Autonomous Replication In Yeast Research Articles

Construction of plasmid vectors that facilitate subcloning and recovery of yeast and Escherichia coli DNA fragments

We have constructed a plasmid vector (pNF2) which is a derivative of the multicopy yeast cloning vehicle YEp24. This derivative contains a single BamHI site flanked immediately on each side by SalI sites. The latter site was selected because it appears to be infrequent in yeast nuclear DNA. Thus, DNA fragments produced by partial digestion with enzymes (such as Sau3A) that cut at frequent intervals and leave single-stranded ends that have sequence homology with BamHI sites, can be conveniently subcloned into this site. Such fragments can then be excised intact by digestion with SalI enzyme. Plasmid pNF2 also contains the kanamycin-resistance ( kan R) gene derived from Tn903 and confers resistance in yeast to the antibiotic G418. pNF2 was converted into an integrating vector (pNF3) by deleting a 2.2-kb EcoRI fragment containing a sequence that determines autonomous replication in yeast. Further deletion of a HindIII fragment containing the yeast URA3 gene converts the plasmid into one containing only pBR322 sequences plus the kan R gene (pNF4).

Identification of a sequence responsible for periodic synthesis of yeast histone 2A mRNA.

Sequences required for the regulated expression of a yeast histone 2A (H2A) gene have been investigated by using fusions between this gene and the Escherichia coli lacZ gene. Fusions containing the entire spacer region in which divergent transcription of the H2A and H2B genes is initiated result in low-level constitutive synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in yeast. Regulated expression (which is characterized by periodic synthesis during the S phase of the cell cycle) is restored when a 1.3-kilobase HindIII fragment containing a small region of the 3' end of the H2B gene is present in either orientation. The regulatory activity in this region appears to be coincident with a sequence that supports autonomous replication in yeast.

Centromeric DNA from Saccharomyces cerevisiae

We have isolated 29 kilobase-pairs of DNA encompassing the centromere-linked gene, TRP1, of Saccharomyces cerevisiae. Two DNA sequences within the isolated region, ARS1 and ARS4, allow autonomous replication of chimeric DNA molecules upon introduction into yeast. Yeast strains transformed by ARS-containing DNA molecules are unstable; they lose the transformed phenotype and, concomitantly, the hybrid molecules at high frequency. Another function, CEN4, stabilizes the ARS-containing hybrids. CEN4 allows proper segregation of hybrid molecules during mitosis and meiosis. Like its predecessor. CEN3 (Clarke & Carbon, 1980 b). CEN4 behaves genetically like a yeast centromere. The centromere-linked sequences we isolated from chromosome IV of yeast are not highly repeated and contain transcriptionally active DNA sequences. We have also isolated other CEN sequences using an enrichment procedure based on the enhanced mitotic stability of molecules carrying both CEN and ARS. Together, the ARS and CEN functions allow DNA molecules to predominantly behave as independent yeast linkage groups. However, improper disjunction of these ARS- CEN hybrids occurs in 3 to 14% of the mitotic cell divisions and ⩽3% of the meiotic divisions while the segregation of other yeast chromosomes, including chromosome IV, is normal. No more than 2% of these monovalent circular chromosomes undergo precocious segregation of sister chromatids during the first meiotic division rather than the second. Thus functional yeast chromosomes have been reconstructed by piecing together fragments of centromeric DNA with fragments that allow autonomous replication in yeast.

Isolation of Physarum DNA segments that support autonomous replication in yeast.

Hybrid plasmids containint the bacterial resistance-transfer factor pBR322 and the yeast leu2+ gene have been used to isolate DNA fragments of Physarum that are capable of initiating DNA replication in a yeast host. Five of forty hybrid plasmids containint Physarum sequences transform leu2- yeast to Leu+ at high frequency. The resulting Leu+ transformants are characterized by phenotypic instability. Supercoiled plasmid molecules containing pBR322 sequences can be detected in the transformed yeast, indicating that the transforming DNA replicates autonomously. Plasmid DNA isolated from Leu+ yeast can transform leuB bacteria. The hybrid plasmid recovered from the Leu+ bacterial transformants is identical to the original plasmid, indicating structural integrity is maintained during passage through the yeast host. These hybrid plasmids containing Physarum sequences have the same characteristics as those containing autonomously replicating yeast chromosomal sequences. As the temporal sequence of DNA replication is particularly accessible to study in Physarum plasmodia, the functional significance of these segments should be amenable to study.

Eukaryotic DNA segments capable of autonomous replication in yeast.

A selective scheme is presented for isolating sequences capable of replicating autonomously in the yeast Saccharomyces cerevisiae. YIp5, a vector that contains the yeast gene ura3, does not transform a ura3 deletion mutant to Ura+. Hybrid YIp5-Escherichia coli DNA molecules also fail to produce transformants. However, collections of molecular hybrids between YIp5 and DNA from any of six eukaryotes tested (S. cerevisiae, Neurospora crassa, Dictyostelium discoideum Ceanhorabditis elegans, Drosophila melanogaster, and Zea mays) do transform the deletion mutant. The Ura+ transformants grow slowly, are unstable under nonselective conditions, and carry the transforming DNA as autonomously replicating, supercoiled circular molecules. Such a phenotype is qualitatively identical to that of strains transformed by molecules containing a yeast chromosomal origin of replication. Thus, these DNA hybrid molecules may contain eukaryotic origins of replication. The isolated sequences may be useful in determiing the signals controlling DNA replication in yeast and in studying both DNA replication and transformation in other eukaryotic organisms.