Preparation of product-specific antisera by gene fusion: antibodies specific for the product of the yeast cell-division-cycle gene CDC28.
Antisera with specificity for the product of a yeast cell-division-cycle (CDC) gene were prepared by immunizing rabbits to a novel hybrid polypeptide. A segment of the yeast gene CDC28 was fused to the Escherichia coli lacZ gene, which encodes beta-galactosidase, by insertion of yeast sequences into the plasmid pBGF1. pBGF1 contains the lac promoter-operator and most of the lacZ gene. An EcoRI site, 16 codons upstream from the carboxyterminus of the beta-galactosidase coding region, served as a convenient splicing site for the heterologous sequences. To insure that an open reading frame be maintained between the two gene segments for some portion of the fusions, the CDC28-encoding segments were first subjected to limited digestion with nuclease BAL31 to produce random junction points. A hybrid polypeptide encoded by such a continuous open reading frame was purified from E. coli by preparative SDS-polyacrylamide gel electrophoresis and used to immunize rabbits. The resulting antisera were shown to have specificity for CDC28 gene product synthesized by cell-free translation of yeast mRNA.
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002453:Cell Cycle; D002455:Cell Division; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004271:DNA, Fungal / Q000276:immunology*; D005796:Genes; D007106:Immune Sera; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D001616:beta-Galactosidase / Q000235:genetics
D000906:Antibodies; D000936:Antigen-Antibody Complex; D004271:DNA, Fungal; D007106:Immune Sera; D004262:DNA Restriction Enzymes; D001616:beta-Galactosidase