Process for the in vivo recombination of DNA sequences having mismatched bases

#The present invention relates to a process for the in vivo recombination of homologous DNA sequences but having a considerable proportion of mismatched bases which can range particularly up to 30%.

#It is thus possible to recombine individual genes, at the level of cells or of organisms of different species and genera having common ancestors.

#The present invention relates also to a process for the production of organisms recombined by crosses and recombination in vivo of different organisms, species and/or genera, as well as a process for the in vivo production of hybrid genes and hence of hybrid proteins coded by the latter.

#FIG. 1 represents a restriction pattern of three intergeneric recombinants from Escherichia coli Hfr and Salmonella typhimurium F.sup.- crosses.

#Conjugation between E. coli Hfr and S. typhimurium F.sup.-.

#Conjugations were made between wild type E. coli Hfr and DNA restriction F.sup.- mutant of S. typhimurium which were deficient for mismatch repair (mutL, mutS, mutH or mutU).

#The transfer origin of the E. coli Hfr strain is at 76.5 min. on the map and xylose is the first marker be injected (78 '). The F.sup.- Salmonella are double auxotrophs for methionine (met A and met E) and cannot ferment xylose (xyl.sup.-). They are resistant to streptomycin. The Hfr E. coli is met.sup.+ and xyl.sup.+ and sensitive to streptomycin. It is also auxotroph for leucine and threonine which allows a triple counterselection after conjugation. E. coli Hfr and S. typhimurium F.sup.- mut.sup.+, mutL, mutS, mutH or mutU deficient for DNA restriction are grown in rich medium (LB). When they reach the log phase (1 to 5 10.sup.8 /ml) 1 ml of Hfr and 1 ml of F.sup.- are mixed and immediately filtered on a millipore filter. This filter is then incubated at 37.degree. C. on a prewarmed rich medium plate. The bacteria are allowed to conjugate for 40 min. and the filter is then put in 1 ml of MgSO.sub.4 10.sup.-2 M and vigorously vortexed for 1' to resuspend the bacteria and separate the conjugants. Aliquots are then plated on selective minimum medium 63 (ref Miller J. H. 72 Experiments in Molecular Genetics (old spring Harbor Laboratory N.Y.) without methionine and with glucose (0.4%) to select for met.sup.+ recombinants and with xylose as sole carbon source (0.4% and methionine (100 .mu.g/ml) to select for xyl.sup.+ recombinants. The two selective media contain neither threonine nor leucine but contain streptomycin (25 .mu.g/ml) to assure a triple counterselection against the Hfr strain. Plates are incubated at 37.degree. C. 40 h, 60 h and 88 h and the recombinant clones counted and studied. A control experiment with the homospecific conjugation Hfr E. coli X F.sup.- E. coli is done in the same conditions. The recA mutation has been moreover introduced into the F.sup.- Salmonella (The recA protein is an indispensable protein for homologous recombination). The results obtained with the mutL and MutS strains for which the effect is the biggest are indicated in Table 1. The frequency per Hfr donor strain of F.sup.- recipients which become xyl+ are indicated in the Table 1 (after subtraction of the revertants obtained with the recipient alone incubation 60 h). Equivalent results have been obtained for the met marker.