The long-term (Phase II) objective of this proposal is the molecular characterization (cDNA cloning, expression) of murine and human genes coding for a growth factor for mature B-lymphocytes (m-BC-GF). Our approach will be to construct a cDNA library from sized, active, m-BC-GF mRNA and to isolate a full-length m-BC-GF cDNA by hybridization with an oligonucleotide probe deduced from protein sequence information derived from purified m-BC-GF protein. During Phase I of this proposal, m-BC-GF activity will be purified from supernates produced by a cloned murine lymphoma cell line (EL-4 lx cell line) recently developed in our laboratory. mRNA coding for m-BC-GF activity in vitro will also be prepared from the EL-4 lx cell line and size fractionated in preparation for cDNA cloning. Once murine m-BC-GF cDNAs are isolated, they will be expressed in appropriate yeast and E. coli vectors. Recombinant m-BC-GF will then be tested for biological activity in murine model systems of B-cell deficiency. Finally, murine cDNAs for m-BC-GF will be used as probes for identification by Southern blot hybridization of analogous human cDNAs. m-BC-GF could be of value in clinical restoration of antibody responsiveness in cases of viral, chemotherapeutic, or radiation induced immune suppression, and for the in vitro generation of human B-lymphocytes and/or hybridomas.
