Patent Application: US-2922898-A

Abstract:
the present invention relates to a novel process for the production of transgenic organisms or transgenic cells , to transgenic orgaisms or transgenic cells obtainable by the process of the present invention , to the use of vectors comprising dna encoding a recombination promoting enzymes for curing impairments caused by environmental influences in plants or plant cells and for gene therapy in mammals or mammalian cells , and to novel vectors .

Description:
nuclear proteins concentrate in the nucleus irrespective of their size . “ nuclear localisation signals ”, short stretches of amino acids , are believed to be responsible for targeting of nuclear proteins ( for reviews see dingwall and laskey , 1986 ; silver , 1991 ). the size of the e . coli reca monomer is close to the exclusion limit of eukaryotic nuclear pores ; cytoplasmic ssdna / reca filaments would most probably be excluded . reca protein is unlikely to contain nuclear localisation signals and therefore might be excluded from the nucleus and hence from its target . in order to cover this eventuality reca protein was expressed in plants as an example of a eukaryotic organism not only in its authentic form but also fused to a nuclear localisation signal . first the reca gene was modified to remove most bacterial sequences from its up - and downstream untranslated regions . in a second step , the coding sequence was attached 5 ′ to the nuclear localisation sequence of the sv40 large t - antigen ( kalderon et al ., 1984 ) yielding a fusion protein ( nt - reca ). to optimise nt - reca translation , a leader sequence derived from the rubisco ssu gene ( cashmore , 1983 ) encoding a translation initiation codon was fused 5 ′ to the nt - reca coding sequence . both reca and nt - reca sequences were placed under transcriptional control of the cauliflower mosaic virus ( camv ) 35s promoter and supplied 3 ′ with a eukaryotic polyadenylation signal ( fig1 ). finally , the genes were inserted into a binary vector suitable for agrobacterium - mediated plant transformation using a sulfonamide resistance gene ( sul r ) to select transformed plants ( fig1 ). in plasmid ps / reca the orientation of the reca transgene relative to the sul r gene was such that both genes are transcribed in opposite directions . in plasmid ps / nt - reca the nt - reca and the sul r genes , however , are transcribed in the same direction . for biochemical characterisation , the nt - reca fusion protein was also expressed in e . coli . for this purpose plasmid pev / nt - reca was constructed in which nt - reca was fused to an artificial ribosome binding site ( fig1 ). modified reca genes were derived from plasmid pdr1453 ( sancar and rupp , 1979 ). the plasmid was digested with the restriction enzyme sacii , the ends made blunt with dna polymerase i large fragment and the amino - terminal part of the reca gene subcloned as a sacii / ecori fragment in plasmid puc18 , which had been cut with ecori and smai yielding plasmid preca - 1 . the same plasmid was digested with hinfi , the ends rendered blunt and the carboxy - terminal part of the reca gene subcloned into puc19 ( ecori / smai ) as a hinfi / ecori fragment ( preca - 2 ). the amino - terminal part was further modified . a bstxi / ecori and a taqi / bstxi fragment obtained from preca - 1 encoding the amino - terminal part of reca without its initiation codon and two complementary oligonucleotides ( 5 ′ ggg gac tcc tcc taa gaa gaa gcg taa ggt tat ggc gat3 ′ ( seq id no : 1 ) and 5 ′ cga tcg cca taa cct tac gct tct tct tag gag gag tcc cc3 ′ ( seq id no : 2 )) encoding the missing codons as well as the sv40 nuclear localisation sequence were inserted into plasmid puc18 which was had been digested with ecori and smai , yielding plasmid preca - 3 . the dna sequence of relevant junctions confirmed the expected structures of the constructs . for expression of nt - reca in plants , the leader sequence and the codons encoding the first 4 amino acids of the rubisco ssu gene were fused to the reca gene . plasmid psp64 / tpnptii ( wassmann et al ., 1986 ) contains the amino - terminal part of the ssu gene . this plasmid was digested with ecorv and sali and a smai / ecori fragment derived from preca - 3 carrying the amino - terminal portion of the reca gene and a ecori / sali