The immediate objectives of the research are fivefold: 1. Separation, characterization, and purification of G1cNAcT-2 (UDP-G1cNAc:nLcOse4Cer-(Beta1-3)-N-acetylglucosaminyltransferase) and G1cNAcT-3 (UDP-G1cNAc:i-core(Beta1-6)N-acetylglucosaminyltransferase) present in the neutral, detergent-solubilized super natant from p-1798 mouse T-lymphoma (estrogen-induced). 2. Purification and kinetic studies of Ga1T-4 (UDP-Gal:LcOse3Cer(Beta1-4)galactosyltransferase) from the 0.1% Triton CF-54-solubilized supernatant fraction of p-1798 mouse T-lymphoma. 3. Completion of the biosynthesis in vitro of the I/i biantennary structure from previously synthesized I/i core glycolipid, using purified GalT-4 and G1cNAcT-2. 4. Characterization of FucT-2 (GDP-Fuc:nLcOse4Cer(Alpha1-2)fucosyltransferase) and FucT-3 (GDP-Fuc:LcOse3Cer(Alpha1-3)fucosyltransferase) from Beta-lymphoma (TEPC-15 alkane-induced, and ABLS-140 Abelson virus-induced). Determination of exact chemical linkages in the biosynthetic products (FucAlpha-Gal-G1cNAc-Gal-G1c-Cer and FucAlpha-G1cNAc-Gal-G1c-Cer) obtained from TEPC-15 and ABLS-140. 5. Screening of the mouse genomic library, prepared from a partial Eco RI digestion of AJ mouse genomic DNA (Shotgun in Charon 4a) with a 32P-labeled cDNA insert of GalT-4. Indirect assays for the translation products for particular GalT-4 DNA sequences, using radioiodinated monoclonal antibodies against GalT-4. Our overall goal is to develop an understanding of the specificities of glycosyltransferases in mouse lymphoreticular systems using one T-lymphoma (P-1798) and two B-lymphomas (TEPC-15 and ABLS-140) as model systems.
