Patent Application: US-22919302-A

Abstract:
the present invention relates generally to isolated genes which encode polypeptides involved in cellulose biosynthesis in plants and transgenic plants expressing same in sense or antisense orientation , or as ribozymes , co - suppression or gene - targeting molecules . more particularly , the present invention is directed to a nucleic acid molecule isolated from arabidopsis thaliana , oryza sativa , wheat , barley , maize , brassica spp ., gossypium hirsutum and eucalyptus spp . which encode or an enzyme which is important in cellulose biosynthesis , in particular the cellulose synthase enzyme and homologues , analogues and derivatives thereof and uses of same in the production of transgenic plants expressing altered cellulose biosynthetic properties .

Description:
the derived amino acid sequences encoded by the cdnas listed in table 8 , is provided in fig8 and 9 and seq id nos : 8 and 10 herein . fig1 a schematic representation of the important features of the rsw1 polypeptide which are conserved within a . thaliana and between a . thaliana and other plant species . in addition to the species indicated in fig1 , the present inventors have also identified maize , wheat , and barley and brassica spp . cellulose biosynthetic genes by homology search . accordingly , the present invention extends to cellulose genes and cellulose biosynthetic polypeptides as hereinbefore defined , derived from any plant species , including a . thaliana , cotton , rice , wheat , barley , maize , eucalyptus spp ., brassica spp . pinus spp ., populus spp ., picea spp ., hemp , jute and flax , amongst others . arabidopsis thaliana double - stranded cdna and cdna libraries were prepared using the capfinder cdna kit ( clontech ). rna was isolated from arabidopsis thaliana columbia rsw1 mutant plants grown in sterile conditions for 21 days . the full - length rsw1 mutant nucleotide sequence was generated by sequencing two amplified dna fragments spanning the rsw1 mutant gene . the 5 ′- end sequence of the cdna ( comprising the 5 ′- untranslated region and exons 1 – 11 ) was amplified using the primer combination 2280 - f / csp1 - r ( example 7 ). the 3 ′- end sequence was amplified using the primers est1 - f and cs3 - r set forth below : 1 . primer est1 - f : 5 ′ aatgcttcttgttgccaaagca 3 ′ ( see seq id no : 5 ) 2 . primer cs3 - r : 5 ′ gacatggaatcaccttaactgcc 3 ′ ( see seq id no : 5 ) wherein primer est1 - f corresponds to nucleotide positions 1399 – 1420 of seq id no : 5 ( within exon 8 ) and primer cs3 - r is complementary to nucleotides 3335 – 3359 of seq id no : 5 ( within the 3 ′- untranslated region of the wild - type transcript ). the full - length sequence of the mutant rsw1 transcript is set forth herein as seq id no : 11 . whilst not being bound by any theory or mode of action , a single nucleotide substitution in the rsw1 mutant nucleotide sequence ( nucleotide position 1716 in seq id no : 11 ), relative to the wild - type rsw1 nucleotide sequence ( nucleotide position 1646 in seq id no : 5 ), resulting in ala549 being substituted with val549 in the mutant polypeptide , may contribute to the altered activity of the rsw1 polypeptide at non - permissive temperatures such as 31 ° c . additional amino acid substitutions are also contemplated by the present invention , to alter the activity of the rsw1 polypeptide , or to make the polypeptide temperature - sensitive . one example of transgenic plants in which cellulose production is inhibited is provided by the expression of an antisense genetic construct therein . antisense technology is used to target expression of a cellulose gene ( s ) to reduce the amount of cellulose produced by transgenic plants . by way of exemplification , an antisense plant transformation construct has been engineered to contain the t20782 cdna insert ( or a part thereof ) in the antisense orientation and in operable connection with the camv 35s promoter present in the binary plasmid prd410 ( datla et al . 1992 ). more particularly , the t20782 cdna clone , which comprises the 3 ′- end of the wild - type rsw1 gene , was digested with xbai and kpni and cloned into the kanamycin - resistant derivative of pgem3zf (−), designated as plasmid , pjkkmf (−). the rsw1 sequence was sub - cloned , in the antisense orientation , into the binary vector prd410 as a xbai / saci fragment , thereby replacing the β - glucuronidase ( gus or uida ) gene . this allows the rsw1 sequence to be transcribed in the antisense orientation under the control of the camv 35s promoter . the antisense rsw1 binary plasmid vector was transferred to agrobacterium tumefaciens strain agl1 , by triparental mating and selection on rifampicin and kanamycin , as described by lazo et al . ( 1991 ). the presence of the rsw1 insert in transformed a . tumefaciens cells was confirmed by southern hybridization analysis ( southern , 1975 ). the construct was shown to be free of deletion or rearrangements prior to transformation of plant tissues , by back - transformation into escherichia coli strain jm 101 and restriction digestion analysis . eight pots , each containing approximately 16 a . thaliana ecotype columbia plants , were grown under standard conditions . plant tissue was transformed with the antisense rsw1 binary plasmid by vacuum infiltration as described by bechtold et al . ( 1993 ). infiltration media contained 2 . 