fragment from preca - 2 containing the carboxy - terminal portion inserted to it to yield preca - 4 . the complete nt - reca gene was excised from preca - 4 by digestion with hindiii and sali . the hindiii ends were made blunt and the fragment inserted into plasmid pdh51 ( pietrzak et al ., 1986 ), which had been modified to contain an additional hindiii site upstream of the unique ecori site . this step fused the camv 35s promoter and the polyadenylation signal , respectively , to the nt - reca gene . for expression of reca in plants , the smai / ecori fragment from preca - 3 carrying the amino - terminal portion of the reca gene and the ecori / sali fragment from preca - 2 containing the carboxy - terminal portion were inserted into the modified pdh51 plasmid via corresponding sites . to obtain plasmid pev / nt - reca for expression in e . coli , the bamhi / ecori fragment from preca - 3 carrying the amino - terminal portion of the reca gene and the ecori / sali fragment from preca - 2 containing its carboxy - terminal portion were inserted into plasmid pevvfr1 ( crowl et al ., 1985 ). the binary vector carrying a sul r selectable marker gene was constructed as follows : plasmid pjit119 ( guerineau et al ., 1990 ) was digested with hindiii , the ends filled in with dna polymerase i large fragment , and the hindiii / sali fragment carrying the sul r gene inserted into plasmid pdh51 , which had been digested with smai and sali . to obtain ps001 , the sul r gene fused to the camv 35s promoter and polyadenylation signal was exchanged for the methotrexate resistance gene in pm001 ( reiss et al ., 1994 ) via the ncoi and ssti sites . the reca and nt - reca gene , respectively , was exised by digestion with hindiii and was inserted into the unique hindiii site of plasmid ps001 leading to plasmids ps / reca and ps / nt - reca . in order to determine whether fusion of the nuclear localisation signal had any influence on the biochemical properties of reca , a number of reactions were tested : ( i ) in e . coli , reca confers uv resistance directly via recombinational repair of replication blocks and indirectly by mediating induction of sos responses including increased expression of reca itself ( roberts et al ., 1978 ). to test the function of nt - reca plasmid pev / nt - reca , in which the nt - reca gene was transcribed from the phage λp l promoter , was introduced into the e . coli reca - strain dh5α . in particular , plasmid pev / nt - reca was transformed into e . coli strain dh5α ( supe44 , δlacu169 ( φ80laczδm15 ), hsdr17 , reca1 , enda1 , gyra96 , thi - 1 , rela1 , gibco / brl ) which harboured a plasmid named 537 ( strebel et al ., 1986 ) encoding a heat inducible lambda repressor gene . cultures were grown at 28 ° c . in lb medium supplemented with ampicillin ( 100 μg / ml ) and kanamycin ( 25 μ / ml ). expression was induced by a temperature shift to 42 ° c . after an additional 2 hour growth and expression period , the cells were harvested by centrifugation and washed in 250 mm tris / hcl ph7 . 5 , 25 % ( w / v ) sucrose . high levels of expression , by thermal inactivation of the λci857 repressor , turned out to be lethal . however , the cells were viable and uv - tolerant at 28 ° c ., indicating that nt - reca was functional . purification of nt - reca was as described for reca by cox et al . ( 1981 ) with the modifications of griffith and shores ( 1985 ), yielding 20 mg nt - reca from 2 g of cells . the protein was stored in 20 mm tris / hcl ph7 . 5 , 1 mm edta , 1 mm dtt buffer containing 20 % ( v / v ) glycerol at − 20 ° c . the protein concentration was determined according to bradford ( 1976 ). the purity and identity of nt - reca protein was verified by sds polyacrylamide gelelectrophoresis , coomassie blue staining , and western blotting using antibody arm414 , an antibody produced according to conventional procedures . the preparation contained a single protein visible in the coomassie blue stain . this protein reacted with the anti - reca antibody in the western blot . using these criteria , the nt - reca preparation was of equal purity with a preparation of reca protein which had been purified according to the same protocol from a nalidixic - acid - induced e . coli cells harbouring plasmid pdr1453 . ( ii ) atpase , ssdna binding , and strand - exchange activities were