162.
Mycobacterium tuberculosis
39,725
17-Jun.-1998


rxa00488
1641
GB_BA1:MTCY78
33818
Z77165
Mycobacterium tuberculosis H37Rv complete genome; segment 145/162.
Mycobacterium tuberculosis
39,451
17-Jun.-1998




GB_BA2:U00015
42325
U00015
Mycobacterium leprae cosmid B1620.
Mycobacterium leprae
39,178
01-Mar.-1994




GB_BA1:SCAJ10601
4692
AJO10601
Streptomyces coellcolor A3(2) DNA for whiD and whiK loci.
Streptomyces coelicolor
60,835
17-Sep.-1998


rxa00489
1245
GB_BA2:U00015
42325
U00015
Mycobacterium leprae cosmid B1620.
Mycobacterium leprae
38,041
01-Mar.-1994




GB_HTG2:HS225E12
126464
AL031772
Homo sapiens chromosome 6 clone RP1-225E12 map q24, *** SEQUENCING
Homo sapiens
36,756
03-Dec.-1999







IN PROGRESS ***, in unordered pieces.




GB_HTG2:HS225E12
126464
AL031772
Homo sapiens chromosome 6 clone RPi-225E12 map q24, *** SEQUENCING
Homo sapiens
36,756
03-Dec.-1999







IN PROGRESS ***, in unordered pieces.


rxa00533
1155
GB_BA1:CGLYS
2803
X57226
C. glutamicum lysC-alpha, lysC-beta and asd genes for aspartokinase-alpha
Corynebacterium glutamicum
99,913
17-Feb.-1997







and -beta subunits, and aspartate beta semialdehyde dehydrogenase,







respectively (EC 2.7.2.4; EC 1.2.1.11).




GB_BA1:CGCYSCASD
1591
X82928
C.glutamicum aspartate-semialdehyde dehydrogenase gene.
Corynebacterium glutamicum
99,221
17-Feb.-1997




GB_PAT:A07546
2112
A07546
Recombinant DNA fragment (Pstl-XhoI).
synthetic construct
99,391
30-Jul.-1993


rxa00534
1386
GB_BA1:CGLYS
2803
X57226
C. glutamicum lysC-alpha, lysC-beta and asd genes for aspartokinase-alpha
Corynebacterium glutamicum
99,856
17-Feb.-1997







and -beta subunits, and aspartate beta semialdehyde dehydrogenase,







respectively (EC 2.7.2.4; EC 1.2.1.11).




GB_BA1:CORASKD
2957
L16848
Corynebacterium flavum aspartokinase (ask), and aspartate-semialdehyde
Corynebacterium flavescens
98,701
11-Jun.-1993







dehydrogenase (asd) genes, complete cds.




GB_PAT:E14514
1643
E14514
DNA encoding Brevibacterium aspartokinase.
Corynebacterium glutamicum
98,773
28-Jul.-1999


rxa00536
1494
GB_BA1:CGLEUA
3492
X70959
C.glutamicum gene leuA for isopropylmalate synthase.
Corynebacterium glutamicum
100,000
10-Feb.-1999




GB_BA1:MTV025
121125
AL022121
Mycobacterium tuberculosis H37Rv complete genome; segment 155/162.
Mycobacterium tuberculosis
68,003
24-Jun.-1999




GB_BA1:MTU88526
2412
U88526
Mycobacterium tuberculosis putative alpha-isopropyl malate synthase (leuA)
Mycobacterium tuberculosis
68,185
26-Feb.-1997







gene, complete cds.


rxa00537
2409
GB_BA2:SCD2S
41622
AL118514
Streptomyces coelicolor cosmid D25.
Streptomyces coelicolor A3(2)
63,187
21-Sep.-1999




GB_BA1:MTCY7H7A
10451
Z95618
Mycobacterium tuberculosis H37Rv complete genome: segment 39/162.
Mycobacterium tuberculosis
62,401
17-Jun.-1998




GB_BA1:MTU34956
2462
U34956
Mycobacterium tuberculosis phosphoribosylformylglycinamgdine synthase
Mycobacterium tuberculosis
62,205
28-Jan.-1997







(purL) gene, complete cds.


rxa00541
792
GB_PAT:I92052
2115
I92052
Sequence 19 from patent U.S. Pat. No. 5726299.
Unknown.
98,359
01-Dec.-1998




GB_BA1:MLCBS
38109
Z95151
Mycobacterium leprae cosmid B5.
Mycobacterium leprae
62,468
24-Jun.-1997




GB_BA1:MTCY369
36850
Z80226
Mycobacterium tuberculosis H37Rv complete genome; segment 36/162.
Mycobacterium tuberculosis
60,814
17-Jun.-1998


rxa00558
1470
GB_BA1:BAPURF
1885
X91252
B.ammoniagenes purF gene.
Corynebacterium
66,095
5-Jun.-1997








ammoniagenes




GB_BA1:MLU15182
40123
U15182
Mycobacterium leprae cosmid B2266.
Mycobacterium leprae
64,315
09-Mar.-1995




GB_BA1:MTCY7H7A
10451
Z95618
Mycobacterium tuberculosis H37Rv complete genome; segment 39/162.
Mycobacterium tuberculosis
64,863
17-Jun.-1998


rxa00579
1983
GB_PAT:AR016483
2104
AR016483
Sequence 1 from patent U.S. Pat. No. 5776740.
Unknown.
98,810
05-Dec.-1998




EM_PAT:E11273
2104
E11273
DNA encoding serine hydroxymethyl transferase.
Corynebacterium glutamicum
98,810
08-Oct.-1997










(Rel. 52.










Created)




GB_PAT:E12594
2104
E12594
DNA encoding senne hydroxymethyltransferase from Brevibacterium flavum.
Corynebacterium glutamicum
98,810
24-Jun.-1998


rxa00580
1425
GB_PAT:E12594
2104
E12594
DNA encoding serine hydroxymethyltransferase from Brevibacterium flavum.
Corynebacterium glutamicum
99,368
24-Jun.-1998




GB_PAT:AR016483
2104
AR016483
Sequence 1 from patent U.S. 5776740.
Unknown.
99,368
05-Dec.-1998




EM_PAT:E11273
2104
E11273
DNA encoding serine hydroxymethyl transferase.
Corynebacterium glutamicum
99,368
08-Oct.-1997










(Rel. 52,










Created)


rxa00581
1092
GB_PAT:E12594
2104
E12594
DNA encoding serine hydroxymethyltransferase from Brevibacterium fiavum.
Corynebacterium glutamicum
37,071
24-Jun.-1998




EM_PAT:E11273
2104
E11273
DNA encoding serine hydroxymethyl transferase.
Corynebacterium glutamicum
37,071
08-Oct.-1997










(Rel. 52.










Created)




GB_PAT:AR018483
2104
AR016483
Sequence 1 from patent U.S. 5776740.
Unknown.
37,071
05-Dec.-1998


rxa00584
1248
GB_BA1:CORAHPS
2570
L07603
Corynebacterium glutamicum 3-deoxy-D-arabinoheptulosonate-7-phosphate
Corynebacterium glutamicum
98,236
26-Apr.-1993







synthase gene, complete cds.




GB_BA1:AOPCZA361
37941
AJ223998
Amycolatopsis orlentalis cosmid PCZA361.
Amycolatopsis orientalis
54,553
29-Mar.-1999




GB_BA1:D90714
14358
D90714
Escherichia coli genomic DNA. (16.8-17.1 min).
Escherichia coli
53,312
7-Feb.-1999


rxa00618
1230
GB_EST19:AA802737
280
AA802737
GM06236.5prime GM Drosophila melanogaster ovary BlueScript Drosophila
Drosophila melanogaster
39,928
25-Nov.-1998







melanogaster cDNA clone GM06236 5prime, mRNA sequence.




GB_EST28:AI534381
581
A1534381
SD07186.5prime SD Drosophila melanogaster Schneider L2 cell culture pOT2
Drosophila melanogaster
41,136
18-Mar.-1999







Drosophila melanogaster cDNA clone SD07186 5prime similar to X89858: Ani







FBgn0011558 PID:g927407 SPTREMBL:Q24240, mRNA sequence.




GB_IN1:DMANILLIN
4029
X89858
D.melanogaster mRNA for anillin protein.
Drosophila melanogaster
34,398
8-Nov.-1995


rxa00619
1551
GB_BA1:MTCY369
36850
Z80226
Mycobacterium tuberculosis H37Rv complete genome; segment 36/162.
Mycobacterium tuberculosis
62,776
17-Jun.-1998




GB_BA1:MLCB5
38109
Z95151
Mycobacterium leprae cosmid B5.
Mycobacterium leprae
61,831
24-Jun.-1997




GB_PAT:A60305
1845
A60305
Sequences 5 from Patent WO9708323.
unidentified
61,785
06-Mar.-1998


rxa00620
1014
GB_PL2:AF063247
1450
AF063247
Pneumocystis carinii f. sp. ratti enolase mRNA, complete cds.
Pneumocystis carinii f. sp. ratti
41,060
5-Jan.-1999




GB_BA1:STMAPP
2069
M91546
Streptomyces lividans aminopeptidase P (PepP) gene, complete cds.
Streptomyces lividans
37,126
12-Jun.-1993




GB_HTG3:AC008783
214575
AC008763
Homo sapiens chromosome 19 clone CITB-E1_3214H19, *** SEQUENCING
Homo sapiens
40,020
3-Aug.-1999







IN PROGRESS ***, 21 unordered pieces.


rxa00624
810
GB_IN1:CEY4IE3
150841
Z95559
Ceenorhabditis elegans cosmid Y41E3, complete sequence.
Caenorhabditis elegans
36,986
2-Sep.-1999




GB_EST13:AA362167
372
AA362167
E5T71561 Macrophage I Homo sapiens cDNA 5′ end, mRNA sequence.
Homo sapiens
38,378
21-Apr.-1997




GB_IN1:CEY41E3
150641
Z95559
Caenortiabditis elegans cosmid Y41E3, complete sequence.
Caenorhabditis elegans
37,694
2-Sep.-1999


rxa00826
1386
GB_BA1:MTCY369
36850
Z80226
Mycobacterium tuberculosis H37Rv complete genome; segment 36/162.
Mycobacterium tuberculosis
57,971
17-Jun.-1998




GB_BA1:MLCB5
38109
Z95151
Mycobacterium leprae cosmid B5.
Mycobacterium leprae
58,806
24-Jun.-1997




GB_BA1:MLU15187
36138
U15187
Mycobacterium leprae cosmid L296.
Mycobacterium leprae
38,007
09-Mar.-1995


rxa00632
795
GB_BA1:BRLBIOAD
2272
D14083
Brevibacterium flavum genes for 7,8-diaminopelargonic acid aminotransferase
Corynebacterium glutamicum
97,358
3-Feb.-1999







and dethioblotin synthetase, complete cds.




GB_PAT:E04041
675
E04041
DNA sequence coding for desthioblotinsynthetase.
Corynebacterium glutamicum
98,074
29-Sep.-1997




GB_PAT:E04040
1272
E04040
DNA sequence coding for diamino pelargonic acid aminotransferase.
Corynebacterium glutamicum
93,814
29-Sep.-1997


rxa00633
1392
GB_BA1:BRLBIOAD
2272
D14083
Brevibacterium flavum genes for 7,8-diaminopelargonic acid aminotransferase
Corynebacterium glutamicum
95,690
3-Feb.-1999







and dethiobiotin synthetase, complete cds.




GB_PAT:E04040
1272
E04040
DNA sequence coding for diamino pelargonic acid aminotransferase.
Corynebacterium glutamicum
95,755
29-Sep.-1997




GB_BA2:EHU38519
1290
U38519
Erwinia herbicola adenosylmethionine-8-amino-7-oxononanoate transaminase
Erwinia herbicola
55,564
4-Nov.-1996







(bioA) gene, complete cds.


rxa00688
666
GB_BA1:MTV041
28826
AL021958
Mycobacterium tuberculosis H37Rv complete genome; segment 35/162.
Mycobacterium tuberculosis
60,030
17-Jun.-1998




GB_BA1:BRLSECY
1516
D14162
Brevibacterium flavum gene for SecY protein (complete cds) and gene or
Corynebacterium glutamicum
99,563
3-Feb.-1999







adenylate kinase (partial cds).




GB_BA2:MBU77912
7163
U77912
Mycobacterium bovis MBE5Oa gene, partial cds; and MBE50b, MBE50c,
Mycobacterium bovis
60,030
27-Jan.-1999







preprotein translocase SecY subunit (secY), adenylate kinase (adk),







methionine aminopeptidase (map), RNA polymerase ECF sigma factor







(sigE50), MBE50d, and MBE50e genes, complete cds.


rxa00708
930
GB_BA2:AF157493
25454
AF157493
Zymomonas mobilis ZM4 fosmid clone 42D7, complete sequence.
Zymomonas mobilis
39,116
5-Jul.-1999




GB_PAT:I00836
1853
100836
Sequence 1 from Patent U.S. 4758514.
Unknown.
47,419
21-May-1993




GB_PAT:E00311
1853
E0031
DNA coding of 2,5-diketogluconic acid reductase.
unidentified
47,419
29-Sep.-1997


rxa00717
1083
GB_PAT:I78753
1187
I78753
Sequence 9 from patent U.S. 5693781.
Unknown.
37,814
3-Apr.-1998




GB_PAT:I92042
1187
I92042
Sequence 9 from patent U.S. 5726299.
Unknown.
37,814
01-Dec.-1998




GB_BA1:MTCI125
37432
Z98268
Mycobacterium tuberculosis H37Rv complete genome; segment 76/162.
Mycobacterium tuberculosis
50,647
17-Jun.-1998


rxa00718
831
GB_BA1:MTCI125
37432
Z98268
Mycobacterium tuberculosis H37Rv complete genome; segment 76/162.
Mycobacterium tuberculosis
55,228
17-Jun.-1998




GB_BA1:MTCI125
37432
Z98268
Mycobacterium tuberculosis H37Rv complete genome; segment 76/162.
Mycobacterium tuberculosis
40,300
17-Jun.-1998




GB_GSS12:AQ420755
671
AQ420755
RPCI-11-168G18.TJ RPCI-11 Homo sapiens genomic clone RPCI-11-
Homo sapiens
35,750
23-Mar.-1999







168G18, genomic survey sequence.


rxa00727
1035
GB_HTG3:AC008332
118545
AC008332
Drosophila melanogaster chromosome 2 clone BACR48D10 (D867) RPCI-98
Drosophila melanogaster
40,634
6-Aug.-1999







48.D.10 map 34A-34A strain y; cn bw sp, *** SEQUENCING IN PROGRESS







***, 78 unordered pieces.




GB_HTG3:AC008332
118545
AC008332
Drosophila melanogaster chromosome 2 clone BACR48D10 (D867) RPCI-98
Drosophila melanogaster
40,634
6-Aug.-1999







48.D.10 map 34A-34A strain y; cn bw sp, *** SEQUENCING IN







PROGRESS***, 78 unordered pieces.




GB_HTG3:AC008332
118545
AC008332
Drosophila melanogaster chromosome 2 clone BACR48D10 (D867) RPCI-98
Drosophila melanogaster
33,888
6-Aug.-1999







48.D.10 map 34A-34A strain y; cn bw sp, *** SEQUENCING IN







PROGRESS***, 78 unordered pieces.


rxa00766
966
GB_HTG2:AC006789
83823
AC006789
Caenorhabditis elegans clone Y49F6, *** SEQUENCING IN PROGRESS ***, 2
Caenorhabditis elegans
36,737
25-Feb.-1999







unordered pieces.




GB_HTG2:AC006789
83823
AC006789
Ceenorhabditis elegans clone Y49F6, *** SEQUENCING IN PROGRESS ***, 2
Caenortiabditis elegans
36,737
25-Feb.-1999







unordered pieces.




GB_BA1:D90810
20476
D90810
E.coli genomic DNA, Kohara clone #319(37.4-37.8min.).
Escherichia coli
36,526
29-May-1997


rxa00770
1293
GB_BA1:MT1V043
68848
AL022004
Mycobacterium tuberculosis H37Rv complete genome; segment 40/162.
Mycobacterium tuberculosis
66,193
24-Jun.-1999




GB_BA1:MLU15182
40123
U15182
Mycobacterium leprae cosmid N2266.
Mycobacterium leprae
61,443
09-Mar.-1995




GB_BA2:SCD25
41622
AL118514
Streptomyces coelicolor cosmid D25.
Streptomyces coelicolor A3(2)
59,938
21-Sep.-1999


rxa00779
1056
GB_HTG1:CER08A5
51920
Z82281
Caenorhabditls elegans chromosome V clone R08A5, *** SEQUENCING IN
Caenortiabditis elegans
64,896
14-Oct.-1998







PROGRESS ***, in unordered pieces.




GB_HTG1;CER08A5
51920
Z82281
Ceenorhabditis elegans chromosome V clone R08A5, SEQUENCING IN
Caenorhabditis elegans
64,896
14-Oct.-1998







PROGRESS ***, in unordered pieces.




