Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation.

Abstract

The CDC8 gene, whose product is required for DNA replication in Saccharomyces cerevisiae, has been isolated on recombinant plasmids. The yeast vector YCp5O bearing the yeast ARSI, CEN4, and URA3 sequences, to provide for replication, stability, and selection, respectively, was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to.grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping. This fragment lies 1 kilobase downstream from the well-characterized sup4 gene, a gene known to be genetically linked to CDC8, thus confirming that the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the lack of CDC8 gene product. The CDC8 protein of Saccharomyces cerevisiae is essential for chromosomal replication in vivo and is required for cell division. When the temperature-sensitive cdc8 mutant is grown at the permissive temperature, 23°C, and then shifted to the restrictive temperature, 36°C, cells accumulate that contain a nucleus located at the isthmus between parent cell and bud, but which do not divide. The first wave of DNA synthesis after shift up does not occur (8). The product of the CDC8 gene is apparently required throughout the period of DNA synthesis, since synchronized cultures of cells defective in the gene cease nuclear DNA replication when shifted to 36°C within the S period (9). Mitochondrial DNA replication also ceases at the nonpermissive temperature in cdc8 mutants (22), and replication of the 2-,um circle plasmid is defective in cdc8 mutants (17).Recently the CDC8 protein has been purified to homogeneity, using in vitro replication systems (1, 15). The purified protein binds to singlestranded DNA and stimulates DNA polymerase I activity on single-stranded DNA templates. The CDC8 protein may be identical to protein C, discovered by Chang et al. (4).To carry out detailed biochemical and functional characterization of an enzyme involved in DNA replication, it is necessary to obtain large quantities of purified protein. In Escherichia coli, one way to overcome the problem of low yield of replication proteins is to clone the gene coding for a given protein into temperatureinducible bacteriophage lambda vectors or into a high-copy-number plasmid. Overproduction of the replication proteins DNA polymerase I (12), DNA ligase (23), and dnaC protein (13) has been achieved. Overproduction of the LEU2 gene product in yeast strains carrying the LEU2 gene on an autonomously replicating, high-copy-number plasmid has also been achieved (10). Therefore, the same approach used in E. coli is applicable in yeast.In this co...

RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination.

“…The effect of the RADI gene on recombination of the his3Y3' and his3A5' duplication in the radl A rad52A double mutant strain was further examined by transforming it with plasmids carrying the wild-type RAD1 gene or different radl mutant alleles. The RAD1 gene or mutant radl alleles were cloned into plasmid YCp5O, which is a single-copy, autonomously replicating yeast plasmid, because of the yeast CEN4 and ARS1 genes (14). As expected, the RAD1 gene on the single-copy CEN plasmid pRR117 raises the HIS3+ recombinant frequency in the radlA radS2A mutant to the rad52A level (Table 3).…”

Mutations Altering the Mitochondrial-Cytoplasmic Distribution of Mod5p Implicate the Actin Cytoskeleton and mRNA 3′ Ends and/or Protein Synthesis in Mitochondrial Delivery

“…Two plasmids, pT3 and pT7, that complemented the temperature-sensitive growth phenotype of mdp2-1 were found by screening a singlecopy YCp50 library (43). The plasmids contained overlapping 6-and 4-kbp inserts which hybridized to chromosome VI and the clone containing the ACT1 gene which codes for actin (27,52).…”

Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae.

“…Although the isolation of homologous genomic fragments capable of complementing cdc7 from two independently constructed genomic libraries suggests that the cloned fragments carry the CDC7 gene itself, the observation that the effects of many conditional lethal mutations in yeasts can be alleviated by extragenic suppression (20) made it necessary to demonstrate further that the plasmids contained the authentic CDC7 gene within the cloned genomic fragment. The genomic fragment from pMP101 was first subcloned into the vector YIp5, which carries the yeast URA3 gene in PBR322.…”