analysed with highly purified nt - reca protein produced by heat induction of a recal e . coli strain carrying pev / nt - reca . purified nt - reca preparations were shown to contain small amounts ( less than 5 %) of a protein of the molecular weight of authentic reca presumably resulting from processing by an unspecific protease or from translation starting at an internal initiation codon in nt - reca . the atpase activities of nt - reca and authentic reca , both purified from e . coli cultures by the same procedure ( griffith and shores , 1985 ), were assayed in parallel in the presence and absence of ssdna ( shibata et al ., 1981 ). essentially , atpase activity was determined as described by shibata et al . ( 1981 ) except that [ 3 h ] atp was substituted by [ 14 c ] atp ( amersham , specific activity 5 × 10 12 ci / mol ). the basal atpase activity of authentic reca was found to be low but to be stimulated 20 - fold by the addition of ssdna , as expected ( see roca and cox , 1990 ). in contrast , the basal atpase activity of nt - reca was found to be much higher and stimulated proportionally less by the addition of ssdna ( fig2 ). ( iii ) binding of purified nt - reca to ssdna was assayed using gel retardation assays . a fixed quantity of ssdna was incubated with increasing amounts of purified reca and nt - reca protein in the presence of ysatp to prevent dissociation of the complexes . in particular , binding of nt - reca and reca to ssdna was determined in a total volume of 20 μl 25 mm tris / acetate , 4 mm mgcl 2 , 1 mm dtt , 20 μm nucleotide ssdna from a derivative of phage m13mp18 , described by wada et al . ( 1994 ), and 2 m μ γsatp . different quantities of protein ( 0 , 20 , 50 , and 100 pmol ) were incubated for 30 min at 37 ° c . with this mixture . for deproteinisation , sds and edta were added to final concentrations of 1 % ( w / v ) and 10 mm , respectively ( riddles and lehmann , 1985 ). no differences in binding kinetics were observed ( fig3 ). ( iv ) reca and nt - reca proteins promoted strand - exchange between linear dsdna and circular ssdna ( menetski et al ., 1990 ) with the same kinetics ( fig4 ). the strand - exchange reaction was performed exactly as described by menetski et al . ( 1990 ). closed circular ssdna and bg1i linearised dsdna prepared from a derivative of phage m13mp18 ( wada et al ., 1994 ) were used as substrates . these tests therefore indicated that nt - reca exhibited the activities expected of a reca protein . agrobacteria harbouring binary vectors carrying the reca and nt - reca transgenes were used to infect tobacco leaf disks : plasmids ps / reca and ps / nt - reca were transferred to agrobacterium tumefaciens strain gv3101 / pmp90rk ( koncz and schell , 1986 ) via electroporation , and the resulting strains ( plants transgenic for reca were designated g64 and the nt - reca transgenic plants g63 ) used to inoculate leaf disks made from sterile tobacco sr1 plants according to published procedures ( koncz and schell , 1986 ). transformed shoots were selected on sulfadiazine ( 100 mg / l ). plants were regenerated and tested for rooting on sulfadiazine ( 100 mg / l ). transgenic plants were grown to maturity in the green house and seeds harvested . the inheritance of the transgenes was tested by germination of seeds in the presence of sulfadiazine on the same media . sr1hph2 plants were grown from seedlings which were selected on hygromycin ( 15 mg / l ) under sterile conditions . g63 and g64 plants were crossed to sr1hph2 plants in the green house . siblings harbouring the reca and hph2 transgenes were selected by growth of seedlings in the presence of both sulfadiazine ( 100 mg / l ) and hygromycin ( 15 mg / l ) under sterile conditions . plants were grown to maturity without further selection . individual transgenic plants were numbered consecutively . shoots which rooted on selective media containing sulfonamide were considered to be resistant and were selected for further analysis . the presence of reca transgenes was confirmed by southern blots . for southern hybridisations , total dna prepared from leaves ( murray and thompson , 1980 ) was restricted with ecori , and the fragments separated by agarose gel electrophoresis and blotted to a nylon membrane ( zetaprobe , biorad ). the membrane was hybridised according to the manufacturers