GB_PL2:AF078693
1492
AF078693
Chlamydomonas reinhardtii putative O-acetylserine(thiol)lyase precursor
Chlamydomonas reinhardtii
57,970
3-Nov.-1999







(Croys-1A) mRNA, nuclear gene encoding organellar protein, complete cds.


rxa00780
669
GB_BA1:MTCY98
31225
Z83880
Mycobacterium tuberculosis H37Rv complete genome; segment 103/162.
Mycobacterium tuberculosis
54,410
17-Jun.-1998




GB_BA1:AVINIFREG
7099
M60090
Azotobacter chroococcum nifU, nifS, nifV, nifP, nifW, nifZ and nifM genes,
Azotobacter chroococcum
51,729
26-Apr.-1993







complete cds.




GB_BA2:AF001780
6701
AF001780
Cyanothece PCC 8801 NifP (nifP), nitrogenase (nifB), FdxN (fdxN), NifS (nifS)
Cyanothece PCC8801
36,309
08-Mar.-1999







and NifU (nifU) genes, complete cds, and NifH (nlfH) gene, partial cds.


rxa00838
1023
GB_EST1:Z30506
329
Z30506
ATT52430 AC16H Arabidopsis thaliana cDNA clone TA1306 3′, mRNA
Arabidopsis thaliana
44,308
11-Mar.-1994







sequence.




GB_PL2:AC006258
110469
AC006258
Arabidopsis thaliana BAC F18G18 from chromosome V near 60.5 cM,
Arabidopsis thaliana
35,571
28-Dec.-1998







complete sequence.




GB_EST37:AI998439
455
AI998439
701545695 A. thaliana, Columbia Col-0, rosette-2 Arabidopsis thaliana cDNA
Arabidopsis thaliana
36,044
8-Sep.-1999







clone 701545695, mRNA sequence.


rxa00863
867
GB_BA1:BLDAPAB
3572
Z21502
B.lactofermentum dapA and dapB genes for dihydrodipicolinate synthase and
Corynebacterium glutamicum
99,539
16-Aug.-1993







dihydrodiplcolinate reductase.




GB_PAT:E16749
2001
E16749
gDNA encoding dihydrodipicolinate synthase (DDPS).
Corynebacterium glutamicum
99,539
28-Jul.-1999




GB_PAT:E14520
2001
E14520
DNA encoding Brevibacterium dihydrodipicolinic acid synthase.
Corynebacterium glutamicum
99,539
28-Jul.-1999


rxa00864
873
GB_BA1:BLDAPAB
3572
Z21502
B.lactofermentum dapA and dapB genes far dihydrodipicolinate synthase and
Corynebacterium glutamicum
99,885
16-Aug.-1993







dihydrodipicolinate reductase.




GB_BA1:CGDAPB
1902
X67737
C.glutamicum dapB gene for dihydrodipicolinate reductase.
Corynebacterium glutamicum
100,000
1-Apr.-1993




GB_PAT:E14520
2001
E14520
DNA encoding Brevibacterium dihydrodipicolinic acid synthase.
Corynebacterium glutamicum
100,000
28-Jul.-1999


rxa00865
1026
GB_BA1:BLDAPAB
3572
Z21502
B.lactofermentum dapA and dapB genes for dihydrodipicolinate synthase and
Corynebacterium glutamicum
100,000
16-Aug.-1993







dihydrodiplcolinate reductase.




GB_PAT:E16752
1411
E16752
gDNA encoding dihydrodipicolinate reductase (DDPR).
Corynebacterium glutamicum
99,805
28-Jul.-1999




GB_PAT:AR038113
1411
AR038113
Sequence 18 from patent U.S. Pat. No. 5804414.
Unknown.
99,805
29-Sep.-1999


rxa00867
650
GB_BA1:MTV002
56414
AL008967
Mycobacterium tuberculosis H37Rv complete genome; segment 122/162.
Mycobacterium tuberculosis
39,179
17-Jun.-1998




GB_BA1:MLCB22
40281
Z98741
Mycobacterium leprae cosmid B22.
Mycobacterium leprae
39,482
22-Aug.-1997




GB_BA1:SAU19858
2838
U19858
Streptomyces antibioticus guanosine pentaphosphate synthetase (gpsl) gene,
Streptomyces antibioticus
69,706
25-Oct.-1996







complete cds.


rxa00873
779
GB_BA1:SCO001206
9184
AJ001206
Streptomyces coelicolor A3(2), glycogen metabolism cluster II.
Streptomyces coelicolor
63,415
29-Mar.-1999




GB_BA1:SCO001205
9589
AJ001205
Streptomyces coelicolor A3(2) glycogen metabolism clusterI.
Streptomyces coelicolor
61,617
29-Mar.-1999




GB_BA1:D78198
2304
D78198
Pimelobacter sp. DNA for trehalose synthase, complete cds.
Pimelobacter sp.
60,594
5-Feb.-1999


rxa00884
1263
GB_BA1:MTCY253
41230
Z81368
Mycobacterium tuberculosis H37Rv complete genome; segment 106/162.
Mycobacterium tuberculosis
37,785
17-Jun.-1998




GB_BA1:MSGY222
41156
AD000010
Mycobacterium tuberculosis sequence from clone y222.
Mycobacterium tuberculosis
38,006
03-Dec.-1996




GB_GSS15:AQ654600
468
AQ654600
Sheared DNA-1O14.TF Sheared DNA Trypanosoma brucei genomic clone
Trypanosoma brucei
33,974
22-Jun.-1999







Sheared DNA-1O14, genomic survey sequence.


rxa00891
1102
GB_BA1:MTCI418B
11700
Z96071
Mycobacterium tuberculosis H37Rv complete genome; segment 7/162.
Mycobacterium tuberculosis
63,297
18-Jun.-1998




GB_BA1:SC0001206
9184
AJ001206
Streptomyces coelicolorA3(2), glycogen metabolism cluster II.
Streptomyces coelicolor
61,965
29-Mar.-1999




GB_BA1:SC0001205
9589
AJ001205
Streptomyces coelicolor A3(2) glycogen metabolism clusterI.
Streptomyces coelicolor
61,727
29-Mar.-1999


rxa00952
963E
EM_PAT:E10963
3118
E10963
gDNA encoding tryptophan synthase.
Corynebacterium glutamicum
99,6888
08-Oct.-1997










(Rel. 52.










Created)




GB_BA1:BLTRP
7725
X04960
Brevibacterium lactofermentum tryptophan operon.
Corynebacterium glutamicum
98,847
10-Feb.-1999




GB_PAT.E01688
7725
E01688
Genomic DNA of trp operon of prepibacterium latophelmentamn.
unidentified
98,428
29-Sep.-1997


rxa00954
644
GB_PAT:E01375
7726
E01375
DNA sequence of tryptophan operon.
Corynebacterium glutamicum
98,758
29-Sep.-1997




GB_PAT:E01688
7725
E01688
Genomic DNA of trp operon of prepibacterium latophelmentamn.
unidentified
98,758
29-Sep.-1997




GB_BA1:BLTRP
7725
X04960
Brevibacterium lactofermentum tryptophan operon.
Corynebacterium glutamicum
98,758
10-Feb.-1999


rxa00955
1545
GB_PAT:E01375
7726
E01375
DNA sequence of tryptophan operon.
Corynebacterium glutamicum
98,372
29-Sep.-1997




GB_BA1:BLTRP
7725
X04960
Brevibacterium lactofermentum tryptophan operon.
Corynebacterium glutamicum
98,372
10-Feb.-1999




GB_PAT.E01688
7725
E01688
Genomic DNA of trp operon of prepibacterium latophelmentamn.
unidentified
98,242
29-Sep.-1997


rxa00958
1237
EM_PAT:E10963
3118
E10963
gDNA encoding tryptophan synthase.
Corynebacterium glutamicum
98,949
08-Oct.-1997










(Rel. 52,










Created)




GB_BA1:BLTRP
7725
X04960
Brevibacterium lactofermentum tryptophan operon.
Corynebacterium glutamicum
99,107
10-Feb.-1999




GB_PAT:E01375
7726
E01375
DNA sequence of tryptophan operon.
Corynebacterium glutamicum
98,945
29-Sep.-1997


rxa00957
1677
GB_BA1:BLTRP
7725
X04960
Brevibacterium lactofermentum tryptophan operon.
Corynebacterium glutamicum
99,165
10-Feb.-1999




GB_PAT:E01375
7726
E01375
DNA sequence of tryptophan operon.
Corynebacterium glutamicum
98,927
29-Sep.-1997




GB_PAT:E01688
7725
E01688
Genomic DNA of trp operon of prepibacterium latophelmentamn.
unidentified
98,867
29-Sep.-1997


rxa00958
747
GB_BA1:BLTRP
7725
X04960
Brevibacterium lactofermentum tryptophan operon.
Corynebacterium glutamicum
98,792
10-Feb.-1999




GB_PAT:E01375
7726
E01375
DNA sequence of tryptophan operon.
Corynebacterium glutamicum
98,792
29-Sep.-1997




GB_PAT:E01688
7725
E01688
Genomic DNA of trp operon of prepibacterium latophelmentamn.
unidentified
98,658
29-Sep.-1997


rxa00970
1050
GB_BA1:CGHOMTHR
3885
Y00546
Corynebacterium glutamicum hom-thrB genes for homoserine dehydrogenase
Corynebacterium glutamicum
99,905
12-Sep.-1993







and homoserine kinase.




GB_PAT:109077
3685
109077
Sequence 1 from Patent WO 8809819.
Unknown.
99,810
02-Dec.-1994




GB_PAT:E01358
2615
E01358
DNA encoding for homosenne dehydrogenase(HDH)and homoserine
Corynebacterium glutamicum
97,524
29-Sep.-1997







kinase(HK).


rxa00972
1458
GB_PAT:E16755
3579
E16755
gDNA encoding diaminopimelate decarboxylase (DDC) and arginyl-tRNA
Corynebacterium glutamicum
99,931
28-Jul.-1999







synthase.




GB_PAT:AR038110
3579
AR038110
Sequence 15 from patent U.S. 5804414.
Unknown.
99,931
29-Sep.-1999




GB_PAT:E14508
3579
E14508
DNA encoding Brevibacterium diaminopimelic pk acid decarboxylase and
Corynebacterium glutamicum
99,931
28-Jul.-1999







arginyl-tRNA synthase.


rxa00981
753
GB_OV:GGA245684
512
AJ245664
Gallus gallus partial mRNA for ATP-citrate lyase (ACL gene).
Gallus gallus
37,538
28-Sep.-1999




GB_PL2:AC007887
159434
AC007887
Genomic sequence for Arabidopsis thaliana pk BAC F1504 from chromosome I,
Arabidopsis thaliana
37,600
04-Oct.-1999







complete sequence.




GB_GSS1:CNS00RNW
542
AL087338
Arabidopsis thaliana genome survey sequence T7 end of SAC F14D7 of IGF
Arabidopsis thaliana
41,264
28-Jun.-1999







library from strain Columbia of Arabidopsis thallana, genomic survey







sequence.


rxa00989
1644
GB_BA1:MTV008
63033
AL021246
Mycobacterium tuberculosis H37Rv complete genome; segment 108/162.
Mycobacterium tuberculosis
40,773
17-Jun.-1998




GB_BA1:SCVALSFP
3619
Y13070
S.coelicolor valS, fpgs, ndk genes.
Streptomyces coelicolor
58,119
03-Mar.-1998




GB_BA1:MTV008
63033
AL021246
Mycobactrium tuberculosis H37Rv complete genome; segment 108/162.
Mycobacterium tuberculosis
38,167
17-Jun.-1998


rxa00997
705
GB_BA2:CGU31225
1817
U31225
Corynebacterium glutamicum L-proline:NADP+5-oxidoreductase (proC) gene,
Corynebacterium glutamicum
40,841
2-Aug.-1996







complete cds.




GB_HTG1:CEY39C12
282838
AL009026
Caenorhabditis elegans chromosome IV clone Y39C12, *** SEQUENCING IN
Caenorhabditis elegans
36,416
26-OCT-1999







PROGRESS ***, In unordered pieces.




GB_IN1:CEB0001
39416
Z69634
Caenorhabditis elegans cosmid B0001, complete sequence.
Caenorhabditis elegans
36,416
2-Sep.-1999


rxa01019
1110
GB_HTG2AC005052
144734
AC005052
Homo sapiens clone RG038K21, *** SEQUENCING IN PROGRESS ***, 3
Homo sapiens
39,172
12-Jun.-1998







unordered pieces.




GB_HTG2:AC005052
144734
AC005052
Homo sapiens clone RG038K21, *** IN PROGRESS ***, 3
Homo sapiens
39,172
12-Jun.-1998







unordered pieces.




GB_GSS9:AQ171808
512
AQ171808
HS_3179_A1_G03_T7 CIT Approved Human Genomic Sperm Library D
Homo sapiens
34,661
17-Oct.-1998







Homo sapiens genomic clone Plate = 3179 Col = 5 Row = M, genomic survey sequence.


rxa01026
1782
GB_BA1:SCIC2
42210
AL031124
Streptomyces coelicolor cosmid 1C2.
Streptomyces coelicolor
68,275
15-Jan.-1999




GB_BA1:ATLEUCD
2982
X84647
A.teichomyceticus leuC and leuD genes.
Actinoplanes teichomyceticus
65,935
04-Oct.-1995




GB_BA1:MTV012
70287
AL021287
Mycobacterium tuberculosis H37Rv complete genome; segment 132/162.
Mycobacterium tuberculosis
40,454
23-Jun.-1999


rxa01027
1131
GB_BA1:MLCB637
44882
Z99263
Mycobacterium leprae cosmid B637.
Mycobacterium leprae
38,636
17-Sep.-1997




GB_BA1:MTCY349
43523
Z83018
Mycobacterium tuberculosis H37Rv complete genome; segment 131/162.
Mycobacterium tuberculosis
51,989
17-Jun.-1998




GB_BA1:SPUNGMUTX
1172
Z21702
S.pneumoniae ung gene and mutX genes encoding uracil-DNA glycosylase
Streptococcus pneumoniae
38,088
15-Jun.-1994







and 8-oxodGTP nucleoside triphosphatase.


rxa01073
954
GB_BA1:BACOUTB
1004
M15811
Bacillus subtills outB gene encoding a sporulation protein, complete cds.
Bacillus subtilis
53,723
26-Apr.-1993




GB_PR4:AC007938
167237
AC007938
Homo sapiens clone UWGC:djs201 from 7q31, complete sequence.
Homo sapiens
34,322
1-Jul.-1999




GB_PL2:ATAC006282
92577
AC006282
Arabidopsis thaliana chromosome II BAC F13K3 genomic sequence, complete
Arabidopsis thaliana
36,181
13-Mar.-1999







sequence.


rxa01079
2226
GB_BA2:AF112535
4363
AF112535
Corynebacterium glutamicum putative glutaredoxin NrdH (nrdH), Nrdl (nrdl),
Corynebacterium glutamicum
99,820
5-Aug.-1999







and ribonucleotide reductase alpha-chain (nrdE) genes, complete cds.




GB_BA1:CANRDFGEN
6054
Y09572
Corynebacterium ammonlagenes nrdH, nrdl, nrdE, nrdF genes.
Corynebacterium
75,966
18-Apr.-1998







ammoniagenes




GB_BA1:MTV012
70287
AL021287
Mycobacterium tuberculosis H37Rv complete genome; segment 132/162.
Mycobacterium tuberculosis
38,296
23-Jun.-1999


rxa01080
567
GB_BA2:AF112535
4363
AF112535
Corynebacterium glutamicum putative glutaredoxin NrdH (nrdH), Nrdl (nrdl),
Corynebacterium glutamicum
100,000
5-Aug.-1999







and ribonucleotide reductase alpha-chain (nrdE) genes, complete cds.




GB_BA1:CANRDFGEN
6054
Y09572
Corynebacterium ammoniagenes nrdH, nrdl, nrdE, nrdF genes.
Corynebacterium
65,511
18-Apr.-1998








ammoniagenes




GB_BA1:STNRD
4894
X73226
S.typhimurium nrdEF operon.
Salmonella typhimurium
52,477
03-Mar.-1997


rxa01087
999
GB_IN2:AF083412
1093
AF063412
Limnadia lenticulans elongation factor 1-alpha mRNA, partial cds.
Limnadia lenticulans
43,750
29-Mar.-1999




GB_PR3:HS24M15
134539
Z94055
Human DNA sequence from PAC 24M15 on chromosome 1. Contains
Homo sapiens
37,475
23-Nov.-1999







tenascin-R (restnctin), EST.




GB_IN2:ARU85702
1240
U85702
Anathix ralla elongation factor-1 alpha (EF-1a) gene, partial cds.
Anathix ralla
37,319
16-Jul.-1997


rxa01095
857
GB_BA1:MTCY01B2
35938
Z95554
Mycobacterium tuberculosis H37Rv complete genome; segment 72/162.
Mycobacterium tuberculosis
43,243
17-Jun.-1998




GB_HTG5:AC011632
175917
AC011632
Homo sapiens clone RP11-3N13, WORKING DRAFT SEQUENCE, 9
Homo sapiens
36,471
19-Nov.-1999







unordered pieces.




