Biomedical subjects

R K Clyne

Publications and source records attributed to R K Clyne.

8 recordsLinked to original sources

PubMed2000

Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.

It has been proposed but never proven that cohesion between sister chromatids distal to chiasmata is responsible for holding homologous chromosomes together while spindles attempt to pull them toward opposite poles during metaphase of meiosis I. Meanwhile, the mechanism by which disjunction of homologs is triggered at the onset of anaphase I has remained a complete mystery. In yeast, cohesion between sister chromatid arms during meiosis depends on a meiosis-specific cohesin subunit called Rec8, whose mitotic equivalent, Sccl, is cleaved at the metaphase to anaphase transition by an endopeptidase called separin. We show here that cleavage of Rec8 by separin at one of two different sites is necessary for the resolution of chiasmata and the disjunction of homologous chromosomes during meiosis.

Amino Acid Sequence↗

PubMed1997

Identification of autonomously replicating sequence (ARS) elements in eukaryotic cells.

Autonomously replicating sequence (ARS) elements were first identified in the budding yeast Saccharomyces cerevisiae as chromosomal DNA fragments that promoted high frequency of transformation and extrachromosomal maintenance of plasmid DNA. These specific sequence elements were subsequently shown to function as origins of DNA replication. Detailed analysis of the structure and function of ARS elements has been limited largely to S. cerevisiae and more recently the fission yeast Schizosaccharomyces pombe. Characterization of ARS activity in other eukaryotes is far less complete. Here we describe the ARS assay developed in yeast and its application to the study of origin function in other eukaryotes. Other available methods for detecting autonomous replication in these systems are also presented.

Animals↗

PubMed1997

The Schizosaccharomyces pombe rad11+ gene encodes the large subunit of replication protein A.

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein present in all eukaryotes. In vitro studies have implicated RPA in simian virus 40 DNA synthesis and nucleotide excision repair, but little direct information is available about the in vivo roles of the protein. We report here the cloning of the largest subunit of RPA (rpa1+) from the fission yeast Schizosaccharomyces pombe. The rpa1+ gene is essential for viability and is expressed specifically at S phase of the cell cycle. Genetic analysis revealed that rpa1+ is the locus of the S. pombe radiation-sensitive mutation rad11. The rad11 allele exhibits pleiotropic effects consistent with an in vivo role for RPA in both DNA repair and DNA synthesis. The mutant is sensitive to both UV and ionizing radiation but is not defective in the DNA damage-dependent checkpoint, consistent with the hypothesis that RPA is part of the enzymatic machinery of DNA repair. When incubated in hydroxyurea, rad11 cells initially arrest with a 1C DNA content but then lose viability coincident with reentry into S phase, suggesting that DNA synthesis is aberrant under these conditions. A significant fraction of the mutant cells subsequently undergo inappropriate mitosis in the presence of hydroxyurea, indicating that RPA also plays a role in the checkpoint mechanism that monitors the completion of S phase. We propose that RPA is required to maintain the integrity of replication complexes when DNA replication is blocked. We further suggest that the rad11 mutation leads to the premature breakdown of such complexes, thereby preventing recovery from the hydroxyurea arrest and eliminating a signal recognized by the S-phase checkpoint mechanism.

Alleles↗

PubMed1995

Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe.

ARS (autonomously replicating sequence) elements are DNA fragments that can function as origins of DNA replication in yeast. We report the first fine-structure analysis of ars1, an ARS element of the fission yeast Schizosaccharomyces pombe. Characterization of a series of nested deletion mutations indicated that the minimal fragment of DNA encompassing ars1 is surprisingly large. No fragment < 650 bp retained significant ARS activity. Analysis of deletion and substitution mutations scanning the entire minimal ars1 identified a single essential 50 bp fragment (segment 1). Only one other 50 bp mutation reduced activity as much as 5-fold and most deletions were without effect. Thus, the minimal ars1 is composed of two general types of genetic elements, a small segment that is absolutely required for efficient ARS activity and a much larger region that is tolerant of internal structural alterations. Higher resolution analysis of segment 1 defined a critical 30 bp A/T-rich segment which appears to contain redundant genetic elements. Schizosaccharomyces pombe ars1 promoted high frequency transformation in the budding yeast S.cerevisiae but this heterologous activity was not dependent on segment 1. Our analysis indicates that the functional elements required for ARS function in S.pombe and S.cerevisiae are clearly different.

Base Sequence↗

PubMed1994

Replication and transcription. Silence of the ORCs.

The origin recognition complex, a multi-protein complex known to bind to replication origins, has now been implicated in transcriptional silencing, providing another link between DNA replication and transcription.

Base Sequence↗

PubMed1994

Reversal of multidrug resistance in murine fibrosarcoma cells by thioxanthene flupentixol.

The purpose of this study was to identify calcium channel and calmodulin antagonists effective in increasing the cytotoxic effects of several chemotherapeutic drugs against UV-2237 murine fibrosarcoma MDR cells. Among 8 compounds tested at nontoxic concentrations, flupentixol, a piperazine-substituted thioxanthene, was the most potent in enhancing the cytotoxicity of anticancer drugs commonly associated with the multidrug resistant (MDR) phenotype, such as Adriamycin, actinomycin D, vinblastine, and vincristine, but not 5-fluorouracil, a drug usually unaffected by MDR. The chemosensitizing effects of flupentixol were produced by increasing intracellular drug accumulation via a mechanism unrelated to the binding of the plasma membrane P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

Overcoming murine tumor cell resistance to vinblastine by presentation of the drug in multilamellar liposomes consisting of phosphatidylcholine and phosphatidylserine.

We determined whether vinblastine (VLB) encapsulated within multilamellar vesicle-liposomes (MLV) would reverse target cell resistance to the drug exhibited by the UV-2237M murine fibrosarcoma and its Adriamycin (ADR)-selected multidrug resistant (MDR) variants. Resistant fibrosarcoma cells were grown in medium containing 1 and 10 micrograms/ml ADR to yield the MDR lines UV-2237M/ADRR (ADR-1) and UV-2237M/ADRRR (ADR-10), respectively. VLB encapsulated in MLV composed of phosphatidylcholine (PC) and phosphatidylserine (PS) (7:3 molar ratio) was hydrophobic, occupied an internal space equivalent of 6.13 microliters/mumol, and was stable in medium at 37 degrees C for up to 6 days. The 50% inhibitory concentrations (IC50) of VLB were 2, 25, and 70 ng/ml for the parent, ADR-1, and ADR-10 cell lines, respectively. VLB in MLV significantly enhanced sensitivity of tumor cells to VLB. The respective IC50 of liposomal VLB were 0.5, 5.7, and 12 ng/ml for the parent, ADR-1, and ADR-10 lines. MLV containing saline were not toxic to the cells. These data indicate that presentation of VLB entrapped in PC:PS MLV provides a method to overcome tumor cell resistance to this drug.

Animals↗

PubMedDate not supplied

Granulocyte-macrophage colony-stimulating factor (GM-CSF) in the management of cancer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the four major colony-stimulating factors (CSFs) that regulate hematopoiesis. GM-CSF can stimulate a single bone marrow stem cell to proliferate and differentiate into mature neutrophils, eosinophils, granulocytes or macrophages. The outcome of recent clinical trials indicates that GM-CSF has the prospect of being clinically effective in augmenting the recovery of hematopoiesis in recipients of autologous bone marrow transplantation, in cancer patients suffering from the hematopoietic toxicity associated with chemotherapy and radiation therapy, and in patients with acquired immunodeficiency syndrome (AIDS).

Colony-Stimulating Factors↗