# Patent

## Title
Means and methods for regulating gene expression

## Abstract

The invention relates to means and methods for regulating gene expression and production of proteinaceous molecules. The invention provides a method for producing a proteinaceous molecule in a cell comprising selecting a cell for its suitability for producing the proteinaceous molecule, providing a nucleic acid encoding the proteinaceous molecule with a nucleic acid comprising a STAR (STabilizing Anti-Repression) sequence, expressing the resulting nucleic acid in the cell and collecting the proteinaceous molecule. Providing at least one STAR sequence to a nucleic acid encoding a proteinaceous molecule will enhance production (yield) of the proteinaceous molecule by a host cell, increase the proportion of host cells with acceptable expression levels, and/or increase stability of a gene expression level.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 11/012,546, filed Dec. 14, 2004, now U.S. Pat. No. 7,267,965, which application is a continuation of International Patent Application No. PCT/NL03/00410, filed May 30, 2003, published in English as International Patent Publication No. WO 03/106674 on Dec. 24, 2003, which claims the benefit under 35 U.S.C. §119 of European Patent Application No. EP 02077344.6, filed Jun. 14, 2002, the entirety of each of which are hereby incorporated by reference.


STATEMENT ACCORDING TO 37 C.F.R §1.52(e)(5)—SEQUENCE LISTING SUBMITTED ON COMPACT DISC
Pursuant to 37 C.F.R. §1.52(e)(1)(iii), a compact disc containing an electronic version of the Sequence Listing has been submitted concomitant with this application, the contents of which are hereby incorporated by reference. The compact disc contains the file “P60555PC00 final.txt” which is 496 KB, and created on Dec. 14, 2004. A second compact disk is submitted and is an identical copy of the first compact disc (labeled, “copy 1” and “copy 2,” respectively).
TECHNICAL FIELD
The invention relates to the fields of medicine and cellular biology. More specifically, the invention relates to means and methods for regulating gene expression, and production of proteinaceous molecules.
BACKGROUND OF THE INVENTION
Polypeptide production at industrial scale currently provides many biologically active polypeptides for a variety of uses, including diagnostic and therapeutic pharmaceuticals, industrial catalysts and nourishment. Polypeptides are produced in a variety of host systems, including transgenic animals and plants, microbes, and cultured mammalian cells. In most cases, the host system is modified by recombinant DNA techniques, for instance resulting in introduction into the host cell of a transgene which encodes a polypeptide of interest. Such a transgene typically includes elements that influence the transcription, translation, and/or processing of the transgene's polypeptide coding sequence. A recombinant host is then identified and isolated which has a suitable yield of a polypeptide of interest, and the cell population of this recombinant host is increased to an extent that it can produce the required amount of polypeptide.
The choice of the host system depends on a number of factors including: (1) the nature and intended use of a polypeptide, and (2) the cost of production. For production of biopharmaceuticals, e.g., therapeutic proteins such as hormones, cytokines, and antibodies, the host system of choice is usually cultured mammalian cells. Considerations with respect to product use and production cost with host cells will be discussed below.
(1) For in vivo therapeutic use, a therapeutic protein must not only have the correct biological activity to alter the course of a disease. It must also do no harm. Most therapeutic proteins are exported from the cell by the secretory pathway. Secreted proteins are modified by a series of post-translational events, including glycosylation, disulfide bond formation, and proteolytic processing. The post-translational modification systems vary among different species and cell types in their detailed mechanisms of action. As a result, the same polypeptide chain can be detectably different when it is produced in different host cells. These differences can be analytical, such as differences in physical properties such as molecular mass, net electrical charge, carbohydrate composition, or structure. The differences can also be functional, affecting for instance the biological activity of the protein itself (catalytic activity, ligand binding characteristics, etc.), and/or its in vivo properties (immunogenicity, biological half life, biodistribution, etc.). Functional differences can, therefore, affect both function and possible side effect(s) of a therapeutic protein. Host cell lines that produce proteins with low efficacy are not suitable for commercial exploitation. Furthermore, host cells which produce modified protein that involves significant side effects in a patient should not be used. These factors are becoming increasingly important considerations during selection of a host cell line for production of a therapeutic protein.
(2) Therapeutic protein production in host cells is an intrinsically costly process. Current methods for industrial production of such proteins often perform poorly, resulting in products that are prohibitively expensive. Poor performance can be due to limitations of protein expression systems and host cell lines currently in use. These limitations mostly have a few specific causes, including (a) failure to identify and isolate recombinant host cell lines that have suitable productivity of proteins (poor predictability), (b) silencing, during the industrial production cycle, of the transgenes that encode proteins (poor stability), and (c) low or incorrect post-translational processing and secretory capacity of the host cell line. These limitations will be considered separately below.
(a) Conventional methods furnish only low frequencies of recombinant host cells that have suitable yields of proteins. Identifying and isolating these rare recombinant cell lines is a laborious and expensive process. The poor predictability of conventional methods means that often a recombinant host cell line is selected for production that has sub-optimal productivity characteristics, simply because a superior recombinant cell line was not identified and isolated during the selection process.
(b) Transgenes are often subject to silencing during cultivation of recombinant host cells. Silencing acts by suppressing transcription of a transgene. Detailed mechanisms of silencing are still not known, and different conventional methods are prone to different kinds of silencing phenomena. With one phenomenon, an individual transgene is silenced by formation of transcriptionally refractory heterochromatin at the transgenic locus. Heterochromatin formation is influenced by the position of genomic integration of a transgene (“position effects” (Boivin and Dura, 1998)). Transgene integration occurs more or less at random. Since most of the genome is heterochromatin, most transgene loci are prone to silencing due to position effects.
A second transgene-silencing phenomenon can occur when two or more copies of a transgene are integrated into a genome during construction of a recombinant cell line. Formation of tandem transgene repeats often occurs during the initial integration step. Furthermore, in order to increase product yield, many recombinant host cell lines are engineered after the integration step to amplify the copy number of a transgene, which also results in tandem transgene repeats (Kaufman, 1990). Tandem repeats and other configurations of multiple transgene copies are particularly prone to silencing (“repeat-induced gene silencing” (Garrick et al., 1998)).
In case that a genome contains multiple copies of a transgene, the yield can also decline via another phenomenon than transcriptional silencing. The number of copies of the transgene can decline during cultivation of a recombinant host cell line. The productivity of such cell lines at the time of selection for use is correlated with a transgene copy number, and consequently as copies of a transgene are lost, the yield declines (Kaufman, 1990).
(c) Different cell types in a mammalian organism have different capacities for post-translational modification and secretion of proteins. The functions of some cell types include production of large quantities of secreted proteins; examples include lymphocytes (producing immunoglobulins), hepatocytes (producing serum proteins), and fibroblasts (producing extracellular matrix proteins). These cell types are favorable sources for deriving host cell lines for production of secreted heterologous proteins. More favorable is the use of a cell line whose progenitor organismal cell type secretes a protein or class of proteins of interest. For example, it is particularly favorable to express recombinant monoclonal antibodies in lymphocytes (or host cells derived from lymphocytes), erythropoietin in hepatocytes (or host cells derived from hepatocytes), and blood clotting factors (e.g., Factor VIII and van Willebrand's factor) in endothelial cells (or host cells derived from endothelial cells).
The use of specific cell types (or cell lines derived therefrom) for production of their affiliated proteins is favorable because such specific cell types will carry out proper post-translational modifications of produced proteins. However, specific cell types often do not have high secretory capacities. For example, cells of the central nervous system, such as neurons, have low intrinsic protein secretion capacities. These cells do secrete proteins, however, including neurotrophins. Neurotrophins regulate the fate and shape of neuronal cells during fetal and juvenile development. Moreover, they influence patterns of neuronal degeneration and regeneration in adults (Bibel and Barde, 2000). Production of neurotrophins for therapeutic applications has considerable biopharmaceutical value (e.g., AxokineTM, recombinant ciliary neurotrophic factor from Regeneron). In order to produce heterologous neurotrophins with post-translational modifications (and hence functional properties) that match the naturally-occurring proteins, expression in host cells derived from the central nervous system is favorable. However, production of polypeptides such as neurotrophins in host cell lines such as those derived from neural tissue is inefficient using conventional methods. The predictability of identifying high-expressor isolates from these types of cell lines is often poor; the yield of proteins from such cell lines is generally low, and production levels are characteristically unstable.
Another drawback to a use of specific host cells for production of affiliated proteins is that it is usually difficult to isolate cell lines with favorable biotechnological characteristics. These characteristics for instance include the mode and rate of growth, and the ease of introduction of a transgene. Consequently, various general host cell lines have been established. Examples of these include CHO cells from Chinese hamster ovary (ATCC (American Type Culture Collection) CCL-61), BHK cells from baby hamster kidney (ATCC CCL-10), and Vero cells from African green monkey kidney (ATCC CCL-81). These “general purpose” host cell lines are widely used for production of a number of heterologous proteins. A disadvantage of general purpose cell lines is that the post-translational modifications of heterologous proteins produced by them often differ from the post-translational modifications of the naturally occurring proteins. These differences can have functional consequences resulting in side effects, as discussed above.
Table 1 lists a number of proteins that are currently in use or under development for biopharmaceutical applications. It also lists the tissue or cell type in which each protein is normally produced in the human body. These 24 proteins (or protein classes) come from a wide range of cells and tissue, ranging from highly secretory cells (hepatocytes, endothelial cells) to cells with low secretory capacity (e.g., neural tissue). Currently, neither general-purpose host cells nor specific host cells have qualities that enable optimal expression of the broad spectrum of biopharmaceutically important secreted proteins.
Hence, protein production by conventional host cell lines involves a lot of disadvantages and complications, for instance with respect to yield and post-translational modifications. There is a need in the art for improved protein production in recombinant host cell lines.
SUMMARY OF THE INVENTION
The present invention provides a method for producing a proteinaceous molecule in a cell comprising selecting a cell for its suitability for producing the proteinaceous molecule, providing a nucleic acid encoding the proteinaceous molecule with a nucleic acid comprising a STAR (STabilizing Anti-Repression) sequence, expressing the resulting nucleic acid in the cell and collecting the proteinaceous molecule.
The STAR sequence has to be operably linked to the nucleic acid encoding the proteinaceous molecule in order to be effective. In one embodiment of the invention, one STAR element is used. Preferably however, more than one STAR element is used. In a particularly preferred embodiment, the nucleic acid encoding the proteinaceous molecule is provided with two STAR sequences; one STAR sequence at the 5′ side of the coding sequence of the nucleic acid and one STAR sequence at the 3′ side of the coding sequence of the nucleic acid.
Description of STAR Elements
New transcription regulatory elements were disclosed by the present inventors, which are named STAR sequences (See EP 01202581.3). STAR sequences are nucleic acid sequences that comprise a capacity to influence transcription of genes in cis. Typically, although not necessarily, the STAR sequences do not code by themselves for a functional protein.
A STAR sequence has a gene transcription modulating quality in at least one type of cell. A STAR sequence is capable of enhancing gene transcription resulting in a higher yield, increasing the proportion of transgene-comprising host cells with acceptable expression levels, and/or increasing stability of transgenes in recombinant cell lines.
In EP 01202581.3 a method of detecting, and optionally selecting, a STAR sequence is provided, comprising providing a transcription system with a variety of a fragment-comprising vectors, the vectors comprising i) an element with a gene-transcription repressing quality, and ii) a promoter directing transcription of a reporter gene, the method further comprising performing a selection step in the transcription system in order to identify the STAR sequence. In a preferred embodiment, the fragments are located between i) the element with a gene-transcription repressing quality, and ii) the promoter directing transcription of the reporter gene. RNA polymerase initiates the transcription process after binding to a specific sequence, called the promoter, that signals where RNA synthesis should begin. A STAR sequence can enhance transcription from the promoter in cis, in a given cell type and/or a given promoter.
Methods disclosed in EP 01202581.3 have been used to isolate STAR elements from the human genome. Isolated human STAR elements have been placed in DNA vectors so as to flank transgene expression units, and the vectors have subsequently been integrated into host cell genomes. Transgene expression in these recombinant host cells has been compared to expression in similar host cells in which the expression units are not flanked by STAR elements. The results show that STAR elements have at least one of three consequences for production of (heterologous) proteinaceous molecule (also referred to as (heterologous) protein): (1) they increase the predictability of identifying host cell lines that express a proteinaceous molecule at industrially acceptable levels; (2) they result in host cell lines with increased protein yields; and/or (3) they result in host cell lines that exhibit more stable protein production during prolonged cultivation. Each of these attributes is discussed in more detail below:
(1) Increased predictability: Integration of transgene expression units can occur at random positions throughout the host cell genome. However, much of the genome is transcriptionally silent heterochromatin. When the expression units include STAR elements flanking the transgene, the position of integration has a reduced effect on expression. The STAR elements impair the ability of adjacent heterochromatin to silence the transgene. Consequently, the proportion of transgene-containing host cells with acceptable expression levels is increased.
(2) Yield: The levels of protein expression in primary populations of recombinant host cells, directly after transgene integration, have been surveyed. The expression level of individuals in the populations varies. However, when the transgenes are protected by STAR elements, the variability is reduced. This reduced variability is most conspicuous in that fewer clones are recovered that have low levels of expression. Furthermore, the populations with STAR elements commonly have individuals with strikingly high expression. These high-yielding individuals are favorable for production of proteinaceous molecules.
(3) Increased stability: STAR elements increase the stability of transgenes in recombinant host cell lines by ensuring that the transgenes are not transcriptionally silenced during prolonged cultivation. Comparative trials show that, under conditions in which transgenes that are not protected by STAR elements are progressively silenced (5 to 25 passages in cultivation), STAR element-protected transgenes continue to be expressed at high levels. This is an advantage during industrial production of proteinaceous molecules, during which cell cultivation continues for prolonged periods, from a few weeks to many months.
Hence, a STAR sequence can enhance expression of a heterologous proteinaceous molecule. In addition, a STAR sequence can enhance expression of a naturally produced proteinaceous molecule.
Transcription can be influenced through a direct effect of the STAR sequence (or the protein(s) binding to it) on the transcription of a particular promoter. Transcription can however, also be influenced by an indirect effect, for instance because the STAR sequence affects the function of one or more other regulatory elements. A STAR sequence can also comprise a stable gene transcription quality. Frequently, expression levels drop dramatically with increasing numbers of cell divisions. With the methods disclosed in EP 01202581.3 it is possible to detect and optionally select a DNA sequence that is capable of at least in part preventing the dramatic drop in transcription levels with increasing numbers of cell divisions. Strikingly, fragments comprising a STAR sequence can be detected and optionally selected with a method EP 01202581.3, in spite of the fact that the method does not necessarily measure long term stability of transcription.
A STAR sequence is suitable for enhancing the level of transcription of a gene of interest in a host cell. If, together with a gene of interest, a STAR sequence is also introduced into host cells, more clones can be detected that express more than a certain amount of the gene of interest. As used herein, such host cells are termed “host cells with acceptable expression levels.”
Furthermore, if, together with a gene of interest, a STAR sequence is also introduced into host cells, a higher yield of produced proteinaceous molecules can be obtained, while gene expression level is also more stable than in the absence of such STAR sequences. Preferably, a STAR sequence derived from a plant and/or vertebrate is used. More preferably a human STAR sequence is used.
Sequences comprising a STAR sequence can be found by using a functional assay, as described above. However, once a collection of such sequences has been identified, bioinformatics can be used to find other STAR sequences. Several methods are available in the art to extract sequence identifiers from a family of DNA sequences sharing a certain common feature. Such sequence identifiers can subsequently be used to identify sequences that share one or more identifiers. Sequences sharing such one or more identifiers are likely to be a member of the same family of sequences, i.e., are likely to share the common feature of the family. By the present inventors a large number of sequences comprising STAR activity (so-called STAR sequences) were used to obtain sequence identifiers (patterns) which are characteristic for sequences comprising STAR activity. These patterns can be used to determine whether a test sequence is likely to contain STAR activity. A method for detecting the presence of a STAR sequence within a nucleic acid sequence of about 50-5000 base pairs is thus provided, comprising determining the frequency of occurrence in the sequence of at least one sequence pattern and determining that the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least one sequence comprising a STAR sequence. In principle any method is suited for determining whether a sequence pattern is representative of a STAR sequence. Many different methods are available in the art. Preferably, the step of determining that the occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least one sequence comprising a STAR sequence comprises, determining that the frequency of occurrence of at least one sequence pattern significantly differs between at least one STAR sequence and at least one control sequence. In principle any significant difference is discriminative for the presence of a STAR sequence. However, in a particularly preferred embodiment, the frequency of occurrence of at least one sequence pattern is significantly higher in at least one sequence comprising a STAR sequence compared to at least one control sequence.
A considerable number of sequences comprising a STAR sequence have been identified by the present inventors. It is possible to use these sequences to test how efficient a pattern is in discriminating between a control sequence and a sequence comprising a STAR sequence. Using so-called discriminant analysis it is possible to determine on the basis of any set of STAR sequences in a species, the most optimal discriminative sequence patterns or combination thereof. Thus, preferably, at least one of the patterns is selected on the basis of optimal discrimination between at least one sequence comprising a STAR sequence and a control sequence.
In a preferred embodiment, the frequency of occurrence of a sequence pattern in a test nucleic acid is compared with the frequency of occurrence in a sequence known to contain a STAR sequence. In this case, a pattern is considered representative for a sequence comprising a STAR sequence if the frequencies of occurrence are similar. In a preferred embodiment, another criterion is used. The frequency of occurrence of a pattern in a sequence comprising a STAR sequence is compared to the frequency of occurrence of the pattern in a control sequence. By comparing the two frequencies it is possible to determine for each pattern thus analyzed, whether the frequency in the sequence comprising the STAR sequence is significantly different from the frequency in the control sequence. In this embodiment, a sequence pattern is considered to be representative of a sequence comprising a STAR sequence, if the frequency of occurrence of the pattern in at least one sequence comprising a STAR sequence is significantly different from the frequency of occurrence of the same pattern in a control sequence. By using larger numbers of sequences comprising a STAR sequence the number of patterns for which a statistical difference can be established increases, thus enlarging the number of patterns for which the frequency of occurrence is representative for a sequence comprising a STAR sequence. Preferably, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least two sequences comprising a STAR sequence; more preferably, in at least five sequences comprising a STAR sequence; and, even more preferably, in at least ten sequences comprising a STAR sequence. More preferably, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least 20 sequences comprising a STAR sequence. In a particularly preferred embodiment, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least 50 sequences comprising a STAR.
The patterns that are indicative for a sequence comprising a STAR sequence are also dependent on the type of control nucleic acid used. The type of control sequence used is preferably selected on the basis of the sequence in which the presence of a STAR sequence is to be detected. In a preferred embodiment, the control sequence comprises a random sequence comprising a similar AT/CG content as at least one sequence comprising a STAR sequence. In another preferred embodiment, the control sequence is derived from the same species as the sequence comprising the STAR sequence. For instance, if a test sequence is scrutinized for the presence of a STAR sequence, active in a plant cell, then preferably the control sequence is also derived from a plant cell. Similarly, for testing for STAR activity in a human cell, the control nucleic acid is preferably also derived from a human genome. In a preferred embodiment, the control sequence comprises between 50% and 150% of the bases of at least one sequence comprising a STAR sequence. In a particularly preferred embodiment, the control sequence comprises between 90% and 110% of the bases of at least one sequence comprising a STAR sequence. More preferably, between 95% and 105%.
A pattern can comprise any number of bases larger than two. Preferably, at least one sequence pattern comprises at least five, more preferably at least six, bases. In another embodiment, at least one sequence pattern comprises at least eight bases. In a preferred embodiment, the at least one sequence pattern comprises a pattern listed in Table 6 and/or Table 7. A pattern may consist of a consecutive list of bases. However, the pattern may also comprise bases that are interrupted one or more times by a number of bases that are not or only partly discriminative. A partly discriminative base is, for instance, indicated as a purine.
Preferably, the presence of STAR activity is verified using a functional assay. Several methods are presented herein to determine whether a sequence comprises STAR activity. STAR activity is confirmed if the sequence is capable of performing at least one of the following functions: (i) at least in part inhibiting the effect of sequence comprising a gene transcription repressing element of the invention, (ii) at least in part blocking chromatin-associated repression, (iii) at least in part blocking activity of an enhancer, (iv) conferring upon an operably linked nucleic acid encoding a transcription unit compared to the same nucleic acid alone, (iv-a) a higher predictability of transcription, (iv-b) a higher transcription, and/or (iv-c) a higher stability of transcription over time.
The large number of sequences comprising STAR activity identified by the present inventors open up a wide variety of possibilities to generate and identify sequences comprising the same activity in kind not necessarily in amount. For instance, it is well within the reach of a skilled person to alter the sequences identified in the present invention and test the altered sequence for STAR activity. Such altered sequences are, therefore, also part of the present invention. Alteration can include deletion, insertion and mutation of one or more bases in the sequences.
Sequences comprising STAR activity were identified in stretches of 400 bases. However, it is expected that not all of these 400 bases are required to retain STAR activity. Methods to delimit the sequences that confer a certain property to a fragment of between 400 and 5000 bases are well known. The minimal sequence length of a fragment comprising STAR activity is estimated to be about 50 bases.
Table 6 (SEQ ID NOS:177-342) and Table 7 (SEQ ID NOS:343-1072) list patterns of six bases that have been found to be over represented in nucleic acid molecules comprising STAR activity. This over representation is considered to be representative for a STAR sequence. The tables were generated for a family of 65 STAR sequences (SEQ ID NOS:1-65). Similar tables can be generated starting from a different set of STAR sequences, or from a smaller or larger set of STAR sequences. A pattern is representative for a STAR sequence if it is over represented in the STAR sequence compared to a sequence not comprising a STAR element. This can be a random sequence. However, to exclude a non relevant bias, the sequence comprising a STAR sequence is preferably compared to a genome or a significant part thereof. Preferably, a genome of a vertebrate or plant, more preferably, a human genome. A significant part of a genome is, for instance, a chromosome. Preferably the sequence comprising a STAR sequence and the control sequence are derived from nucleic acid of the same species.
The more STAR sequences are used for the determination of the frequency of occurrence of sequence patterns, the more representative for STARs the patterns are that are over- or under-represented. Considering that many of the functional features that can be expressed by nucleic acids are mediated by proteinaceous molecules binding to them, it is preferred that the representative pattern is over-represented in the STAR sequences. Such over-represented pattern can be part of a binding site for such a proteinaceous molecule. Preferably, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least two sequences comprising a STAR sequence; more preferably, in at least five sequences comprising a STAR sequence; and, even more preferably, in at least ten sequences comprising a STAR sequence. More preferably, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least 20 sequences comprising a STAR sequence. In a particularly preferred embodiment, the frequency of occurrence is representative of the frequency of occurrence of at least one sequence pattern in at least 50 sequences comprising a STAR. Preferably, the sequences comprising a STAR sequence comprises at least one of the sequences depicted in the sequences comprising STAR1-STAR65 (SEQ ID NOS:1-65), sequences comprising STAR66 and testing set (SEQ ID NOS:66-84), and sequences comprising Arabidopsis STAR A1-A35 (SEQ ID NOS:85-119) (hereinafter SEQ ID NOS:1-119).
