Patent Application: US-46209295-A

Abstract:
a method of inducibly enhancing the constitutive expression of a dna sequence of interest is described in which plant cells are transformed with a dna sequence of interest that is operably joined to a plant ubiquitin regulatory region comprised of a heat shock element , a promoter , a transcription start site , an intron , and a translation start site . when monocot or dicot plant cells are subjected to permissive heat shock temperatures , the level of expression of the dna sequence of interest is enhanced .

Description:
the following definitions are provided in order to remove ambiguities as to the intent or scope of their usage in the specification and claims . expression refers to the transcription and / or translation of a structural gene . promoter refers to the nucleotide sequences at the 5 &# 39 ; end of a structural gene which direct the initiation of transcription . promoter sequences are necessary , but not always sufficient , to drive the expression of a downstream gene . in general , eukaryotic promoters include a characteristic dna sequence homologous to the consensus 5 &# 39 ;- tataat - 3 &# 39 ; ( tata ) box about 10 - 30 bp 5 &# 39 ; to the transcription start ( cap ) site , which , by convention , is numbered + 1 . bases 3 &# 39 ; to the cap site are given positive numbers , whereas bases 5 &# 39 ; to the cap site receive negative numbers , reflecting their distance from the cap site . another promoter component , the caat box , is often found about 30 to 70 bp 5 &# 39 ; to the tata box and has homology to the canonical form 5 - ccaat - 3 &# 39 ; ( r . breathnach and p . chambon ( 1981 ) ann . rev . biochem . 50 : 349 - 383 ). in plants the caat box is sometimes replaced by a sequence known as the agga box , a region having adenine residues symmetrically flanking the triplet g ( ort ) ng ( j . messing et al . ( 1983 ), in genetic engineering of plants , t . kosuge , c . meredith and a . hollaender ( eds . ), plenum press , pp . 211 - 227 ). other sequences conferring regulatory influences on transcription can be found within the promoter region and extending as far as 1000 bp or more from the cap site . regulatory control refers to the modulation of gene expression induced by dna sequence elements located primarily , but not exclusively , upstream of ( 5 &# 39 ; to ) the transcription start site . regulation may result in an all - or - nothing response to environmental stimuli , or it may result in variations in the level of gene expression . in this invention , the heat shock regulatory elements function to enhance transiently the level of downstream gene expression in response to sudden temperature elevation . placing a structural gene under the regulatory control of a pomoter or a regulatory element means positioning the structural gene such that the expression of the gene is controlled by these sequences . in general , promoters are found positioned 5 &# 39 ; ( upstream ) to the genes that they control . thus , in the construction of heterologous promoter / structural gene combinations , the promoter is preferably positioned upstream to the gene and at a distance from the transcription start site that approximates the distance between the promoter and the gene it controls in its natural setting . as is known in the art , some variation in this distance can be tolerated without loss of promoter function . similarly , the preferred positioning of a regulatory element with respect to a heterologous gene placed under its control reflects its natural position relative to the structural gene naturally regulates . again , as is known in the art , some variation in this distance can be accommodated . promoter function during expression of a structural gene under its regulatory control can be tested at the transcriptional stage using dna - rna hybridization assays (&# 34 ; northern &# 34 ; blots ) and at the translational stage using specific functional assays for the protein synthesized ( for example , by enzymatic activity or by immunoassay of the protein ). structural gene is that portion of a gene comprising a dna segment encoding a protein , polypeptide or a portion thereof , and excluding the 5 &# 39 ; sequence which drives the initiation of transcription . the structural gene may be one which is normally found in the cell or one which is not normally found in the cellular location wherein it is introduced , in which case it is termed a heterologous gene . a heterologous gene may be derived in whole or in part from any source known to the art , including a bacterial genome or episome , eukaryotic , nuclear or plasmid dna , cdna , viral dna or chemically synthesized dna . a structural gene may contain one or more modifications in either the coding or the untranslated regions which could affect the biological activity or the chemical structure of the expression product , the rate of expression or the manner of expression control . such modifications include , but are not limited to , mutations , insertions , deletions and substitutions of one or more nucleotides . the structural gene may constitute an uninterrupted coding sequence or it may include one or more introns , bound by the appropriate splice junctions . the structural gene may be a composite of segments derived from a plurality of sources , naturally occurring or synthetic . the structural gene may also encode a fusion protein . it is contemplated that the introduction into plant tissue of recombinant dna molecules containing the promoter / structural gene / polyadenylation signal complex will include constructions wherein the structural gene and its promoter are each derived from different plant species . plant ubiquitin regulatory system refers to the approximately 1 kb nucleotide sequence 5 &# 39 ; to the translation start site of the maize ubiquitin gene and comprises sequences that direct initiation of transcription , regulation of transcription , control of expression level , induction of stress genes and enhancement of expression in response to stress . the regulatory system , comprising