HUMAN CORD-BLOOD KAPPA REPERTOIRE

To determine the V kappa gene utilization in cord blood, we made libra ries of Ig kappa sequences from two cord blood cDNA samples. The rearr anged sequences were amplified using random amplification of cDNA ends PCR, ensuring unbiased amplification of all V kappa genes. Although t he human kappa locus contains similar to 38 potentially functional V g enes, we observed that similar to 75% of the 146 sequences from our tw o samples used only nine V kappa genes. Using leader-specific primers, we also amplified V kappa I and V kappa III rearrangements from genom ic DNA from one of these individuals. Nonproductive rearrangements giv e an approximation of the relative frequency of gene rearrangement. So me of the genes that were overused in the cDNA libraries were also obs erved to rearrange frequently, but others did not show high rearrangem ent frequencies, suggesting that cellular selection caused their incre ase in the periphery. Surprisingly, we observed a high frequency of re arrangements using L9, which has been reported to be a defective V kap pa gene. Sequence analysis of the unrearranged gene revealed two new f unctional alleles of this gene. We observed that N nucleotides were pr esent in 29% of the productive sequences from cord blood DNA and RNA. To determine the actual rate of N region addition, we analyzed V-J jun ctions of rearrangements of two nonfunctional V genes. Forty-six perce nt of those cord blood sequences contained N regions. In comparison, 5 7% of junctions of the rearranged nonfunctional gene from adult PBMC c ontained N regions. Finally, we observed that CDR3 length heterogeneit y was more pronounced for V kappa III genes than for all of the other V kappa families.