GB_HTG5:AC011632
175917
AC011632
Homo sapiens clone RP11-3N13, WORKING DRAFT SEQUENCE, 9
Homo sapiens
36,836
19-Nov.99







unordered pieces.


rxa01097
477
GB_BA2:AF030405
774
AF030405
Corynebacterium glutamicum cyclase (hisF) gene, complete cds.
Corynebacterium glutamicum
100,000
13-Nov.-1997




GB_BA2:AF030405
774
AF030405
Corynebacterium glutamicum cydase (hisF) gene, complete cds.
Corynebacterium glutamicum
41,206
13-Nov.-1997


rxa01098
897
GB_BA2:AF030405
774
AF030405
Corynebacterium glutamicum cyclase (hisF) gene, complete cds.
Corynebacterium glutamicum
97,933
13-Nov.-1997




GB_BA1:MSGY223
42061
AD000019
Mycobacterium tuberculosis sequence from clone y223.
Mycobacterium tuberculosis
40,972
10-Dec.-1996




GB_BA1:MLCB1610
40055
AL049913
Mycobacterium leprae cosmid B1610.
Mycobacterium leprae
61,366
27-Aug.-1999


rxa01100
861
GB_BA2:AF051846
738
AF051846
Corynebacterium glutamicum phosphoribosylformimino-5-amino-1
Corynebacterium glutamicum
97,154
12-Mar.-1998







phosphonbosyl-4-imidazolecarboxamlde isomerase (hisA) gene,







complete cds.




GB_BA2:AF060558
636
AF060558
Corynebacterium glutamicum glutamine amidotransferase (hisH) gene,
Corynebacterium glutamicum
95,455
29-Apr.-1998







complete cds.




GB_HTG1:HSDJ140A9
221755
AL109917
Homo sapiens chromosome 1 clone RP1-140A9, *** SEQUENCING IN
Homo sapiens
30,523
23-Nov.-1999







PROGRESS ***, in unordered pieces.


rxa01101
756
GB_BA2:AF060558
636
AF060558
Corynebacterium glutamicum glutamine amidotransferase (hisH) gene,
Corynebacterium glutamicum
94,462
29-Apr.-1998







complete cds.




GB_BA1:SC4G6
36917
AL096884
Streptomyces coelicolor cosmid 4G6.
Streptomyces coelicolor A3(2)
38,378
23-Jul.-1999




GB_BA1:STMHISOPA
3981
M31628
S.coelicolor histidine biosynthesis operon encoding hisD, partial cds., and
Streptomyces coelicolor
60,053
26-Apr.-1993







hisC, hisB, hisH, and hisA genes, complete cds.


rxa01104
729
GB_BA1:STMHISOPA
3981
M31628
S.coellcolor histidine biosynthesis operon encoding hisD, partial cds., and
Streptomyces coelicolor
58,333
26-Apr.-1993







hisO, hisB, hisH, and hisA genes, complete cds.




GB_BA1:SC4G6
36917
AL096884
Streptomyces coelicolor cosmid 4G6.
Streptamyces coelicolor A3(2)
39,045
23-Jul.-1999




GB_BA1:MTCY336
32437
Z95586
Mycobacterium tuberculosis H37Rv complete genome; segment 70/162.
Mycobacterium tuberculosis
60,364
24-Jun.-1999


rxa01105
1221
GB_BA1:MTCY336
32437
Z95586
Mycobacterium tuberculosis H37Rv complete genome; segment 70/162.
Mycobacterium tuberculosis
60,931
24-Jun.-1999




GB_BA1:M5GY223
42061
AD000019
Mycobacterium tuberculosis sequence from clone y223.
Mycobacterium tuberculosis
36,851
10-Dec.-1996




GB_BA1:MLCB1610
40055
AL049913
Mycobacterium leprae cosmid B1610.
Mycobacterium leprae
60,902
27-Aug.-1999


rxa01106
1449
GB_BA1:M5GY223
42061
AD000019
Mycobacterium tuberculosis sequence from clone y223.
Mycobacterium tuberculosis
37,233
10-Dec.-1996




GB_BA1:MSHISCD
2298
X65542
M.smegmatis genes hisD and hisC for histidinol dehydrogenase and histidinol-
Mycobacterium smegmatis
60,111
30-Jun.-1993







phosphate aminotransferase, respectively.




GB_BA1:MTCY336
32437
Z95586
Mycobacterium tuberculosis H37Rv complete genome; segment 70/162.
Mycobacterium tuberculosis
58,420
24-Jun.-1999


rxa01145
1137
GB_BA1:CORAIA
4705
L09232
Corynebacterium glutamicum acetohydroxy acid synthase (ilvB) and (ilvN)
Corynebacterium glutamicum
100,000
23-Feb.-1995







genes, and acetohydroxy acid isomeroreductase (ilvC) gene, complete cds.




GB_BA1:BRLILVCA
1364
D14551
Brevibacterium flavum ilvC gene for acetohydroxy acid isomeroreductase,
Corynebacterium glutamicum
99,560
3-Feb.-1999







complete ods.




GB_PAT:E08232
1017
E08232
DNA encoding acetohydroxy-acid isomeroreductase.
Corynebacterium glutamicum
99,803
29-Sep.-1997


rxa01162
1449
GB_PAT:A60299
2869
A60299
Sequence 18 from Patent WO9706261.
Aspergillus niger
38,675
06-Mar.-1998




GB_PR3:H524E5
35506
Z82185
Human DNA sequence from Fosmid 24E5 on chromosome 22q11.2-qter
Homo sapiens
36,204
23-Nov.-1999







contains parvalbumin, ESTs, STS.




GB_PR3:AC005265
43900
AC005265
Homo sapiens chromosome 19, cosmid F19750, complete sequence.
Homo sapiens
38,363
6-Jul.-1998


rxa01208
848
GB_HTG2:AC004965
323792
AC004965
Homo sapiens clone DJ1 106H14, *** SEQUENCING IN PROGRESS ***, 42
Homo sapiens
36,058
12-Jun.-1998







unordered pieces.




GB_HTG2:AC004965
323792
AC004985
Homo sapiens clone DJ1106H14, *** SEQUENCING IN PROGRESS ***, 42
Homo sapiens
36,058
12-Jun.-1998







unordered pieces.




GB_PL2:TAU55859
2397
U55859
Triticum aestivum heat shock protein 80 mRNA, complete cds.
Triticum aestivum
37,269
1-Feb.-1999


rxa01209
1528
GB_HTG3:AC011469
113436
AC011469
Homo sapiens chromosome 19 clone CIT-HSPC 475D23, *** SEQUENCING
Homo sapiens
40000
07-Oct.-1999







IN PROGRESS ***, 31 unordered pieces.




GB_HTG3:AC011469
113436
AC011469
Homo sapiens chromosome 19 clone CIT-HSPC_475D23, *** SEQUENCING
Homo sapiens
40,000
07-Oct.-1999







IN PROGRESS ***, 31 unordered pieces.




GB_PL1:AB010077
77380
AB010077
Arabidopsis thaliana genomic DNA, chromosome 5, P1 clone: MYH19,
Arabidopsis thaliana
36,803
20-Nov.-1999







complete sequence.


rxa01215
1098
GB_BA1:MTCY10G2
38970
Z92539
Mycobacterium tuberculosis H37Rv complete genome; segment 47/162.
Mycobacterium tuberculosis
37,047
17-Jun.-1998




GB_IN1:LEIPRPP
1887
M76553
Leishmanla donovani phosphoribosylpyrophosphate synthetase gene,
Leishmania donovani
50,738
7-Jun.-1993







complete cds.




GB_HTG2:HSJ799D16
130149
AL050344
Homo sapiens chromosome 1 clone RP4-799D16 map p34.3-36.1, ***
Homo sapiens
38,135
29-Nov.-1999







SEQUENCING IN PROGRESS ***, In unordered pieces.


rxa01239
2558
GB_BA1:MTCY48
35377
Z74020
Mycobacterium tuberculosis H37Rv complete genome; segment 69/162.
Mycobacterium tuberculosis
38,139
17-Jun.-1998




GB_PR2:AB029032
8377
AB029032
Homo sapiens mRNA for KIAA1 109 protein, partial cds.
Homo sapiens
39,394
4-Aug.-1999




GB_GSS9:AQ107201
355
AQ107201
HS_3098_A1_C03_T7 CIT Approved Human Genomic Sperm Library D Homo
Homo sapiens
41,408
28-Aug.-1998







sapiens genomic clone Plate = 3098 Col = 5 Row = E, genomic survey sequence.


rxa01253
873
GB_PL2:F508
99923
AC005990
Arabidopsis thaliana chromosome 1 BAC P508 sequence, complete
Arabidopsis thaliana
36,118
23-Dec.-1998







sequence.




GB_PL2:F508
99923
AC005990
Arabidopsis thaliana chromosome 1 BAC F508 sequence, complete
Arabidopsis thaliana
35,574
23-Dec.-1998







sequence.




GB_IN1:CELCO6G1
31205
U41014
Caenorhabditls elegans cosmid CO6G1.
Caenorhabditis elegans
38,560
30-Nov.-1995


rxa01321
1044
GB_GSS14:AQ518843
441
AQ518843
HS_5106_A1_D10_SP6E RPCI-11 Human Male BAC Library Home sapiens
Homo sapiens
41,121
05-May-1999







genomic clone Plate = 682 Col = 19 Row = G, genomic survey sequence.




GB_HTG2:AC007473
194859
AC007473
Drosophila melanogaster chromosome 2 clone BACR38D12 (D590) RPCI-1998
Drosophila melanogaster
40,634
2-Aug.-1999







38.D.12 map 48A-48B strain y; cn bw sp, **** SEQUENCING IN PROGRESS







***, 60 unordered pieces.




GB_HTG4:AC011696
115847
AC011696
Drosophila melanogaster chromosome 2 clone BACR35F01 (D1156) RPCI-98
Drosophila melanogaster
38,290
26-Oct.-1999







35.F.1 map 48A-48C strain y: cn bw sp, SEQUENCING IN PROGRESS







***, 108 unordered pieces.


rxa01352
706
GB_PL2ATAC005167
83260
AC005187
Arabidopsis thaliana chromosome II BAC F12A24 genomic sequence,
Arabidopsis thaliana
34,311
15-Oct.-1998







complete sequence.




GB_PL2:ATAC005825
97380
AC005825
Arabidopsis thaliana chromosome II BAC T24121 genomic sequence. complete
Arabidopsis thaliana
34,311
12-Apr.-1999







sequence.




GB_HTG3:AC011150
127222
AC011150
Homo sapiens clone 4_K_17, LOW-PASS SEQUENCE SAMPLING.
Homo sapiens
37,722
01-Oct.-1999


rxa01360
259
GB_EST32:A1725583
728
A1725583
BNLGHI12371 Six-day Cotton fiber Gossypium hirsutum cDNA 5′ similar to
Gossypium hirsutum
38,492
11-Jun.-1999







(U86081) root hair defective 3 [Arabidopsis thallana], mRNA sequence.




GB_PR2:HS227P17
82951
Z81007
Human DNA sequence from PAC 227P17, between markers DX56791
Homo sapiens
39,738
23-Nov.-1999







andDXS8038 on chromosome X contains CpG island, EST.




GB_EST34:AV171099
173
AV171099
AV171099 Mus musculus head C576L16J 14, 17 day embryo Mus musculus
Mus musculus
46,237
6-Jul.-1999







cDNA clone 3200002M11, mRNA sequence.


rxa01361
629
GB_RO:AB008915S1
530
AB008915
Mus musculus mGpi1 gene, exon 1.
Mus musculus
45,574
28-Sep.-1999




GB_EST22:AI050532
293
AI050532
uc83d10.y1 Sugano mouse kidney mkia Mus musculus cDNA clone
Mus musculus
44,097
9-Jul.-1998







IMAGE:1432243 5′ similar to TR:O35120 O35120 MGPI1P.;, mRNA







sequence.




GB_RO:AB008895
3062
AB008895
Mus musculus mRNA for mGpi1p, complete cds.
Mus musculus
41,316
23-Nov.-1997


rxa01381
944
GB_PL1:AB005237
87835
AB005237
Arabidopsis thaliana genomic DNA, chromosome 5, P1 clone: MJJ3,complete
Arabidopsis thaliana
36,606
20-Nov.-1999







sequence.




GB_GSS5:AQ766840
491
AQ766840
HS_2026_A2_C09_T7C CIT Approved Human Genomic Sperm Library D
Homo sapiens
37,916
28-Jul.-1999







Homo sapiens genomic clone Plate = 2026 Col = 18 Row = E,







genomic survey sequence.




GB_BA1:MTV043
68848
AL022004
Mycobacterium tuberculosis H37Rv complete genome; segment 40/162.
Mycobacterium tuberculosis
37,419
24-Jun.-1999


rxa01393
993
GB_BA1:CGLYSEG
2374
X96471
C. glutamicum lysE and lysG genes.
Corynebacterium glutamicum
34,831
24-Feb.-1997




GB_BA1:SC5A7
40337
AL031107
Streptomyces coelicolor cosmid 5A7.
Streptomyces coelicolor
35,138
27-Jul.-1998




GB_PR3:AC004054
112184
AC004054
Homo sapiens chromosome 4 clone B220G8 map 4q21, complete sequence.
Homo sapiens
37,277
9-Jul.-1998


rxa01394
822
GB_BA1:CGLYSEG
2374
X96471
C.glutamicum lysE and lysG genes.
Corynebacterium glutamicum
100,000
24-Feb.-1997




GB_GSS5:AQ769223
500
AQ769223
HS_3155_B2_G10_T7C CIT Approved Human Genomic Sperm Library D
Homo sapiens
38,400
28-Jul.-1999







Homo sapiens genomic clone Plate=3155 Col=20 Row=N, genomic survey







sequence.




GB_BA1:CGLYSEG
2374
X96471
C.glutamicum lysE and lysG genes.
Corynebacterium glutamicum
33,665
24-Feb.-1997


rxa01416
630
GB_BA1:SC3C3
31382
ALO31231
Streptomyces coelicolor cosmid 3C3.
Streptomyces coelicolor
62,726
10-Aug.-1998




GB_BA1:MLCB22
40281
Z98741
Mycobacterium leprae cosmid B22.
Mycobacterium leprae
39,159
22-Aug.-1997




GB_BA1:MTV002
56414
AL008967
Mycobacterium tuberculosis H37Rv complete genome; segment 122/162.
Mycobacterium tuberculosis
37,340
17-Jun.-1998


rxa01442
1347
GB_BA1:D90827
18886
D90827
E.coli genomic DNA, Kohara clone #336(41.2-41.6 min.).
Escherichia coli
58,517
21-Mar.-1997




GB_BA1:D90828
14590
D90828
E.coli genomic DNA, Kohara clone #336gap(41.6-41.9 min.).
Escherichia coli
56,151
21-Mar.-1997




GB_BA2:AE000279
10855
AE000279
Escherichia coli K-12 MG1655 section 169 of 400 of the complete genome.
Escherichia coli
56,021
12-Nov.-1998


rxa01446
1413
GB_BA1.SCH10
39524
AL049754
Streptomyces coelicolor cosmid H10.
Streptomyces coelicolor
39,037
04-May-1999




GB_BA1:MTY13E10
35019
Z95324
Mycobacterium tuberculosis H37Rv complete genome: segment 18/162.
Mycobacterium tuberculosis
40,130
17-Jun.-1998




GB_BA1:MLCB4
36310
AL023514
Mycobacterium leprae cosmid B4.
Mycobacterium leprae
37,752
27-Aug.-1999


rxa01483
1395
GB_BA1:MTCY98
31225
Z83860
Mycobacterium tuberculosis H37Rv complete genome; segment 103/162.
Mycobacterium tuberculosis
39,057
17-Jun.-1998




GB_BA1:MSGB_1229CS
30670
L78812
Mycobacterium leprae cosmid B1229 DNA sequence.
Mycobacterium leprae
54,382
15-Jun.-1996




GB_BA2:AF027507
5168
AF027507
Mycobacterium smegmatis dGTPase (dgt), and pnmase (dnaG) genes,
Mycobacterium smegmatis
52,941
16-Jan.-1998







complete cds; tRNA-Asn gene, complete sequence.


rxa01486
757
GB_BA1:MTV002
58414
AL008967
Mycobacterium tuberculosis H37Rv complete genome; segment 122/162.
Mycobacterium tuberculosis
40,941
17-Jun.-1998




GB_BA1:MLCB22
40281
Z98741
Mycobacterium leprae cosmid B22.
Mycobacterium leprae
38,451
22-Aug.-1997




GB_BA1:SC3C3
31382
AL031231
Streptomyces coelicolor cosmid 3C3.
Streptomyces coelicolor
61194
10-Aug.-1998


rxa01489
1146
GB_BA1:CORFADS
1547
D37967
Corynebacterium ammonlagenes gene for FAD synthetase, complete cds.
Corynebacterium
58,021
8-Feb.-1999








ammoniagenes




GB_BA1:MLCB22
40281
Z98741
Mycobacterium leprae cosmid 822.
Mycobacterium leprae
38,414
22-Aug.-1997




GB_BA1:SC10A7
39739
AL078618
Streptomyces coelicolor cosmid 10A7.
Streptomyces coelicolor
36,930
9-Jun.-1999


rxa01491
774
GB_BA1:MTV002
56414
AL008967
Mycobacterium tuberculosis H37Rv complete genome; segment 122/162.
Mycobacterium tuberculosis
37,062
17-Jun.-1998




GB_EST13:AA356956
255
AA356956
EST65614 Jurkat T-cells III Homo sapiens cDNA 5 end, mRNA sequence.
Homo sapiens
37,647
21-Apr.-1997




GB_OV:OMDNAPROI
7327
X92380
O.mossamblcus prolactin I gene.
Tilapia mossambica
38,289
19-Oct.-1995


rxa01508
1662
GB_INI:CEF28C12
14653
Z93380
Caenorhabditis elegans cosmid F28C12, complete sequence.
Caenorhabditis elegans
37,984
23-Nov.-1998




GB_IN1:CEF28C12
14653
Z93380
Caenorhabdltis elegans cosmid F28C12, complete sequence.
Caenorhabditis elegans
38,469
23-Nov.-1998


rxa01512
723
GB_BA1:SCE9
37730
AL049841
Streptomyces coelicolor cosmid E9.
Streptomyces coelicolor
39,021
19-May-1999




GB_BA1:MAU88875
840
U88875
Mycobacterium avium hypoxanthine-guanine phosphoribosyl transferase gene,
Mycobacterium avium
57,521
05-Mar.-1997







complete cds.