both promoter and regulatory functions , is the dna sequence providing regulatory control or modulation of gene expression . a structural gene placed under the regulatory control of the plant ubiquitin regulatory system means that a structural gene is positioned such that the regulated expression of the gene is controlled by the sequences comprising the ubiquitin regulatory system . polyadenylation signal refers to any nucleic acid sequence capable of effecting mrna processing , usually characterized by the addition of polyadenylic acid tracts to the 3 &# 39 ;- ends of the mrna precursors . the polyadenylation signal dna segment may itself be a composite of segments derived from several sources , naturally occurring or synthetic , and may be from a genomic dna or an mrna - derived cdna . polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5 &# 39 ;- aataa - 3 &# 39 ;, although variation of distance , partial &# 34 ; readthrough &# 34 ; and multiple tandem canonical sequences are not uncommon ( j . messing et al . supra ). it should be recognized that a canonical &# 34 ; polyadenylation signal &# 34 ; may in fact cause transcriptional termination and not polyadenylation per se ( c . montell et al . ( 1983 ) nature 305 : 600 - 605 ) plant tissue includes differentiated and undifferentiated tissues of plants , including , but not limited to roots , shoots , leaves , pollen , seeds , tumor tissue and various forms of cells in culture , such as single cells , protoplasts , embryos and callus tissue . the plant tissue may be in planta or in organ , tissue or cell culture . homology , as used herein , refers to identity or near identity of nucleotide and / or amino acid sequences . as is understood in the art , nucleotide mismatches can occur at the third or wobble base in the codon without causing amino acid substitutions in the final polypeptide sequence . also , minor nucleotide modifications ( e . g ., substitutions , insertions or deletions ) in certain regions of the gene sequence can be tolerated and considered insignificant whenever such modifications result in changes in amino acid sequence that do not alter the functionality of the final product . it has been shown that chemically synthesized copies of whole , or parts of , gene sequences can replace the corresponding regions in the natural gene without loss of gene function . homologs of specific dna sequences may be identified by those skilled in the art using the test of cross - hybridization of nucleic acids under conditions of stringency as is well understood in the art ( as described in hames and higgens ( eds .) ( 1985 ) nucleic acid hybridization , irl press , oxford , uk ). extent of hemology is often measured in terms of percentage of identity between the sequences compared . thus , in this disclosure it will be understood that minor sequence variation can exist within homologous sequences . derived from is used herein to mean taken , obtained , received , traced , replicated or descended from a source ( chemical and / or biological ). a derivative may be produced by chemical or biological manipulation ( including but not limited to substitution , addition , insertion , deletion , extraction , isolation , mutation and replication ) of the original source . chemically , synthesized , as related to a sequence of dna , means that the component nucleotides were assembled vitro . manual chemical synthesis of dna may be accomplished using well established procedures ( m . caruthers ( 1983 ) in methodology of dna and rna sequencing , weissman ( ed . ), praeger publishers ( new york ) chapter 1 ), or automated chemical synthesis can be performed using one of a number of commercially available machines . heat shock elements refer to dna sequences that regulate gene expression in response to the stress of sudden temperature elevations . the response is seen as an immediate albeit transitory enhancement in level of expression of a downstream gene . the original work on heat shock genes was done with drosophila but many other species including plants ( t . barnett et al . ( 1980 ) dev . genet . 1 : 331 - 340 ) exhibited analogous responses to stress . the essential primary component of the heat shock element was described in drosophila to have the consensus sequence 5 &# 39 ;- ctggaat - ttctaga - 3 &# 39 ; and to be located in the region between residues - 66 through - 47 bp upstream to the transcriptional start site ( h . pelham and m . bienz ( 1982 ) supra ). a chemically synthesized oligonucleotide copy of this consensus sequence can replace the natural sequence in conferring heat shock inducibility . in other systems , multiple heat shock elements were identified within the promoter region . for example , rochester et al . ( 1986 ) supra recognized two heat shock elements in the maize hsp 70 gene . leader sequence refers to a dna sequence comprising about 100 nucleotides located between the transcription start site and the translation start site . embodied within the leader sequence is a region that specifies the ribosome binding site . introns or intervening sequences refer in this work to those regions of dna sequence that are transcribed along with the coding sequences ( exons ) but are then removed in the formation of the mature mrna . introns may occur anywhere within a transcribed sequence -- between coding sequences of the same or different genes , within the coding sequence of a gene , interrupting and splitting its amino acid sequences , and within the promoter region ( 5 &# 39 ; to the translation start site ). introns in the primary transcript are excised and the coding sequences are simultaneously and precisely ligated to form the mature mrna . the junctions of introns and exons form the splice sites . the base sequence of an intron begins with gu and ends with ag . the same splicing signal is found in many higher eukaryotes . the present invention relates to the development of a recombinant vector useful for the expression of dna coding segments in plant cells . the vector herein described employs a maize ubiquitin promote to control