Patent Application: US-10252898-A

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 .

Description:
isolation of a gene required for male fertility in arabidopsis thaliana 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 : 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 : ac12 ( 5 ′ agatgctgctacccaatcttttgtgc 3 ′) ( seq id no : 2 ). the results were as follows 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 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 ( fig1 ) 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 . 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 ) ( fig2 ). 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 fig3 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 fig3 ) 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 fig3 ); 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 ( fig4 ). 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 fig5 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 fig3 ). the genotypes of 7 revertant plants or sectors were determined and are presented in fig5 . 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 . 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 ). 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 ). 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 ( fig7 ). 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 . 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 . 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 . 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 ( fig7 ). 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 . 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 , fig3 ) and 3 ′ 2 , 5 ′ aatgtactactactactacttaggac 3 ′ ( seq id no : 10 ) ( positions 3001 - 2976 , fig3 ). 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 ( fig7 ). 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 ( fig8 ). 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 . 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 ( fig8 ). 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 ( fig8 ). 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 . 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 fig9 . 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 ). 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 ). 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 . 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 fig1 . 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 ( fig1 ). this indicates that this cdna is a good candidate for the maize orthologue of the arabidopsis ms41a gene . 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 ( fig1 ) is likely to encode a functional orthologue of ms41 - a . 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 ). class a comprising 5 clones ( z9 , z23 , z27 , z35 and z36 ); 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 fig1 ). 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 fig1 . 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 fig1 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 fig1 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 fig1 ). 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 . 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 . met ser pro pro ser ala thr ala gly asp ile asn his arg glu val asp pro thr ile trp arg ala cys ala gly ala ser val gln ile pro val leu his ser arg val tyr tyr phe pro gln gly his val glu his cys ile ile thr ser ile gln leu leu ala asp pro val thr asp glu asn asn lys val thr thr phe ala lys ile leu thr pro ser asp ala val thr asp ile his gly ala val trp asp phe arg his ile tyr arg gly thr pro arg arg his leu leu thr thr gly trp ser lys 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