GB_BA1:MTY15C10
33050
Z95436
Mycobacterium tuberculosis H37Rv complete genome; segment 154/162.
Mycobacterium tuberculosis
40,086
17-Jun.-1998


rxa01514
711
GB_BA1:MTCY7H7B
24244
Z95557
Mycobacterium tuberculosis H37Rv complete genome; segment 153/162.
Mycobacterium tuberculosis
43,343
18-Jun.-1998




GB_BA1:MLCB2548
38916
AL023093
Mycobacterium leprae cosmid 82548.
Mycobacterium leprae
38,177
27-Aug.-1999




GB_PL1:EGGTPCHI
242
Z49757
E.gracills mRNA for GTP cyclohydrolase I (core region).
Euglena gracilis
64,876
20-Oct.-1995


rxa01515
975
GB_BA1:ECOUW93
338534
U14003
Escherichia coil K-12 chromosomal region from 92.8 to 00.1 minutes.
Escherichia coli
38,943
17-Apr.-1996




GB_BA1:EC0UW93
338534
U14003
Escherichia coli K-12 chromosomal region from 92.8 to 00.1 minutes.
Escherichia coli
37,500
17-Apr.-1996




GB_BA1:MTCY49
39430
Z73966
Mycobacterium tuberculosis H37Rv complete genome; segment 93/162.
Mycobacterium tuberculosis
38,010
24-Jun.-1999


rxa01516
513
GB_IN1:DME238847
5419
AJ238847
Drosophila melanogaster mRNA for drosophila dodeca-satellite protein 1
Drosophila melanogaster
36,346
13-Aug.-1999







(DDP-1).




GB_HTG3:AC009210
103814
AC009210
Drosophila melanogaster chromosome 2 clone BACR01106 (D1054) RPCI-98
Drosophila melanogaster
37,897
20-Aug.-1999







01.1.6 map 55D-55D strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***,







86 unordered pieces.




GB_IN2:AF132179
4842
AF132179
Drosophila melanogaster clone LD21677 unknown mRNA.
Drosophila melanogaster
36,149
3-Jun.-1999


rxa01517
600
GB_PL2:F6H8
82596
AF178045
Arabidopsis thaliana SAC F6H8.
Arabidopsis thaliana
35,846
19-Aug.-1999




GB_PL2:AF038831
647
AF038831
Sorosporium saponarlae intemal transcribed spacer 1, 5.8 S ribosomal RNA
Sorosporium saponariae
40,566
13-Apr.-1999







gene, and internal transcribed spacer 2, complete sequence.




GB_PL2:ATAC005957
108355
AC005957
Arabidopsis thaliana chromosome II BAC T15J14 genomic sequence,
Arabidopsis thaliana
38,095
7-Jan.-1999







complete sequence.


rxa01521
921
GB_BA1:ANANIFBH
5936
J0511
Anabaena sp. (clone AnH20.1) nitrogen fixation operon nifB, fdxN, nifS, nifU,
Anabaena sp.
38,206
26-Apr.-1993







and nifH genes, complete cds.




GB_PR2:AC002461
197273
AC002461
Human BAC clone RG204l16 from 7q31, complete sequence.
Homo sapiens
36,623
20-Aug.-1997




GB_PR2:AC002461
197273
AC002461
Human BAC clone RG204l16 from 7q31, complete sequence.
Homo sapiens
34,719
20-Aug.-1997


rxa01528
651
GB_RO:MM437P9
165901
AL049866
Mus musculus chromosome X, clone 437P9.
Mus musculus
37,500
29-Jun.-1999




GB_PR3:AC005740
186780
AC005740
Homo sapiens chromosome 5p, BAC clone 50g21 (LBNL H154), complete
Homo sapiens
37,031
01-Oct.-1998







sequence.




GB_PR3:AC005740
186780
AC005740
Homo sapiens chromosome 5p, BAC clone 50g21 (LBNL H154), complete
Homo sapiens
38,035
01-Oct.-1998







sequence.


rxa01551
1998
GB_BA1:MTCY22G10
35420
Z84724
Mycobacterium tuberculosis H37Rv complete genome; segment 21/162.
Mycobacterium tuberculosis
38,371
17-Jun.-1998




GB_BA2:ECOUW89
176195
U00006
E. coli chromosomal region from 89.2 to 92.8 minutes.
Escherichia coli
38,064
17-Dec.-1993




GB_BA1:SCQ11
15441
AL096823
Streptomyces coelicolor cosmid Q11.
Streptomyces coelicolor
60,775
8-Jul.-1999


rxa01561
1053
GB_IN1:CEY62H9A
47396
AL032630
Caenorhabditis elegans cosmid Y62H9A, complete sequence.
Caenorhabditis elegans
38,514
2-Sep.-1999




GB_PR4:HSU51003
3202
U51003
Homo sapiens DLX-2 (DLX-2) gene, complete cds.
Homo sapiens
37,730
07-Dec.-1999




GB_OM:PIGDAO1
395
M18444
Pig D-amino acid oxidase (DAO) gene, exon 1.
Sus scrofa
39,340
27-Apr.-1993


rxa01599
1785
GB_BA1:MTCI125
37432
Z98268
Mycobacterium tuberculosis H37Rv complete genome; segment 76/162.
Mycobacterium tuberculosis
63,300
17-Jun.-1998




GB_BA1:U00021
39193
U00021
Mycobacterium leprae cosmid L247.
Mycobacterium leprae
36,756
29-Sep.-1994




GB_BA1:MLCB1351
38936
Z95117
Mycobacterium leprae cosmid B1351.
Mycobacterium leprae
36,756
24-Jun.-1997


rxa01617
795
GB_PR2:HSMTM0
217657
AL034384
Human chromosome Xq28, cosmid clones 7H3, 14D7, C1230, 11E7, F1096,
Homo sapiens
40,811
5-Jul.-1999







A12197, 12G8, A09100; complete sequence bases 1..217657.




GB_PR2:HS13D10
153147
AL021407
Homo sapiens DNA sequence from PAC 13D10 on chromosome 6p22.3-23.
Homo sapiens
38,768
23-Nov.-1999







Contains CpG island.




GB_PR2:HSMTM0
217657
AL034384
Human chromosome Xq28, cosmid clones 7H3, 14D7, C1230, 11E7, F1096,
Homo sapiens
39,018
5-Jul.-1999







A12197, 12G8, A09100; complete sequence bases 1..217657.


rxa01657
723
GB_BA1:MTCY1A10
25949
Z95387
Mycobacterium tuberculosis H37Rv complete genome; segment 117/162.
Mycobacterium tuberculosis
40,656
17-Jun.-1998




GB_EST6:D79278
392
D79278
HUM213DO6B Human aorta polyA+ (TFujiwara) Homo sapiens cDNA clone
Homo sapiens
44,262
9-Feb.-1996







GEN-213D06 5′, mRNA sequence.




GB_BA2:AF129925
10243
AF129925
Thlobacillus ferrooxidans carboxysome operon, complete cds.
Thiobacillus ferrooxidans
40,709
17-May-1999


rxa01660
675
GB_BA1:MTV013
11364
AL021309
Mycobacterium tuberculosis H37Rv complete genome; segment 134/162.
Mycobacterium tuberculosis
40,986
17-Jun.-1998




GB_RO:MMFVI
6480
X97719
M.musculus retrovirus restriction gene Fy1.
Mus musculus
35,364
29-Aug.-1996




GB_PAT:A67508
6480
A67508
Sequence 1 from Patent W09743410.
Mus musculus
35,364
05-May-1999


rxa01678
651
GB_VI:TVU95309
600
U95309
Tula virus O64 nucleocapsid protein gene, partial cds.
Tula virus
41,894
28-Oct.-1997




GB_VI:TVU95303
600
U95303
Tula virus O52 nucleocapsid protein gene, partial cds.
Tula virus
41,712
28-Oct.-1997




GB_VI:TVU95302
600
U95302
Tula virus O24 nucleocapsid protein gene, partial cds.
Tula virus
39,576
26-Oct.-1997


rxa01679
1359
GB_EST5:H91843
362
H91843
ys81e01.s1 Soares retina N2b4HR Homo sapiens cDNA clone IMAGE:221208
Homo sapiens
39,157
29-Nov.-1995







3′ similar to gb:X63749_ma1 GUANINE NUCLEOTIDE-BINDING PROTEIN







G(T), ALPHA-1 (HUMAN);, mRNA sequence.




GB_STS:G26925
362
G26925
human STS SHGC-30023, sequence tagged site.
Homo sapiens
39,157
14-Jun.-1996




GB_PL2:AF139451
1202
AF139451
Gossypium robinsonii Ce1A2 pseudogene, partial sequence.
Gossypium robinsonii
38,910
1-Jun.-1999


rxa01690
1224
GB_BA1:SC1C2
42210
AL031124
Streptomyces coelicolor cosmid 1C2.
Streptomyces coelicolor
60,644
15-Jan.-1999




GB_EST22:A1064232
493
A1064232
GH04563.5prime GH Drosophila melanogaster head pOT2 Drosophila
Drosophila melanogaster
38,037
24-Nov.-1998







melanogaster cDNA clone GH04563 5prime, mRNA sequence.




GB_IN2:AF117896
1020
AF117896
Drosophila melanogaster neuropeptide F (npf) gene, complete cds.
Drosophila melanogaster
36,122
2-Jul.-1999


rxa01692
873
GB_BA2:AF067123
1034
AF067123
Lactobacillus reuteri cobalamin biosynthesis protein J (cbiJ) gene, partial cds;
Lactobacillus reuteri
48,079
3-Jun.-1998







and uroporphyrin-III C-methyltransferase (sumT) gene, complete cds.




GB_RO:RATNFHPEP
3085
M37227
Rat heavy neurofilament (NF-H) polypeptide, partial cds.
Rattus norvegicus
37,093
27-Apr.-1993




GB_RO:RSNFH
3085
X13804
Rat mRNA for heavy neurofilament polypeptide NF-H C-terminus.
Rattus sp.
37,093
14-Jul.-1995


rxa01698
1353
GB_8A2:AF124600
4115
AF124600
Corynebacterium glutamicum chorismate synthase (aroC), shikimate kinase
Corynebacterium glutamicum
100,000
04-May-1999







(aroK), and 3-dehydroqulnate synthase (aroB) genes, complete cds; and







putative cytoplasmic peptidase (pepQ) gene, partial cds.




GB_BA1:MTCY159
33818
Z83863
Mycobacterium tuberculosis H37Rv complete genome; segment 111/162.
Mycobacterium tuberculosis
36,323
17-Jun.-1998




GB_BA1.MSGB937CS
38914
L78820
Mycobacterium leprae cosmid B937 DNA sequence.
Mycobacterium leprae
62,780
15-Jun.-1996


rxa01699
693
GB_BA2:AF124600
4115
AF124600
Corynebacterium glutamicum chorismate synthase (aroC), shikimate kinase
Corynebacterium glutamicum
100,000
04-May-1999







(aroK), and 3-dehydroquinate synthase (aroB) genes, complete cds; and







putative cytoplasmic peptidase (pepQ) gene, partial cds.




GB_BA2:AF016585
41097
AF016585
Streptomyces caelestis cytochrome P-450 hydroxylase homolog (nidi) gene,
Streptomyces caelestis
40,260
07-Dec.-1997







partial cds; polyketide synthase modules 1 through 7 (nidA) genes, complete







cds; and N-methyltransferase homolog gene, partial cds.




GB_EST9:C19712
399
C19712
Rice panicle at ripening stage Oryza sativa cDNA clone E10821_1A,
Oryza sativa
45,425
24-Oct.-1996







mRNA sequence.


rxa01712
805
GB_EST21:M952466
278
AA952466
TENS1404 T. cruzi epimastigote normalized cDNA Library Trypanosoma cruzi
Trypanosoma cruzi
40,876
29-Oct.-1998







cDNA clone 1404 5′, mRNA sequence.




GB_EST21:M952466
278
AA952466
TENS1404 T. cruzi epimastigote normalized cDNA Library Trypanosoma cruzi
Trypanosoma cruzi
41,367
29-Oct.-1998







cDNA clone 1404 5′, mRNA sequence.


rxa01719
684
GB_HTG1:HSDJ534K7
154416
AL109925
Homo sapiens chromosome 1 clone RP4-534K7, *** SEQUENCING IN
Homo sapiens
35,651
23-Nov.-1999







PROGRESS ***, in unordered pieces.




GB_HTG1:HSDJ534K7
154416
AL109925
Homo sapiens chromosome 1 clone RP4-534K7, *** SEQUENCING IN
Homo sapiens
35651
23-Nov.-1999







PROGRESS ***, in unordered pieces.




GB_EST27:AI447108
431
AI447108
mq91e08.x1 Stratagene mouse heart (#937316) Mus musculus cDNA clone
Mus musculus
39,671
09-Mar.-1999







IMAGE:586118 3′, mRNA sequence.


rxa01720
1332
GB_PR4:AC006322
179640
AC006322
Homo sapiens PAC clone DJ1060B11 from 7q11.23-q21.1, complete
Homo sapiens
35,817
18-Mar.-1999







sequence.




GB_PL2:TM018A10
106184
AF013294
Arabidopsis thaliana BAC TM018A10.
Arabidopsis thaliana
35,698
12-Jul.-1997




GB_PR4:AC006322
179640
AC006322
Homo sapiens PAC clone DJ106OB11 from 7q11.23-q21.1, complete
Homo sapiens
37,243
18-Mar.-1999







sequence.


rxa01746
876
GB_EST3:R46227
443
R46227
yg52a03.s1 Soares infant brain 1NIB Homo sapiens cDNA clone
Homo sapiens
42,812
22-May-1995







IMAGE:36000 3′, mRNA sequence.




GB_EST3:R46227
443
R46227
yg52a03.s1 Soares infant brain 1NIB Homo sapiens cDNA clone
Homo sapiens
42,655
22-May-1995







IMAGE:36000 3′, mRNA sequence.


rxa01747
1167
GB_BA1:MTCY190
34150
Z70283
Mycobacterium tuberculosis H37Rv complete genome: segment 98/162.
Mycobacterium tuberculosis
59,294
17-Jun.-1998




GB_BA1:MLCB22
40281
Z98741
Mycobacterium leprae cosmid B22.
Mycobacterium leprae
57,584
22-Aug.-1997




GB_BA1:5C5F7
40024
AL096872
Streptomyces coelicolor cosmid 5F7.
Streptomyces coelicolor A3(2)
61,810
22-Jul.-1999


rxa01757
924
GB_EST21:AA918454
416
AA918454
om38c02.s1 Soares NFL_T_GBC_S1 Homo sapiens cDNA clone
Homo sapiens
39,655
23-Jun.-1998







IMAGE:1543298 3′similar to WP:F28F8.3 CE09757 SMALL NUCLEAR







RIBONUCLEOPROTEIN E;, mRNA sequence.




GB_EST4:H34042
345
H34042
EST110563 Rat PC-12 cells, NGF-treated (9 days) Rattus sp. cDNA clone
Rattus sp.
35,942
2-Apr.-1998







RPNB181 5′ end, mRNA sequence.




GB_EST20:M899038
450
AA899038
NCP6G8T7 Perithecial Neurospora crassa cDNA clone NP6G8 3′ end, mRNA
Neurospora crassa
40,000
12-Apr.-1998







sequence.


rxa01807
915
GB_BA1:AP000063
185300
AP000063
Aeropyrum pemix genomic DNA, section 6/7.
Aeropyrum pemix
40,067
22-Jun.-1999




GB_HTG4:AC010694
115857
AC010694
Drosophila melanogaster clone RPCI98-6H2, *** SEQUENCING IN
Drosophila melanogaster
35,450
16-Oct.-1999







PROGRESS ***, 75 unordered pieces.




GB_HTG4:AC010694
115857
AC010694
Drosophila melanogaster clone RPCI98-6H2, *** SEQUENCING IN
Drosophila melanogaster
35,450
16-Oct.-1999







PROGRESS ***, 75 unordered pieces.


rxa01821
401
GB_BA1:CGL007732
4460
AJ007732
Corynebacterium glutamicum 3′ ppc gene, secG gene, amt gene, ocd gene
Corynebacterium glutamicum
100,000
7-Jan.-1999







and 5′ soxA gene.