5 % ( w / v ) sucrose and plants were infiltrated for 2 min until a vacuum of approximately 400 mm hg was obtained . the vacuum connection was shut off and plants allowed to sit under vacuum for 5 min . approximately 34 , 000 t1 seed was screened on ms plates containing 50 μg / ml kanamycin , to select for plants containing the antisense rsw1 construct . of the t1 seed sown , 135 kanamycin - resistant seedlings were identified , of which 91 were transferred into soil and grown at 21 ° c . under a long - day photoperiod ( 16 hr light ; 8 hr dark ). of the 91 transgenic lines , 19 lines were chosen for further analysis which had anther filaments in each flower which were too short to deposit pollen upon the stigma and , as a consequence , required hand - pollination to obtain t2 seed therefrom . t2 seed from 14 of these 19 lines was plated out onto vertical hoaglands plates containing kanamycin to determine segregation ratios . between five and ten seed were plated per transgenic line . control seeds , including a . thaliana columbia containing the binary vector pbin19 ( bevan , 1984 ) and segregating 3 : 1 for kanamycin resistance , and the rsw1 mutant transformed with the nptii gene , also segregating 3 : 1 for kanamycin resistance , were grown under the same conditions . kanamycin - resistant plants were transferred to soil and grown at 21 ° c . under long days , until flowering . untransformed arabidopsis thaliana columbia plants were also grown under similar conditions , in the absence of kanamycin . a comparison of the morphology of antisense rsw1 plants grown at 21 ° c ., to mutant rsw1 plants grown at the non - permissive temperature ( i . e . 31 ° c .) has identified a number of common phenotypes . for example , the antisense plants exhibit reduced fertility , inflorescence shortening and have short anthers , compared to wild - type plants , when grown at 21 ° c . these phenotypes are also observed in mutant rsw1 plants grown at 31 ° c . these results suggest that the antisense construct in the transgenic plants may be targeting the expression of the wild - type rsw1 gene at 21 ° c . fig7 shows the reduced inflorescence ( bolt ) height in antisense 35s - rsw1 plants compared to wild - type a . thaliana columbia plants grown under identical conditions . t3 plants which are homozygous for the 35s - rsw1 antisense construct are generated and the content of cellulose therein is determined as described in example 1 . plants expressing the antisense construct are shown to have significantly less cellulose in their cell walls , compared to wild - type plants . additionally , the levels of non - crystalline β - 1 , 4 - glucan and starch are elevated in the cells of antisense plants , compared to otherwise isogenic plant lines which have not been transformed with the antisense genetic construct . total rna was extracted from 0 . 2 g of leaf tissue derived from 33 kanamycin - resistant t1 plants containing the antisense 35s - rsw1 genetic construct , essentially according to longemann et al . ( 1986 ). total rna ( 25 μg ) was separated on a 2 . 2m formaldehyde / agarose gel , blotted onto nylon filters and hybridized to a riboprobe comprising the sense strand sequence of the cdna clone t20782 . to produce the riboprobe , t7 rna polymerase was used to transcribe sense rna from a linearised plasmid template containing t20782 , in the presence of [ α - 32 p ] utp . hybridizations and subsequent washes were performed as described by dolferus et al . ( 1994 ). hybridized membranes were exposed to phosphor screens ( molecular dynamics , usa ). the levels of expression of the rsw1 antisense transcript were determined and compared to the level of fertility observed for the plant lines . as shown in table 9 , the level of antisense gene expression is correlated with the reduced fertility phenotype of the antisense plants . in 13 lines , a very high or high level of expression of the 35s - rsw1 antisense gene was observed and , in 11 of these lines fertility was reduced . only lines 2w and 3e which expressed high to very high levels of antisense mrna , appeared to be fully