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R H Haynes

Publications and source records attributed to R H Haynes.

At least 19 recordsLinked to original sources
PubMed2000

Glycine metabolism in Candida albicans: characterization of the serine hydroxymethyltransferase (SHM1, SHM2) and threonine aldolase (GLY1) genes.

Genes encoding the mitochondrial (SHM1) and cytosolic (SHM2) serine hydroxymethyltransferases, and the L-threonine aldolase gene (GLY1) from Candida albicans were cloned and sequenced. All three genes are involved in glycine metabolism. The C. albicans Shm1 protein is 82% identical to that from Saccharomyces cerevisiae and 56% identical to that from Homo sapiens. The corresponding identities for the Shm2 proteins are 68% and 53%. The Gly1 protein shares significant identity with the S. cerevisiae L-threonine aldolase (55%) and also with threonine aldolases from Aeromonas jandiae (36%) and Escherichia coli (36%). Genetic ablation experiments show that GLY1 is a non-essential gene in C. albicans and that L-threonine aldolase plays a lesser role in glycine metabolism than it does in S. cerevisiae. GenBank Accession Nos of the C. albicans SHM1 and SHM2 are AF009965 and AF009966, respectively. Accession No. for C. albicans GLY1 is AF009967.

Amino Acid Sequence↗
PubMed1997

A consensus sequence for a functional human endogenous retrovirus K (HERV-K) dUTPase.

Amino acid sequence comparisons have revealed that a potential dUTPase gene is encoded by the retrovirus HERV-K, a defective multicopy virus that is transmitted vertically in humans. This gene is distinct from the human cellular dUTPase gene and thus two potential sources of the enzyme exist in human cells. dUTPases characterized from various sources each contain five conserved amino acid sequence motifs that form the active site of the enzyme. The protein sequence of the putative HERV-K dUTPase deduced from previous DNA sequence data from one proviral clone (HERV-K10) shows marked deviations at highly conserved residues in four of five of these motifs. Therefore, the reported DNA sequence may represent a mutated form of the viral dUTPase gene. To address this possibility, we cloned and sequenced 22 copies of the HERV-K dUTPase gene from human DNA. The results of this analysis indicate that variations evident in the HERV-K10 dUTPase amino acid sequence represent mutations of the wild-type viral DNA sequence. A version of the HERV-K dUTPase gene that corresponds to the ancestral, wild-type DNA sequence was constructed and adapted for expression in Escherichia coli. The resulting enzyme was found to exhibit properties similar to those of dUTPases isolated from other systems. A possible role of the HERV-K dUTPase in human disease is discussed.

Amino Acid Sequence↗
PubMed1997

dUTP pyrophosphatase as a potential target for chemotherapeutic drug development.

Aberrant dUTP metabolism plays a critical role in the molecular mechanism of cell killing induced by inhibitors of dihydrofolate reductase and thymidylate synthase. While considerable effort has been directed towards discovering new, more potent inhibitors of these two enzymes, little attention has been given dUTP pyrophosphatase (dUTPase)--the key modulator of cellular dUTP levels--as a potential target for chemotherapeutic drug development. Recent studies have provided evidence that dUTPase is vital for cellular and, in some cases, viral DNA replication. Furthermore, some retroviruses encode dUTPases--a fact which suggests that cellular dUTP metabolism may be more important than previously realized. Here, we briefly review current knowledge of cellular and viral dUTPases and discuss the potential of these enzymes as targets for cancer chemotherapeutic and anti-viral drug development.

Animals↗
PubMed1996

HIV and human endogenous retroviruses: an hypothesis with therapeutic implications.

The enzyme dUTP pyrophosphatase (dUTPase, EC 3.6.1.23) is essential for cellular DNA replication and cell viability by virtue of its role in reducing the availability of dUTP as a substrate for DNA polymerases. Several members of the onco- and lentivirus families of retroviruses encode dUTPases and mutant strains of these viruses defective in this enzyme exhibit suboptimal replication kinetics. Among the lentiviruses there exists a surprising phylogenetic discontinuity in the distribution of dUTPase genes: non-primate viruses (EIAV, CAEV, FIV, visna) contain such genes whereas the primate viruses (HIVs, SIVs) do not. The reason for this difference is unknown. We suggest the following explanation: (1) the nuclear and mitochondrial compartmentalization of the mammalian dUTPase, combined with the cytoplasmic location of ribonucleotide reductase, leads to the net synthesis of dUTP, together with dCTP, dGTP and dATP in the cytoplasm; (2) this combination of dNTPs serves as a "toxic cocktail" for viral replication by virtue of its ability to promote the synthesis of uracil-substituted DNA; (3) many viruses have adapted to this challenge by encoding dUTPases that are free of normal cellular regulatory constraints; and (4) the fortuitous expression of a dUTPase encoded by one or more human endogenous retroviruses (HERVs) has led to the evolutionary loss of the putative ancestral dUTPase gene of primate lentiviruses. Thus, we propose that efficient replication of HIV in humans depends upon expression of a dUTPase encoded by an endogenous retrovirus. If this proposal is correct, then the entry of HIV into target cells is necessary, but not sufficient, for replication of the virus in humans.