STAR activity is a feature shared by the sequences listed in SEQ ID NOS:1-119. However, this does not mean that they must all share the same identifier sequence. It is very well possible that different identifiers exist. Identifiers may confer this common feature onto a fragment containing it, though this is not necessarily so.
By using more sequences comprising STAR activity for determining the frequency of occurrence of a sequence pattern or patterns, it is possible to select patterns that are more often than others present or absent in such a STAR sequence. In this way it is possible to find patterns that are very frequently over- or under-represented in STAR sequences. Frequently, over- or under-represented patterns are more likely to identify candidate STAR sequences in test sets. Another way of using a set of over- or under-represented patterns is to determine which pattern or combination of patterns is best suited to identify a STAR in a sequence. Using so-called discriminative statistics, we have identified a set of patterns that performs best in identifying a sequence comprising a STAR element. In a preferred embodiment, at least one of the sequence patterns for detecting a STAR sequence comprises a sequence pattern GGACCC (SEQ ID NO:464), CCCTGC (SEQ ID NO:816), AAGCCC (SEQ ID NO:270), CCCCCA (SEQ ID NO:298) and/or AGCACC (SEQ ID NO:336). In another embodiment, at least one of the sequence patterns for detecting a STAR sequence comprises a sequence pattern CCCN{16}AGC (SEQ ID NO:415), GGCN{9}GAC (SEQ ID NO:536), CACN{13}AGG (SEQ ID NO:761), and/or CTGN{4}GCC (SEQ ID NO:839).
A list of STAR sequences can also be used to determine one or more consensus sequences therein. The invention, therefore, also provides a consensus sequence for a STAR element. This consensus sequence can of course be used to identify candidate STAR elements in a test sequence.
Moreover, once a sequence comprising a STAR element has been identified in a vertebrate it can be used by means of sequence homology to identify sequences comprising a STAR element in other species belonging to vertebrate. Preferably a mammalian STAR sequence is used to screen for STAR sequences in other mammalian species. Similarly, once a STAR sequence has been identified in a plant species, it can be used to screen for homologous sequences with similar function in other plant species. The invention in one aspect provides a STAR sequence obtainable by a method according to the invention. Further provided is a collection of STAR sequences. Preferably, the STAR sequence is a vertebrate or plant STAR sequence. More preferably, the STAR sequence is a mammalian STAR sequence or an angiosperm (monocot, such as rice or dicot, such as Arabidopsis). More preferably, the STAR sequence is a primate and/or human STAR sequence.
A list of sequences comprising STAR activity can be used to determine whether a test sequence comprises a STAR element. There are, as mentioned above, many different methods for using such a list for this purpose. In a preferred embodiment, the invention provides a method for determining whether a nucleic acid sequence of about 50-5000 base pairs comprises a STAR sequence, the method comprising: generating a first table of sequence patterns comprising the frequency of occurrence of the patterns in a collection of STAR sequences of the invention; generating a second table of the patterns comprising the frequency of occurrence of the patterns in at least one reference sequence; selecting at least one pattern of which the frequency of occurrence differs between the two tables; determining, within the nucleic acid sequence of about 50-5000 base pairs, the frequency of occurrence of at least one of the selected patterns; and determining whether the occurrence in the test nucleic acid is representative of the occurrence of the selected pattern in the collection of STAR sequences. Alternatively, determining comprises determining whether the frequency of occurrence in the test nucleic acid is representative of the frequency occurrence of the selected pattern in the collection of STAR sequences. Preferably, the method further comprises determining whether the candidate STAR comprises a gene transcription modulating quality using a method of the invention. Preferably, the collection of STARS comprises sequence as depicted in SEQ ID NOS:1-119. In another aspect, the invention provides an isolated and/or recombinant nucleic acid sequence comprising a STAR sequence obtainable by a method of the invention.
As mentioned above, a STAR sequence can exert its activity in a directional way, i.e., more to one side of the fragment containing it than to the other. Moreover, STAR activity can be amplified in amount by multiplying the number of STAR elements. The latter suggests that a STAR element may comprise one or more elements comprising STAR activity. Another way of identifying a sequence capable of conferring STAR activity on a fragment containing it comprises selecting from a vertebrate or plant sequence, a sequence comprising STAR activity and identifying whether the selected sequence and sequences flanking the selected sequence are conserved in another species. Such conserved flanking sequences are likely to be functional sequences. In one aspect, the invention, therefore, provides a method for identifying a sequence comprising a STAR element comprising selecting a sequence of about 50 to 5000 base pairs from a vertebrate or plant species comprising a STAR element and identifying whether sequences flanking the selected sequence in the species are conserved in at least one other species. The invention, therefore, further provides a method for detecting the presence of a STAR sequence within a nucleic acid sequence of about 50-5000 base pairs, comprising identifying a sequence comprising a STAR sequence in a part of a chromosome of a cell of a species and detecting significant homology between the sequence and a sequence of a chromosome of a different species. Preferably, the species comprises a plant or vertebrate species, ideally a mammalian species. The invention also provides a method for detecting the presence of a STAR element within a nucleic acid sequence of about 50-5000 base pairs of a vertebrate or plant species, comprising identifying whether a flanking sequence of the nucleic acid sequence is conserved in at least one other species.
It is important to note that methods of the invention for detecting the presence of a sequence comprising a STAR sequence using bioinformatical information are iterative in nature. The more sequences comprising a STAR sequence are identified with a method of the invention, the more patterns are found to be discriminative between a sequence comprising a STAR sequence and a control sequence. Using these newly found discriminative patterns, more sequences comprising a STAR sequence can be identified, which, in turn, enlarges the set of patterns that can discriminate and so on. This iterative aspect is an important aspect of methods provided in the present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagram illustrating the pSelect plasmid used for isolating STAR elements. The zeocin resistance gene is under control of the SV40 promoter, and is upstream of the SV40 polyadenylation site. Upstream of the SV40 promoter is a tandem array of lexA operator sites. Between the lexA operators and the SV40 promoter is a cloning site; test DNAs (e.g., size-fractionated genomic DNA) are cloned into the BamHI site. The plasmid also has the hygromycin resistance gene (hygro) for selection of transformed cells, the EBNA-1 and oriP sequences for plasmid replication in mammalian cells, and the ampicillin resistance gene (ampR) and ori sequence for propagation in Escherichia coli. 
FIG. 2 is a diagram depicting the pSDH-CSP plasmid used for testing STAR activity. The Secreted Alkaline Phosphatase (SEAP) reporter gene is under control of the CMV promoter, and the puromycin resistance selectable marker (puro) is under control of the SV40 promoter. Flanking these two genes are multiple cloning sites (MCSI and MCSII) into which STAR elements can be cloned. The plasmid also has an origin of replication (ori) and ampicillin resistance gene (ampR) for propagation in Escherichia coli. 
FIG. 3 is a graph showing that STAR6 (SEQ ID NO:6) and STAR8 (SEQ ID NO:8) improve predictability and yield of transgene expression in U-2 OS cells. Expression of luciferase from the CMV promoter by U-2 OS cells transfected with pSDH-CMV, pSDH-CMV-STAR6, or pSDH-CMV-STAR8 was determined. The STAR-containing constructs confer greater predictability and elevated yield relative to the pSDH-CMV construct alone.
FIG. 4 is a graph illustrating that STAR6 (SEQ ID NO:6), but not STAR8 (SEQ ID NO:8), improves predictability and yield of transgene expression in CHO cells. Expression of SEAP from the CMV promoter by CHO cells transfected with pSDH-CSP, pSDH-CSP-STAR6, or pSDH-CSP-STAR8 was determined. The STAR6-containing constructs confer greater predictability and elevated yield relative to the pSDH-CSP construct alone, identifying STAR6 (SEQ ID NO:6) as a promiscuous STAR element. In contrast, the STAR8-containing constructs do not consistently increase yield or predictability relative to the pSDH-CSP construct, suggesting that STAR8 (SEQ ID NO:8) is a cell line-specific STAR element.
FIG. 5 is a graph depicting that STAR6 (SEQ ID NO:6) and STAR49 (SEQ ID NO:49) improve predictability and yield of transgene expression. Expression of SEAP from the CMV promoter by CHO cells transfected with pSDH-CSP, pSDH-CSP-STAR6, or pSDH-CSP-STAR49 was determined. The STAR-containing constructs confer greater predictability and elevated yield relative to the pSDH-CSP construct alone.
FIG. 6 is a graph showing the minimal essential sequences of STAR10 (SEQ ID NO:10) and STAR27 (SEQ ID NO:27). Portions of the STAR elements were amplified by PCR: STAR10 (SEQ ID NO:10) was amplified with primers E23 (SEQ ID NO:166) and E12 (SEQ ID NO:155) to yield fragment 10A (corresponding approximately to the first 400 nucleotides of SEQ ID NO:10), E13 (SEQ ID NO:156) and E14 (SEQ ID NO:157) to yield fragment 10B (corresponding approximately to the second 400 nucleotides of SEQ ID NO:10), and E15 (SEQ ID NO:158) and E16 (SEQ ID NO:159) to yield fragment 10C (corresponding approximately to the third 400 nucleotides of SEQ ID NO:10). STAR27 (SEQ ID NO:27) was amplified with primers E17 (SEQ ID NO:160) and E18 (SEQ ID NO:161) to yield fragment 27A (corresponding approximately to the first 500 nucleotides of SEQ ID NO:27), E19 (SEQ ID NO:162) and E20 (SEQ ID NO:163) to yield fragment 27B (corresponding to the second 500 nucleotides of SEQ ID NO:27), and E21 (SEQ ID NO:164) and E22 (SEQ ID NO:165) to yield fragment 27C (corresponding approximately to the third 500 nucleotides of SEQ ID NO:27). These sub-fragments were cloned into the pSelect vector. After transfection into U-2 OS/Tet-Off/LexA-HP1 cells, the growth of the cultures in the presence of zeocin was monitored. Growth rates varied from vigorous (+++) to poor (+/−), while some cultures failed to survive zeocin treatment (−) due to absence of STAR activity in the DNA fragment tested.
FIG. 7 is a graph illustrating the STAR element function in the context of the SV40 promoter. pSDH-SV40 and pSDH-SV40-STAR6 were transfected into the human osteosarcoma U-2 OS cell line, and expression of luciferase was assayed with or without protection from gene silencing by STAR6 (SEQ ID NO:6) in puromycin-resistant clones.
FIG. 8 is a graph showing the STAR element function in the context of the Tet-Off promoter. pSDH-Tet and pSDH-Tet-STAR6 were transfected into the human osteosarcoma U-2 OS cell line, and expression of luciferase was assayed with or without protection from gene silencing by STAR6 (SEQ ID NO:6) in puromycin-resistant clones.
FIG. 9 is a schematic diagram of the orientation of STAR elements as they are cloned in the pSelect vector (panel A), as they are cloned into pSDH vectors to preserve their native orientation (panel B), and as they are cloned into pSDH vector in the opposite orientation (panel C).
FIG. 10 is a graph showing directionality of STAR66 (SEQ ID NO:66) function. The STAR66 (SEQ ID NO:66) element was cloned into pSDH-Tet in either the native (STAR66 native) or the opposite orientation (STAR66 opposite) and transfected into U-2 OS cells. Luciferase activity was assayed in puromycin resistant clones.
FIG. 11 is a southern blot showing copy number-dependence of STAR function. Southern blot of luciferase expression units in pSDH-Tet-STAR10, integrated into U-2 OS genomic DNA. Radioactive luciferase DNA probe was used to detect the amount of transgene DNA in the genome of each clone, which was then quantified with a phosphorimager.
FIG. 12 is a graph illustrating copy number-dependence of STAR function. The copy number of pSDH-Tet-STAR10 expression units in each clone was determined by phosphorimagery and compared with the activity of the luciferase reporter enzyme expressed by each clone.
FIG. 13 is a schematic diagram and graphs depicting enhancer-blocking and enhancer assays. The luciferase expression vectors used for testing STARs for enhancer-blocking and enhancer activity are shown schematically. The E-box binding site for the E47 enhancer protein is upstream of a cloning site for STAR elements. Downstream of the STAR cloning site is the luciferase gene under control of a human alkaline phosphatase minimal promoter (mp). The histograms indicate the expected outcomes for the three possible experimental situations (see text). Panel A: Enhancer-blocking assay. Panel B: Enhancer assay.
FIG. 14 is a graph showing enhancer-blocking assay. Luciferase expression from a minimal promoter is activated by the E47/E-box enhancer in the empty vector (vector). Insertion of enhancer-blockers (scs, HS4) or STAR elements (STAR elements 1, 2, 3, 6, 10, 11, 18, and 27; SEQ ID NOS:1, 2, 3, 6, 10, 11, 18 and 27, respectively) block luciferase activation by the E47/E-box enhancer.
FIG. 15 is a graph illustrating enhancer assay. Luciferase expression from a minimal promoter is activated by the E47/E-box enhancer in the empty vector (E47). Insertion of the scs and HS4 elements or various STAR elements (STARs 1, 2, 3, 6, 10, 11, 18, and 27; SEQ ID NOS:1, 2, 3, 6, 10, 11, 18 and 27, respectively) do not activate transcription of the reporter gene.
FIG. 16 illustrates STAR18 (SEQ ID NO:18) sequence conservation between mouse and human. The region of the human genome containing 497 base pair STAR18 (SEQ ID NO:18) is shown (black boxes); the element occurs between the HOXD8 and HOXD4 homeobox genes on human chromosome 2. It is aligned with a region in mouse chromosome 2 that shares 72% sequence identity. The region of human chromosome 2 immediately to the left of STAR18 (SEQ ID NO:18) is also highly conserved with mouse chromosome 2 (73% identity; gray boxes); beyond these region, the identity drops below 60%. The ability of these regions from human and mouse, either separately or in combination, to confer growth on zeocin is indicated: −, no growth; +, moderate growth; ++, vigorous growth; +++, rapid growth.
FIG. 17 is a schematic diagram of bio-informatic analysis workflow. For details, see text.
FIG. 18 is a schematic diagram showing the results of discriminant analysis on classification of the training set of 65 STAR elements. STAR elements that are correctly classified as STARs by Stepwise Linear Discriminant Analysis (LDA) are shown in a Venn diagram. The variables for LDA were selected from frequency analysis results for hexameric oligonucleotides (“oligos”) and for dyads. The diagram indicates the concordance of the two sets of variables in correctly classifying STARs.
FIG. 19 is a graph illustrating that U-2 OS/Tet-Off/lexA-HP1 cells were transfected with candidate Arabidopsis STAR elements and cultivated at low doxycycline concentrations. Total RNA was isolated and subjected to RT-PCR; the bands corresponding to the zeocin and hygromycin resistance mRNAs were detected by Southern blotting and quantified with a phosphorimager. The ratio of the zeocin to hygromycin signals is shown for transfectants containing zeocin expression units flanked by 12 different Arabidopsis STAR elements, the Drosophila scs element, or no flanking element.
FIG. 20 is a schematic diagram and graph illustrating that STAR elements improve GFP expression in CHO cells. The ppGIZ and ppGIZ-STAR7 plasmids used for testing STAR activity are shown. The expression unit comprises (from 5′ to 3′) a transgene (encoding for the GFP protein), an IRES, and a selectable marker (zeo, conferring zeocin resistance) under control of the CMV promoter. The expression unit has the SV40 transcriptional terminator at its 3′ end (t). The entire cassette with the expression unit is either flanked by STAR7 (SEQ ID NO:7) elements (STAR7-shielded) or not (Control). The constructs are transfected to CHO-K1 cells. Stable colonies are expanded and the GFP signal is determined on a XL-MCL Beckman Coulter flow cytometer. For each independent colony the mean of the GFP signal is plotted. This is taken as measure for the level of GFP expression. The results in FIG. 20 show that in CHO cells the STAR7-shielded construct confers greater predictability and elevated GFP expression relative to the ppGIZ control construct alone.
FIG. 21 is a schematic diagram and graph showing that STAR elements improve GFP expression in NSO cells. The ppGIZ and ppGIZ-STAR7 plasmids used for testing STAR activity are shown as in FIG. 20. The constructs are transfected to NSO cells. Stable colonies are expanded and the GFP signal is determined on a XL-MCL Beckman Coulter flow cytometer. For each independent colony the mean of the GFP signal is plotted. This is taken as measure for the level of GFP expression. The results in FIG. 21 show that in NSO cells the STAR7-shielded (SEQ ID NO:7) construct confers greater predictability and elevated GFP expression relative to the ppGIZ control construct alone.
FIG. 22 is a schematic diagram and graph depicting that STAR elements improve GFP expression in 293 cells. The ppGIZ and ppGIZ-STAR7 plasmids used for testing STAR activity are shown as in FIG. 20. The constructs are transfected to 293 cells. Stable colonies are expanded and the GFP signal is determined on a XL-MCL Beckman Coulter flow cytometer. For each independent colony, the mean of the GFP signal is plotted. This is taken as measure for the level of GFP expression. The results in FIG. 22 show that in 293 cells the STAR7-shielded (SEQ ID NO:7) construct confers greater predictability and elevated GFP expression relative to the ppGIZ control construct alone.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
Quality
The term “quality” in relation to a sequence refers to an activity of the sequence.
STAR and STAR Sequence
The terms “STAR,” “STAR sequence” or “STAR element,” as used herein, refer to a DNA sequence comprising one or more of the mentioned gene transcription modulating qualities.
DNA Sequence
The term “DNA sequence” as used herein, unless otherwise specified, does not refer to a listing of specific ordering of bases, but rather to a physical piece of DNA. A transcription quality with reference to a DNA sequence refers to an effect that the DNA sequence has on transcription of a gene of interest. “Quality” as used herein refers to detectable properties or attributes of a nucleic acid or protein in a transcription system.
Proteinaceous Molecule
By a “proteinaceous molecule” is meant herein a molecule comprising amino acids. At least a part of the amino acids are bound to each other to form a peptide. Preferably, the proteinaceous molecule comprises a polypeptide. In this application, the term “proteinaceous molecule” also includes “polypeptide.”
Essentially the Same Properties
By “essentially the same properties” is meant that the properties are essentially the same in kind, not necessarily in amount. For instance, if a proteinaceous molecule has essentially the same properties as a pharmaceutically active compound, the proteinaceous molecule also displays such pharmaceutical activity in kind, not necessarily in amount.
Naturally Occurring Proteinaceous Molecule of the Same Kind
By a “naturally occurring proteinaceous molecule of the same kind” is meant a proteinaceous molecule with the same primary structure, which is naturally produced in vivo, not influenced by human interference. Examples comprise an antibody produced in vivo by a lymphocyte and erythropoietin produced in vivo by a hepatocyte.
Host Cell, Host Cell Line
As used herein, the terms “host cell” and “host cell line” refer to a cell and to homogeneous populations thereof that are capable of expressing a nucleic acid encoding a proteinaceous molecule.
Recombinant Host Cell, Recombinant Host Cell Line
The terms “recombinant host cell” and “recombinant host cell line” refer to a host cell and to homogeneous populations thereof into which a nucleic acid has been introduced.
Expression
As used herein, the term “expression” refers to production of a proteinaceous molecule, encoded by a nucleic acid. The production, for instance, involves transcription of a DNA sequence, translation of the corresponding mRNA sequence, and/or posttranslational modification. In case of secreted proteins, it can also refer to the processes of transcription, translation, and/or post-translational modification (e.g., glycosylation, disulfide bond formation, etc.), followed by exocytosis. In the case of multimeric proteins, it can include assembly of the multimeric structure from the polypeptide monomers.
Silencing
The term “silencing” refers to diminution of a level of expression of a gene or genes, including transgenes, typically over time. The expression level can be diminished but still detectable, or diminished below the threshold of detection.
Enhanced Expression
As used herein, “enhanced expression” of a gene encoding a proteinaceous molecule, or enhanced production of a proteinaceous molecule, can either involve a higher yield of the proteinaceous molecule, a higher proportion of host cells with acceptable expression levels, and/or a higher stability of a gene expression level.
Affiliated Proteinaceous Molecule of a Cell
By an “affiliated proteinaceous molecule of a cell” is meant a proteinaceous molecule which is naturally produced by the kind of cell in the organism from which the cell is derived.
For instance, erythropoietin is an affiliated proteinaceous molecule of a hepatocyte, or of a hepatocyte-derived cell line. Likewise, an antibody is an affiliated proteinaceous molecule of a lymphocyte, or of a lymphocyte-derived cell line, typically of a B-cell or a B-cell derived cell line.
Specific Host Cell Line
A “specific host cell line” is a host cell line derived from a cell which normally expresses a particular proteinaceous molecule (or class of proteinaceous molecules) in the organism from which the cell is derived.
Heterologous STAR Sequence
The term “heterologous STAR sequence” is herein used to define a STAR sequence that is, for example, obtained from a different cell type (from the same species or organism) or is obtained from a different species or organism (either from the same cell type or a different cell type) compared to the cell in which it is used.
Stable
“Stable” means that the observed transcription level is not significantly changed over at least 30 cell divisions. A stable quality is useful in situations wherein expression characteristics should be predictable over many cell divisions. Typical examples are cell lines transfected with foreign genes. Other examples are transgenic animals and plants and gene therapies. Very often, introduced expression cassettes function differently after increasing numbers of cell divisions or plant or animal generations. A stable quality preferably comprises a capacity to maintain gene transcription in subsequent generations of a transgenic plant or animal. Of course, in the case where expression is inducible, the quality comprises the quality to maintain inducibility of expression in subsequent generations of a transgenic plant or animal.
Acceptable Expression Level
An “acceptable expression level” means an acceptable expression level for commercial exploitation. Whether or not a certain expression level is acceptable for commercial exploitation often depends on the kind of proteinaceous molecule that is produced. Acceptable expression levels of different kinds of proteinaceous molecules often involve different amounts of produced proteinaceous molecule.
A STAR sequence, a collection of STAR sequences, and/or a nucleic acid comprising a STAR sequence obtainable by a method of the invention, is of course suitable for use in a method of the invention for producing a proteinaceous molecule in a cell. In one aspect, the invention, therefore, provides a method for producing a proteinaceous molecule in a cell comprising selecting a cell for its suitability for producing the proteinaceous molecule, providing a nucleic acid encoding the proteinaceous molecule with a nucleic acid comprising a STAR sequence, expressing the resulting nucleic acid in the cell and collecting the proteinaceous molecule, wherein the nucleic acid comprising a STAR sequence is obtainable by a method of the invention for identifying and obtaining a nucleic acid comprising a STAR sequence. The nucleic acid comprising a STAR sequence can be identified and obtained using at least one pattern that is representative for sequences comprising STAR activity. Preferably, the nucleic acid comprising a STAR sequence is identified and obtained using at least one pattern as depicted in Table 6 (SEQ ID NOS:177-342) or Table 7 (SEQ ID NOS:343-1072).
A cell can be selected for its suitability for producing a proteinaceous molecule in many different ways. For instance, it can be determined whether the cell is competent of nucleic acid uptake. A nucleic acid encoding a proteinaceous molecule is preferably easily introduced into the cell. Furthermore it can be determined whether the cell secretes produced proteinaceous molecule. Secreted proteinaceous molecule can usually be easily collected. Collecting proteinaceous molecules that are not secreted usually involves sacrificing at least part of a culture. This implicates separating a proteinaceous molecule of interest from other cell components, optionally starting up a new culture, etc. This is more cumbersome. Hence, in a preferred embodiment, a method of the invention is provided wherein the proteinaceous molecule is secreted by the cell.
In another preferred embodiment, a method of the invention is provided wherein the cell is selected for its capability of post-translationally modifying the proteinaceous molecule, such that the proteinaceous molecule has essentially the same properties as a naturally occurring proteinaceous molecule of the same kind. As has been explained above, different kinds of cells display different post-translational modifications. As a result, the same proteinaceous molecule can be modified differently when produced in different host cells. These differences can affect the properties of such proteinaceous molecules, such as for instance pharmaceutical properties. It is, therefore, highly preferred to select a cell which produces a proteinaceous molecule with essentially the same properties as its naturally occurring counterpart. This does not necessarily mean that the host cell has to display exactly the same kind of post-translational modifications, as long as the produced proteinaceous molecule has essentially the same properties. A proteinaceous molecule can be produced that is physically different from its natural counterpart, but which is functionally essentially the same.
In one embodiment, of the invention, however, a cell is selected for its capability of post-translationally modifying the proteinaceous molecule in essentially the same way as the proteinaceous molecule is post-translationally modified in nature.
In one aspect, the invention provides a method of the invention wherein the proteinaceous molecule comprises an affiliated proteinaceous molecule of the cell.
It is especially preferred to choose a host cell, or a cell line derived from a cell, which normally produces a proteinaceous molecule of interest in the organism from which the cell is derived. These cells are capable of carrying out post-translational modifications of their affiliated proteinaceous molecules such that the resulting proteinaceous molecule has the same kind of properties in kind, not necessarily in amount, as a proteinaceous molecule of the same kind which is normally present in the organism. Such specific cells are naturally adapted for production of the affiliated proteinaceous molecule. Apart from other activities of STAR sequences it is also possible to at least partly solve a problem of low production of proteinaceous molecules, which often occurs with such specific cells. Providing at least one STAR sequence to a nucleic acid encoding such proteinaceous molecule will enhance production (yield) of the proteinaceous molecule by the specific cell, increase the proportion of host cells with acceptable expression levels, and/or increase stability of a gene expression level.
In another preferred embodiment, a method of the invention is disclosed wherein the cell is selected for suitable growth in a suspension culture. This facilitates culturing of the cell, and collection of produced proteinaceous molecule, especially when the proteinaceous molecule is secreted into the suspension. More preferably, the cell is selected for suitable growth in a serum-free culture, since serum can contain contaminants and pathogens. Such contaminants/pathogens often have to be separated from produced proteinaceous molecule. This requires an extra step, which consumes time and chemicals, with a potential loss of produced proteinaceous molecule. Moreover, a possibility of a presence of pathogens involves a potential risk for employees. If a pathogen has been present in a culture, a produced proteinaceous molecule is not allowed to be used anymore for commercial and/or medical applications.
In yet another preferred embodiment, the cell is selected for the presence of an adenovirus Early Region 1 (E1) sequence. The presence of an adenoviral E1 sequence enhances protein production in a cell. Hence, an adenoviral E1 sequence is suitable for host cells engineered for protein production.
The invention also provides a method for producing a proteinaceous molecule comprising:

