ANALYSIS OF HOMOLOGY-DIRECTED RECOMBINATION IN VDJ JUNCTIONS FROM CYTOPLASMIC IG(-) PRE-B-CELLS OF NEWBORN MICE

We previously showed that most VD and DJ gene combinations from newbor n surface Ig(-) pre-B cells had one to three predominant junctions, al l of which occurred at the sites of short sequence homologies between the two coding ends. Because the majority of sequences that are presen t in pre-B cells are in-frame, however, the possibility existed that t he frequency of occurrence of predominant IgH junctions was skewed by proliferation of pre-B cells with productive rearrangements. In this s tudy, we analyzed cytoplasmic Ig(-) pre-B cells, because these cells s hould not yet be subject to such selection. Two-thirds of the rearrang ements from this population in the adult were out-of-frame, suggesting that these rearrangements are unbiased. In newborn clg(-) pre-B cells , DJ junctions still showed the same predominant sequences as slg(-) p re-B cells, but there was less use of predominant junctions in VD junc tions for three of four different V-H genes analyzed. For those three V-H genes, an average of 30% of the sequences were in-frame. When only the in-frame rearrangements from these clg(-) newborn cells were anal yzed, frequencies of predominant VD junctions were comparable to those in slg(-) pre-B cells. For sequences using the V(H)S107/V11 gene, how ever, 67% of the junctions were created at the site of the same dinucl eotide in the V gene, and as a result, 73% of the sequences were in-fr ame. Thus homology-directed recombination does not initially produce a s much junctional homogeneity as anticipated in all VD combinations, a lthough it is a frequently used mechanism in the early fetal/neonatal gene rearrangements.