Patent Application: US-98690607-A

Abstract:
the invention relates to methods for directing integration of a nucleic acid of interest towards homologous recombination and uses thereof . the present invention discloses factors involved in integration of a nucleic acid by illegitimate recombination which provides a method of directing integration of a nucleic acid of interest to a predetermined site , whereby the nucleic acid has a homology at or around the predetermined site , in a eukaryote with a preference for non - homologous recombination comprising steering an integration pathway towards homologous recombination . furthermore , the invention provides a method of directing integration of a nucleic acid of interest to a subtelomeric and / or telomeric region in a eukaryote with a preference for non - homologous recombination .

Description:
the yeast strains that were used are listed in table 1 . yeast mutants isogenic to the haploid yph250 strain were constructed using the one - step disruption method ( rothstein , 1991 ). a 1987 bp fragment from the yku70 locus was amplified by pcr using the primers hdflp1 5 ′- gggattgctttaaggtag - 3 ′ ( seq id no : 1 ) and hdflp2 5 ′- caaataccctaccctacc - 3 ′ ( seq id no : 2 ). the pcr product was cloned into pt7blue ( novagen ) to obtain pt7blueyku70 . an 1177 bp ecorv / hindiii fragment from the yku70 orf was replaced by a 2033 bp hindiii / smai leu2 - containing fragment from pjj283 ( jones and prakash , 1990 ), to form pt7blueyku70 :: leu2 . in order to obtain yku70 disruptants , leu + colonies were selected after transformation of yph250 with a 2884 bp ndei / smai fragment from pt7blueyku70 :: leu2 . the expand ™ high fidelity system ( boehringer mannheim ) was used according to the supplied protocol to amplify a 3285 bp fragment from the lig4 locus with primers dnl4p1 5 ′- cgtaagattcgccgagtatag - 3 ′ ( seq id no : 3 ) and dnl4p2 5 ′- cgtttcaaatgggaccacagc - 3 ′ ( seq id no : 4 ). the pcr product was cloned into pgemt ( promega ), resulting in pgemtlig4 . a 1326 bp bamhi / xhoi fragment from pjj215 ( jones and prakash , 1990 ) containing the his3 gene was inserted into the bamhi and xhoi sites of pic20r , resulting in pic20rhis3 . a 782 bp ecori fragment from the lig4 orf was replaced with a 1367 bp ecori his3 - containing fragment from pic20rhis3 to construct pgemtlig4 :: his3 . in order to obtain lig4 disruptants , his + colonies were selected after transformation of yph250 with a 3854 bp ncoi / noti fragment from pgemtlig4 :: his3 . in order to obtain rad50 disruptants , yph250 was transformed with an ecori / bglii fragment from pnky83 , and ura + colonies were selected ( alani et al ., 1989 ). a rad50 :: hisg strain was obtained by selecting ura − colonies on selective medium containing 5 - foa . similarly , rad51 disruptants were obtained after transformation of yph250 with a rad51 :: leu2 xbai / psti fragment from pdg152 and selection of leu + colonies ( schiestl et al ., 1994 ). the trp1 marker in psm21 ( schild et al ., 1983 ) was replaced with a bglii / xbai leu2 - containing fragment from pjj283 ( jones and prakash , 1990 ). this resulted in psm21leu2 . leu + rad52 disruptant colonies were selected after transformation of yph250 with the rad52 :: leu2 bamhi fragment from psm21leu2 . disruption constructs were transformed to yph250 by the lithium acetate transformation method as described ( gietz et al ., 1992 ; schiestl et al ., 1993 ). disruption of yku70 , lig4 , rad50 , rad51 and rad52 was confirmed by pcr and southern blot analysis . to construct psdm8000 , a 1513 bp pvuii / ecorv fragment carrying the kanmx marker was obtained from pfa6a ( wach et al ., 1994 ) and was ligated into the unique hpai site of psdm14 ( offringa , 1992 ). psdm8001 was made in three cloning steps . a 1476 bp bamhi / ecori fragment carrying the kanmx marker was obtained from pfa6a and ligated into bamhi - and ecori - digested pic20h to form pic20hkanmx . the kanmx marker was inserted between the pda1 flanks by replacement of a 2610 bp bglii fragment from puc4e1α10 ( steensma et al ., 1990 ) with a 1465 bglii fragment from pic20hkanmx . a 3721 bp xhoi / kpni fragment