Preparation of product-specific antisera by gene fusion: antibodies specific for the product of the yeast cell-division-cycle gene CDC28.
Gene
; 20(2): 255-65, 1982 Dec.
Article
em En
| MEDLINE
| ID: mdl-6299892
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
/
DNA Fúngico
/
Genes
Idioma:
En
Ano de publicação:
1982
Tipo de documento:
Article