Recombinant DRB sequences produced by mismatch repair of heteroduplexes during cloning in Escherichia coli.
Recombinant chimeric sequences originating from a mixture of the sequences of two different alleles are frequently found after amplification and cloning in Escherichia coli of exon 2 of the major histocompatibility complex (MHC) DRB genes. Several authors have suggested that the recombinant molecules result from in vitro recombination during PCR; nevertheless, a clear experimental demonstration of this hypothesis is lacking. In order to understand the mechanism producing the chimeric sequences, we set up a simple experiment based on the different restriction patterns of parental and recombinant sequences. Our data demonstrate that in the analysed case most of the recombinant variants were not produced by in vitro recombination during PCR, but were the result of the mismatch repair of heteroduplex molecules during cloning in E. coli. The high mutation rate in the alpha-helix region of DRB expressed genes, both after cloning in E. coli and after the germ-line differentiation process in vertebrates, suggests that the observed mutations are the result of similar gene conversion processes, probably favoured by chi-dependent microrecombination events.
Longeri
M
M
Istituto di Zootecnica, Faculty of Veterinary Medicine, University of Milan, Italy. maria.longeri@unimi.it
Zanotti
M
M
Damiani
G
G
D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D002417:Cattle / Q000235:genetics*; D003001:Cloning, Molecular; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics; D006579:Heterozygote; D006649:Histocompatibility Antigens / Q000235:genetics*; D006720:Homozygote; D008969:Molecular Sequence Data
D006649:Histocompatibility Antigens