GB_RO:RATALGL
7601
M24108
Rattus norvegicus (clone A2U42) alpha2u globulin gene, exons 1-7.
Rattus norvegicus
38,692
15-Dec.-1994




GB_OV:APIGY2
1381
X78272
Anas platyrhynchos (Super M) IgY upsilon heavy chain gene, exon 2.
Anas platyrhynchos
36,962
15-Feb.-1999


rxa01835
654
GB_EST30:A1629479
353
A1629479
486101D10.x1 486- leaf pilmordia cDNA library from Hake lab Zea mays
Zea mays
38,109
26-Apr.-1999







cDNA, mRNA sequence.




GB_STS:G48245
515
G48245
SHGC-62915 Human Homo sapiens STS genomic, sequence tagged site.
Homo sapiens
37,021
26-Mar.-1999




GB_GSS3:B49052
515
B49052
RPCI11-4I12.TV RPCI-11 Homo sapiens genomic clone RPCI-11-4I12,
Homo sapiens
37,021
8-Apr.-1999







genomic survey sequence.


rxa01850
1470
GB_BA2:ECOUW67_0
110000
U18997
Escherichia coil K-12 chromosomal region from 67.4 to 76.0 minutes.
Escherichia coli
37,196
U18997




GB_BA2:AE000392
10345
AE000392
Escherichia coli K-12 MG1655 section 282 of 400 of the complete genome.
Escherichia coli
38,021
12-Nov.-1998




GB_BA2:U32715
13136
U32715
Haemophilus Influenzae Rd section 30 of 163 of the complete genome.
Haemophilus influenzae Rd
39,860
29-May-1998


rxa01878
1002
GB_HTG1:CEY64F11
177748
Z99776
Caenorhabditis elegans chromosome IV clone Y64F11, *** SEQUENCING IN
Caenorhabditis elegans
37,564
14-Oct.-1998







PROGRESS ***, in unordered pieces.




GB_HTG1:CEYB4F11
177748
Z99776
Caenorhabditis elegans chromosome IV clone Y64F11, *** SEQUENCING IN
Caenorhabditis elegans
37,564
14-Oct.-1998







PROGRESS ***, in unordered pieces.




GB_HTG1:CEY64F11
177748
Z99776
Caenorhabditis elegans chromosome IV clone Y64F11, *** SEQUENCING IN
Caenorhabditis elegans
37,576
14-Oct.-1998







PROGRESS ***, in unordered pieces.


rxa01892
852
GB_BA1:MTCY274
39991
Z74024
Mycobacterium tuberculosis H37Rv complete genome; segment 126/162.
Mycobacterium tuberculosis
35,910
19-Jun.-1998




GB_BA1:MLCB250
40603
Z97369
Mycobacterium leprae cosmid B250.
Mycobacterium leprae
64,260
27-Aug.-1999




GB_BA1:MSGB1529CS
36985
L78824
Mycobacterium leprae cosmid Bi 529 DNA sequence.
Mycobacterium leprae
64,260
15-Jun.-1996


rxa01894
978
GB_BA1:MTCY274
39991
Z74024
Mycobacterium tuberculosis H37Rv complete genome; segment 126/162.
Mycobacterium tuberculosis
37,229
19-Jun.-1998




GB_IN1:CELF46H5
38886
U41543
Caenorhabditis elegans cosmid F46H5.
Caenorhabditis elegans
38,525
29-Nov.-1996




GB_HTG3:AC009204
115633
AC009204
Drosophila melanogaster chromosome 2 clone BACRO3E19 (D1033) RPCI-98
Drosophila melanogaster
31,579
18-Aug.-1999







03.E.19 map 36E-37C strain y; cn bw sp, *** SEQUENCING IN PROGRESS







***, 94 unordered pieces.


rxa01920
1125
GB_BA2:AF112536
1798
AF112536
Corynebacterium glutamicum ribonucleotide reductase beta-chain (nrdF)
Corynebacterium glutamicum
99,733
5-Aug.-1999







gene, complete cds.




GB_BA1:CANRDFGEN
6054
Y09572
Corynebacterium ammoniagenes nrdH, nrdl, nrdE, nrdF genes.
Corynebacterium
70,321
18-Apr.-1998








ammoniagenes




GB_BA2:AF050168
1228
AF050168
Corynebacterium ammoniagenes ribonucleoside diphosphate reductase small
Corynebacterium
72,082
23-Apr.-1998







subunit (nrdF) gene, complete cds.
ammoniagenes


rxa01928
960
GB_BA1:CGPAN
2164
X96580
C.glutamicum panD, panC &xyIB genes.
Corynebacterium glutamicum
100,000
11-May-1999




GB_PL1:AP000423
154478
AP000423
Arabidopsls thaliana chloroplast genomic DNA, complete sequence,
Chloroplast Arabidopsis
35,917
15-Sep.-1999







strain:Columbia.
thaliana




GB_PL1:AP000423
154478
AP000423
Arabidopsis thaliana chloroplast genomic DNA, complete sequence,
Chloroplast Arabidopsis
33,925
15-Sep.-1999







strain:Columbia.
thaliana


rxa01929
938
GB_BA1:CGPAN
2164
X96580
C.glutamicum panB, panC &xylB genes.
Corynebacterium glutamicum
100,0001
1-May-1999




GB_BA1:XCU33548
8429
U33548
Xanthomonas campestris hrpB pathogenicity locus proteins HrpBl, HrpB2,
Xanthomonas campestris pv.
38,749
19-Sep.-1996







HrpB3, HrpB4, HrpB5, HrpB6, HrpB7, HrpB8, HrpAl, and ORF62
vesicatona







genes, complete cds.




GB_BA1:XANHRPB6A
1329
M99174
Xanthomonas campestris hrpB6 gene, complete cds.
Xanthomonas campestns
39,305
14-Sep.-1993


rxa01940
1059
GB_IN2:CFU43371
1060
U43371
Crithidla fasciculata inosine-undine preferring nucleoside hydrolase (IUNH)
Crithidia fasciculata
61,417
18-Jun.-1996







gene, complete cds.




GB_BA2:AE001467
11601
AE001467
Helicobacter pylon, strain J99 section 28 of 132 of the complete genome.
Helicobacter pylon J99
38,560
20-Jan.-1999




GB_RO:AF175967
3492
AF175967
Homo sapiens Leman coiled-coil protein (LCCP) mRNA, complete cds.
Mus musculus
40,275
26-Sep.-1999


rxa02022
1230
GB_BA1:CGDAPE
1966
X81379
C.glutamicum dapE gene and orf2.
Corynebacterium glutamicum
100,000
8-Aug.-1995




GB_BA1:CGDNMROP
2612
X85965
C.glutamicum ORF3 and aroP gene.
Corynebacterium glutamicum
38,889
30-Nov.-1997




GB_BA1:APU47055
6469
U47055
Anabaena PCC7I2O nitrogen fixation proteins (nifE, nifN, nifX, nifW) genes,
Anabaena PCC7120
36,647
17-Feb.-1996







complete cds, and nitrogenase (nifK) and hesA genes, partial cds.


rxa02024
859
GB_BA1:MTCI364
29540
Z93777
Mycobacterium tuberculosis H37Rv complete genome; segment 52/162.
Mycobacterium tuberculosis
59,415
17-Jun.-1998




GB_BA1:MSGB_1912CS
38503
L01536
M. leprae genomic dna sequence, cosmid b1912.
Mycobacterium leprae
57,093
14-Jun.-1996




GB_BA1:MLU15180
38675
U15180
Mycobacterium leprae cosmid B1756.
Mycobacterium leprae
57,210
09-Mar.-1995


rxa02027


nca02031


rxa02072
1464
GB_BA1:CGGDHA
2037
X72855
C. glutamicum GDHA gene.
Corynebacterium glutamicum
99,317
24-May-1993




GB_BA1:CGGDH
2037
X59404
Corynebacterium glutamicum , gdh gen for glutamate dehydrogenase.
Corynebacterium glutamicum
94,387
30-Jul.-1999




GB_BA1:PAE18494
1628
Y18494
Pseudomonas aeruginosa gdhA gene, strain PAC1.
Pseudomonas aeruginosa
62,247
6-Feb.-1999


rxa02085
2358
GB_BA1:MTCY22G8
22550
Z95585
Mycobacterium tuberculosis H37Rv complete genome: segment 49/162.
Mycobacterium tuberculosis
38,442
17-Jun.-1998




GB_BA1:MLCB33
42224
Z94723
Mycobacterium leprae cosmid B33.
Mycobacterium leprae
56,486
24-Jun.-1997




GB_BA1:ECOUW85
91414
M87049
E. coil genomic sequence of the region from 84.5 to 86.5 minutes.
Escherichia coli
52,127
29-May-1995


rxa02093
927
GB_EST14:M448146
452
AA448146
zw82h01.r1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE:782737 5′,
Homo sapiens
34,163
4-Jun.-1997







mRNA sequence.




GB_EST17:M641937
444
AA641937
ns18b10.r1 NCI_CGAPG_CB1 Homo sapiens cDNA clone IMAGE:1 183963 5′,
Homo sapiens
35,586
27-Oct.1997







mRNA sequence.




GB_PR3:AC003074
143029
AC003074
Human PAC clone DJ0596009 from 7p15, complete sequence.
Homo sapiens
31,917
6-Nov.-1997


rxa02106
1179
GB_BA1:SC1A6
37620
AL023496
Streptomyces coelicolor cosmid 1A6.
Streptomyces coelicolor
35,818
13-Jan.-1999




GB_PR4:AC005553
179651
AC005553
Homo sapiens chromosome 17, clone hRPK.112_J_9, complete sequence.
Homo sapiens
34,274
31-Dec.-1998




GB_EST3:R49746
397
R49746
yg71g10.rl Soares infant brain 1NIB Homo sapiens cDNA clone
Homo sapiens
41,162
18-May-1995







IMAGE:38768 5′ similar to gb:V00567 BETA-2-MICROGLOBULIN







PRECURSOR (HUMAN);, mRNA sequence.


rxa02111
1407
GB_BA1:SC6G10
36734
AL049497
Streptomyces coelicolor cosmid 6G10.
Streptomyces coelicolor
50,791
24-Mar.-1999




GB_BA1:U00010
41171
U00010
Mycobacterium leprae cosmid B1170.
Mycobacterium leprae
37,563
01-Mar.-1994




GB_BA1:MTCY336
32437
Z95586
Mycobacterium tuberculosis H37Rv complete genome; segment 70/162.
Mycobacterium tuberculosis
39,504
24-Jun.-1999


rxa02112
960
GB_HTG3:AC010579
157658
AC010579
Drosophila melanogaster chromosome 3 clone BACR09D08 (D1101) RPCI-98
Drosophila melanogaster
37,909
24-Sep.-1999







09.D.8 map 96F-96F strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***,







121 unordered pieces.




GB_GSS3:B09839
1191
B09839
T12A12-Sp6 TAMU Arabidopsis thaliana genomic clone T12A12, genomic
Arabidopsis thaliana
37,843
14-May-1997







survey sequence.




GB_HTG3:AC010579
157658
AC010579
Drosophila melanogaster chromosome 3 clone BACRO9D08 (D1101) RPCI-98
Drosophila melanogaster
37,909
24-Sep.-1999







09.D.8 map 96F-1996F strain y; cn bw sp, *** SEQUENCING IN PROGRESS ***,







121 unordered pieces.


rxa02134
1044
GB_BA1:SCSECYDNA
6154
X83011
S.coelicolor secY locus DNA.
Streptomyces coelicolor
36,533
02-Mar.-1998




GB_EST32:AI731596
568
A1731596
BNLGHi10185 Six-day Cotton fiber Gossypium hirsutum cDNA 5′ similar to
Gossypium hirsutum
33,451
11-Jun-1999







(AC004005) putative nbosomal protein L7 [Arabidopsis thaliana], mRNA







sequence.




GB_BA1:SCSECYDNA
6154
X83011
S.coelicolor secY locus DNA.
Streptomyces coelicolor
36,756
02-Mar.-1998


rxa02135
1197
GB_PR3:HS525L6
168111
AL023807
Human DNA sequence from clone RP3-525L6 on chromosome 6p22.3-23
Homo sapiens
34,365
23-Nov.-1999







Contains CA repeat, STSs, GSSs and a CpG Island, complete sequence.




GB_PL2:ATF21P8
85785
AL022347
Arabidopsis thaliana DNA chromosome 4, BAC clone F21P8 (ESSA project).
Arabidopsis thaliana
34,325
9-Jun.-1999




GB_PL2:U89959
106973
U89959
Arabidopsis thaliana BAC T7123, complete sequence.
Arabidopsis thaliana
33,874
26-Jun.-1998


rxa02136
645
GB_PL2:ATAC005819
57752
AC005819
Arabidopsis thaliana chromosome II BAC T3A4 genomic sequence, complete
Arabidopsis thaliana
34,123
3-Nov.-1998







sequence.




GB_PL2:F15K9
71097
AC005278
Arabldopsis thaliana chromosome 1 BAC F15K9 sequence, complete
Arabidopsis thaliana
31,260
7-Nov.-1998







sequence.




GB_PL2:U89959
106973
U89959
Arabidopsis thaliana BAC T7123, complete sequence.
Arabidopsis thaliana
34,281
26-Jun.-1998


rxa02139
1962
GB_BA1:MTCY190
34150
Z70283
Mycobacterium tuberculosis H37Rv complete genome; segment 98/162.
Mycobacterium tuberculosis
62,904
17-Jun.-1998




GBBA1:MSGB_1554CS
36548
L78814
Mycobacterium leprae cosmid 51554 DNA sequence.
Mycobacterium leprae
36,648
15-Jun.-1996




GBBA1:MSGB_1551CS
36548
L78813
Mycobacterium leprae cosmid B1551 DNA sequence.
Mycobacterium leprae
36,648
15-Jun.-1996


rxa02153
903
GB_BA2:AF049897
9196
AF049897
Corynebacterium glutamicum N-acetylglutamylphosphate reductase (argC),
Corynebacterium glutamicum
99,104
1-Jul.-1998







omithine acetyltransferase (argJ), N-acetylglutamate kinase (argB),







acetylomithine transaminase (argD), omithine carbamoyltransferase







(argF), arginine repressor (argR), arglninosuccinate synthase (argG), and







argininosuccinate lyase (argH) genes, complete cds.




GB_BA1:AF005242
1044
AF005242
Corynebacterium glutamicum N-acetylglutamate-5-semialdehyde
Corynebacterium glutamicum
99,224
2-Jul.-1997







dehydrogenase (argC) gene, complete cds.




GB_BA1:CGARGCJBD
4355
X86157
C.glutamicum argC, argJ, argB, argD, and argF genes.
Corynebacterium glutamicum
100,000
25-Jul.-1996


rxa02154
414
GB_BA2:AF049897
9196
AF049897
Corynebacterium glutamicum N-acetylglutamylphosphate reductase (argC),
Corynebacterium glutamicum
98,551
1-Jul.-1998







omithine acetyltransferase (argJ), N-acetylglutamate kinase (argB),







acetylomithine transaminase (argD), omithine carbamoyltransferase (argF),







arginine repressor (argR), argininosuccinate synthase (argG), and







argininosuccinate lyase (argH) genes, complete cds.




GB_BA1:AF005242
1044
AF005242
Corynebacterium glutamicum N-acetylglutamate-5-semialdehyde
Corynebacterium glutamicum
98,477
2-Jul.-1997







dehydrogenase (argC) gene, complete cds.




GB_BA1:CGARGCJBD
4355
X86157
C.glutamicum argC, argJ, argB, argD, and argF genes.
Corynebacterium glutamicum
100,000
25-Jul.-1996


rxa02155
1287
GB_BA1:CGARGCJBD
4355
X86157
C.glutamicum argC, argJ, argB, argD, and argF genes.
Corynebacterium glutamicum
99,767
25-Jul.-1996




GB_BA2:AF049897
9196
AF049897
Corynebacterium glutamicum N-acetylglutamylphosphate reductase (argC),
Corynebacterium glutamicum
99,378
1-Jul.-1998







omithine acetyltransferase (argJ), N-acetylglutamate kinase (argS),







acetylomithine transaminase (argD), ornithine carbamoyltransferase (argF),







arginine repressor (argR), argininosuccinate synthase (argG), and







argininosuccinate lyase (argH) genes, complete cds.




GB_BA1:MSGB1133CS
42106
L78811
Mycobacterium leprae cosmid Bi 133 DNA sequence.
Mycobacterium leprae
55,504
15-Jun.-1996


rxa02156
1074
GB_BA2:AF049897
9196
AF049897
Corynebacterium glutamicum N-acetylglutamylphosphate reductase (argC),
Corynebacterium glutamicum
100,000
1-Jul.-1998







omithine acetyltransferase (argJ), N-acetylglutamate kinase (argB),







acetylomithine transaminase (argD), omithine carbamoyltransferase (argF),







arginine repressor (argR), argininosuccinate synthase (argG), and







argininosuccinate lyase (argH) genes, complete cds.




