We have examined at the molecular level the CDR3 and adjacent regions in peripheral blood B lymphocytes ofnormal individuals. A total of 111 sequences (12-28 sequences from six individuals) were obtained after cloning of the polymerase chain reaction-amplified segments into plasmids or phage. The average length of the VDJ joining was 109 nucleotides, with a range from 79 to 151. Approximately 75% of the sequences were in frame when translated into amino acids. Among the JH segments, JH4 was found most frequently (in 52.5% of the sequences), and JH1 and JH2 segments the least frequently (-I% of the clones). A polymorphic JH6 gene with a one-codnn deletion accompanied by a base change was present in two ofsix patients. Preferential breakpoints were found for JH2, J. 3, J 4, and Jx5, although the breakpoints of JH6 were distributed more heterogenously.

In-90% of the cases, significant homology of the D regions with published D sequences was found. Preferential usage of a particular coding frame was observed in in-frame sequences utilizing DA, D21/9, and DMl segments. However, in general, all coding frames of germline D genes were used to generate CDR3s. Eight sequences that have a DN1-like D sequence with two base changes at the same positions were identified, suggesting the likely existence of a new germ line D gene belonging to the DN families. Using probes specific for a particular CDR3, the frequency of a specific B cell clone in the peripheral blood of normal individuals was estimated to be at most as high as 1/20,000.