Patent Application: US-21329398-A

Abstract:
the present invention is based on the isolation and characterization of a plant cell dna sequence encoding for a retinoblastoma protein . such finding is based on the structural and functional properties of the plant retinoblastoma protein as possible regulator of the cellular cycle , of the cellular growth and of the plant cellular differentiation . for this reason , among other aspects , it is claimed the use of retinoblastoma protein or the dna sequence which encodes for it in the growing control of vegetable cells , plants and / or vegetable virus , as well as the use of vectors , cells , plants or animals , or animal cells modified through the manipulation of the control route based on plant retinoblastoma protein .

Description:
the present invention will now be illustrated further by reference to the following non - limiting examples . further embodiments falling within the scope of the claims attached hereto will occur to those skilled in the light of these . fig1 . the sub - figure a shows the relative lengths of the present zmrb1 protein and the human retinoblastoma proteins . the sub - figure b shows the alignment of the amino acids sequences of the pocket a and pocket b of the zmrb1 with that of the xenopus , chicken , rat and three human protein ( rb , p107 and p130 ) ( seq id nos : 6 - 19 ). fig2 . this figure is a map of the main characteristics of the wdv virus and the pwori vector derived from wdv and the positions of the deletions and mutations used in order to establish that the lxcxe motif is required for its replication in plants cells . total rna was isolated from maize root and mature leaves by grinding the material previously frozen in liquid nitrogen essentially as described in soni et al ( 1995 ). the major and minor p75zmrb1 mrnas were is identified by hybridization to a random - primed 32p - labelled psti internal fragment ( 1 . 4 kb ). a portion of a maize cdna library ( 106 pfu ) in 1zapii ( stratagene ) was screened by subsequent hybridization to 5 ′- labelled oligonucleotides designed to be complementary to a known est sequence of homologue maize of p130 . these otigonucleotides were 5 ′- aatagacacatcgatcaa / g ( seq id no : 3 ) ( m . 5m , nt positions 1411 - 1438 ) and 5 ′- gtaatgataccaacatgg ( seq id no : 4 ) ( m . 3c , nt positions 1606 - 1590 ) ( isogen biosciences ). after the second round of screening , pbluescript sk -( pbs ) phagemids from positive clones were isolated by in vivo excision with exassist helper phage ( stratagene ) according to protocols recommended by the manufacturer . dna sequencing was carried out using a sequenasetm kit ( usb ). the 5 ′- end of the mrnas encoding p75zmrb1 was determined by race - pcr . poly - a + mrna was purified by chromatography on oligo - dt - cellulose ( amersham ). the first strand was synthesized using oligonucleotide dral35 ( 5 ′- gatttaaaatcaagctcc ( seq id no : 5 ), nt positions 113 - 96 ). after denaturation at 90 ° c . for 3 min , rna was eliminated by rnase treatment , the cdna recovered and 5 ′- tailed with terminal transferase and datp . then a pcr fragment was amplified using primer dral35 and the linker - primer ( 50 bp ) of the stratagene cdna synthesis kit . one of the positive clones so produced contained a ˜ 4 kb insert that , according to restriction analysis , extended both 5 ′ and 3 ′ of the region contained in the expressed sequence tag used . the nucleotide sequence corresponding to the longest cdna insert ( 3747 bp ) is shown in seq id no . 1 . this zmrb1 cdna contains a single open reading frame capable of encoding a protein of 683 amino acids ( predicted mr 75247 , p75zmrb1 ) followed by a 1646 bp 3 ′- untranslated region . untranslated regions of similar length have been also found in mammalian rb cdnas ( lee , w .- l . et al , science 235 , 1394 ( 1987 ); bernards , r . et al , proc . natl . acad . sci . usa 86 , 6474 ( 1989 )). northern analysis indicates that maize cells derived from both root meristems and mature leaves contain a major message , ˜ 2 . 7 ± 0 . 2 kb in length . in addition , a minor ˜ 3 . 7 ± 0 . 2 kb message also appears . heterogeneous transcripts have been detected in other species ( destrée , o . h . j . et al , dev . biol . 153 , 141 ( 1992 )). plasmid pworiδδ was constructed by deleting in pwori most of the sequences encoding wdv proteins ( sanz and gutierrez , unpublished ). plasmid p35s . rb1 was constructed by inserting the camv 35s promoter ( obtained from pwdv3 : 35sgus ) upstream of the zmrb1 cdna in the pbs vector . plasmid p35s . 130 was constructed by introducing the complete coding sequence of human p130 instead of zmrb1 sequences into p35s . rb1 . plasmid p35 . a + b was constructed by substituting sequences encoding the wdv repa and repb orfs instead of zmrb1 in p35s . rb1 plasmid . ( see soni , r . and murray , j . a . h . anal . biochem . 218 , 474 - 476 ( 1994 )). the sequence around the methionine codon at nucleotide position 31 contains a consensus translation start ( kozak , m . j . mol . biol . 