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PMID: 6287231 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA.

Molecular and cellular biology ·Vol. 2 ·No. 3 ·1982-03-00 ·Pages 221-32

Zakian VA, Scott JF

Abstract

Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.

MeSH Terms
Base Sequence Chromatin/analysis Chromosomes Cloning, Molecular DNA Restriction Enzymes DNA, Circular/analysis,biosynthesis DNA, Recombinant Meiosis Mitosis Plasmids Saccharomyces cerevisiae/genetics Transformation, Genetic
Chemicals
Chromatin DNA, Circular DNA, Recombinant DNA Restriction Enzymes
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Zakian V A
Scott J F
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.2.3.221-232.1982
Published
1982-03-00
Pages
221-32
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC369780
Subset
IM
Grants
NIGMS NIH HHS · GM26938 · United States
NIGMS NIH HHS · GM27000 · United States
External Links
PubMed source DOI: 10.1128/mcb.2.3.221-232.1982 PMC Full Text View journal details
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