from this construct was inserted into the xhoi and kpni sites of psdm14 . the binary vectors psdm8000 and psdm8001 were introduced into agrobacterium tumefaciens lba1119 by electroporation ( den dulk - ras and hooykaas , 1995 ). cocultivations were performed as described earlier with slight modifications ( bundock et al ., 1995 ). agrobacterium was grown overnight in lc medium . the mix of agrobacterium and s . cerevisiae cells was incubated for nine days at 20 ° c . g418 - resistant s . cerevisiae strains were selected at 30 ° c . on ypad medium containing geneticin ( 200 μg / ml ) ( life technologies / gibco brl ). chromosomal dna was isolated using qiagen &# 39 ; s genomic tips g / 20 per manufacturer &# 39 ; s protocol . 1 - 2 μg of genomic dna was digested with ecori , clai , psti or hindiii and run on a 1 % tbe - gel . nonradioactive southern blotting was performed . the membrane was hybridized with a digoxigenine - labeled kanmx probe to determine the size of t - dna / genomic dna fragments ( ecori and clai for rb - containing fragments and psti and hindiii for lb - containing fragments ). the kanmx probe , a 792 bp internal fragment of the kanmx marker , was made by pcr using primers kanmxp1 5 ′- agactcacgtttcgaggcc - 3 ′ ( seq id no : 5 ) and kanmxp2 5 ′- tcaccgaggcagttccatag - 3 ′ ( seq id no : 6 ) and a nonradioactive dna labeling and detection kit ( boehringer mannheim ). the enzyme showing the smallest band on blot was used for vectorette pcr in order to amplify the smallest junction sequence of t - dna and genomic dna . vectorette pcr was performed as described ( genomewww . stanford . edu / group / botlab / protocols / vectorette . html ). the expand ™ high fidelity system ( boehringer mannheim ) was used to amplify fragments larger than 2 . 5 kb , whereas staq dna polymerase ( sphaeroq ) was used for amplification of fragments smaller than 2 . 5 kb . primers kanmxp3 5 ′- tcgcaggtctgcagcgaggagc - 3 ′ ( seq id no : 7 ) and kanmxp4 5 ′- tcgcctcgacatcatctgcccag - 3 ′ ( seq id no : 8 ) were used to amplify rb / genomic dna and lb / genomic dna junction sequences , respectively . vectorette pcr products were cloned in pgemteasy ( promega ) and sequenced using the t7 polymerase sequencing kit ( pharmacia ) according to the manufacturer &# 39 ; s protocol . in order to obtain sequences flanking the rb and lb , primers kanmxp5 5 ′- tcacatcatgcccctgagctgc - 3 ′ ( seq id no : 9 ) and kanmxp4 were used , respectively . it was previously demonstrated that agrobacterium tumefaciens is able to transfer its t - dna not only to plants but also to another eukaryote , namely , the yeast saccharomyces cerevisiae ( bundock et al ., 1995 ). t - dna carrying homology with the yeast genome was shown to become integrated by homologous recombination . t - dna lacking any homology with the yeast genome was integrated randomly into the genome by ir , like in plants ( bundock et al ., 1995 ; bundock and hooykaas , 1996 ). the t - dna used in these experiments carried the s . cerevisiae ura3 gene for selection of ura + colonies after t - dna transfer to the haploid yeast strain rsy12 ( ura3δ ). however , in this system , only yeast strains could be used in which the ura3 gene had been deleted to avoid homology between the incoming t - dna and the s . cerevisiae genome . we wanted to set up a system in which t - dna transfer to any yeast strain could be studied . therefore , two new binary vectors were constructed using the dominant marker kanmx ( wach et al ., 1994 ), which confers resistance against geneticin ( g418 ). the t - dna of psdm8000 carries only the kanmx marker . since this kanmx marker consists of heterologous dna , lacking any homology with the s . cerevisiae genome , we would expect this t - dna to integrate by ir at a random position in the yeast genome . to be able to compare this with t - dna integration by homologous recombination , psdm8001 was constructed . the t - dna of psdm8001 carries the kanmx marker flanked by sequences from the s . cerevisiae pda1 locus . the pda1 sequences have been shown to mediate the integration of t - dna by hr at the pda1 locus on chromosome v ( bundock et al ., 1995 ). cocultivations between agrobacterium strains carrying psdm8000 and psdm8001 , respectively , and the haploid yeast strains yph250 and jkm115 , respectively , were carried out as described in the experimental part . g418 - resistant colonies were obtained at low frequencies for yph250 ( 1 . 