    
    
        providing a host cell with a nucleic acid comprising a STAR sequence;
        selecting a cell with enhanced expression of a proteinaceous molecule; and
        collecting the proteinaceous molecule.
    
    


In one embodiment, the method is performed with a host cell whose genome has not been modified by human interference. The method then results in enhanced expression of a proteinaceous molecule which is encoded by the genome of the host cell. The proteinaceous molecule may be normally expressed by the host cell in the organism from which the cell is derived, but it may also normally be subject to silencing, resulting in little or no expression in the host cell under normal conditions. Introduction of at least one STAR sequence can at least in part inhibit silencing of a gene of interest induced by gene-transcription repressing chromatin. Expression of a proteinaceous molecule is enhanced by introduction of a STAR sequence.
In another embodiment, the host cell is transfected with a nucleic acid of interest. Such nucleic acid for instance, encodes a heterologous proteinaceous molecule which is not naturally encoded by the genome of the host cell. Introduction of a STAR sequence also enhances expression of such heterologous proteinaceous molecule.
The STAR sequence can be introduced randomly into the genome of the host cell, using methods known in the art (for instance calcium precipitation, transfection with a vector comprising a nucleic acid of interest, use of a gene delivery vehicle, etc). If a STAR sequence is introduced near a nucleic acid sequence encoding a proteinaceous molecule, it is capable of enhancing expression of the proteinaceous molecule. Cells expressing a desired proteinaceous molecule can be isolated from cultures with randomly inserted STAR sequences.
Preferably, the STAR sequence is introduced into the host cell by homologous recombination. A nucleic acid comprising a STAR sequence can be provided with an additional sequence. The additional sequence can be chosen such that it is at least in part homologous to a nucleic acid sequence in the host cell which is known to be present in vicinity of a gene encoding a proteinaceous molecule of interest. If a nucleic acid comprising a STAR sequence and such additional sequence is provided to the host cell, it can be incorporated into the host cell's genome by homologous recombination at the site with the (partly) homologous nucleic acid sequence. As a result, the STAR sequence is introduced in vicinity of the gene encoding the proteinaceous molecule of interest. Expression of the proteinaceous molecule is then enhanced by the introduced STAR sequence.
A preferred embodiment of the present invention provides a method of the invention wherein the STAR sequence comprises a species-specific STAR sequence. More preferably, the STAR sequence comprises a cell type-specific STAR sequence.
Two types of STAR elements have been identified. Promiscuous STAR elements are able to function in more than one host cell line. For example, STAR6 (SEQ ID NO:6) increases the predictability, yield, and stability of a transgene in both the U-2 OS human osteosarcoma cell line and in CHO (Chinese hamster ovary) cells. Other STAR elements are species-specific and/or cell type-specific; for example, STAR8 (SEQ ID NO:8) increases the predictability, yield, and stability of transgenes in U-2 OS cells, but not in CHO cells (see Examples 2 and 3 and FIGS. 3 and 4).
If a certain type of host cell (line) is chosen for expression of a proteinaceous molecule (for instance, because it is known to possess a preferred post-translational modification system) a STAR sequence which is naturally present in the cell can be used in a method of the invention. Such STAR sequence is referred to as a cell type-specific STAR sequence. A STAR sequence which is naturally present in a species from which the cell is derived can also be used. Such STAR sequence is referred to as a species-specific STAR sequence. A species-specific STAR sequence may be naturally present in the cell type, although this is not necessary.
A known species-specific STAR sequence or cell-type specific STAR sequence can be used in a method of the invention. Alternatively, a (previously unknown) species-specific STAR sequence or cell-type specific STAR sequence can be detected and isolated by a method as described by the present inventors (EP 01202581.3). The use of a species-specific STAR sequence or cell type-specific STAR sequence is preferred because such sequence is especially active in the host cell and is adapted to the specific circumstances within the cell. For instance, such cell type-specific STAR sequence may interact with a protein which is not present in some other cell-types. In that case, the cell type-specific STAR sequence will be less capable—if at all—of enhancing expression in cells lacking the protein. A species-specific or cell type-specific STAR element often has functional characteristics that are superior to promiscuous STAR elements. Furthermore, a cell line-specific STAR element can satisfy product safety or ethical considerations for use of the host cell line.
A promiscuous STAR sequence is particularly useful if no tissue specific or cell-type specific STAR sequence is known. In that case a known promiscuous STAR sequence can be used. This saves efforts to detect and isolate a cell-type specific STAR sequence.
Several STAR sequences are listed in SEQ ID NOS:1-119. Hence, in one aspect, a method of the invention is provided wherein the STAR sequence comprises a sequence as depicted in SEQ ID NOS:1-119.
In a preferred embodiment, the invention provides a cell line that comprises at least one heterologous STAR sequence or a functional equivalent and/or a functional fragment thereof. In an even more preferred embodiment, the cell line is a human cell line. The invention provides multiple examples of STAR sequences and also methods of testing STAR sequences and hence, a person skilled in the art is very well capable of obtaining a functional equivalent and/or a functional fragment of a STAR sequence, for example by deletion or mutation. In yet another preferred embodiment, the invention provides a non-human cell line that comprises at least one recombinant STAR sequence derived or obtained from a human cell, i.e., a human STAR sequence. It is clear that the amount of STAR sequences may vary, for example, a cell line according to the invention may comprise two, three, or four, or even more STAR sequences which may either be identical or different from each other.
In one aspect, the invention provides a cell line provided with a nucleic acid comprising a STAR sequence, wherein the cell line is selected for its suitability for producing a proteinaceous molecule. Preferably, a cell line of the invention comprises a vertebrate or plant cell line. A vertebrate cell line is very suitable for producing a human proteinaceous molecule of interest, because vertebrates are phylogenetically close related.
Plant cells are for instance very suitable for vaccine production. Vaccine production in plants can be inexpensive, while the vaccine can be easily delivered to an individual by eating the edible portion of the plant (Mercenier et al., 2001).
A cell line of the invention is particularly suitable for production of a proteinaceous molecule of interest, because the STAR sequence can enhance expression of a gene of interest (higher yield of a proteinaceous molecule, higher proportion of host cells with acceptable expression levels, and/or higher stability of a gene expression level). Methods for generating a cell line are known in the art and many techniques are known to provide a cell with a nucleic acid of interest. Furthermore, many general purpose cell lines are available. Such cell lines can be dedicated to production of a certain proteinaceous molecule using recombinant techniques. Examples of available cell lines include CHO cells from Chinese hamster ovary and BHK cells from baby hamster kidney (as described above).
Another embodiment of the invention provides a cell line provided with a nucleic acid comprising a STAR sequence, wherein the cell line comprises an adenovirus Early Region 1 sequence. As has been described above, an adenoviral E1 sequence enhances cellular protein production. More preferably a cell line of the invention is provided wherein the cell line comprises a U-2 OS osteosarcoma, CHO, 293, HuNS-1 myeloma, WERI-Rb-1 retinoblastoma, BHK, Vero, non-secreting mouse myeloma Sp2/0-Ag 14, non-secreting mouse myeloma NSO, or NCI-H295R adrenal gland carcinoma (ATCC CRL-2128) cell line.
A cell line of the invention is particularly suitable for production of a proteinaceous molecule, because production can be enhanced by one or more STAR sequences (higher yield of a proteinaceous molecule, higher proportion of host cells with acceptable expression levels, and/or higher stability of a gene expression level). A cell line of the invention can comprise promiscuous, species-specific and/or cell type-specific STAR sequences. Furthermore, a cell line of the invention can be used to produce a heterologous proteinaceous molecule, and/or an affiliated proteinaceous molecule.
Hence, a use of a cell line of the invention for the production of a proteinaceous molecule is also herewith provided. Preferably, the proteinaceous molecule comprises an affiliated protein of the cell line.
Of course, a proteinaceous molecule obtainable by a method of the invention is also provided by the present invention.
In one aspect, the invention provides a method for selecting a cell suitable for producing a proteinaceous molecule comprising:

    
    
        providing a nucleic acid encoding the proteinaceous molecule with a nucleic acid comprising a STAR sequence;
        expressing the resulting nucleic acid in the cell; and
        determining whether produced proteinaceous molecule has a desired property.
    
    


The desired property, for instance, comprises a pharmaceutical property. The property can be influenced by post translational modification(s), a configuration of a produced proteinaceous molecule, etc.
In yet another aspect, the invention provides a method for selecting a cell suitable for producing a proteinaceous molecule comprising:

    
    
        providing a host cell with a nucleic acid comprising a STAR sequence;
        selecting a cell with enhanced expression of a proteinaceous molecule; and
        determining whether the proteinaceous molecule has a desired property.
    
    