Animals↗
PubMed1995

Mathematical parameters for quantification of mutational responses in bacteria.

This paper introduces a new parameter, derivable from dose-response data for induced mutagenesis in bacteria, that can be used to quantify mutational responses in short-term tests. We called this parameter the mutational response of the bipartite experimental system (agent plus cells). We defined it as being jointly proportional to the efficiency of the mutagen and the sensitivity of the test. We show how this quantity can be used to rank order chemical carcinogens on the basis of their mutagenicity and to determine the strength of any quantitative correlation that may exist between mutagenicity in bacteria and carcinogenicity in rodents. We find that this particular measure of mutational response for 10 direct-acting monofunctional alkylating agents correlates remarkably well with the rodent carcinogenicity of these chemicals measured in terms of their reciprocal TD50 values.

Alkylating Agents↗
PubMed1993

dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design.

Amino Acid Sequence↗
PubMed1992

Human dUTP pyrophosphatase: cDNA sequence and potential biological importance of the enzyme.

Two functional human dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) cDNAs were isolated from a cDNA expression library by genetic complementation in Escherichia coli. These cDNAs differed in size but exhibited a common overlapping DNA sequence. Contained within this sequence was a single long open reading frame sufficient to encode a polypeptide of 141 amino acids with a calculated molecular mass of 16.6 kDa. The amino acid sequence of this protein exhibits 35% identity with the E. coli dUTPase and 53% identity with the Saccharomyces cerevisiae enzyme. The human dUTPase was found to contain five characteristics amino acid sequence motifs that are common to the dUTPases of E. coli, yeast, and herpesviruses and to dUTPase-like sequences encoded by some retrovirus gag and pol genes. A high degree of amino acid sequence identity (greater than 60%) was also observed between the human dUTPase and the putative pseudoproteases of two poxviruses, indicating that these virus proteins are dUTPases. Northern hybridization analysis reveals that dUTPase is encoded by at least two species of poly(A)+ mRNA and possibly a third, smaller species. All of these mRNAs are present in a variety of human tissues but their relative levels vary between tissues. Southern analysis indicates that the dUTPase gene has been conserved to some extent throughout vertebrate evolution; however, the gene may be very large, or its organization somewhat complex in some systems. We suggest that dUTPase may generally perform an essential role in DNA replication and therefore could serve as a target enzyme for the development of chemotherapeutic compounds.

Amino Acid Sequence↗
PubMed1992

The implantation of life on Mars: feasibility and motivation.

Environmental conditions on Mars are extremely hostile, and would be destructive to any organisms which might arrive there unprotected to-day. However, it is a biocompatible planet. Its unalterable astrophysical parameters would allow the maintenance of a much thicker, warmer carbon dioxide atmosphere than that which currently exists. Though very cold (averaging about -60 degrees C), highly oxidizing and desiccated, Mars may possess substantial quantities of the materials needed to support life--in particular, water and carbon dioxide. A general scenario for implanting life on Mars would include three main phases: (1) robotic and human exploration to determine whether sufficiently large and accessible volatile inventories are available; (2) planetary engineering designed to warm the planet, release liquid water and produce a thick carbon dioxide atmosphere; and (3) if no indigenous Martian organisms emerge as liquid water becomes available, a program of biological engineering designed to construct and implant pioneering microbial communities able to proliferate in the newly clement, though still anaerobic, Martian environment. The process of establishing an ecosystem, or biosphere, on a lifeless planet is best termed 'ecopoiesis.' This new word, derived from Greek, means 'the making of an abode for life.' It is by no means clear whether ecopoiesis on Mars is scientifically possible or technologically achievable. Thus we urge that it be one of the objectives of space research during the next century to assess the feasibility of ecopoiesis on Mars.

Atmosphere↗
PubMed1990

Analysis of interactions between mutagens, I. Heat and ultraviolet light in Saccharomyces cerevisiae.