guidelines , to radioactively labeled probes prepared as described by feinberg and vogelstein ( 1984 ). fragments encoding reca sequences were detected using a fragment from preca - 4 which covered the entire gene . the copy number of inserts was determined using probes specific for border fragments which were derived from the sulr . and reca genes . it was found that 11 of 12 sulfonamide resistant g64 plants carried an intact reca transgene . plants with single copy inserts were selected and shown to transmit the sulfonamide resistance marker to their progeny as a single mendelian trait . in contrast , only three of 36 g63 plants were found to carry an intact nt - reca gene . in one of them , g63 / 19 , the right border of the t - dna was deleted . the deletion resulted in a fusion of the 35s promoter sequences controlling nt - reca expression to plant genomic sequences . other transgenic plant lines harboured either no reca sequences or had rearranged nt - reca genes . the intact nt - reca transgenes in the three independent transgenic plants were shown to be present in single copy and to be inherited as a single mendelian trait . expression of the reca and nt - reca genes was monitored using western blots of protein extracts of leaves probed with a reca - specific monoclonal mouse antibody ( arm414 , ikeda et al ., 1990 ). first , proteins were extracted from leaves of plants grown in sterile culture . leaves were ground with laemmli sample buffer ( laemmli , 1970 ) ( without bromphenol blue ) plus sea sand , using a glass rod in an eppendorf tube . after heat denaturation of the samples for 15 min at 95 ° c ., the extract was cleared by centrifugation and the supernatant used for further analysis . the protein concentration was determined ( bradford , 1976 ) and 50 μg used for electrophoresis on polyacrylamide sds gels according to laemmli ( 1970 ). proteins were transferred to nitrocellulose membranes ( schleicher and schuell , pore size 0 . 45 μm ) as described ( towbin , 1979 ). reca protein was detected using the monoclonal anti - reca antibody arm414 ( ikeda et al ., 1990 ) in a 1 : 200 dilution in tbst buffer after blocking of non - specific protein binding by 5 % non - fat dry milk . a second antibody ( goat anti - mouse , promega ) coupled to alkaline phosphatase and nbt / bcip ( promega ) staining or a second anti - mouse antibody coupled to horse radish peroxidase and chemiluminescence ( ecl , amersham ) was used to develop the blot . all plants with an intact reca or nt - reca gene were thus shown to express reca protein . expression in g64 plants ( reca ) was similar in most transgenics and amounted to about 0 . 1 % of total plant protein , as judged from a comparison of staining intensities with those of a dilution series of purified reca protein . the expression level of nt - reca ranged from 0 . 01 % ( g63 / 17 ) to 0 . 1 % ( g63 / 19 ) of total protein . both reca and nt - reca proteins appeared stable in plant cells and showed the expected molecular weights ( fig5 ). in nt - reca transgenic plants , small amounts of additional proteins were detected with the antibody . since these proteins were of lower molecular weight , they presumably were degradation products of the actual nt - reca protein . the localisation of the reca and nt - reca proteins in the cell was studied by indirect immunbfluorescence . plants with comparable expression levels were selected ( g64 / 2 and g63 / 19 ) and protoplasts or root squashes were prepared . the preparation of protoplasts was done , according to the method of negrutiu ( 1987 ), for immuno - histochemical localisation of reca and nt - reca protein . proteins were fixed with 5 % formaldehyde ( 10 5 protoplasts in 2 ml k3 , 0 . 4m sucrose ) at room temperature . formaldeyde was removed by washing in w5 and chlorophyll extracted with methanol . nonspecific binding was blocked by an incubation of the protoplast preparation in 5 % bsa in tbst for 1 hour . the protoplasts were collected by centrifugation and incubated with anti - reca antibody arm414 ( 1 : 50 ) in tbst . after extensive washing with tbst buffer containing 5 % bsa , the protoplasts were incubated with fitc - labelled anti - mouse antibody ( promega , 1 : 1000 ). unbound antibody was removed by washing with tbst 5 % bsa . nuclei were stained with dapi ( 2 μg / ml ) in the same buffer . the preparation was examined using fluorescence microscopy ( zeiss axiophot ). reca and nt - reca proteins were localised in whole tissue from young plants after fixation of leaves or roots in methanol : acetic acid ( 3 : 1 ) for 1 hour at room temperature . tissue was equilibrated with k3 medium and incubated over night with 0 . 