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Abstract
A nuclear gene (CBP1) of Saccharomyces cerevisiae involved in 5' end processing of the cytochrome b pre-mRNA has been cloned and sequenced. The gene was originally selected by transformation of cbp1 mutants with a recombinant plasmid bank of random fragments of yeast nuclear DNA ligated to a plasmid vector with autonomous replicative function in yeast. The recombinant plasmid pG60/T10 with a nuclear DNA insert of 6.7 kilobase pairs (kb) was used to construct a new plasmid pool with the CBP1 gene on smaller fragments of nuclear DNA. A number of subclones have been isolated with plasmids carrying inserts of 2.4 to 6.1 kb. The plasmid designated pG60/T31 confers respiratory competency to cbp1 mutants and restores their ability to synthesize mature cytochrome b mRNA. The pG60/T31 plasmid has a nuclear DNA insert of 2.4 kb. The sequence of the cloned fragment reveals only one open reading frame capable of coding for a protein. The reading frame is 1962 nucleotides long and codes for a basic polypeptide with a molecular weight of 76,140. A transcript of a size commensurate with the length of the gene has been detected in wild type yeast.
Highlights
NUCLEOTIDESEQUENCE OF A YEAST NUCLEAR GENE ( C B P l ) INVOLVED IN 5’ END PROCESSING OF CYTOCHROME b PRE-mRNA*
The gene was originally selected by transformation of cbpl mutants with a recombinantplasmidbank of random fragments of yeast nuclear DNA ligated to a plasmidvector with autonomous replicative function in yeast
Complementation of cbpl mutants with pG60/T31 has been shown by genetic means to be due to the presence in the plasmid of the wild type copy of the CBPl gene
Summary
NUCLEOTIDESEQUENCE OF A YEAST NUCLEAR GENE ( C B P l ) INVOLVED IN 5’ END PROCESSING OF CYTOCHROME b PRE-mRNA*. In the present study we have used a previously isolated recombinant plasmid capable of complementing cbpl mutants [5] to subclone the wild type gene on a smaller fragment of nuclear DNA, amenable for DNA sequencing.
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