6 × 10 − 7 ) and jkm115 ( 1 . 2 × 10 − 5 ) after t - dna transfer from psdm8000 ( table 2 ). t - dna transfer from psdm8001 - generated g418 - resistant colonies at higher frequencies ( 2 . 4 × 10 − 5 for yph250 and 1 . 8 × 10 4 jkm115 , table 2 ). the ratio of homologous recombination versus illegitimate recombination is determined by comparing the frequencies of g418 - resistant colonies obtained from cocultivations using either psdm8001 or psdm8000 . this showed that a t - dna from psdm8001 was 150 - fold more likely to integrate than a t - dna from psdm8000 in yph250 ( table 2 ). a similar difference was previously seen using t - dnas with the ura3 marker ( bundock and hooykaas , 1996 ). in contrast , t - dna from psdm8001 was only 16 - fold more likely to integrate than a t - dna from psdm8000 in jkm115 . there was no significant difference in the frequency of t - dna transfer to these two yeast strains as was determined by t - dna transfer experiments in which a t - dna that carried the kanmx marker and the yeast 2 micron replicon was employed . therefore , the differences in the frequencies of t - dna integration by hr and ir between the yeast strains yph250 and jkm115 , respectively , most likely contributed to differences in the capacities of their hr and ir recombination machineries . we confirmed by pcr that the t - dna from psdm8001 became integrated at the pda1 locus by homologous recombination ( data not shown ). in order to find out whether the t - dna from psdm8000 had integrated randomly by ir , yeast target sites for integration were determined from eight g418 - resistant yph250 colonies by vectorette pcr ( for detailed description see materials and methods ). chromosomal dna was isolated and digested with a restriction enzyme that cuts within the t - dna . a vectorette was ligated to the digested dna and a pcr was performed using a t - dna - specific primer and a vectorette - specific primer . the pcr product obtained was cloned into pgemteasy and sequenced using a t - dna - specific primer . the position of the t - dna insertion was determined by basic blast search of the yeast genome ( www - genome . stanford . edu / sgd ). we were thus able to map the position of the t - dna insertions of all eight g418 - resistant colonies analyzed . they were present at different positions spread out over the genome . comparison of the t - dna sequence and yeast target site sequences did not reveal any obvious homology . these data show that the t - dna from psdm8000 had integrated via an ir mechanism as expected . the following characteristics have previously been observed for t - dnas integrated by ir : a ) the 3 ′ end of the t - dna is usually less conserved compared to the 5 ′ end , b ) microhomology is sometimes present between the t - dna ends and the target site , c ) integration is often accompanied by small deletions of the target site dna ( matsumoto et al ., 1990 ; gheysen et al ., 1991 ; mayerhofer et al ., 1991 ; bundock and hooykaas , 1996 ). similar characteristics were seen in the currently analyzed eight t - dna insertions . in three strains , we observed microhomology of 2 - 6 bp between the lb and yeast target site ( fig1 , wt . 51 was taken as an example ). in five strains , deletions of 1 - 5 bp of yeast target site dna was found and we observed deletions , varying from 1 - 112 bp , of the 3 ′ end of the t - dna in seven out of eight analyzed strains . in only one strain , the 3 ′ end appeared to be intact . the 5 ′ end of the t - dna was conserved in almost all strains . in only two strains , we observed small deletions of 1 and 2 bp at the 5 ′ end of the t - dna . thus , we can conclude that the t - dna from psdm8000 had integrated via the same ir mechanism described before . a averages of two or more independent experiments are shown . frequencies are depicted as the number