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R H Haynes

Publications and source records attributed to R H Haynes.

At least 37 records · Page 2Linked to original sources
PubMed1987

Molecular characterization of the cell cycle-regulated thymidylate synthase gene of Saccharomyces cerevisiae.

The complete nucleotide sequence of a 1.8-kilobase DNA fragment containing the cell cycle-regulated thymidylate synthase gene (TMP 1) of the yeast Saccharomyces cerevisiae is presented. This analysis has revealed a 912-base pair open reading frame which encodes a 304-amino acid residue protein with a calculated Mr of 35,007. The tmp1-6 and cdc21-1 mutant alleles of this gene also have been sequenced, and both show single base pair changes which would result in different amino acid substitutions. The amino acid sequence of the yeast thymidylate synthase gene derived from the DNA sequence shows considerable homology when compared with the human, mouse, Herpesvirus saimiri, Leishmania major, Leishmania tropica, Escherichia coli, Lactobacillus casei, bacteriophage T4, and Bacillus subtilis phage phi 3T enzymes. Northern blot hybridization reveals that the TMP 1 mRNA is a 1.15-kilobase polyadenylated transcript. A set of consensus yeast mRNA splice sequences appears within the open reading frame of TMP 1, but S1 nuclease protection experiments reveal that splicing of the mRNA does not occur. Disruption of the gene by the introduction of a large insertion did not produce any defect besides the expected dependence on dTMP for growth. Specifically, the viability of the mutants in the presence of dTMP indicates that the protein does not play a significant structural role in a complex of replication enzymes.

Alleles↗
PubMed1987

Suicide and social response in Fiji: a historical survey.

A historical review of suicide in Fiji shows that rates for people of Indian descent have always been higher than those of other groups, yet until now this phenomenon has attracted little official or public attention. Recent changes in the incidence and methods of suicide have led to the introduction of some preventive measures, but the emphasis has been on eliminating some of the methods rather than the reasons for suicide. The continuing high incidence of suicide by hanging involving some sectors of society appears to have been overlooked. Possible causes of this apparent lack of interest are examined, such as the low status historically of Indian immigrants and concepts held about them by other groups in Fiji, and the "invisibility" of groups of people such as rural women and the aged.

Female↗
PubMed1986

Transcription of genes encoding enzymes involved in DNA synthesis during the cell cycle of Saccharomyces cerevisiae.

We have examined the pattern of transcription exhibited by four genes in the dTTP biosynthetic pathway of Saccharomyces cerevisiae. Consistent with the results reported previously by Storms et al. (1984), the TMP1 (or CDC21) gene encoding thymidylate synthase was found to be transcribed in a periodic manner during the cell cycle with maximal mRNA levels occurring just prior to the onset of DNA replication. Three other genes in this pathway DCD1, DUT1 and DFR1 encoding dCMP deaminase, dUTP pyrophosphatase and dihydrofolate reductase, respectively, exhibited relatively constant levels of transcription throughout the cell cycle. These results, particularly for DFR1, are in marked contrast with those obtained in other eukaryotic systems which have suggested that, in general, genes encoding enzymes involved in DNA precursor synthesis are subject to cell cycle regulation. Thus, periodic transcription is not a property common to all genes involved in DNA replication in this eukaryote.

Cell Cycle↗
PubMed1986

Inhibition of DNA replication in Saccharomyces cerevisiae by araCMP.

Cytosine arabinoside (araC), a potent inhibitor of DNA replication in mammalian cells, was found to be completely ineffective in Saccharomyces cerevisiae. The 5' monophosphate derivative, araCMP, is toxic and effectively inhibits both nuclear and mitochondrial DNA synthesis in this organism. Although wild-type strains can be inhibited by araCMP, dTMP permeable (tup-) strains were found to be much more sensitive to the analogue. In vivo labelling experiments indicate that araC enters yeast cells; however, it is extensively catabolized by deamination and breakage of the glycosidic bond. In addition, the analogue is not efficiently phosphorylated in S. cerevisiae owing to an apparent lack of deoxynucleoside kinase activity. These results provide further evidence that deoxyribonucleotides can be synthesized only through de novo pathways in this organism. Finally, araCMP was found to be recombinagenic in S. cerevisiae which suggests, together with other previous studies, that, in general, inhibition of DNA synthesis in yeast promotes mitotic recombination events.

Arabinonucleotides↗
PubMed1986

Hyperresistance to DNA damaging agents in yeast.

In order to study resistance to DNA damaging agents, yeast DNA segments conferring hyperresistance in this organism to such genotoxic agents were selected for among yeast cells transformed by a yeast genome library based on the multi-copy vector plasmid YEp13. Genetic variants hyperresistant to 4-nitroquinoline-N-oxide, formaldehyde, and alkylating agents were isolated and the respective hyperresistance determinants shown to co-segregate with the vector plasmid. Phenotypical characterization indicated different degrees of resistance, few cases of cross-resistance and differing structural stability of the cloned DNA. By transfer to E. coli and subsequent retransformation of yeast a number of plasmids was shown to stably carry the genetic information for hyperresistance.