fertile . in 12 lines which expressed medium levels of antisense mrna , approximately one - half were fertile and one - half appeared to exhibit reduced fertility . in contrast , in 8 plant lines in which only a low or very low level of expression of the antisense 35s - rsw1 genetic construct was observed , a wild - type ( i . e . fertile ) phenotype was observed for all but one transgenic line , line 2r . data presented in table 9 and fig7 indicate that the phenotype of the cellulose - deficient mutant rsw1 may be reproduced by expressing antisense rsw1 genetic constructs in transgenic plants . to confirm reduced cellulose synthesis and / or deposition in transgenic plants expressing the antisense rsw1 gene , the level of cellulose is measured by the 14 c incorporation assay or as acetic / nitric acid insoluble material as described in example 1 and compared to cellulose production in otherwise isogenic wild - type plants . cellulose production in the transgenic plants is shown to be significantly reduced compared to wild - type plants . the severity of phenotype of the transgenic plants thus produced varies considerably , depending to some extent upon the level of inhibition of cellulose biosynthesis . to identify rsw1 related nucleotide sequences in rice , a genetic sequence database was searched for nucleotide sequences which were closely - related to one or more of the arabidopsis thaliana rsw1 nucleotide sequences described in the preceding examples . rice est s0542 ( maff dna bank , japan ) was identified , for which only a partial nucleotide sequences was available . additionally , before the instant invention , there was no probable function attached to the rice est s0542 sequence . the present inventors have obtained the complete nucleotide sequence of clone s0542 and derived the amino acid sequence encoded therefor . the s0542 cdna is only 1741 bp in length and appears to be a partial cdna clone because , although it comprises 100 bp of 5 ′- untranslated sequence and contains the atg start codon , it is truncated at 3 ′- end and , as a consequence encodes only the first 547 amino acid residues of the rice rsw1 or rsw1 - like polypeptide . based upon the length of the corresponding arabidopsis thaliana rsw1 polypeptide ( 1081 amino acids ), the rice rsw1 sequence set forth in seq id no : 14 appears to contain approximately one - half of the complete amino acid sequence . the n - terminal half of the rice rsw1 amino acid sequence is approximately 70 % identical to the arabidopsis thaliana rsw1 polypeptide set forth in seq id no : 6 , with higher homology ( approximately 90 %) occurring between amino acid residues 271 – 547 of the rice sequence . these data strongly suggest that s0542 is the rice homologue of the a . thaliana rsw1 gene . alignments of rice , a . thaliana and cotton rsw1 amino acid sequences are presented in fig9 and 10 . to isolate full - length cdna clones and genomic clone equivalents of s0542 ( this study and maff dna bank , japan ) or d48636 ( pear et al ., 1996 ), cdna and genomic clone libraries are produced using rice mrna and genomic dna respectively , and screened by hybridisation using the s0542 or d48636 cdnas as a probe , essentially as described herein . positive - hybridising plaques are identified and plaque - purified , during further rounds of screening by hybridisation , to single plaques . the rice clones are sequenced as described in the preceding examples to determine the complete nucleotide sequences of the rice rsw1 genes and derived amino acid sequences therefor . those skilled in the art will be aware that such gene sequences are useful for the production of transgenic plants , in particular transgenic cereal plants having altered cellulose content and / or quality , using standard techniques . the present invention extends to all such genetic sequences and applications therefor . a 32 p - labeled rsw1 pcr fragment was used to screen approximately 200 , 000 cdna clones in a cotton fibre cdna library . the rsw1 pcr probe was initially amplified from arabidopsis thaliana wild type cdna using the primers 2280 - f and csp1 - r described in the preceding examples , and then re - amplified using the primer combination 2370 - f / csp1 - r , also described in the preceding examples . hybridisations were carried out under low stringency conditions at 55 ° c . six putative positive - hybridising plaques were identified in the first screening round . using two further rounds of screening by hybridisation , four of these plaques were purified to single plaques . three plaques hybridise very strongly to the rsw1 probe while the fourth plaque hybridises less intensely . we conclude that the positive - hybridising plaques which have been purified are strong candidates