GB_BA1:CGARGCJBD
4355
X86157
C.glutamicum argC, argJ, argB, argD, and argF genes.
Corynebacterium glutamicum
100,000
25-Jul.-1996




GB_BA2:AE001818
10007
AE001816
Thermotoga maritima section 128 of 136 of the complete genome.
Thermotoga mantima
50,238
2-Jun.-1999


rxa02157
1296
GB_BA2:AF049897
9196
AF049897
Corynebacterium glutamicum N-acetylglutamylphosphate reductase (argC),
Corynebacterium glutamicum
99,612
1-Jul.-1998







omithlne acetyltransferase (argJ), N-acetylglutamate kinase (argB),







acetylomithine transaminase (argD), omithine carbamoyltransferase (argF),







arglnine repressor (argR), argininosuccinate synthase (argG), and







arglninosuccinate lyase (argH) genes, complete cds.




GB_BA1:CGARGCJBD
4355
X86157
C.glutamicum argC, argJ, argB, argD, and argF genes.
Corynebacterium glutamicum
99,612
25-Jul.-1996




GB_BA1:MTCY06H11
38000
Z85982
Mycobacterium tuberculosis H37Rv complete genome: segment 73/162.
Mycobacterium tuberculosis
57,278
17-Jun.-1998


rxa02158
1080
GB_BA2:AF049897
9196
AF049897
Corynebacterium glutamicum N-acetylglutamylphosphate reductase (argC),
Corynebacterium glutamicum
100,000
1-Jul.-1998







omithine acetyltransferase (argJ), N-acetylglutamate kinase (argB),







acetylomithine transaminase (argD), omithine carbamoyltransferase (argF),







arginine repressor (argR), argininosuccinate synthase (argG), and







argininosuccinate lyase (argH) genes, complete cds.




GB_BA2:AF031518
2045
AF031518
Corynebacterium glutamicum omithine carbamolytransferase (argF) gene,
Corynebacterium glutamicum
99,898
5-Jan.-1999







complete cds.




GB_BA1:CGARGCJBD
4355
X86157
C.glutamicum argC, argJ, argB, argO, and argF genes.
Corynebacterium glutamicum
100,000
25-Jul.-1996


rxa02159
636
GB_BA2:AF049897
9196
AF049897
Corynebacterium glutamicum N-acetylglutamylphosphate reductase (argC),
Corynebacterium glutamicum
99,843
1-Jul.-1998







omithine acetyltransferase (argJ), N-acetylglutamate kinase (argB),







acetylomithine transaminase (argO), omithine carbamoyltransferase (argF),







arginine repressor (argR), argininosuccinate synthase (argG), and







argininosuccinate lyase (argH) genes, complete cds.




GB_BA2:AF031518
2045
AF031518
Corynebacterium glutamicum ornithine carbamolytransferase (argF) gene,
Corynebacterium glutamicum
88,679
5-Jan.-1999







complete cds.




GB_BA2:AF041436
516
AF041436
Corynebacterium glutamicum arginine repressor (argR) gene, complete cds.
Corynebacterium glutamicum
100,000
5-Jan.-1999


rxa02160
1326
GB_BA2:AF049897
9196
AF049897
Corynebacterium glutamicum N-acetylglutamylphosphate reductase (argC),
Corynebacterium glutamicum
99,774
1-Jul.-1998







omithine acetyltransferase (argJ), N-acetylglutamate kinase (argB),







acetylornithine transaminase (argD), ornithine carbamoyltransferase (argF),







arginine repressor (argR), argininosuccinate synthase (argG), and







argininosucanate lyase (argH) genes, complete cds.




GB_BA2:AF030520
1206
AF030520
Corynebacterium glutamicum argininosuccinate synthetase (argG) gene,
Corynebacterium glutamicum
99,834
19-Nov.-1997







complete cds.




GB_BA1:SCARGGH
1909
Z49111
S.clavuligerus argG gene and argH gene (partial).
Streptomyces clavuligerus
65,913
22-Apr.-1996


rxa02162
1554
GB_BA2:AF049897
9196
AF049897
Corynebacterium glutemicum N-acetylglutamylphosphate reductase (argC),
Corynebacterium glutamicum
88,524
01-Jul.-1998







ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB),







acetylornithine transaminase (argD), ornithine carbamoyltransferase (argF),







arginine repressor (argR), arglninosuccinate synthase (argG), and







arginlnosuccinate lyase (argH) genes, complete cds.




GB_BA2:AF048764
1437
AF048764
Corynebacterium glutamicum argininosuccinate lyase (argH) gene, complete
Corynebacterium glutamicum
87,561
1-Jul.-1998







cds.




GB_BA1:MTCY06H11
38000
Z85982
Mycobacterium tuberculosis H37Rv complete genome; segment 73/162.
Mycobacterium tuberculosis
64,732
17-Jun.-1998


rxa02176
1251
GB_BA1:MTCY3I
37630
Z73101
Mycobacterium tuberculosis H37Rv complete genome; segment 41/162.
Mycobacterium tuberculosis
36,998
17-Jun.-1998




GB_BA1:CGGLTG
3013
X66112
C. glutamicum glt gene for citrate synthase and ORF,
Corynebacterium glutamicum
39,910
17-Feb.-1995




GB_PL2:PGU65399
2700
U65399
Basidlomycete CECT 20197 phenoloxidase (pox1) gene, complete cds.
basidiomycete CECT 20197
38,474
19-Jul.-1997


rxa02189
861
GB_PR3:AC002468
115888
AC002468
Human Chromosome 15q26.1 PAC clone pDJ4l7d7, complete sequence.
Homo sapiens
35,941
16-Sep.-1998




GB_BA1:MSGB1970CS
39399
L78815
Mycobacterium leprae cosmid B1970 DNA sequence.
Mycobacterium leprae
40,286
15-Jun.-1996




GB_PR3:AC002468
115888
AC002468
Human Chromosome 15q26.1 PAC clone pDJ4l7d7, complete sequence.
Homo sapiens
33,689
16-Sep.-1998


rxa02193
1701
GB_BA1:BRLASPA
1987
D25316
Brevibacterium flavum aspA gene for aspartase, complete cds.
Corynebacterium glutamicum
99,353
6-Feb.-1999




GB_PAT:E04307
1581
E04307
DNA encoding Brevibacterium flavum aspartase.
Corynebacterium glutamicum
99,367
29-Sep.-1997




GB_BA1:ECOUW93
338534
U14003
Escherichia coli K-12 chromosomal region from 92.8 to 00.1 minutes.
Escherichia coli
37,651
17-Apr.-1996


rxa02194
968
GB_BA2:AF050166
840
AF050166
Corynebacterium glutamicum ATP phosphoribosyltransferase (hisG) gene,
Corynebacterium glutamicum
98,214
5-Jan.-1999







complete cds.




GB_BA1:BRLASPA
1987
D25316
Brevibacterium flavum aspA gene for aspartase, complete cds.
Corynebacterium glutamicum
93,805
6-Feb.-1999




GB_PAT:E08649
188
E08649
DNA encoding part of aspartase from coryneform bacteria.
Corynebacterium glutamicum
100,000
29-Sep.-1997


rxa02195
393
GB_BA2:AF086704
284
AF086704
Corynebacterium glutamicum phosphoribosyl-ATP-pyrophosphohydrolase
Corynebacterium glutamicum
100,000
8-Feb.-1999







(hisE) gene, complete cds.




GB_BA1:EAY17145
6019
Y17145
Eubacterium acidaminophilum grdR, grdI, grdH genes and partial Idc, grdT
Eubacterium acidaminophilum
39075
5-Aug.-1998







genes.




GB_STS:G01195
332
G01195
fruit fly STS Dm1930 clone DS06959 T7.
Drosophila melanogaster
35,542
28-Feb.-1995


rxa02197
551
GB_BA1:MTCY261
27322
Z97559
Mycobacterium tuberculosis H37Rv complete genome; segment 95/162.
Mycobacterium tuberculosis
33,938
17-Jun.-1998




GB_BA1:ML0B2533
40245
AL035310
Mycobacterium leprae cosmid B2533.
Mycobacterium leprae
65,517
27-Aug.-1999




GB_BA1:U00017
42157
U00017
Mycobacterium leprae cosmid B2126.
Mycobacterium leprae
36,770
01-Mar.-1994


rxa02198
2599
GB_BA1:U00017
42157
U00017
Mycobacterium leprae cosmid B2126.
Mycobacterium leprae
38,674
01-Mar.-1994




GB_BA1:MLCB2533
40245
AL035310
Mycobacterium leprae cosmid B2533.
Mycobacterium leprae
65,465
27-Aug.-1999




GB_BA1:MTCY261
27322
Z97559
Mycobacterium tuberculosis H37Rv complete genome; segment 95/162.
Mycobacterium tuberculosis
37,577
17-Jun.-1998


rxa02208
1025
GB_BA1:U00017
42157
U00017
Mycobacterium leprae cosmid B2126.
Mycobacterium leprae
59,823
01-Mar.-1994




GB_BA1:AP000063
185300
AP000063
Aeropyrum pemix genomic DNA, section 6/7.
Aeropyrum pemix
39,442
22-Jun.-1999




GB_YR4:AC006236
127593
A0006236
Homo sapiens chromosome 17, clone hCIT.162_E_12, complete sequence.
Homo sapiens
37,191
29-Dec.-1998


rxa02229
948
GB_BA1:MSGY154
40221
AD000002
Mycobacterium tuberculosis sequence from clone y154.
Mycobacterium tuberculosis
53,541
03-Dec-1996




GB_BA1:MTCY154
13935
Z98209
Mycobacterium tuberculosis H37Rv complete genome; segment 121/162.
Mycobacterium tuberculosis
40,407
17-Jun.-1998




GB_BA1:U00019
36033
U00019
Mycobacterium leprae cosmid B2235.
Mycobacterium leprae
40,541
01-Mar.-1994


nca02234
3462
GB_BA1:MSGB937CS
38914
L78820
Mycobacterium leprae cosmid B937 DNA sequence.
Mycobacterium leprae
66,027
15-Jun.-1996




GB_BA1:MTCY2B12
20431
Z81011
Mycobacterium tuberculosis H37Rv complete genome; segment 61/162.
Mycobacterium tuberculosis
71,723
18-Jun.-1998




GB_BA2:U01072
4393
U01072
Mycobacterium bovis BOG orotidine-5-monophosphate decarboxylase (uraA)
Mycobacterium bovis
67,101
22-Dec-1993







gene.


rxa02235
727
GB_BA1:M5U91572
960
U91572
Mycobacterium smegmatis carbamoyl phosphate synthetase (pyrAB) gene,
Mycobacterium smegmatis
60,870
22-Mar.-1997







partial cds and orotidine 5-monophosphate decarboxylase (pyrF) gene,







complete cds.




GB_HTG3:AC009364
192791
AC009364
Homo sapiens chromosome 7, ***SEQUENCING IN PROGRESS ***, 57
Homo sapiens
37,994
1-Sep.-1999







unordered pieces.




GB_HTG3:AC009364
192791
AC009364
Homo sapiens chromosome 7, *** SEQUENCING IN PROGRESS ***, 57
Homo sapiens
37,994
1-Sep.-1999







unordered pieces.


rxa02237
693
GB_BA1:MTCY21B4
39150
Z80108
Mycobacterium tuberculosis H37Rv complete genome; segment 62/162.
Mycobacterium tuberculosis
55,844
23-Jun.-1998




GB_BA2:AF077324
5228
AF077324
Rhodococcus equi strain 103 plasmid RE-VP1 fragment f.
Rhodococcus equi
41,185
5-Nov.-1998




GB_EST22:AU017763
586
AU017763
AU017763 Mouse two-cell stage embryo cDNA Mus musculus cDNA clone
Mus musculus
38,616
19-Oct-1998







J0744A04 3′, mRNA sequence.


rxa02239
1389
GB_BA1:MTCY21B4
39150
Z80108
Mycobacterium tuberculosis H37Rv complete genome: segment 62/162.
Mycobacterium tuberculosis
56,282
23-Jun.-1998




GB_HTG3:AC010745
193862
AC010745
Homo sapiens clone NH0549D18, *** SEQUENCING IN PROGRESS ***, 30
Home sapiens
36,772
21-Sep.-1999







unordered pieces.




GB_HTG3:AC010745
193862
AC010745
Homo sapiens clone NH0549D18, *** SEQUENCING IN PROGRESS ***, 30
Homo sapiens
36,772
21-Sep.-1999







unordered pieces.


rxa02240
1344
EM_PAT:E09855
1239
E09855
gDNA encoding 5-adenosylmethionine synthetase.
Corynebacterium glutamicum
99,515
07-Oct.-1997










(Rel. 52,










Created)




GB_PAT:A37831
5392
A37831
Sequence 1 from Patent W09408014.
Streptomyces pristinaespiralis
63,568
05-Mar.-1997




GB_BA2:AF117274
2303
AF117274
Streptomyces spectabilis flavoprotein homolog Dfp (dfp) gene, partial cds; and
Streptomyces spectabilis
65,000
31-Mar.-1999







S-adenosylmethionine synthetase (metK) gene, complete cds.


rxa02246
1107
EM_BA1:AB003693
5589
AB003693
Corynebacterium ammoniagenes DNA for rib operon, complete cds.
Corynebacterium
52,909
03-Oct.-1997








ammoniagenes

(Rel. 52,










Created)




GB_PAT:E07957
5589
E07957
gDNA encoding at least guanosine triphosphate cyclohydrolase and riboflavin
Corynebacterium
52,909
29-Sep.-1997







synthase.
ammoniagenes




GB_PAT:I32742
5589
I32742
Sequence 1 from patent U.S. Pat. No. 5589355.
Unknown.
52,909
6-Feb.-1997


rxa02247
756
GB_PAT:I32743
2689
I32743
Sequence 2 from patent U.S. Pat. No. 5589355.
Unknown.
57,937
6-Feb.-1997




EM_BA1:AB003693
5589
AB003693
Corynebacterium ammoniagenes DNA for rib operon, complete cds.
Corynebacterium
57,937
03-Oct.-1997








ammoniagenes

(Rel. 52,










Created)




GB_PAT:I32742
5589
I32742
Sequence 1 from patent U.S. Pat. No. 5589355.
Unknown.
57,937
6-Feb.-1997


rxa02248
1389
GB_PAT:I32742
5589
132742
Sequence 1 from patent U.S. Pat. No. 5589355.
Unknown.
61,843
6-Feb.-1997




EM_BA1:AB003693
5589
AB003693
Corynebacterium ammoniagenes DNA for rib operon, complete cds.
Corynebacterium
61,843
03-Oct.-1997








ammoniagenes

(Rel. 52.










Created)




GB_PAT:E07957
5589
E07957
gDNA encoding at least guanosine triphosphate cyclohydrolase and riboflavin
Corynebacterium
61,843
29-Sep.-1997







synthase.
ammoniagenes


rxa02249
600
GB_PAT:E07957
5589
E07957
gDNA encoding at least guanosine triphosphate cyclohydrolase and riboflavin
Corynebacterium
64,346
29-Sep.-1997







synthase.
ammoniagenes




GB_PAT:I32742
5589
I32742
Sequence 1 from patent U.S. 5589355.
Unknown.
64,346
6-Feb.-1997




GB_PAT:I32743
2689
I32743
Sequence 2 from patent U.S. 5589355.
Unknown.
64,346
6-Feb.-1997


nca02250
643
GB_PAT:E07957
5589
E07957
gDNA encoding at least guanosine triphosphate cyclohydrolase and riboflavin
Corynebacterium
56,318
29-Sep.-1997







synthase.
ammoniagenes




GB_PAT:I32742
5589
I32742
Sequence 1 from patent U.S. Pat. No. 5589355.
Unknown.
56,318
6-Feb.-1997




EM_BA1:AB003693
5589
AB003693
Corynebacterium ammoniagenes DNA for rib operon, complete cds.
Carynebacterium
56,318
03-Oct.-1997








ammoniagenes

(Rel. 52.










Created)


rxa02262
1269
GB_BA1:CGL007732
4460
AJ007732
Corynebacterium glutamicum 3′ppc gene, secG gene, amt gene, ocd gene
Corynebacterium glutamicum
100,000
7-Jan.-1999







and 5′ soxA gene.




GB_BA1:CGAMTGENE
2028
X93513
C. glutamicum amt gene.
Corynebacterium glutamicum
100,000
29-May-1996




GB_VI:HEHCMVCG
229354
X17403
Human cytomegalovirus strain AD169 complete genome.
human herpesvirus 5
38,651
10-Feb.-1999


rxa02263
488
GB_BA1:CGL007732
4460
AJ007732
Corynebacterium glutamicum 3′ ppc gene, secG gene, amt gene, ocd gene
Corynebacterium glutamicum
100,000
7-Jan.-1999







and 5′ soxA gene.