9 % cellulase in the same medium . after incubation in acetic acid for 5 min , the tissue was transferred to a slide and squashed . staining with anti - reca antibody was as described above for protoplasts . in g63 / 19 plants , fitc - fluorescence was found almost exclusively in the nucleus ( fig6 ). some staining was visible also in the chloroplasts . in contrast , in g64 / 2 cells the nuclei were not particularly stained . however , there seemed to be a weak preference for association with the nucleus and concentration in the region around the nucleus ( fig6 ). only background staining was observed in non - transgenic sr1 tobacco plants . it can be concluded that the sv40 nuclear localisation sequence in nt - reca leads to efficient accumulation of this protein in the nucleus of the plant cell . expression of reca and nt - reca leads to increased resistance to mitomycin c to analyse the effect of mitomycin c on plant growth , a quantitative and reproducible assay was used . the system described by lebel et al . ( 1993 ) which allows to follow the fate of single plant cells was developed further to obtain greater sensitivity to mitomycin c and a monotonic survival - dose - response curve . in this system protoplasts were prepared from sterile - grown tobacco sr1 plants and parallel preparations treated with various concentrations of mitomycin c . subsequently the protoplasts were cultivated in a bead - type culture in the presence of mitomycin c . untreated protoplasts actively divided and formed microcalli within a period of 4 to 8 weeks . the experimental details were as follows : protoplasts were prepared from leaves of axenically grown plants as described by negrutiu ( 1987 ), with some modifications . cut leaves ( 3 g ) were digested in 50 ml k3 , 0 . 4 m sucrose , 1 mg / l naa , 0 . 2 mg / l kinetin , 0 . 6 % cellulase onozuka r10 ( serva ), 0 . 3 % macerozyme r10 ( serva ) in 145mm petri dishes at 22 ° c ., in the dark , for 16 hours . protoplasts were purified by filtration through steel sieves ( 250 μm and 100 μm mesh width ) and washed once in w5 medium . protoplasts were suspended in 1 ml of mamg buffer ( 0 . 5 m mannitol , 15 mm mgcl 2 , 0 . 1 % mes , ph5 . 7 ), counted under a light microscope and diluted to a final concentration of 10 6 cells / ml with k3 , 0 . 4 m sucrose . a 1 ml aliquot of protoplast solution was diluted with 9 ml k3 , 0 . 4 m sucrose medium and mitomycin c added from a stock solution , to the final concentrations indicated in fig7 . after incubation for 2 days in the dark at 22 ° c ., the protoplasts were cultivated using the bead - type technique of shillito et al . ( 1983 ) with some modifications . protoplasts were embedded by dilution with an equal volume of media containing 0 . 8 % low - melting - point agarose ( fmc ), as a gelling agent , and grown on a solid support carrier system ( paper filter discs ) in 20 ml liquid media . cultures were grown for approximately 4 weeks with weekly changes of media . survival was scored when the microcalli reached sizes of 2 to 4 mm . plants were regenerated from representative samples . these plants showed no obvious growth abnormalities . in a typical control experiment , 10 % to 20 % of the protoplasts plated grew to microcalli . increasing concentrations of mitomycin c progressively inhibited the formation of microcalli ( fig7 ). no growth ( less than 10 − 3 % of control values ) was observed at concentrations of 40 μ / ml and above . the survival curve showed a low - dose shoulder suggesting the presence of repair mechanisms leading to resistance to mitomycin c followed by a semi - logarithmic region at high doses of mitomycin c causing damage which can no longer be repaired by the endogenous repair mechanisms . protoplasts of a plant homozygous for the reca transgene ( g64 / 2 ) were slightly but significantly more resistant to the toxic effect of mitomycin c than control cells . at concentrations of 40 μg / ml mitomycin c more than 0 . 