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Biomedical subjects

R C von Borstel

Publications and source records attributed to R C von Borstel.

At least 19 recordsLinked to original sources
PubMed2001

A rapid assay for mitochondrial DNA damage and respiratory chain inhibition in the yeast Saccharomyces cerevisiae.

There is a need for a rapid assay to identify agents that damage mitochondria because the mitochondrion may be an important target for numerous environmental mitotoxins. Certainly at least one chemotherapeutic regimen (CHOP therapy) that includes doxorubicin can induce cardiomyopathy through mitochondrial genotoxicity in cardiac muscle cells. Yeast cells (1.5 x 10(6)-10(7)) in water are spread on a YEPD plate, and, when the suspension of cells has dried, a small well (12 mm diameter) is cut into the agar; 200-400 microl of a solution of the presumptive mitochondrial genotoxin is placed in the well, and the plates are incubated for 2 days. The genotoxin forms a concentration gradient through the agar and affects the growing cells. An overlay containing tetrazolium chloride is added, and the plates are incubated for 6-24 hr. Respiring cells turn red, and nonrespiring cells, with damaged DNA or inhibited respiratory chains, that are adjacent to the well, are white. A white ring, or a more lightly colored red ring, around the well indicates the presence of cells with lowered respiratory activity which may be fully reversible when the mitochondrial genotoxin is removed. In preliminary experiments, doxorubicin (= adriamycin) shows strong activity with this assay; cyclophosphamide is negative, and 4-hydroxycyclophosphamide, a metabolite of cyclophosphamide, is weakly positive. Ethidium bromide, methotrexate, 5-fluorouracil, and 5-fluorocytosine also are mitochondrial genotoxins. Antifungal agents similar to 5-fluorocytosine and anthelmintic compounds such as pyrvinium iodide can be powerful mitochondrial genotoxins.

Biological Assay↗
PubMed1999

An experimental solution for the Luria-Delbrück fluctuation problem in measuring hypermutation rates.

A cell line harboring all trans-acting elements necessary for hypermutation was transfected with a plasmid harboring the major cis-acting elements plus a green fluorescent protein gene containing a premature chain-termination codon. Transfected cells do not fluoresce unless the stop codon reverts. When a sizable cell population is purged of revertants by sorting, the frequency of mutants increases linearly with time, and there is no Luria-Delbrück fluctuation effect. Moreover, as mutant frequencies seemed to vary less than cell numbers in replicate cultures, it is suggested that hypermutation might not be coupled closely to cell division.

Cell Line, Transformed↗
PubMed1998

Janus carcinogens and mutagens.

Janus carcinogens are carcinogenic agents that, under differing conditions of cell type or dose, can instead act as anticarcinogens. Studies by Haseman and Johnson [J.K. Haseman, F.M. Johnson, Analysis of rodent NTP bioassay data for anticarcinogenic effects, Mutat. Res. , 350 (1996) 131-142], have demonstrated that many chemicals that are carcinogenic for one tissue type can have anticarcinogenic action on another tissue type. As Magni et al. [G.E. Magni, R.C. von Borstel, S. Sora, Mutagenic action during meiosis and antimutagenic action during mitosis by 5-aminoacridine in yeast, Mutat. Res., 1 (1964) 227-230] have shown in 1964, this principle holds true for chemical mutagens as well, that is 9-aminoacridine is an antimutagen in the vegetative cell and a mutagen in the sporulating cell. The conclusion can be drawn that two established carcinogens, tobacco and ionizing radiation, are indeed Janus carcinogens. In their review of 'ambiguous carcinogens' (their name), Weinberg and Storer [A.M. Weinberg, J.B. Storer, Ambiguous carcinogens and their regulation, Risk Anal., 5 (1985) 151-156], pointed out that tobacco can be classified as an ambiguous carcinogen. The strong carcinogenicity and anticarcinogenicity of tobacco smoke and/or tobacco itself (i.e., chewing tobacco) may be due to components in the mixture, not that of a single carcinogenic chemical that also may be anticarcinogenic. Kondo [S. Kondo, Health Effects of Low-Level Radiation, Kinki Univ. Press, Osaka, Japan and Medical Physics Publishing, Madison, WI, 1995, 213 pp.] has compiled data that demonstrate that human populations who survive exposures to ionizing radiation generally live longer and have less cancer than unirradiated human populations, and this Janus phenomenon goes beyond the more trivial concept of increased sensitivity to radiation of rapidly dividing tumor cells. Thiabendazole is an interesting compound in that it is both aneugenic and antimutagenic, and yet it does not appear to be a carcinogen or a mutagen. It is discussed here because aneugenesis and antimutagenesis are at extremes of the mutagenic spectrum. In general, mutagenic or carcinogenic actions usually are at least partially understood at a molecular level, whereas antimutagenic and anticarcinogenic actions usually are not. It is possible there may be numerous specific mechanisms underlying the Janus activity of different chemicals.

Animals↗
PubMed1998

Topical reversion at the HIS1 locus of Saccharomyces cerevisiae. A tale of three mutants.

Mutants of the HIS1 locus of the yeast Saccharomyces cerevisiae are suitable reporters for spontaneous reversion events because most reversions are topical, that is, within the locus itself. Thirteen mutations of his1-1 now have been identified with respect to base sequence. Revertants of three mutants and their spontaneous reversion rates are presented: (1) a chain termination mutation (his1-208, née his1-1) that does not revert by mutations of tRNA loci and reverts only by intracodonic suppression; (2) a missense mutation (his1-798, née his1-7) that can revert by intragenic suppression by base substitutions of any sort, including a back mutation as well as one three-base deletion; and (3) a -1 frameshift mutation (his1-434, née his1-19) that only reverts topically by +1 back mutation, +1 intragenic suppression, or a -2 deletion. Often the +1 insertion is accompanied by base substitution events at one or both ends of a run of A's. Missense suppressors of his1-798 are either feeders or nonfeeders, and at four different locations within the locus, a single base substitution encoding an amino acid alteration will suffice to turn the nonfeeder phenotype into a feeder phenotype. Late-appearing revertants of his1-798 were found to be slowly growing leaky mutants rather than a manifestation of adaptive mutagenesis. Spontaneous revertants of his1-208 and his1-434 produced no late-arising colonies.

