Patent Abstract:
This invention relates to a gene associated with male fertility, labeled Ms41-A, and a recessive mutant form thereof, labelled ms41-A, which confers male sterility. The Ms41-A gene is isolated from Arabidopsis, while the related gene Zm41-A is isolated from maize. Male-sterile plants are useful for the production of hybrid plants by sexual hybridization.

Full Description:
This application is a continuation of PCT International Application No. PCT/GB96/03191, filed Dec. 20, 1996, designating the United States of America and claiming priority of Great Britain Patent Application No. 9526218.4, filed Dec. 21, 1995. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to recombinant, isolated and other synthetic DNA useful in male-sterility systems for plants. In particular, the invention relates to a gene associated with male fertility, labelled Ms41-A, and a recessive mutant form thereof, labelled ms41-A, which confers male sterility. Male-sterile plants are useful for the production of hybrid plants by sexual hybridisation. 
     Hybrid plants have the advantages of higher yield and better disease resistance than their parents, because of heterosis or hybrid vigour. Crop uniformity is another is advantage of hybrid plants when the parents are extensively homozygous; this leads to improved crop management. Hybrid seed is therefore commercially important and sells at a premium price. 
     Producing a hybrid plant entails ensuring that the female parent does not self-fertilise. There have been many prior proposals, mechanical, chemical and genetic, for preventing self-pollination. Among the genetic methods is the use of anther-specific genes or their promoters to disrupt the normal production of pollen grains. An anther-specific promoter, for example, can be used to drive a “male-sterility DNA” at the appropriate time and in the right place. Male sterility DNAs include those coding for lytic enzymes, including those that lyse proteins, nucleic acids and carbohydrates. Glucanases are enzymes which break down carbohydrates. 
     WO-A-9302197 describes recombinant or isolated DNA encoding a glucanase called callase. 
     Aarts et al, ( Nature , 363:715-717 (7993)) have described a gene required for male fertility, isolated from Arabidopsis, which has been labelled Ms2. 
     We have now identified and isolated from Arabidopsis another gene linked to male fertility. This gene has been labelled Ms41-A. Its mutant , recessive, form is labelled ms41-A and is capable of conferring male sterility. This gene would appear to offer advantages over Ms2 when used to produce male sterile plants. 
     SUMMARY OF THE INVENTION 
     Thus, in a first aspect the present invention provides recombinant or isolated Nucleic acid which: 
     a) encodes the Ms41-A protein from Arabidopsis; 
     b) encodes a Ms41-A like protein; 
     c) encodes the ms41-A protein from Arabidopsis; 
     d) encodes a ms41-A like protein; 
     e) comprises a promoter sequence which regulates expression of the Ms41-A protein from Arabidopsis or a promoter sequence which regulates expression of a Ms41-A like protein; or 
     f) hybridises under stringent conditions to Nucleic acid a), b), c), d) or e) or would do so but for the degeneracy of the genetic code. 
     In one embodiment of a) above, the Nucleic acid encodes a protein having an amino acid squence as shown in FIG.  4 . Although FIG. 4 relates only to a protein of 
     Arabidopsis, those skilled in the art will readily be able to identify equivalent proteins from other members of the family Brassicaceae or indeed similar proteins from other commercially important plant families, ie Ms41-A like proteins. 
     In turn the equivalent genes may be identified by hybridisation studies, restriction fragment length polymorphism (RFLP), degenerate PCR and other methods known in the art. Genes or other DNA sequences, whether natural, engineered or synthetic, encoding closely equivalent proteins may for example hybridise under stringent conditions (such as at approximately 35° C. to 65° C. in a salt solution of approximately 0.9 molar) to the Arabidopsis gene, or fragments of it of, for example, 10, 20, 50 or 100 nucleotides. A 15-20 nucleotide probe would be appropriate under many circumstances. 
     In the context of the present invention, “Nucleic acid which encodes” includes all nucleic acid, eg DNA sequences which will, when expressed, give rise to the protein. Examples of such DNA sequences include, but are not limited to, ones which comprise non-coding regions, e.g introns, sequences which include leader sequences and/or signal sequences, or simply comprise a coding sequence for the protein. The skilled person will also appreciate that, due to codon degeneracy, there will, for example, be a number of DNA sequences capable of coding for the Ms41-A protein or a Ms41-A like protein. 
     In general, the Nucleic acid of the invention will comprise at least a direct coding sequence for the protein as well as a promoter and transcription termination sequence. The promoter can itself comprise only chose sequences, or elements, necessary for the correct initiation of transcription (which regions can be described as transcription initiation regions, or instance), or, alternatively, it can include regions or sequence which are not directly involved in the initiation of transcription, i.e. a complete promoter can be employed. 
     A preferred coding sequence described in this specification is from Arabidopsis and can be isolated by methods known in the art, for example by (a) synthesising cDNA from mRNA isolated from Arabidopsis, (b) isolating this cDNA. This cDNA can, in turn, be used (c) as a probe to identify regions of the plant genome of a chosen member of another plant species, eg Maize, that encode mRNA of interest and (d) identifying the upstream (5′) regulatory regions that contain the promoter of this DNA. 
     A particularly preferred DNA sequence is that shown in FIG. 3, and more particularly, the sequence shown in FIG. 3 which commences with the base pair labelled 1, as will subsequently be described in the examples. Those skilled in the art will, with the information given in this specification, be able to identify with sufficient precision the coding regions and to isolate and/or recombine DNA containing them. 
     The Nucleic acid of the invention can be used to confer male sterility on plants. For instance, the recessive form of the gene, ie ms41-A can be used to transform a plant. Alternatively, the dominant form, ie Ms41-A can be downregulated in some way. 
     As discussed herein, the Nucleic acid can include a promoter, and to increase the likelihood of male sterility being conferred it is possible to use promoters which drive expression in particular plant tissues which are involved in the control of fertility. Examples of such promoters are those which are tapetum-specific, for example a Brassicaceae A3 or A9 promoter, described in WO-A-9211379, and the A6 promoter described in WO-A-9302197. Both WO-A-9211379 and WO-A-9302197 are hereby incorporated by reference. 
     Because of the natural specificity of the regulation of expression of the Ms41-A or Ms41-A like gene, it is not necessary for the Ms41-A promoter to be linked to specific disrupter DNA to provide a useful male-sterility system (although it can be); non-specific disrupter DNA can be used. 
     Ms41-A like promoters from other plant species, eg from Maize, and modified Ms41-A promoters can be used, and if necessary located or identified and isolated as described above or the Ms41-A coding sequences, mutatis murandis. Ms41-A or Ms41-A like promoter-containing DNA in accordance with the invention can, as indicated above, be used to confer male sterility on plants, particularly those belonging to the family Brassicaceae, in a variety of ways as will be discussed below. In an important embodiment of the invention, therefore, a promoter as described above is operatively linked to DNA which, when expressed, causes male sterility. 
     Since an effective sterility system is complete, propagation of the seed parent must proceed either by asexual means or via the pollination of the male-sterile by an isogenic male-fertile line, and the subsequent identification or selection of male sterile plants among the offspring. Where vegetative propagation is practical, the present invention forms a complete system for hybrid production. Where fertility restoration is necessary to produce a seed crop, the present invention forms the basis of a new male sterility system. In some seed crops where the level of cross pollination is high, seed mixtures may enable restoration to be bypassed. The male sterility will be particularly useful in crops where restoration of fertility is not required, such as in the vegetable Brassica spp., and such other edible plants as lettuce, spinach, and onions. 
     Nucleic acid in accordance with the invention and incorporating the Ms41-A or Ms41-A like promoter can drive male sterility DNA thereby producing male sterile plants, which can be used in hybrid production. 
     A construct comprising a promoter operatively linked to a male sterility DNA can be transformed into plants (particularly those of the genus Brassica, but also other genera such as Nicotiana and Hordeum) by methods which may be well known in themselves. This transformation results in the production of plants, the cells of which contain a foreign chimeric DNA sequence composed of the promoter and a male sterility DNA. Male-sterility DNA encodes an RNA, protein or polypeptide which, when produced or over-produced in a stamen cell of the plant, prevents the normal development of the stamen cell. The Ms41-A or Ms41-A like promoter may be used to drive a variety of male sterility DNA sequences which code for RNAs, proteins or polypeptides which bring about the failure of mechanisms to produce viable male gametes. The invention is not limited by the sequence driven, but a number of classes and particular examples of male sterility promoter-drivable sequences are preferred. 
     For example, the drivable male sterility DNA may encode a lytic enzyme. The lytic enzyme may cause degradation of one or more biologically important molecules, such as macromolecules including nucleic acid, protein (or glycoprotein), carbohydrate and (in some circumstances) lipid. 
     Ribonuclease (such as RNase T1 and barnase) are examples of enzymes which cause lysis of RNA. Examples of enzymes which lyse DNA include exonucleases and endonucleases, whether site-specific such as EcoRI or non-site-specific. 
     Actinidin is an example of a protease, DNA coding for which can be suitable male sterility DNA. Other examples include papain zymogen and papain active protein. 
     Lipases whose corresponding nucleic acids may be useful as male sterility DNAs include phospholipase A 2 . 
     Male sterility DNA does not have to encode a lytic enzyme. Other examples of male sterility DNA encode enzymes which catalyse the synthesis of phytohormones, such as isopentyl transferase, which is involved in cytokinin synthesis, and one or more of the enzymes involved in the synthesis of auxin. DNA coding for a lipoxygenase or other enzymes having a deleterious effect may also be used. 
     As mentioned above, one way to confer male sterility will be to downregulate the Ms41-A or Ms41-A like gene. This could he achieved by the use of antisense DNA. Introducing the coding region of a gene in the reverse orientation to that found in nature can result in the down-regulation of the gene and hence the production of less or none of the gene product. The RNA transcribed from antisense DNA is capable of binding to, and destroying the function of, a sense RNA version of the sequence normally found in the cell thereby disrupting function. 
     It is not crucial for antisense DNA solely to be transcribed at the time when the natural sense transcription product is being produced. Antisense RNA will in general only bind with its sense complementary strand, and so will only have its toxic effect when the sense RNA is transcribed. Antisense DNA corresponding to some or all of the DNA encoding the Ms41-A or Ms41-A like gene product may therefore be produced not only while the gene is being expressed. Such antisense DNA may be expressed constitutively, under the control of any appropriate promoter. 
     It is also the case that one may wish to restore male fertility in later generations, this can also be achieved using antisense nucleic acid, eg nucleic acid which is antisense For a DNA molecule encoding ms41-A. 
     Thus, in a second aspect, the present invention provides Antisense nucleic acid which includes a transcribable strand of DNA complementary to at least a part of a DNA molecule of the invention. 
     In one embodiment of this aspect the antisense nucleic acid is under the control of a constitutive promoter, such as the CaMV35S promoter. 
     A still further example of male sterility DNA encodes an RNA enzyme (known as a ribozyme) capable of highly specific cleavage against a given target sequence (Haseloff and Gerlach  Nature  334 585-591 (1988)). Like antisense DNA, ribozyme DNA (coding in this instance for a ribozyme which is targeted against the RNA encoded by the Ms41-A or Ms41-A like gene) does not have to be expressed only at the time of expression of the Ms41-A or Ms41-A like gene. Again, it may be possible to use any appropriate promoter to drive ribozyme-encoding DNA, including one which is adapted for constitutive expression. 
     According to a further aspect of the invention, there is therefore provided DNA encoding a ribozyme capable of specific cleavage of RNA encoded by a DNA molecule of the invention. Such ribozyme-encoding DNA would be useful in conferring male sterility on members of, eg the family Brassicaceae. 
     In addition, there are other useful methods which can be employed for the downregulation of the Ms41-A or Ms41-A like DNA sequences. Some examples of these are as follows: 
     i) expression of an antibody or antibodies, domains or fragments thereof against the Ms41-A or a Ms41-A like protein; 
     ii) expression of mutant versions of the Ms41-A or of a Ms41-A like protein which may interfere with the function of the normal protein; 
     iii) by creation of mutations in the Ms41-A sequence or the the Ms41-a like sequence with the result that mutant plants can be used in the recessive AMS system as hereinbefore described; and 
     iv) expression of mRNA binding proteins that will interfere specifically with Ms41-A or Ms41-A like transcription. 
     In preferred embodiments of DNA sequences of this invention 3′ transcription regulation signals, including a polyadenylation signal, may be provided. Preferred 3′ transcription regulation signals are derived from the Cauliflower Mosaic Virus 35S gene. It should be recognised that other 3′ transcription regulation signals could also be used. 
     Recombinant DNA in accordance with the invention may be in the form of a vector. The vector may for example be a plasmid, cosmid or phage. Vectors will frequently include one or more selectable markers to enable selection of cells transfected (or transformed: the terms are used interchangeably in this specification) with them and, preferably, to enable selection of cells harbouring vectors incorporating heterologous DNA. Appropriate start and stop signals will generally be present. Additionally, if the vector is intended for expression, sufficient regulatory sequences to drive expression will be present; however, DNA in accordance with the invention will generally be expressed in plant cells, and so microbial host expression would not be among the primary objectives of the invention, although it is not ruled out. Vectors not including regulatory sequences are useful as cloning vectors. 
     Cloning vectors can be introduced into  E. coli  or another suitable host which facilitate their manipulation. According to another aspect of the invention, there is therefore provided a host cell transfected or transformed with DNA as described above. 
     DNA in accordance with the invention can be prepared by any convenient method involving coupling together successive nucleotides, and/or ligating oligo- and/or poly-nucleotides, including in vitro processes, but recombinant DNA technology forms the method of choice. 
     Ultimately, DNA in accordance with the invention (whether (i) Ms41-A gene, ms41-A gene, Ms41-A like gene or ms41-A like gene (ii) antisense DNA to any option listed in i), ribozyme DNA targeted to RNA for any option listed in i) or DNA comprising a promoter as described herein used to drive expression of a disrupter sequence, eg encoding Barnase) will be introduced into plant cells, by any suitable means. 
     According to a further aspect of the invention, there is provided a plant cell including DNA in accordance with the invention as described above. 
     Preferably, DNA is transformed into plant cells using a disarmed Ti-plasmid vector and carried by Agrobacterium by procedures known in the art, for example as described in EP-A-0116718 and EP-A-0270822. Alternatively, the foreign DNA could be introduced directly into plant cells using an electrical discharge apparatus. This method is preferred where Agrobacterium is ineffective, for example where the recipient plant is monocotyledenous. Any other method that provides for the stable incorporation of the DNA within the nuclear DNA of any plant cell of any species would also be suitable. This includes species of plant which are not currently capable of genetic transformation. 
     Preferably DNA in accordance with the invention also contains a second chimeric gene (a “marker” gene) that enables a transformed plant containing the foreign DNA to be easily distinguished from other plants that do not contain the foreign DNA. Examples of such a marker gene include antibiotic resistance (Herrera-Estrella et al.  EMBO J . 2, 987-995 (1983)), herbicide resistance (EP-A-0242246) and glucuronidase (GUS) expression (EP-A-0344029). Expression of the marker gene is preferably controlled by a second promoter which allows expression in cells other than the tapetum, thus allowing selection of cells or tissue containing the marker at any stage of regeneration of the plant. The preferred second promoter is derived from the gene which encodes the 35S subunit of Cauliflower Mosaic Virus (CaMV) coat protein. However any other suitable second promoter could be used. 
     A whole plant can be regenerated from a single transformed plant cell, and the invention therefore provides transgenic plants (or parts of them, such as propagating material) including DNA in accordance with the invention as described above. The regeneration can proceed by known methods. When the transformed plant flowers it can be seen to be male sterile by the inability to produce viable pollen. Where pollen is produced it can be confirmed to be non-viable by the inability to effect seed set on a recipient plant. 
     Preferred features of each aspect of the invention are as for each other aspect mutatis mutandis. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG.  1 : shows a Southern Blot of HindIII-cut genomic DNA from 21 ms41-A plants demonstrating linkage of the 35S-Ac element to ms41-A; 
     FIG.  2 : shows a schematic diagram of the region containing the MS41-A locus cloned in lambda MSE3. The position of insertion of the 35S-Ac is indicated; B, BamHI; E, EcoRI; H, HindIII; S, SaeI; 
     FIG.  3 : (SEQ ID NO:11) shows the genomic DNA sequence of the MS41-A gene. The sequence is numbered from the putative transcriptional start point of the MS41-A message. The predicted amino-acid sequence of MS41-A is shown together with the restriction sites; 
     FIG.  4 : (SEQ ID NO:12) shows the predicted amino acid sequence of MS41-A; 
     FIG.  5 : (SEQ ID NOS:13-17) shows the oligonucleocides used to examine excision events of 35S-Ac from the ms41-A locus; 
     FIG.  6 : shows DNA sequences left by 35S-Ac excision events at the ms41-A locus; 
     FIG.  7 : shows a diagram of the MS41-A promoter-GUS and MS41-A promoter-Barnase chimeric genes; 
     FIG.  8 : shows a diagram of the MS41-A promoter-antisense MS41-A and CaMV 35S promoter-antisense and sense MS41-A chimeric genes; 
     FIG.  9 : (SEG ID NOS:18-25) shows sequence alignments of proteins related to MS41-A; 
     FIG.  10 : (SEG ID NOS:26-27) shows a partial DNA sequence and predicted amino acid translation of Zm41-A; 
     FIG.  11 : shows a dendrogram of MS41-A related sequences; 
     FIG.  12 : (SEQ ID NO:28) shows the nucleotide sequence of the Z31 Zm41-A gene. The portion of the sequence corresponding to putative coding region is shown in bold type capital lettters. ♦ indicates putative first methionine deduced in frame with cDNA Zm41-A and 5′RACE products. * indicates the start of the longest 5′RACE product. ▾ indicates the start of Zm41-A cDNA. 12 exons are present and the translation is stopped in exon 11, the stop codon is TGA (□). Non spliced DNA present in some RACE products is underlined; 
     FIG.  13 : snows restriction maps of Z31, Z33 and Z35 genomic clones isolated with cDNA of Zm41-A. EI, HIII, NI and SI indicate restiction sites of endonucleases EcoRI, HindIII, NcoI and SalI, respectively. * indicates the start of the longest RACE product. ▾ indicates the start of Zm41-A cDNA. Dotted lines indicate homologous regions and Δ indicates deletions; 
     FIG.  14 : (SEQ ID NOS:29-30) shows clustal V alignment between the protein deduced from the Zm41-A cDNA and from the genomic longest open reading frame of Z31; 
     FIG.  15 : (SEQ ID NO:31) shows the nucleotide sequence of the Z33 Zm41-A gene. The portion of the sequence corresponding to DNA transcription is shown in bold type capital letters. Non spliced DNA present in some RACE products is underlined. This gene is truncated and only exons 3,5 and 6 are present; and 
     FIG.  16 : (SEQ ID NO:32) shows the nucleotide sequence of the Z35 Zm41-A gene. The portion of the sequence corresponding to DNA transcription is shown in bold type capital letters. Non spliced DNA present in some RACE products is underlined. This gene is truncated and only exons 3,4,5 and 6 are present. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     EXAMPLE 1 
     Isolation of a Gene Required for Male Fertility in  Arabidopsis thaliana    
     i) Isolation and Phenotype of the ms41-A Male Sterile Mutant. 
     The method used to identify a gene required for male fertility in  Arabidopsis thaliana  was transposon tagging. This method is a powerful technique for isolating genes which encode unknown products, allowing genes identified only by their mutant phenotype, to be cloned.  Arabidopsis thaliana  is a widely used model species that is an ideal plant for transposon tagging of genes, since it is a transformable diploid with a very small genome. Thus the chance of tagging desired genes is maximised. Additionally Arabidopsis is a Brassicaceae and is thus very closely related to important crop plants such as  Brassica napus  (Oil Seed Rape). 
     Transposon tagging was achieved by transformation of C24 Arabidopsis roots with modified autonomous Ac elements from Maize: D Ac and 35S Ac inserted into the leader of the GUS reporter gene in the reverse orientation (Constructs described in Finnegan et al.,  Plant Molecular Biology , 22:625-633 (1993) (As this work was in progress the first reports of gene tagging with similar Ac elements in heterologous plant species were published; a pH controlling gene from Petunia: Chuck et al.,  Plant Cell , 5:371-378 (1993)); the Arabidopsis DRL1 locus: Bancroft et al.,  Plant Cell , 5:631-638 (1993)) and the  Arabidopsis Albino  gene (Long et al.,  Proceedings of the National Academy of Sciences U.S.A ., 90:10370-10374 (1993)). 
     Transformed plants were regenerated and the T2 progeny analysed for GUS activity and by molecular analysis. This demonstrated that the 35S Ac transposed quite efficiently (in 30% to 40% of progeny). The T3 progeny families derived from 279 selected T1 plants were then visually screened for mutants affected in male sterility. 
     A few fertility-reduced or sterile plants were recovered, some possessing additional abnormalities. A male sterile mutant (ms41-A) which appeared in family 41 had collapsed anthers with empty locules. Only one sterile plant was recovered from more than 2000 T3 siblings in this family. After cross-pollination with wild type pollen, elongation of siliques was observed, confirming that female fertility is unaffected by the mutation. 
     From the above cross 21 F1 individuals were grown and allowed to self pollinate to produce F2 seed ; all the F1 plants were completely fertile suggesting that the mutation is recessive. The first analysis of 6 different F2 populations confirmed the recessive character of the mutation, as male sterility reappeared in a small proportion of each F2 population, with all other siblings presenting a wild type phenotype. Moreover, the vegetative development of the male sterile plants was identical to wild type C24 Arabidopsis. The observed frequency of male transmission of the mutation suggests a non-classical mendelian inheritance for a single recessive mutation—the frequencies of mutant plants in the F2 populations were: 16.8; 13.0; 11.9; 12.7; 15.4 and 17.0%. The expected frequency of mutant plants is 25% or a 3 to 1 ratio of wild type to mutant plants. In this case there is a ratio of approximately 7 to 1 wild type to mutant plants. A homogeneity test on the data of the 6 F2 populations presented concludes that there is homogenous transmission of the male sterile phenotype (Chi square with 5 degrees of freedom=8.69, 0.10&lt;P&lt;0.20). 
     Proof of reduced transmission of Ms41-A through the male gametophyte was obtained by genetic mapping of Ms41-A. The hypothesis was that markers genetically linked to Ms41-A but present on the homologous chromosome (in repulsion) on a F1 cross with an Ms41-A plant should be over-represented in the derived F2 population. The F1 crosses were made with 5 tester lines, one for each chromosome, constructed by Marteen Korneef (described in; O&#39;Brian S. T. (ed) Genetic maps of complex genomes, Book 6, Plant Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 94-97 (1990)), and linkage of Ms41-A was demonstrated with markers on the lower part of chromosome 1. Compiled recombination data of 2 populations (476 and 540 individuals) were analysed by the Maps Maker software version 2 (Lander et al.,  Genetics , 121:174-181 (1987))). 
     Ms41-A is between apetala 1 (8.1 cM) and glabra 2 (9.8 cM) and 40.2 cM away from than chlorina 1. In the first F2 population, the deficit of Ms41-A plants was observed as before (14.7% of plants were male sterile) and it was correlated with the expected increase of apetala 1 and glabra 2 plants (29% and 31.5% respectively); the most distal marker, chlorina 1 behaves quite normally (22.3%). In the second F2, where the penetrance of the Ms41-A is less affected (18.3%), the over representation is not as prevalent (as expected); only the proportion of glabra 2 plants appears co be slightly increased (27.2%). 
     Microscopic observations of microsporogenesis in the male sterile Ms41-A plants revealed that the tetrads release abnormal microspores which degenerate rapidly. By aniline blue staining the tetrads appear abnormal with irregular shaped cells and with great variation in cell size. Moreover there is a mixed population of meiocytes, dyads (a stage not usually observed in Arabidopsis) and tetrads in the same anther. The defect apparently lies just before or during meiosis. Cytological observations on fixed young anther buds reinforce this finding, since at meiosis the meiocytes are affected but the tapetum behaves normally. No differences were observed cytologically between the Ms41-A heterozygote and wild type plants. 
     One other gene required for male-fertility (also in Arabidopsis) has been described previously (Aarts et al.,  Nature , 363:715-717 (1993)). Plants with a mutation in this gene (Ms2) were grown together with Ms41-A plants. In certain conditions , especially after the plants had been flowering for a long time the ms2 but not the Ms41-A plants reverted to male fertility. 
     ii) Linkage of a Transposed 35S Ac with the Mutant Phenotype 
     To determine if the Ms41-A mutation was due to the insertion of a 35S-Ac element, HindIII-cut DNA from five Ms41-A F1 individuals was analysed by Southern blotting using a 5′Ac fragment (2.5 Kb EcoR I fragment from pBGS335RI (Finnegan et al.,  Plant Molecular Biology , 22: 625-633 (1993)) as a probe. Two identical Ac bands were present in the five mutant plants: 
     the internal Ac Hind III 1.6 kb band and 
     a junction 3′ Ac band of approximately 2.8 kb, which differs from the expected non-transposed 35S Ac (2.1 kb). 
     This indicates the presence of only one 35S Ac element which has transposed in the parental male sterile plant, or more likely in its parents. To determine linkage between this 35S Ac element and the Ms41-A phenotype, 24 Ms41-A plants from each of 6 different F2 populations were analysed by PCR for the presence of the Ac element using oligonucleocides: 
     5′ H (5′AAGGATCCTGGCAAAGACATAAATC 3′) (SEQ ID NO:1) and 
     Ac12 (5′AGATGCTGCTACCCAATCTTTTGTGC 3′) (SEQ ID NO:2). The results were as follows 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 F2 41-A-A 
                 23 positives out of 24 
               
