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