4-Nitroquinoline-1-oxide↗
PubMed1986

Induction of intrachromosomal recombination in yeast by inhibition of thymidylate biosynthesis.

The biosynthesis of thymidylate in the yeast Saccharomyces cerevisiae can be inhibited by antifolate drugs. We have found that antifolate treatment enhances the formation of leucine prototrophs in a haploid strain of yeast carrying, on the same chromosome, two different mutant leu2 alleles separated by Escherichia coli plasmid sequences. That this effect is a consequence of thymine nucleotide depletion was verified by the finding that provision of exogenous thymidylate eliminates the increased production of Leu+ colonies. DNA hybridization analysis revealed that recombination, including reciprocal exchange, gene conversion and unequal sister-chromatid crossing over, between the duplicated genes gave rise to the induced Leu+ segregants. Although gene conversion unaccompanied by crossing over was responsible for the major fraction of leucine prototrophs, events involving reciprocal exchange exhibited the largest increase in frequency. These data show that recombination is induced between directly repeated DNA sequences under conditions of thymine nucleotide depletion. In addition, the results of this and previous studies are consistent with the possibility that inhibition of thymidylate biosynthesis in yeast may create a metabolic condition that provokes all forms of mitotic recombination.

Genotype↗
PubMed1986

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Amino Acid Sequence↗
PubMed1985

A possible role for deoxyribonucleotide pool imbalances in carcinogenesis.

Thymine nucleotide pool alterations, produced by attack on non-DNA primary targets, induce a variety of chromosome and chromatid aberrations. Specifically, in lower eukaryotes, thymidylate deprivation and excess are recombinagenic and dTMP depletion also produces DNA strand breakage. In higher eukaryotes, imbalances in thymine nucleotide pools provoke chromosome breaks and rearrangements, and inhibition of thymidylate biosynthesis causes morphological and oncogenic transformation in vitro. Thus, chromosomal rearrangements induced by dTMP deprivation may be the critical changes that lead to oncogenic transformation in response to thymine nucleotide depletion.

Animals↗
PubMed1985

Mating-type switching in yeast is induced by thymine nucleotide depletion.

Thymidylate biosynthesis was inhibited in a haploid heterothallic strain of Saccharomyces cerevisiae. When the treated cells were mixed with a haploid strain of the same mating-type, there was an increase in the recovery of diploid colonies. Genetic and biochemical analyses demonstrated that the diploid clones arose as a consequence of induced mating-type interconversion.

Crosses, Genetic↗
PubMed1985

Analysis of non-linearities in frequency curves for UV-induced mitotic recombination in wild-type and excision-repair-deficient strains of yeast.

Frequency curves for UV-induced mitotic recombination often are linear at low doses. As dose increases, these curves either increase at higher powers of dose and/or reach a maximum induced frequency and then decline. Similar dose-response patterns have been observed previously for mutation. The non-linearities can arise from higher order effects inherent in the molecular mechanisms of mutagenesis and/or from 'delta-effects' (Eckardt and Haynes, 1977a), i.e., differential probabilities of clone formation for mutant and non-mutant cells. Previously, we have shown that one can distinguish between these two possibilities by plotting the ratio of the induced mutant yield to the linear component of frequency as a function of dose (Haynes et al., 1985). In this study, we have used this ratio, a quantity we call 'apparent survival', to analyse the non-linear regions of the dose-response curves for UV-induced mitotic crossing-over and gene conversion in wild-type (RAD) and excision-repair-deficient (rad3) strains of yeast. Plots of apparent survival versus dose reveal the existence of a positive, non-linear component associated with UV-induced gene conversion in RAD, but not rad3, cells. A high dose decline in frequency, which is observed for UV-induced recombination in both strains, can be attributed to delta-effects.

Cycloheximide↗
PubMed1984

Isolation of a Saccharomyces cerevisiae mutant strain deficient in deoxycytidylate deaminase activity and partial characterization of the enzyme.

Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes. A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase. We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP. The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.

Alleles↗
PubMed1984

Suicide in Fiji: a preliminary study.

A survey of police and medical records in a province of Fiji indicated high rates of suicide for Indian women under 30 and for Indian men over that age, especially amongst families engaged in sugar-cane farming. Indian suicide rates were high during the Indenture period (1879-1920), and are still higher amongst Indians than amongst native Fijians: almost 90% of all suicides are Indian, though this group comprises only 50% of Fiji's total population. Age/sex/ethnic distribution, methods, and precipitating factors of recorded suicides were examined for one province of Vanua Levu during the period 1979-82. The increasing use of toxic herbicides, especially paraquat, as a vehicle for suicide is discussed.

Adolescent↗