As has been discussed above, the nucleic acid comprising a STAR sequence can be randomly introduced into the genome of the host cell. Preferably, however, the nucleic acid sequence is introduced into the genome of the host cell by homologous recombination.
The invention is further explained in the following examples. The examples do not limit the invention in any way. They merely serve to clarify the invention.
EXAMPLES
Example 1
Method for Isolation of Star Elements from the Human Genome
STAR elements are identified and cloned from human genomic DNA based on their ability to block the spread of transcriptional repression from DNA binding sites for repressor proteins in a test vector, as described in this example. The method described in this example is applicable in principle to any mammalian cell line, for isolation of both promiscuous and cell line-specific STAR elements.
A Method to Isolate Human Star Elements Functional in U-2 Os Cell
Materials and Methods
Plasmids and strains. The selection vector for STAR elements, pSelect-SV40-zeo (“pSelect,” FIG. 1) was constructed as follows: the pREP4 vector (Invitrogen V004-50) was used as the plasmid backbone. It provides the Epstein Barr oriP origin of replication and EBNA-1 nuclear antigen for high-copy episomal replication in primate cell lines; the hygromycin resistance gene with the thymidine kinase promoter and polyadenylation site, for selection in mammalian cells; and the ampicillin resistance gene and colE1 origin of replication for maintenance in Escherichia coli. The vector contains four consecutive LexA operator sites between XbaI and NheI restriction sites (Bunker and Kingston, 1994). Embedded between the LexA operators and the NheI site is a polylinker consisting of the following restriction sites: HindIII-AscI-BamHI-AscI-HindIII. Between the NheI site and a SalI site is the zeocin resistance gene with the SV40 promoter and polyadenylation site, derived from pSV40/Zeo (Invitrogen V502-20); this is the selectable marker for the STAR screen.
Gene libraries were constructed by Sau3AI digestion of human genomic DNA, either purified from placenta (Clontech 6550-1) or carried in bacterial/P1 (BAC/PAC) artificial chromosomes. The BAC/PAC clones contain genomic DNA from the 1q12 cytogenetic region (clones RP1154H19 and RP3328E19), from the HOX cluster of homeotic genes (clones RP1167F23, RP1170019, and RP11387A1), or from human chromosome 22 (Research Genetics 96010-22). The DNAs were size-fractionated, and the 0.5-2 kb size fraction ligated into BamHI-digested pSelect vector, by standard techniques (Sambrook et al., 1989).
The construction of the host strains has been described (van der Vlag et al., 2000). Briefly, they are based on the U-2 OS human osteosarcoma cell line (American Type Culture Collection HTB-96). U-2 OS was stably transfected with the pTet-Off plasmid (Clontech K1620-A), encoding a protein chimera consisting of the Tet-repressor DNA binding domain and the VP16 transactivation domain. The cell line was subsequently stably transfected with fusion protein genes containing the LexA DNA binding domain, and the coding regions of HP1, MeCP2, or HPC2 (three Drosophila proteins that repress gene expression when tethered to DNA). The LexA-repressor genes are under control of the Tet-Off transcriptional regulatory system (Gossen and Bujard, 1992).
Library screening and STAR element characterization. The gene libraries in pSelect were transfected into U-2 OS/Tet-Off/LexA-repressor cell lines by calcium phosphate precipitation (Graham and van der Eb, 1973, Wigler et al., 1978) as recommended by the supplier of the transfection reagent (Life Technologies). Transfected cells were cultured under hygromycin selection (25 μg/ml) and tetracycline repression (doxycycline, 10 ng/ml) for one week (50% confluence). Then the doxycycline concentration was reduced to 0.1 ng/ml to induce the LexA-repressor genes, and after two days zeocin was added to 250 μg/ml. The cells are cultured for a further four to five weeks, until the control cultures (transfected with empty pSelect) were killed by the zeocin.
Zeocin-resistant colonies from the library transfection were propagated, and plasmid DNA isolated and rescued into E. coli by standard techniques (Sambrook et al., 1989). The candidate STAR elements in the rescued DNA were analyzed by restriction endonuclease mapping (Sambrook et al., 1989), and tested for STAR activity (zeocin resistance) after re-transfection to U-2 OS/Tet-Off/LexA-repressor cells and lowering the doxycycline concentration.
The human genomic DNA inserts in these plasmids were sequenced by the dideoxy method (Sanger et al., 1977) using a Beckman CEQTM2000 automated DNA sequencer, using the manufacturer's instructions. Briefly, DNA was purified from E. coli using QIAprep® Spin Miniprep and Plasmid Midi Kits (QIAGEN® 27106 and 12145, respectively). Cycle sequencing was carried out using custom oligonucleotides corresponding to the pSelect vector (primers D89 (SEQ ID NO:149) and D95 (SEQ ID NO:154); all oligonucleotides are described in Table 2), in the presence of dye terminators (CEQTM Dye Terminator Cycle Sequencing Kit, Beckman 608000). Assembled STAR DNA sequences were located in the human genome using BLAST (Basic Local Alignment Search Tool (Altschul et al., 1990); worldwideweb.ncbi.nlm.nih.gov/BLAST/).
Results
The screens of human genomic DNA have yielded 66 STAR elements; the lengths and chromosomal locations of these elements are tabulated in SEQ ID NO:1-SEQ ID NO:66. They confer zeocin resistance on U-2 OS host cells when placed between LexA-repressor binding sites and the zeocin resistance gene. Their anti-repression activity was demonstrated both in the initial screen and upon re-transfection (demonstrating that the anti-repression activity is due to the STAR element and not to somatic acquisition of zeocin resistance). The STAR elements correspond to known and unique sequences in the human genome, as demonstrated by BLAST searches (Table 3). In some cases, the cloned element is a chimera of two unlinked genomic loci (e.g., STAR3 (SEQ ID NO:3), Table 3). They range in length from 500 to 2361 base pairs in length.
Example 2
Predictability and Yield is Improved by Promiscuous Star Elements in More than One Host Cell Line
STAR elements function to block the effect of transcriptional repression influences on transgene expression units. These repression influences can be due to heterochromatin (“position effects”) or to adjacent copies of the transgene (“repeat-induced gene silencing”). Two of the benefits of STAR elements for heterologous protein production are increased predictability of finding high-expressing primary recombinant host cells and increased yield during production cycles. These benefits are illustrated in this example.
Materials and Methods
Construction of the pSDH vectors and STAR-containing derivatives: The pSDH-Tet vector was constructed by polymerase chain reaction amplification (PCR) of the luciferase open reading frame from plasmid pREP4-HSF-Luc (van der Vlag et al., 2000) using primers C67 (SEQ ID NO:136) and C68 (SEQ ID NO:137), and insertion of the SacII/BamHI fragment into SacII/BamHI-digested pUHD10-3 (Gossen and Bujard, 1992). The luciferase expression unit was re-amplified with primers C65 (SEQ ID NO:134) and C66 (SEQ ID NO:135), and re-inserted into pUHD10-3 in order to flank it with multiple cloning sites (MCSI and MCSII). An AscI site was then introduced into MCSI by digestion with EcoRI and insertion of a linker (comprised of annealed oligonucleotides D93 (SEQ ID NO:152) and D94 (SEQ ID NO:153)). The CMV promoter was amplified from plasmid pCMV-Bsd (Invitrogen K510-01) with primers D90 (SEQ ID NO:150) and D91 (SEQ ID NO:151), and used to replace the Tet-Off promoter in pSDH-Tet by SalI/SacII digestion and ligation to create vector pSDH-CMV. The luciferase open reading frame in this vector was replaced by SEAP (Secreted Alkaline Phosphatase) as follows: vector pSDH-CMV was digested with SacII and BamHI and made blunt; the SEAP open reading frame was isolated from pSEAP-basic (Clontech 6037-1) by EcoRI/SalI digestion, made blunt and ligated into pSDH-CMV to create vector pSDH-CS. The puromycin resistance gene under control of the SV40 promoter was isolated from plasmid pBabe-Puro (Morgenstern and Land, 1990) by PCR, using primers C81 (SEQ ID NO:138) and C82 (SEQ ID NO:139). This was ligated into vector pGL3-control (BamHI site removed) (Promega E1741) digested with NcoI/XbaI to create pGL3-puro. pGL3-puro was digested with BglII/SalI to isolate the SV40-puro resistance unit, which was made blunt and ligated into NheI digested, blunt-ended pSDH-CS. The resulting vector, pSDH-CSP, is shown in FIG. 2. All cloning steps were carried out following the instructions provided by the manufacturers of the reagents used, according to methods known in the art (Sambrook et al., 1989).
STAR elements were inserted into MCSI and MCSII in two steps, by digestion of the STAR element and the pSDH-CSP vector with an appropriate restriction enzyme, followed by ligation. The orientation of STAR elements in recombinant pSDH vectors was determined by restriction mapping, and in all cases verified by DNA sequence analysis using primers C85 (SEQ ID NO:140), E42 (SEQ ID NO:168), and E25 (SEQ ID NO:167) (Table 2; see Example 1).
Transfection and culture of U-2 OS cells with pSDH-CMV plasmids: The human osteosarcoma U-2 OS cell line (ATCC #HTB-96) was cultured in Dulbecco's Modified Eagle Medium+10% Fetal Calf Serum containing glutamine, penicillin, and streptomycin (supra) at 37° C./5% CO2. Cells were co-transfected with the pSDH-CMV vector and its derivatives containing STAR6 (SEQ ID NO:6) or STAR8 (SEQ ID NO:8) in MCSI and MCSII (along with plasmid pBabe-Puro) using SuperFect® (supra). Puromycin selection was complete in two weeks, after which time individual puromycin resistant U-2 OS/pSDH-CMV clones were isolated at random and cultured further.
Luciferase assay: Luciferase activity (Himes and Shannon, 2000) was assayed in resuspended cells according to the instructions of the assay kit manufacturer (Roche 1669893), using a luminometer (Turner 20/20TD). Total cellular protein concentration was determined by the bicinchoninic acid method according to the manufacturer's instructions (Sigma B-9643), and used to normalize the luciferase data.
Transfection and culture of CHO cells with pSDH-CSP plasmids: The Chinese Hamster Ovary cell line CHO-K1 (ATCC CCL-61) was cultured in HAMS-F12 medium+10% Fetal Calf Serum containing 2 mM glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomycin at 37° C./5% CO2. Cells were transfected with recombinant pSDH-CSP vectors using SuperFect® (QIAGEN®) as described by the manufacturer. Briefly, cells were seeded to culture vessels and grown overnight to 70-90% confluence. SuperFect® reagent was combined with plasmid DNA (linearized in this example by digestion with PvuI) at a ratio of 6 microliters per microgram (e.g., for a 10 cm Petri dish, 20 micrograms DNA and 120 microliters SuperFect®) and added to the cells. After overnight incubation, the transfection mixture was replaced with fresh medium, and the transfected cells were incubated further. After overnight cultivation, 5 micrograms/ml puromycin was added. Puromycin selection was complete in two weeks, after which time individual puromycin resistant CHO/pSDH-CSP clones were isolated at random and cultured further.
Secreted Alkaline Phosphatase (SEAP) assay: SEAP activity (Berger et al., 1988, Henthorn et al., 1988, Kain, 1997, Yang et al., 1997) in the culture medium of CHO/pSDH-CSP clones was determined as described by the manufacturer (Clontech Great EscAPe kit #K2041). Briefly, an aliquot of medium was heat inactivated at 65° C., then combined with assay buffer and CSPD chemiluminescent substrate and incubated at room temperature for ten minutes. The rate of substrate conversion was then determined in a luminometer (Turner 20/20TD). Cell density was determined by counting trypsinized cells in a Coulter ACT10 cell counter. Luminescence units were converted into picograms SEAP based on a SEAP positive control calibration curve, and normalized to cell number.
Results
Recombinant U-2 OS cell clones containing the pSDH-CMV vector, or a pSDH-CMV plasmid containing STAR6 (SEQ ID NO:6) (Table 3), were cultured for three weeks. The luciferase activity in the host cells was then determined, and is expressed as relative luciferase units (FIG. 3), normalized to total cell protein. The recombinant U-2 OS clones with STAR6 (SEQ ID NO:6) flanking the expression units had higher yields than the STAR-less clones: the STAR6 clones had maximal luciferase expression levels five-fold higher than the STAR-less clones. The STAR6 (SEQ ID NO:6) element conferred greater predictability as well: 15-20% of the clones expressed luciferase at levels comparable to or greater than the STAR-less clone with the highest expression level.
Recombinant CHO cell clones containing the pSDH-CSP vector, or a pSDH-CSP plasmid containing STAR6 (SEQ ID NO:6), were cultured for three weeks. The SEAP activity in the culture supernatants was then determined, and is expressed on the basis of cell number (FIG. 4). As can be seen, clones with the STAR6 (SEQ ID NO:6) element in the expression units were isolated that express two- to three-fold higher SEAP activity than clones whose expression units do not include this STAR element. Furthermore, the number of STAR6-containing (SEQ ID NO:6-containing) clones that express SEAP activity at or above the maximal activity of the STAR-less clones is quite high: 40% of the STAR6 clone populations exceed the highest SEAP expression of the pSDH-CSP clones.
These results demonstrate that, when used with the strong CMV promoter, the STAR6 (SEQ ID NO:6) element increases the yield of this heterologous protein in both of the host cell lines tested. STAR6 (SEQ ID NO:6) also confers increased predictability, as manifested by the large proportion of the clones with yields equal to or greater than the highest yield displayed by the STAR-less clones. Thus, STAR6 (SEQ ID NO:6) is an example of a promiscuous STAR element, able to suppress transgene repression in more than one host cell line. The cell lines used are derived from different species (human and hamster) and different tissue types (bone and ovary), reflecting the broad range of host cells in which this STAR element can be utilized in improving heterologous protein expression.
Example 3
STAR8 (SEQ ID NO:8) is a Cell Line-Specific STAR Element
The patterns of gene expression and epigenetic gene regulation in a host cell line reflect the developmental state of the somatic cells from which they are derived. Furthermore, the biotechnology industry takes advantage of general purpose cell lines from different species according to specific requirements of a heterologous protein production process. Therefore, it is expected that some STAR elements will not function in cell lines other than those in which they are isolated. This expectation has been fulfilled by some of the STAR elements shown in Table 3. One example will be given here.
Materials and Methods
pSDH vector construction, transfection and cultivation of CHO and U-2 OS cell lines, and assay methods for the SEAP and luciferase reporter genes has been described in Example 2.
Results
Recombinant U-2 OS cell clones containing the pSDH-CMV vector, or a pSDH-CMV plasmid containing STAR8 (SEQ ID NO:8) (Table 3), were cultured for three weeks. The luciferase activity in the host cells was then determined, and is expressed as relative luciferase units (FIG. 3), normalized to total cell protein. The recombinant U-2 OS clones with the STAR8 (SEQ ID NO:8) element flanking the expression units had higher yields than the STAR-less clones: the highest expression observed from STAR8 clones was two- to three-fold higher than the expression from STAR-less clones. The STAR8 (SEQ ID NO:8) element conferred greater predictability as well: for this STAR element,  ̃15% of the clones displayed luciferase expression at levels comparable to or greater than the STAR-less clone with the highest expression level.
Recombinant CHO cell clones transfected with the pSDH-CSP vector, or a pSDH-CSP plasmid containing STAR8 (SEQ ID NO:8), were cultured for three weeks. The SEAP activity in the culture supernatants was then determined, and is expressed on the basis of cell number (FIG. 4). As can be seen, one clone with the STAR8 (SEQ ID NO:8) element in the expression unit had a yield approximately two-fold higher than the highest-expressing STAR-less clone. However, the rest of the STAR8 clones expressed very poorly relative to the STAR-less clone population. Since only one individual in the STAR8 population had a good yield, it is probable that the expression unit in this clone was integrated in open, transcriptionally active chromatin, and the high yield does not reflect anti-repression activity of STAR8 (SEQ ID NO:8) in CHO cells. Certainly in the CHO clones transfected with STAR8-containing (SEQ ID NO:8-containing) expression units the predictability is quite poor; of the 17 puromycin-resistant clones, only one clone had a yield of SEAP activity above the background level of expression.
This example demonstrates that good performance of a STAR element in one cell line (in this case, the U-2 OS cell line in which STAR8 (SEQ ID NO:8) was originally isolated) is not an accurate predictor of its performance in other cell lines. STAR8 (SEQ ID NO:8) is thus an example of a cell line-specific STAR element.
Example 4
STAR Elements Functionality in Diverse Cell Line
Materials and Methods
Cell lines including the U-2 OS osteosarcoma and CHO (Chinese hamster ovary) cell lines (supra), the 293 cell line (ATCC CRL-1573) derived from human embryonal kidney (immortalized by adenovirus 5 transfection), the HuNS-1 myeloma (ATCC CRL-8644) and the WERI-Rb-1 retinoblastoma cell line (ATCC HTB-169), the NCI-H295R adrenal gland carcinoma (ATCC CRL-2128), and the non-secreting mouse myelomas Sp2/0-Ag 14 and NSO are examined according to the previous examples.
Example 5
STAR Elements Improve the Stability of Transgene Expression
During cultivation of recombinant host cells, it is common practice to maintain antibiotic selection. This is intended to prevent transcriptional silencing of the transgene, or loss of the transgene from the genome by processes such as recombination. However it is undesirable for production of heterologous proteins, for a number of reasons. First, the antibiotics that are used are quite expensive, and contribute significantly to the unit cost of the product. Second, for biopharmaceutical use, the protein must be demonstrably pure, with no traces of the antibiotic in the product. One advantage of STAR elements for heterologous protein production is that they confer stable expression on transgenes during prolonged cultivation, even in the absence of antibiotic selection; this property is demonstrated in this example.
Materials and Methods
The U-2 OS cell line was transfected with the plasmid pSDH-Tet-STAR6 and cultivated as described in Example 2. Individual puromycin-resistant clones were isolated and cultivated further in the absence of doxycycline. At weekly intervals the cells were transferred to fresh culture vessels at a dilution of 1:20. Luciferase activity was measured at periodic intervals as described in Example 2. After 15 weeks, the cultures were divided into two replicates; one replicate continued to receive puromycin, while the other replicate received no antibiotic for the remainder of the experiment (25 weeks total).
Results
Table 4 presents the data on luciferase expression by an expression unit flanked with STAR6 (SEQ ID NO:6) during prolonged growth with or without antibiotic. As can be seen, the expression of the reporter transgene, luciferase, remains stable in the U-2 OS host cells for the duration of the experiment. After the cultures were divided into two treatments (plus antibiotic and without antibiotic) the expression of luciferase was essentially stable in the absence of antibiotic selection. This demonstrates the ability of STAR elements to protect transgenes from silencing or loss during prolonged cultivation. It also demonstrates that this property is independent of antibiotic selection. Therefore, production of heterologous proteins is possible without incurring the costs of the antibiotic or of difficult downstream processing.
Example 6
Minimal Essential Sequences of STAR Elements
STAR elements are isolated from the genetic screen described in Example 1. The screen uses libraries constructed with human genomic DNA that was size-fractionated to approximately 0.5-2 kilobases (supra). The STAR elements range from 500 to 2361 base pairs (Table 3). It is likely that, for many of the STAR elements that have been isolated, STAR activity is conferred by a smaller DNA fragment than the initially isolated clone. It is useful to determine these minimum fragment sizes that are essential for STAR activity, for two reasons. First, smaller functional STAR elements would be advantageous in the design of compact expression vectors, since smaller vectors transfect host cells with higher efficiency. Second, determining minimum essential STAR sequences permits the modification of those sequences for enhanced functionality. Two STAR elements have been fine-mapped to determine their minimal essential sequences.
Materials and Methods
STAR10 (SEQ ID NO:10) (1167 base pairs) and STAR27 (SEQ ID NO:27) (1520 base pairs) have been fine-mapped. They have been amplified by PCR to yield sub-fragments of approximately equal length (FIG. 6 legend). For initial testing, these have been cloned into the pSelect vector at the BamHI site, and transfected into U-2 OS/Tet-Off/LexA-HP1 cells as described in Example 1. After selection for hygromycin resistance, LexA-HP1 was induced by lowering the doxycycline concentration. Transfected cells were then incubated with zeocin to test the ability of the STAR fragments to protect the SV40-Zeo expression unit from repression due to LexA-HP1 binding.
Results
In this experiment STAR10 (SEQ ID NO:10) and STAR 27 (SEQ ID NO:27) confer good protection against gene silencing, as expected (FIG. 6). This is manifested by robust growth in the presence of zeocin.
Of the three STAR10 (SEQ ID NO:10) sub-fragments, 10A ( ̃400 base pairs, corresponding to approximately the first 400 nucleotides of SEQ ID NO:10) confers on transfected cells vigorous growth in the presence of zeocin, exceeding that of the full-length STAR element. Cells transfected with pSelect constructs containing the other two sub-fragments do not grow in the presence of zeocin. These results identify the  ̃400 base pair 10A fragment as encompassing the DNA sequence responsible for the anti-repression activity of STAR10 (SEQ ID NO:10).
STAR27 (SEQ ID NO:27) confers moderate growth in zeocin to transfected cells in this experiment (FIG. 6). One of the sub-fragments of this STAR, 27B ( ̃500 base pairs, corresponding to approximately the second 500 nucleotides of SEQ ID NO:27), permits weak growth of the host cells in zeocin-containing medium. This suggests that the anti-repression activity of this STAR is partially localized on sub-fragment 27B, but full activity requires sequences from 27A (corresponding to approximately the first 500 nucleotides of SEQ ID NO:27) and/or 27C (corresponding to approximately the third 500 nucleotides of SEQ ID NO:27) (each  ̃500 base pairs) as well.
Example 7
STAR Elements Function in the Context of Various Transcriptional Promoters
Transgene transcription is achieved by placing the transgene open reading frame under control of an exogenous promoter. The choice of promoter is influenced by the nature of the heterologous protein and the production system. In most cases, strong constitutive promoters are preferred because of the high yields they can provide. Some viral promoters have these properties; the promoter/enhancer of the cytomegalovirus immediate early gene (“CMV promoter”) is generally regarded as the strongest promoter in common biotechnological use (Boshart et al., 1985, Doll et al., 1996, Foecking and Hofstetter, 1986). The simian virus SV40 promoter is also moderately strong (Boshart et al., 1985, Foecking and Hofstetter, 1986) and is frequently used for ectopic expression in mammalian cell vectors. The Tet-Off promoter is inducible: the promoter is repressed in the presence of tetracycline or related antibiotics (doxycycline is commonly used) in cell-lines which express the tTA plasmid (Clontech K1620-A), and removal of the antibiotic results in transcriptional induction (Deuschle et al., 1995, Gossen and Bujard, 1992, Izumi and Gilbert, 1999, Umana et al., 1999).
Materials and Methods
The construction of the pSDH-Tet and pSDH-CMV vectors is described in Example 2. pSDH-SV40 was constructed by PCR amplification of the SV40 promoter (primers D41 (SEQ ID NO:142) and D42 (SEQ ID NO:143)) from plasmid pSelect-SV40-Zeo (Example 1), followed by digestion of the PCR product with SacII and SalI. The pSDH-CMV vector was digested with SacII and SalI to remove the CMV promoter, and the vector and SV40 fragment were ligated together to create pSDH-SV40. STAR6 (SEQ ID NO:6) was cloned into MCSI and MCSII as described in Example 2. The plasmids pSDH-Tet, pSDH-Tet-STAR6, pSDH-Tet-STAR7, pSDH-SV40 and pSDH-SV40-STAR6 were co-transfected with pBabe-Puro into U-2 OS using SuperFect® as described by the manufacturer. Cell cultivation, puromycin selection, and luciferase assays were carried out as described in Example 2.
Results
FIGS. 3, 7, and 8 compare the expression of the luciferase reporter gene from three different promoters: two strong and constitutive viral promoters (CMV and SV40), and the inducible Tet-Off promoter. All three promoters were tested in the context of the STAR6 (SEQ ID NO:6) element in U-2 OS cells. The results demonstrate that the yield and predictability from all three promoters are increased by STAR6 (SEQ ID NO:6). As described in Examples 2 and 5, STAR6 (SEQ ID NO:6) is beneficial in the context of the CMV promoter (FIG. 3). Similar improvements are seen in the context of the SV40 promoter (FIG. 7): the yield from the highest-expressing STAR6 clone is two- to three-fold greater than the best pSDH-SV40 clones, and six STAR clones (20% of the population) have yields higher than the best STAR-less clones. In the context of the Tet-Off promoter under inducing (low doxycycline) concentrations, STAR6 (SEQ ID NO:6) also improves the yield and predictability of transgene expression (FIG. 8): the highest-expressing STAR6 clone has a 20-fold higher yield than the best pSDH-Tet clone, and nine STAR6 clones (35% of the population) have yields higher than the best STAR-less clone. It is concluded that this STAR element is versatile in its transgene-protecting properties, since it functions in the context of various biotechnologically useful promoters of transcription.
Example 8
STAR Element Function can be Directional
While short nucleic acid sequences can be symmetrical (e.g., palindromic), longer, naturally-occurring sequences are typically asymmetrical. As a result, the information content of nucleic acid sequences is directional and the sequences themselves can be described with respect to their 5′ and 3′ ends. The directionality of nucleic acid sequence information affects the arrangement in which recombinant DNA molecules are assembled using standard cloning techniques known in the art (Sambrook et al., 1989). STAR elements are long, asymmetrical DNA sequences, and have a directionality based on the orientation in which they were originally cloned in the pSelect vector. In the examples given above, using two STAR elements in pSDH vectors, this directionality was preserved. This orientation is described as the native or 5′-3′ orientation, relative to the zeocin resistance gene (see FIG. 9). In this example the importance of directionality for STAR function is tested in the pSDH-Tet vector. Since the reporter genes in the pSDH vectors are flanked on both sides by copies of the STAR element of interest, the orientation of each STAR copy must be considered. This example compares the native orientation with the opposite orientation (FIG. 9).
Materials and Methods
The STAR66 (SEQ ID NO:66) element was cloned into pSDH-Tet as described in Example 2. U-2 OS cells were co-transfected with plasmids pSDH-Tet-STAR66-native and pSDH-Tet-STAR66-opposite, and cultivated as described in Example 2. Individual clones were isolated and cultivated; the level of luciferase expression was determined as described (supra).
Results
The results of the comparison of STAR66 (SEQ ID NO:66) activity in the native orientation and the opposite orientation are shown in FIG. 10. When STAR66 (SEQ ID NO:66) is in the opposite orientation, the yield of only one clone is reasonably high (60 luciferase units). In contrast, the yield of the highest-expressing clone when STAR66 (SEQ ID NO:66) is in the native orientation is considerably higher (100 luciferase units) and the predictability is much higher, as well: seven clones of the native-orientation population (30%) express luciferase above the level of the highest-expressing clone from the opposite-orientation population, and 15 of the clones in the native-orientation population (60%) express luciferase above ten relative luciferase units. Therefore, it is demonstrated that STAR66 (SEQ ID NO:66) function is directional.
Example 9
Transgene Expression in the Context of STAR Elements is Copy Number-Dependent
Transgene expression units for heterologous protein expression are generally integrated into the genome of the host cell to ensure stable retention during cell division. Integration can result in one or multiple copies of the expression unit being inserted into the genome; multiple copies may or may not be present as tandem arrays. The increased yield demonstrated for transgenes protected by STAR elements (supra) suggests that STAR elements are able to permit the transgene expression units to function independently of influences on transcription associated with the site of integration in the genome (independence from position effects (Boivin and Dura, 1998)). It suggests further that the STAR elements permit each expression unit to function independently of neighboring copies of the expression unit when they are integrated as a tandem array (independence from repeat-induced gene silencing (Garrick et al., 1998)). Copy number-dependence is determined from the relationship between transgene expression levels and copy number, as described in the example below.
Materials and Methods
U-2 OS cells were co-transfected with pSDH-Tet-STAR10 and cultivated under puromycin selection as described (supra). Eight individual clones were isolated and cultivated further. Then cells were harvested, and one portion was assayed for luciferase activity as described (supra). The remaining cells were lysed and the genomic DNA purified using the DNeasy® Tissue Kit (QIAGEN® 69504) as described by the manufacturer. DNA samples were quantitated by UV spectrophotometry. Three micrograms of each genomic DNA sample were digested with PvuII and XhoI overnight as described by the manufacturer (New England Biolabs), and resolved by agarose gel electrophoresis. DNA fragments were transferred to a nylon membrane as described (Sambrook et al., 1989), and hybridized with a radioactively labeled probe to the luciferase gene (isolated from BamHI/SacII-digested pSDH-Tet). The blot was washed as described (Sambrook et al., 1989) and exposed to a phosphorimager screen (Personal F/X, BioRad). The resulting autoradiogram (FIG. 11) was analyzed by densitometry to determine the relative strength of the luciferase DNA bands, which represents the transgene copy number.
Results
The enzyme activities and copy numbers (DNA band intensities) of luciferase in the clones from the pSDH-Tet-STAR10 clone population is shown in FIG. 12. The transgene copy number is highly correlated with the level of luciferase expression in these pSDH-Tet-STAR10 clones (r=0.86). This suggests that STAR10 (SEQ ID NO:10) confers copy number-dependence on the transgene expression units, making transgene expression independent of other transgene copies in tandem arrays and independent of gene-silencing influences at the site of integration.
Example 10
STAR Elements Function as Enhancer Blockers but not Enhancers
Gene promoters are subject to both positive and negative influences on their ability to initiate transcription. An important class of elements that exert positive influences are enhancers. Enhancers are characteristically able to affect promoters even when they are located far away (many kilobase pairs) from the promoter. Negative influences that act by heterochromatin formation (e.g., Polycomb group proteins) have been described above, and these are the target of STAR activity. The biochemical basis for enhancer function and for heterochromatin formation is fundamentally similar, since they both involve binding of proteins to DNA. Therefore, it is important to determine whether STAR elements are able to block positive influences as well as negative influences, in other words, to shield transgenes from genomic enhancers in the vicinity of the site of integration. The ability to shield transgenes from enhancer activity ensures stable and predictable performance of transgenes in biotechnological applications. This example examines the performance of STAR elements in an enhancer-blocking assay.
Another feature of STAR activity that is important to their function is the increased yield they confer on transgenes (Example 2). STARs are isolated on the basis of their ability to maintain high levels of zeocin expression when heterochromatin-forming proteins are bound adjacent to the candidate STAR elements. High expression is predicted to occur because STARs are anticipated to block the spread of heterochromatin into the zeocin expression unit. However, a second scenario is that the DNA fragments in zeocin-resistant clones contain enhancers. Enhancers have been demonstrated to have the ability to overcome the repressive effects of Polycomb-group proteins such as those used in the method of the STAR screen (Zink and Paro, 1995). Enhancers isolated by this phenomenon would be considered false positives, since enhancers do not have the properties claimed here for STARs. In order to demonstrate that STAR elements are not enhancers, they have been tested in an enhancer assay.
The enhancer-blocking assay and the enhancer assay are methodologically and conceptually similar. The assays are shown schematically in FIG. 13. The ability of STAR elements to block enhancers is performed using the E47/E-box enhancer system. The E47 protein is able to activate transcription by promoters when it is bound to an E-box DNA sequence located in the vicinity of those promoters (Quong et al., 2002). E47 is normally involved in regulation of B and T lymphocyte differentiation (Quong et al., 2002), but it is able to function in diverse cell types when expressed ectopically (Petersson et al., 2002). The E-box is a palindromic DNA sequence, CANNTG (Knofler et al., 2002). In the enhancer-blocking assay, an E-box is placed upstream of a luciferase reporter gene (including a minimal promoter) in an expression vector. A cloning site for STAR elements is placed between the E-box and the promoter. The E47 protein is encoded on a second plasmid. The assay is performed by transfecting both the E47 plasmid and the luciferase expression vector into cells; the E47 protein is expressed and binds to the E-box, and the E47/E-box complex is able to act as an enhancer. When the luciferase expression vector does not contain a STAR element, the E47/E-box complex enhances luciferase expression (FIG. 13A, situation 1). When STAR elements are inserted between the E-box and the promoter, their ability to block the enhancer is demonstrated by reduced expression of luciferase activity (FIG. 13A, situation 2); if STARs cannot block enhancers, luciferase expression is activated (FIG. 13A, situation 3).
The ability of STAR elements to act as enhancers utilizes the same luciferase expression vector. In the absence of E47, the E-box itself does not affect transcription. Instead, enhancer behavior by STAR elements will result in activation of luciferase transcription. The assay is performed by transfecting the luciferase expression vector without the E47 plasmid. When the expression vector does not contain STAR elements, luciferase expression is low (FIG. 13B, situation 1). If STAR elements do not have enhancer properties, luciferase expression is low when a STAR element is present in the vector (FIG. 13B, situation 2). If STAR elements do have enhancer properties, luciferase expression will be activated in the STAR-containing vectors (FIG. 13B, situation 3).
Materials and Methods
The luciferase expression vector was constructed by inserting the E-box and a human alkaline phosphatase minimal promoter from plasmid mu-E5+E2×6-cat(x) (Ruezinsky et al., 1991) upstream of the luciferase gene in plasmid pGL3-basic (Promega E1751), to create pGL3-E-box-luciferase (gift of W. Romanow). The E47 expression plasmid contains the E47 open reading frame under control of a beta-actin promoter in the pHBAPr-1-neo plasmid; E47 in constitutively expressed from this plasmid (gift of W. Romanow).
STAR elements 1, 2, 3, 6, 10, 11, 18, and 27 (SEQ ID NOS:1, 2, 3, 6, 10, 11, 18, and 27, respectively) have been cloned into the luciferase expression vector. Clones containing the Drosophila scs element and the chicken beta-globin HS4-6× core (“HS4”) element have been included as positive controls (they are known to block enhancers, and to have no intrinsic enhancer properties (Chung et al., 1993, Kellum and Schedl, 1992)), and the empty luciferase expression vector has been included as a negative control. All assays were performed using the U-2 OS cell line. In the enhancer-blocking assay, the E47 plasmid was co-transfected with the luciferase expression vectors (empty vector, or containing STAR or positive-control elements). In the enhancer assay, the E47 plasmid was co-transfected with STARless luciferase expression vector as a positive control for enhancer activity; all other samples received a mock plasmid during co-transfection. The transiently transfected cells were assayed for luciferase activity 48 hours after plasmid transfection (supra). The luciferase activity expressed from a plasmid containing no E-box or STAR/control elements was subtracted, and the luciferase activities were normalized to protein content as described (supra).
Results
FIG. 14 shows the results of the enhancer-blocking assay. In the absence of STAR elements (or the known enhancer-blocking elements scs and HS4), the E47/E-box enhancer complex activates expression of luciferase (“vector”); this enhanced level of expression has been normalized to 100. Enhancer activity is blocked by all STAR elements tested. Enhancer activity is also blocked by the HS4 and scs elements, as expected (Bell et al., 2001, Gerasimova and Corces, 2001). These results demonstrate that in addition to their ability to block the spreading of transcriptional silencing (negative influences), STAR elements are able to block the action of enhancers (positive influences).
FIG. 15 shows the results of the enhancer assay. The level of luciferase expression due to enhancement by the E47/E-box complex is set at 100 (“E47”). By comparison, none of the STAR elements bring about significant activation of luciferase expression. As expected, the scs and HS4 elements also do not bring about activation of the reporter gene. Therefore, it is concluded that at least the tested STAR elements do not possess enhancer properties.
Example 11
Star Elements are Conserved Between Mouse and Human
BLAT analysis of the STAR DNA sequence against the human genome database (http://genome.ucsc.edu/cgi-bin/hgGateway) reveals that some of these sequences have high sequence conservation with other regions of the human genome. These duplicated regions are candidate STAR elements; if they do show STAR activity, they would be considered paralogs of the cloned STARs (two genes or genetic elements are said to be paralogous if they are derived from a duplication event (Li, 1997)).
BLAST analysis of the human STARs against the mouse genome (http://www.ensembl.org/Mus_musculus/blastview) also reveals regions of high sequence conservation between mouse and human. This sequence conservation has been shown for fragments of 15 out of the 65 human STAR elements. The conservation ranges from 64% to 89%, over lengths of 141 base pairs to 909 base pairs (Table 5). These degrees of sequence conservation are remarkable and suggest that these DNA sequences may confer STAR activity within the mouse genome as well. Some of the sequences from the mouse and human genomes in Table 5 could be strictly defined as orthologs (two genes or genetic elements are said to be orthologous if they are derived from a speciation event (Li, 1997)). For example, STAR6 (SEQ ID NO:6) is between the SLC8A1 and HAAO genes in both the human and mouse genomes. In other cases, a cloned human STAR has a paralog within the human genome, and its ortholog has been identified in the mouse genome. For example, STAR3a is a fragment of the 15q11.2 region of human chromosome 15. This region is 96.9% identical (paralogous) with a DNA fragment at 5q33.3 on human chromosome 5, which is near the IL12B interleukin gene. These human DNAs share approximately 80% identity with a fragment of the 11B2 region on mouse chromosome 11. The 11B2 fragment is also near the (mouse) IL12B interleukin gene. Therefore, STAR3a and the mouse 11B2 fragment can be strictly defined as paralogs.