expression of an inserted dna coding segment . the transcriptional regulatory sequences may be combined with an extrachromosomal replication system for a predetermined host . other dna sequences having restriction sites for gene insertion may be added to provide a vector for the regulated transcription and translation of the inserted genes in said host . the vector may also include a prokaryotic replication system allowing amplification in a prokaryotic host , markers for selection and other dna regions . this would allow large quantities of the vector to be grown in well characterized bacterial systems prior to transforming a plant or mammalian host . the principles for construction of a vector having proper orientation of the promoter and coding sequences with respect to each other are matters well - known to those skilled in the art . in some situations it may be desirable to join the promoter system to a desired structural gene and to introduce the resultant construct dna directly into a host . methods for such direct transfers include , but are not limited to , protoplast transformation , electroporation , direct injection of dna into nuclei and co - transformation by calcium precipitation . this invention comprises the first report of an isolated and characterized plant ubiquitin promoter . the maize ubiquitin promoter as described in the present work includes the rna polymerase recognition and binding sites , the transcriptional initiation sequence ( cap site ), regulatory sequences responsible for inducible transcription and an untranslatable intervening sequence ( intron ) between the transcriptional start site and the translational initiation site . two overlapping heat shock consensus promoter sequences are situated 5 &# 39 ; (- 214 and - 204 ) of the transcriptional start site . an exon of 83 nucleotides is located immediately adjacent to the cap site and is followed by a large ( approximately 1 kb ) intron . the ubiquitin promoter along with the ubiquitin structural gene can be isolated on two approximately 2 kb pst1 fragments of the maize genome ( fig1 ). the entire fragment can be used to show promoter function by monitoring expression of mrna or protein . introduction of a heterologous gene downstream of the ubiquitin translation initiation codon will result in the expression of a fused protein . insertion of a heterologous gene ( having its own start and stop codons ) between the ubiquitin promoter and translation initiation codon will result in the expression of the native polypeptide corresponding to the inserted gene . the insertion of the desired structural gene is most conveniently accomplished with the use of blunt - ended linkers at the ends of the gene . alternatively , the ubiquitin gene fragment may be restricted , particularly at a site immediately preceding the start of the structural gene or at a site preceding the transcription start site . for example , in the present invention the promoter fragment was derived from the ubiquitin gene as an approximately 2 kb pst1 fragment . to ensure that the promoter fragment is devoid of the translational initiation codon , the fragment containing the 5 &# 39 ; flanking region may be selectively digested with double stranded exonuclease under controlled conditions to remove a desired number of nucleotide pairs . it is desirable to remove the ubiquitin translation initiation codon so that translation of the inserted gene will commence at its own start site . the isolated ( and shortened ) promoter fragment may then be inserted into the vector using linkers or homopolymer tailing to introduce desired restriction sites compatible with the remaining regions of the vector . in general , the promoter fragment may be cleaved with specific restriction enzymes and the resultant shortened dna fragments tested for promoter function and compared to that of the intact promoter . in addition , dna codons may be added and / or existing sequences may be modified to give derivative dna fragments retaining promoter functions . the resulting dna constructs may be useful as cloning vehicles for a structural gene of interest in a plant host . in this invention , the structural gene encoding cat under control of either the maize ubiquitin promoter or the cauliflower mosaic virus promoter was expressed in both oat and tobacco cells . when the ubiquitin promoter was employed , a greater degree of expression was obtained with the monocot host than with the dicot host ; however , a higher level of expression was obtained with dicot than with monocot host when the cauliflower mosaic virus promoter was utilized . the differential in expression levels reflects the inherent inequality of different promoters as well as basic cellular differences in regulation of expression and processing between monocots and dicots . to date , it is not predictable , routine or obvious that a monocot promoter will operate in a dicot host cell . a wide variety of structural genes may be introduced into the subject dna cloning vectors for the production of desired proteins , such as enzymes , hormones and the like . in addition , dna constructs of this type can be used for the enhanced production of dna derived from a particular gene , as well as for enhanced production of mrna which can be used to produce cdna . such vectors carrying specific dna sequences find wide application and are quite versatile ; for example , they can be used for amplification in bacteria as well as for expression in higher cells which allow for additional cellular functions . an advantage of utilizing higher eukaryotic recombinant systems to produce commercially medical and agriculturally desirable proteins is that they ensure correct post - translational modifications which may otherwise be difficult to duplicate in prokaryotic and lower eukaryotic hosts . in this invention the maize ubiquitin promoter was shown to function in oat and tobacco , as examples of monocots and dicots , respectively , and it is conceivable that this promoter can function in yet other cells . such systems include , by way of example , and