               
                   
                 F2 41-A-B 
                  5   ″ 
               
               
                   
                 F2 41-A-C 
                 23   ″ 
               
               
                   
                 F2 41-A-D 
                 10   ″ 
               
               
                   
                 F2 41-A-E 
                 24   ″ 
               
               
                   
                 F2 41-A-F 
                  3   ″ 
               
               
                   
                   
               
             
          
         
       
     
     If the Ac element is linked to Ms41-A all male sterile plants should have the Ac element, however if the Ac is not linked only ¾ of Ms41-A plants should have the Ac element. The results obtained indicate complete linkage only in the 41-A-E population. The lack of linkage in the other populations may be due to frequent imprecise excision of the Ac element from the Ms41-A locus leaving a mutation in Ms41-A. 
     To confirm linkage, the most stable population, 41-A-E, was analysed by Southern blotting with a probe that contained both a region of the transposed Ac element and 3′ flanking plant DNA. To generate this probe DNA from a Ms41-A plant was digested with SspI, religated and amplified by PCR using Ac oligonucleotides: 
     Ac 11 (5′CGTATCGGTTTTCGATTACCGTATT 3′) (SEQ ID NO:3) and 
     Ac 12 (5′AGATGCTGCTACCCAATCTTTTGTGC 3′) (SEQ ID NO:4). The 1.1 kb inverse PCR (IPCR) fragment generated contained 500 bp of Ac and the remainder consisted of 3′ flanking Arabidopsis DNA. 
     DNA from plants of the F2 population 41-A-E was digested with HindIII and probed with the 3′ IPCR fragment. 21 new F2 mutant individuals and 28 male fertile F2 plants were analysed, the selfed progenies of the latter were checked for the presence of mutant plants revealing that 15 of the 28 were heterozygous for Ms41-A. All of the 21 mutant plants (FIG. 1) and those heterozygotes segregating the mutation in the F3 showed the same transposed 35S Ac revealed by the 2.8 kb specific band and the Ac internal 1.6 kb band. A 3.3 kb band, corresponding to the wild type allele is detectable in most of the F2 mutants; this is probably due to to somatic excision of Ac and confirms that the transposed Ac element is still active. These results confirm that the 35S Ac is located in or in the vicinity of the Ms41-A gene. 
     iii) Genomic Clones and cDNAs of the Ms41-A Gene 
     Two different genomic libraries—one MboI partial library in EMBL 3A ( Clontech) and one HindIII partial in Lambda Dash II ( T. Pelissier, S.Tutois and G. Picard, unpublished) were screened with the 3′ IPCR cloned product. Four different clones spanning the mutated region, were characterised by Southern analysis. One of them, lambda MSE3, which spans the transposon insertion site, was used for fine mapping. It contains the IPCR hybridising fragments detected on a genomic Southern (HindIII 3.3 kb, SspI 1.8 kb and PstI 4 kb). The entire plant DNA insert in MSE3 is contained on 4 SalI fragments; S1 (5 kb), S2 ( 4.9 kb), S3 (4.3 kb) and S4 (2.3 kb) (FIG.  2 ). The S3 fragment contains the plant DNA from the IPCR product. 
     After sequencing the IPCR product to determine the plant sequence 3′ of the Ac element, more than 5000 bp of genomic sequence was obtained from MSE3 (3100 bp from the 5′ Ac flanking region and 1900 bp at the 3′). The genomic sequence is presented in FIG.  3  and is indexed according to the putative transcription initiation site determined by 5′ RACE (see below). One of the SalI sites of the fragment S3 is positioned at 2061 bp the other one is situated 5′ upstream an EcoRI site (−1753 bp) and has not been sequenced. The transposon is inserted at position +318 bp. 
     To identify mRNAs expressed in the region of the transposon insertion site, three Arabidopsis cDNA libraries were probed with either the S1 or S3 fragments; a developing flower buds library (young buds) (Weigel et al.,  Cell , 69:843-859 (1992)), a library from flowers at late stages (after stage 10) (Hofte et al.,  Plant Journal , 4:1051-1061 (1993)) and an immature siliques library (Giraudat et al.,  Plant Cell , 4:1251-1261 (1992)). 
     Two classes of cDNAs were recovered with the S3 fragment as a probe and characterised. 
     a 1.9 kb cDNA (W11), isolated from the developing flower buds library. Its 3′ end is located 1.5 kb upstream of the 3′ 35S Ac end, suggesting that it is not linked to the Ms41-A phenotype. Sequencing of the extremities revealed that the EcoRI site (−1753 bp in FIG. 3) is present in the 3′ part of this mRNA. 
     a 0.8 kb cDNA (G6), isolated from the immature siliques library but also present in the developing flower buds library. Comparison of G6 and genomic sequences shows that the transposon insertion site is 1440 bp upstream of the 5′ end of the longest G6 cDNA (861 bp). In addition, the lack of a methionine codon in the 5′ sequence of G6 indicated that this cDNA was not full-length. Further attempts at obtaining longer cDNAs from the three libraries were unsuccessful. 
     Another cDNA (A6) of approximately 1 Kb was isolated using the S1 fragment as a probe. It maps downstream of the G6 message. 
     Out of the 3 transcription units in the vicinity of the transposon insertion site, the best candidate for the Ms41-A mRNA was that corresponding to G6. To obtain a full-length G6 cDNA, primers were designed to the 5′ end of the longest G6 cDNA and used in a 5′ RACE reaction (5′ AmpliFinder kit, Clontech). This proved unsuccessful, probably due to the 5′ end of G6 lying far upstream of the longest cDNA obtained. Therefore primers were designed to regions of the genomic sequence that were upstream of the 5′ end of the longest G6 cDNA. These, in combination with primers designed to the G6 cDNA, were used in RT-PCR reactions to define the extent of the G6 transcribed region. Results obtained suggested that the G6 message was at least 1 kb longer than the longest G6 cDNA obtained, and that the upstream sequence contained an intron of about 450 bp. 
     The G6 transcriptional start site was finally mapped by 5′ RACE using primers Z3 
     (5′TTATCATCAACATCGCCATCGAATCTGCCG 3′, (SEQ ID NO:5) positions 494-464 bp in FIG.  3 ); 
     and W1 (5′AAAGTAGTAAACCCTAGAG 3′, (SEQ ID NO:6) positions 279-260 bp). RT-PCR was then used to recover a nearly full-length G6 message. Comparison of the G6 and genomic sequences shows that the first ATG is situated at position 157 bp; thus G6 putatively encodes a protein of 584 amino acids (FIG.  4 ). Over the region of overlap the cDNA and genomic DNA sequences were identical. This deduced protein has no significant homology to proteins of known function on the Genebank, EMBL and NBRF databases. The coding sequence consists of three exons, the first of which has been disrupted by the insertion of the 35 Ac element at amino acid position 54 in the Ms41-A mutant. This is strong evidence that G6 corresponds to Ms41-A. Final confirmation was obtained by analysis of phenotypes and DNA sequences around the Ac insertion site in Ms41-A progeny plants in which the 35S Ac element has excised. 
     To induce somatic exision of the 35S Ac element, plants were regenerated from liquid root cultures from single individuals derived from two different test-crosses. These crosses where between plants (A and B) that had only one Ac element but were still male sterile due to imprecise exision of the other Ac element, and male fertile plants that were heterozygous for Ms41-A: 35S Ac. This material was chosen because of the higher percentage of male sterile plants (40% instead of 20%, 50% instead of 25%?) than in a normal F2 population. Regenerants from clones representing male sterile plants were scored for male fertility. Numerous completely fertile plants were obtained from some individuals, however from 5 different regenerated plants from 4 different individuals, 7 different “revertant siliques” were obtained. 
     DNA from revertant plants or from progeny from “revertant siliques” was analysed by PCR for excision of the Ac element and PCR products cloned to determine the sequence left by the Ac element (footprint). The oligonucleotides presented in FIG. 5 were used:Ac 11 with W2 for the presence of the 3′ junction, Ac 14 with G6 5′-11 for the 5′ junction and W2 with G6 5′-11 or with Z3 for the excision allele(s). The PCR fragments derived from W2 with G6 5′-11 or with Z3 were cloned in the pGEM-T vector (Promega) and sequenced for all revertants. Previously junction products were sequenced confirming the presence of the typical target duplicated sequence of 8 base pairs:CTCCTCTC (positions 311 to 318 in FIG.  3 ). 
     The genotypes of 7 revertant plants or sectors were determined and are presented in FIG.  5 . For all of them an allele restoring the open reading frame is observed which is the same as the wild type in 4 cases , a 3 bp insertion in 2 cases and a 6 bp insertion in one case. Footprints destroying the coding phase are observed in different revertants and also in the female parents (2 different 7 bp insertions and 2 different 5 bp insertion, and one with the addition of a 9 bp insertion which also introduces an in frame, TGA, stop codon). Their presence is always associated with segregation of male sterile individuals in the progeny. These results demonstrate that the Ms41-A protein has a determinant role in male fertility and that the Ms41-A gene has been tagged with the 35S Ac element. 
     iv) Ms41-A Genetic Mapping 
     Classical genetic mapping of Ms41-A with visual phenotypic markers has been described previously in section i) of this example. It places the Ms41-A locus near the bottom of chromosome 1. To determine if the Ms41-A mutation has been isolated previously in Arabidopsis the mutation was mapped more precisely using recombinant inbred lines made by Caroline Dean (Lister et al.,  Plant Journal , 4:745-750 (1993)). This method requires the identification of restriction enzyme fragment length polymorphisms (RFLPs) between the two parental lines (Columbia and Landsburg erecta) which are in, or near the Ms41-A locus. Polymorphisms were not found in Ms41-A or 5′ of it, however the downstream cDNA, 6A, gives a HhaI polymorphism. Results, processed by MapMaker version 2, have positioned Ms41-A near the marker m532 (1.3 cM) and marker g17311 (4.6 cM). Those RFLP markers are situated on chromosome 1 close to the ADH locus, and map in the vicinity of glabrous 2 and apetala 1 on the integrated Arabidopsis genetic map (Hauge et al.,  Plant Journal , 3:745-754 (1993)). 
     Ms41-A is a new male-sterile mutant. It is not allelic to ms1 (Van der Veen and Wirtz,  Euphytica , 17: 371-XXX (1968)) ms3, ms5, ms10, ms11 or ms12 (Chaudhury 1993). It is also different to the Ms2 gene (Aarts et al., supra). 
     v) Abundance of the Ms41-A Message 
     Ms41-A is expressed in 7 day old seedlings, in young floral buds and in immature siliques (cDNA libraries and RT-PCR data). The mRNA could not be detected in these tissues by Northern blotting using poly A+ mRNA which had been used successfully in RT-PCR analysis for the Ms41-A message. Thus the Ms41-A message appears to be of very low abundance; approximately 10 fold lower than another message required for male ferility in Arabidopsis, Ms2, in the same cDNA library (1 out of 12000 plaques for Ms2 (Aarts et al., supra) versus 1 out of 125000 for Ms41-A). 
     EXAMPLE 2 
     Isolation of the Ms41-A Promoter and Fusion to the β-Glucuronidase (GUS) Resorter Gene 
     To attempt to determine the extent of utility of the Ms41-A promoter in male sterilty systems putative Ms41-A promoter fragments were linked to the reporter gene GUS and transformed into Arabidopsis and tobacco. This will reveal more precisely the spatial and temporal expression patterns of the Ms41-A gene and determine whether the low abundance of the Ms41-A transcript is due to weak expression or transcript instability. 
     Two promoter fragments, −903 (Hind III) to +79 (Short promoter) and −1753 (EcoR I) to +79 (Long promoter), have been fused to the GUS gene (transcriptional fusions) to produce the binary vectors pBIOS 176 and pBIOS 177 (FIG.  7 ). 
     These plasmids were constructed as follows: 
     The primers Y7 (positions −1799 to −1782 in FIG. 3) 
     5′CCTAACTTTCTTTGCGGC 3′ (SEQ ID NO:7) 
     and W3 Xba (positions 84 to 59 in FIG. 3) 
     5′ GATCTAGACCGTGATGTCTTAGAAGG 3′ (SEQ ID NO:8) 
     were used in a PCR to recover a 1883 bp Ms41-A promoter fragment. This was cloned into the vector pGEM-T (Promega) forming p511. This plasmid was introduced into a dam, dcm minus  E.coli  strain (SCS 110) thus allowing the XbaI restriction enzyme to cleave the XbaI site. The 985 bp HindIII, XbaI fragment of p511 was cloned between the HindIII and XbaI sites of pBI121 (replacing the 35S CaMV promoter of this plasmid) forming plasmid pBIOS176. The 1853 bp EcoRI, XbaI fragment of p511 was cloned between the EcoRI and XbaI sites of pBIOS4 (a derivative of pBI121), replacing the 35S CaMV promoter of this plasmid, forming plasmid pBIOS177. 
     To construct pBIOS4, pBI121 was digested with EcoRI, the ends filled using Klenow polymerase and then religated forming pBIOS5. This plasmid was digested with HindIII, the ends filled using Klenow and an EcoRI sinker ligated into the destroyed HindIII site, forming pBIOS4. 
     pSIOS176 and pBIOS177 were transformed into Arabidopsis and tobacco. The larger promoter fragment is predicted to is contain the entire Ms41-A promoter region since the EcoRI site lies with the 3′ end of the W11 transcript. 
     Arabidopsis Results: 
     a) Short promoter: Histochemical staining reveals that GUS activity is observed in most tissues and is especially high in callus, (strong blue staining is detectable after a few hours in X-GLUC (5-bromo-4-chloro-3-indolyl glucuronide). 
     b) Long promoter: GUS activity was seen in callus, but no obvious blue staining was observed in the vegetative parts of primary transformants. However 75% of the 40 transformants had significant GUS activity in anthers. In the floral buds observed, GUS expression is detected just after the breakdown of the callose wall (floral stage 10); expression appears to be located initially in the tapetum and subsequently in the microspores. GUS activity is still present in mature pollen. However it is possible that there is also GUS activity in the microsporocytes and tetrad microspores since the GUS substrate may not pentrate the thick callose wails surrounding the microsporocytes and tetrads. 
     Similar staining experiments were done with plants containing the 3 tapetum-specific promoter fusions—TA29 (Koltunow et al.,  Plant Cell , 2:1201-1224 (1990)), A6 (Hird et al.,  Plant Journal , 4:1023-1033 (1993)) and A9 (Paul et al.,  Plant Molecular Biology , 19:611-622 (1992)) and with the microspore/pollen promoter LAT 52 (Twell et al.,  Molecular and General Genetics , 217:240-245 (1989)). 
     A9 is definitely the earliest and with the A6 promoter, GUS is expressed when tetrads are visible; by contrast the TA 29 promoter gives expression at roughly at the same time as Ms41-A; the latter also shows earlier expression in microspores than LAT 52. In seedlings of 5 out of 7 transformed plants, very low levels of GUS expression is detected in aerial parts. 
     Tobacco Results: 
     a) Short promoter: GUS expression appears to be constitutive. 
     b) Long promoter: Results were similar to those observed in Arabidopsis, ie expression is largly confined to the tapetum, microspores and pollen of the anther. Very low GUS expression was seen in the aerial parts of seedlings, however no expression was detected in callus. 
     It appears that expression from the long promoter matches that of the Ms41-A gene, with very low level “constitutive” expression. Expression in the anther is much stronger than predicted by the abundance of Ms41-A transcript in floral parts indicating that the Ms41-A message may be very unstable. Higher level constitutive expression observed from the short promoter suggests that there a constitutive silencer is present in the upstream region of the promoter between posititions −1635 to −900 bp. The conserved pattern of expression of the long promoter between tobacco and Arabidopsis suggests that the long promoter will be useful in male sterility systems in a wide range of plant species. Examples 3 and 4 below demonstrate the use of the long Ms41-A promoter in male sterility systems. 
     EXAMPLE 3 
     Expression of Barnase from the Ms41-A Promoter in Tobacco and Maize 
     The timing of expression of the Ms41-A promoter in the tapetum is similar to that seen from the tobacco TA29 promoter, thus fusion to cytotoxins such as Dipthera toxin A (Thorsness et al.,  Developmental Biology , 143: 173-184 (1991)) and Barnase (Mariani et al.,  Nature , 347: 737-741 (1990)) will ablate the anther tapetum leading to complete male sterility. Thus the long Ms41-A promoter is linked to Barnase. A 1 kb XbaI, HindIII (filled) fragment encoding Barnase is excised from pWP127 (Paul et al., supra) and cloned between the XbaI and SstI (filled) sites of pBIOS177 forming pBIOS 177-Barnase (FIG.  7 ). 
     This plasmid is used to regenerate tobacco and Maize transformants that are male sterile. Although the weak “consitutive” expression of the Ms41-A promoter should prevent recovery of such plants, it is likely that these plants have reduced Ms41-A promoter expression. Thus no significant expression of Barnase occurs in vegetative tissues whereas expression is sufficient to cause tapetal cell death and male sterility. 
     EXAMPLE 4 
     Expression of Antisense Ms41-A from the Ms41-A Promoter in Arabidoysis 
     The Ms41-A promoter can be used to downregulate the expression of genes essential for tapetal function thus causing complete male sterility. Downregulation can be achieved by expression from the Ms41-A promoter of antisense or sense fragments of the target gene or by expression of ribozymes which will cleave the target gene transcript. Such a target gene is Ms41-A. To construct an Ms41-A promoter- Ms41-A antisense chimeric gene, RT-PCR is used to generate a 1923 bp Ms41-A fragment from young Arabidopsis floral buds mRNA. The primers used are: 
