Immunoglobulin (Ig) variable (V) region genes are assembled in precursor B (pre-B) lymphocytes from multiple germline segments. The heavy-chain V-region gene is composed of variable (VH), diversity (D) and joining (JH) segments; kappa (K) and lambda (lambda) light-chain V-region genes have analogous VL and JL segments. Assembly of Ig V-gene segments, as well as those of the highly related T-cell receptor, is regulated at several levels and shows both stage and tissue specificity; for example Ig heavy-chain V-gene assembly precedes that of Ig light chains during B-cell differentiation. Joining of all classes of V-gene segments involves conserved recognition sequences that are probably targets for a common recombinase. Evidence has been presented suggesting that rearrangement of specific classes of segments is regulated by modulation of their accessibility to the recombinase. To elucidate mechanisms which control V-region gene assembly, we have investigated the effect of flanking gene expression on the frequency at which introduced V-gene segments are assembled in pre-B cell lines. Our findings suggest that transcription may play a direct role in the regulation of immunoglobulin V-gene assembly.

Recently, we reported that thymocytes expressing the CD8 molecule on their surface can give rise to CD4+CD8+ double-positive and CD4+ single-positive progeny following intrathymic transfer into an irradiated host mouse. Thymcoytes expressing a high density of CD8, referred to as CD8hi, and those expressing a low density of the molecule, CD8lo, were both able to differentiate in vivo. In this study we examined the ability of these CD8+ thymocytes populations and of CD4-CD8- double-negative thymocytes to change their phenotype during brief in vitro culture. CD8+ thymocytes were prepared by anti-CD4 plus complement lysis followed by positive selection of the survivors on anti-CD8-coated plates. After 16 h of culture, greater than 60% of CD8+ thymocytes became double-positive. Both CD8hi and CD8lo cells were able to show this in vitro change: about 30% of the former and about 80% of the latter became double-positive. In contrast to this, double-negative thymocytes which had been depleted of cells expressing low densities of CD8 did not show such a phenotypic conversion in vitro. Further panning experiments suggested that all of the CD8+ thymocytes actually express a low surface density of the CD4 molecule which is undetectable in our cytofluorometric assays.

The role of T cell receptor (TcR) gamma genes in T cell development has not been determined. To extend our understanding of the repertoire of TcR gamma expression, we prepared a cDNA library from CD4-CD8- adult BALB/c thymocytes and cloned and sequenced 15 TcR gamma genes from this cDNA library. We found that two clones were transcripts of the unrearranged C gamma 2 gene and that three clones terminated in the J gamma 2 region. Nine of the remaining clones were V gamma 1.2 J gamma 2 C gamma 2 genes and five of these were in frame. Only one clone corresponded to C gamma 1 and resulted from an in-frame V gamma 2 J gamma 1 C gamma 1 join. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the gamma chain proteins from the surface of both BALB/c and C57BL/6 adult CD4-CD8- thymocytes did not detect the 32-kDa V gamma 1.2 C gamma 2 protein, but did detect the 35-kDa V gamma C gamma 1 protein. These results suggest that despite the abundance of full-length functional V gamma 1.2 C gamma 2 transcripts in the thymocyte subset, the protein product is not expressed on the cell surface as the predicted 32-kDa gamma protein. Finally, our analysis of the V-J joining of the gamma genes reveals both flexibility at the V-J junction and extensive N-region nucleotide addition that lead to diversity of the predicted protein sequence.