without limitation , other cells from which ubiquitin genes have been isolated and found to be highly conserved , for example , other monocots in addition to maize , dicots other than tobacco , lower eukaryotic organisms such as yeast and mammalian cells . the screening of cellular systems suitable for use with the maize ubiguitin promoter can be accomplished according to the teaching herein , without undue experimentation . the construction of vectors suitable for the expression of a dna coding segment in individual systems has been well documented . shuttle vectors capable of replication in more than one host have also been described , for example , shuttle expression vectors for both yeast and mammalian cells , for plants and animal cells and for plants and bacterial cells . in addition , it will be understood that ubiquitin genes from any other system , that are similar to the maize ubiquitin gene in functioning as a plant promoter , may be employed as the source for the ubiquitin promoter sequence . the present invention also relates to the utilization of the maize ubiquitin promoter as a heat shock promoter . two heat shock consensus sequences are located upstream of the maize ubiquitin gene at positions - 214 and - 204 . in many eukaryotes , naturally occurring and chemically - synthesized sequences homologous to the heat shock consensus sequence have been shown to regulate the induction of gene expression . although the ubiquitin promoter contains sequences that are identified as being those of heat shock elements , the promoter is distinguished from classical heat shock promoters ( 1 ) in having a nontranslated intron 3 &# 39 ; to the transcription start site and ( 2 ) in regulating ubiquitin expression constitutively as well as inductively . the functional relationship between heat shock elements and the presence of a large intron within the promoter region is unknown to prior art . the nucleotide distance between these characteristic features and also the directionality and orientation of one element with respect to the other are presumed in the present work to be variable , as long as the basic promoter function of the derivative regulatory fragments remains active . the presence of an intron in the promoter region has been related to the relative stability of the unprocessed mrna transcript and , indirectly , to the level of protein synthesized ( callis et al . ( 1987 ) genes and development 1 : 1183 - 1200 ). constitutively expressed ubiquitin mrna has been reported to be maintained at stable levels in chicken embryo fibroblasts , whereas ubiquitin mrna formed in response to stress has a half - life of approximately 1 . 5 to 2 h . in yeast four distinct ubiquitin - coding loci have been described . constitutively expressed ubiquitin is encoded by one or more of three of the ubiquitin genes , two of which contain an approximately 400 bp intron immediately within the coding region . the fourth ubiquitin gene , devoid of a nontranslated intron but comprising multiple heat shock elements , functions primarily in inducing ubiquitin expression in response to stress . it has been shown that the latter ubiquitin gene does not act constitutively but rather is turned on in response to heat shock or stress signal ( e . ozkaynak et al . ( 1987 ) embo j . 6 : 1429 - 1439 ). in maize , ubiquitin is encoded by a small multigene family . in this invention is presented the nucleotide sequence of one of the ubiquitin genes . a large ( approximately 1 kb ) intron between the transcriptional and the translational start sites as well as nucleotide sequences corresponding to consensus heat shock sequences are found within the maize ubiquitin promoter region . these two regions of specialization most probably are involved in ubiquitin synthesis and in regulating the ubiquitin level in response to external influences . the functional relationship between the intron and the heat shock elements encompassed within the ubiquitin promoter is unknown . it is reported in this invention that the maize ubiquitin promoter regulates the synthesis of mrna both under normal and under heat shock conditions and that changes in the regulation of transcription account for the enhancement in ubiquitin synthesis after heat shock . the following examples are offered by way of illustration and not by way of limitation . zea mays inbred line b73 was grown in moist vermiculite for 4 to 5 days at 25 ° c . in the dark . the portion of the seedlings from the mesocotyl node to the shoot tip was harvested , frozen in liquid nitrogen and stored at - 80 ° c . total cellular rna was extracted from frozen tissue using the guanidine thiocyanate procedure . poly ( a )+ rna was isolated from total cellular rna by passage over a poly u - sephadex ( bethesda research laboratories , gaithersburg , md .) column . total or poly ( a )+ rna was electrophoresed in 1 . 5 % agarose gels containing 3 % ( wt / vol ) formaldehyde . rna was transferred to gene screen ™ ( dupont ) by capillary blotting using 25 mm sodium phosphate ( ph6 . 5 ). blots were prehybridized in 50 % formamide , 5xssc , 100 μg denatured salmon dna , 40 mm sodium phosphate ( ph6 . 8 ), 0 . 5 % bsa and 1 % sds . blots were hybridized in 50 % formamide , 5xssc , 100 μg / ml denatured salmon dna , 40 mm sodium phosphate ( ph6 . 8 ) and 10 % dextran sulfate . double stranded cdna was synthesized from poly ( a )+ rna by a modification of the method of murray et al . ( 1983 ) plant mol . biol . 2 : 75 - 84 . oligo ( dc )- tailing of the double - stranded cdna and annealing of oligo ( dc )- tailed cdna with oligo ( dg )- tailed pbr322 were performed using standard technology . the annealed dna was transformed into e . coli hb101 and plated directly onto nitrocellulose filters ( millipore , hatf ; 0 . 45 μm ) on l - agar plates containing tetracycline ( 15 μg / ml ). a number of cdnas representing potentially light - regulated mrnas were obtained by screening a cdna library by differential hybridization . several of these cdnas were selected and further screened by rna blot analysis to confirm light regulation . one cdna clone , p6t7 . 