     W3 Bam, 5′ CGGATCCTTCTAAGACATCACG 3′ (SEQ ID NO:9) (positions 54-75, FIG. 3) and 3′2, 5′ AATGTACTACTACTACTACTTAGGAC 3′ (SEQ ID NO:10) (positions 3001-2976, FIG.  3 ). 
     This PCR fragment is cloned into pGEM-T forming p542, such that the 5′ end of MS41-A is adjacent to the ApaI site of pGEM-T (FIG.  7 ). The MS41-A Spel, ApaI (filled using T4 DNA polymerase) fragment is cloned between the XbaI and SstI (filled) sites of pBIOS177, thus replacing the GUS gene of pBIOS177 and forming pBIOS182 (FIG.  8 ). This plasmid is used to transform Arabidopsis. A proportion of transformants are male sterile with a phenotype that resembled that of the original Ms41-A mutant. Examples 5 and 7 below describe the use of the Ms41-A transcribed region in male sterility systems. 
     EXAMPLE 5 
     Expression of a 35S CAMV Promoter- Ms41-A Antisense Chimeric Gene and a 35S CaMV Promoter Ms41-A Sense Chimeric Gene in Arabidopsis 
     As described in Example 4, downregulation of the Ms41-A gene by expression of Ms41-A antisense fragments, sense fragments or ribozymes, each driven from the Ms41-A promoter will lead to male sterility. However any promoter that has the appropriate pattern of expression, ie is active in microsporocyte and/or tapetal cells of the anther at the time of Ms41-A expression, may be used to downregulate Ms41-A and cause male sterility. Thus a CaMV 35S promoter is linked to an antisense Ms41-A fragment and to a sense Ms41-A fragment. The antisense construct is obtained by cloning the ApaI (filled), SpeI p542 MS41-A fragment between the XbaI and SstI (filled) sites of pBIOS4 forming pBIOS188 (FIG.  8 ). 
     The sense construct is obtained by cloning the ApaI (filled), SstI p542 MS41-A fragment between the Smal and SstI sites of pBIOS4 forming pBIOS186 (FIG.  8 ). These plasmids are transformed into Arabidopisis. A proportion of the antisense and sense transformants are male sterile with a phenotype similar to that of the original Ms41-A mutant plant. 
     EXAMPLE 6 
     Isolation of a Ms41-A Orthologue from Maize 
     Most methods to use the coding region of the Ms41-A in a male sterilty system require the isolation of the orthologous sequence either from the crop species of interest or from a close evolutionary relative. Such methods include antisense and sense supression and the use of ribozymes. The degree of evolutionary conservation between orthologous protein sequences is variable and is probably dependant on constraints on protein function. Athough orthologous protein sequences may be highly conserved, codon usage may be quite different, producing orthologous mRNA sequences that may have low homology. Thus, in order co downregulate the Maize version of Ms41-A, it is probably necessary to isolate the Maize version of Ms41-A. Given the Arabidiopsis Ms41-A mRNA sequence, several approaches are possible for the isolation of the Maize orthologue. Some of which are outlined below: 
     The Ms41-A cDNA can be used as a probe on a Maize Northern or Southern at low stringency to see if a mRNA or genomic band hybridises. This was unsucessful indicating that these sequences are widely diverged. The Arabidopsis sequence can be used as a probe in more closely related species and the orthologues in turn used as further probes until the version in Maize is identified. The cloning and sequencing of such orthologues may also result in the identification of conserved areas that can be used in a degenerate PCR approach. 
     Antibodies to Ms41-A may also be useful since protein sequences and epitopes are generally more conserved than RNA/DNA sequences. 
     The approach used was to screen the Genebank and EST (Expressed Sequence Tag) databases for sequences that showed homology to the Arabidopsis Ms41-A DNA sequence. Four groups of sequences were identified according to the degree of sequence similarity. Alignments of these sequences are presented in FIG.  9 . 
     Group 1 
     This group contains the Arabidopsis Ms41-A cDNA and an EST sequence from rice OSS2204 (D40316) which was cloned from a shoot cDNA library (prepared from etiolated 8 day old seedlings). 
     Group 2 
     In this group are two pairs of almost identical Arabidopsis EST sequences (ATTS3975 (Z37232) and T43470) and (T21748 and R30405) which are presumably derived from the same transcripts and can be considered as two sequences. The R30405, T21748 and T43470 cDNAs were isolated from a library prepared using a mixture of RNA from various tissues. The ATTS3975 cDNA is from a library prepared from cell suspension culture. In addition, in this group is a rice cDNA isolated from a root cDNA library (seedling stage) OSR1187 (D24087). 
     Group 3 
     In this group are 3 EST sequences and 1 cDNA sequence ATTS1074 (isolated from a cycling cells cDNA library). A partial EST sequence for ATTS 1074 is on the database (Z25611) and after identification of this sequence as similar to Ms-41A the cDNA clone was obtained and the sequence completed. The other 3 sequences are all identical or almost identical to the ATTS1074 sequence. 
     The cDNA clones R65265 and T44526 were isolated from a mixed RNA library. ATTS2424 is a 3′ sequence EST sequence from the same cDNA clone as ATTS1074, this clone (TAI231) was isolated from a cDNA library prepared from a cell suspension culture containing cycling cells. 
     Group 4 
     This group contains sequences of 4 closely related plant transcription factors; Viviparous-1 from maize (McCarty et al.,  Cell , 66:895-905 (1991)) and rice (Hattori et al.,  Plant Molecular Biology , 24:805-810 (1994)), ABI 3 from Arabidopsis (Giraudat et al.,  Plant Cell , 4:1251-1261 (1992)) and a  Phaseolus vulgaris  embryo-specific acidic transcriptional activator PvAlf (Bobb er al.,  Plant Journal  In press (1995)). 
     There is some amino-acid similarity between a region in the N-terminal of the Ms41-A protein and the proposed DNA binding domain of maize Viviparous-1. This region is highly conserved between the 4 transcription factors (&gt;80% amino-acid identity between all 4 sequences). This suggests that the Ms-41A protein may have DNA binding activity, although the MS41-A protein might be sorted via the ER, perhaps to be secreted, since Ms41-A has a putative signal peptide and 6 putative N glycosylation sites. 
     The most closely related sequence to Ms41-A identified by this analysis is the rice OSS2204 sequence. This was obtained from the rice sequencing project and used to probe a Maize cDNA library made in Lambda UniZap (Stratagene) from polyA+ RNA. isolated from pre-meiotic to meiotic-stage male inflorescences. The cDNA isolated, Zm41-A, is approximately 2.2 kb in length and has a poly A tail at it&#39;s 3′ end. Approximately 300 bp of 5′ prime sequence is shown in FIG.  10 . 
     This sequence shows strong similarity to the rice OSS2204 cDNA sequence (84% identity) but is only 53% identical to the Arabidopsis sequence. The ORF indicated underneath the DNA sequence is similar to both the proposed OSS2204 ORF (89% identical, 94% similar) and the Arabidopsis Ms41-A protein sequence (54% identical, 65% similar). 
     A dendrogram of the Ms41-A related sequences indicates that the Zm41-A sequence falls into group 1 (FIG.  11 ). This indicates that this cDNA is a good candidate for the maize orthologue of the Arabidopsis MS41A gene. 
     EXAMPLE 7 
     Expression of an Actin Promoter- Zm41-A Antisense Chimeric Gene in Maize 
     The Zm41-A cDNA is linked in an antisense orientation to a rice actin promoter. The entire Zm41-A cDNA is excised from pBluescript SK− (Stratagene) as an XhoI (filled), PstI fragment and cloned into PstI, SmaI-cut pCOR113 (McElroy et al.,  Molecular and General Genetics , 231: 150-160). This plasmid is used to transform Maize by a particle bombardment technique. A proportion of the transformants are male-sterile with a phenotype similar to that of the Arabidopsis Ms41-A mutant. This suggests that the Zm41-A sequence is the functional orthologue of Ms41-A and indicates that any sequence that falls within group 1 (FIG. 11) is likely to encode a functional orthologue of Ms41-A. 
     EXAMPLE 8 
     Molecular Characterisation of Zm41-A Gene(s) 
     a) Zm41-A Gene Transcription 
     BY RT-PCR this transcript has been shown to be abundant in anther RNA; in leaf and tassel RNA populations it is detected at a lower level. 
     After comparison of the maize and Arabidopsis sequences it was thought that the cDNA was unlikely to be a full length clone. With the “Marathon cDNA amplification” kit (Clontech, Palo Alto, Calif., USA) 5′RACE experiments were conducted on mRNA extracted from maize anthers at the meiosis stage, which yielded additional 5′ sequence. Two types of 5′RACE products were obtained and sequenced, the first contained approximately 150 bp of additional 5′ sequence as well as a 108 bp insertion at position 244 in the cDNA. The second RACE product contained approximately 130 bp of additional 5′ sequence. It is believed that the first RACE product may be the result of differential or incomplete splicing of the transcript resulting in a 36 amino acid insertion in the predicted peptide sequence as well as the 52 additional amino acids at the N terminal of the protein. Even with these additional sequences the full length transcript is likely to be longer at the 5′ end, based on comparison with the Arabidopsis protein and the maize genomic sequence. 
     b) Isolation of and Characterisation of Maize Genes which are Orthologs to Ms41-A 
     The Zm41-A cDNA was used to screen two different maize genomic lambda libraries. The first was a commercial library (Clontech, Palo Alto, Calif., USA) elaborated with DNA fragments from maize line 373 plantlets. DNA was partially digested with MboI enzyme and the fragments were cloned into the BamHI site of EMBL-3 (Frischauf et al,  J.Mol.Biol ., 170:827 (1983)). The insert DNA can be excised from the clone by the enzyme SalI. The second was a lambda library kindly provided by R. Mache (Universite Joseph Fourrier, URA 1178, Grenoble, France) elaborated with DNA fragments from the Mo 17 maize line. DNA was partially digested with the enzyme MboI and the fragments were cloned into the BamHI site of EMBL-4 (Frischaul et al, supra). The insert DNA was excised by the enzyme EcoRI. The genomic libraries screening was performed following the instructions of Sambrook et al (Molecular cloning: a laboratory manual, cold Spring Harbour Laboratory Press, New York, 1989). 10 6  recombinant Lambda per library were screened and three rounds of screening were performed. Fourteea positive lambda clones were isolated one of which was obtained from the library provided by R. Mache. 
     DNA from positive lambda clones was extracted and purified using Qiagen columns (Chatsworth, Calif., USA) according to the manufacturer&#39;s instructions. Then the clones were characterised by Southern analysis ( J.Mol.Biol ., 98:503-517 (1975)) in order to establish classes. DNAs from the Clontech library were restricted with HindIII and EcoRI and double restricted with HindIII/SalI. DNAs from the Mache library were restricted with HindIII and EcoRI and double restricted with HindIII/EcoRI. DNA fragments were separated on agarose gel, denatured and blotted onto Hybond N +  membrane (Amersham, Buckinghamshire, UK). The blots were hybridised with  32 P-labelled Zm41-A cDNA isolated after digestion with BamHI and XhoI (the resulting fragment is 2.1 kb long). 
     Ten lambda clones were different and were distributed in three classes: 
     class A comprising 5 clones (Z9, Z23, Z27, Z35 and Z36); 
     class B comprising 4 clones (Z7, Z28, Z29 and Z33); and 
     class c with only one clone, Z31, isolated from the R. Mache library. 
     In order to study the sequence of these three classes, the sub-cloning of three different genomic phages (Z31, Z33 and Z35) in the plasmid pBSII SK +  (Stratagene, Lajolla, Calif., USA) was performed according to the classical cloning method (Sambrook et al, supra). Hybridizing fragments were firstly slected. After the sequencing of the fragments&#39; extremities with universal primers, oligonucleotides were designed and the sequencing was chieved using the walking primer method. 
     With the clone Z31, 7.8 kb of continuous sequence data were obtained (see FIG.  12 ). To determine the complete gene structure, we have sequenced the entire Zm41-A cDNA. This is 2109 bp in length and encodes a putative peptide of 587 amino acids. The comparison between the genomic sequence and the cDNA and 5′RACE sequences indicated that this gene contains at least 12 exons. The insertion reported in the longest RACE products corresponds to the end of intron 4. Thus, the two families of cDNAs might be explained by the presence of two splicing sites in this intron. In the genomic sequence upstream of the end of the RACE products, there was detected the continuation of the open reading frame of 270 bp before an initiation codon at a NcoI restriction site. AAsuming that this initiation site is the right one, the length of the fragment which might contain the promoter sequence was 2.7 kb from the HindIII site where the sequence starts to the NcoI site. Therefore the translation of the Zm41-A Z31 gene should give a putative protein of 736 amino acids. The Z31 gene structure is depicted in FIG.  13 . 
     With the addition of the unspliced sequence (homologous to the end of intron 4) a longer protein might be obtained. Indeed, the longest open reading frame deduced from the genomic sequence Z31 including this insertion sequence exhibits two stop codons in frame. It is also worthwhile noting that there is a clear polymorphism here since the RACE products do not show these stop codons. The mis-splicing phenomenon may be a regulatory mechanism for the expression of the the Zm41-A related proteins as has recently been demonstrated in maize for another gene (Burr et al,  The Plant Cell , 8:1249-1259 (1996)). Therefore, either this gene codes for two proteins (736 aa and 131 aa) or it codes for the 736 aa and 772 aa proteins. 
     Moreover, a slight difference was observed between the Zm41-A cDNA and the Z31 genomic sequence in exon ten where a small addition is present (15 bp replaced by 36 bp); this is also in agreement with genetic polymorphism between maize lines. The maize lines used to study the mRNA and the genomic sequence are divergent (A188, B73 and Mo17 respectively). In FIG. 14 there is provided the alignment of the Z31 protein (736 aa) deduced from the longest open reading frame, with the protein deduced from the Zm41-A cDNA (587 aa). We found 15 amino acid changes as well as an additional 7 amino acids for the Z31 protein, these additional amino acids being located at position 556 of the Zm41-A cDNA protein. 
     For the other two genes, Z33 and Z35, 2.9 Kb and 5.8 Kb were respectively sequenced (see FIGS.  15  and  16 ). Z35 contains exon 3 in part and the complete exons 4, 5 and 6 from the Zm41-A cDNA. Z33 is similar to Z35 but it has a deletion of exon 4 and the 3′ end of exon 3 the two have the insertion sequence found in the longest 5′ RACE products. In addition, the comparison of the Z33 and Z35 sequences indicates at two deletions in the Z33 gene with respect to the Z35 gene. The first one is 686 bp long and starts in the 3′ end of exon 3 and extends to the end of exon 4 (with reference to the Z31 gene structure). The latter is located upstream of the sequence homologous to Z31 and the Zm41-A cDNA and is 808 bp long (see FIG.  13 ). Moreover, these two genes differed in their 3′ sequenced regions. 
     Due to the high level of conservation between these 3 sequences it is possible that the Z35 gene derived from Z31 via genetic rearrangements, deletions and/or insertions. Z33 has subsequent deletions from Z35. 
     EXAMPLE 9 
     Genetic Mapping of Zm41-A loci 
     58 single seed descent (SSD) maize lines derived from the cross A188×HD7 (Murigneux et al,  Theor.Appl.Genet ., 87:278-287 (1993)) were used for genetic mapping by RFLP technology. Hybridisation was performed with radiolabelled Zm41-A cDNA (BamHI-XhoI fragment, 2.1 Kb) on blots containing DNA from SSD lines and parental lines, digested with HindIII or EcoRI. Linkage analysis with the other RFLP markers mapped on this population was done using the Mapmaker version 2.0 computer program for Macintosh (Lander et al,  Genomics , 1:174-181 (1987)) and map distances were calculated with Kosambi function. 
     Many polymorphic bands between parental lines were revealed: one or two major bands and a few faint bands. Three loci, named Zm41-A.A, Zm41-A.B and Zm41-A.C were found located on two different chromosomes. Zm41-A.A locus corresponding to major bands, was located on the long arm of chromosome 6 at 26 cM from the RFLP marker umc132 and at 2 cM from the rflp marker umc62 (Maize Genetics Cooperation Newsletters (MNL) (August 1995) 69:248). Zm41-A.B and Zm41-A.C loci, corresponding to faint bands were located on chromosome 2 and were separated from each other by 19 cM. The Zm41-A.B locus lies near the centomere between umc131 (6 cM) and umc055 (3 cM) markers (MNL, supra). The Zm41-A.C locus was on the longchromosomic arm between umc055 (16 cM) and umc022 (6 cM) (MNL, supra). According to the mutant maize genetic map, no obvious male sterile mutant is mapped in those regions. One dominant male sterile mutant, Ms21, discovered in 1950 has been assigned on chromosome 6 but not very precisely. This mutation gives sterility only in the presence of the sks1 mutation. Interestingly, this mutation maps on chromosome 2, in the vicinity of the Zm41-A.B. Hybridisation on the blots containing DNA from SSD lines, with a Z31 gene specific probe, demonstrated that the Z31 gene corresponds to the Zm41-A.A locus on chromosome 6. 
     