GB_BA1:CGL007732
4460
AJ007732
Corynebacterium glutamicum 3′ ppc gene, secG gene, amt gene, ocd gene
Corynebacterium glutamicum
37,526
7-Jan.-1999







and 5′ soxA gene.


rxa02272
1368
EM_PAT:E09373
1591
E09373
Creatinine deiminase gene.
Bacillus sp.
96,928
08-Oct.-1997










(Rel. 52,










Created)




GB_BA1:D38505
1591
D38505
Bacillus sp. gene for creatinine deaminase, complete cds.
Bacillus sp.
96,781
7-Aug.-1998




GB_HTG2:AC008595
146070
AC008595
Homo sapiens , *** SEQUENCING IN PROGRESS ***, 4 unordered pieces.
Homo sapiens
36,264
20-Feb.-1999


rxa02281
1545
GB_GSS12:AQ411010
551
AQ411010
HS_2257_B1_H02_MR CIT Approved Human Genomic Sperm Library D
Homo sapiens
36,197
17-Mar.-1999







Homo sapiens genomic clone Plate = 2257 Col = 3 Row = P,







genomic survey sequence.




GB_EST23:AI128823
363
AI128623
qa62c01.si Scares_fetal_heart_NbHH19W Home sapiens cDNA clone
Home sapiens
37,017
05-Oct.-1998







IMAGE:1691328 3′, mRNA sequence.




GB_PL2:ATAC007019
102335
AC007019
Arabidopsis thaliana chromosome II BAC F7D8 genomic sequence, complete
Arabidopsis thaliana
33,988
16-Mar.-1999







sequence.


rxa02299
531
GB_BA2:AF116184
540
AF116184
Corynebacterium glutamicum L-aspartate-alpha-decarboxylase precursor
Corynebacterium glutamicum
100,000
02-May-1999







(panD) gene, complete cds.




GB_GSS9:AQ164310
507
AQ184310
HS_2171_A2E01MR CIT Approved Human Genomic Sperm Library D
Homo sapiens
37,278
16-Oct.-1998







Homo sapiens genomic clone Plate = 2171 Col = 2 Row = 1,







genomic survey sequence.




GB_VI:MH68TKH
4557
X93468
Murine herpesvirus type 68 thymidine kinase and glycoprotein H genes.
murine herpesvirus 68
40,288
3-Sep.-1996


rxa02311
813
GB_HTG4:AC006091
176878
AC006091
Drosophila melanogaster chromosome 3 clone BACR48G05 (D475) RPCI-98
Drosophila melanogaster
36,454
27-Oct.-1999







48.G.5 map 91F1-91F13 strain y; cn bwsp, *** SEQUENCING IN PROGRESS







***, 4 unordered pieces.




GB_HTG4:AC006091
176878
AC006091
Drosophila melanogaster chromosome 3 clone BACR48G05 (D475) RPCI-98
Drosophila melanogaster
36,454
27-Oct.-1999







48.G.5 map 91F1-91F13 strain y; cn bw sp, *** SEQUENCING IN PROGRESS







***, 4 unordered pieces.




GB_BA2:RRU65510
16259
U65510
Rhodospirillum rubrum CO-induced hydrogenase operon (cooM, cooK, cooL,
Rhodospirillum rubrum
37,828
9-Apr.-1997







cooX, cooLJ, cooH) genes, iron sulfur protein (cooF) gene, carbon monoxide







dehydrogenase (cooS) gene, carbon monoxide dehydrogenase







accessory proteins (cooC, cooT, cooJ) genes, putative transcriptional activator







(cooA) gene, nicotinate-nucleotide pyrophosphorylase (nadC) gene, complete







cds, L-aspartate oxidase (nadB) gene, and alkyl hydroperoxide







reductase (ahpC) gene, partial cds.


rxa02315
1752
GB_BA1:MSGY224
40051
AD000004
Mycobacterium tuberculosis sequence from clone y224.
Mycobacterium tuberculosis
49,418
03-Dec.-1996




GB_BA1:MTY25D10
40838
Z95558
Mycobacterium tuberculosis H37Rv complete genome; segment 28/162.
Mycobacterium tuberculosis
49,360
17-Jun.-1998




GB_BA1:MSGY224
40051
AD000004
Mycobactertum tuberculosis sequence from clone y224.
Mycobacterium tuberculosis
38,150
03-Dec.-1996


rxa02318
402
GB_HTG3:AC011348
111083
AC011348
Homo sapiens chromosome 5 clone CIT-HSPC_303E13, *** SEQUENCING
Homo sapiens
35,821
06-Oct.-1999







IN PROGRESS ***, 3 ordered pieces.




GB_HTG3:AC011348
111083
AC011348
Homo sapiens chromosomes 5 clone CIT-HSPC_303E13, *** SEQUENCING
Homo sapiens
35,821
06-Oct.-1999







IN PROGRESS ***, 3 ordered pieces.




GB_HTG3:AC011412
89234
AC011412
Homo sapiens chromosome 5 clone CIT978SKB_81K21, *** SEQUENCING
Homo sapiens
36,181
06-Oct.-1999







IN PROGRESS ***, 3 ordered pieces.


rxa02319
1080
GB_BA1:M5GY224
40051
AD000004
Mycobacterium tuberculosis sequence from clone y224.
Mycobacterium tuberculosis
37,792
03-Dec.-1996




GB_BA1:MTY25D10
40838
Z95558
Mycobacterium tuberculosis H37Rv complete genome; segment 28/162.
Mycobacterium tuberculosis
37,792
17-Jun.-1998




GB_EST23:AI117213
476
AI117213
ub83h02.rl Soares 2NbMT Mus musculus cDNA clone IMAGE:1395123
Mus musculus
35,084
2-Sep.-1998







5′, mRNA sequence.


rxa02345
1320
GB_BA1:BAPURKE
2582
X91189
B. ammonlagenes purK and purE genes.
Corynebacterium
61,731
14-Jan.-1997








ammoniagenes




GB_BA1:MTCY71
42729
Z92771
Mycobacterium tuberculosis H37Rv complete genome; segment 141/162.
Mycobacterium tuberculosis
39,624
10-Feb.-1999




GB_BA1:MTCY71
42729
Z92771
Mycobacterium tuberculosis H37Rv complete genome; segment 141/162.
Mycobacterium tuberculosis
39,847
10-Feb.-1999


rxa02350
618
GB_BA1:BAPURKE
2582
X91189
B. ammoniagenes purK and purE genes.
Corynebacterium
64,286
14-Jan.-1997








ammoniagenes




GB_PL1:SC130KBXV
129528
X94335
S. cerevislae 130 kb DNA fragment from chromosome XV.
Saccharomyces cerevisiae
36,617
15-Jul.-1997




GB_PLI:SCXVORFS
50984
X90518
S. cerevlslae DNA of 51 Kb from chromosome XV right arm.
Saccharomyces cerevisiae
36,617
1-Nov.-1995


rxa02373
1038
GB_PAT:E00311
1853
E00311
DNA coding of 2,5-diketogluconic acid reductase.
unidentified
56,123
29-Sep.-1997




GB_PAT:I06030
1853
I08030
Sequence 4 from Patent EP 0305608.
Unknown.
56,220
02-Dec.-1994




GB_PAT:I00836
1853
I00836
Sequence 1 from Patent U.S. Pat. No. 4758514.
Unknown.
56,220
21-May-1993


rxa02375
1350
GB_BA2:CGU31230
3005
U31230
Corynebacterium glutamicum Obg protein homolog gene, partial cds, gamma
Corynebacterium glutamicum
99,332
2-Aug.-1996







glutamyl kinase (proB) gene, complete cds, and (unkdh) gene, complete cds.




GB_HTG3:AC009946
169072
AC009946
Homo sapiens clone NH0012C17, *** SEQUENCING IN PROGRESS ***, 1
Homo sapiens
36,115
8-Sep.-1999







unordered pieces.




GB_HTG3:AC009946
169072
AC009946
Homo sapiens clone NH0012C17, tmSEQUENCING IN PROGRESS ***, 1
Homo sapiens
36,115
8-Sep.-1999







unordered pieces.


rxa02380
777
GB_BA1:MTCY253
41230
Z81368
Mycobacterium tuberculosis H37Rv complete genome; segment 106/162.
Mycobacterium tuberculosis
38,088
17-Jun.-1998




GB_HTG4:AC010658
120754
AC010858
Drosophila melanogaster chromosome 3L175C1 clone RPCI98-3B20, ***
Drosophila melanogaster
35,817
16-Oct.-1999







SEQUENCING IN PROGRESS ***, 78 unordered pieces.




GB_HTG4:AC010658
120754
AC010658
Drosophila melanogaster chromosome 3L175C1 clone RPCI98-3B20, ***
Drosophila melanogaster
35,817
16-Oct.-1999







SEQUENCING IN PROGRESS ***, 78 unordered pieces.


rxa02382
1419
GB_BA1:CGPROAGEN
1783
X82929
C.glutamicum proA gene.
Corynebacterium glutamicum
98,802
23-Jan.97




GB_BA1:MTCY428
26914
Z81451
Mycobacterium tuberculosis H37Rv complete genome; segment 107/162.
Mycobacterium tuberculosis
38,054
17-Jun.-1998




GB_BA2:CGU31230
3005
U31230
Corynebacterium glutamicum Obg protein homolog gene, partial cds, gamma
Corynebacterium glutamicum
98,529
2-Aug.-1996







glutamyl kinase (proB) gene, complete cds, and (unkdh) gene, complete cds.


rxa02400
693
GB_BA1:CGACEA
2427
X75504
C.glutamicum aceA gene and thiX genes (partial).
Corynebacterium glutamicum
100,000
9-Sep.-1994




GB_PAT:I86191
2135
I86191
Sequence 3 from patent U.S. Pat. No. 5700661.
Unknown.
100,000
10-Jun.-1998




GB_PAT:I13693
2135
I13693
Sequence 3 from patent U.S. Pat. No. 5439822.
Unknown.
100,000
26-Sep.-1995


rxa02432
1098
GB_GSS15:AQ606842
574
AQ606842
HS_5404_B2_E07_T7A RPCI-11 Human Male BAC Library Homo sapiens
Homo sapiens
39,716
10-Jun.-1999







genomic clone Plate = 980 Col = 14 Row = J, genomic survey sequence.




GB_EST1:T05804
406
T05804
EST03693 Fetal brain, Stratagene (cat#936206) Homo sapiens cDNA clone
Homo sapiens
37,915
30-Jun.-1993







HFBDG63 similar to EST containing Alu repeat, mRNA sequence.




GB_PL1:AB006699
77363
AB006699
Arabidopsis thaliana genomic DNA, chromosome 5, P1 clone: MDJ22,
Arabidopsis thaliana
35,526
20-Nov.-1999







complete sequence.


rxa02458
1413
GB_BA2:AF114233
1852
AF114233
Corynebacterium glutamicum 5-enolpyruvylshikimate 3-phosphate synthase
Corynebacterium glutamicum
100,000
7-Feb.-1999







(aroA) gene, complete cds.




GB_EST37:AW013061
578
AW013061
ODT-0033 Winter flounder ovary Pleuronectes americanus cDNA clone ODT-
Pleuronectes americanus
39,175
10-Sep.-1999







0033 5′similar to FRUCTOSE-BISPHOSPHATE ALDOLASE B (LIVER),







mRNA sequence.




GB_GSS15:AQ650027
728
AQ650027
Sheared DNA-5L2.TF Sheared DNA Trypanosoma brucei genomic clone
Trypanosoma brucei
39,281
22-Jun.-1999







Sheared DNA-5L2, genomic survey sequence.


rxa02469
1554
GB_BA1:MTCY359
36021
Z83859
Mycobacterium tuberculosis H37Rv complete genome; segment 84/162.
Mycobacterium tuberculosis
39,634
17-Jun.-1998




GB_BA1:MLCB1788
39228
AL008609
Mycobacterium leprae cosmid B1788.
Mycobacterium leprae
59,343
27-Aug.-1999




GB_BA1:SCAJ10601
4692
AJ010601
Streptomyces coelicolor A3(2)-DNA for whiD and whiK loci.
Streptomyces coelicolor
48,899
17-Sep.-1998


rxa02497
1050
GB_BA2:CGU31224
422
U31224
Corynebacterium glutamicum (ppx) gene, partial cds.
Corynebacterium glutamicum
96,445
2-Aug.-1996




GB_BA1:MTCY20G9
37218
Z77162
Mycobacterium tuberculosis H37Rv complete genome; segment 25/162.
Mycobacterium tuberculosis
59,429
17-Jun.-1998




GB_BA1:SCE7
16911
AL049819
Streptomyces coelicolor cosmid E7.
Streptomyces coelicolor
39,510
10-May-1999


rxa02499
933
GB_BA2:CGU31225
1817
U31225
Corynebacterium glutamicum L-proline:NADP+5-oxidoreductase (proC) gene,
Corynebacterium glutamicum
97,749
2-Aug.-1996







complete cds.




GB_BA1:NG17PILA
1920
X13965
Neisserla gonorrhoeae pilA gene.
Neisserla gonorrhoeae
43,249
30-Sep.-1993




GB_HTG2:AC007984
129715
AC007984
Drosophila melanogaster chromosome 3 clone BACRO5C10 (D781) RPCI-98
Drosophila melanogaster
33,406
2-Aug.-1999







05.C.10 map 97D-1997E strain y; cn bw sp, *** SEQUENCING IN PROGRESS







***, 87 unordered pieces.


rxa02501
1188
GB_BA1:MTCY2OG9
37218
Z77162
Mycobacterium tuberculosis H37Rv complete genome; segment 25/162.
Mycobacterium tuberculosis
39,357
17-Jun.-1998




GB_BA1:U00018
42991
U00018
Mycobacterium leprae cosmid B2168.
Mycobacterium leprae
51,768
01-Mar.-1994




GB_VI:HE1CG
152261
X14112
Herpes simplex virus (HSV) type 1 complete genome.
human herpesvirus 1
39,378
17-Apr.-1997


rxa02503
522
GB_PR3:AC005328
35414
AC005328
Homo sapiens chromosome 19, cosmid R26660, complete sequence.
Homo sapiens
39,922
28-Jul.-1998




GB_PR3:AC005545
43514
AC005545
Homo sapiens chromosome 19, cosmid R26634, complete sequence.
Homo sapiens
39,922
3-Sep.-1998




GB_PR3:AC005328
35414
AC005328
Homo sapiens chromosome 19, cosmld R26660, complete sequence.
Homo sapiens
34,911
28-Jul.-1998


rxa02504
681
GB_BA1:MTCY20G9
37218
Z77162
Mycobacterium tuberculosis H37Rv complete genome; segment 25/162.
Mycobacterium tuberculosis
54,940
17-Jun.-1998




GB_PR3:AC005328
35414
AC005328
Homo sapiens chromosome 19, cosmid R26660, complete sequence.
Homo sapiens
41,265
28-Jul.-1998




GB_PR3:AC005545
43514
AC005545
Homo sapiens chromosome 19, cosmid R26634, complete sequence.
Homo sapiens
41,265
3-Sep.-1998


rxa02516
1386
GB_BA1:MLCLS36
36224
Z99125
Mycobacterium leprae cosmid L536.
Mycobacterium leprae
37,723
04-Dec.-1998




GB_BA1:U00013
35881
U00013
Mycobacterium leprae cosmid B1496.
Mycobacterium leprae
37,723
01-Mar.-1994




GB_BA1:MTV007
32806
AL021184
Mycobacterium tuberculosis H37Rv complete genome; segment 64/162.
Mycobacterium tuberculosis
61,335
17-Jun.98


rxa02517
570
GB_BA1:MLCL536
36224
Z99125
Mycobacterium leprae cosmid L536.
Mycobacterium leprae
37,018
4-Dec.-1998




GB_BA1:U00013
35881
U00013
Mycobacterium leprae cosmid B1496.
Mycobacterium leprae
37,018
01-Mar.-1994




GB_BA1:SCC22
22115
AL096839
Streptomyces coelicolor cosmid C22.
Streptomyces coelicolor
37,071
12-Jul.-1999


rxa02532
1170
GB_OV:AF137219
831
AF137219
Amia calva mixed lineage leukemia-like protein (MII) gene, partial cds.
Amia calva
36,853
7-Sep.-1999




GB_EST30:AI645057
301
A1645057
vs52a10.yl Stratagene mouse Tcell 937311 Mus musculus cDNA clone
Mus musculus
41860
29-Apr.-1999







IMAGE:1149882 5′, mRNA sequence.




GB_EST20:AA822595
429
AA822595
vs52a10.r1 Stratagene mouse Tcell 937311 Mus musculus cDNA clone
Mus musculus
42,353
17-Feb.-1998







IMAGE:1149882 5′, mRNA sequence.


rxa02536
879
GB_HTG2:AF130866
118874
AF130866
Homo sapiens chromosome 8 clone PAC 172N13 map 8q24,***
Homo sapiens
40,754
21-Mar.-1999







SEQUENCING IN PROGRESS ***, in unordered pieces.




GB_HTG2:AF130866
118874
AF130866
Homo sapiens chromosome 8 clone PAC 172N13 map 8q24, ***
Homo sapiens
40,754
21-Mar.-1999







SEQUENCING IN PROGRESS ***, in unordered pieces.