2b1 , while not representing a red - light regulated mrna , was of interest because it hybridized with three poly ( a )+ rnas of different size and abundance . nick translated p6t7 . 2b1 hybridized strongly with the 2100 nucleotide and 1600 nucleotide mrnas , but only weakly with the 800 nucleotide transcript . however , hybridization of northern blots with a single stranded 32 p - labeled rna generated by sp6 polymerase transcription of linearized pca210 , a plasmid constructed by subcloning the cdna insert of p6t7 . 2b1 into psp64 , readily detected all three transcripts . since rna - rna hybrids are known to be more thermally stable than dna - rna hybrids , single stranded rna probes rather than nick translated dna probes were used in northern blot hybridizations . again , the 1600 base transcript was found to be about 3 fold less abundant than the 2100 base transcript as determined from northern blots , regardless of whether the blot was hybridized with nick translated dna or single strand rna probes . the smallest transcript was about half as abundant as the 2100 base mrna in blots hybridized with rna probes . restriction fragments were subcloned into m13mp18 and / or mp19 and sequenced by the dideoxynucleotide chain termination method . analysis of the sequence of the clone revealed a single long open reading frame of 818 bp terminating in a taa stop codon . the national biomedical research foundation library was searched using the d fast p program for protein sequences homologous with the deduced amine acid sequence . greater than 95 % hemology was found between the deduced amine acid sequence of the maize cdna clone and the sequences of bovine and human ubiquitin . high molecular weight maize dna was isolated from frozen maize seedling . dna was partially digested with sau3a , size fractionated and cloned into the bamh1 sites of charon 35 ( loenen et al . ( 1983 ) gene 26 : 171 - 179 ). a library of about 2 × 10 6 pfu was screened for recombinant phage containing sequences homologous to the ubiquitin cdna clone by in situ plaque hybridization using a ubiquitin cdna clone as a hybridization probe . recombinant phage were purified from broth lysates and phage dna was isolated using standard techniques . restriction endonuclease digestions were carried out according to manufacturers &# 39 ; specifications . isolated , high molecular weight maize dna was digested with ecor1 , hind111 and sac1 , fractionated on 0 . 7 % agarose gels and the dna fragments were transferred to gene screen plus ™ ( dupont ). filters were prehybridized for 6 - 8 h at 65 ° c . in 6xssc ( 1xssc = 0 . 15m nacl , 0 . 025m na citrate ), 5x denhardt &# 39 ; s medium , 100 μg / ml denatured , sonicated salmon dna , 20 μg / ml polyadenylic acid , 10 mm disodium edta and 0 . 5 % sds . filters were hybridized at 65 ° c . in fresh buffer with 32 p labeled plasmid dna ( pca210 ). autoradiography was carried out at - 80 ° c . using kodak x - omat ar film and one dupont cronex lightningplus intensifying screen . in each digest , 8 to 10 restriction fragments hybridized with the nick translated pca210 probe , suggesting that ubiquitin is coded by a small multigene family . evidence that ubiquitin is encoded by a small multigene family has also been reported for xenopus , barley and yeast . two or three fragments in each digest hybridized strongly with the probe , whereas the remainder of the fragments hybridized weakly . the differences in hybridization intensities may reflect different sequence homology such that the cdna probe hybridizes preferentially to the gene from which it was derived . ubiquitin genes from yeast and xenopus have been characterized and have six and at least twelve ubiquitin repeats , respectively . maize genes corresponding to the three transcripts detected on northern blots may have seven , five and one or two ubiquitin repeats in the 2 . 1 , 1 . 6 and 0 . 8 kb mrnas , respectively . the maize ubiquitin gene described in this invention codes for seven repeats . thus , the difference in hybridization intensity observed on southern blots may be a result of the restriction fragments containing a different number of ubiquitin repeats . the ubiquitin cdna clone did not contain ecor1 and hind111 sites . however , the maize ubiquitin genes may contain introns which are cut by the restriction endonucleases used in the genomic digests . this could result in ubiquitin exons being on different fragments and could account for the differential hybridization intensities observed in the southern blots . dideoxynucleotide chain termination sequencing was performed using klenow fragments of dna polymerase 1 ( boehringer mannheim ). a 1 . 85 kb pst1 fragment of the genomic clone 7 . 2b1 ( see fig1 b ) homologous to the cdna clone p6t7 . 2b . 1 and the 2 kb pst1 fragment immediately upstream , termed ac3 # 9m13rf , were subcloned in both orientations into m13mp19 . recombinant phage rf dna was prepared as for plasmid dna . unidirectional progressive deletion clones for sequencing both strands of these pst1 fragments were prepared . exonuclease 111 and exonuclease v11 were obtained from new england biolabs and bethesda research laboratories , respectively . computer analysis of dna sequences was performed using programs made available by the university of wisconsin genetics computer group . the transcription start site of the ubiquitin gene and the 3 &# 39 ; junction of the intron and exon in the 5 &# 39 ; untranslated region of the gene were determined by s1 nuclease mapping . fragments suitable for s1 probes were prepared as follows . the ubiquitin dna was digested with either bgl11 or xho1 . these were then labeled with 32 p using - 32 p atp ( 6000 ci / mmole , new - england nuclear , boston , mass .) and t4 polynucleotide kinase ( new england biolabs ). subsequent digestion of the bgl11 and xho1 kinased fragments with pst1 and ecor1 , respectively , generated a 946 bp pst1 - bgl11 fragment and a 643 bp ecor1xho1 fragment . these fragments were separated from the other end - labeled fragments by electrophoresis through a 5 % polyacrylamide gel . slices containing the 946 bp pst1 - bgl11 and the 643 bp ecor1 - xho1 fragments were cut out of the gel and the labeled dnas were eluted from the gel . end - labeled dna fragment ( 10 - 20 fmole ) was hybridized with 2 μg of poly ( a )+ rna in 30 μl of buffer containing 80 % deionized formamide , 0 . 