for comprising cotton rsw1 gene sequences or rsw1 - like gene sequences . furthermore , the cotton cdnas may encode the catalytic subunit of cellulose synthase , because the subunit protein architecture of cellulose synthase appears to be highly conserved among plants as highlighted in the preceding example . furthermore , a southern blot of cotton genomic dna digested with bglii was hybridised with the 5 ′ end of the rsw1 cdna , under low stringency hybridisation conditions at 55 ° c . results are presented in fig1 . these data demonstrate that rsw1 - related sequences exist in the cotton genome . the cotton cdna clones described herein are sequenced as described in the preceding examples and used to produce transgenic cotton plants having altered fibre characteristics . the cdnas are also used to genetically alter the cellulose content and / or quality of other plants , using standard techniques . putative eucalyptus spp . cellulose synthase catalytic subunit gene fragments were obtained by amplification using pcr . dna primers were designed to conserved amino acid residues found in the arabidopsis thaliana rsw1 and 12c4 amino acid sequences . three primers were used for pcr . the primers are listed below : pcsf - i 5 ′- a a / g a a g a t i g a c / t t a c / t c / t t i a a a / g g a c / t a a - 3 ′( seq id no : 34 ) pcsr - ii 5 ′- a t i g t i g g i g t i c g / t a / g t t c / t t g a / t / g / c c t / g a / t / c / g c c - 3 ′ ( seq id no : 35 ) pcsf - 115 ′- g c i a t g a a a / g a / c g i g a i t a c / t g a a / g g a - 3 ′( seq id no : 36 ) using standard pcr conditions ( 50 ° c . annealing temperature ) and solutions , the primer sets pcsf - i / pcsr - ii and pcsf - ii / pcsr - ii were used to amplify genetic sequences from pooled eucalyptus spp . cdna . in the first reaction primers pcsf - i and pcsr - ii were used to generate a fragment approximately 700 bp in length . in the second pcr reaction , which used primers pcsf - ii and pcsr - ii , a fragment estimated to 700 bp was obtained . the sizes of the pcr fragments are within the size range estimated for the corresponding arabidopsis thaliana sequences . we conclude that the amplified eucalyptus spp . pcr fragments are likely to be related to the arabidopsis thaliana rsw1 gene and may encode at least a part of the eucalyptus spp . cellulose synthase catalytic subunit . the eucalyptus spp . pcr clones described herein are sequenced as described in the preceding examples and used to isolate the corresponding full - length eucalyptus spp . cdnas and genomic gene equivalents . those skilled in the art will be aware that such gene sequences are useful for the production of transgenic plants , in particular transgenic eucalyptus spp . plants having altered cellulose content and / or quality , using standard techniques . the present invention extends to all such genetic sequences and applications therefor . the properties of plant cell walls depend on the carbohydrates , proteins and other polymers of which they are composed and the complex ways in which they interact . increasing the quantities of non - crystalline β - 1 , 4 - glucan in cell walls affects those wall properties which influence mechanical , nutritional and many other qualities as well as having secondary consequences resulting from the diversion of carbon into non - crystalline glucan at the expense of other uses . to illustrate one of these effects , we investigated the ability of the non - crystalline glucan to hydrogen bond to other wall components particularly cellulose in the way that has been shown to be important for wall mechanics . hemicelluloses such as xyloglucans cross - link cellulose microfibrils by hydrogen bonding to the microfibril surface ( levy et al , 1991 ). since the β - 1 , 4 - glucan backbone of xyloglucan is thought to be responsible for hydrogen bonding ( with the xylose , galactose and fucose substitutions limiting the capacity to form further hydrogen bonds ) we can expect the non - crystalline β - 1 , 4 - glucan also to have a capacity to hydrogen bond and cross link cellulose . the effectiveness of strong alkalis in extracting xyloglucans is thought to relate to their disruption of the hydrogen bonds with cellulose ( hayashi and maclachlan , 1984 ). to demonstrate that the non - crystalline β - 1 , 4 - glucan forms similar associations with the cellulose microfibrils , we examined whether the 4 m koh fraction , extracted from shoots of the rsw1 mutant and from wild type rsw1 plants , contained non - crystalline glucan in addition to xyloglucan . the non - crystalline glucan was separated from xyloglucan in the 4 m koh extract by dialysing the neutralised extract against distilled water and centrifuging at 14000 g for 1 hour . the pellet was