GB_PL1:ATT12J5
84499
AL035522
Arabidopsis thaliana DNA chromosome 4, BAC clone T12J5 (ESSAII project).
Arabidopsis thaliana
35,063
24-Feb.-1999


rxa02550
1434
GB_BA1:MTCY279
9150
Z97991
Mycobacterium tuberculosis H37Rv complete genome; segment 17/162.
Mycobacterium tuberculosis
37,773
17-Jun.-1998




GB_BA1:MSGB1970CS
39399
L78815
Mycobacterium leprae cosmid B1970 DNA sequence.
Mycobacterium leprae
39,024
15-Jun.-1996




GB_BA2:SC2H4
25970
AL031514
Streptomyces coelicolor cosmid 2H4.
Streptomyces coelicolor A3(2)
37,906
19-Oct.-1999


rxa02559
1026
GB_BA1:MTV004
69350
AL009198
Mycobacterium tuberculosis H37Rv complete genome; segment 144/162.
Mycobacterium tuberculosis
47,358
18-Jun.-1998




GB_PAT:I28684
5100
I28684
Sequence 1 from patent U.S. Pat. No. 5573915.
Unknown.
39,138
6-Feb.-1997




GB_BA1:MTU27357
5100
U27357
Mycobacterium tuberculosis cyclopropane mycolic acid synthase (cma1) gene,
Mycobacterium tuberculosis
39,138
26-Sep.-1995







complete cds.


rxa02622
1683
GB_BA2:AE001780
11997
AE001780
Thermotoga maritima section 92 of 136 of the complete genome.
Thermotoga maritima
44,914
2-Jun.-1999




GB_OV:AF064564
49254
AF064564
Fugu rubripes neurofibromatosis type 1 (NF1), A-kinase anchor protein
Fugu rubripes
39,732
17-Aug.-1999







(AKAP84), BAW protein (BAW), and WSB1 protein WSB1) genes, complete







cds.




GB_OV:AF064564
49254
AF064564
Fugu rubripes neurofibromatosis type 1 (NF1), A-kinase anchor protein
Fugu rubripes
36,703
17-Aug.-1999







(AKAP84), BAW protein (BAW), and WSB1 protein (WSB1) genes, complete







cds.


rxa02623
714
GB_GSS5:AQ818728
444
AQ818728
HS_5268_A1_G09_SP6E RPCI-11 Human Male BAC Library Homo sapiens
Homo sapiens
38,801
26-Aug.-1999







genomic clone Plate = 844 Col = 17 Row = M, genomic survey sequence.




GB_HTG5:AC011083
198586
AC011083
Homo sapiens chromosome 9 clone RP11-111M7 map 9, WORKING DRAFT
Homo sapiens
35,714
19-Nov.-1999







SEQUENCE, 51 unordered pieces.




GB_GSS6:AQ826948
544
AQ826948
HS_5014_A2_C12_T7A RPCI-11 Human Male BAC Library Homo sapiens
Homo sapiens
39,146
27-Aug.-1999







genomic clone Plate = 590 Col = 24 Row = E, genomic survey sequence.


rxa02629
708
GB_VI:BRSMGP
462
M86652
Bovine respiratory syncytial virus membrane glycoprotein mRNA, complete
Bovine respiratory syncytial
37,013
28-Apr.-1993







cds.
virus




GB_VI:BRSMGP
462
M86652
Bovine respiratory syncytial virus membrane glycoprotein mRNA, complete
Bovine respiratory syncytial
37,0132
8-Apr.-1993







cds.
virus


rxa02645
1953
GB_PAT:A45577
1925
A45577
Sequence 1 from Patent WO9519442.
Corynebacterium glutamicum
39,130
07-Mar.-1997




GB_PAT:A45581
1925
A45581
Sequence 5 from Patent WO9519442.
Corynebacterium glutamicum
39,130
07-Mar.-1997




GB_BA1:CORILVA
1925
L01508
Corynebacterium glutamicum threonine dehydratase (ilvA) gene, complete
Corynebacterium glutamicum
39,130
26-Apr.-1993







cds.


rxa02646
1392
GB_BA1:CORILVA
1925
L01508
Corynebacterium glutamicum threonine dehydratase (ilvA) gene, complete
Corynebacterium glutamicum
99,138
26-Apr.-1993







cds.




GB_PAT:A45585
1925
A45585
Sequence 9 from Patent WO9519442.
Corynebacterium glutamicum
99,066
07-Mar.-1997




GB_PAT:A45583
1925
A45583
Sequence 7 from Patent WO9519442.
Corynebacterium glutamicum
99,066
07-Mar.-1997


rca02648
1326
GB_OV:ICTCNC
2049
M83111
Ictalurus punctatus cyclic nucleotide-gated channel RNA sequence.
Ictalurus punctatus
38,402
24-May-1993




GB_EST11:AA265464
345
AA265464
mx91c06.r1 Soares mouse NML Mus musculus cDNA clone IMAGE:693706
Mus musculus
38,655
20-Mar.-1997







5′, mRNA sequence.




GB_GSS8:AQ006950
480
AQ006950
CIT-HSP-2294E14.TR CIT-HSP Homo sapiens genomic clone 2294E14,
Homo sapiens
36,074
27-Jun.-1998







genomic survey sequence.


rxa02653


rxa02687
1068
GB_BA1:CORPHEA
1088
M13774
C.glutamicum pheA gene encoding prephenate dehydratase, complete cds.
Corynebacterium glutamicum
99,715
26-Apr.-1993




GB_PAT:E04483
948
E04483
DNA encoding prephenate dehydratase.
Corynebacterium glutamicum
98,523
29-Sep.-1997




GB_PAT:E06110
948
E06110
DNA encoding prephenate dehydratase.
Corynebacterium glutamicum
98,523
29-Sep.-1997


rxa02717
1005
GB_PL1:HVCH4H
59748
Y14573
Hordeum vulgare DNA for chromosome 4H.
Hordeum vulgare
36,593
25-Mar.-1999




GB_PR2:HS310H5
29718
Z69705
Human DNA sequence from cosmid 310H5 from a contig from the tip of the
Homo sapiens
36,089
22-Nov.-1999







short arm of chromosome 16, spanning 2Mb of 16p13.3. Contains EST and







CpG island.




GB_PR3:AC004754
39188
AC004754
Homo sapiens chromosome 16, cosmid clone RT286 (LANL), complete
Homo sapiens
36,089
28-May-1998







sequence.


rxa02754
1461
GB_HTG2:AC008223
130212
AC008223
Drosophila melanogaster chromosome 3 clone BACR16I18 (D815) RPCI-98
Drosophila melanogaster
32,757
2-Aug.-1999







16.I.18 map 95A-1995A strain y: cn bw sp, *** SEQUENCING IN PROGRESS***,







101 unordered pieces.




GB_HTG2:AC008223
130212
AC008223
Drosophila melanogaster chromosome 3 clone BACR16I18 (D815) RPCI-98
Drosophila melanogaster
32,757
2-Aug.-1999







16.I.18 map 95A-95A strain y; cn bw sp, *** SEQUENCING IN PROGRESS







***, 101 unordered pieces.




GB_BA1:MTCY71
42729
Z92771
Mycobacterium tuberculosis H37Rv complete genome: segment 141/162.
Mycobacterium tuberculosis
37,838
10-Feb.-1999


rxa02758
1422
GB_HTG5:AC011678
171967
AC011678
Homo sapiens clone 14_B_7, *** SEQUENCING IN PROGRESS ***, 20
Homo sapiens
35,331
5-Nov.-1999







unordered pieces.




GB_HTG5:AC011678
171967
AC011678
Homo sapiens clone 14_B_7, *** SEQUENCING IN PROGRESS ***, 20
Homo sapiens
33,807
5-Nov.-1999







unordered pieces.




GB_BA2:AF084070
23183
AF064070
Burkholderia pseudomallei putative dihydroorotase (pyrC) gene, partial cds;
Burkholderia pseudomallei
36,929
20-Jan.-1999







putative 1-acyl-sn-glycerol-3-phosphate acyltransferase (pIsC), putative







diadenosine tetraphosphatase (apaH), complete cds; type II 0-antigen







biosynthesis gene cluster, complete sequence; putative undecaprenyl







phosphate N-acetylglucosaminyltransferase, and putative UDP-glucose 4-







epimerase genes, complete cds; and putative galactosyl transferase gene,







partial cds.


rxa02771
678
GB_BA2:AF038651
4077
AF038651
Corynebacterium glutamicum dipeptide-binding protein (dciAE) gene, partial
Corynebacterium glutamicum
99,852
14-Sep.-1998







cds; adenine phosphonbosyltransferase (apt) and GTP pyrophosphokinase







(rel) genes, complete cds; and unknown gene.




GB_IN1:CELT19B4
37121
U80438
Caenorhabditis elegans cosmid T19B4.
Caenorhabditis elegans
43,836
04-Dec.-1996




GB_EST3G:AV193572
360
AV193572
AV193572 Yuji Kohara unpublished cDNA:Strain N2 hermaphrodite embryo
Caenorhabditis elegans
48,588
22-Jul.-1999







Caenorhabditis elegans cDNA clone yk618h8 5′. mRNA sequence.


rxa02772
1158
GB_BA2:AF038651
4077
AF038651
Corynebacterium glutamicum dipeptide-binding protein (dciAE) gene, partial
Corynebacterium glutamicum
99,914
14-Sep.-1998







cds; adenine phosphonbosyltransferase (apt) and GTP pyrophosphokinase







(rel) genes, complete cds; and unknown gene.




GB_BA1:MTCY227
35946
Z77724
Mycobacterium tuberculosis H37Rv complete genome: segment 114/162.
Mycobacterium tuberculosis
38,339
17-Jun.-1998




GB_BA1:U00011
40429
U00011
Mycobacterium leprae cosmid B1177.
Mycobacterium leprae
38,996
01-Mar.-1994


rxa02790
1266
GB_BA1:MTCY159
33818
Z83863
Mycobacterium tuberculosis H37Rv complete genome; segment 111/162.
Mycobacterium tuberculosis
37,640
17-Jun.-1998




GB_PR4.AC006581
172931
AC006581
Homo sapiens 12p21 BAC RPCI11-259O18 (Roswell Park Cancer Institute
Homo sapiens
37,906
3-Jun.-1999







Human BAC Library) complete sequence.




GB_PR4:AC006581
172931
AC006581
Homo sapiens 12p21 BAC RPCI11-259O18 (Roswell Park Cancer Institute
Homo sapiens
35,280
3-Jun.-1999







Human BAC Library) complete sequence.


rxa02791
951
GB_BA1:MTCY159
33818
Z83863
Mycobacterium tuberculosis H37Rv complete genome; segment 111/162.
Mycobacterium tuberculosis
39,765
17-Jun.-1998




GB_OV:CHKCEK2
3694
M35195
Chicken tyrosine kinase (cek2) mRNA, complete cds.
Gallus gallus
38,937
28-Apr.-1993




GB_BA1:MSASDASK
5037
Z17372
M.smegmatis asd, ask-alpha, and ask-beta genes.
Mycobacterium smegmatis
38,495
9-Aug.-1994


rxa02802
1194
GB_EST24:AI223401
169
AI223401
qg48g01.x1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE:1838448
Homo sapiens
40,828
27-Oct.-1998







3′ similar to WP:C25D7.8 CE08394;, mRNA sequence.




GB_EST24:AI223401
169
AI223401
qg48g01.x1 Soares_testis_NHT Homo sapiens cDNA clone IMAGE:1838448
Homo sapiens
40,828
27-Oct.-1998







3′ similar to WP:C25D7.8 CE08394;, mRNA sequence.


rxa02814
494
GB_BA1:MTCY7D11
22070
Z95120
Mycobacterium tuberculosis H37Rv complete genome; segment 138/162.
Mycobacterium tuberculosis
58,418
17-Jun.-1998




GB_BA1:MTCY7D11
22070
Z95120
Mycobacterium tuberculosis H37Rv complete genome; segment 138/162.
Mycobacterium tuberculosis
40,496
17-Jun.-1998




GB_PR1:HSAJ2962
778
AJ002962
Homo sapiens mRNA for hB-FABP.
Homo sapiens
39,826
8-Jan.-1998


rxa02843
608
GB_BA1:CGAJ4934
1160
AJ004934
Corynebacterium glutamicum dapD gene, complete CDS.
Corynebacterium glutamicum
100,000
17-Jun.-1998




GB_BA1:MTCI364
29540
Z93777
Mycobacterium tuberculosis H37Rv complete genome; segment 52/162.
Mycobacterium tuberculosis
37,710
17-Jun.-1998




GB_BA1:MLU15180
38675
U15180
Mycobacterium leprae cosmid B1756.
Mycobacterium leprae
39,626
09-Mar.-1995


rxs03205
963
GB_BA1:BLSIGBGN
2906
Z49824
B. lactofermentum orf1 gene and sigB gene.
Corynebacterium glutamicum
98,854
25-Apr.-1996




GB_EST21:AA980237
377
M980237
ua32a12.r1 Soares_mammary_gland_NbMMG Mus musculus cDNA clone
Mus musculus
41,489
27-May-1998







IMAGE:1348414 5′ similar to TR:Q61025 061025 HYPOTHETICAL 15.2 KD







PROTEIN. ;, mRNA sequence.




GB_EST23:AI158316
371
AI158316
ud27c05.r1 Soares_thymus_2NbMT Mus musculus cDNA clone
Mus musculus
38,005
30-Sep.-1998







IMAGE:14471 12 5′ , mRNA sequence.


rxs03223
1237
GB_IN1:LMFL2743
38368
AL031910
Leishmania major Friedlin chromosome 4 cosmid L2743.
Leishmania major
39,869
15-Dec.-1999




GB_PR3:HSDJ61B2
119666
AL096710
Human DNA sequence from clone RP1-61B2 on chromosome 6p11.2-12.3
Homo sapiens
34,930
17-Dec.-1999







Contains isoforms 1 and 3 of BPAG1 (bullous pemphigoid antigen 1







(230/240 kD), an exon of a gene similar to murine MACF cytoskeletal protein,







STSs and GSSs, complete sequence.




GB_PR3:HSDJ61B2
119666
AL096710
Human DNA sequence from clone RP1-61B2 on chromosome 6p11.2-12.3
Homo sapiens
34,634
17-Dec.-1999







Contains isoforms 1 and 3 of BPAG1 (bullous pemphigoid antigen 1







(230/240 kD), an exon of a gene similar to murine MACF cytoskeletal protein,







STSs and GSSs, complete sequence.

## Claims

1. An isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, or a full complement thereof.

2. An isolated nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a full complement thereof.

3. An isolated nucleic acid molecule comprising a nucleotide sequence which is at least 90% identical to the entire nucleotide sequence of SEQ ID NO:1, wherein the nucleic acid molecule encodes a polypeptide having an O-acetylhomoserine sulfhydrylase activity, or a full complement thereof.

4. An isolated nucleic acid molecule consisting of a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1, or a full complement thereof.

5. An isolated nucleic acid molecule comprising a fragment of at least 25 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1, wherein the nucleotide sequence of SEQ ID NO:1 encodes a polypeptide having an O-acetylhomoserine sulfhydrylase activity, or a full complement thereof.

6. An isolated nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence which is at least 90% identical to the entire amino acid sequence of SEQ ID NO:2, wherein the polypeptide has an O-acetylhomoserine sulfhydrylase activity, or a full complement thereof.

7. An isolated nucleic acid molecule which hybridizes to the complement of the nucleotide sequence of SEQ ID NO:1 in 6× sodium chloride/sodium citrate (SSG) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C., wherein said nucleic acid molecule encodes a polypeptide having an O-acetylhomoserine sulfhydrylase activity, or a full complement thereof.

8. An isolated nucleic acid molecule comprising the nucleic acid molecule of any one of claims 1, 2, 3, 4, 5, 6, and 7 and a nucleotide sequence encoding a heterologous polypeptide.

9. A vector comprising the nucleic acid molecule of any one of claims 1, 2, 3, 4, 5, 6, and 7.

10. The vector of claim 9, which is an expression vector.

11. An isolated host cell transfected with the expression vector of claim 10.

12. The host cell of claim 11, wherein said cell is a microorganism.

13. The host cell of claim 12, wherein said cell belongs to the genus Corynebacterium or Brevibacterium.

14. A method of producing a polypeptide comprising culturing the host cell of claim 11 in an appropriate culture medium to, thereby, produce the polypeptide.

15. A method for producing an amino acid, comprising culturing the host cell of claim 11, such that the amino acid is produced.

16. The method of claim 15, wherein said cell is cultured in the presence of a sulfur source.

17. The method of claim 15, wherein said method further comprises the step of recovering the amino acid.

18. The method of claim 15, wherein said amino acid is methionine or lysine.

19. The method of claim 15, wherein said cell belongs to the genus Corynebacterium or Brevibacterium.

20. The method of claim 15, wherein said cell is selected from the group consisting of: Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium lilium, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium acetophilum, Corynebacterium ammoniagenes, Corynebacterium fujiokense, Corynebacterium nitrilophilus, Brevibacterium ammoniagenes, Brevibacterium butanicum, Brevibacterium divaricatum, Brevibacterium flavum, Brevibacterium healii, Brevibacterium ketoglutamicum, Brevibacterium ketosoreductum, Brevibacterium lactofermentum, Brevibacterium linens, Brevibacterium paraffinolyticum.

21. A method for producing an amino acid, comprising culturing a cell whose genomic DNA has been altered by the inclusion of the nucleic acid molecule of any one of claims 1, 2, 3, 4, 5, 6 and 7.