4m sodium chloride , 40 mm pipes ( ph6 . 4 ) and 1 mm edta ( ph8 . 0 ). the nucleic acid solution was heated to 80 ° c . for 15 min to denature the dna and then incubated at 42 ° c . for about 16 h . ice - cold s1 digestion buffer ( 300 μl ) containing 280 mm sodium chloride , 50 mm sodium acetate ( ph4 . 6 ), 4 . 5 mm zinc sulfate and 20 μg / ml single stranded dna was added and the dna digested with 250 units / ml of s1 nuclease ( new england nuclear ). the reaction was stopped with 75 μl of s1 termination mix containing 2 . 5m ammonium acetate and 50 mm edta . the products of the s1 nuclease digestion were then separated on a 6 % polyacrylamide / 8m urea gel and visualized by autoradiography . the end points of the s1 protected fragments in the ubiquitin sequence were determined by comparison with a sequence ladder generated by maxam / gilbert base modification - cleavage reactions carried out on the end labeled fragments used as s1 probes . the dna sequence of the maize ubiquitin - 1 gene , 7 . 2b1 , is shown in fig2 . the sequence is composed of 899 bases upstream of the transcription start site , 1992 bases of 5 &# 39 ; untranslated and intron sequences , and 1999 bases encoding seven ubiquitin protein repeats preceding 249 bases of 3 &# 39 ; sequence . a &# 34 ; tata &# 34 ; box is located at - 30 and two overlapping heat shock elements are located at - 214 and - 204 . the dna sequence of the coding and 3 &# 39 ; regions of the ubiquitin - 1 gene from maize , 7 . 2b1 , is also presented in fig3 . the derived amine acid sequence of maize ubiquitin is shown at the top and the nucleotide sequence of the seven ubiquitin repeats is aligned underneath . a schematic of the organization of the seven complete ubiquitin units in the genomic dna is shown in fig1 c . the derived amine acid sequences of all of the ubiquitin repeats are identical ( fig3 ). the terminal ( seventh ) ubiquitin repeat contains an additional 77th amine acid , glutamine , prior to the taa stop codon . this additional amine acid is not found in mature ubiquitin , and is apparently removed during processing . the 77th amine acid of the final repeat in the human gene is valine , while in the two chicken genes , it is tyrosine and asparagine . yeast and barley also have an extra amine acid , asparagine and lysine , respectively ; however an extra amino acid was not found in the xenopus gene . this extra amino acid has been proposed to function as a block to conjugation of unprocessed polyubiquitin to target proteins . a polyadenylation signal ( aataat ) is present in the 3 &# 39 ; untranslated sequence , 113 bp from the stop codon . all seven repeats encode the identical amino acid sequence , whereas the nucleotide sequence of the repeats varies by as many as 39 nucleotides . this is similar to what has been reported for the nucleotide sequence homologies between ubiquitin coding repeats of other ubiquitin genes . about 80 % of the nucleotide mismatches between ubiquitin repeats are at the third ( wobble ) base in the - codon . alternate codon usage for leucine ( 5 codons ), serine ( 3 codons ) and arginine ( 3 codons ) account for the remaining nucleotide mismatches . the amino acid sequence for maize ubiquitin is identical to that determined for two other higher plants , oat and barley . the sequence differs from the sequence reported for yeast by two amino acids ; alanine for serine substitutions at positions 28 and 57 . the maize sequence is also slightly different from that reported for ubiquitin from all animals ; substitutions by serine for proline at position 19 , aspartate for glutamate at position 24 and alanine for serine at position 57 . thus , based on sequence , there appear to be three types of ubiquitin : plant , animal and yeast . example 2 : construction of plasmid pub - cat comprising the maize ubiquitin promoter and a structural gene the procedure used for construction of the ubiquitin gene upstream region - chloramphenicol acetyl transferase ( cat ) gene fusion is outlined in fig4 . the bamh1 - hind111 restriction fragment containing the cat gene and the nopaline synthase ( nos ) 3 &# 39 ; untranslated region and polyadenylation signal of pnos - cat ( fromm et al . ( 1985 ) proc . natl . acad . sci . 82 : 5824 - 5828 ) was subcloned into bamh1 and hind1 digested puc18 . this construct was termed pub - cat . an approximately 2 . 0 kb pst1 fragment immediately upstream of the ubiquitin polyprotein coding region of the maize ubiquitin gene 7 . 2 b1 war subcloned into m13mp19 . this segment of dna spans nucleotides - 899 to 1092 of the maize ubiquitin sequence documented in fig2 . this recombinant dna was termed ac3 # 9m13rf and contains the ubiquitin promoter , 5 &# 39 ; untranslated leader sequence and about 1 kb intron , labeled ubi - 5 &# 39 ; in fig4 . the ubiquitin promoter - cat reporter gene fusion was constructed by blunt ending with t 4 dna polymerase the 2 . 0 kb pst1 fragment of ac3 # 9m13rf and cloning this fragment into sma1 - digested puc18 - cat . this construct was termed pub - cat . leaves ( 2g ) of 5 - to 6 - day old etiolated oat seedlings were finely chopped with a razor blade . the tissue was rinsed several times with digestion medium ( 3 mm mes , ph5 . 7 , 10 mm calcium chloride , 0 . 5m mannitol and 2 mg / ml arginine ) and then incubated for 4 h at room temperature with 20 ml digestion medium containing 2 % cellulase . the tissue was shaken occasionally to release protoplasts . the material was filtered through a 63 μm mesh and centrifuged 5 min at 50 xg . the supernatant fluid was removed and the protoplasts were washed two times with digestion medium and then resuspended in electroporation buffer to give 0 . 