shown to be a pure β - 1 , 4 - glucan by using the methods for monosaccharide analysis , methylation analysis and enzyme digestion used to characterise the glucan in the ammonium oxalate fraction ( see example 1 ). table 10 shows the presence of substantial quantities of glucan recovered in pure form in the pellet from 4 m koh fractions extracted from the overproducing rsw1 mutant of arabidopsis thaliana . these data also demonstrate the presence of smaller quantities of non - crystalline β - 1 , 4 - glucan in the 4 m koh fraction from wild type plants , compared to rsw1 , particularly when grown at 31 ° c . the monosaccharide composition of the supernatant remaining after centrifugation was determined after tfa hydrolysis . these data , and data from methylation analysis , are consistent with the supernatant being a relatively pure xyloglucan . the supernatant was free of glucan , because no glucose could be released by the endocellulase /- glucosidase mixture that released glucose from β - 1 , 4 - glucan . the presence of both non - crystalline β - 1 , 4 - glucan and xyloglucan in the 4 m koh fraction , when taken together with the implications from structural predictions ( levy et al . 1991 ), is consistent with some of the non - crystalline β - 1 , 4 - glucan in the wall hydrogen bonding to cellulose microfibrils in similar fashion to the β - 1 , 4 - glucan backbone of xyloglucan . the cross linking provided when xyloglucans and other hemicelluloses bind to two or more microfibrils is an important determinant of the mechanical properties of cellulosic walls ( hayashi , 1989 ). the effects of increasing the amounts of non - crystalline β - 1 , 4 - glucan in walls are likely to be greatest in walls which otherwise possess relatively low levels of cross linking as a result of high ratios of cellulose : hemicelluloses . such conditions are common in secondary walls including those of various fibres , and the cellulose : hemicellulose ratio is particularly high in cotton fibres . the effects on wall mechanical properties of overproducing non - crystalline glucan are shown by transforming plants with the mutant allele of rsw1 ( seq id no : 11 ) operably under the control of either the rsw1 promoter derived from seq id no : 3 or seq id no : 4 or alternatively , an appropriate constitutive promoter such as the camv 35s promoter . production of non - crystalline glucan is quantified by fractionating the cell walls using the methods described above to show in particular that non - crystalline glucan is recovered in the 4 m koh fraction . mechanical properties of the cell walls are measured using standard methods for fibre analysis to study parameters such as stress - strain curves , and breaking strain , amongst other properties . three strategies are employed to over - express cellulose synthase in arabidopsis thaliana plants . in the first strategy , the camv 35s promoter sequence is operably connected to the full - length cellulose synthase cdna which is obtainable by primer extension of seq id no : 1 . this is achievable by cloning the full - length cdna encoding cellulose synthase , in the sense orientation , between the camv 35s promoter or other suitable promoter operable in plants and the nopaline synthase terminator sequences of the binary plasmid pbi121 . in the second strategy , the coding part of the genomic gene is cloned , in the sense orientation , between the camv 35s promoter and the nopaline synthase terminator sequences of the binary plasmid pbi121 . in the third strategy , the 23h12 binary cosmid clone or the derivative prsw1 , containing the cellulose synthase gene sequence operably under the control of the cellulose synthase gene promoter and terminator sequences is prepared in a form suitable for transformation of plant tissue . for agrobacterium - mediated tissue transformation , binary plasmid constructs discussed supra are transformed into agrobacterium tumefaciens strain agl 1 or other suitable strain . the recombinant dna constructs are then introduced into wild type arabidopsis thaliana plants ( columbia ecotype ), as described in the preceding examples . alternatively , plant tissue is directly transformed using the vacuum infiltration method described by beshtold et al . ( 1993 ). the transgenic plants thus produced exhibit a range of phenotypes , partly because of position effects and variable levels of expression of the cellulose synthase transgene . cellulose content in the transgenic plants and isogenic untransformed control plants is determined by the 14 c incorporation assay or as acetic / nitric acid insoluble material as described in example 1 . in general , the level of cellulose deposition and rates of cellulose biosynthesis in the transgenic plants are significantly