Richard T. Surosky

The phenotype of the minichromosome maintenance mutant mcm3 is characteristic of mutants defective in DNA replication.

MCM3 is an essential gene involved in the maintenance of minichromosomes in yeast cells. It encodes a protein of 971 amino acids that shows striking homology to the Mcm2 protein. We have mapped the mcm3-1 mutation of the left arm of chromosome V approximately 3 kb centromere proximal of anp1. The mcm3-1 mutant was found to be thermosensitive for growth. Under permissive growth conditions, it was defective in minichromosome maintenance in an autonomously replicating sequence-specific manner and showed an increase in chromosome loss and recombination. Under nonpermissive conditions, mcm3-1 exhibited a cell cycle arrest phenotype, arresting at the large-bud stage with an undivided nucleus that had a DNA content of nearly 2n. These phenotypes are consistent with incomplete replication of the genome of the mcm3-1 mutant, possibly as a result of limited replication initiation at selective autonomously replicating sequences leading to cell cycle arrest before mitosis. The phenotype exhibited by the mcm3 mutant is very similar to that of mcm2, suggesting that the Mcm2 and Mcm3 protein may play interacting roles in DNA replication.

Isolation and characterization of the centromere from chromosome V (CEN5) of Saccharomyces cerevisiae.

We have cloned a functional centromeric DNA sequence from Saccharomyces cerevisiae. Using the 2 mu chromosome-loss mapping technique and meiotic tetrad analysis, we have identified this DNA sequence as the centromere of chromosome V (CEN5). The CEN5 sequence has been localized on an 1,100-base-pair BamHI-BglII restriction fragment. Plasmids containing CEN5 and an autonomously replicating sequence are mitotically stable in S. cerevisiae and segregate in a Mendelian fashion during meiosis.

Site-directed chromosomal rearrangements in yeast.

Publisher Summary This chapter describes an extension of gene disruption or gene replacement, which allows the replacement of large regions of the genome with small DNA fragments. Using this technique, a series of large chromosomal deletions and a circular derivative of a chromosome has been generated. Diploid strains are used in the construction of large chromosomal deletions, because extensive deletions are lethal in haploid cells. The chapter describes a homozygous diploid for mutation in the Z gene. Three criteria are used to identify the desired deletions: (1) other markers in the deleted region of the chromosome are lost; (2) diploids sporulate to produce only two viable spores; and (3) the restriction map determined by genomic blot analysis agrees with the predicted restriction map of the desired deletion. The chapter discusses deletions on one arm of the chromosome, the physical analysis of deletion chromosomes, deletions on both arms of the chromosome, the deletion derivatives of chromosome III, and the construction of ring chromosomes.

Host Factors in Nuclear Plasmid Maintenance in Saccharomyces Cerevisiae

In yeast, the initiation of DNA replication on chromosomes is believed to occur at specific sequences known as autonomously replicating sequences (ARSs). We previously isolated a number of mutants that are defective in the maintenance of minichromosomes. Analysis of these mutants suggests that although ARSs differ greatly from one another in their primary sequences, they appear to share a common enzyme complex for the initiation of DNA replication. However, this initiation enzyme complex probably binds with differential affinity to different ARSs. This idea is corroborated by our identification of an ARS-binding protein that binds to different ARSs with different efficiencies.