       
         
           
             32 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              1
AAGGATCCTG GCAAAGACAT AAATC                                           25 
           
           
             
               26 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              2
AGATGCTGCT ACCCAATCTT TTGTGC                                          26 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              3
CGTATCGGTT TTCGATTACC GTATT                                           25 
           
           
             
               26 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              4
AGATGCTGCT ACCCAATCTT TTGTGC                                          26 
           
           
             
               30 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              5
TTATCATCAA CATCGCCATC GAATCTGCCG                                      30 
           
           
             
               19 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              6
AAAGTAGTAA ACCCTAGAG                                                  19 
           
           
             
               18 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              7
CCTAACTTTC TTTGCGGC                                                   18 
           
           
             
               26 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              8
GATCTAGACC GTGATGTCTT AGAAGG                                          26 
           
           
             
               22 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              9
CGGATCCTTC TAAGACATCA CG                                              22 
           
           
             
               26 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              10
AATGTACTAC TACTACTACT TAGGAC                                          26 
           
           
             
               5336 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               unknown 
             
             
               CDS 
               1957..3018 
             
             
               CDS 
               3487..4173 
             
             
               CDS 
               4736..4741 
             
             
               intron 
               3019..3486 
             
             
               intron 
               4174..4735 
             
              11
TCCTAACTTT CTTTGCGGCA TTTCTTATAA TACTTCGTCA GTTTTCAGAA TTCTTAAATC     60
TTTTTGCTGT GTTCTTATAA AGAAACATCA TCTATTAAAG TTGTCTTCGT TTGGATTTGG    120
TTTTGATGAC TTTGGGAAAT ATTTATGTTT AAGAAGGTTT CATTGGTCAT TGACTTTTAT    180
ATATTATATC GTAACCATGA TGTGATAGTG GGCCTTAGAT CAACAAACAT GCGAAAAACA    240
GAAGCAGAGG CCCGTTTCAA CGGAGCATAA TAAATTGCAT TCTCTGTCTT TTGTTTTTAG    300
GTTTTTTTTT TAACTGATAG ATGTGCCGTC GAAAATAATA TTGATATTTA AAAATTCACA    360
ACAAACATTC TTAACTGACC CACCCATCTA TCTGCTATTC CCACGCGCCA AGGAAAATAA    420
TAATAATAGC GAAATTGATT TTACATTTAT TTATTGATAG ATAATTTGTG TATTGTTAAG    480
ATTAACAGAT TTTAAGGGAT TAAAGTGGAA AAGGTAAACC GAAGACAACT TGCCATTTAC    540
TGATTTACAA CAATCCAAAT TTAAAAACAA ATGGTCCCAG TTTTTAGGGT TGTCACTTAA    600
ATTTATCGAA ATATTTACAC TTTAATTGGG TAAAACATAA TGGACAGAAA AACAAATATT    660
GTGACAAACA AAAAAACATG TTTTCACCAA GAAAAACAAA AACAAAAAAG ATGTAAAGCT    720
TTTCTTACAT CTGTACAAAA TAAAAGCAGA CGAAATTGTA CTTTATTTTC CTTATTAAAT    780
TGTCGGTATG TTTTATATGT TGTGAAAAGT AGAATGGATA ACCAAATAAA AATTACTGCA    840
TCTTAATAAA GTTGGTTCAA CCGGTTTAAA ATGTATTTTT TTAGTGTTAA CAACTTAAAG    900
CTTTTTTCGA TTATCGAATT GCAACAAACA AATATATTAA CAGAAAAAAG GAATCATGTA    960
TCTATTTCAA TATCCTGTTT TTTTTCTTCC ATTTGGATAT TTAGATCTTT TTCTGAATTT   1020
ATCTTGTTCT TAAATTAAAA CAGAAAAAAA GATTAAAAGT AAGACAGCTT GCTAATGGCA   1080
ACCGCAACAA ACAAGATAAT TTTGAAACGG ATCCACTTGG ATTTTCTTTG ATTTTGTAGA   1140
AAAATTGACA AATTGCTTTT GTATAAAAAC AAAAAATGTA CCGTAAAAAC ACACACATAA   1200
AAAATAAAAA GTGATAATGA CAAACAAATA AAGAGGTATT TTTCTTTTAT CTACTAATGT   1260
GATTATAAAA AAATCGACAT TGAAAATTTC AACACATCTT TTTCGCCAAA ACCTGAAAAT   1320
GGTCTTATTA TAACATAAAT TAGTTTTTTT GTCTTTCTAT TATATATTCA ATAACTCATC   1380
CCAACTTGAA CAAACCTATA AGTTCCGTAG TGTTCTTTTC TGTTGTGACA AAAAATACTA   1440
GCTAACGAGG GATAAGCACA AAAACATGAT TAATGTTTCT CTAATCATTC TAAAAATCTA   1500
CAGGAATATT CCCTTTTCAG TTTTTTCTTT CTTAAATGCA TTTCTTAGTT CTTCATAATT   1560
CAGTGAGTTT TAATAACAAT AATAAAAAAA AGAGCATCAT TAATTGAACC TAAAAATAAT   1620
GGGAAGAAAA ACCAAAAAGA TAGAGAGTAA GATGCACGCG CTAAAGATCG AACGGTTAAT   1680
AGAATCAGGT TAGTGAAGAG AGATATTAAA AGTTTGTTGT CGTGTGGCAA AAACTATAAT   1740
TTCCTTCACA CAAACAAAAA AAATAAAATC AAACACAAAA TCCCGTAGCA TCGTAACAGT   1800
AATTCGCTAT TATCTCCTCA CCCTCCGCTT TCGCTTCCCT TCTCTGCCCG TTTCAATTCC   1860
TTCTAAGACA TCACGGTCTC TCTCTATAAA AACAGTACCT ACCTCTTCTT CTTCTTCTTC   1920
ATTCGCTGAC TTCGTTTACA CTGAAAACAA ATACCTATGT CACCGCCGTC GGCAACCGCC   1980
GGTGACATCA ACCACCGTGA AGTAGACCCG ACGATCTGGC GCGCTTGTGC TGGAGCCTCC   2040
GTCCAGATCC CTGTCCTTCA CTCTAGGGTT TACTACTTTC CACAAGGTCA CGTTGAGCAC   2100
TGTTGCCCTC TCCTCTCTAC TCTTCCTTCC TCCACCTCGC CGGTTCCATG TATCATCACT   2160
TCAATCCAGT TGCTCGCCGA TCCGGTTACC GACGAGGTCT TTGCTCACCT TATTCTTCAA   2220
CCGATCACGC AGCAGCAGTT TACTCCGACT AATTATTCAC GATTCGGCAG ATTCGATGGC   2280
GATGTTGATG ATAACAACAA GGTGACTACC TTCGCCAAAA TTCTCACGCC TTCTGATGCT   2340
AACAATGGAG GTGGCTTCTC CGTTCCTCGT TTCTGTGCTG ATTCCGTCTT CCCTCTGCTT   2400
AATTTTCAAA TCGATCCACC GGTTCAGAAG CTCTACGTCA CTGATATCCA TGGAGCTGTT   2460
TGGGATTTCA GGCATATCTA TCGCGGTACA CCGAGGCGTC ACTTGCTAAC AACGGGATGG   2520
AGTAAGTTTG TCAATAGCAA GAAGCTCATC GCTGGAGATT CGGTTGTGTT TATGAGAAAA   2580
TCTGCAGATG AGATGTACAT CGGTGTTAGG CGAACTCCGA TCTCAAGCAG CGACGGAGGA   2640
AGTAGCTATT ACGGAGGAGA TGAGTATAAC GGTTACTACA GTCAGAGTAG CGTTGCCAAG   2700
GAAGATGATG GGAGTCCGAA GAAGACGTTT AGGAGATCTG GGAATGGTAA GTTGACTGCT   2760
GAGGCTGTAC GATCGATCAA TAGAGCGTCT CAGGGATTAC CGTTTGAGGT GGTGTTTTAT   2820
CCGGCTGCTG GATGGTCTGA GTTTGTTGTG AGAGCTGAAG ATGTTGAGTC TTCAATGTCT   2880
ATGTATTGGA CTCCTGGGAC TCGAGTCAAG ATGGCTATGG AGACTGAAGA TTCTTCTCGG   2940
ATCACATGGT TTCAAGGCAT CGTTTCCTCT ACTTATCAGG AGACCGGTCC ATGGCGTGGA   3000
TCTCCATGGA AGCAGCTTCA GGTATATGAT GTTTTTGAAA TGGTCTTTGC TCTTCTTATC   3060
TCTGTGATGT TGAGTTAATG GAACAATTCA GAATCGATCT TGTATCTGTT GTGTGCAAGC   3120
CTTTAAGATG ATGTTTAAGT CTCATCCTGG TTATTCAAAT GTCAATTGGG TTTTGAATGT   3180
TGTTTTGATT GCTGTGTTGT TTGTTTTGAA GCTAAATATT GGAAACAGGA TAAGTTAANT   3240
CATACGAAAA TGAATGTTCT GTCTCAGATT CATCTTCTAT AAGATGGAAT TGAAACTGGA   3300
AGATTTGGCT TAGTATTGTN TGTNTTGAGC GTCCGTGATG TAGAGTTGTT TTCATTATCC   3360
TTCTTTGGCC ACGCATTGTA CATTGTGTTT GTTAAACTAG AGTTCCTCTG ATTAGTCTTA   3420
TGAGATACTC CTTTTTTGCC AATATATTCT ACTTCCTCTG ATTAGTTCCT TTGTTTTTAA   3480
CTTGCGTAGA TCACATGGGA TGAACCTGAG ATTCTGCAAA ACGTGAAGAG GGTGAATCCA   3540
TGGCAAGTGG AAATTGCTGC ACATGCAACT CAACTGCATA CCCCTTTCCC TCCAGCAAAG   3600
AGGTTGAAGT ATCCACAACC CGGAGGAGGG TTCTTGAGTG GAGATGATGG AGAAATCCTT   3660
TATCCTCAAA GTGGACTGTC TAGTGCAGCA GCACCTGATC CAAGTCCTTC TATGTTCTCG   3720
TATTCTACAT TTCCTGCTGG CATGCAGGGA GCCAGGCAAT ATGATTTTGG GTCTTTCAAT   3780
CCAACCGGAT TCATTGGAGG AAATCCTCCC CAGCTATTCA CCAATAACTT CTTAAGTCCG   3840
CTTCCTGATT TGGGAAAAGT GTCGACTGAG ATGATGAACT TTGGCAGTCC GCCATCAGAT   3900
AACTTATCGC CTAATAGCAA CACCACTAAT CTGTCCTCTG GAAATGACCT GGTTGGAAAC   3960
CGAGGCCCCC TTTCAAAGAA AGTTAACTCG ATTCAGTTGT TTGGCAAGAT CATTACCGTG   4020
GAGGAGCATT CTGAGAGCGG TCCTGCAGAG TCTGGCTTGT GTGAAGAGGA TGGCAGCAAA   4080
GAGTCCAGCG ACAATGAGAC ACAGTTGTCC TTATCACATG CTCCTCCAAG CGTGCCTAAA   4140
CATTCCAACA GCAACGCAGG TTCTAGCTCC CAAGGTATAT TCCGATCTCT CTCAAGTACA   4200
ATAATCAATT GAATCAGTTG CTATAAGCTT TTATTACTGT TTTGCACAAG GCAATTTCTC   4260
TTCCTTTCCC ATGAACTATA TTATGTAGAG TAGGAAACAC AATCATGATT TCTGATATGA   4320
CTTGACTGAT GATGATACTT GTNAAAACTA TCTATATATC TCTTCAGTAA TCAGTCGCCT   4380
TGAGGTAATT GGAATTTGGA ACTTGAACAT TACTTGGATT TTAACTTTTC AATAGCATAA   4440
GCNTTCCTGT TTCATCATAT ATGTTTCACT ATACTTGTAT GCTTTTATTA CTGCTGATAT   4500
TTACTATTCC TGCTATTTTT TTTGGGTCTC GTTAACGGTA ATAAGGACAC AGAATTGGCT   4560
CTTTTATCCA TCAGAACTAG ACATTACTGT ACAAGTAGAT GAAGAATTAT GTGGTTCCAT   4620
TACAAATTTA ATTTGCAGAA AGCTTGAAGC TGCTGCTTAT AGACGATTAT AATGTTGGAA   4680
GATCCTGAAG CTTGGAATGA TTTGTACTTT TCTTTTGTTT GTGTGTGTTT TGACAGGTTA   4740
AAAAGTGAAA GAAGTGGTGG ATCTTTGCTG GAATCTCCAA GTCCTAAGTA GTAGTAGTAG   4800
TACATTATAT ATAATTCTGT TGTTTCTGCA ATTGACTTTT CTCTGGCTTT TCTTTGCCAC   4860
GTGACGATTC CGGTTTTTAC TTTCTTTCTT TTTTTTTTAT CAATTTCTCA GACACATTTG   4920
ATGAACATCT CGCTCTCATC TAATCGTTAA CTATTTTTAT TGGGGTAAAT GTCTGGATTT   4980
GTCTTACCTA AACATGTTTT AAGACTGATG TTTATGCAGA GTGAAAACAG TAAATAATTT   5040
AATGCTTTAT TCAATCCCTA TGCAATGGAT CTCAACTTAA CGGCGCCAAC CAGAGAGTTT   5100
TACTAACTGT CTTTTGCTTT TAGTTAATAT TCCTAATAAA TAAAAAGACT GCCAATAATA   5160
AAATCGGACC ATTTTTATTC TCATAATAAA TAAAAGAAGC TCAAGGGAGG TCCCTCCTAC   5220
ACTTTTCTGA CTCCTTTATG TTCTGTTCTC TGTGATTCAT TAACGGATCA GCTATAGCAT   5280
TTCCAATTTG TCAGTAAGTT AGGGTTGGTT TGGATTAGCT AATAGCTACC AATGAG       5336 
           