In order to test the hypothesis that STAR activity is shared between regions of high sequence conservation in the mouse and human genome, one of the human STARs with a conserved sequence in mouse, STAR18 (SEQ ID NO:18), has been analyzed in greater detail. The sequence conservation in the mouse genome detected with the original STAR18 clone extends leftward on human chromosome 2 for about 500 base pairs (FIG. 16; left and right relate to the standard description of the arms of chromosome 2). In this example, we examine whether the region of sequence conservation defines a “naturally occurring” STAR element in human that is more extensive in length than the original clone. We also examine whether the STAR function of this STAR element is conserved between mouse and human.
Materials and Methods
The region of mouse/human sequence conservation around STAR18 (SEQ ID NO:18) was recovered from human BAC clone RP11-387A1 by PCR amplification, in three fragments: the entire region (primers E93 (SEQ ID NO:171) and E94 (SEQ ID NO:172)), the leftward half (primers E93 (SEQ ID NO:171) and E92 (SEQ ID NO:170)), and the rightward half (primers E57 (SEQ ID NO:169) and E94 (SEQ ID NO:172)). The corresponding fragments from the homologous mouse region were recovered from BAC clone RP23-400H17 in the same fashion (primers E95 (SEQ ID NO:173) and E98 (SEQ ID NO:176), E95 (SEQ ID NO:173) and E96 (SEQ ID NO:174), and E97 (SEQ ID NO:175) and E98 (SEQ ID NO:176), respectively). All fragments were cloned into the pSelect vector and transfected into a U-2 OS/Tet-Off/LexA-HP1 cell line (supra). Following transfection, hygromycin selection was carried out to select for transfected cells. The LexA-HP1 protein was induced by lowering the doxycycline concentration, and the ability of the transfected cells to withstand the antibiotic zeocin (a measure of STAR activity) was assessed by monitoring cell growth.
Results
The original STAR18 clone was isolated from Sau3AI digested human DNA ligated into the pSelect vector on the basis of its ability to prevent silencing of a zeocin resistance gene. Alignment of the human STAR18 clone (497 base pairs) with the mouse genome revealed high sequence similarity (72%) between the orthologous human and mouse STAR18 (SEQ ID NO:18) regions. It also uncovered high similarity (73%) in the region extending for 488 base pairs immediately to the left of the Sau3AI site that defines the left end of the cloned region (FIG. 16). Outside these regions the sequence similarity between human and mouse DNA drops below 60%.
As indicated in FIG. 16, both the human and the mouse STAR18 (SEQ ID NO:18) elements confer survival on zeocin to host cells expressing the lexA-HP1 repressor protein. The original 497 base pair STAR18 clone and its mouse ortholog both confer the ability to grow (FIG. 16, a and d). The adjacent 488 base pair regions of high similarity from both genomes also confer the ability to grow, and in fact their growth phenotype is more vigorous than that of the original STAR18 clone (FIG. 16, b and e). When the entire region of sequence similarity was tested, these DNAs from both mouse and human confer growth, and the growth phenotype is more vigorous than the two sub-fragments (FIG. 16, c and f). These results demonstrate that the STAR activity of human STAR18 (SEQ ID NO:18) is conserved in its ortholog from mouse. The high sequence conservation between these orthologous regions is particularly noteworthy because they are not protein-coding sequences, leading to the conclusion that they have some regulatory function that has prevented their evolutionary divergence through mutation.
This analysis demonstrates that cloned STAR elements identified by the original screening program may in some cases represent partial STAR elements, and that analysis of the genomic DNA in which they are embedded can identify sequences with stronger STAR activity.
Example 12
STAR Elements Contain Characteristic DNA Sequence Motifs
STAR elements are isolated on the basis of their anti-repression phenotype with respect to transgene expression. This anti-repression phenotype reflects underlying biochemical processes that regulate chromatin formation which are associated with the STAR elements. These processes are typically sequence-specific and result from protein binding or DNA structure. This suggests that STAR elements will share DNA sequence similarity. Identification of sequence similarity among STAR elements will provide sequence motifs that are characteristic of the elements that have already been identified by functional screens and tests. The sequence motifs will also be useful to recognize and claim new STAR elements whose functions conform to the claims of this patent. The functions include improved yield and stability of transgenes expressed in eukaryotic host cells.
Other benefits of identifying sequence motifs that characterize STAR elements include: (1) provision of search motifs for prediction and identification of new STAR elements in genome databases, (2) provision of a rationale for modification of the elements, and (3) provision of information for functional analysis of STAR activity. Using bio-informatics, sequence similarities among STAR elements have been identified; the results are presented in this example.
Bio-Informatic and Statistical Background
Regulatory DNA elements typically function via interaction with sequence-specific DNA-binding proteins. Bio-informatic analysis of DNA elements, such as STAR elements whose regulatory properties have been identified, but whose interacting proteins are unknown, requires a statistical approach for identification of sequence motifs. This can be achieved by a method that detects short DNA sequence patterns that are over-represented in a set of regulatory DNA elements (e.g., the STAR elements) compared to a reference sequence (e.g., the complete human genome). The method determines the number of observed and expected occurrences of the patterns in each regulatory element. The number of expected occurrences is calculated from the number of observed occurrences of each pattern in the reference sequence.
The DNA sequence patterns can be oligonucleotides of a given length, e.g., six base pairs. In the simplest analysis, for a six-base-pair oligonucleotide (hexamer) composed of the four nucleotides (A, C, G, and T) there are 46=4096 distinct oligonucleotides (all combinations from AAAAAA (SEQ ID NO:121) to TTTTTT (SEQ ID NO:122)). If the regulatory and reference sequences were completely random and had equal proportions of the A, C, G, and T nucleotides, then the expected frequency of each hexamer would be 1/4096 ( ̃0.00024). However, the actual frequency of each hexamer in the reference sequence is typically different than this due to biases in the content of G:C base pairs, etc. Therefore, the frequency of each oligonucleotide in the reference sequence is determined empirically by counting, to create a “frequency table” for the patterns.
The pattern frequency table of the reference sequence is then used to calculate the expected frequency of occurrence of each pattern in the regulatory element set. The expected frequencies are compared with the observed frequencies of occurrence of the patterns. Patterns that are “over-represented” in the set are identified; for example, if the hexamer ACGTGA (SEQ ID NO:123) is expected to occur five times in 20 kilobase pairs of sequence, but is observed to occur 15 times, then it is three-fold over-represented. Ten of the 15 occurrences of that hexameric sequence pattern would not be expected in the regulatory elements if the elements had the same hexamer composition as the entire genome. Once the over-represented patterns are identified, a statistical test is applied to determine whether their over-representation is significant, or may be due to chance. For this test, a significance index, “sig,” is calculated for each pattern. The significance index is derived from the probability of occurrence of each pattern, which is estimated by a binomial distribution. The probability takes into account the number of possible patterns (4096 for hexamers). The highest sig values correspond to the most overrepresented oligonucleotides (van Helden et al., 1998). In practical terms, oligonucleotides with sig ≧0 are considered as over-represented. A pattern with sig ≧0 is likely to be over-represented due to chance once (=100) in the set of regulatory element sequences. However, at sig ≧1 a pattern is expected to be over-represented once in ten (=101) sequence sets, sig ≧2 once in 100 (=102) sequence sets, etc.
The patterns that are significantly over-represented in the regulatory element set are used to develop a model for classification and prediction of regulatory element sequences. This employs Discriminant Analysis, a so-called “supervised” method of statistical classification known to one of ordinary skill in the art (Huberty, 1994). In Discriminant Analysis, sets of known or classified items (e.g., STAR elements) are used to “train” a model to recognize those items on the basis of specific variables (e.g., sequence patterns such as hexamers). The trained model is then used to predict whether other items should be classified as belonging to the set of known items (e.g., is a DNA sequence a STAR element). In this example, the known items in the training set are STAR elements (positive training set). They are contrasted with sequences that are randomly selected from the genome (negative training set) which have the same length as the STAR elements. Discriminant Analysis establishes criteria for discriminating positives from negatives based on a set of variables that distinguish the positives; in this example, the variables are the significantly over-represented patterns (e.g., hexamers).
When the number of over-represented patterns is high compared to the size of the training set, the model could become biased due to over-training. Over-training is circumvented by applying a forward stepwise selection of variables (Huberty, 1994). The goal of Stepwise Discriminant Analysis is to select the minimum number of variables that provides maximum discrimination between the positives and negatives. The model is trained by evaluating variables one-by-one for their ability to properly classify the items in the positive and negative training sets. This is done until addition of new variables to the model does not significantly increase the model's predictive power (i.e., until the classification error rate is minimized). This optimized model is then used for testing, in order to predict whether “new” items are positives or negatives (Huberty, 1994).
It is inherent in classification statistics that for complex items such as DNA sequences, some elements of the positive training set will be classified as negatives (false negatives), and some members of the negative training set will be classified as positives (false positives). When a trained model is applied to testing new items, the same types of misclassifications are expected to occur.
In the bio-informatic method described here, the first step, Pattern Frequency Analysis, reduces a large set of sequence patterns (e.g., all 4096 hexamers) to a smaller set of significantly over-represented patterns (e.g., 100 hexamers); in the second step, Stepwise Discriminant Analysis reduces the set of over-represented patterns to the subset of those patterns that have maximal discriminative power (e.g., five to ten hexamers). Therefore, this approach provides simple and robust criteria for identifying regulatory DNA elements such as STAR elements.
DNA-binding proteins can be distinguished on the basis of the type of binding site they occupy. Some recognize contiguous sequences; for this type of protein, patterns that are oligonucleotides of length six base pairs (hexamers) are fruitful for bio-informatic analysis (van Helden et al., 1998). Other proteins bind to sequence dyads: contact is made between pairs of highly conserved trinucleotides separated by a non-conserved region of fixed width (van Helden et al., 2000). In order to identify sequences in STAR elements that may be bound by dyad-binding proteins, frequency analysis was also conducted for this type of pattern, where the spacing between the two trinucleotides was varied from 0 to 20 (i.e., XXXN{0-20}XXX where X's are specific nucleotides composing the trinucleotides, and N's are random nucleotides from 0 to 20 base pairs in length). The results of dyad frequency analysis are also used for Linear Discriminant Analysis as described above.
Materials and Methods
Using the genetic screen described in the original patent application, sixty-six (66) STAR elements were initially isolated from human genomic DNA and characterized in detail (Table 3). The screen was performed on gene libraries constructed by Sau3AI digestion of human genomic DNA, either purified from placenta (Clontech 6550-1) or carried in bacterial/P1 (BAC/PAC) artificial chromosomes. The BAC/PAC clones contain genomic DNA from regions of chromosome 1 (clones RP1154H19 and RP3328E19), from the HOX cluster of homeotic genes (clones RP1167F23, RP1170019, and RP11387A1), or from human chromosome 22 (Research Genetics 96010-22). The DNAs were size-fractionated, and the 0.5-2 kb size fraction was ligated into BamHI-digested pSelect vector, by standard techniques (Sambrook et al., 1989). pSelect plasmids containing human genomic DNA that conferred resistance to zeocin at low doxycycline concentrations were isolated and propagated in Escherichia coli. The screens that yielded the STAR elements of Table 3 have assayed approximately 1-2% of the human genome.
The human genomic DNA inserts in these 66 plasmids were sequenced by the dideoxy method (Sanger et al., 1977) using a Beckman CEQTM2000 automated DNA sequencer, using the manufacturer's instructions. Briefly, DNA was purified from E. coli using QIAprep® Spin Miniprep and Plasmid Midi Kits (QIAGEN® 27106 and 12145, respectively). Cycle sequencing was carried out using custom oligonucleotides corresponding to the pSelect vector (primers D89 (SEQ ID NO:149) and D95 (SEQ ID NO:154), Table 2), in the presence of dye terminators (CEQTM Dye Terminator Cycle Sequencing Kit, Beckman 608000). Assembled STAR DNA sequences were located in the human genome (database builds August and December 2001) using BLAT (Basic Local Alignment Tool (Kent, 2002); http://genome.ucsc.edu/cgi-bin/hgGateway; Table 3). In aggregate, the combined STAR sequences comprise 85.6 kilobase pairs, with an average length of 1.3 kilobase pairs.
Sequence motifs that distinguish STAR elements within human genomic DNA were identified by bio-informatic analysis using a two-step procedure, as follows (see FIG. 17 for a schematic diagram). The analysis has two input datasets: (1) the DNA sequences of the STAR elements (STAR1-STAR65 (SEQ ID NOS:1-65) were used; Table 3); and (2) the DNA sequence of the human genome (except for chromosome 1, which was not feasible to include due to its large size; for dyad analysis a random subset of human genomic DNA sequence ( ̃27 Mb) was used).
Pattern Frequency Analysis
The first step in the analysis uses RSA-Tools software (Regulatory Sequence Analysis Tools; http://www.ucmb.ulb.ac.be/bioinformatics/rsa-tools/; references (van Helden et al., 1998, van Helden et al., 2000, van Helden et al., 2000)) to determine the following information: (1) the frequencies of all dyads and hexameric oligonucleotides in the human genome; (2) the frequencies of the oligonucleotides and dyads in the 65 STAR elements; and (3) the significance indices of those oligonucleotides and dyads that are over-represented in the STAR elements compared to the genome. A control analysis was done with 65 sequences that were selected at random from the human genome (i.e., from 2689×103 kilobase pairs) that match the length of the STAR elements of Table 3.
Discriminant Analysis
The over-represented oligonucleotides and dyads were used to train models for prediction of STAR elements by Linear Discriminant Analysis (Huberty, 1994). A pre-selection of variables was performed by selecting the 50 patterns with the highest individual discriminatory power from the over-represented oligos or dyads of the frequency analyses. These pre-selected variables were then used for model training in a Stepwise Linear Discriminant Analysis to select the most discriminant combination of variables (Huberty, 1994). Variable selection was based on minimizing the classification error rate (percentage of false negative classifications). In addition, the expected error rate was estimated by applying the same discriminant approach to the control set of random sequences (minimizing the percentage of false positive classifications).
The predictive models from the training phase of Discriminant Analysis were tested in two ways. First, the STAR elements and random sequences that were used to generate the model (the training sets) were classified. Second, sequences in a collection of 19 candidate STAR elements (recently cloned by zeocin selection as described above) were classified. These candidate STAR elements are listed in Table 8 (SEQ ID NOS:66-84).
Results
Pattern frequency analysis was performed with RSA-Tools on 65 STAR elements, using the human genome as the reference sequence. One hundred sixty-six (166) hexameric oligonucleotides were found to be over-represented in the set of STAR elements (sig ≧0) compared to the entire genome (Table 6). The most significantly over-represented oligonucleotide, CCCCAC (SEQ ID NO:177), occurs 107 times among the 65 STAR elements, but is expected to occur only 49 times. It has a significance coefficient of 8.76; in other words, the probability that its over-representation is due to random chance is 1/108.76, i.e., less than one in 500 million.
Ninety-five of the oligonucleotides have a significance coefficient greater than one, and are, therefore, highly over-represented in the STAR elements. Among the over-represented oligonucleotides, their observed and expected occurrences, respectively, range from 6 and 1 (for oligo 163, CGCGAA (SEQ ID NO:339), sig=0.02) to 133 and 95 (for oligo 120, CCCAGG (SEQ ID NO:296), sig=0.49). The differences in expected occurrences reflect factors such as the G:C content of the human genome. Therefore, the differences among the oligonucleotides in their number of occurrences is less important than their over-representation; for example, oligo 2 (CAGCGG (SEQ ID NO:178)) is 36/9=four-fold over-represented, which has a probability of being due to random chance of one in fifty million (sig=7.75).
Table 6 also presents the number of STAR elements in which each over-represented oligonucleotide is found. For example, the most significant oligonucleotide, oligo 1 (CCCCAC (SEQ ID NO:177)), occurs 107 times, but is found in only 51 STARs, i.e., on average it occurs as two copies per STAR. The least abundant oligonucleotide, number 166 (AATCGG (SEQ ID NO:342)), occurs on average as a single copy per STAR (thirteen occurrences on eleven STARs); single-copy oligonucleotides occur frequently, especially for the lower-abundance oligos. At the other extreme, oligo 4 (CAGCCC (SEQ ID NO:527)) occurs on average three times in those STARs in which it is found (37 STARs). The most widespread oligonucleotide is number 120 (CCCAGG (SEQ ID NO:296)), which occurs on 58 STARs (on average twice per STAR), and the least widespread oligonucleotide is number 114 (CGTCGC (SEQ ID NO:290)), which occurs on only six STARs (and on average only once per STAR).
Results of dyad frequency analysis are given in Table 7. Seven hundred thirty (730) dyads were found to be over-represented in the set of STAR elements (sig ≧0) compared to the reference sequence. The most significantly over-represented dyad, CCCN{2}CGG (SEQ ID NO:343), occurs 36 times among the 65 STAR elements, but is expected to occur only seven times. It has a significance coefficient of 9.31; in other words, the probability that its over-representation is due to chance is 1/109.31, i.e., less than one in two billion.
Three hundred ninety-seven (397) of the dyads have a significance coefficient greater than 1, and are, therefore, highly over-represented in the STAR elements. Among the over-represented dyads, their observed and expected occurrences, respectively, range from 9 and 1 (for five dyads (numbers 380, 435, 493, 640, and 665)) to 118 and 63 (for number 30 (AGGN{2}GGG (SEQ ID NO:372)), sig=4.44).
The oligonucleotides and dyads found to be over-represented in STAR elements by pattern frequency analysis were tested for their discriminative power by Linear Discriminant Analysis. Discriminant models were trained by step-wise selection of the best combination among the 50 most discriminant oligonucleotide (Table 6) or dyad (Table 7) patterns. The models achieved optimal error rates after incorporation of four (dyad) or five variables. The discriminative variables from oligo analysis are numbers 11, 30, 94, 122, and 160 (Table 6); those from dyad analysis are numbers 73, 194, 419, and 497 (Table 7).
The discriminant models were then used to classify the 65 STAR elements in the training set and their associated random sequences. The model using oligonucleotide variables classifies 46 of the 65 STAR elements as STAR elements (true positives); the dyad model classifies 49 of the STAR elements as true positives. In combination, the models classify 59 of the 65 STAR elements as STAR elements (91%; FIG. 18). The false positive rates (random sequences classified as STARs) were seven for the dyad model, eight for the oligonucleotide model, and 13 for the combined predictions of the two models (20%). The STAR elements of Table 3 that were not classified as STARs by LDA are STAR7, STAR22, STAR35, STAR44, STAR46, and STAR65 (SEQ ID NOS:7, 22, 35, 44, 46 and 65, respectively). These elements display stabilizing anti-repressor activity in functional assays, so the fact that they are not classified as STARs by LDA suggests that they represent another class (or classes) of STAR elements.
The models were then used to classify the 19 candidate STAR elements in the testing set listed in Table 8. The dyad model classifies 12 of these candidate STARs as STAR elements, and the oligonucleotide model classifies 14 as STARs. The combined number of the candidates that are classified as STAR elements is 15 (79%). This is a lower rate of classification than obtained with the training set of 65 STARs; this is expected for two reasons. First, the discriminant models were trained with the 65 STARs of Table 3, and discriminative variables based on this training set may be less well represented in the testing set. Second, the candidate STAR sequences in the testing set have not yet been fully characterized in terms of in vivo function, and may include elements with only weak anti-repression properties.
This analysis demonstrates the power of a statistical approach to bio-informatic classification of STAR elements. The STAR sequences contain a number of dyad and hexameric oligonucleotide patterns that are significantly over-represented in comparison with the human genome as a whole. These patterns may represent binding sites for proteins that confer STAR activity; in any case they form a set of sequence motifs that can be used to recognize STAR element sequences.
Using these patterns to recognize STAR elements by Discriminant Analysis, a high proportion of the elements obtained by the genetic screen of the invention are in fact classified as STARs. This reflects underlying sequence and functional similarities among these elements. An important aspect of the method described here (pattern frequency analysis followed by Discriminant Analysis) is that it can be reiterated; for example, by including the 19 candidate STAR elements of Table 8 with the 66 STAR elements of Table 3 into one training set, an improved discriminant model can be trained. This improved model can then be used to classify other candidate regulatory elements as STARs. Large-scale in vivo screening of genomic sequences using the method of the invention, combined with reiteration of the bio-informatic analysis, will provide a means of discriminating STAR elements that asymptotically approaches 100% recognition and prediction of elements as the genome is screened in its entirety. These stringent and comprehensive predictions of STAR function will ensure that all human STAR elements are recognized, and are available for use in improving transgene expression.
Example 13
Cloning and Characterization of STAR Elements from Arabidopsis thaliana 
Transgene silencing occurs in transgenic plants at both the transcriptional and post-transcriptional levels (Meyer, 2000, Vance and Vaucheret, 2001). In either case, the desired result of transgene expression can be compromised by silencing; the low expression and instability of the transgene results in poor expression of desirable traits (e.g., pest resistance) or low yields of recombinant proteins. It also results in poor predictability: the proportion of transgenic plants that express the transgene at biotechnologically useful levels is low, which necessitates laborious and expensive screening of transformed individuals for those with beneficial expression characteristics. This example describes the isolation of STAR elements from the genome of the dicot plant Arabidopsis thaliana for use in preventing transcriptional transgene silencing in transgenic plants. Arabidopsis was chosen for this example because it is a well-studied model organism: it has a compact genome, it is amenable to genetic and recombinant DNA manipulations, and its genome has been sequenced (Bevan et al., 2001, Initiative, 2000, Meinke et al., 1998).
Materials and Methods
Genomic DNA was isolated from Arabidopsis thaliana ecotype Columbia as described (Stam et al., 1998) and partially digested with MboI. The digested DNA was size-fractionated to 0.5-2 kilobase pairs by agarose gel electrophoresis and purification from the gel (QIAquick® Gel Extraction Kit, QIAGEN® 28706), followed by ligation into the pSelect vector (supra). Transfection into the U-2 OS/Tet-Off/LexA-HP1 cell line and selection for zeocin resistance at low doxycycline concentration was performed as described (supra). Plasmids were isolated from zeocin resistant colonies and re-transfected into the U-2 OS/Tet-Off/LexA-HP1 cell line.
Sequencing of Arabidopsis genomic DNA fragments that conferred zeocin resistance upon re-transfection was performed as described (supra). The DNA sequences were compared to the sequence of the Arabidopsis genome by BLAST analysis ((Altschul et al., 1990); URL http://www.ncbi.nlm.nih.gov/blast/Blast).
STAR activity was tested further by measuring mRNA levels for the hygromycin- and zeocin-resistance genes in recombinant host cells by reverse transcription PCR (RT-PCR). Cells of the U-2 OS/Tet-Off/lexA-HP1 cell line were transfected with pSelect plasmids containing Arabidopsis STAR elements, the Drosophila scs element, or containing no insert (supra). These were cultivated on hygromycin for two weeks at high doxycycline concentration, then the doxycycline concentration was lowered to 0.1 ng/ml to induce the lexA-HP1 repressor protein. After ten days, total RNA was isolated by the RNeasy® mini kit (QIAGEN® 74104) as described by the manufacturer. First-strand cDNA synthesis was carried out using the RevertAidTM First Strand cDNA Synthesis kit (MBI Fermentas 1622) using oligo(dT)18 primer as described by the manufacturer. An aliquot of the cDNA was used as the template in a PCR reaction using primers D58 (SEQ ID NO:145) and D80 (SEQ ID NO:148) (for the zeocin marker), and D70 (SEQ ID NO:146) and D71 (SEQ ID NO:147) (for the hygromycin marker), and Taq DNA polymerase (Promega M2661). The reaction conditions were 15-20 cycles of 94° C. for one minute, 54° C. for one minute, and 72° C. for 90 seconds. These conditions result in a linear relationship between input RNA and PCR product DNA. The PCR products were resolved by agarose gel electrophoresis, and the zeocin and hygromycin bands were detected by Southern blotting as described (Sambrook et al., 1989), using PCR products produced as above with purified pSelect plasmid as template. The ratio of the zeocin and hygromycin signals corresponds to the normalized expression level of the zeocin gene.
Results
The library of Arabidopsis genomic DNA in the pSelect vector comprised 69,000 primary clones in E. coli, 80% of which carried inserts. The average insert size was approximately 1000 base pairs; the library, therefore, represents approximately 40% of the Arabidopsis genome.
A portion of this library (representing approximately 16% of the Arabidopsis genome) was transfected into the U-2 OS/Tet-Off/LexA-HP1 cell line. Hygromycin selection was imposed to isolate transfectants, which resulted in 27,000 surviving colonies. These were then subjected to zeocin selection at low doxycycline concentration. Putative STAR-containing plasmids from 56 zeocin-resistant colonies were rescued into E. coli and re-transfected into U-2 OS/Tet-Off/LexA-HP1 cells. Forty-four of these plasmids (79% of the plasmids tested) conferred zeocin resistance on the host cells at low doxycycline concentrations, demonstrating that the plasmids carried STAR elements. This indicates that the pSelect screen in human U-2 OS cells is highly efficient at detection of STAR elements from plant genomic DNA.
The DNA sequences of these 44 candidate STAR elements were determined. Thirty-five of them were identified as single loci in the database of Arabidopsis nuclear genomic sequence (Table 9; SEQ ID NO:85-SEQ ID NO:119). Four others were identified as coming from the chloroplast genome, four were chimeras of DNA fragments from two loci, and one was not found in the Arabidopsis genome database.
The strength of the cloned Arabidopsis STAR elements was tested by assessing their ability to prevent transcriptional repression of the zeocin-resistance gene, using an RT-PCR assay. As a control for RNA input among the samples, the transcript levels of the hygromycin-resistance gene for each STAR transfection were assessed too. This analysis has been performed for 12 of the Arabidopsis STAR elements. The results (FIG. 19) demonstrate that the Arabidopsis STAR elements are superior to the Drosophila scs element (positive control) and the empty vector (“SV40”; negative control) in their ability to protect the zeocin-resistance gene from transcriptional repression. In particular, STAR-A28 (SEQ ID NO:112) and STAR-A30 (SEQ ID NO:114) enable two-fold higher levels of zeocin-resistance gene expression than the scs element (normalized to the internal control of hygromycin-resistance gene mRNA) when the lexA-HP1 repressor is expressed.
These results demonstrate that the method of the invention can be successfully applied to recovery of STAR elements from genomes of other species than human. Its successful application to STAR elements from a plant genome is particularly significant because it demonstrates the wide taxonomic range over which the method of the invention is applicable and because plants are an important target of biotechnological development.
Example 14
STAR Elements Function in CHO Cells
STAR elements function to block the effect of transcriptional repression influences on transgene expression units. Two of the benefits of STAR elements for heterologous protein production are an increased predictability to find high-expressing primary recombinant host cells as well as increased protein production or yield in these cells. Importantly, the disclosed STAR elements are human DNA sequences, isolated in the human U-2 OS osteosarcoma cell line. It is, therefore, an important question whether the human STAR elements are functional in a) cell lines derived from species other than man, and/or in b) human cell lines other than the U-2 OS osteosarcoma cell line. In this example the functionality of STAR 7 (SEQ ID NO:7) in (CHO) Chinese hamster ovary are illustrated.
Material and Methods
The STAR7 (SEQ ID NO:7) element is tested in the ppGIZ-STAR7 vector (FIG. 20). The construction of the pPlug&Play-GFP-ires-Zeo (ppGIZ) vector is described below. Plasmid pGFP (Clontech 6010-1) is modified by insertion of a linker at the BsiWI site to yield pGFP-link. The linker (made by annealing oligonucleotides 5′ GTACGGATATCAGATCTTTAATTAAG 3′ (SEQ ID NO:124) and 5′ GTACCTTAATTAAAGATCTGATATCC 3′ (SEQ ID NO:125)) introduces sites for the PacI, BglII, and EcoRV restriction endonucleases. This creates the multiple cloning site MCSII for insertion of STAR elements. Then primers 5′ ATCAGATCTGGCGCGCCATTTAAATCGTC TCGCGCGTTTCGGTGATGACGG 3′ (SEQ ID NO:126) and 5′ AGGCGGATCCGAATG TATTTAGAAAAATAAACAAATAGGGG 3′ (SEQ ID NO:127) are used to amplify a region of 0.37 kb from pGFP, which is inserted into the BglII site of pIRES (Clontech 6028-1) to yield pIRES-stuf. This introduces sites for the AscI and SwaI restriction endonucleases at MCSI, and acts as a “stuffer fragment” to avoid potential interference between STAR elements and adjacent promoters. pIRES-stuf is digested with BglII and FspI to liberate a DNA fragment composed of the stuffer fragment, the CMV promoter, the IRES element (flanked by multiple cloning sites MCS A and MCS B), and the SV40 polyadenylation signal. This fragment is ligated with the vector backbone of pGFP-link produced by digestion with BamHI and StuI, to yield pIRES-link.
The open reading frames of the zeocin-resistance gene is inserted into the BamHI/NotI sites of MCS B in pIRES-link as follows: the zeocin-resistance ORF is amplified by PCR with primers 5′ GATCGGATCCTTCGAAATGGCCAAGTTGACCAGTGC 3′ (SEQ ID NO:128) and 5′ AGGCGCGGCCGCAATTCTCAGTCCTGCTCCTC 3′ (SEQ ID NO:129) from plasmid pEM7/zeo, digested with BamHI and NotI, and ligated with BamHI/NotI-digested pIRES-link to yield pIRES-link-zeo. The GFP reporter ORF was introduced into pIRES-link-zeo by amplification of phr-GFP-1 with primers 5′ GATCGAATTCTCGCGAATGGTGAGCAAGCAGATCCTGAAG 3′ (SEQ ID NO:130) and 5′ AGGCGAATTCACCGGTGTTTAAACTTACACCCACTCGTGCAGGCTGCCCAGG 3′ (SEQ ID NO:131), and insertion of the EcoRI-digested GFP cassette into the EcoRI site in MCS A of the pIRES-link-zeo plasmid. This created the ppGIZ (for ppGFP-IRES-zeo). STAR7 (SEQ ID NO:7) is cloned into the SalI site (5′) and into the PacI site (3′).
Transfection and Culture of CHO Cells
The Chinese Hamster Ovary cell line CHO-K1 (ATCC CCL-61) is cultured in HAMS-F12 medium+10% Fetal Calf Serum containing 2 mM glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomycin at 37° C./5% CO2. Cells are transfected with the plasmids using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Briefly, cells are seeded to culture vessels and grown overnight to 70-90% confluence. Lipofectamine reagent is combined with plasmid DNA at a ratio of 7.5 microliters per 3 microgram (e.g., for a 10 cm Petri dish, 20 micrograms DNA and 120 microliters Lipofectamine) and added after a 30-minute incubation at 25° C. to the cells. After a six-hour incubation, the transfection mixture is replaced with fresh medium, and the transfected cells are incubated further. After overnight cultivation, cells are trypsinized and seeded into fresh petri dishes with fresh medium with zeocin added to a concentration of 100 μg/ml and the cells are cultured further. When individual colonies become visible (approximately ten days after transfection) medium is removed and replaced with fresh medium without zeocin. Individual clones are isolated and transferred to 24-well plates in medium with zeocin. Expression of the GFP reporter gene is assessed approximately three weeks after transfection.
The tested constructs consist of a bicistronic gene with the GFP gene, an IRES and the Zeocin resistance gene under control of the CMV promoter, but either with or without STAR7 (SEQ ID NO:7) element to flank the entire construct (FIG. 20). The constructs are transfected to CHO-K1 cells. Stable colonies are expanded before the GFP signal is determined on a XL-MCL Beckman Coulter flow cytometer. The mean of the GFP signal is taken as measure for the level of GFP expression and this is plotted in FIG. 20.
Results
FIG. 20 shows that flanking a GFP reporter gene that is under the control of the CMV promoter results in a higher number of CHO colonies that express significantly higher levels of GFP protein, as compared to the control without STAR7 (SEQ ID NO:7) element. The STAR7 (SEQ ID NO:7) element, therefore, conveys a higher degree of predictability of transgene expression in CHO cells. The highest GFP expression level in STAR-shielded CHO colonies is also higher than in STAR-less control colonies. In addition, when the tested colonies were further grown for another 30 days without Zeocin in the culture medium, the GFP expression levels in the STAR-shielded colonies remained equally high, whereas the GFP expression levels in the STAR-less colonies dropped to at least below 50% of the original values. It is, therefore, concluded that STAR7 (SEQ ID NO:7) is able to convey higher as well as more stable expression levels to a transgene in CHO cells, this being a cell line derived from another species than man.
Example 15
STAR Elements Function in NSO Cells
STAR elements function to block the effect of transcriptional repression influences on transgene expression units. Two of the benefits of STAR elements for heterologous protein production are an increased predictability to find high-expressing primary recombinant host cells, as well as increased protein production or yield in these cells. Importantly, the disclosed STAR elements are human DNA sequences, isolated in the human U-2 OS osteosarcoma cell line. It is, therefore, an important question whether the human STAR elements are functional in a) cell lines derived from species other than man, and/or in b) human cell lines other than the U-2 OS osteosarcoma cell line. In this example the functionality of STAR 7 (SEQ ID NO:7) in non-secreting mouse myeloma (NSO) cells are illustrated.
Materials and Methods
The tested constructs are the same as described in Example 14. NSO (Non-Secreting mouse myeloma) cells (ECACC 85110503) are suspension cells that are cultured in RPMI 1640 medium+10% Fetal Calf Serum containing 2 mM glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomycin at 37° C./5% CO2. Cells are transfected with the plasmids using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Briefly, cells are seeded to culture vessels and grown overnight to 4×105/ml. Lipofectamine reagent is combined with plasmid DNA at a ratio of 3 microliters per microgram DNA (e.g., for a 10 cm Petri dish, 20 micrograms DNA and 60 microliters Lipofectamine) and added after 30 minutes incubation at 25° C. temperature to the cells. After overnight incubation, the transfection mixture is replaced with fresh medium and the transfected cells are incubated further. After another overnight incubation, zeocin is added to a concentration of 100 μg/ml and the cells are cultured and further incubated for three days. Then the cells are seeded in 96-well plates in such dilutions that one well will contain  ̃1 cell. After ten days growing colonies are transferred to 24-well plates.
Results
FIG. 21 shows that flanking a GFP reporter gene that is under the control of the CMV promoter results in a higher number of NSO colonies that express significantly higher levels of GFP protein, as compared to the control without STAR7 (SEQ ID NO:7) element. The STAR7 (SEQ ID NO:7) element, therefore, conveys a higher degree of predictability of transgene expression in NSO cells. The highest GFP expression level in STAR-shielded NSO colonies is also higher than in STAR-less control colonies. It is, therefore, concluded that STAR7 (SEQ ID NO:7) is able to convey higher expression levels to a transgene in NSO cells, this being a cell line derived from another species than man.
Example 16
STAR Elements Function in Human 293 Cells
STAR elements function to block the effect of transcriptional repression influences on transgene expression units. Two of the benefits of STAR elements for heterologous protein production are an increased predictability to find high-expressing primary recombinant host cells as well as increased protein production or yield in these cells. Importantly, the disclosed STAR elements are human DNA sequences, isolated in the human U-2 OS osteosarcoma cell line. It is, therefore, an important question whether the human STAR elements are functional in a) cell lines derived from species other than man, and/or in b) human cell lines other than the U-2 OS osteosarcoma cell line. In this example, the functionality of STAR7 (SEQ ID NO:7) in human 293 cells are illustrated.
Materials and Methods
The tested constructs are the same as described in Example 14. The 293 cell line (ATCC CRL-1573) is derived from human embryonal kidney (immortalized by adenovirus 5 transfection) and is cultured in Dulbecco's Modified Eagle Medium+10% Fetal Calf Serum containing 2 mM glutamine, 100 U/ml penicillin, and 100 micrograms/ml streptomycin at 37° C./5% CO2. Cells are transfected with the plasmids using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Selection and propagation of the 293 colonies are as described in Example 14 for U-2 OS cells.
Results
FIG. 22 shows that flanking a GFP reporter gene that is under the control of the CMV promoter results in a higher number of 293 colonies that express significantly higher levels of GFP protein, as compared to the control without STAR7 (SEQ ID NO:7) element. The STAR7 (SEQ ID NO:7) element, therefore, conveys a higher degree of predictability of transgene expression in 293 cells. The highest GFP expression level in STAR-shielded colonies is also higher than in STAR-less control colonies. It is, therefore, concluded that STAR7 (SEQ ID NO:7) is able to convey higher expression levels to a transgene in 293 cells, this being another human cell line, distinct from the human U-2 OS cell line.