5 ml of protoplast suspension per electroporation . the electroporation buffer consisted of : 10 mm hepes , ph7 . 2 , 150 mm sodium chloride , 4 mm calcium chloride and 0 . 5m mannitol . protoplasts ( 0 . 5 ml ) in electroporation buffer were mixed on ice with 0 . 5 ml of electroporation buffer containing 40 μg plasmid dna plus 100 μg sonicated salmon dna . the protoplasts were electroporated on ice with a 350 volt , 70 msec pulse . the protoplasts were incubated another 10 min on ice , then diluted into 10 ml murashige - skoog ( ms ) medium and incubated at room temperature for 24 h . protoplasts were pelleted by centrifugation for 5 min at 50 xg . the supernatant fluid was removed and the protoplasts washed once with ms medium . the protoplast pellet was resuspended in 200 μl buffer a ( 0 . 25m tris , ph7 . 8 , 1 mm edta , 1 mm β - mercaptoethanol ) and transferred to a microcentrifuge tube . protoplasts were disrupted by sonication for 5 - 10 sec at the lowest setting . protoplast debris was pelleted by centrifugation for 5 min at 4 ° c . the supernatant fluid was removed , heated to 65 ° c . for 10 min and stored at - 20 ° c . aliquots ( 100 μl ) of the electroporated protoplast extract ( extract of cells transformed with recombinant dna ) were added to 80 μl of buffer a and 20 μl of a mix of 20 μl 14 c - chloramphenicol ( 40 - 60 mci / mm ), 2 mg acetyl coa and 230 μl buffer a . the reaction was incubated for 90 min at 37 ° c . the reaction products were extracted with 600 μl ethyl acetate and were concentrated by evaporating the ethyl acetate and resuspending in 10 μl ethyl acetate . the reaction products were separated by thin layer chromatography using chloroform : methanol ( 95 : 5 , v / v ) solvent and were detected by autoradiography . transformation of host cells was determined by measuring the amount of enzymatic activity expressed by the structural gene contained within the promoter gene fusion construct . in this example , the structural gene encoding chloramphenicol acetyl transferase was employed in the dna construct . to test the efficacy of the promoter utilized in the recombinant dna fusion construct , parallel electroporations were carried out , utilizing either the maize ubiquitin promoter - cat gene fusion puc - cat ( described herein and in fig4 ) and pcamv - cat , a cauliflower mosaic virus 35s promoter - cat gene fusion ( fromm et al . ( 1985 ) proc . natl . acad . sci . usa 82 : 5824 - 5828 ) obtained from v . walbot , stanford university . as illustrated in fig5 in oat protoplasts the ubiquitin promoter is &# 34 ; stronger &# 34 ; than the camv promoter , as judged by the amount of enzymatic activity expressed . to heat shock , 4 to 5 day old etiolated seedlings were transferred to an incubator at 42 ° c . and harvested 1 , 3 and 8 h after transfer . total rna ( 7 μg ) was isolated , denatured and electrophoresed through a 1 . 5 % agarose 3 % formaldehyde gel . the rna was transferred to gene screen and probed with single stranded rna transcribed from linearized pca210 using sp6 rna polymerase . ( the recombinant plasmid , pca210 , was constructed by subcloning the 975 bp insert of p6t7 . 2b1 into psp64 ( promega ) so that sp6 rna polymerase synthesized a rna probe specific for hybridization with ubiquitin mrna .) after autoradiography , the bands were cut out and the amount of radioactivity bound to the filter was determined by liquid scintillation . from analysis of the northern blots , levels of three ubiquitin transcripts were determined . one hour after transfer to 42 ° c ., the level of the 2 . 1 kb transcript increased 2 . 5 to 3 fold . an approximately 2 fold increase was observed for the 1 . 6 kb transcript , however , no increase was seen for the 0 . 8 kb transcript . by three hours after transfer of the seedlings to elevated temperature , the levels of the two largest ubiquitin transcripts had returned to the level observed in unshocked tissue and remained at those levels for at least another five hours . the transitory nature of ubiquitin during the heat shock response in maize may indicate that ubiquitin has a specialized role in heat shock and that only brief periods of increased levels of ubiquitin are required . the nucleotide sequence of the maize ubiquitin gene is presented in fig2 . within the promoter region are nucleotide sequences homologous to the consensus heat shock sequence that has been shown to confer stress inducibility when placed upstream of heterologous promoters ( pelham ( 1982 ) supra ). the consensus sequence for the drosophila heat shock element is and is generally found approximately 26 bases upstream of the transcriptional start site . located within 900 bases 5 &# 39 ; to the transcriptional start site of the maize ubiquitin promoter are two overlapping heat shock sequences : the ubiquitin promoter from chicken embryo fibroblasts was also found to contain two overlapping heat shock consensus promoter sequences : the 5 &# 39 ; flanking region of the yeast ubiquitin gene ub14 ( e . ozkaynak et al . ( 1987 ) supra ) comprises an 18 kb , rotationally symmetric ( palindromic ) sequence , 5 &# 39 ;- ttctagaacgttctagaa - 3 , 365 bases upstream of the translation start site . the middle 14 bases ( underlined ) of this 18 bp sequence contain an exact homology to the rotationally symmetric consensus ` heat shock box ` nucleotide sequence starting at approximately 284 nucleotides upstream of the presumed transcription start site . the relative position of the heat shock sequence with respect to the transcriptional initiation codon and its ultimate consequence on the magnitude of the induction response to heat shock or other stress remains largely unknown , although it has been suggested ( u . bond et al . ( 1986 ) supra ) that the further a heat shock element is located 5 &# 39 ; from the transcriptional start site , the smaller is the level of induction in response to stress . in this invention it is assumed that a heat shock sequence may be arbitrarily positioned at different loci within the ubiquitin promoter and that it may be chemically altered in sequence or be replaced with a synthetic homologous