           
             
               584 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
              12
Met Ser Pro Pro Ser Ala Thr Ala Gly Asp Ile Asn His Arg Glu Val
1               5                   10                  15
Asp Pro Thr Ile Trp Arg Ala Cys Ala Gly Ala Ser Val Gln Ile Pro
            20                  25                  30
Val Leu His Ser Arg Val Tyr Tyr Phe Pro Gln Gly His Val Glu His
        35                  40                  45
Cys Cys Pro Leu Leu Ser Thr Leu Pro Ser Ser Thr Ser Pro Val Pro
    50                  55                  60
Cys Ile Ile Thr Ser Ile Gln Leu Leu Ala Asp Pro Val Thr Asp Glu
65                  70                  75                  80
Val Phe Ala His Leu Ile Leu Gln Pro Ile Thr Gln Gln Gln Phe Thr
                85                  90                  95
Pro Thr Asn Tyr Ser Arg Phe Gly Arg Phe Asp Gly Asp Val Asp Asp
            100                 105                 110
Asn Asn Lys Val Thr Thr Phe Ala Lys Ile Leu Thr Pro Ser Asp Ala
        115                 120                 125
Asn Asn Gly Gly Gly Phe Ser Val Pro Arg Phe Cys Ala Asp Ser Val
    130                 135                 140
Phe Pro Leu Leu Asn Phe Gln Ile Asp Pro Pro Val Gln Lys Leu Tyr
145                 150                 155                 160
Val Thr Asp Ile His Gly Ala Val Trp Asp Phe Arg His Ile Tyr Arg
                165                 170                 175
Gly Thr Pro Arg Arg His Leu Leu Thr Thr Gly Trp Ser Lys Phe Val
            180                 185                 190
Asn Ser Lys Lys Leu Ile Ala Gly Asp Ser Val Val Phe Met Arg Lys
        195                 200                 205
Ser Ala Asp Glu Met Tyr Ile Gly Val Arg Arg Thr Pro Ile Ser Ser
    210                 215                 220
Ser Asp Gly Gly Ser Ser Tyr Tyr Gly Gly Asp Glu Tyr Asn Gly Tyr
225                 230                 235                 240
Tyr Ser Gln Ser Ser Val Ala Lys Glu Asp Asp Gly Ser Pro Lys Lys
                245                 250                 255
Thr Phe Arg Arg Ser Gly Asn Gly Lys Leu Thr Ala Glu Ala Val Arg
            260                 265                 270
Ser Ile Asn Arg Ala Ser Gln Gly Leu Pro Phe Glu Val Val Phe Tyr
        275                 280                 285
Pro Ala Ala Gly Trp Ser Glu Phe Val Val Arg Ala Glu Asp Val Glu
    290                 295                 300
Ser Ser Met Ser Met Tyr Trp Thr Pro Gly Thr Arg Val Lys Met Ala
305                 310                 315                 320
Met Glu Thr Glu Asp Ser Ser Arg Ile Thr Trp Phe Gln Gly Ile Val
                325                 330                 335
Ser Ser Thr Tyr Gln Glu Thr Gly Pro Trp Arg Gly Ser Pro Trp Lys
            340                 345                 350
Gln Leu Gln Ile Thr Trp Asp Glu Pro Glu Ile Leu Gln Asn Val Lys
        355                 360                 365
Arg Val Asn Pro Trp Gln Val Glu Ile Ala Ala His Ala Thr Gln Leu
    370                 375                 380
His Thr Pro Phe Pro Pro Ala Lys Arg Leu Lys Tyr Pro Gln Pro Gly
385                 390                 395                 400
Gly Gly Phe Leu Ser Gly Asp Asp Gly Glu Ile Leu Tyr Pro Gln Ser
                405                 410                 415
Gly Leu Ser Ser Ala Ala Ala Pro Asp Pro Ser Pro Ser Met Phe Ser
            420                 425                 430
Tyr Ser Thr Phe Pro Ala Gly Met Gln Gly Ala Arg Gln Tyr Asp Phe
        435                 440                 445
Gly Ser Phe Asn Pro Thr Gly Phe Ile Gly Gly Asn Pro Pro Gln Leu
    450                 455                 460
Phe Thr Asn Asn Phe Leu Ser Pro Leu Pro Asp Leu Gly Lys Val Ser
465                 470                 475                 480
Thr Glu Met Met Asn Phe Gly Ser Pro Pro Ser Asp Asn Leu Ser Pro
                485                 490                 495
Asn Ser Asn Thr Thr Asn Leu Ser Ser Gly Asn Asp Leu Val Gly Asn
            500                 505                 510
Arg Gly Pro Leu Ser Lys Lys Val Asn Ser Ile Gln Leu Phe Gly Lys
        515                 520                 525
Ile Ile Thr Val Glu Glu His Ser Glu Gly Ser Pro Ala Glu Ser Gly
    530                 535                 540
Leu Cys Glu Glu Asp Gly Ser Lys Glu Ser Ser Asp Asn Glu Thr Gln
545                 550                 555                 560
Leu Ser Leu Ser His Ala Pro Pro Ser Val Pro Lys His Ser Asn Ser
                565                 570                 575
Asn Ala Gly Ser Ser Ser Gln Gly
            580 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              13
CGTATCGGTT TTCGATTACC GTATT                                           25 
           
           
             
               25 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              14
CGTTTCCGTT TCCGTTTACC GTTTT                                           25 
           
           
             
               16 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              15
TGCTTGTGCT GGAGCC                                                     16 
           
           
             
               31 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              16
GTTATCATCA ACATCGCCAT CGAATCTGCC G                                    31 
           
           
             
               18 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               unknown 
             
              17
CTGCTGCTGC GTGATCGG                                                   18 
           
           
             
               83 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..83 
               /note= “Figure 9, sequence of
               ZmVP1” 
             
              18
Leu Leu Gln Lys Val Leu Lys Gln Ser Asp Val Gly Ser Leu Gly Arg
1               5                   10                  15
Ile Val Leu Pro Lys Lys Glu Ala Glu Val His Leu Pro Glu Leu Lys
            20                  25                  30
Thr Arg Asp Gly Ile Ser Ile Pro Met Glu Asp Ile Gly Thr Ser Arg
        35                  40                  45
Val Trp Asn Met Arg Tyr Arg Phe Trp Pro Asn Asn Lys Ser Arg Met
    50                  55                  60
Tyr Leu Leu Glu Asn Thr Gly Glu Phe Val Arg Ser Asn Glu Leu Gln
65                  70                  75                  80
Glu Gly Asp 
           
           
             
               67 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..67 
               /note= “Figure 9, sequence of
               OSR1187” 
             
              19
Glu Lys Arg Leu Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu Val
1               5                   10                  15
Ile Pro Lys Gln Xaa Ala Glu Arg Tyr Phe Xaa Leu Gly Gly Gly Asp
            20                  25                  30
Ser Gly Xaa Lys Xaa Leu Leu Leu Ser Xaa Glu Asp Glu Ser Gly Lys
        35                  40                  45
Pro Trp Arg Phe Arg Tyr Ser Tyr Trp Thr Ser Ser Gln Ser Tyr Val
    50                  55                  60
Leu Xaa Lys
65 
           
           
             
               131 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..131 
               /note= “Figure 9, sequence of
               ATTS3975” 
             
              20
Leu Arg Lys His Thr Tyr Asn Glu Glu Leu Glu Gln Ser Lys Arg Arg
1               5                   10                  15
Arg Asn Gly Asn Gly Asn Met Thr Arg Thr Leu Leu Thr Ser Gly Leu
            20                  25                  30
Ser Asn Asp Gly Val Ser Thr Thr Gly Phe Arg Ser Ala Glu Ala Leu
        35                  40                  45
Phe Glu Lys Ala Val Thr Pro Ser Asp Val Gly Lys Leu Asn Arg Leu
    50                  55                  60
Val Ile Pro Lys His His Ala Glu Lys His Phe Pro Leu Pro Ser Ser
65                  70                  75                  80
Asn Val Ser Val Lys Gly Val Leu Leu Asn Phe Glu Asp Val Asn Gly
                85                  90                  95
Lys Val Trp Arg Phe Arg Tyr Ser Tyr Trp Asn Ser Ser Gln Ser Tyr
            100                 105                 110
Val Leu Thr Lys Gly Trp Ser Arg Phe Val Lys Glu Lys Asn Leu Arg
        115                 120                 125
Ala Gly Asp
    130 
           
           
             
               512 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..512 
               /note= “Figure 9, sequence of
               At41a” 
             
              21
Leu Ala Asp Pro Val Thr Asp Glu Val Phe Ala His Leu Ile Leu Gln
1               5                   10                  15
Pro Ile Thr Gln Gln Gln Phe Thr Pro Thr Asn Tyr Ser Arg Phe Gly
            20                  25                  30
Arg Phe Asp Gly Asp Val Asp Asp Asn Asn Lys Val Thr Thr Phe Ala
        35                  40                  45
Lys Ile Leu Thr Pro Ser Asp Ala Asn Asn Gly Gly Gly Phe Ser Val
    50                  55                  60
Pro Arg Phe Cys Ala Asp Ser Val Phe Pro Leu Leu Asn Phe Gln Ile
65                  70                  75                  80
Asp Pro Pro Val Gln Lys Leu Tyr Val Thr Asp Ile His Gly Ala Val
                85                  90                  95
Trp Asp Phe Arg His Ile Tyr Arg Gly Thr Pro Arg Arg His Leu Leu
            100                 105                 110
Thr Thr Gly Trp Ser Lys Phe Val Asn Ser Lys Lys Leu Ile Ala Gly
        115                 120                 125
Asp Ser Val Val Phe Met Arg Lys Ser Ala Asp Glu Met Tyr Ile Gly
    130                 135                 140
Val Arg Arg Thr Pro Ile Ser Ser Ser Asp Gly Gly Ser Ser Tyr Tyr
145                 150                 155                 160
Gly Gly Asp Glu Tyr Asn Gly Tyr Tyr Ser Gln Ser Ser Val Ala Lys
                165                 170                 175
Glu Asp Asp Gly Ser Pro Lys Lys Thr Phe Arg Arg Ser Gly Asn Gly
            180                 185                 190
Lys Leu Thr Ala Glu Ala Val Arg Ser Ile Asn Arg Ala Ser Gln Gly
        195                 200                 205
Leu Pro Phe Glu Val Val Phe Tyr Pro Ala Ala Gly Trp Ser Glu Phe
    210                 215                 220
Val Val Arg Ala Glu Asp Val Glu Ser Ser Met Ser Met Tyr Trp Thr
225                 230                 235                 240
Pro Gly Thr Arg Val Lys Met Ala Met Glu Thr Glu Asp Ser Ser Arg
                245                 250                 255
Ile Thr Trp Phe Gln Gly Ile Val Ser Ser Thr Tyr Gln Glu Thr Gly
            260                 265                 270
Pro Trp Arg Gly Ser Pro Trp Lys Gln Leu Gln Ile Thr Trp Asp Glu
        275                 280                 285
Pro Glu Ile Leu Gln Asn Val Lys Arg Val Asn Pro Trp Gln Val Glu
    290                 295                 300
Ile Ala Ala His Ala Thr Gln Leu His Thr Pro Phe Pro Pro Ala Lys
305                 310                 315                 320
Arg Leu Lys Tyr Pro Gln Pro Gly Gly Gly Phe Leu Ser Gly Asp Asp
                325                 330                 335
Gly Glu Ile Leu Tyr Pro Gln Ser Gly Leu Ser Ser Ala Ala Ala Pro
            340                 345                 350
Asp Pro Ser Pro Ser Met Phe Ser Tyr Ser Thr Phe Pro Ala Gly Met
        355                 360                 365
Gln Gly Ala Arg Gln Tyr Asp Phe Gly Ser Phe Asn Pro Thr Gly Phe
    370                 375                 380
Ile Gly Gly Asn Pro Pro Gln Leu Phe Thr Asn Asn Phe Leu Ser Pro
385                 390                 395                 400
Leu Pro Asp Leu Gly Lys Val Ser Thr Glu Met Met Asn Phe Gly Ser
                405                 410                 415
Pro Pro Ser Asp Asn Leu Ser Pro Asn Ser Asn Thr Thr Asn Leu Ser
            420                 425                 430
Ser Gly Asn Asp Leu Val Gly Asn Arg Gly Pro Leu Ser Lys Lys Val
        435                 440                 445
Asn Ser Ile Gln Leu Phe Gly Lys Ile Ile Thr Val Glu Glu His Ser
    450                 455                 460
Glu Ser Gly Pro Ala Glu Ser Gly Leu Cys Glu Glu Asp Gly Ser Lys
465                 470                 475                 480
Glu Ser Ser Asp Asn Glu Thr Gln Leu Ser Leu Ser His Ala Pro Pro
                485                 490                 495
Ser Val Pro Lys His Ser Asn Ser Asn Ala Gly Ser Ser Ser Gln Gly
            500                 505                 510 
           
           
             
               123 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..123 
               /note= “Figure 9, sequence of
               OSS2204” 
             
              22
Ala Val Lys Arg Leu Ala Arg Ile Pro His Met Phe Cys Lys Thr Leu
1               5                   10                  15
Thr Ala Ser Asp Thr Ser Thr His Gly Gly Phe Ser Val Pro Arg Arg
            20                  25                  30
Ala Ala Glu Asp Cys Phe Pro Pro Leu Asp Tyr Ser Leu Gln Arg Pro
        35                  40                  45
Phe Gln Glu Leu Val Ala Lys Asp Leu His Gly Thr Glu Trp Arg Phe
    50                  55                  60
Arg His Ile Tyr Arg Gly Gln Pro Arg Arg His Leu Leu Thr Thr Gly
65                  70                  75                  80
Trp Ser Gly Phe Ile Asn Lys Lys Lys Leu Val Ser Gly Asp Cys Ser
                85                  90                  95
Ala Ile Pro Gln Glu Val Lys Met Glu Asn Phe Asp Trp Gly Val Arg
            100                 105                 110
Arg Ala Ala Gln Leu Lys Asn Ala Ile Ser Phe
        115                 120 
           
           
             
               107 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..107 
               /note= “Figure 9, sequence of
               Zm41a” 
             
              23
Asp Gly Ser Ala Glu Asp Gly Val Arg Lys Gly Glu Thr Val Lys Gln
1               5                   10                  15
Arg Phe Ser Arg Met Pro His Met Phe Cys Lys Thr Leu Thr Ala Ser
            20                  25                  30
Asp Thr Ser Thr His Gly Gly Phe Ser Val Pro Arg Arg Ala Ala Glu
        35                  40                  45
Asp Cys Phe Pro Pro Leu Asp Tyr Ser Gln Gln Arg Pro Ser Gln Glu
    50                  55                  60
Leu Val Ala Lys Asp Leu His Gly Thr Glu Trp Arg Phe Arg His Ile
65                  70                  75                  80
Tyr Arg Gly Gln Pro Arg Arg His Leu Leu Thr Thr Gly Trp Ser Ala
                85                  90                  95
Phe Val Asn Lys Lys Lys Leu Val Ser Gly Asp
            100                 105 
           
           
             
               72 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..72 
               /note= “Figure 9, sequence of
               T21748” 
             
              24
Leu Asn Arg Leu Val Ile Pro Lys Gln His Ala Glu Lys His Phe Pro
1               5                   10                  15
Leu Pro Ser Pro Ser Pro Ala Val Thr Lys Gly Val Leu Ile Asn Phe
            20                  25                  30
Glu Asp Val Asn Arg Lys Val Trp Arg Phe Arg Tyr Ser Tyr Trp Asn
        35                  40                  45
Ser Ser Gln Ser Tyr Val Leu Thr Lys Gly Trp Ser Arg Phe Val Lys
    50                  55                  60
Glu Lys Asn Leu Arg Ala Gly Asn
65                  70 
           
           
             
               461 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..461 
               /note= “Figure 9, sequence of
               ATTS1074” 
             
              25
Gly Phe Ser Gly Phe Leu Arg Asp Asp Glu Ser Thr Thr Thr Thr Ser
1               5                   10                  15
Lys Leu Met Met Met Lys Arg Asn Gly Asn Asn Asp Gly Asn Ala Ala
            20                  25                  30
Ala Thr Gly Arg Val Arg Val Glu Ala Val Ala Glu Ala Val Ala Arg
        35                  40                  45
Ala Ala Cys Gly Gln Ala Phe Glu Val Val Tyr Tyr Pro Arg Ala Ser
    50                  55                  60
Thr Pro Glu Phe Cys Val Lys Ala Ala Asp Val Arg Ser Ala Met Arg
65                  70                  75                  80
Ile Arg Trp Cys Ser Gly Met Arg Phe Lys Met Ala Phe Glu Thr Glu
                85                  90                  95
Asp Ser Ser Arg Ile Ser Trp Phe Met Gly Thr Val Ser Ala Val Gln
            100                 105                 110
Val Ala Asp Pro Ile Arg Trp Pro Asn Ser Pro Trp Arg Leu Leu Gln
        115                 120                 125
Val Ala Trp Asp Glu Pro Asp Leu Leu Gln Asn Val Lys Arg Val Ser
    130                 135                 140
Pro Trp Leu Val Glu Leu Val Ser Asn Met Pro Thr Ile His Leu Ser
145                 150                 155                 160
Pro Phe Ser Pro Arg Lys Lys Ile Arg Ile Pro Gln Pro Phe Glu Phe
                165                 170                 175
Pro Phe His Gly Thr Lys Phe Pro Ile Phe Ser Pro Gly Phe Ala Asn
            180                 185                 190
Asn Gly Gly Gly Glu Ser Met Cys Tyr Leu Ser Asn Asp Asn Asn Asn
        195                 200                 205
Ala Pro Glu Gly Ile Gln Gly Ala Arg Gln Ala Gln Gln Leu Phe Gly
    210                 215                 220
Ser Pro Ser Pro Ser Leu Leu Ser Asp Leu Asn Leu Ser Ser Tyr Thr
225                 230                 235                 240
Gly Asn Asn Lys Leu His Ser Pro Ala Met Phe Leu Ser Ser Phe Asn
                245                 250                 255
Pro Arg His His His Tyr Gln Ala Arg Asp Ser Glu Asn Ser Asn Asn
            260                 265                 270
Ile Ser Cys Ser Leu Thr Met Gly Asn Pro Ala Met Val Gln Asp Lys
        275                 280                 285
Lys Lys Ser Val Gly Ser Val Lys Thr His Gln Phe Val Leu Phe Gly
    290                 295                 300
Gln Pro Ile Leu Thr Glu Gln Gln Val Met Asn Arg Lys Arg Phe Leu
305                 310                 315                 320
Glu Glu Glu Ala Glu Ala Glu Glu Glu Lys Gly Leu Val Ala Arg Gly
                325                 330                 335
Leu Thr Trp Asn Tyr Ser Leu Gln Gly Leu Glu Thr Gly His Cys Lys
            340                 345                 350
Val Phe Met Glu Ser Glu Asp Val Gly Arg Thr Leu Asp Leu Ser Val
        355                 360                 365
Ile Gly Ser Tyr Gln Glu Leu Tyr Arg Lys Leu Ala Glu Met Phe His
    370                 375                 380
Ile Glu Glu Arg Ser Asp Leu Leu Thr His Val Val Tyr Arg Asp Ala
385                 390                 395                 400
Asn Gly Val Ile Lys Arg Ile Gly Asp Glu Pro Phe Ser Asp Phe Met
                405                 410                 415
Lys Ala Thr Lys Arg Leu Pro Ile Lys Met Asp Ile Gly Gly Asp Asn
            420                 425                 430
Val Arg Lys Thr Trp Ile Thr Gly Ile Arg Thr Gly Glu Asn Gly Ile
        435                 440                 445
Asp Ala Ser Thr Lys Thr Gly Pro Leu Ser Ile Phe Ala
    450                 455                 460 
           
