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PMID: 3023902 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.

Molecular and cellular biology ·Vol. 6 ·No. 5 ·1986-05-00 ·Pages 1711-21

McIntosh EM, Haynes RH

Abstract

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

MeSH Terms
Amino Acid Sequence Base Sequence DCMP Deaminase/genetics DNA Restriction Enzymes Genes Genes, Fungal Nucleic Acid Hybridization Nucleotide Deaminases/genetics Plasmids Saccharomyces cerevisiae/enzymology,genetics Sequence Homology, Nucleic Acid Transcription, Genetic
Chemicals
DNA Restriction Enzymes Nucleotide Deaminases DCMP Deaminase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
McIntosh E M
Haynes R H
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.6.5.1711-1721.1986
Published
1986-05-00
Pages
1711-21
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC367699
Subset
IM
Databases
GENBANK
M13010
External Links
PubMed source DOI: 10.1128/mcb.6.5.1711-1721.1986 PMC Full Text View journal details
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