Expression of bovine beta-1,4-galactosyltransferase cDNA in COS-7 cells.
A bovine beta-1,4-galactosyltransferase (GT; EC 2.4.1.90) cDNA in an Okayama-Berg vector, pLsGT, was constructed from a partial cDNA clone and a genomic fragment. We report that the cDNA sequence of pLsGT, in a transient expression assay in COS-7 cells, codes for an enzymatically active GT protein. There is an approximately 12-fold increase in the GT activity in pLsGT-transfected cells compared to cells transfected with the antisense bovine GT construct, pLasGT, or pSV2Neo or mock-transfected cells. The increased activity is correlated with the increase in bovine GT mRNA, which is distinguishable from COS GT mRNA with a 3'-end-specific probe of pLsGT. The expressed GT activity is modulated by alpha-lactalbumin, which changes the acceptor specificity to glucose to synthesize lactose. Polyclonal antibody raised against SDS/PAGE-purified bovine milk GT and a monoclonal antibody (mAb 4-10) directed against a synthetic peptide corresponding to the amino-terminal region of the protein encoded by pLsGT bind the expressed protein, and the resulting immunoprecipitates exhibit GT enzymatic activity.
Masibay
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Division of Cancer Biology and Diagnosis, National Cancer Institute, Bethesda, MD 20892.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005260:Female; D005796:Genes; D007700:Kinetics; D007788:Lactose Synthase / Q000235:genetics*; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000235:genetics* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D010957:Plasmids; D011247:Pregnancy; D015183:Restriction Mapping; D014162:Transfection
D004247:DNA; D007788:Lactose Synthase; D009237:N-Acetyllactosamine Synthase