           
             
               323 base pairs 
               nucleic acid 
               double 
               linear 
             
             
               DNA (genomic) 
             
             
               unknown 
             
             
               CDS 
               3..323 
             
              26
AG GAC GGC AGC GCC GAG GAC GGC GTA CGG AAG GGG GAA ACC GTG AAG        47
   Asp Gly Ser Ala Glu Asp Gly Val Arg Lys Gly Glu Thr Val Lys
     1               5                  10                  15
CAG CGG TTC TCG CGG ATG CCG CAC ATG TTC TGC AAG ACG CTC ACG GCC       95
Gln Arg Phe Ser Arg Met Pro His Met Phe Cys Lys Thr Leu Thr Ala
                 20                  25                  30
TCC GAC ACC AGC ACG CAC GGG GGT TTC TCC GTG CCG CGC CGC GCC GCC      143
Ser Asp Thr Ser Thr His Gly Gly Phe Ser Val Pro Arg Arg Ala Ala
             35                  40                  45
GAG GAC TGC TTC CCG CCT CTG GAC TAC AGC CAG CAG CGA CCG TCG CAG      191
Glu Asp Cys Phe Pro Pro Leu Asp Tyr Ser Gln Gln Arg Pro Ser Gln
         50                  55                  60
GAG CTT GTG GCC AAG GAT TTG CAC GGA ACC GAG TGG AGG TTC CGC CAC      239
Glu Leu Val Ala Lys Asp Leu His Gly Thr Glu Trp Arg Phe Arg His
     65                  70                  75
ATT TAT CGA GGG CAG CCC CGC AGA CAC CTT TTA ACC ACT GGA TGG AGT      287
Ile Tyr Arg Gly Gln Pro Arg Arg His Leu Leu Thr Thr Gly Trp Ser
 80                  85                  90                  95
GCC TTT GTC AAC AAG AAG AAG CTT GTC TCA GGG GAC                      323
Ala Phe Val Asn Lys Lys Lys Leu Val Ser Gly Asp
                100                 105 
           
           
             
               107 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               unknown 
             
              27
Asp Gly Ser Ala Glu Asp Gly Val Arg Lys Gly Glu Thr Val Lys Gln
  1               5                  10                  15
Arg Phe Ser Arg Met Pro His Met Phe Cys Lys Thr Leu Thr Ala Ser
             20                  25                  30
Asp Thr Ser Thr His Gly Gly Phe Ser Val Pro Arg Arg Ala Ala Glu
         35                  40                  45
Asp Cys Phe Pro Pro Leu Asp Tyr Ser Gln Gln Arg Pro Ser Gln Glu
     50                  55                  60
Leu Val Ala Lys Asp Leu His Gly Thr Glu Trp Arg Phe Arg His Ile
 65                  70                  75                  80
Tyr Arg Gly Gln Pro Arg Arg His Leu Leu Thr Thr Gly Trp Ser Ala
                 85                  90                  95
Phe Val Asn Lys Lys Lys Leu Val Ser Gly Asp
            100                 105 
           
           
             
               7815 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               unknown 
             
              28
AAGCTTTAGT GACTAGTGAG AGTGATTTGT TGTGTTCTTT TGAGCTCTTG CGCTTGGATT     60
GCTTTCTTCT TTCTCATTCT TTCTTGAGAT CAATACTCAC TTGTAACCGA GGCAAGAGAC    120
ACCAATTGTG TGGTGGTCCT TGCGGGTAAG TTTTGTTCCC GGTTGATTTG AGAAGAGAAA    180
GCTCACTCGG TCCGAGGGAC CGTTTGAAAG AGGGAAGGGG TTGAAAAAGA CCCGGCCTTT    240
GTGGCCTCCT CAATGGGGAG TAGGTTTGCG AAAACCGAAC CTCGGTAAAA CAAATCCGCG    300
TGTCACACTT CTTATCTGCT TGCGATTTGT TTTTCACCCT CTCTCGCGGA CTCGATTATA    360
TTTCTAACGC TAACCCGACT TGTAGTTGTG ATTAACTTTG TAAATTTCAG TTTCGCCCTA    420
TTCACCCCCC TCTATGCGAC TTTCAGTAGT TCATCTATCC CATGTTTTAC CCCTATTTGC    480
TTGGATCTGA GCTGATTGCG ACTTAGAGAC TAAACTGCTG AACTTATGAA CCTGTGAATA    540
AAATACTAAG TAAACTAGTT AGTCCGAATG TTTGTGATAG TCATCAAGCA CCAAAATCAA    600
TATAAAAATG GTTTAAGGCC AATTTCCTTT CGCAAAGATA TGGAATGTCA TAACCCGTCA    660
ATCCTTCATG TAACAATGGT CGTGCGTTCC CTCAACCATA CAAAGGGACA TGGCCGCACT    720
GAAAAGGCAG ACACACATAG TTTTACATAT TTTCTACGCT AGCACAATAG CCTCGTTCTC    780
CACTCTGCAA CTCACGAAAA CAGTAACAAA AACTTCAACA ACATACTAGG CATATTTTCT    840
CTCCAAACTG GTCTAAAAAC TCTCTTCAAA CTCACTTCGA GCAAGGTAAT CGGGACATTA    900
GCACCGCAAT CCCTTTCCTA AACTCCAATC TACTTGTCAT GGGGTTGAAA TCACGATAGT    960
TAATGTGCTA GGTAAGGGGT ATGGCGCGTT GCATTTAGCT TTCGATGGGA TTCGATCGTT   1020
TTCCCATGAC GCCTTCACTC TCGAAACCAA TGTCACATTT TGAGATCTTG GACTTTGTTT   1080
CCACCAAGGG ATTGCGCCAT GCAGCTCCTC ACCCGCGTCC GGACGGTAGC ACACGAGAGG   1140
AACCATGAAG GCGCCTTCGA CATGCGGGCC TTGGATGGGT CGACGAAAAA GGTCCTAGGT   1200
TCGCGGCCTA ATGTCGCACG ACCCGATGCT TCGTACAAGG TCTATAGAAC GGTTAGGGCA   1260
TAAGGGCACC TAGTTCAAAA AACTCAAAAG GGCCCAACCG AGGGTAGGGT TGGCAGCGGG   1320
CGACGAAGCG AATATAGCCG CACGTGCCAC CACACAAAAT GAGGGCTAAT CTTCGATGTC   1380
GCACCGTCTA GGACCCATCA TGCATGTAGG ATCCATCTTC GATGTCAATC ACGATCCCTC   1440
ATGCTCTTAC GACCTCCTCG ACACGCCTTC GTGCGATTGC TAGAGGACAT TGTCGACGGA   1500
ATATCCCCTC TTTGCCCTGT GACTTGGATG ATTATACATT TATGGTAGGT GTTATGGATG   1560
TTATAAATGG ATGTATATGA ATGTGTGTGT ATCTATGTGT TGTGGATGAA TATAAATAAT   1620
TATTTTCTAA CTGGTAAGAA TCATTTCTGG TGACTAGGTT CAGTCGATAA AAATTAGTAT   1680
GTCTAATTTG TGTATTATGT CTATGAAAAT TAGTTAATTT TAGTTTATTA ATCTTCAAAA   1740
GTTACAGACC GACGAAAACT AGACTATCAG TCACAACTGG TAAGAAGGAA CAACGACAAC   1800
AGAGATGCCA AGTTACTGGC TTACTGCAGC AAGCTACCGT TTTCTGCCCG CGTGTACATT   1860
GAAGCACAGG TGCGTCTACA CTCTACGCTC TCGAGTCCAA TATAAAAATA GACTGTTGGG   1920
CACCTATTGT ACCCGTACCC CTGTTCCTGC TCCTGCCGCA GTACTGAATT CTGCTGCTGC   1980
TACACTCCTC TGTCCGCATC CATCCACGTC TCTCTCCTCT GCCGCCCGCC TGCGCCACCC   2040
ATCACTGTGC GCGTCTCCCG CATCGTCCGC TCTCTTTCTT TTTCACCCTT TCCCGGCCCA   2100
TCTTCTCTTT TTACATCTGC AACGGCAGGC CGGCTGCGGC AGCGGCAGCG GCAGCTGACC   2160
AGTGACCGAC CACCCCCACA CCACTCCGGC GCCCCAATCC TCCCCCTTCT TCTTTTTCAC   2220
TACTACTACT GTACTGCACG GTCGCCAAGC GCCAGAACGC AGTGGAGAAC GGGGGGCAGG   2280
ACTCCAACAA GCGTTGATTT CTGCCGGCAC GCACGGCACG GGCACGGGCA CGGGCACGGG   2340
CGTCCCCCCT CACTCACGCA CCCTGCGTCT TTTCCGGCTG CCGCTGCTGG CTGGCTGGCT   2400
CTGGCTCACA GCTACAGGCT ACAGTGACCG CCACGCAACC CACACTGTCT CTGTCCTCGT   2460
CTCCCTCTCC CCTCCTAGCT CTAGCTGGAT AGGTGGGCTC TGGGGAGGAG GAGGAGGGTA   2520
GCTAGGTAGT AGCTGCCTAT AGGCCTCGGC CCCCATTCAT GGCCATTACC ACGATGTGTC   2580
ACCCCACCAC ACCGCCCTCT CCGATGCTGC CTCCCTCATG ATAACCCTCT CCCTGGTGGT   2640
TGTTCTTTGC CTTGTTGCCG TGCAGCCTCC ACCCCCACCC TCCTCATTAA TCACTTGCTA   2700
GCTCCCTGCC TCCCTCCCGG CTCCCGCTCC CCCTTCTCGT GCTTCGCGCC CCCGCAGCAG   2760
CCATGGCGGG GATCGACCTC AACGACACCG TGGAGGAGGA CGAGGAGGAG GCGGAGCCCG   2820
GCAACGCCTG CTCCCAGCAG AGCCGGACCA GCTCCGCGGC CACGTTCCCG CCGCCGCCGC   2880
CGAACCAGCC GAGGCCGAGC GCCGCGGTGT GCCTCGAGCT GTGGCACGCC TGCGCCGGCC   2940
CCGTCGCGCC GCTGCCGAGG AAAGGGAGCG TCGTGGTGTA CCTCCCGCAG GGACACATCG   3000
AGCACCTCGG CGACGCCGCG GCCGCCGGCG GAGGCGCGCC GCCGCCCGTC GCCCTGCCGC   3060
CCCACGTCTT CTGCCGCGTC GTCGACGTCA CTCTCCATGT GCGCGCGCCG GTTCCTACTC   3120
AATGCGTGCG TGTGTGGATT GCCCGTGCCG GTGTGCGGCT TCCACTGACT CTGTCCCTCT   3180
TGCGCTCGTT GCAGGCGGAC GCGTCCACGG ACGAGGTGTA CGCCCAGCTC GCCCTCGTCG   3240
CCGAGAACGA GGTGCGCGCA AGCCACAGTG CTCCACCGGC ATTGGATTCG GCTTGGTTTT   3300
CTCCTTGCGT CCACAGAGAC GAGATTTGGG CTGATTTGGT GTTTCTTGTG GCGCTTGCTT   3360
CGTGCAGGAT GTCGCGAGGC GGCTGCGCGG ACGGTCGGAG GACGGCAGCG CCGAGGACGG   3420
CGACGAAGGG GAAACCGTGA AGCAGCGGTT CTCGCGGATG CCGCACATGT TCTGCAAGAC   3480
GCTCACGGCC TCCGACACCA GCACGCACGG CGGCTTCTCC GTGCCACGCC GCGCCGCCGA   3540
GGACTGCTTC CCGCCTCTGG TACGCTTGCG TTGGCTTGGA AAGCTTCCAT CTTTTGGGTG   3600
CCCGGGTGCT GCTCTCAAGT GCGATTCTGA ATCATCTGCT CTTGGGGCGT GCAGGACTAC   3660
AGCCAGCAGC GACCGTCGCA GGAGCTTGTG GCCAAGGATT TGCACGGAAC CGAGTGGAGG   3720
TTCCGCCACA TTTATCGAGG TACATGAACA AATAATGAGA TACAAGACGA GCACATCTAC   3780
CTATTTCTTT AGCAAACTTA TGTGCTTGCT CGCCCTGAAT CATTCAGTGT CAGCGAATGA   3840
TGTCAATGGC TGCACTTCAG TTGGTGATTG TTAGCGTTTT TTTACAGGAT TTGCATTACT   3900
TGTTTGGATT GAGCACTTGG GAATGCTTCA TCTTTGCTCA CTTAAGTCCA GGATTTGAAG   3960
TCATTGTTCA GTCACTCTTT TGCTATATAT GTCACCATTA TGTGATCAGA ACTACTAATG   4020
GTTATATGTT GAGAGAGATA TACAAACTAT GTCAATGTTT CCTGCTGTCT GCATTTGCAA   4080
CCTTGTGCGC TATGCTCAGC ATTTCTCATG TCATTGGTTA GTTATTGTAG TCGTACTTAA   4140
AATTTACCAT TTTGTCCATG AAAAATCATC TGATTATATG TTCAGGAGTT CTGGTCCCGT   4200
TTTAAGGAAT GTAAAAGAAC AAACATGAGA AGCTATGTCA TGTGTGGTCC TTGGTTTCTG   4260
ATGAATCTGC ATCTGAATGT GATGCAGGGC AGCCCCGCAG ACACCTTTTA ACCACTGGAT   4320
GGAGTGCCTT TGTCAACAAG AAGAAGCTTG TCTCAGGGGA CGCCGTACTA TTTTTGAGGT   4380
AGGCCACAGC TAACATTGGA GATAATTATC ACATGTTGGT GTTGGCCCTT TCTGAAGATT   4440
CCTCATAATT TTCAGGGGTG ATAATGGGGA GCTAAGACTT GGAGTGCGCC GTGCAGCTCA   4500
GCTTAAAAAT GGATCTGCTT TTCCAGCTCT TTATAACCAG TGCTTAAATC TTGGTTCACT   4560
ACCTAATGTT GCACATGCTG TGGCCACCAA AAGTGTGTTC CACATCTACT ACAACCCCAG   4620
GTGATGATGA ATATAGCGGT TTCACTTTAA TGCTTTTGCA TGTTCAATTG TTCATGTTGT   4680
TGGCACTCTT TTAGATGATG TGAACTGAAA TGTGCTATTA ACTATACTCT TTCAATTGAC   4740
GGCGATTTGA AATTGTGTCA TTTTGTGTGA TATCATTTCC TGAGTTGTTT CGAACTATGT   4800
AATTCATGAT TCTTACTGCA ATTCAACATT AAGTGATATA TAATTACTTT TTGAATTGAT   4860
ATTGTCACTT ACATTTGGAC CCTTCAATAT AATATAGTTC CACAGCTCTT TTTTTAGATA   4920
TCATGACAAG TACGCAAGTA GATCTTTGGT TCCTTATGTA TCTCATGTGC ATTTTTACCT   4980
TCTTGGACCC TGATGTGTTG CTGCAAGCCT TACCTTTTTA TCCACCAACA ATGATGGCCC   5040
TGATGGCAAT TATTGCTTTC CAAAAATCTT ACAGATTAAG CCAATCTGAA TTCATTATAC   5100
CATTTTCGAA GTTTATCAAG AGCTTCAGTC AACCATTTTC TGCTGGTTCG AGGTTCAAAG   5160
TGAAATATGA GAGTGATGAT GCTTCTGAAA GAAGGTTGGT GTGCTACAGT TCTCATCTTT   5220
TACATAGATT TATGATGGTT GACACATGAG AGTATTATGC AGATGCACAG GGATCATAGC   5280
AGGAATTGGT GATGCTGACC CCATGTGGCG TGGTTCGAAA TGGAAATGTT TGATGGTATG   5340
TTGCCTTTTA AGCTTTAATG ATTCACTTTC TGTATAACTT TTCAGGTGGT AAATTTGTGT   5400
TACATATGAA AATAATCCAT GTTAGATACA TGTTGAATAT AACATGTTTC TTTATACAGA   5460
ACACTAGGCG TGTGCATCAT GTAGCTGCCG TTGCCATCTA TTTGCACTAT TTGCTTGCTA   5520
ATAAACCAAT AAGCAATCTT GCATATCTAT CCAATAATAC AATGCACAAC AAATGTTGAA   5580
AATTGCAATT GAGAGCCTAC TATGCATCCC GTGCTCCCTG AGCTGTCTCT GTTTGATGTA   5640
CAAGTTTAAT TGTAATGACA CATTTTTTTT GCATGTAAGT AGTTCTCCTT CTCCAGAGCA   5700
CATTCTTTGA TGAGCCTCAT CTTAGAGGCA TGTTGTATCT TTATCTAAAA GAGACTGCCT   5760
TGTGCCAGCC TGGTTTCCTT GATCAGGGCT CTAAGTAAAT AAGTTCATTT CATTTTGGTT   5820
TCTTATTGCC CTGCCCCTGA GTGCACATTG TAGGGGTACA TAATACCCTC TTGACTTAGT   5880
AAGCCAGTTC TAAATTGCCG CAATCTTAAT CCTCTTGATG ACCTTACATA TTTTGTATAT   5940
AAACCAATGG TTCATTTTTG CAGGTTCGAT GGGATGACGA TGTAGATTTT CGTCAACCAA   6000
ACAGGATTTC TCCTTGGGAG ATTGAGCTGA CTAGTTCAGT TTCAGGATCT CACATGTCTG   6060
CACCAAATGC AAAGAGACTG AAACCATGTC TTCCCCATGT TAATCCAGAC TACCTAGTTC   6120
CAAGTATGCC CTGTTCTGCC CAGATGTTCG CTTAATGATT ATTTTGTTAG CTTCCGTCAT   6180
GAATAATATT TTCATTTTGA TAGATGGAAG CGGTCGTCCT GATTTTGCGG AATCTGCCCA   6240
ATTCCACAAG GTCTTGCAAG GTCAAGAATT ACTGGGTTAT AGAACTCATG ACAATGCTGC   6300
TGTTGCAACT TCTCAGCCAT GCGAAGCAAC GAACATGCAG TACATTGATG AACGAAGTTG   6360
CTCCAACGAT GCGAGTAACA TTATCCCGGG GGTTCCAAGA ATTGGTGTCA GAACACCACT   6420
CGGAAGCCCT AGGTTTTCCT ACCGTTGCTC AGGCTTTGGG GAGTCTCCAA GATTCCAAAA   6480
GGTCTTGCAA GGTCAAGAAG TATTTCATCC CTACAGAGGA ACTCTGGTCG ATGCAAGCTT   6540
GAGTAATAGT GGCTTCCATC AGCAAGATGG TTCTCATGTG CCTACTCAGG CCAGCAAGTG   6600
GCACGCACAG CTACATGGAT GTGCTTTTCG TGGCCAACAA GCACCAGCTG TTCCATCTCA   6660
ATCCTCATCC CCACCATCTG TCCTGATGTT TCAACGAGGT GATCCAAAGA TGTCCCCATT   6720
TGAATTTGGG CATTTCCACG TGAATAAGAA AGAGGATAGA CGCGCAATGT TTGTCCATGC   6780
TGGAGGCATC GGAGGAACTG AGCAAACGAC GATGCTCCAG GCTCATCATG TTTCTGGAGG   6840
AACGGGAAAC AGAGATGTGA CCGTTGAGAA ATCTCATCCC GCTGTTGCCG CTGCTTCAGA   6900
CAACAGGGAA GTTAGCAAAA ACAGTTGCAA AATATTTGGC ATATCTTTGA CCGAGAAGGT   6960
TCCAGCAATG AAAGAAAAGG GCTGTGGTGA CATCAACACC AACTATCCAT CCCCCTTCCT   7020
GTCTTTGAAG CAACAAGTGC CGAAATCGCT GGGCAACAGC TGTGCCACCG TGAGTGTCCT   7080
ACACCATGTA GCACCCTTGA TGTCTTTCTC GAGTGAAGTA ACTCTTAACT ATTATAAAAT   7140
CCTGCACGTT CATGAGCAGA GGCCTGTTGT TGCTAGGGTG ATTGACGTTT CAACAGTGGA   7200
TATGATGATC TGATGTATTG GAAAACTGTC CTGGAGGTGA AGTCATGCTA GTACCACCTC   7260
TGTCTTCATG CTAGTGACCA TGAACAGCAT CAAAGCATTT TAAGCTGACT GTTCTTAAGC   7320
ACATCGCTTA TTGTTGTTGC CTTGTGTTTT TGCAGGCTGT GTTGCGTAGT GTGGACAGTG   7380
TCGGTTTGAT GGTTCGGTAT CGTGAAGACG GGATTTGATT GAGGATCTGG CCAGATTTGT   7440
ATCCTAGTTG TAGCTGTTAG AGCACTTTGT ATGACAACCG TGAGTGCTCC GTGTTATCAG   7500
CACTAGTTGC TGCTCACAAC TTGCCTCTAT GTTCATAATC TGTATGCCAT GTCAGACCCA   7560
TTTATAGAGG GTTTGTTTGC TTGGCATAGT TCTAGACTTA AAGCATTATT ATGAGAACAA   7620
ATTTGCTCTG CACCGTATCT TTCTTACTTT CAAGTTGGCA ACGGATTAAC GGTGGAGGAG   7680
ATGATCTGAG AGGTTAGTTG TGCGACGTAT TAATGGTGTT ACATATATTA TGCTTAGGAG   7740
CATTCTGCCA GCTCATTTAT CATATACATG TCAGCACTTG ATTTGTTAAG TGTAGTTAGT   7800
AGCCTTGCAC TTTGG                                                    7815 
           
