A dramatic change in the antibody repertoire of isogeneic Xenopus is associated with the developmental transition from the larval to the adult form. To define this process in molecular genetic terms, VH genes will be identified in isogeneic """"""""clonal"""""""" animals using heterologous cross-hybridization, the extent of the VH gene family will be assessed and genomic organization will be established with segment specific probes (JH and constant region) derived from VH+ cDNAs as well as by DNA sequencing (D regions). The organization of the VH locus will be studied in lambda vectors and in newly developed cosmid vectors, which exhibit stable replication characteristics. Microscale mRNA isolation and high efficiency cDNA cloning in lambda will permit the identification of immunoglobulin mRNAs from antigen binding and polyclonally activated cells of both adult and developmentally blocked, larval animals. These will be characterized at the sequence level and their corresponding germline structures will be identified using synthetic probe techniques. Extended linkage maps will be constructed and the relationships between developmental expression, gene structure and chromosomal organization will be analyzed. Flanking region sequence comparisons will be used to determine whether specific DNA patterns can be correlated with developmental expression. Adult and larval genes will be injected into fertilized eggs and their regulation during development studied using gene specific probes. The ability to microinject large numbers of eggs with fusion gene constructs will greatly facilitate the study of essential regulatory sequences. Studies will be extended to include light chain genes. Overall we expect to characterize the developmental regulation of the antibody repertoire at the gene level and identify the control mechanisms that function in this system. Such findings have far reaching significance in terms of our understanding the developmental regulation of eukaryotic gene expression and the ontogenesis of adaptive immunity.