of g418 - resistant colonies divided by the output number of yeast cells ( cells / ml ). b the frequency of t - dna integration by ir ( psdm8000 ) divided by the frequency of t - dna integration by hr ( psdm8001 ). c the ratio of ir / hr in the mutant strain divided by the ratio of ir / hr in the wild - type strain . the observation that the t - dna from psdm8000 integrates by ir into the yeast genome allowed us to use this system to study the effect of host factors on the process of integration . many proteins involved in various forms of dna recombination have been identified in yeast . in order to determine the roles of a representative set of these enzymes in t - dna integration , we compared t - dna transfer and integration in wild - type yeasts with that of strains carrying disruptions of the genes encoding several recombination proteins . the rad51 , rad52 , ku70 , rad50 and lig4 genes were deleted from yph250 using the one step disruption method . yeast strains carrying deletions in mre11 , xrs2 and sir4 in the jkm115 background were kindly provided by dr . j . haber . the results of cocultivations with these yeast strains are shown in table 2 . in rad51 and rad52 mutants , which are impaired in homologous recombination , the frequency of t - dna integration by hr was sixteen - and nine - fold lower , respectively , than observed for the wild - type ( table 2 ). this showed that rad51 and rad52 play a role in t - dna integration by homologous recombination . in the ir defective ku70 , rad50 , lig4 , mre11 , xrs2 and sir4 mutants , the frequency of t - dna integration by hr did not differ significantly from that observed for wild - type ( table 2 ). this showed that these genes do not play a role in t - dna integration by homologous recombination . the frequency of t - dna integration by ir in a rad51 mutant did not differ significantly from that observed for wild - type , whereas in a rad52 mutant , the frequency was about two - fold higher ( table 2 ). this showed that rad51 and rad52 are not involved in t - dna integration by ir . the product of the rad52 gene may compete with ir - enzymes for the t - dna and thereby inhibits integration by ir to some extent . strikingly , in rad50 , mre11 , xrs2 , lig4 and sir4 mutants , the frequency of t - dna integration by ir was reduced dramatically : 20 - to more than 40 - fold ( table 2 ). t - dna integration by ir seemed to be completely abolished in the ku70 mutant . we did not obtain any g418 - resistant colonies from several cocultivation experiments . this strongly suggests that ku70 plays an important role in random t - dna integration in yeast . since t - dna integration by hr is normal in these mutants , these results clearly show that the yeast genes ku70 , rad50 , mre11 , xrs2 , lig4 and sir4 are involved in t - dna integration by illegitimate recombination . 3 . chromosomal distribution of integrated t - dna copies in ir - defective s . cerevisiae . from several cocultivation experiments with the rad50 , mre11 , xrs2 , lig4 and sir4 mutants , we obtained a small number of g418 - resistant colonies . the t - dna structure was determined for a number of these lines . to this end , chromosomal dna was isolated from these g418 - resistant colonies and subjected to vectorette pcr to amplify junction sequences of genomic dna and t - dna . pcr products were cloned and sequenced . the yeast sequences linked to the t - dna were used in a blast search at www - genome . stanford . edu / sgd to determine the t - dna integration sites . strikingly , analysis of lb / genomic dna junctions revealed that in two out of three rad50 , four out of six mre11 and two xrs2 strains analyzed , t - dnas had integrated in telomeres or subtelomeric regions ( rad50k . 1 , rad50k . 6 , mre11k . 8 , mre11k . 11 , mre11k . 14 , mre11k . 17 , xrs2k . 1 and xrs2k . 17 ; table 3 and fig1 ). s . cerevisiae telomeres generally consist of one or more copies of the y ′ element followed by telomerase - generated c ( 1 - 3 ) a / tg ( 1 - 3 ) repeats ( zakian , 1996 ). in two rad50 strains , two mrell strains and one xrs2 strain , the lb was found to be fused to this typical telomerase - generated c ( 1 - 3 ) a / tg ( 1 - 3 ) repeat ( rad50k . 