greater than for untransformed control plants . furthermore , in some cases , co - supression leads to mimicry of the rsw1 mutant phenotype . the nucleotide sequence of the rsw1 gene contained in 23h12 is mutated using site - directed mutagenesis , at several positions to alter its catalytic activity or substrate affinity or glucan properties . in one example , the rsw1 gene is mutated to comprise one or more mutations present in the mutant rsw1 allele . the mutated genetic sequences are cloned into binary plasmid described in the preceding examples , in place of the wild - type sequences . plant tissue obtained from both wild - type arabidopsis thaliana ( columbia ) plants and a . thaliana rsw1 plants is transformed as described herein and whole plants are regenerated . control transformations are performed using the wild - type cellulose synthase gene sequence . plants transformed with genetic constructs described in example 15 ( and elsewhere ) are categorised initially on the basis of number of transgene copies , to eliminate variability arising therefrom . plants expressing single copies of different transgenes are analysed further for cell wall components , including cellulose , non - crystalline β - 1 , 4 - glucan polymer , starch and carbohydrate content . cellulose content in the transgenic plants is determined by the 14 c incorporation assay as described in example 1 . cell walls are prepared , fractionated and the monosaccharide composition of individual fractions determined as in example 1 . transgenic plants expressing the rsw1 mutant allele exhibit a higher level of non - crystalline , and therefore extractable , β - 1 , 4 - glucan in cell walls compared to plants expressing an additional copy of the wild - type rsw1 allele . thus , it is possible to change the crystallinity of the p - 1 , 4 - glucan chains present in the cell wall by mutation of the wild - type rsw1 allele . transgenic plants are also analysed to determine the effect of mutagenesis of the rsw1 gene on the level of starch deposited in their roots . the quantity of starch present in material prepared from the crude wall fraction is determined using the anthrone / h 2 so 4 method described in example 1 . the data show that mutating the rsw1 gene to the mutant rsw1 allele increases starch deposition . this demonstrates that the gene can be used to alter the partitioning of carbon into carbohydrates other than cellulose . the cell wall composition of transgenic plant material is also analysed . wild type and rsw1 and transgenic seedlings are grown for 2 d at 21 ° c . and then kept for a further 5 d at either 21 ° c . or 31 ° c . with transfer to 31 ° c . when the seed has scarcely germinated , the wall composition at final harvest largely reflects the operation of the mutated rsw1 gene product at its restrictive temperature . cell wall fractionation is carried out in similar fashion to that described for the 14 c - experiment ( example 1 ) and the monosaccharide composition of each fraction is quantified by gc / ms after hydrolysis with trifluoroacetic acid or , in the case of crystalline cellulose , h 2 so 4 . in some transgenic plants in which the rsw1 gene is mutated , the monosaccharide composition is comparable to that observed for homozygous rsw1 plants , at least in some cases , confirming that there is a major reduction in the quantity of crystalline cellulose in the final , acid insoluble fraction . thus , mutation of the rsw1 gene can be performed to produce changes in the composition of plant cell walls . chemical modification of the rsw1 gene to manipulate cellulose production and plant cell wall content . as demonstrated in the preceding examples , the rsw1 gene is involved in cellulose production and the manipulation of cell wall content . in the present example , to identify novel phenotypes and gene sequences important for the normal functioning of the cellulose synthase gene , the rsw1 gene is modified in planta , using the chemical mutagen ems . the mutant plants are identified following germination and the modified rsw1 genes are isolated and characterised at the nucleotide sequence level . a sequence comparison between the mutant gene sequences and the wild type sequence reveals nucleotides which encode amino acids important to the normal catalytic activity of the cellulose synthase enzyme , at least in arabidopsis thaliana plants . this approach thus generates further gene sequences of utility in the modification of cellulose content and properties in plants . five pieces of evidence make a compelling case that the rsw1 gene product encodes the catalytic subunit of cellulose synthase : 1 . the rsw1 mutation selectively inhibits cellulose synthesis and promotes accumulation of a non - crystalline