1 % of the cells survived and grew to microcalli ( fig7 ). however , no reca transgenic cells ( less than 10 − 3 % of control values ) grew at mitomycin c concentrations of 50 μg / ml and above . in contrast , more than 0 . 1 % nt - reca transgenic protoplasts ( homozygous g63 / 19 ) were able to grow and regenerate at concentrations of up to 60 μ / ml mitomycin c , the highest concentration tested ( fig7 ). intrachromosomal recombination of a chromosomal marker is stimulated by reca and nt - reca plants usually contain large amounts of repetitive dna sequences . recombination within these sequences apparently played a role in genome evolution ( for review see : flavell , 1982 ). to study the process of intrachromosomal recombination in plants , peterhans et al . ( 1990 ) have developed a transgenic system . a pair of deletion derivatives of the selectable marker gene neomycin phosphotransferase ( nptii , beck et al ., 1982 ) were stably integrated into the tobacco genome . the deletions removed portions of either the 5 ′ or the 3 ′ end of the gene rendering it non - functional . the segments in line sr1hph2 ( peterhans et al ., 1990 ) were oriented as direct repeats with a 352 - bp homologous overlap , interrupted by a functional hygromycin phosphotransferase gene ( van den elzen et al ., 1985 ). in this line , the basic module was present in three tightly linked copies in the genome . intrachromosomal recombination events which lead to restoration of a functional nptii gene can easily be detected by selection of kanamycin resistant cells in tissue culture . to study the influence of reca and nt - reca expression on intrachromosomal recombination , line sr1hph2 containing the defective nptii genes was crossed respectively to the homozygous lines g64 / 2 and g63 / 19 . progeny plants carrying the reca respectively nt - reca genes as well as the defective nptii genes were selected by germinating seeds on hygromycin and sulfonamide . plants resistant to both antibiotics were grown under sterile culture conditions without further selection and leaf mesophyll protoplasts were prepared as described in example 4 . to determine the number of intrachromosomal recombination events , cultures were grown for 6 to 8 weeks in the presence of 100 μ / ml kanamycin after embedding . to determine the regeneration frequency , protoplasts were grown under identical conditions without kanamycin . from representative samples of microcalli , plants were regenerated to verify resistance to kanamycin . protoplasts were plated and cultured until microcalli appeared . the number of protoplasts forming microcalli in the absence of selection ( regeneration frequency ) was determined for each batch of protoplasts and found to be about 20 % to 30 % for all protoplast preparations . the number of protoplasts regenerating in the presence of kanamycin was determined . the frequency of intrachromosomal recombination was calculated from the number of microcalli which grew on kanamycin versus the total number of calli appearing on non - selective media . 20 kanamycin - resistant microcalli were selected at random for regeneration into plants . all regenerated plants formed roots on kanamycin - containing medium , confirming that calli which grew in the presence of kanamycin were indeed resistant to the antibiotic . the frequency of intrachromosomal recombination was found to be 1 . 04 × 10 − 5 in the control line sr1hph2 . in contrast , the frequencies in g64 / 2 x sr1hph2 and g63 / 19 x sr1hph2 were found to be 5 . 37 × 10 − 5 and 10 . 3 × 10 − 5 , respectively ( fig8 ). these data show that the reca protein , especially if targeted to the nucleus , is able to interact with the plant chromosome and the host recombination machinery and to markedly increase the level of intrachromosomal somatic recombination . enhancement of gene targeting frequencies in plants by ectopic nt - reca expression a chimeric target locus was generated which consists of the seed specific high molecular weight glutenin ( hmw ) promoter ( colot et al ., 1987 ) and additional sequences consisting of pbr322 and a selectable marker conferring methotrexate resistance in plants . to obtain this construct , the hmw promoter was cloned as an ecori / bamhi fragment into the binary vector pmn001 ( reiss et al ., 1994 ). the resulting plasmid was transferred via electroporation