196 , 947 ( 1987 )). to determine whether the cdna contained the full - length zmrb1 coding region , the 5 ′- end of the mrnas was amplified by race - pcr using an oligonucleotide derived from a region close to the putative initiator aug , which would produce a fragment of ˜ 150 bp . the results are consistent with the zmrb1 cdna clone containing the complete coding region . the zmrb1 protein contains segments homologous to the a and b subdomains of the “ pocket ” that is present in all members of the rb family . these subdomains are separated by a non - conserved spacer . zmrb1 also contains non - conserved n - terminal and c - terminal domains . overall , zmrb1 shares ˜ 28 - 30 % amino acid identity (˜ 50 % similarity ) with the rb family members ( hannon , g . j ., demetrick , d . & amp ; beach , d . genes dev . 7 , 2378 ( 1993 ); cobrinik , d ., whyte , p ., peeper , d . s ., jacks , t . & amp ; weinberg , r . a . ibid ., p . 2392 ( 1993 ). ewen , m . e ., xing , y . lawrence , j . b . and livingston , d . m . cell 66 , 1155 ( 1991 ))( lee w . l . et al , science 235 , 1394 ( 1987 ); bernards et al , proc . natl . acad . sci . usa 86 , 6974 ( 1989 )), with the a and b subdomains exhibiting the highest homology (˜ 50 - 64 %). interestingly , amino acid c706 in human rb , critical for its function ( kaye , f . j ., kratzke r . a ., gerster , j . l . and horowitz , j . m . proc . natl . acad . sci . usa 87 , 6922 ( 1990 )), is also conserved in maize p75zmrb1 . zmrb1 contains 16 consensus sites , sp or tp for phosphorylation by cyclins dependant kinases ( cdks ) with one of the 5 ′- tail of the sub - domain a and several in the c - terminal area which are potential sites of phosphorylation . a nucleic acid preferred group which encodes proteins in which one or more of these sites are changed or deleted , making the protein more resistant to the phosphorylation and thus , to its functionality , for example linking to e2f or similar . this can be easily carried out by means of mutagenesis conducted by means of pcr . replication of wheat dwarf geminivirus ( wdv ) is dependent upon an intact lxcxe motif of the viral repa protein . this motif can mediate interaction with a member of the human rb family , p130 , in yeasts . therefore , the inventors investigated whether p75zmrb1 could complex with wdv repa by using the yeast two - hybrid system ( fields , s . and song , o . nature 340 , 245 - 246 ( 1989 )). yeast cells were co - transformed with a plasmid encoding the fusion gal4bd - repa protein and with plasmids encoding different gal4ad fusion protein . the gal4ad - p75zmrb1 fusion could also complex with gal4bd - repa to allow growth of the recipient yeast cells in the absence of histidine . this interaction was slightly stronger than that seen with the human p130 protein . repa could also bind to some extent to a n - terminally truncated form of p75zmrb1 . the role of the lxcxe motif in repa - p75zmrb1 interaction was assessed using a point mutation in wdv repa ( e198k ) which we previously showed to destroy interaction with human p130 . co - transformation of zmrb1 with a plasmid encoding the fusion gal4bd - repa ( e198k ) indicated that the interaction between repa and p75zmrb1 occurred through the lxcxe motif . in this respect , the e198k mutant of wdv repa behaves similarly to analogous point mutants of animal virus oncoproteins ( moran , e ., zerler , b ., harrison , t . m . and mathews , m . b . mol . cell biol . 6 , 3470 ( 1986 ); cherington , v . et al ., ibid ., p . 1380 ( 1988 ); lillie , j . w ., lowenstein , p . m ., green , m . r . and green , m . cell 50 , 1091 ( 1987 ); decarpio , j . a . et al ., ibid ., p . 275 ( 1988 )). specific interaction between maize p75zmrb1 and wdv repa in the yeast two - hybrid system ( fields et al ) relied on the ability to reconstitute a functional gal4 activity from two separated gal4 fusion proteins containing the dna binding domain ( gal4bd ) and the activation domain ( gal4ad ). yeast hf7c cells were co - transformed with a plasmid expressing the gal4bd - repa or the gal4bd - repa ( e198k ) fusions and the plasmids expressing the gal4ad alone ( vec ) or fused to human p130 , maize p75 ( p75zmrb1 ) or a 69 amino acids n - terminal deletion of p75 ( p75zmrb1 - dn ). cells were streaked on plates with or without histidine according to the distribution shown in the upper left corner . the ability to grow in the absence of histidine depends on the functional reconstitution of a gal4 activity upon interaction of the fusion proteins , since this triggers expression of the his3 gene which is under the control of a gal4 responsive element . the growth characteristics of these yeast co - transformants correlate with the levels of b - galactosidase activity . procedures for two - hybrid analysis are described in xie et al ( 1995 ). the gal4ad - zmrb1 fusions were construed in the pgad424 vector . geminivirus dna replication requires the cellular dna replication machinery as well as other s - phase specific factors ( davies , j . w . and stanley , j . trends genet . 5 , 77 ( 1989 ); lazarowitz , s . crit . rev . plant sci . 