A new mathematical approach to the description of interaction data (Ager and Haynes, 1987) is applied here to the interaction between heat and ultraviolet light (UV) in Saccharomyces cerevisiae. A strong synergism for cell killing is found to be associated with large increases in gene conversion (of up to 8-fold), and mutation (of up to 14-fold). Analysis of the interaction data for both wild-type and repair-deficient strains indicates that the heat-UV synergism arises via the inhibition of two different repair pathways. Unambiguous conclusions regarding the molecular mechanisms by which these repair processes are inhibited cannot be drawn on the basis of dose-response data alone. However, this approach does enable one to make well defined, empirical comparisons of the nature and kinetics of such interactions.

Gene Conversion↗
PubMed1990

Analysis of interactions between mutagens, II. Ethyl methanesulfonate and ultraviolet light in Saccharomyces cerevisiae.

The results of this study indicate the existence of a strong interaction between ethyl methanesulfonate (EMS) and ultraviolet light (UV) for cell killing in the yeast Saccharomyces cerevisiae. Conversely, mutation and gene conversion frequencies observed for the combined treatment of EMS and UV do not deviate significantly from that expected on the basis of simple additivity. Studies involving repair-deficient mutants (rad mutants) reveal that the synergistic interaction for cell killing depends on RAD52 function (recombinational repair), but not on RAD3 function (excision repair). On the basis of this analysis, the interaction between EMS and UV in S. cerevisiae might arise from the inhibition of double-strand break repair by one, or both agents.

Cell Survival↗
PubMed1990

Essay: should we implant life on Mars?

The question of whether or not we should implant life on Mars has at its core another question. Could we implant life on Mars? As interesting as the ethical considerations of the proposition are--and we shall look at these--they are moot unless the concept itself is feasible.

Ecosystem↗
PubMed1989

Mutagenesis and mathematics: the allure of numbers.

This paper sets out the "formal," "empirical," and "mechanistic" equations that my colleagues and I have developed for the description and analysis of dose-response data on the lethal and genetic effects of mutagens in microorganisms. These three types of equations are interrelated inasmuch as they are all based ultimately on the use of the Poisson distribution in the formal definition of lethal and mutational hit functions. Explicit mathematical expressions for these functions can be written down in either empirical or mechanistic terms. The empirical equations are obtained simply by writing the hit functions as finite polynomials with adjustable coefficients. The mechanistic equations are based on the assumptions of the "DNA damage-repair hypothesis." The mathematical formulation of this hypothesis entails an important change in the definition of the word "hit" from that used in the classical hit/target theory of radiation biology. The theoretical and practical applications of these various equations in mutation research are summarized briefly and their merits are assessed in light of recent advances in our understanding of the biochemical basis of mutagenesis.

DNA Damage↗
PubMed1988

Quantitative aspects of the interactive killing effects between X rays and other mutagens in microorganisms.

Recently we presented a mathematical description of the synergistic interaction which occurs when Escherichia coli B/r is exposed to both X rays and 254 nm ultraviolet light (D. D. Ager and R. H. Haynes, Radiat. Res. 110, 129-141 (1987]. Here we extend this approach to other bacteria and describe a graphical technique which can be used to determine the nature and relative importance of second and third degree terms in the function h(x, y), which describes the dose dependence of such effects. In most cases, interaction functions appear to be dominated, in the biologically interesting dose range, by a second degree term in the product, xy, of the doses of the two agents. We find that the magnitudes of these interactions vary among the organisms examined and can be surprisingly large. Finally, we show that the simple xy dependence observed for most interactions does not carry any unambiguous implications with respect to previous speculations on the mechanisms of these effects.

Bacteria↗
PubMed1987

Molecular characterization of the Saccharomyces cerevisiae dihydrofolate reductase gene (DFR1).

The complete nucleotide sequence of a 1957 bp DNA fragment containing the dihydrofolate reductase gene (DFR1) of the yeast Saccharomyces cerevisiae is presented. Within this region a single open reading frame of 633 base pairs was found which is capable of encoding a 211 amino acid residue protein with a calculated Mr of 24,233. The amino acid sequence derived from the yeast DFR1 gene shows limited homology with sequences from both eukaryotic and non-eukaryotic DHFR enzymes. Northern blot hybridization reveals that the mRNA from this gene is a 900 base polyadenylated transcript. Yeast strains containing the cloned DFR1 gene on multicopy number shuttle vector plasmids show dramatically enhanced methotrexate resistance. Consensus DNA sequences responsible for RNA polymerase II interaction and general amino acid control in S. cerevisiae are located within the 5'-noncoding region with respect to the open reading frame. The DNA fragment containing these sequences has been shown to be necessary for DFR1 gene expression in both S. cerevisiae and E. coli.

Amino Acid Sequence↗