to agrobacterium ( gv3101pmp90rk , koncz and schell , 1986 ) and the resulting strains were used to generate transgenic tobacco sr1 plants by leaf disk infection according to published methods ( marton et al ., 1982 ; de block et al ., 1984 ; marton , 1984 ; horsch et al ., 1985 ). these plants were characterized by southern blotting and one line which contained the chimeric target locus in single copy designated b18 / 4 . this line expressed npt ii in seeds , but no activity was detected in leaves using a sensitive enzymatic assay ( reiss et al ., 1984 ). when callus was induced on leaf disks from these plants , green living material readily developed on methotrexate , but no callus formed on kanamycin . these plants were crossed to g63 / 19 sr1 plants expressing the nt - reca protein ( reiss et al ., 1996 ). a repair construct was made which would lead to constitutive expression of npt ii upon homologous recombination with the b18 / 4 target . this construct was identical to the one used to generate b18 / 4 , but contained a promoter expressed in all tissues , the camv 35s promoter , inserted into the bamhi site between the hmw promoter and the npt ii gene and the npt ii gene was the non - functional variant d42 which contained a carboxyterminal deletion shown to yield no active protein in e . coli ( beck et al ., 1982 ). this plasmid was introduced into agrobacterium ( gv3101pmp90rk ) as described above to yield strain g125 . to analyze gene targeting , siblings obtained from g63 / 19 plants crossed to b18 / 4 were selected on methotrexate and sulfonamide to select for the presence of both sets of transgenes . leaf disks were prepared and transformed with agrobacterium strain g125 which harboured the repair construct . in control transformations , 500 calli were obtained with g125 on a total of 58 sr1 leaf disks after selection on methotrexate . this demonstrated that g125 was fully functional . in total , 189 leaf disks from b18 / 4 × g63 / 19 were infected with g125 and 21 kanamycin resistance calli obtained after selection on kanamycin . to determine whether the resistant calli derived from potential gene targeting events , total genomic dna was prepared from 9 of them . the dna was amplified by pcr with a primer pair specific for an intact npt ii gene under 35s promoter control ( primer 1 homologous to the − 90 region of the 35s promoter : 5 ′ gtg gat tga tgt gat atc tcc3 ′ ( seq id no : 3 ); primer 2 homologous to sequences of npt ii deleted in d42 : 5 ′ ccg ctc aga aga act cgt ca3 ′ ( seq id no : 4 )). a fragment of the size predicted for amplification of the restored nptii gene with this primer pair was obtained in six calli . no amplification product was obtained with dna from the parental b18 / 4 line and the residual 3 calli . these results indicate the presence of an intact npt ii gene under 35s promoter control in 6 of the 9 calli investigated . the npt ii gene in the 3 kanamycin resistant , but pcr negative calli most likely was activated by somaclonal variation , not by gene targeting . the frequency of restoration of an intact npt ii gene expressed in leaf tissue by transformation with g125 can be calculated therefore as follows : the transformation frequency in the control experiment was 500 transformants per 58 leaf disks or 8 . 6 / leaf disk . thus , it was to be expected that in a total of 189 b18 / 4 × g63 / 19 leaf disks transformed , approximately 189 × 8 , 6 = 1625 transformants were generated without selection on kanamycin . a number of 21 kanamycin resistant calli was detected . therefore , these calli appeared with a frequency of 21 / 1625 which approximately equals 1 . 3 %. in a representative sample of 9 , 6 contained a restored npt ii gene . therefore the frequency of targeting in this experiment was 0 . 87 %. transgenic target loci similar to the system described here were used previously . the targeting frequencies observed with those target loci using agrobacterium - mediated transformation were in the order of 10 − 4 ( offringa et al ., 1990 ). although these experiments might not be directly comparable , the large increase in frequencies observed in our experiments indicate a stimulatory role of reca expression . beck , e ., ludwig , g ., auerswald , e . a ., reiss , b ., and schaller , h . 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