sequence , so long as the modified promoter sequence retains ubiquitin promoter function , which comprises the initiation , direction and regulation of transcription under stress and non - stress conditions . biochemical techniques required to insert and / or delete nucleotides and to manipulate the resultant dna fragments are well known to those skilled in the art of genetic redesigning . example 4 : presence of heat shock sequence ( s ) and a large intron within the ubiquitin promoter the ubiquitin promoter from maize is characterized structurally by the presence of two overlapping heat shock sequences approximately two hundred bp upstream of the transcriptional start site and that of a large ( approximately 1 kb ) intron between the transcriptional start site and the translational initiation codon . this promoter structure is very similar to that reported ( u . bond et al . ( 1986 ) supra ) for the ubiquitin promoter from chicken embryo fibroblasts in which two overlapping heat shock sequences are located approximately 350 bp upstream of the transcriptional start site and a 674 bp intron is contained between the transcriptional and translational initiation codons . recently ( e . ozkaynak et al . ( 1987 ) supra ), the nucleotide sequence of the promoter region from yeast ubiquitin ub14 gene was determined and found to contain a heat shock sequence approximately 280 bp upstream of the transcriptional start site ; but this yeast ubiquitin promoter was devoid of a large intron between the transcription and translation initiation sites . however , two other yeast ubiquitin genes , which did contain introns , were found to be lacking sequences homologous to the pelham &# 34 ; heat shock box &# 34 ; sequence . ubiquitin promoters have been shown to up - regulate expression of ubiquitin in response to heat shock in yeast , chicken embryo fibroblasts and maize . in all three systems , the level ubiquitin mrna is elevated after heat shock treatment and the increase in ubiquitin level was determined in maize and chicken embryo fibroblasts to be approximately 3 fold . this enhancement in ubiquitin expression in response to heat shock is significantly less than that obtained with other heat shock genes . it was found in chicken embryo fibroblasts that the levels of ubiquitin mrna in cells exposed to 45 ° c . increased by 2 . 5 fold over a 2 . 5 h period , whereas the levels of hsp70 mrna increased 10 fold under the same heat shock conditions . moreover , the relative instability of ubiquitin mrna during recovery of cells from a 3 h heat shock ( half - life of approximately 1 . 5 to 2 h ) was also found to differ significantly from that of hsp70 mrnas which were found to be stable . it is interesting to note that in contrast to ubiquitin promoters , hsp70 genes do not contain large introns between the transcriptional and translational initiation codons . another difference between the ubiquitin promoter and other heat shock promoters is that ubiquitin is expressed both constitutively and inductively , whereas expression of classical heat shock proteins occurs predominantly in response to heat shock or other stress . this invention allows skilled workers knowledgeable in the art to modify ubiquitin promoter with respect to the composition / sequence and position of both the intron and the heat shock sequences in order to alter constitutive and / or inductive expression of ubiquitin . also , standard recombinant technology may be employed to reposition , as well as to chemically alter the nucleotide sequences within the maize ubiquitin promoter region in such a fashion as to retain or improve the promoter function of the resultant modified dna . testing for ubiquitin promoter function may be carried out as taught in example 2 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 2 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 3840 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : cds ( b ) location : 1993 .. 3591 ( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 533 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : protein ( xi ) sequence description : seq id no : 2 : metglnilephevallysthrleuthrglylysthrilethrleuglu151015valgluserseraspthrileaspasnvallysalalysileglnasp202530lysgluglyileproproaspglnglnargleuilephealaglylys354045glnleugluaspglyargthrleualaasptyrasnileglnlysglu505560serthrleuhisleuvalleuargleuargglyglymetglnilephe65707580vallysthrleuthrglylysthrilethrleugluvalgluserser859095aspthrileaspasnvallysalalysileglnasplysgluglyile100105110proproaspglnglnargleuilephealaglylysglnleugluasp115120125glyargthrleualaasptyrasnileglnlysgluserthrleuhis130135140leuvalleuargleuargglyglymetglnilephevallysthrleu145150155160thrglylysthrilethrleugluvalgluserseraspthrileasp165170175asnvallysalalysileglnasplysgluglyileproproaspgln180185190glnargleuilephealaglylysglnleugluaspglyargthrleu195200205alaasptyrasnileglnlysgluserthrleuhisleuvalleuarg210215220leuargglyglymetglnilephevallysthrleuthrglylysthr225230235240ilethrleugluvalgluserseraspthrileaspasnvallysala245250255lysileglnasplysgluglyileproproaspglnglnargleuile260265270phealaglylysglnleugluaspglyargthrleualaasptyrasn275280285ileglnlysgluserthrleuhisleuvalleuargleuargglygly290295300metglnilephevallysthrleuthrglylysthrilethrleuglu305310315320valgluserseraspthrileaspasnvallysalalysileglnasp325330335lysgluglyileproproaspglnglnargleuilephealaglylys340345350glnleugluaspglyargthrleualaasptyrasnileglnlysglu355360365serthrleuhisleuvalleuargleuargglyglymetglnilephe370375380vallysthrleuthrglylysthrilethrleugluvalgluserser385390395400aspthrileaspasnvallysalalysileglnasplysgluglyile405410415proproaspglnglnargleuilephealaglylysglnleugluasp420425430glyargthrleualaasptyrasnileglnlysgluserthrleuhis435440445leuvalleuargleuargglyglymetglnilephevallysthrleu450455460thrglylysthrilethrleugluvalgluserseraspthrileasp465470475480asnvallysalalysileglnasplysgluglyileproproaspgln485490495glnargleuilephealaglylysglnleugluaspglyargthrleu500505510alaasptyrasnileglnlysgluserthrleuhisleuvalleuarg515520525leuargglyglygln530__________________________________________________________________________