TABLE 1







Biopharmaceutical Proteins, Their Tissue or Cell Type of Origin










Protein
Tissue/Somatic Cells
Cell Lines (ATCC #)1
Indications





alpha-1 Antitrypsin
Liver, leukocytes
Hep G2 (HB-8065)
Cystic fibrosis, emphysema


alpha-Galactosidase A; -Glucosidase
Fibroblasts
WI 38 (CCL-75)
Fabry disease; Pompe's





disease


Antibodies (monoclonal,
Lymphocytes
Transfectomas
Various therapeutic


single-chain, etc)


strategies


Antithrombin III
Liver
Hep G2 (HB-8065)
Thrombophilia


Calcitonin
Thyroid (parafollicular cells)
TT (CRL-1803)
Osteoporosis


Ciliary neurotrophic factor
Neural tissue (e.g., astrocytes)
HCN-1A (CRL-10442)
Motor neuron disease


Epidermal Growth Factor
Kidney
G-401 (CRL-1441)
Wound healing


Erythropoietin
Liver, kidney
Hep G2 (HB-8065),
Anemia




G-401 (CRL-1441)


Factors VII, VIII, IX
Endothelial cells
HUV-EC-C (CRL-1730)
Hemophilia


Famoxin (recombinant gAcrp30)
Adipocytes
NA2
Obesity


Fibroblast growth factor (basic)
Cerebral cortex, hypothalamus
HCN-1A (CRL-10442)
Wound healing, angiogenesis


Gastric lipase
Pancreas
BxPC-3 (CRL-1687)
Pancreatic insufficiency,





cystic fibrosis


Glucocerebrosidase
Macrophages
U-937 (CRL-1593.2)
Gaucher disease


Granulocyte macrophage-colony
T-lymphocytes
J.CaM1.6 (CRL-2063)
Chemotherapy neutropenia


stimulatin factor


Human growth hormone
Pituitary gland
HP75 (CRL-2506)
Growth retardation, Turner's


(somatotropin)


syndrome


Human serum albumin
Liver (hepatocytes)
Hep G2 (HB-8065)
Blood replacement (surgery,





burns)


Insulin
Pancreas (Islet beta cells)
BxPC-3 (CRL-1687)
Diabetes


Interferons alpha
Leukocytes
WBC264-9C (HB-8902)
Cancer, hepatitis C


Interferons beta
Fibroblasts
WI 38 (CCL-75)
Multiple sclerosis


Interleukin-2, -4, -10
T-lymphocytes
J.CaM1.6 (CRL-2063)
Cancer, rheumatoid arthritis,





hepatitis


Interleukin-18
Monocytes and macrophages
U-937 (CRL-1593.2)
Cancer, bacterial infections


Interleukin-1 Receptor Antagonist
Epithelium
HBE4-E6/E7 (CRL-2078)
Rheumatoid arthritis


Soluble Tumor Necrosis Factor
Placenta, spleen, fibroblasts
BeWo (CCL-98)
Rheumatoid arthritis,


receptor


multiple sclerosis


van Willebrand's factor
Endothelial cells
HUV-EC-C (CRL-1730)
Hemophilia





1These cell lines are offered only as examples of cultured cells corresponding to the tissues and somatic cells; ATCC #: American Type Culture Collection accession number


2NA: Not Available; adipocytes can be differentiated from various other cell types














TABLE 2







Oligonucleotides used for polymerase chain



reactions (PCR primers) or DNA mutagenesis


(SEQ ID NOS:134-176)









SEQ ID NO:
Number
Sequence













134
C65
AACAAGCTTGATATCAGATCTGCTAGCTTGGT





CGAGCTGATACTTCCC


 


135
C66
AAACTCGAGCGGCCGCGAATTCGTCGACTTTA




CCACTCCCTATCAGTGATAGAG


 


136
C67
AAACCGCGGCATGGAAGACGCCAAAAACATAA




AGAAAGG


 


137
C68
TATGGATCCTAGAATTACACGGCGATCTTTCC


 


138
C81
AAACCATGGCCGAGTACAAGCCCACGGTGCG




CC


 


139
C82
AAATCTAGATCAGGCACCGGGCTTGCGGGTCA




TGC


 


140
C85
CATTTCCCCGAAAAGTGCCACC


 


141
D30
TCACTGCTAGCGAGTGGTAAACTC


 


142
D41
GAAGTCGACGAGGCAGGCAGAAGTATGC


 


143
D42
GAGCCGCGGTTTAGTTCCTCACCTTGTCG


 


144
D51
TCTGGAAGCTTTGCTGAAGAAAC


 


145
D58
CCAAGTTGACCAGTGCC


 


146
D70
TACAAGCCAACCACGGCCT


 


147
D71
CGGAAGTGCTTGACATTGGG


 


148
D80
GTTCGTGGACACGACCTCCG


 


149
D89
GGGCAAGATGTCGTAGTCAGG


 


150
D90
AGGCCCATGGTCACCTCCATCGCTACTGTG


 


151
D91
CTAATCACTCACTGTGTAAT


 


152
D93
AATTACAGGCGCGCC


 


153
D94
AATTGGCGCGCCTGT


 


154
D95
TGCTTTGCATACTTCTGCCTGCCTC


 


155
E12
TAGGGGGGATCCAAATGTTC


 


156
E13
CCTAAAAGAAGATCTTTAGC


 


157
E14
AAGTGTTGGATCCACTTTGG


 


158
E15
TTTGAAGATCTACCAAATGG


 


159
E16
GTTCGGGATCCACCTGGCCG


 


160
E17
TAGGCAAGATCTTGGCCCTC


 


161
E18
CCTCTCTAGGGATCCGACCC


 


162
E19
CTAGAGAGATCTTCCAGTAT


 


163
E20
AGAGTTCCGGATCCGCCTGG


 


164
E21
CCAGGCAGACTCGGAACTCT


 


165
E22
TGGTGAAACCGGATCCCTAC


 


166
E23
AGGTCAGGAGATCTAGACCA


 


167
E25
CCATTTTCGCTTCCTTAGCTCC


 


168
E42
CGATGTAACCCACTCGTGCACC


 


169
E57
AGAGATCTAGGATAATTTCG


 


170
E92
AGGCGCTAGCACGCGTTCTACTCTTTTCCTAC




TCTG


 


171
E93
GATCAAGCTTACGCGTCTAAAGGCATTTTATA




TAG


 


172
E94
AGGCGCTAGCACGCGTTCAGAGTTAGTGATCC




AGG


 


173
E95
GATCAAGCTTACGCGTCAGTAAAGGTTTCGTA




TGG


 


174
E96
AGGCGCTAGCACGCGTTCTACTCTTTCATTAC




TCTG


 


175
E97
CGAGGAAGCTGGAGAAGGAGAAGCTG


 


176
E98
CAAGGGCCGCAGCTTACACATGTTC
















TABLE 3







STAR elements of the invention, including genomic location and


length (SEQ ID NOS: 1-66)










STAR
SEQ ID NO:
Location1
Length













1
1
2q31.1
750


2
2
7p15.2
916


3
3
15q11.2 and 10q22.2
2132


4
4
1p31.1 and 14q24.1
1625


5
5
20q13.32
1571


6
6
2p21
1173


7
7
1q34
2101


8
8
9q32
1839


9
9
10p15.3
1936


10
10
Xp11.3
1167


11
11
2p25.1
1377


12
12
5q35.3
1051


13
13
9q34.3
1291


14
14
22q11.22
732


15
15
1p36.31
1881


16
16
1p21.2
1282


17
17
2q31.1
793


18
18
2q31.3
497


19
19
6p22.1
1840


20
20
8p13.3
780


21
21
6q24.2
620


22
22
2q12.2
1380


23
23
6p22.1
1246


24
24
1q21.2
948


25
25
1q21.3
1067


26
26
1q21.1
540


27
27
1q23.1
1520


28
28
22q11.23
961


29
29
2q13.31
2253


30
30
22q12.3
1851


31
31
9q34.11 and 22q11.21
1165


32
32
21q22.2
771


33
33
21q22.2
1368


34
34
9q34.14
755


35
35
7q22.3
1211


36
36
21q22.2
1712


37
37
22q11.23
1331


38
38
22q11.1 and 22q11.1
~1000


39
39
22q12.3
2331


40
40
22q11.21
1071


41
41
22q11.21
1144


42
42
22q11.1
735


43
43
14q24.3
1231


44
44
22q11.1
1591


45
45
22q11.21
1991


46
46
22q11.23
1871


47
47
22q11.21
1082


48
48
22q11.22
1242


49
49
Chr 12 random clone, and
1015




3q26.32


50
50
6p21.31
2361


51
51
5q21.3
2289


52
52
7p15.2
1200


53
53
Xp11.3
1431


54
54
4q21.1
981


55
55
15q13.1
501


56
56
includes 3p25.3
741


57
57
4q35.2
1371


58
58
21q11.2
1401


59
59
17 random clone
872


60
60
4p16.1 and 6q27
2068


61
61
7p14.3 and 11q25
1482


62
62
14q24.3
1011


63
63
22q13.3
1421


64
64
17q11.2
1414


65
65
7q21.11 = 28.4
1310


66
66
20q13.33 and 6q14.1
~2800





1Chromosomal location is determined by BLAST search of DNA sequence data from the STAR elements against the human genome database. The location is given according to standard nomenclature referring to the cytogenetic ideogram of each chromosome; e.g., 1p2.3 is the third cytogenetic sub-band of the second cytogenetic band of the short arm of chromosome 1 (http://www.ncbi.nlm.nih.gov/Class/MLACourse/Genetics/chrombanding.html). F, forward sequencing reaction result; R, reverse sequencing reaction result.













TABLE 4







STAR elements convey stability over time on transgene


expression1










Cell Divisions2
Luciferase Expression3












STAR6 (SEQ ID NO: 6)
42
18,000


plus puromycin
60
23,000



84
20,000



108
16,000


STAR6 (SEQ ID NO: 6)
84
12,000


without puromycin4
108
15,000



144
12,000





1Plasmid pSDH-Tet-STAR6 was transfected into U-2 OS cells, and clones were isolated and cultivated in doxycycline-free medium as described in Example 1. Cells were transferred to fresh culture vessels weekly at a dilution of 1:20.


2The number of cell divisions is based on the estimation that in one week the culture reaches cell confluence, which represents ~6 cell divisions.


3Luciferase was assayed as described in Example 1.


4After 60 cell divisions the cells were transferred to two culture vessels; one was supplied with culture medium that contained puromycin, as for the first 60 cell divisions, and the second was supplied with culture medium lacking antibiotic.













TABLE 5







Human STAR elements and their putative mouse orthologs and


paralogs












NUMBER
STAR
Human1
Mouse2
Similarity3
SEQ ID NO:















1
 1
2q31.1
2D
600 bp 69%
1


2
 2
7p15.2
6B3
909 bp 89%
2


3
 3a
5q33.3
11B2
248 bp 83%
3


4
 3b
10q22.2
14B
1. 363 bp 89%
3






2. 163 bp 86%


5
 6
2p21
17E4
437 bp 78%
6


6
12
5q35.3
11b1.3
796 bp 66%
12


7
13
9q34.3
2A3
753 bp 77%
13


8
18
2q31.3
2E1
497 bp 72%
18


9
36
21q22.2
16C4
166 bp 79%
36


10
40
22q11.1
6F1
1. 270 bp 75%
40






2. 309 bp 70%


11
50
6p21.31
17B1
1. 451 bp 72%
50






2. 188 bp 80%






3. 142 bp 64%


12
52
7p15.2
6B3
1. 846 bp 74%
52






2. 195 bp 71%


13
53
Xp11.3
XA2
364 bp 64%
53


14
54
4q21.1
5E3
1. 174 bp 80%
54






2. 240 bp 73%






3. 141 bp 67%






4. 144 bp 68%


15
61a
7p14.3
6B3
188 bp 68%
61





1Cytogenetic location of STAR element in the human genome.


2Cytogenetic location of STAR element ortholog in the mouse genome.


3Length of region(s) displaying high sequence similarity, and percentage similarity. In some cases more than one block of high similarity occurs; in those cases, each block is described separately. Similarity <60% is not considered significant.






The patterns are ranked according to significance coefficient. These were determined using RSA-Tools with the sequence of the human genome as reference. Patterns that comprise the most discriminant variables in Linear Discriminant Analysis are indicated with an asterisk. (SEQ ID NOS:177-342)








TABLE 6







Oligonucleotide patterns (6 base pairs) over-represented in



STAR elements.



















Number of





Oligonucleotide
Observed
Expected
Significance
matching
SEQ ID


Number
sequence
occurrences
occurrences
coefficient
STARs
NO:

















1
CCCCAC
107
49
8.76
51
177



 


2
CAGCGG
36
9
7.75
23
178


 


3
GGCCCC
74
31
7.21
34
179


 


4
CAGCCC
103
50
7.18
37
180


 


5
GCCCCC
70
29
6.97
34
181


 


6
CGGGGC
40
12
6.95
18
182


 


7
CCCCGC
43
13
6.79
22
183


 


8
CGGCAG
35
9
6.64
18
184


 


9
AGCCCC
83
38
6.54
40
185


 


10
CCAGGG
107
54
6.52
43
186


 


11
GGACCC *
58
23
6.04
35
187


 


12
GCGGAC
20
3
5.94
14
188


 


13
CCAGCG
34
10
5.9
24
189


 


14
GCAGCC
92
45
5.84
43
190


 


15
CCGGCA
28
7
5.61
16
191


 


16
AGCGGC
27
7
5.45
17
192


 


17
CAGGGG
86
43
5.09
43
193


 


18
CCGCCC
43
15
5.02
18
194


 


19
CCCCCG
35
11
4.91
20
195


 


20
GCCGCC
34
10
4.88
18
196


 


21
GCCGGC
22
5
4.7
16
197


 


22
CGGACC
19
4
4.68
14
198


 


23
CGCCCC
35
11
4.64
19
199


 


24
CGCCAG
28
8
4.31
19
200


 


25
CGCAGC
29
8
4.29
20
201


 


26
CAGCCG
32
10
4
24
202


 


27
CCCACG
33
11
3.97
26
203


 


28
GCTGCC
78
40
3.9
43
204


 


29
CCCTCC
106
60
3.87
48
205


 


30
CCCTGC *
92
50
3.83
42
206


 


31
CACCCC
77
40
3.75
40
207


 


32
GCGCCA
30
10
3.58
23
208


 


33
AGGGGC
70
35
3.55
34
209


 


34
GAGGGC
66
32
3.5
40
210


 


35
GCGAAC
14
2
3.37
13
211


 


36
CCGGCG
17
4
3.33
12
212


 


37
AGCCGG
34
12
3.29
25
213


 


38
GGAGCC
67
34
3.27
40
214


 


39
CCCCAG
103
60
3.23
51
215


 


40
CCGCTC
24
7
3.19
19
216


 


41
CCCCTC
81
44
3.19
43
217


 


42
CACCGC
33
12
3.14
22
218


 


43
CTGCCC
96
55
3.01
42
219


 


44
GGGCCA
68
35
2.99
39
220


 


45
CGCTGC
28
9
2.88
22
221


 


46
CAGCGC
25
8
2.77
19
222


 


47
CGGCCC
28
10
2.73
19
223


 


48
CCGCCG
19
5
2.56
9
224


 


49
CCCCGG
30
11
2.41
17
225


 


50
AGCCGC
23
7
2.34
17
226


 


51
GCACCC
55
27
2.31
38
227


 


52
AGGACC
54
27
2.22
33
228


 


53
AGGGCG
24
8
2.2
18
229


 


54
CAGGGC
81
47
2.18
42
230


 


55
CCCGCC
45
21
2.15
20
231


 


56
GCCAGC
66
36
2.09
39
232


 


57
AGCGCC
21
6
2.09
18
233


 


58
AGGCCC
64
34
2.08
32
234


 


59
CCCACC
101
62
2.05
54
235


 


60
CGCTCA
21
6
2.03
17
236


 


61
AACGCG
9
1
1.96
9
237


 


62
GCGGCA
21
7
1.92
14
238


 


63
AGGTCC
49
24
1.87
36
239


 


64
CCGTCA
19
6
1.78
14
240


 


65
CAGAGG
107
68
1.77
47
241


 


66
CCCGAG
33
14
1.77
22
242


 


67
CCGAGG
36
16
1.76
25
243


 


68
CGCGGA
11
2
1.75
8
244


 


69
CCACCC
87
53
1.71
45
245


 


70
CCTCGC
23
8
1.71
20
246


 


71
CAAGCC
59
32
1.69
40
247


 


72
TCCGCA
18
5
1.68
17
248


 


73
CGCCGC
18
5
1.67
9
249


 


74
GGGAAC
55
29
1.63
39
250


 


75
CCAGAG
93
58
1.57
49
251


 


76
CGTTCC
19
6
1.53
16
252


 


77
CGAGGA
23
8
1.5
19
253


 


78
GGGACC
48
24
1.48
31
254


 


79
CCGCGA
10
2
1.48
8
255


 


80
CCTGCG
24
9
1.45
17
256


 


81
CTGCGC
23
8
1.32
14
257


 


82
GACCCC
47
24
1.31
33
258


 


83
GCTCCA
66
38
1.25
39
259


 


84
CGCCAC
33
15
1.19
21
260


 


85
GCGGGA
23
9
1.17
18
261


 


86
CTGCGA
18
6
1.15
15
262


 


87
CTGCTC
80
49
1.14
50
263


 


88
CAGACG
23
9
1.13
19
264


 


89
CGAGAG
21
8
1.09
17
265


 


90
CGGTGC
18
6
1.06
16
266


 


91
CTCCCC
84
53
1.05
47
267


 


92
GCGGCC
22
8
1.04
14
268


 


93
CGGCGC
14
4
1.04
13
269


 


94
AAGCCC *
60
34
1.03
42
270


 


95
CCGCAG
24
9
1.03
17
271


 


96
GCCCAC
59
34
0.95
35
272


 


97
CACCCA
92
60
0.93
49
273


 


98
GCGCCC
27
11
0.93
18
274


 


99
ACCGGC
15
4
0.92
13
275


 


100
CTCGCA
16
5
0.89
14
276


 


101
ACGCTC
16
5
0.88
12
277


 


102
CTGGAC
58
33
0.88
32
278


 


103
GCCCCA
67
40
0.87
38
279


 


104
ACCGTC
15
4
0.86
11
280


 


105
CCCTCG
21
8
0.8
18
281


 


106
AGCCCG
22
8
0.79
14
282


 


107
ACCCGA
16
5
0.78
13
283


 


108
AGCAGC
79
50
0.75
41
284


 


109
ACCGCG
14
4
0.69
7
285


 


110
CGAGGC
29
13
0.69
24
286


 


111
AGCTGC
70
43
0.64
36
287


 


112
GGGGAC
49
27
0.64
34
288


 


113
CCGCAA
16
5
0.64
12
289


 


114
CGTCGC
8
1
0.62
6
290


 


115
CGTGAC
17
6
0.57
15
291


 


116
CGCCCA
33
16
0.56
22
292


 


117
CTCTGC
97
65
0.54
47
293


 


118
AGCGGG
21
8
0.52
17
294


 


119
ACCGCT
15
5
0.5
11
295


 


120
CCCAGG
133
95
0.49
58
296


 


121
CCCTCA
71
45
0.49
39
297


 


122
CCCCCA *
77
49
0.49
42
298


 


123
GGCGAA
16
5
0.48
14
299


 


124
CGGCTC
29
13
0.47
19
300


 


125
CTCGCC
20
8
0.46
17
301


 


126
CGGAGA
20
8
0.45
14
302


 


127
TCCCCA
95
64
0.43
52
303


 


128
GACACC
44
24
0.42
33
304


 


129
CTCCGA
17
6
0.42
13
305


 


130
CTCGTC
17
6
0.42
14
306


 


131
CGACCA
13
4
0.39
11
307


 


132
ATGACG
17
6
0.37
12
308


 


133
CCATCG
17
6
0.37
13
309


 


134
AGGGGA
78
51
0.36
44
310


 


135
GCTGCA
77
50
0.35
43
311


 


136
ACCCCA
76
49
0.33
40
312


 


137
CGGAGC
21
9
0.33
16
313


 


138
CCTCCG
28
13
0.32
19
314


 


139
CGGGAC
16
6
0.3
10
315


 


140
CCTGGA
88
59
0.3
45
316


 


141
AGGCGA
18
7
0.29
17
317


 


142
ACCCCT
54
32
0.28
36
318


 


143
GCTCCC
56
34
0.27
36
319


 


144
CGTCAC
16
6
0.27
15
320


 


145
AGCGCA
16
6
0.26
11
321


 


146
GAAGCC
62
38
0.25
39
322


 


147
GAGGCC
79
52
0.22
42
323


 


148
ACCCTC
54
32
0.22
33
324


 


149
CCCGGC
37
20
0.21
21
325


 


150
CGAGAA
20
8
0.2
17
326


 


151
CCACCG
29
14
0.18
20
327


 


152
ACTTCG
16
6
0.17
14
328


 


153
GATGAC
48
28
0.17
35
329


 


154
ACGAGG
23
10
0.16
18
330


 


155
CCGGAG
20
8
0.15
18
331


 


156
ACCCAC
60
37
0.12
41
332


 


157
CTGGGC
105
74
0.11
50
333


 


158
CCACGG
23
10
0.09
19
334


 


159
CGGTCC
13
4
0.09
12
335


 


160
AGCACC *
54
33
0.09
40
336


 


161
ACACCC
53
32
0.08
38
337


 


162
AGGGCC
54
33
0.08
30
338


 


163
CGCGAA
6
1
0.02
6
339


 


164
GAGCCC
58
36
0.02
36
340


 


165
CTGAGC
71
46
0.02
45
341


 


166
AATCGG
13
4
0.02
11
342









The patterns are ranked according to significance coefficient. These were determined using RSA-Tools with the random sequence from the human genome as reference. Patterns that comprise the most discriminant variables in Linear Discriminant Analysis are indicated with an asterisk. (SEQ ID NOS:343-1072)







TABLE 7







Dyad patterns over-represented in STAR elements.