1 , rad50k . 6 , mre11k . 14 , mre11k . 17 and xrs2k . 1 ; fig1 ). besides , we also found one t - dna insertion in a ty ltr element in the mre11 mutant and two insertions in the rdna region , present in chromosome xii , in the mre11 and rad50 mutants ( mre11k . 5 , mre11k . 4 and rad50k . 5 , respectively ; table 3 and fig1 ). the 3 ′ end of the t - dna was truncated in all strains . deletions of 3 - 11 bp of the 3 ′ end of the t - dna were observed ( fig1 ). microhomology between the 3 ′ end of the t - dna and yeast target site was only found in two lines ( 5 bp in mre11k . 4 and 4 bp in mre11k . 14 ; fig1 ). for the t - dna copies present at the yeast telomeres , the rb / genomic dna junction sequences could not be obtained from these strains using vectorette pcr . this was only possible for the rad50 and mrell strains carrying the t - dna in the rdna region on chromosome xii . in both strains , the rb was intact and no homology between the 5 ′ end of the t - dna and the yeast target site was found ( data not shown in fig1 ). previously , target sites for t - dna integration in the genome of s . cerevisiae strain rsy12 were determined ( bundock and hooykaas , 1996 ; bundock , 1999 ). in four out of 44 strains analyzed , t - dna copies were integrated in rdna , ty ltr elements ( in two strains ) and a subtelomerically located y ′ element , respectively . in addition , we determined the position of t - dna integration in ten s . cerevisiae yph250 strains . we did not find any t - dna insertions in rdna , ltr elements or subtelomeric / telomeric regions amongst these ten . pooling all insertions analyzed in wild - type ( 54 ), in two out of 54 strains analyzed ( 4 %), insertions were found in a ty ltr element and in two other strains in the rdna repeat ( 2 %) and a subtelomeric region ( 2 %), respectively . in contrast , we report here that t - dna in yeast strains mutated in rad50 , mre11 or xrs2 t - dna integrates preferentially in ( sub ) telomeric regions ( eight out of eleven lines : ˜ 73 %) of rad50 , mre11 and xrs2 mutants ( table 3 ). from the remaining strains , two t - dnas were present in rdna and one in a ty ltr element , respectively . apparently , the rdna repeat is also a preferred integration site in these mutants (˜ 18 % vs . ˜ 2 % in the wild - type ). telomeres consist of a large array of telomerase - generated c ( 1 - 3 ) a / tg ( 1 - 3 ) repeats (˜ 350 bp ). in the subtelomeric regions , two common classes of y ′ elements , 6 . 7 and 5 . 2 kb , can be found ( in most strains , chromosome i does not contain y ′) ( zakian and blanton , 1988 ), making the average size of these regions ˜ 6 . 0 kb . thus , the yeast genome contains ( 16 × 2 × 0 . 35 )+( 15 × 2 × 6 . 0 )= 191 kb of subtelomeric / telomeric sequences . the yeast genome is 12 , 052 kb in size , which means that only 1 . 6 % of the genome consists of subtelomeric / telomeric sequences . in accordance with this , we observed in only 2 % of the wild - type strains t - dna copies inserted in a subtelomeric region , which we would expect on the basis of random t - dna integration . in contrast , in the rad50 , mre11 and xrs2 mutants , 73 % of the t - dna insertions were found in the ( sub ) telomeric region . analysis of seven lines revealed that in the sir4 mutant t - dna was integrated randomly into the yeast genome . so , although sir4 has an effect on the efficiency of t - dna integration by ir , the pattern of t - dna distribution in the transformants seems similar as in the wild - type strain . in the sir4 mutant t - dna , integration by ir was characterized by truncation of the 3 ′ end of the t - dna , deletions at the target site and microhomology between the lb and the target site ( data not shown ); this was observed for t - dna integration by ir in the wild - type . these results clearly show that in the rad50 , mre11 and xrs2 mutants , the t - dna , if integrated at all , becomes preferentially inserted in telomeres or subtelomeric regions and that the genomic distribution of integrated t - 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