           
             
               736 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..736 
               /note= “Figure 14, sequence of Z31” 
             
              29
Met Ala Gly Ile Asp Leu Asn Asp Thr Val Glu Glu Asp Glu Glu Glu
1               5                   10                  15
Ala Glu Pro Gly Asn Ala Cys Ser Gln Gln Ser Arg Thr Ser Ser Ala
            20                  25                  30
Ala Thr Phe Pro Pro Pro Pro Pro Asn Gln Pro Arg Pro Ser Ala Ala
        35                  40                  45
Val Cys Leu Glu Leu Trp His Ala Cys Ala Gly Pro Val Ala Pro Leu
    50                  55                  60
Pro Arg Lys Gly Ser Val Val Val Tyr Leu Pro Gln Gly His Ile Glu
65                  70                  75                  80
His Leu Gly Asp Ala Ala Ala Ala Gly Gly Gly Ala Pro Pro Pro Val
                85                  90                  95
Ala Leu Pro Pro His Val Phe Cys Arg Val Val Asp Val Thr Leu His
            100                 105                 110
Ala Asp Ala Ser Thr Asp Glu Val Tyr Ala Gln Leu Ala Leu Val Ala
        115                 120                 125
Glu Asn Glu Asp Val Ala Arg Arg Leu Arg Gly Arg Ser Glu Asp Gly
    130                 135                 140
Ser Ala Glu Asp Gly Asp Glu Gly Glu Thr Val Lys Gln Arg Phe Ser
145                 150                 155                 160
Arg Met Pro His Met Phe Cys Lys Thr Leu Thr Ala Ser Asp Thr Ser
                165                 170                 175
Thr His Gly Gly Phe Ser Val Pro Arg Arg Ala Ala Glu Asp Cys Phe
            180                 185                 190
Pro Pro Leu Asp Tyr Ser Gln Gln Arg Pro Ser Gln Glu Leu Val Ala
        195                 200                 205
Lys Asp Leu His Gly Thr Glu Trp Arg Phe Arg His Ile Tyr Arg Gly
    210                 215                 220
Gln Pro Arg Arg His Leu Leu Thr Thr Gly Trp Ser Ala Phe Val Asn
225                 230                 235                 240
Lys Lys Lys Leu Val Ser Gly Asp Ala Val Leu Phe Leu Arg Gly Asp
                245                 250                 255
Asn Gly Glu Leu Arg Leu Gly Val Arg Arg Ala Ala Gln Leu Lys Asn
            260                 265                 270
Gly Ser Ala Phe Pro Ala Leu Tyr Asn Gln Cys Leu Asn Leu Gly Ser
        275                 280                 285
Leu Pro Asn Val Ala His Ala Val Ala Thr Lys Ser Val Phe His Ile
    290                 295                 300
Tyr Tyr Asn Pro Arg Leu Ser Gln Ser Glu Phe Ile Ile Pro Phe Ser
305                 310                 315                 320
Lys Phe Ile Lys Ser Phe Ser Gln Pro Phe Ser Ala Gly Ser Arg Phe
                325                 330                 335
Lys Val Lys Tyr Glu Ser Asp Asp Ala Ser Glu Arg Arg Cys Thr Gly
            340                 345                 350
Ile Ile Ala Gly Ile Gly Asp Ala Asp Pro Met Trp Arg Gly Ser Lys
        355                 360                 365
Trp Lys Cys Leu Met Val Arg Trp Asp Asp Asp Val Asp Phe Arg Gln
    370                 375                 380
Pro Asn Arg Ile Ser Pro Trp Glu Ile Glu Leu Thr Ser Ser Val Ser
385                 390                 395                 400
Gly Ser His Met Ser Ala Pro Asn Ala Lys Arg Leu Lys Pro Cys Leu
                405                 410                 415
Pro His Val Asn Pro Asp Tyr Leu Val Pro Asn Gly Ser Gly Arg Pro
            420                 425                 430
Asp Phe Ala Glu Ser Ala Gln Phe His Lys Val Leu Gln Gly Gln Glu
        435                 440                 445
Leu Leu Gly Tyr Arg Thr His Asp Asn Ala Ala Val Ala Thr Ser Gln
    450                 455                 460
Pro Cys Glu Ala Thr Asn Met Gln Tyr Ile Asp Glu Arg Ser Cys Ser
465                 470                 475                 480
Asn Asp Ala Ser Asn Ile Ile Pro Gly Val Pro Arg Ile Gly Val Arg
                485                 490                 495
Thr Pro Leu Gly Ser Pro Arg Phe Ser Tyr Arg Cys Ser Gly Phe Gly
            500                 505                 510
Glu Ser Pro Arg Phe Gln Lys Val Leu Gln Gly Gln Glu Val Phe His
        515                 520                 525
Pro Tyr Arg Gly Thr Leu Val Asp Ala Ser Leu Ser Asn Ser Gly Phe
    530                 535                 540
His Gln Gln Asp Gly Ser His Val Pro Thr Gln Ala Ser Lys Trp His
545                 550                 555                 560
Ala Gln Leu His Gly Cys Ala Phe Arg Gly Gln Gln Ala Pro Ala Val
                565                 570                 575
Pro Ser Gln Ser Ser Ser Pro Pro Ser Val Leu Met Phe Gln Arg Gly
            580                 585                 590
Asp Pro Lys Met Ser Pro Phe Glu Phe Gly His Phe His Val Asn Lys
        595                 600                 605
Lys Glu Asp Arg Arg Ala Met Phe Val His Ala Gly Gly Ile Gly Gly
    610                 615                 620
Thr Glu Gln Thr Thr Met Leu Gln Ala His His Val Ser Gly Gly Thr
625                 630                 635                 640
Gly Asn Arg Asp Val Thr Val Glu Lys Ser His Pro Ala Val Ala Ala
                645                 650                 655
Ala Ser Asp Asn Arg Glu Val Ser Lys Asn Ser Cys Lys Ile Phe Gly
            660                 665                 670
Ile Ser Leu Thr Glu Lys Val Pro Ala Met Lys Glu Lys Gly Cys Gly
        675                 680                 685
Asp Ile Asn Thr Asn Tyr Pro Ser Pro Phe Leu Ser Leu Lys Gln Gln
    690                 695                 700
Val Pro Lys Ser Leu Gly Asn Ser Cys Ala Thr Val His Glu Gln Arg
705                 710                 715                 720
Pro Val Val Ala Arg Val Ile Asp Val Ser Thr Val Asp Met Met Ile
                725                 730                 735 
           
           
             
               587 amino acids 
               amino acid 
               single 
               linear 
             
             
               peptide 
             
             
               unknown 
             
             
               Peptide 
               1..587 
               /note= “Figure 14, sequence of
               Zm41-A” 
             
              30
Asp Gly Ser Ala Glu Asp Gly Asp Glu Gly Glu Thr Val Lys Gln Arg
1               5                   10                  15
Phe Ser Arg Met Pro His Met Phe Cys Lys Thr Leu Thr Ala Ser Asp
            20                  25                  30
Thr Ser Thr His Gly Gly Phe Ser Val Pro Arg Arg Ala Ala Glu Asp
        35                  40                  45
Cys Phe Pro Pro Leu Asp Tyr Ser Gln Gln Arg Pro Ser Gln Glu Leu
    50                  55                  60
Val Ala Lys Asp Leu His Gly Thr Glu Trp Arg Phe Arg His Ile Tyr
65                  70                  75                  80
Arg Gly Gln Pro Arg Arg His Leu Leu Thr Thr Gly Trp Ser Ala Phe
                85                  90                  95
Val Asn Lys Lys Lys Leu Val Ser Gly Asp Ala Val Leu Phe Leu Arg
            100                 105                 110
Gly Asp Asn Gly Glu Leu Arg Leu Gly Val Arg Arg Ala Ala Gln Leu
        115                 120                 125
Lys Asn Gly Ser Ala Phe Pro Ala Leu Tyr Asn Gln Cys Ser Asn Leu
    130                 135                 140
Gly Ser Leu Pro Asn Val Ala His Ala Val Ala Thr Lys Ser Val Phe
145                 150                 155                 160
His Ile Tyr Tyr Asn Pro Arg Leu Ser Gln Ser Glu Phe Ile Ile Pro
                165                 170                 175
Phe Ser Lys Phe Ile Lys Ser Phe Ser Gln Pro Phe Ser Val Gly Ser
            180                 185                 190
Arg Phe Lys Val Arg Tyr Glu Ser Asp Asp Ala Ser Glu Arg Arg Cys
        195                 200                 205
Thr Gly Ile Ile Ala Gly Ile Gly Asp Ala Asp Pro Met Trp Arg Gly
    210                 215                 220
Ser Lys Trp Lys Cys Leu Met Val Arg Trp Asp Asp Asp Val Asp Phe
225                 230                 235                 240
Arg Gln Pro Asn Arg Ile Ser Pro Trp Glu Ile Glu Leu Thr Ser Ser
                245                 250                 255
Val Ser Gly Ser His Met Ser Ala Pro Asn Ala Lys Arg Leu Lys Pro
            260                 265                 270
Cys Leu Pro His Val Asn Pro Asp Tyr Leu Val Pro Asn Gly Ser Gly
        275                 280                 285
Arg Pro Asp Phe Ala Glu Ser Ala Gln Phe His Lys Val Leu Gln Gly
    290                 295                 300
Gln Glu Leu Leu Gly Tyr Arg Thr His Asp Asn Ala Ala Val Ala Thr
305                 310                 315                 320
Ser Gln Pro Cys Glu Ala Thr Asn Met Gln Tyr Ile Asp Glu Arg Ser
                325                 330                 335
Cys Ser Asn Asp Ala Ser Asn Ile Ile Pro Gly Val Pro Arg Ile Gly
            340                 345                 350
Val Arg Thr Pro Leu Gly Ser Pro Arg Phe Ser Tyr Arg Cys Ser Gly
        355                 360                 365
Phe Gly Glu Ser Pro Arg Phe Gln Lys Val Leu Gln Gly Gln Glu Ile
    370                 375                 380
Phe His Pro Tyr Arg Gly Thr Leu Val Asp Ala Ser Leu Ser Asn Thr
385                 390                 395                 400
Gly Phe His Gln Gln Asp Gly Ser His Val Pro Thr Gln Ala Ser Lys
                405                 410                 415
Trp His Ala Gln Leu His Gly Cys Ala Phe Arg Gly Pro Gln Ala Pro
            420                 425                 430
Ala Val Pro Ser Gln Ser Ser Ser Pro Pro Ser Val Leu Met Phe Gln
        435                 440                 445
Arg Gly Asp Pro Lys Met Ser Pro Phe Glu Phe Gly His Phe His Val
    450                 455                 460
Asn Lys Lys Glu Asp Arg Arg Pro Met Phe Val His Ala Gly Gly Ile
465                 470                 475                 480
Gly Gly Thr Glu Gln Thr Thr Met Leu Gln Ala His His Val Ser Gly
                485                 490                 495
Gly Thr Gly Asn Arg Asp Val Thr Val Glu Lys Ser His Pro Ala Val
            500                 505                 510
Ala Thr Ala Ser Asp Asn Arg Glu Phe Ser Lys Asn Ser Cys Lys Ile
        515                 520                 525
Phe Gly Ile Ser Leu Thr Glu Lys Val Pro Ala Met Lys Glu Lys Gly
    530                 535                 540
Cys Gly Asp Ile Asn Thr Asn Ile Asn Thr Asn Tyr Pro Lys Ser Leu
545                 550                 555                 560
Gly Asn Ser Cys Ala Thr Val His Glu Gln Arg Pro Val Val Gly Arg
                565                 570                 575
Val Ile Asp Val Ser Thr Val Asp Met Met Ile
            580                 585 
           
           
             