11 , 327 ( 1992 )). consistent with this requirement , geminivirus infection appears to drive non - proliferating cells into s - phase , as indicated by the accumulation of the proliferating cell nuclear antigen ( pcna ), a protein which is not normally present in the nuclei of differentiated cells ( nagar , s ., pedersen , t . j ., carrick , k . m ., hanley - bowdoin , l . and robertson , d . plant cell 7 , 705 ( 1995 )). the inventors finding that efficient wdv dna replication requires an intact lxcxe motif in repa coupled with the discovery of a plant homolog of rb supports the model that , as in animal cells , sequestration of plant rb by viral repa protein promotes inappropriate entry of infected cells into s - phase . therefore , one way to investigate the function of p75zmrb1 was to measure geminivirus dna replication in cells transfected with a plasmid bearing the zmrb1 sequences under a promoter functional in plant cells , an approach analogous to that previously used in human cells ( uzvolgi , e . et al ., cell growth diff 2 , 297 ( 1991 )). accumulation of newly replicated viral plasmid dna was impaired in wheat cells transfected with plasmids expressing p75zmrb1 or human p130 , when expression of wdv replication protein ( s ) is directed either by the wdv promoter or by the camv 35s promoter . since wdv dna replication requires an s - phase cellular environment , interference with viral dna replication by p75zmrb1 and human p130 strongly evidences a role for retinoblastoma protein in the control of the g1 / s transition in plants . the existence of a plant rb homolog implies that despite their ancient divergence , plant and animal cells use , at least in part , similar regulatory proteins and pathways for cell cycle control . two lines of evidences reinforce this model . first , a gene encoding a protein that complements specifically the g1 / s , but not the g2 / m transition of the budding yeast cdc28 mutant has been identified in alfalfa cells ( hirt , h ., páy , a ., bögre , l ., meskiene , i . and heberle - bors , e . plant j . 4 , 61 ( 1993 )). second , plant homologs of d - type cyclins have been isolated from arabidopsis and these , like their mammalian relatives , contain lxcxe motifs . in concert with plant versions of cdk4 and cdk6 , plant d - type cyclins may regulate passage through g1 phase by controlling the phosphorylation state of rb - like proteins . in animal cells , the rb family has been implicated in tumor suppression and in the control of differentiation and development . thus , p75zmrb1 could also play key regulatory roles at other levels during the plant cell life . one key question that is raised by the existence of rb homologs in plant cells is whether , as in animals disruption of the rb pathway leads to a tumor - prone condition . in this regard , the inventors have noted that the virb4 protein encoded by the ti plasmids of both agrobacterium tumefaciens and a . rhyzogenes contains an lxcxe motif . although the virb4 protein is required for tumor induction ( hooykas , p . j . j . and beijersbergen , a . g . m . annu . rev . phytopathol . 32 , 157 ( 1994 ), the function of its lxcxe motif in this context remains to be examined . geminivirus infection is not accompanied by tumor development in the infected plant , but in some cases an abnormal growth of enactions has been observed ( g . dafalla and b . gronenborn , personal communication ). inhibition of wheat dwarf geminivirus ( wdv ) dna replication by zmrb1 or human p130 in cultured wheat cells was carried out as follows . a . wheat cells were transfected , as indicated , with pwori ( xie et al . 1995 ) alone ( 0 . 5 μg ), a replicating wdv - based plasmid which encodes wdv proteins required for viral dna replication , and with control plasmid pbs ( 10 μg ) or p35s . rb1 ( 10 μg ), which encodes zmrb1 sequences under the control of the camv 35s promoter . total dna was purified one and two days after transfection , equal amounts fractionated in agarose gels and ethidium bromide staining and viral pwori dna identified by southern hybridization . plasmid dna represents exclusively newly - replicated plasmid dna since it is fully resistant to dpni digestion and sensitive to mbol . note that the mboi - digested samples were run for about half of the length than the undigested samples . b . to test the effect of human p130 on wdv dna replication , wheat cells were co - transfected with pwori ( 0 . 5 μg ) and plasmids pbs ( control ), p35s . rb1 or p35s . 130 ( 10 μg in each case ). replication of the test plasmid ( pwori ) was analyzed two days after transfection and was detected as described in part a using ethidium bromide staining ; and southern hybridization . c . to test the effect of zmrb1 or human p130 on wdv dna replication when expression of viral proteins was directed by the camv 35s promoter , the test plasmid pworiδδ ( which does not encode functional wdv replication proteins but replicates when they are provided by a different plasmid , i . e . pwori ) was used . wheat cells were co - transfected , as indicated , with pworiδδ ( 0 . 25 μg ), pwori ( 0 . 25 μg ), p35s . a + b ( 6 μg ), p35s . rb1 ( 10 μg ) and / or p35s . 130 ( 10 μg ). replication of the test plasmid ( pworiδδ ) was analyzed 36 hours after transfection and was detected as described in part a using ethidium bromide staining ; southern hybridization . plasmids pwori ( m1 ) and pworiδδ ( m2 ; sanz and gutierrez , unpublished ), 100 pg in each case , were used as markers . suspension cultures of wheat cells , transfection by 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