| Rabbits preferentially use a single V;To determine whether rabbit RAD51 is involved in somatic gene conversion of IgH genes, I examined its expression in appendix. I found that RAD51 mRNA levels are increased 2- to 5-fold in appendix of six week-old rabbits relative to the levels observed in appendix from two week-old rabbits, a time at which somatic gene conversion is not detectable.;To determine whether the increase in RAD51 expression in the appendix was found specifically in B lymphocytes, I sorted IgM;To determine whether the rabbit gene could function in gene conversion, I used a S. cerevisiae rad51 mutant (yeast) that is unable to switch mating-type by gene conversion. No mating-type switching occurred in cells expressing either rabbit RAD51 in the antisense orientation or vector alone.;To this end, I cloned the rabbit RAD51 cDNA by the polymerase chain reaction (PCR) and further, I used this clone as a probe to screen a genomic library to isolate the gene. Nucleotide sequence analysis showed that rabbit RAD51 was 65% identical to S. cerevisiae RAD51, suggesting that the function of RAD51 would also be conserved. Importantly, the region of the gene sharing the most identity (71%) is required for homologous recombination activity of S. cerevisiae RAD51.;Lastly, the expression of several genes that participate in recombinational DNA repair are DNA damage-inducible. To determine whether rabbit RAD51 is also DNA damage-inducible, I irradiated the rabbit cell line, 55D1, with 6 and 7 Gray ;In summary, I demonstrated that RAD51 protein levels increase in B lymphocytes from the appendix, a tissue where somatic gene conversion of IgH genes occurs, at a time when diversification is occurring. Further, rabbit RAD51 can function in gene conversion in yeast. These observations strongly suggest that rabbit RAD51 is involved in somatic gene conversion of rabbit IgH genes. (Abstract shortened by UMI.). |