               2582 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               unknown 
             
              31
GAATTCAAGG GAGAAGATGA TTTATCAGCA GGCTCTATGA GCACAGCTGC AAAGTCAAGA     60
CATAATTCTT GGGCCTCTGC AGGTGATTCT CACCCCTACT CTGACATTGC TTGCCCTTCA    120
AAAATATTCA GTCAAGACAA AAAAGAACTT ACTAATCAAA TGTCATTATC AGTCAATACT    180
TTAAGATAAG TAGAATCGAT GTCCCATACG ACATTCTAGC CACGCACTTA AACATGTGCC    240
AGATATGTTC AGATCTTGTG ATTCAACAGA CCTCGACGCC GACTTTCATG TATATCTTTT    300
AGGTTGAAGC TTTTGCTTAG TTCAGTGTTG CTATCAGAAA GCTAAAATTA TTTTCTTGCC    360
ACCTCCTCTG CATTTTTTAC TGCTTCAGCT CCTGGTGCTT CTAATCGAGT ACTATAGAAA    420
GCATCTCCCT TGATAAATCG TTGTGTGCAA ATATAGGGTG CTTATATAAT CCATCATTAG    480
AGTATGAGGC GTGCTTTATT CTATGTGCTT CCCACAAAAA GAGTAGCCTA TTATAAACTT    540
TGTATTAGAG CACATGACGT TCTAAGTTTT GACCACATTT CTCTACTATT ATATTGCAGC    600
CATAAAGATT CAATTTTTAT GTTGGGCACC ATAAAGATGT TTGGCACCAT TCTTCCCAAA    660
CATTTATCTA CTATTATAAT GCATGCTTTA TTCAATTTTT AGTATTGTTA GGGGTGAAGT    720
CTTAGTCTCA AGATAGCATA TTGTTGTTTG CCTACTCCGA CGACTCTGAC GAGGCTGCTG    780
CCCCGCGCCA GGAGGGAGGT CAAGAAGCCT AAGAAGCCCA AGGTGAAGCA ACGATTCTCG    840
CGGATGCCGC ACATGTTCTG TAAGACGCTC ACGGCCTCCG ACACCAGCAC ACACGTCGGC    900
TTCTCCGTGT CGCGCAAGGA CAGAGCAAGC TATGTCATGT GAAGCTATGT CATGTGTGGT    960
CCTTGGTTTC TGATGAATAT GCATATGAAT GTGATGCAGG GCAGCCCCGC AGACACCTTT   1020
TAACCACTGG ATGGAGTGCC TTTGTCAACA AGAAGAAGCT TGTCTCAAGG GACGCCGTAC   1080
TATTTTTGAG GTAGGCCACA ACTAACATTG GAGATAATTA TCACATGTTG GTGTTGGCCC   1140
TTTCTGAAGG TTCCTCATAA TTTTCAGGGG TGATAATGGG GAGCTAAGAC TTGGAGTGCG   1200
CCGTGCAGCT CAGCTTAAAA ATGGATCTGC TTTTCCAGCT CTTTATAACC AGTGCTCAAA   1260
TCTTGGTTCA CTACCTAATG TTGCACATGC TGTGGCCACC AAAAGTGTGT TCCACATCTA   1320
CTACAACCCT AGGTGATGAT GAATATAGCG GTTTCACTTT AATGTTTTTG CATGTTCAAT   1380
TGTTCATGTG GTTGGCACTC TTTTAGATGA TGTGAATTGA AATGTGCTTA TTAACTACTC   1440
TTTCAATTGA CGGGGAATTT GAAATTGTGT CATTGTGTGT GATATCATTT CCTGAGTTGT   1500
TTCGAGCTAT GTAATTCATG ATTCTTACTG CAATTCAACA TTAAGTGATA TATAATTACT   1560
TTTTGAATTG ATATTGTCAC TTACATTTGG ACCCTTCAAT ATAAATCTTT CCAATTAATG   1620
CTCTTTTTAT CCACTCTTTG TTGTCAAGTT TCTGCAATTT AGAAGTATGC TTTCTTTTGT   1680
ATTTAATTCT TTTTAGGCCA CAGATTGTTA TTTCTTCATG CCATAATTTC TCTGTTTTAT   1740
TAGTCATAGT AACAGAAATA TTTTTCAATT GTTGTGGCGG CTGGCCTTGA CTGCTATGGC   1800
GGTGGCCGGA CTGGCCAGCG ATGGCGGTGG CCGGATAGCA CCGCGAGAGC AACGTCCAGA   1860
GGCTAGCAGT TCGTTGGTTG TTGAGATTTG TACCAATGAT TATCTATATT TAGAGTTGTT   1920
GTTGGATACA CCCATCCATT TAGTCCTTGT CTATCTTTTA CACAACCATC TAAACTATAA   1980
ATTTAGCTAG GATTATAAAT AAGCTGTTGG AGTTGCTCTT AGGTGGCTCC TCCAATATAG   2040
GATTAGTCCA TTTTTCTACA AACTTTGATG TGAATTGAGT TTCTGCCAAT CATGTTATAT   2100
ATGCATATGT GATGTGAATT GAGATTCATT GAGCAACACA AGGATTCTGT GTTGGAGATG   2160
GGGTCTTAAT ATTTCTATCA TGTAATATCT TTTGGTAGCT TGCATCATAT TAATAAAATA   2220
TCTTTGGTGG CCTCAGGTCT GGTGGTAATG CTTATGTGAT TGGTGATTCT GCAAAGCCTG   2280
AGCAGAAGTG GCACGCCTAC TATGCCACTA CTGAGCACCC CTGAGGAGCT TGTTGTTACT   2340
CTTAACATGT GCATGACTGG GCTGGACAAG AAGAGAGCTT CTGTCTTCTT CTAGGCTTCT   2400
GCTGATGGTT ACACATCTTG TGCTAAGGAG ATGACCAAGC TCTCAGGTAT CTCGGACATT   2460
ATCCTATAGA CAGAGATCTG CGACTAATTT GTTAGGTTGG TTCTTCATCA TTTTGTAGAT   2520
GCCCTTCCTT CTCGCTACAT GAACTAACTA ATGACAGAGG GTGGAAGTGA CCCATGAAGC   2580
TT                                                                  2582 
           
           
             
               5872 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               DNA (genomic) 
             
             
               unknown 
             
              32
GTCGACCTGC AGGTCAACGG ATCTATTGAA CCAGCAGTCT TTGCAATTGA GATTTGACTG     60
CCGGATTTGG TTTCAGCATG GATGCACCAC CCCACATCAT GTGGTTCTAG AGCATATAGT    120
GGTCTTGTAG CGCCTAAAAG TTTTAGTAGC ATCAAATGTC AGAAATATAT CTTCATCTCC    180
AGAAAATATT AGTACTTCAT AGGATGAAAA TTGTTCAACC TGAAATAATT TATTTCTTGC    240
ATCCTTCAGG TTGTATGCGA AACCACTAGA TTGAATAATT CAAGAAATCT ACAGAGGCAG    300
TCGTGAACAA CTATATATGC GCAAGATTGA GCCTAAGGTT TGTAGACCCT TTAATTCATA    360
CAAGGGCATT GCCATTTCCC CCGTAATTTC GATGCAGCTC CTTTAGCCAT ATAACAATGA    420
AAACCAACGA TCCTGCAATC CTGAAAGGGT GAATTTATGG GAGAAGCGTA CAACTCCTTT    480
AGCCAATGAT TCCAATGAAG CACCAGCCTA CAAGAATAAG ATAGATAAAT TAACAGGGTA    540
TAAAAATGAT ACTAATCACA TGTAGTAAAA GAAACTTAAT CCTTCCACTG CATCACGTAT    600
ATGTGAGTGC TCCCTGGTTT TTCATTACAG TCTTGTGATT TCCATTTTAT GCTCGATGTA    660
GGTATAGGCA TCTGATGGAG GACGTTTTGT CTCTACTCCC GCATGTGAAG AAGGACAACC    720
AGGACAAGGT CGAGTCCAAG CAGAGCAAGG GGAACACGCT GAACAAGTTG CTTGAGTTCA    780
GGAGCTGCTT CAGCTGCCTT TCTTCGAGGT ATAGATATTC TACTGTGCCT CCACACAGCT    840
GGTGGAAATT TTGTTATCAT AGATACGATG GCGGCTGCTT ACATGTGGGA ATCTTACACT    900
GTATAAGTCA GTGGCGCAAA TCAAATCTCC AACTTGGGTT TGGTCCACCT TTCGTGAAAT    960
GAATGTTTTC TGGGCTTTCA GGTATTGAGT AAGGAGCTCC CATTTTGCTC TGGTGCCAAA   1020
TTCTCTACTA GGCAATTGAC GTTTTTACTG CATTTGTGAC ATCTGCCTTC CCACAATTAT   1080
AATTGTTCAA TATATGTATG CATTAGACTT ATCAATTTTA TTAACTTATT GAATTGTATG   1140
TGCATGAAGT TTTTTCTTTC ATGTATTACA CCACATGACA TAGTTCTTTA ACTAATGGCA   1200
GTGTACCTTT TTTAACCTTT AGATGGCTAA ATTCAAGGGA GAAGATGATT TATTAGCAGG   1260
CTCTATGAGC ACAGCTGCAC AGTCAAGACA TAATTCTTGG GCCTCTGCAG GTGATTCTCA   1320
CCCCTACGCT GACATTGCTT GGCCTTCAAA AATATTCAGT CAAGACAAAA AGAACTTACT   1380
AATCAAATGT CATTATCAGT CAATACTTTA AGATAAGTAG AATCGATGTC CCATACGACA   1440
TTCTAGCCAC GCACTTAAAC ATGTGCCAGA TATGTTCAGA TCTTGTGATT CAGCAGACCT   1500
TGACGCCGAG CGGGCCTCCG CGGAGGCAGT AGCCAGATCT GGCCATTGAG TGCCCCGACG   1560
CCGCTGCTTA CTCATCCATC GCCGCGGTGA CCTGCTCCCC CTCGGGCATA TCTGTCCATT   1620
GACACCAAGC ATGTTCTTTC CTGAACTGTT CTAAAAGTTC AGTTTCATGG TTGTTTATTC   1680
TTTTGATCAG GAAGGAGAGA AAGGGAGAAT CAGTTAGAAG AAAGAAGAGT CTGAAAGCTG   1740
AGTAATTTAC CTCAACTTTA CTACCCATGT TATTAAGATC TATTGATGAT CGTCCCACTT   1800
ACTCCTATGA TGCACAGACT TAATGGATCA TGGACTGACA TATTTATCAC GGGTTTTGGG   1860
TTGTCTTCCT TCCCAGTTTT GTTTTACCAG TGGAGACACG AAGATTGGAG GACATAAGGG   1920
CGCAACACAG GACTACAGCG AGGGGGAAGG CCAGATCAAG CAGGAGACAA CAAGAGGTGG   1980
GTTGCTGCTC ATTCACAATT TGATATGTTT GTTTTTTCGT TGTTATAGCT GAACTGCACA   2040
TGCAGTTTGA AACATGTTGT TACTGATGTG TTTGTCTATT ACAGGATGTG ATAGATGGTG   2100
ATCTCTGTGA GCAGTATCCC TCCCTCCTAG CTGATATGCA GAGGAAGATT GCTGATGAGC   2160
TGGACAGAAG TCCGACGCCT GCAGCACTGC TTGGTGAGGA TTGCCAAGGA GGAAGACTAG   2220
AACAAGCAAG AGCAGCGTTA ATCAGTGACA GAGCATGATG CCATCCAGAT GGGACAAGAT   2280
AAGTAAGCAG TCTTATATAG TCTGCCCACT CGAGTTTTGT ATATATTTTA GGTTGAAGCT   2340
TTTGCTTAGT TCAGTGTTGC TATCGGAAAG CTAAAATTAT TTTCTTGCCA CCTCCTCTGC   2400
ATTGTTTTGC TGCTTCAGCT CCTGGTGCTT CTAATCGAGT ACTATAGAAA GCATCTCTCT   2460
TGATAAATCG TTGTGTGCAA ATATAGGGTG CTTATATAAT CCATCATTAG AGTATGAGGC   2520
GTGTTTTATT CTGTGTGCTT CCCACAAAAA AGAGTAGCCT ATTATAAACT TTGTATTAGA   2580
GCACATGACG TTCTAAGTTT TGACCACATT TCTCTACTAT TATAATGCAG CCATAAAGAT   2640
TCAATTTTTA TGTTGGGCAC CATAAAGATG TTTGGCACCA TTCTTCCCAA ACATTTATCT   2700
ACTATTATAA TGTGTGCTTT ATTCAATTTT TAGTATTGTT AGGGGTGAAG TCTTAGTCTC   2760
AAGATAGCAT ATTGTTGTTT GCCTACTCCG ACGACTCTGA CGAGGCTGCT GCCCCGCGCC   2820
AGGAGGGAGG TCAAGAAGCC TAAGAAGCCC AAGGTGAAGA AGCCCAAGGT GAAGCAACGA   2880
TTCTCGTGGA TGCCGCACAT GTTCTGCAAG ACGCTCATGG CCTCCGACAC CAGCATGCAC   2940
GTCGGCTTCT CTGTGCTGNG CCGCTCCGCC GAGGACTGCT TCCCGCCTCT AGTACGCTTG   3000
CGTTGGNTTG GAAAGCTTCC ATCTTTTCGG TGCCCGGGTG CTGCTCTCAA GGTGTGATTC   3060
TGAATCATCT GCTCTTGGGG CGTGCAGGAC TACAGCCAGC AGCGATCGTC GCAGGAGCTT   3120
GTGGCCAAGG ATTTGCACGG AACCGAGTGG AGGTTCCGCC ACATTTATCG AGGTACATGA   3180
ACAAATACTG AGATACAAGC CGAGCACATC TACCTATTTC TTTAGCAAAC TTATGTGCTT   3240
GCTCGCCCTG AATCATTCAG TGTCAGCGAA TGATGTCAAT GGCTGCACTT CAGTTGATGA   3300
CTGTTAGCGC TTTTTACAGG ATTTGCATTA CTTGTTTGGA TTGAGCACTT AGGAATGCTT   3360
CATCTTTGCT CACTTAAGTC CAGGATTTGA AGTCATTGTT CAGCCACTCT TTTGCTATAT   3420
ATGTCACCAT TATGTGATCA GAACTAATAA TGGTTATATG TCGAGAGAGA TATACAAACT   3480
ATGTCAATGT TTCCTGTTGT CTGCATTTGC AGCCTTGTGC GCTATGCTCA GCATTTCTCA   3540
TGTCATTGGT TAGTTATTGT AGTTGTACTT AAAAATTACC ATTTTGTCCA TGAAAAATCA   3600
TCTGATTATA TGTTCAGGAG TTCTGGTCCC GTTTAAAGGA ATGTAAAAGA ACAAACATGA   3660
GAAGCTATGT CATGTGTGGT CCTTGGTTTC TGATGAATAT GCATCTGAAT GTGATGCAGG   3720
GCAGCCCCAC AGACACCTTT TAACCACTGG ATGGAGTGCC TTTGTCAACA AGAAGCTTGT   3780
CTCAAGGGAC GCCGTACTAT TTTTGAGGTA GGCCACAACT AACATTGGAG ATAATTATCA   3840
CATGTTGGTG TTGGCCCTTT CTGAAGGTTC CTCGTAATTT TCAGGGGTGA TAATGGGGAG   3900
CTAAGACTTG GAGTGCGCCG TGCAGCTCAG CTTAAAAATG GATCTGCTTT TCCAGCTCTT   3960
TATAACCAGT GCTCAAATCT TGGTTCACTA CCTAATGTTG CACATGCTGT GGCCACCAAA   4020
AGTGTGTTCC ACATCTACTA CAACCCCAGG TGATGATGAA TATAGCGGTT TCACTTTAAT   4080
GCTTTTGCAT GTTCAATTGT TCATGTTGTT GGCACTCTTT TAGATGATGT GAACTGAAAT   4140
GTGCTTATTA ACTACTCTTT CAATTGACGG GGATTTGAAA TTGTGTCATT GTGTGTGATA   4200
TCATTTCCTG AGTTGTTTCG AGCTATGTAA TTCATGATTC TTACTGCAAT TCAACATTAA   4260
GTGATATATA ATTACTTTTT GAATTGATAT TGTCACTTAC ATTTGGACCC TTCAATATAA   4320
ATCTTTCCAA TTATTGCTCT TTTTATCCAC TCTTTGTTGT CAAGTTTCTG CAATTTAGAA   4380
GTATGCTTTC TTTTGTATTT AATTCTTTTT AGGCCACAAA TTGTTATTTC TTCATGCCAT   4440
AATTTCTCTG TTTTATTAGT CATAGTAACA GAAATATTTT TCAATTGTTG TGGCGGCTAG   4500
CCTTGACTGC TATGGCGGTG GCCGGACTGG CCTGAGATGG CGGTGGCCGG ATAGCACCGC   4560
GAGAGCAACG TCCAGAGGCT AGCAGTTCAT TGGTTGTTGA GATTTGTACC AATGATTATC   4620
TATATTTAGA GTTGTTGTTG GATACACCCA TCCATTTAGT CCTTGTTTAT CTTTTACACA   4680
GCCATCTAAA CTCTAAATTT AGCTAGGATT ATAAATAAGC TGTTGGATGC TCTTAGGTGG   4740
CTCCTCCAAT ATAGGATTAG TCCATTTTTC TACAGATGGG GTGATAGCAT GCACATTCTA   4800
GCATACACAT GCCCTTGGCC TGGTAATGCT TGGATTTTTT TCTCACGCAA AAGAATATAC   4860
CGGTTCGTTG AATTATGTGA TGTCATTTTC TACTTTTCTG TTTTTTAGCC GATCATCCGA   4920
AGGCTAATGA ATATTACCCT GACCCAAGAT TAGTAGCATA TGTTGTACCC TATGCACCTA   4980
TCCTATCGTG GTATCACTAA TCCTTCTAAA TTTGATATCA TCTTATCTGA TTCAGCTTGT   5040
TACTTGATTT AATTTGGCTC CTTGTTAACA GTACGGATGC TGCAAAAAAT TCCCTGAGGA   5100
GAAAGGTTGA AATCTTAAAA TTGAAGCCTC ATTGGTCCAA AGCTTACTTC TATTTGTGGG   5160
ATGAGGTGCG TTATTTTACC TTTTCTGCTA TGTCCTGATT TCAGGGGACA CCAGTGCAGA   5220
TGCATGTAGG GAGAAACTTG TTGCAGTTAC AGAAATGGTT TCCAATATCT ACTCTTGCAA   5280
TTGAAGATAT GGAGTTACTC CTTGGGTTCT CCTTTTAGTT TTATTATGCT CGTCCAGTAG   5340
ACATGCTCCT GTAGTAAACT TATATTCATG CTTGTAATTC CATTTACAAT GTGAATATTG   5400
TGTATAGTAG CCATGACATG ATAATAGATT GTTAGGGTCA CTCATCAAAT ATTACTATGT   5460
GCCGTCACAA ATATGGGCAC TCCACTAGGG TTTAGGGTTT TACCTGTTGT GCCCAGTTAG   5520
GGTCACTCAT CAAATATTAC AGAGGGTATG TTCCATTTAC AGTTGGAGTA GATACGCATG   5580
ACGGGGGCGC ACATGAGTTA TTAGTCTTGT CGGGATCTCA TGAGTCTGAT TGACGTATTT   5640
CGGATGGCTC TCGACGTGCG GGTCGACGAC GGAACACTTG CAGCGCCCAT GTTCGGATGC   5700
AGCGACAGCC TCCTTGTGTC TTCGAACTCG CGACGAGAGA GAGTGGTATT CAGGACTGCT   5760
TGCTTACAGG AGAGAAATAA GCTAATTTCT CAGAATCTTA GAAGCTGATT TTACAACAGG   5820
ATTGCTTGCT TACAGAGTTG ATCAACTAAA AAAGCGCTAT GGTTCAGAAT TC           5872

Technology Classification (CPC): 2