Observed
Expected
Significance
SEQ ID



Number
Dyad sequence
occurrences
occurrences
coefficient
NO:
















1
CCCN{2}CGG
36
7
9.31
343



 


2
CCGN{6}CCC
40
10
7.3
344


 


3
CAGN{0}CGG
36
8
7.13
345


 


4
CGCN{15}CCC
34
8
6.88
346


 


5
CGGN{9}GCC
33
7
6.82
347


 


6
CCCN{9}CGC
35
8
6.72
348


 


7
CCCN{1}GCG
34
8
6.64
349


 


8
CCCN{0}CAC
103
48
6.61
350


 


9
AGCN{16}CCG
29
6
5.96
351


 


10
CCCN{4}CGC
34
8
5.8
352


 


11
CGCN{13}GGA
26
5
5.77
353


 


12
GCGN{16}CCC
30
7
5.74
354


 


13
CGCN{5}GCA
25
5
5.49
355


 


14
CCCN{14}CCC
101
49
5.43
356


 


15
CTGN{4}CGC
34
9
5.41
357


 


16
CCAN{12}GCG
28
6
5.37
358


 


17
CGGN{11}CAG
36
10
5.25
359


 


18
CCCN{5}GCC
75
33
4.87
360


 


19
GCCN{0}CCC
64
26
4.81
361


 


20
CGCN{4}GAC
19
3
4.78
362


 


21
CGGN{0}CAG
33
9
4.76
363


 


22
CCCN{3}CGC
32
8
4.67
364


 


23
CGCN{1}GAC
20
3
4.58
365


 


24
GCGN{2}GCC
29
7
4.54
366


 


25
CCCN{4}GCC
76
34
4.53
367


 


26
CCCN{1}CCC
103
52
4.53
368


 


27
CCGN{13}CAG
33
9
4.5
369


 


28
GCCN{4}GGA
64
27
4.48
370


 


29
CCGN{3}GGA
26
6
4.46
371


 


30
AGGN{2}GGG
118
63
4.44
372


 


31
CACN{5}GCG
22
4
4.42
373


 


32
CGCN{17}CCA
27
6
4.39
374


 


33
CCCN{9}GGC
69
30
4.38
375


 


34
CCTN{5}GCG
28
7
4.37
376


 


35
GCGN{0}GAC
19
3
4.32
377


 


36
GCCN{0}GGC
40
7
4.28
378


 


37
GCGN{2}CCC
26
6
4.27
379


 


38
CCGN{11}CCC
32
9
4.17
380


 


39
CCCN{8}TCG
23
5
4.12
381


 


40
CCGN{17}GCC
30
8
4.12
382


 


41
GGGN{5}GGA
101
52
4.11
383


 


42
GGCN{6}GGA
71
32
4.1
384


 


43
CCAN{4}CCC
96
48
4.1
385


 


44
CCTN{14}CCG
32
9
4.09
386


 


45
GACN{12}GGC
45
16
4.07
387


 


46
CGCN{13}CCC
30
8
4.04
388


 


47
CAGN{16}CCC
92
46
4.02
389


 


48
AGCN{10}GGG
75
35
3.94
390


 


49
CGGN{13}GGC
30
8
3.93
391


 


50
CGGN{1}GCC
30
8
3.92
392


 


51
AGCN{0}GGC
26
6
3.9
393


 


52
CCCN{16}GGC
64
28
3.89
394


 


53
GCTN{19}CCC
67
29
3.87
395


 


54
CCCN{16}GGG
88
31
3.81
396


 


55
CCCN{9}CGG
30
8
3.77
397


 


56
CCCN{10}CGG
30
8
3.76
398


 


57
CCAN{0}GCG
32
9
3.75
399


 


58
GCCN{17}CGC
26
6
3.74
400


 


59
CCTN{6}CGC
27
7
3.73
401


 


60
GGAN{1}CCC
63
27
3.71
402


 


61
CGCN{18}CAC
24
5
3.7
403


 


62
CGCN{20}CCG
21
4
3.69
404


 


63
CCGN{0}GCA
26
6
3.69
405


 


64
CGCN{20}CCC
28
7
3.69
406


 


65
AGCN{15}CCC
67
30
3.65
407


 


66
CCTN{7}GGC
69
31
3.63
408


 


67
GCCN{5}CGC
32
9
3.61
409


 


68
GCCN{14}CGC
28
7
3.59
410


 


69
CAGN{11}CCC
89
45
3.58
411


 


70
GGGN{16}GAC
53
21
3.57
412


 


71
CCCN{15}GCG
25
6
3.57
413


 


72
CCCN{0}CGC
37
12
3.54
414


 


73
CCCN{16}AGC *
67
30
3.54
415


 


74
AGGN{9}GGG
96
50
3.52
416


 


75
CGCN{12}CTC
28
7
3.46
417


 


76
CACN{8}CGC
23
5
3.43
418


 


77
CCAN{7}CCG
31
9
3.42
419


 


78
CGGN{1}GCA
25
6
3.41
420


 


79
CGCN{14}CCC
29
8
3.4
421


 


80
AGCN{0}CCC
76
36
3.4
422


 


81
CGCN{13}GTC
18
3
3.37
423


 


82
GCGN{3}GCA
26
7
3.35
424


 


83
CGGN{0}GGC
34
11
3.35
425


 


84
GCCN{14}CCC
68
31
3.33
426


 


85
ACCN{7}CGC
21
4
3.32
427


 


86
AGGN{7}CGG
33
10
3.31
428


 


87
CCCN{16}CGA
22
5
3.3
429


 


88
CGCN{6}CAG
31
9
3.29
430


 


89
CAGN{11}GCG
29
8
3.29
431


 


90
CCGN{12}CCG
19
4
3.26
432


 


91
CGCN{18}CAG
27
7
3.24
433


 


92
CAGN{1}GGG
80
39
3.21
434


 


93
CGCN{0}CCC
32
10
3.2
435


 


94
GCGN{18}GCC
26
7
3.18
436


 


95
CGGN{15}GGC
27
7
3.15
437


 


96
CCCN{15}AGG
72
34
3.14
438


 


97
AGGN{20}GCG
26
7
3.14
439


 


98
CGGN{5}CTC
26
7
3.13
440


 


99
TCCN{17}CGA
23
5
3.12
441


 


100
GCGN{4}CCC
30
9
3.08
442


 


101
CCCN{2}CGC
30
9
3.07
443


 


102
CGTN{3}CAG
28
8
3.06
444


 


103
CCGN{13}GAG
27
7
3.05
445


 


104
CTCN{6}CGC
28
8
3.04
446


 


105
CGCN{4}GAG
21
5
3.03
447


 


106
GCGN{5}GGA
24
6
3.03
448


 


107
CCGN{1}CAG
27
7
3.01
449


 


108
CGCN{11}CCG
18
3
2.99
450


 


109
GCGN{19}CCC
26
7
2.98
451


 


110
CGCN{18}GAA
21
5
2.98
452


 


111
GGGN{19}GGA
78
39
2.95
453


 


112
CCAN{1}CGG
24
6
2.94
454


 


113
CCCN{7}GCG
25
6
2.94
455


 


114
AGGN{10}CCC
84
43
2.92
456


 


115
CCAN{0}GGG
97
52
2.88
457


 


116
CAGN{10}CCC
82
41
2.87
458


 


117
CCGN{18}CCG
19
4
2.86
459


 


118
CCGN{18}GGC
26
7
2.85
460


 


119
CCCN{2}GCG
24
6
2.84
461


 


120
CGCN{1}GGC
25
7
2.83
462


 


121
CCGN{5}GAC
19
4
2.81
463


 


122
GGAN{0}CCC
52
22
2.8
464


 


123
CCCN{1}CCG
29
9
2.78
465


 


124
CCCN{15}ACG
23
6
2.75
466


 


125
AGCN{8}CCC
66
31
2.73
467


 


126
CCCN{3}GGC
60
27
2.71
468


 


127
AGGN{9}CGG
31
10
2.7
469


 


128
CCCN{14}CGC
27
8
2.7
470


 


129
CCGN{0}CCG
19
4
2.7
471


 


130
CGCN{8}AGC
23
6
2.69
472


 


131
CGCN{19}ACC
21
5
2.68
473


 


132
GCGN{17}GAC
17
3
2.66
474


 


133
AGCN{1}GCG
24
6
2.63
475


 


134
CCGN{11}GGC
31
10
2.63
476


 


135
CGGN{4}AGA
26
7
2.63
477


 


136
CGCN{14}CCG
17
3
2.62
478


 


137
CCTN{20}GCG
24
6
2.62
479


 


138
CCAN{10}CGC
26
7
2.61
480


 


139
CCCN{20}CAC
69
33
2.6
481


 


140
CCGN{11}GCC
27
8
2.6
482


 


141
CGCN{18}CCC
26
7
2.59
483


 


142
CGGN{15}CGC
16
3
2.57
484


 


143
CGCN{16}GCC
24
6
2.55
485


 


144
CGCN{20}GGC
23
6
2.54
486


 


145
CGCN{19}CCG
18
4
2.52
487


 


146
CGGN{10}CCA
28
8
2.51
488


 


147
CGCN{17}CCC
26
7
2.51
489


 


148
CGCN{11}ACA
23
6
2.51
490


 


149
CGGN{0}ACC
17
3
2.5
491


 


150
GCGN{10}GCC
24
6
2.49
492


 


151
GCGN{8}GAC
17
3
2.49
493


 


152
CCCN{15}GGG
84
32
2.44
494


 


153
CGGN{16}GGC
27
8
2.44
495


 


154
CGCN{16}CCA
23
6
2.42
496


 


155
GCCN{3}CCC
73
36
2.4
497


 


156
CAGN{4}GGG
94
51
2.4
498


 


157
CCCN{6}GCG
23
6
2.38
499


 


158
CCGN{16}CGC
17
3
2.38
500


 


159
CCCN{17}GCA
61
28
2.37
501


 


160
CGCN{13}TCC
24
6
2.37
502


 


161
GCCN{1}CGC
29
9
2.36
503


 


162
CCGN{19}GAG
26
7
2.35
504


 


163
GGGN{10}GGA
89
48
2.35
505


 


164
CAGN{5}CCG
32
11
2.35
506


 


165
CGCN{3}AGA
19
4
2.32
507


 


166
GCCN{0}GCC
29
9
2.32
508


 


167
CCCN{8}GGC
61
28
2.31
509


 


168
CCTN{6}GCG
22
6
2.29
510


 


169
GACN{6}CCC
48
20
2.29
511


 


170
CGGN{1}CCC
26
8
2.27
512


 


171
CCCN{15}CCG
30
10
2.27
513


 


172
CAGN{9}CCC
84
44
2.26
514


 


173
CGGN{10}GGC
27
8
2.26
515


 


174
CGAN{10}ACG
10
1
2.26
516


 


175
GCGN{3}TCC
21
5
2.26
517


 


176
CCCN{3}GCC
75
38
2.24
518


 


177
GCGN{1}ACC
17
3
2.24
519


 


178
CCGN{9}AGG
27
8
2.23
520


 


179
CGCN{16}CAG
26
8
2.23
521


 


180
GGCN{0}CCC
62
29
2.22
522


 


181
AGGN{12}CCG
26
8
2.19
523


 


182
CCGN{0}GCG
16
3
2.19
524


 


183
CCGN{2}GCC
30
10
2.18
525


 


184
CCGN{11}GTC
19
4
2.17
526


 


185
CAGN{0}CCC
88
47
2.17
527


 


186
CCCN{5}CCG
32
11
2.17
528


 


187
GCCN{20}CCC
66
32
2.15
529


 


188
GACN{2}CGC
18
4
2.14
530


 


189
CGCN{6}CAC
23
6
2.13
531


 


190
AGGN{14}GCG
25
7
2.1
532


 


191
GACN{5}CGC
17
3
2.1
533


 


192
CCTN{19}CCG
29
9
2.1
534


 


193
CCGN{12}GGA
24
7
2.08
535


 


194
GGCN{9}GAC *
44
18
2.08
536


 


195
AGGN{10}GGG
94
52
2.07
537


 


196
CCGN{10}GAG
25
7
2.07
538


 


197
CGCN{6}GGA
20
5
2.06
539


 


198
CGCN{7}AGC
23
6
2.04
540


 


199
CCAN{13}CGG
26
8
2.03
541


 


200
CGGN{6}GGA
25
7
2.03
542


 


201
CGCN{19}GCC
24
7
2.03
543


 


202
CCAN{12}CGC
24
7
2.02
544


 


203
CGGN{1}GGC
41
16
2.02
545


 


204
GCGN{3}CCA
25
7
2.01
546


 


205
AGGN{1}CGC
21
5
2
547


 


206
CTCN{5}CGC
24
7
1.98
548


 


207
CCCN{0}ACG
30
10
1.97
549


 


208
CAGN{17}CCG
29
9
1.96
550


 


209
GGCN{4}CCC
62
30
1.96
551


 


210
AGGN{8}GCG
26
8
1.96
552


 


211
CTGN{1}CCC
88
48
1.94
553


 


212
CCCN{16}CAG
85
46
1.94
554


 


213
CGCN{9}GAC
16
3
1.93
555


 


214
CAGN{6}CCG
29
9
1.92
556


 


215
CGTN{12}CGC
11
1
1.92
557


 


216
CTCN{7}GCC
69
35
1.92
558


 


217
CGCN{19}TCC
22
6
1.92
559


 


218
CCCN{7}GCC
67
33
1.91
560


 


219
CAGN{13}CGG
30
10
1.9
561


 


220
CGCN{1}GCC
27
8
1.9
562


 


221
CGCN{17}CCG
17
4
1.89
563


 


222
AGGN{4}CCC
63
31
1.89
564


 


223
AGCN{10}CGC
21
5
1.89
565


 


224
CCCN{11}CGG
30
10
1.88
566


 


225
CCCN{8}GCC
75
39
1.86
567


 


226
CCGN{1}CGG
22
3
1.86
568


 


227
CCCN{1}ACC
71
36
1.85
569


 


228
CGCN{0}CAG
25
7
1.85
570


 


229
CCGN{19}TGC
23
6
1.82
571


 


230
GCGN{4}CGA
12
2
1.82
572


 


231
CCGN{19}GCC
30
10
1.82
573


 


232
CCAN{10}CCC
85
46
1.81
574


 


233
CAGN{13}GGG
91
51
1.81
575


 


234
AGCN{18}CGG
23
6
1.81
576


 


235
CGAN{8}CGC
11
1
1.81
577


 


236
AGCN{4}CCC
63
31
1.8
578


 


237
GGAN{6}CCC
61
30
1.8
579


 


238
CGGN{13}AAG
23
6
1.8
580


 


239
ACCN{11}CGC
19
5
1.79
581


 


240
CCGN{12}CAG
28
9
1.78
582


 


241
CCCN{12}GGG
76
29
1.77
583


 


242
CACN{17}ACG
22
6
1.76
584


 


243
CAGN{18}CCC
82
44
1.76
585


 


244
CGTN{10}GTC
19
5
1.75
586


 


245
CCCN{13}GCG
23
6
1.75
587


 


246
GCAN{1}CGC
20
5
1.73
588


 


247
AGAN{4}CCG
24
7
1.73
589


 


248
GCGN{10}AGC
22
6
1.72
590


 


249
CGCN{0}GGA
12
2
1.72
591


 


250
CGGN{4}GAC
17
4
1.69
592


 


251
CCCN{12}CGC
26
8
1.68
593


 


252
GCCN{15}CCC
65
33
1.68
594


 


253
GCGN{6}TCC
20
5
1.66
595


 


254
CGGN{3}CAG
33
12
1.65
596


 


255
CCCN{3}CCA
88
49
1.65
597


 


256
AGCN{3}CCC
59
28
1.65
598


 


257
GGGN{16}GCA
65
33
1.65
599


 


258
AGGN{8}CCG
28
9
1.64
600


 


259
CCCN{0}CCG
29
10
1.64
601


 


260
GCGN{5}GAC
16
3
1.64
602


 


261
CCCN{9}ACC
60
29
1.64
603


 


262
CTGN{5}CGC
25
8
1.64
604


 


263
CGCN{14}CTC
23
7
1.64
605


 


264
CGGN{14}GCA
23
7
1.63
606


 


265
CCGN{8}GCC
26
8
1.62
607


 


266
CCGN{7}CAC
23
7
1.62
608


 


267
AGCN{8}GCG
21
6
1.61
609


 


268
CGGN{16}GGA
29
10
1.61
610


 


269
CCAN{12}CCG
26
8
1.61
611


 


270
CGGN{2}CCC
26
8
1.6
612


 


271
CCAN{13}GGG
71
37
1.6
613


 


272
CGGN{15}GCA
21
6
1.6
614


 


273
CGCN{9}GCA
20
5
1.58
615


 


274
CGGN{19}CCA
26
8
1.58
616


 


275
GGGN{15}CGA
20
5
1.57
617


 


276
CCCN{10}CGC
26
8
1.57
618


 


277
CTCN{14}CGC
26
8
1.55
619


 


278
CACN{11}GCG
20
5
1.55
620


 


279
CCGN{2}GGC
24
7
1.55
621


 


280
CTGN{18}CCC
85
47
1.54
622


 


281
GGGN{13}CAC
58
28
1.54
623


 


282
CCTN{15}GGC
62
31
1.54
624


 


283
CCCN{20}CGA
20
5
1.54
625


 


284
CCCN{8}CGA
20
5
1.53
626


 


285
GAGN{7}CCC
61
30
1.53
627


 


286
CGCN{2}CCG
22
6
1.53
628


 


287
CCCN{0}TCC
98
57
1.52
629


 


288
AGCN{0}GCC
21
6
1.52
630


 


289
CCCN{2}TCC
82
45
1.52
631


 


290
CCGN{5}CCC
30
10
1.52
632


 


291
CGCN{13}CGC
16
3
1.51
633


 


292
CCCN{1}CGC
28
9
1.51
634


 


293
GCCN{16}GCA
53
25
1.51
635


 


294
CCCN{16}CCA
84
46
1.5
636


 


295
CCGN{13}CGC
19
5
1.5
637


 


296
CCGN{17}CAG
28
9
1.49
638


 


297
CGGN{18}GGC
26
8
1.49
639


 


298
CCGN{14}AGG
23
7
1.49
640


 


299
CCCN{5}CGG
26
8
1.49
641


 


300
CCCN{6}GGA
58
28
1.49
642


 


301
ACGN{2}CCC
20
5
1.49
643


 


302
CCAN{9}CCG
27
9
1.48
644


 


303
CCCN{19}CCA
78
42
1.48
645


 


304
CAGN{0}GGG
77
41
1.48
646


 


305
AGCN{1}CCC
58
28
1.47
647


 


306
GCGN{7}TCC
27
9
1.46
648


 


307
ACGN{18}CCA
25
8
1.46
649


 


308
GCTN{14}CCC
61
30
1.46
650


 


309
GCGN{14}CCC
23
7
1.46
651


 


310
GCGN{19}AGC
20
5
1.45
652


 


311
CCGN{8}CAG
29
10
1.45
653


 


312
GCGN{6}GCC
22
6
1.45
654


 


313
GCGN{10}GCA
20
5
1.44
655


 


314
CCTN{7}GCC
69
36
1.44
656


 


315
GCCN{13}GCC
54
26
1.42
657


 


316
CCCN{14}GCC
63
32
1.42
658


 


317
CCCN{15}CGG
26
8
1.42
659


 


318
CCAN{13}CGC
23
7
1.42
660


 


319
AGCN{11}GGG
67
35
1.41
661


 


320
GGAN{0}GCC
64
32
1.4
662


 


321
GCCN{3}TCC
61
30
1.4
663


 


322
CCTN{5}GCC
69
36
1.39
664


 


323
CGGN{18}CCC
25
8
1.39
665


 


324
CCTN{3}GGC
59
29
1.38
666


 


325
CCGN{0}CTC
22
6
1.38
667


 


326
AGCN{17}GCG
19
5
1.37
668


 


327
ACGN{14}GGG
20
5
1.37
669


 


328
CGAN{12}GGC
19
5
1.37
670


 


329
CCCN{20}CGC
24
7
1.37
671


 


330
ACGN{12}CTG
24
7
1.36
672


 


331
CCGN{0}CCC
36
14
1.36
673


 


332
CCGN{10}GGA
23
7
1.36
674


 


333
CCCN{3}GCG
21
6
1.36
675


 


334
GCGN{14}CGC
22
3
1.35
676


 


335
CCGN{8}CGC
16
4
1.35
677


 


336
CGCN{10}ACA
22
6
1.34
678


 


337
CCCN{19}CCG
28
10
1.33
679


 


338
CACN{14}CGC
20
5
1.32
680


 


339
GACN{3}GGC
46
21
1.32
681


 


340
GAAN{7}CGC
19
5
1.32
682


 


341
CGCN{16}GGC
21
6
1.31
683


 


342
GGCN{9}CCC
64
33
1.31
684


 


343
CCCN{9}GCC
64
33
1.31
685


 


344
CGCN{0}TGC
26
9
1.3
686


 


345
CCTN{8}GGC
67
35
1.3
687


 


346
CCAN{8}CCC
82
46
1.29
688


 


347
GACN{2}CCC
42
18
1.28
689


 


348
GGCN{1}CCC
54
26
1.27
690


 


349
CGCN{0}AGC
24
7
1.26
691


 


350
AGGN{4}GCG
28
10
1.26
692


 


351
CGGN{6}TCC
22
6
1.25
693


 


352
ACGN{19}GGC
20
5
1.25
694


 


353
CCCN{8}ACG
21
6
1.24
695


 


354
CCCN{18}GCC
62
31
1.24
696


 


355
GCCN{2}CGA
19
5
1.24
697


 


356
CCCN{8}GCG
28
10
1.23
698


 


357
CCCN{0}CTC
76
41
1.23
699


 


358
GCCN{11}CGC
27
9
1.22
700


 


359
AGCN{9}CCC
59
29
1.22
701


 


360
GCTN{0}GCC
71
38
1.21
702


 


361
CGCN{3}CCC
26
9
1.21
703


 


362
CCCN{2}CCC
117
72
1.19
704


 


363
GCCN{9}CGC
23
7
1.19
705


 


364
GCAN{19}CGC
19
5
1.19
706


 


365
CAGN{4}CGG
32
12
1.18
707


 


366
CAGN{2}GGG
80
44
1.17
708


 


367
GCCN{16}CCC
67
35
1.16
709


 


368
GAGN{5}CCC
60
30
1.16
710


 


369
CCTN{16}TCG
20
6
1.16
711


 


370
CCCN{2}GGC
62
32
1.15
712


 


371
GCGN{13}GGA
24
8
1.15
713


 


372
GCCN{17}GGC
66
25
1.15
714


 


373
CCCN{14}GGC
58
29
1.14
715


 


374
AGGN{3}CCG
31
12
1.14
716


 


375
CACN{0}CGC
32
12
1.14
717


 


376
CGGN{18}CAG
28
10
1.14
718


 


377
AGCN{1}GCC
57
28
1.13
719


 


378
CGCN{18}GGC
23
7
1.13
720


 


379
CCCN{5}AGG
64
33
1.11
721


 


380
AACN{0}GCG
9
1
1.11
722


 


381
CCCN{10}CCA
88
50
1.09
723


 


382
CGCN{13}GAG
20
6
1.09
724


 


383
CGCN{7}GCC
25
8
1.08
725


 


384
CCCN{9}CCG
28
10
1.07
726


 


385
CGCN{16}CCC
24
8
1.05
727


 


386
GAAN{13}CGC
18
5
1.05
728


 


387
GGCN{3}CCC
49
23
1.03
729


 


388
TCCN{11}CCA
87
50
1.03
730


 


389
CACN{0}CCC
70
38
1.02
731


 


390
CGCN{16}CCG
15
3
1.02
732


 


391
CGGN{15}AGC
21
6
1.02
733


 


392
CCCN{12}GCG
21
6
1.02
734


 


393
CCCN{9}GAG
59
30
1.01
735


 


394
CCGN{20}TCC
24
8
1.01
736


 


395
CGCN{0}CGC
17
4
1.01
737


 


396
ATGN{7}CGG
20
6
1
738


 


397
GGGN{20}GCA
59
30
1
739


 


398
CGGN{4}GGC
26
9
0.99
740


 


399
CGGN{16}AGC
22
7
0.99
741


 


400
CGGN{5}GGC
25
8
0.99
742


 


401
GCGN{0}GGA
25
8
0.98
743


 


402
GGCN{20}CAC
52
25
0.98
744


 


403
CCCN{9}CCC
97
58
0.97
745


 


404
ACCN{17}GGC
44
20
0.97
746


 


405
CCCN{6}CGA
18
5
0.96
747


 


406
AAGN{10}CGG
26
9
0.96
748


 


407
CGCN{17}CAC
21
6
0.95
749


 


408
CCCN{16}CGG
25
8
0.94
750


 


409
GACN{18}GGC
39
17
0.94
751


 