β - 1 , 4 - glucan ; 2 . the rsw1 mutation removes cellulose synthase complexes from the plasma membrane , providing a plausible mechanism for reduced cellulose accumulation and placing the rsw1 product either in the complexes or interacting with them ; 3 . the d , d , d , qxxrw ( seq id no : 37 ) signature identifies the rsw1 gene product as a processive glycosyl transferase enzyme ( saxena , 1995 ); 4 . the wild type allele corrects the temperature sensitive phenotype of the rsw1 mutant ; and 5 . antisense expression of the rsw1 in transgenic plants grown at 21 ° c . reproduces some of the phenotype of rsw1 which is observed following growth at 31 ° c . consistent with the plasma membrane location expected for a catalytic subunit , the putative 122 kda rsw1 product contains 8 predicted membrane - spanning regions . six of these regions cluster near the c - terminus ( fig1 ), separated from the other two by a domain that is probably cytoplasmic and has the weak sequence similarities to prokaryotic glycosyl transferases ( wong , 1990 ; saxena , 1990 ; matthyse , 1995 ; sofia , 1994 ; kutish , 1996 ). rsw1 therefore qualifies as a member of the large family of arabidopsis thaliana genes whose members show weak similarities to bacterial cellulose synthase . rsw1 is the first member of that family to be rigorously identified as an authentic cellulose synthase . among the diverse genes in a . thaliana , at least two genes show very strong sequence similarities to the rsw1 gene and are most likely members of a highly conserved sub - family involved in cellulose synthesis . the closely related sequences come from cosmid 12c4 , a partial genomic clone cross - hybridising with est t20782 designated ath - a , and from a full length cdna designated ath - b . ath - a resembles rsw1 ( seq id no : 5 ) at its n - terminus whereas ath - b starts 22 amino acid residues downstream [ fig8 and fig9 a – 9j ]. closely related sequences in other angiosperms are the rice est s0542 [ fig9 a – 9j ], which resembles the polypeptides encoded by rsw1 and ath - a and the cotton cela1 gene ( pear , 1996 ) at the n - terminus . the arabidopsis thaliana , rice and cotton genes have regions of very high sequence similarity interspersed with variable regions ( fig9 a – 9j and 10 ). most of the highest conservation among those gene products occurs in their central cytoplasmic domain where the weak similarities to the bacterial cellulose synthase occur . the n - terminal region that precedes the first membrane spanning region is probably also cytoplasmic but shows many amino acid substitutions as well as sequences in rsw1 that have no counterpart in some of the other genes as already noted for cela . an exception to this is a region comprising 7 cysteine residues with highly conserved spacings ( fig1 ). this is reminiscent of regions suggested to mediate protein - protein and protein - lipid interactions in diverse proteins including transcriptional regulators and may account for the striking sequence similarity between this region of rsw1 and two putative soybean bzip transcription factors ( genbank soystf1a and 1b ). in conclusion , the chemical and ultrastructural changes seen in the cellulose - deficient mutant combine with gene cloning and complementation of the mutant to provide strong evidence that the rsw1 locus encodes the catalytic subunit of cellulose synthase . accumulation of non - crystalline β - 1 , 4 - glucan in the shoot of the rsw1 mutant suggests that properties affected by the mutation are required for glucan chains to assemble into microfibrils . whilst not being bound by any theory or mode of action , a key property may be the aggregation of catalytic subunits into plasma membrane rosettes . at the restrictive temperature , mutant synthase complexes disassemble to monomers ( or smaller oligomers ) that are undetectable by freeze etching . at least in the shoot , the monomers seem to remain biosynthetically active but their β - 1 , 4 - glucan products fail to crystallise into microfibrils probably because the chains are growing from dispersed sites . crystallisation into microfibrils , with all its consequences for wall mechanics and morphogenesis , therefore may depend upon catalytic subunits remaining aggregated as plasma membrane rosettes . those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described . it is to be understood that the invention includes all such variations and modifications . the invention also includes all of the steps , features , compositions and compounds referred to or indicated in this specification , individually or collectively , and any and all combinations or any two or more of said steps or features . 2 . ausubel et al . 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