410
GGGN{15}GAC
47
22
0.92
752


 


411
GCCN{4}TCC
66
35
0.92
753


 


412
GGCN{15}CCC
56
28
0.92
754


 


413
CAGN{12}CGC
24
8
0.92
755


 


414
CCAN{3}GCG
22
7
0.91
756


 


415
CCGN{16}GAG
22
7
0.9
757


 


416
AGCN{2}CGC
24
8
0.89
758


 


417
GAGN{4}CCC
54
27
0.89
759


 


418
AGGN{3}CGC
23
7
0.88
760


 


419
CACN{13}AGG *
67
36
0.88
761


 


420
CCCN{4}CAG
88
51
0.88
762


 


421
CCCN{2}GAA
63
33
0.87
763


 


422
CGCN{19}GAG
21
6
0.87
764


 


423
ACGN{18}GGG
21
6
0.87
765


 


424
CCCN{4}GGC
62
32
0.87
766


 


425
CGGN{9}GAG
28
10
0.86
767


 


426
CCCN{3}GGG
66
26
0.86
768


 


427
GAGN{4}GGC
66
35
0.85
769


 


428
CGCN{5}GAG
18
5
0.84
770


 


429
CCGN{20}AGG
24
8
0.84
771


 


430
CCCN{15}CCC
88
51
0.83
772


 


431
AGGN{17}CCG
25
8
0.82
773


 


432
AGGN{6}GGG
89
52
0.82
774


 


433
GGCN{20}CCC
57
29
0.82
775


 


434
GCAN{17}CGC
19
5
0.82
776


 


435
CGAN{11}ACG
9
1
0.81
777


 


436
CGCN{2}GGA
19
5
0.81
778


 


437
CTGN{5}CCC
79
45
0.8
779


 


438
TCCN{20}CCA
77
43
0.8
780


 


439
CCAN{2}GGG
59
30
0.8
781


 


440
CCGN{15}GCG
14
3
0.8
782


 


441
CCAN{5}GGG
69
38
0.79
783


 


442
CGGN{1}TGC
24
8
0.79
784


 


443
CCCN{14}GCG
21
6
0.79
785


 


444
CAGN{0}CCG
27
10
0.79
786


 


445
GCCN{9}TCC
60
31
0.78
787


 


446
AGGN{20}CGC
22
7
0.78
788


 


447
CCCN{6}GAC
42
19
0.77
789


 


448
CGGN{11}CCA
23
7
0.76
790


 


449
GGGN{14}CAC
57
29
0.75
791


 


450
GCAN{15}CGC
19
5
0.74
792


 


451
CGCN{2}ACA
20
6
0.74
793


 


452
ACCN{9}CCC
57
29
0.73
794


 


453
GCGN{9}CGC
20
3
0.73
795


 


454
CAGN{15}GCG
23
7
0.73
796


 


455
CCCN{18}GTC
45
21
0.72
797


 


456
GCGN{3}CCC
24
8
0.72
798


 


457
CGGN{11}GCC
23
8
0.72
799


 


458
CCCN{1}CGG
24
8
0.71
800


 


459
GCCN{4}CCA
70
38
0.71
801


 


460
CCCN{4}CCG
30
12
0.7
802


 


461
CGTN{2}GCA
21
6
0.7
803


 


462
AGCN{7}TCG
18
5
0.69
804


 


463
CCGN{15}GAA
20
6
0.69
805


 


464
ACCN{5}CCC
62
33
0.69
806


 


465
CGCN{14}GAG
19
5
0.68
807


 


466
CCCN{7}CGC
30
12
0.68
808


 


467
GAGN{12}CGC
21
6
0.68
809


 


468
GGCN{17}CCC
58
30
0.67
810


 


469
ACGN{11}CTC
21
7
0.65
811


 


470
ACAN{9}CGG
24
8
0.65
812


 


471
CTGN{7}CCC
82
47
0.65
813


 


472
CCCN{2}GCC
72
40
0.65
814


 


473
CGGN{2}GCA
24
8
0.64
815


 


474
CCCN{0}TGC
83
48
0.64
816


 


475
CGCN{7}ACC
18
5
0.63
817


 


476
GCAN{2}GCC
54
27
0.63
818


 


477
GCGN{8}CCA
20
6
0.63
819


 


478
AGCN{0}CGC
22
7
0.63
820


 


479
GCGN{2}GCA
18
5
0.63
821


 


480
CCGN{2}GTC
18
5
0.62
822


 


481
CCGN{3}ACA
21
7
0.62
823


 


482
ACGN{13}TGG
21
7
0.62
824


 


483
CCAN{8}CGC
23
8
0.62
825


 


484
CCGN{9}GGC
23
8
0.61
826


 


485
CCAN{5}CCG
25
9
0.61
827


 


486
AGGN{3}GGG
97
59
0.61
828


 


487
CAGN{2}GGC
78
45
0.61
829


 


488
CCCN{8}CAG
81
47
0.61
830


 


489
AGCN{5}CAG
80
46
0.6
831


 


490
CGGN{16}GCC
22
7
0.6
832


 


491
GCGN{15}CCC
23
8
0.6
833


 


492
CCCN{11}GCC
59
31
0.59
834


 


493
CGAN{2}ACG
9
1
0.59
835


 


494
CGGN{4}GCC
22
7
0.59
836


 


495
CACN{6}CGC
19
6
0.59
837


 


496
CGGN{5}ACG
11
2
0.59
838


 


497
CTGN{4}GCC *
66
36
0.59
839


 


498
GGGN{18}CGA
18
5
0.59
840


 


499
CCTN{8}CGC
22
7
0.59
841


 


500
GCCN{4}CCC
67
37
0.58
842


 


501
CGGN{10}GCC
22
7
0.58
843


 


502
GCCN{5}GGA
54
27
0.57
844


 


503
ACCN{7}GCG
15
4
0.57
845


 


504
CCCN{8}CGC
24
8
0.57
846


 


505
CAGN{5}CCC
77
44
0.56
847


 


506
CACN{14}GGA
63
34
0.56
848


 


507
CCCN{1}GCC
94
57
0.55
849


 


508
CCCN{5}AGC
67
37
0.55
850


 


509
GGCN{5}GGA
59
31
0.55
851


 


510
CGAN{17}GAG
19
6
0.55
852


 


511
CGCN{7}ACA
18
5
0.54
853


 


512
CCAN{13}CCC
87
52
0.54
854


 


513
CGGN{20}GGC
24
8
0.54
855


 


514
CCCN{17}GCC
58
30
0.53
856


 


515
CCTN{10}CCG
30
12
0.53
857


 


516
CCCN{8}CCG
27
10
0.53
858


 


517
CGCN{3}GAG
18
5
0.52
859


 


518
CGCN{7}AAG
17
5
0.51
860


 


519
CGGN{11}GGA
23
8
0.51
861


 


520
CCGN{15}CCG
15
4
0.51
862


 


521
CCCN{3}GCA
57
30
0.51
863


 


522
CGGN{2}CAG
24
8
0.5
864


 


523
AGGN{2}CCG
24
8
0.5
865


 


524
CCCN{4}CAC
69
38
0.5
866


 


525
GGAN{19}CCC
56
29
0.49
867


 


526
CCCN{8}CAC
68
38
0.49
868


 


527
ACCN{6}CCG
18
5
0.49
869


 


528
CCCN{6}GGC
54
28
0.49
870


 


529
CCCN{6}CCG
29
11
0.48
871


 


530
CGCN{14}GCC
26
9
0.47
872


 


531
CCGN{5}TCC
25
9
0.46
873


 


532
GCCN{6}GCC
55
28
0.46
874


 


533
CGGN{7}GGA
24
8
0.45
875


 


534
GGGN{6}GGA
87
52
0.44
876


 


535
GCCN{12}TCC
60
32
0.44
877


 


536
AGTN{16}CCG
17
5
0.44
878


 


537
GGCN{19}GCC
68
29
0.44
879


 


538
CCGN{3}CCG
22
7
0.44
880


 


539
CCCN{8}ACC
58
31
0.44
881


 


540
CAGN{15}GCC
77
44
0.44
882


 


541
CCCN{17}CGG
24
8
0.44
883


 


542
GCGN{1}CCA
22
7
0.44
884


 


543
CCCN{14}CAG
79
46
0.44
885


 


544
CCCN{8}CCC
89
53
0.44
886


 


545
ACAN{12}GCG
23
8
0.43
887


 


546
AGGN{4}CCG
23
8
0.43
888


 


547
CGCN{13}GCC
23
8
0.43
889


 


548
GAGN{2}CGC
23
8
0.42
890


 


549
CCCN{9}GCG
21
7
0.42
891


 


550
CGCN{17}ACA
17
5
0.42
892


 


551
GCGN{17}CCA
23
8
0.42
893


 


552
AAGN{18}CCG
20
6
0.42
894


 


553
CGCN{1}GGA
18
5
0.41
895


 


554
CCAN{1}CCC
90
54
0.41
896


 


555
CGTN{18}TGC
20
6
0.41
897


 


556
TCCN{14}CGA
17
5
0.41
898


 


557
CACN{5}GGG
56
29
0.4
899


 


558
CCGN{12}GCA
21
7
0.4
900


 


559
CTGN{6}CCC
77
44
0.4
901


 


560
CGGN{8}GGC
32
13
0.4
902


 


561
CCAN{11}GGG
68
38
0.4
903


 


562
ACGN{19}CAA
21
7
0.39
904


 


563
GGGN{20}CCC
72
31
0.39
905


 


564
CGCN{3}CAG
23
8
0.39
906


 


565
AGCN{17}GGG
58
31
0.37
907


 


566
CACN{20}CCG
21
7
0.37
908


 


567
ACGN{17}CAG
24
8
0.37
909


 


568
AGGN{1}CCC
60
32
0.37
910


 


569
CGTN{12}CAC
20
6
0.37
911


 


570
CGGN{9}GGC
23
8
0.37
912


 


571
CGCN{10}GCG
18
3
0.37
913


 


572
CCCN{6}CTC
80
47
0.36
914


 


573
CCGN{10}AGG
23
8
0.36
915


 


574
CCCN{18}CAG
79
46
0.36
916


 


575
AGCN{17}CCG
21
7
0.36
917


 


576
AGCN{9}GCG
18
5
0.36
918


 


577
CCAN{3}GGC
62
34
0.36
919


 


578
CCCN{11}GGC
57
30
0.35
920


 


579
ACGN{5}GCA
23
8
0.35
921


 


580
CCCN{14}CGG
23
8
0.35
922


 


581
CCCN{5}CCA
91
55
0.35
923


 


582
CCGN{1}AGG
22
7
0.34
924


 


583
GGGN{10}GAC
45
22
0.34
925


 


584
CGCN{15}CCA
20
6
0.34
926


 


585
CCTN{19}CGC
22
7
0.34
927


 


586
CGTN{3}CGC
10
2
0.33
928


 


587
AGCN{14}CCG
21
7
0.33
929


 


588
GGCN{2}CGA
17
5
0.33
930


 


589
CAGN{8}CCC
79
46
0.33
931


 


590
CCGN{2}GAC
16
4
0.33
932


 


591
AGCN{19}AGG
70
40
0.32
933


 


592
CCTN{4}GGC
64
35
0.32
934


 


593
CCGN{11}AGC
22
7
0.32
935


 


594
CACN{4}CGC
18
5
0.32
936


 


595
CCGN{1}CCC
30
12
0.31
937


 


596
CTGN{13}GGC
73
42
0.31
938


 


597
CGCN{16}ACC
15
4
0.31
939


 


598
CACN{18}CAG
79
46
0.31
940


 


599
GGCN{8}GCC
68
29
0.29
941


 


600
GGGN{15}GGA
78
46
0.29
942


 


601
CCGN{16}GCC
22
7
0.29
943


 


602
CCGN{20}ACC
18
5
0.29
944


 


603
CGAN{7}CCC
17
5
0.28
945


 


604
CCGN{6}CTC
23
8
0.28
946


 


605
CGGN{10}CTC
22
7
0.28
947


 


606
CAGN{16}CGC
23
8
0.28
948


 


607
CCAN{3}AGG
77
45
0.27
949


 


608
GCCN{18}GCC
52
27
0.27
950


 


609
CGCN{18}GGA
19
6
0.26
951


 


610
CCGN{20}GGC
22
7
0.26
952


 


611
ACAN{10}GCG
17
5
0.26
953


 


612
CGGN{5}CCC
25
9
0.25
954


 


613
CCCN{7}TCC
75
43
0.25
955


 


614
ACGN{10}CGC
10
2
0.25
956


 


615
CCCN{3}TCC
81
48
0.25
957


 


616
CCGN{8}CGG
20
3
0.24
958


 


617
CCAN{15}CGG
22
7
0.24
959


 


618
CCGN{6}CCG
17
5
0.24
960


 


619
CAGN{3}GCG
25
9
0.24
961


 


620
GAGN{1}CCC
62
34
0.24
962


 


621
CCGN{18}TGC
22
7
0.23
963


 


622
CCCN{7}CCA
85
51
0.23
964


 


623
CGGN{3}CCA
24
9
0.23
965


 


624
ACGN{1}CCC
18
5
0.23
966


 


625
CGGN{13}TGA
21
7
0.22
967


 


626
CTCN{6}GGC
53
28
0.22
968


 


627
GCGN{2}GAC
15
4
0.22
969


 


628
GGGN{11}ACC
49
25
0.22
970


 


629
CGCN{4}GGA
17
5
0.22
971


 


630
CCCN{11}CCG
27
10
0.22
972


 


631
CCGN{19}GCA
20
6
0.22
973


 


632
GCGN{0}GCA
20
6
0.21
974


 


633
AGAN{7}CCC
61
33
0.21
975


 


634
CGGN{2}CCA
21
7
0.21
976


 


635
CCCN{7}CCC
89
54
0.21
977


 


636
ACCN{4}GCG
15
4
0.2
978


 


637
CCTN{15}CGC
20
6
0.2
979


 


638
AGCN{9}GTC
44
21
0.2
980


 


639
CCCN{18}CTC
74
43
0.2
981


 


640
CGCN{18}CGA
9
1
0.19
982


 


641
CCCN{15}GCC
62
34
0.18
983


 


642
ACCN{11}GGC
45
22
0.18
984


 


643
AGGN{15}CGC
29
12
0.18
985


 


644
GCGN{0}CCA
27
10
0.18
986


 


645
GCGN{9}AGC
18
5
0.17
987


 


646
GGGN{18}GCA
59
32
0.17
988


 


647
CCCN{17}CAG
77
45
0.17
989


 


648
CCAN{8}CGG
22
8
0.16
990


 


649
CCGN{10}GGC
21
7
0.16
991


 


650
GCAN{0}GCC
76
44
0.16
992


 


651
CAGN{2}CGC
20
6
0.16
993


 


652
CGCN{8}GGC
19
6
0.16
994


 


653
CTGN{17}GGC
65
36
0.16
995


 


654
GGGN{14}ACC
46
23
0.16
996


 


655
CCGN{1}TGC
20
6
0.16
997


 


656
CAGN{8}CGC
22
8
0.15
998


 


657
AAGN{11}CGC
17
5
0.15
999


 


658
CCGN{6}TCC
22
8
0.14
1000


 


659
CCAN{18}CCC
72
42
0.14
1001


 


660
CCAN{0}CCC
84
51
0.14
1002


 


661
GAGN{6}CCC
53
28
0.14
1003


 


662
AGCN{20}GGC
52
27
0.14
1004


 


663
CAGN{0}CGC
21
7
0.14
1005


 


664
CCGN{12}CTC
22
8
0.14
1006


 


665
CGCN{15}ACG
9
1
0.13
1007


 


666
GGCN{17}CGA
15
4
0.13
1008


 


667
CCGN{16}AAG
19
6
0.13
1009


 


668
CGCN{14}TCC
19
6
0.12
1010


 


669
AGGN{7}CGC
20
7
0.12
1011


 


670
CGGN{7}CCC
22
8
0.12
1012


 


671
CGCN{4}GCC
34
15
0.12
1013


 


672
CGAN{6}CCC
17
5
0.12
1014


 


673
CCCN{19}GGA
60
33
0.11
1015


 


674
CCCN{16}GCG
28
11
0.11
1016


 


675
CCAN{7}CGC
20
7
0.11
1017


 


676
CCCN{6}GCC
80
48
0.11
1018


 


677
GCCN{14}TCC
55
29
0.11
1019


 


678
AGGN{14}GCC
64
36
0.1
1020


 


679
CGCN{11}GCC
20
7
0.1
1021


 


680
TCCN{0}GCA
17
5
0.09
1022


 


681
GCGN{8}CCC
27
11
0.09
1023


 


682
CCAN{11}GCG
19
6
0.09
1024


 


683
CACN{4}GGG
51
26
0.09
1025


 


684
CGGN{7}TCC
20
7
0.09
1026


 


685
GCGN{5}GCC
20
7
0.09
1027


 


686
ACGN{12}CAG
26
10
0.09
1028


 


687
CCGN{19}CGC
14
4
0.08
1029


 


688
CGGN{8}TGC
18
5
0.08
1030


 


689
CCCN{1}GAG
65
37
0.07
1031


 


690
GCGN{19}TGA
18
6
0.07
1032


 


691
GGCN{15}GCC
70
31
0.07
1033


 


692
CCGN{7}CCC
27
11
0.07
1034


 


693
ACAN{19}CCC
63
35
0.07
1035


 


694
ACCN{16}GGG
47
24
0.07
1036


 


695
AGAN{1}GGC
64
36
0.07
1037


 


696
GGGN{17}TGA
64
36
0.06
1038


 


697
CAGN{5}GGG
83
50
0.06
1039


 


698
GCCN{13}CGC
22
8
0.06
1040


 


699
GCGN{7}GGA
19
6
0.06
1041


 


700
CAGN{14}CCA
94
58
0.06
1042


 


701
CCGN{4}GTC
16
4
0.06
1043


 


702
CCCN{13}CGC
22
8
0.06
1044


 


703
GCGN{14}ACC
15
4
0.05
1045


 


704
CAGN{20}GGG
81
49
0.05
1046


 


705
CCGN{4}CCC
27
11
0.05
1047


 


706
CGCN{5}GGC
18
6
0.05
1048


 


707
CCTN{6}GGC
57
31
0.05
1049


 


708
AGGN{3}GGC
67
38
0.05
1050


 


709
CGGN{11}CGC
14
4
0.05
1051


 


710
CTGN{18}GGA
77
46
0.04
1052


 


711
CACN{17}CCA
74
43
0.04
1053


 


712
CGGN{3}GAG
22
8
0.04
1054


 


713
CCCN{9}CCA
82
49
0.03
1055


 


714
CCCN{1}ACG
18
6
0.03
1056


 


715
CAGN{1}GCC
72
42
0.03
1057


 


716
AGGN{6}CCG
23
8
0.03
1058


 


717
AGCN{9}GGG
57
31
0.03
1059


 


718
CCCN{7}GGC
54
29
0.02
1060


 


719
CCTN{13}CCC
88
54
0.02
1061


 


720
CCGN{19}TTC
20
7
0.02
1062


 


721
CCCN{7}CCG
27
11
0.02
1063


 


722
CGAN{6}GGC
17
5
0.01
1064


 


723
CGGN{4}CTC
21
7
0.01
1065


 


724
CGGN{0}CGC
13
3
0.01
1066


 


725
CCTN{13}ACG
19
6
0.01
1067


 


726
GGGN{6}CAC
53
28
0.01
1068


 


727
CCCN{16}CGC
21
7
0.01
1069


 


728
CCCN{10}CTC
76
45
0
1070


 


729
CCCN{0}CAG
92
57
0
1071


 


730
GCCN{5}CCC
65
37
0
1072
















TABLE 8







Candidate STAR elements tested by Linear Discriminant Analysis


(SEQ ID NOS: 66-84)










SEQ ID NO:
Candidate STAR
Location1
Length





66
T2 F
20q13.33
~2800


67
T2 R
6q14.1
~2800


68
T3 F
15q12
~2900


69
T3 R
7q31.2
~2900


70
T5 F
9q34.13
ND2


71
T5 R
9q34.13
ND


72
T7
22q12.3
~1200


73
T9 F
21q22.2
~1600


74
T9 R
22q11.22
~1600


75
T10 F
7q22.2
~1300


76
T10 R
6q14.1
~1300


77
T11 F
17q23.3
~2000


78
T11 R
16q23.1
~2000


79
T12
4p15.1
~2100


80
T13 F
20p13
~1700


81
T13 R
1p13.3
~1700


82
T14 R
11q25
~1500


83
T17
2q31.3
ND


84
T18
2q31.1
ND





1Chromosomal location is determined by BLAT search of DNA sequence data from the STAR elements against the human genome database. The location is given according to standard nomenclature referring to the cytogenetic ideogram of each chromosome; e.g., 1p2.3 is the third cytogenetic sub-band of the second cytogenetic band of the short arm of chromosome 1 (http://www.ncbi.nlm.nih.gov/Class/MLACourse/Genetics/chrombanding.html). F, forward sequencing reaction result; R, reverse sequencing reaction result. When the forward and reverse sequencing results mapped to different genomic locations, each sequence was extended to the full length of the original clone (as determined by restriction mapping) based on sequence information from the human genome database.


2ND: Not Determined.













TABLE 9







Arabidopsis STAR elements of the invention, including


chromosome location and length (SEQ ID NOS: 85-119)












STAR
Chromosome
Length, kb
SEQ ID NO:
















A1
I
1.2
85



A2
I
0.9
86



A3
I
0.9
87



A4
I
0.8
88



A5
I
1.3
89



A6
I
1.4
90



A7
II
1.2
91



A8
II
0.8
92



A9
II
0.9
93



A10
II
1.7
94



A11
II
1.9
95



A12
II
1.4
96



A13
II
1.2
97



A14
II
2.1
98



A15
II
1.4
99



A16
II
0.7
100



A17
II
1.5
101



A18
III
1.5
102



A19
III
0.7
103



A20
III
2.0
104



A21
IV
1.8
105



A22
IV
0.8
106



A23
IV
0.6
107



A24
IV
0.5
108



A25
V
0.9
109



A26
V
1.9
110



A27
V
1.1
111



A28
V
1.6
112



A29
V
0.9
113



A30
V
2.0
114



A31
V
2.0
115



A32
V
1.3
116



A33
V
0.9
117



A34
I
0.9
118



A35
II
1.1
119










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## Claims

1. A method for producing a proteinaceous molecule in a cell comprising:
providing a cell selected from the group consisting of: a cell having an adenovirus Early Region 1 (E1) sequence, a HuNS-1 myeloma cell, a 293 cell, a CHO cell, a Vero cell, a WERI-Rb-1 retinoblastoma cell, a BHK cell, a non-secreting mouse myeloma Sp2/0-Ag 14 cell, a non-secreting mouse myeloma NSO cell, and an NCI-H295R adrenal gland carcinoma cell;
wherein said cell comprises an anti-repressor activity sequence operably linked to a nucleic acid sequence encoding a heterologous proteinaceous molecule, wherein said anti-repressor activity sequence comprises SEQ ID NO:44;
expressing the proteinaceous molecule in said cell; and
isolating said proteinaceous molecule.

2. The method of claim 1, wherein said anti-repressor activity sequence is SEQ ID NO:44.

3. The method of claim 1, wherein said cell comprises an adenovirus Early Region 1 (E1) sequence.

4. The method of claim 1, wherein said proteinaceous molecule is secreted by said cell.

5. The method of claim 2, wherein said proteinaceous molecule is secreted by said cell.

6. The method of claim 1, wherein said cell comprises a plurality of the anti-repressor activity sequence operably linked to said nucleic acid sequence encoding the heterologous proteinaceous molecule.

7. The method of claim 6, wherein at least one anti-repressor activity sequence is positioned 5′ of the sequence encoding the proteinaceous molecule and at least one anti-repressor activity sequence is positioned 3′ of the sequence encoding the proteinaceous molecule.

8. The method of claim 1, wherein said cell is a CHO cell.

9. The method of claim 6, wherein said cell is a CHO cell.

10. The method of claim 7, wherein said cell is a CHO cell.

11. A recombinant host cell line, comprising:
a cell selected from the group consisting of: a cell line comprising an adenovirus Early Region 1 (E1) sequence, a HuNS-1 myeloma cell line, a 293 cell line, a CHO cell line, a Vero cell line, a WERI-Rb-1 retinoblastoma cell line, a BHK cell line, a non-secreting mouse myeloma Sp2/0-Ag 14 cell line, a non-secreting mouse myeloma NSO cell line, and an NCI-H295R adrenal gland carcinoma cell line;
said cell comprising an anti-repressor activity sequence operably linked to a nucleic acid sequence encoding a heterologous proteinaceous molecule, wherein said anti-repressor activity sequence comprises SEQ ID NO:44.

12. The cell line of claim 11, wherein said anti-repressor activity sequence is SEQ ID NO:44.

13. The cell line of claim 11, wherein said cell line comprises an adenovirus Early Region 1 sequence.

14. The cell line of claim 11, wherein said cell comprises a plurality of the anti-repressor activity sequence operably linked to said nucleic acid sequence encoding the heterologous proteinaceous molecule.

15. The cell line of claim 11, wherein at least one anti-repressor activity sequence is positioned 5′ of the sequence encoding the proteinaceous molecule and at least one anti-repressor activity sequence is positioned 3′ of the sequence encoding the proteinaceous molecule.

16. The cell line of claim 11, wherein said cell line is a CHO cell line.

17. The cell line of claim 14, wherein said cell line is a CHO cell line.

18. The cell line of claim 15, wherein said cell line is a CHO cell line.