Adenine↗
PubMed1996

Protection and repair of gamma-radiation-induced lesions in mice with DNA or deoxyribonucleoside treatments.

Mice can survive lethal doses of ionizing radiation if deoxyribonucleosides or 'highly polymerized' salmon sperm DNA (Sigma) are administered 30 min to 24 h post-irradiation. DNA is more effective than deoxyribonucleosides in increasing the survival frequency. At supralethal exposures of gamma-irradiation, Deoxyribonucleosides and DNA are equally effective in reversing radiation damage which otherwise leads to chromosome breakage. The micronucleus frequencies in the polychromatic erythrocytes of bone marrow cells from DNA- or deoxyribonucleoside-treated mice were near the unirradiated control values. This reduction in chromosome breakage was approximately 4-fold when compared with the irradiated, saline-treated control. 'Highly polymerized' DNA protects against mortality if administered 48 and 24 h prior to irradiation. This is somewhat comparable to the effectiveness of the growth factors Interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNFalpha) administered prior to irradiation. With survival as criterion, the sensitivity of 4 lines of mice to gamma-irradiation is BALB/c > C3H/OuJ > or = C3H/HeJ > C57B1/6.

Animals↗
PubMed1994

Origins of spontaneous base substitutions.

Although simple to understand, satisfying to the imagination, and compelling as a teaching aid for beginning students, the evidence is mounting that the tautomeric shift is neither a common nor a likely origin for spontaneous base substitutions. Indeed, other sources, such as ionized base mispairings with nonionized bases, wobble of a bases in the DNA, and transient misalignment of bases causing dislocations at pairing sites, have been shown to induce spontaneous base substitutions. On the other hand, among the 4 common bases in DNA, no experimental evidence exists that tautomeric shifts can induce mutations.

Base Composition↗
PubMed1993

The mutator mut7-1 of Saccharomyces cerevisiae.

The mut7-1 mutant of Saccharomyces cerevisiae is a cell-division-cycle mutant, exhibiting temperature-sensitive lethality and enhancement of mutator activity with increases in temperature. The base-sequence alterations in mutants arising in a mut7-1 background differed from the control by there being a higher transversion/transition ratio and by the much increased production of multi-base deletions. The deletions were, in every instance, associated with repeated oligonucleotide sequences (3-8 bases in length), where one of the two sequences was removed during the deletion process. The mutant mut7-1 failed to complement with cdc2, the temperature-sensitive mutant of the locus which encodes DNA polymerase III (delta).

Base Sequence↗
PubMed1992

The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae.

We have constructed a dihydrofolate reductase mutant (dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.

Leucovorin↗
PubMed1992

Base alterations in yeast induced by alkylating agents with differing Swain-Scott substrate constants.

The base alterations induced by four alkylating agents, methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-nitroso-N-methylurea (MNU), and N-nitroso-N-ethylurea (ENU), have been determined at the URA3 locus in the yeast Saccharomyces cerevisiae. The mutagen treatment was carried out on yeast cells in the logarithmic phase of growth. The mutants were selected by their resistance to 7.3 mM-5-fluoroorotic acid at pH 3.8. DNA sequence analysis was carried out by the dideoxy chain termination method. The alkylating agents were selected for their widely differing Swain-Scott substrate constants (s values), which are as follows: MMS, s = 0.83; EMS, s = 0.67; MNU, s = 0.42; ENU, s = 0.26. A higher s value is correlated with a higher ratio of 7-alkylguanine to O6-alkylguanine in native DNA in vitro. 125 forward mutations from URA3----ura3 were sequenced with marked differences in the mutational spectra being observed as the s value changed. Five hotspots were recorded for the four alkylating agents. They were all G.C----A.T transition mutations. There was one common hotspot for all of them; there were two additional ones for the two ethylating agents (ENU and EMS) and two different ones for MNU. Four of the five hotspots have the 5'-GG-3' sequence with the 3'-guanine mutated. It was seen that MMS, which has the highest Swain-Scott substrate constant, yielded the widest array of mutational types. As the substrate constants decreased, the types of mutations became more and more restricted to the G.C----A.T transitions and the A.T----T.A transversions. The transitions are consistent with the concept that mutations arise from O6-alkylation of guanine and alkylation of thymine. The transversions are consistent with the notion of N1-alkylation of adenosine or adenylic acid.

Alkylating Agents↗
PubMed1992

Induction of the cytoplasmic 'petite' mutation by chemical and physical agents in Saccharomyces cerevisiae.

A range of physical and chemical agents induce the mitochondrial 'petite' mutation in the yeast Saccharomyces cerevisiae. DNA intercalating agents as well as chemicals which can interfere with DNA synthesis induce this mutation, but only in growing cells. Many chemical or physical agents that produce a DNA lesion which is not simply reversed can induce various levels of the petite mutation, and may be more effective in non-growing cells. A limited number of chemicals act like ethidium bromide, inducing a high frequency of petites which is partially reversible with increasing concentration or time. The ability of a specific compound to be transported into mitochondria or its affinity for AT base pairs in DNA may determine whether it acts primarily as a nuclear or mitochondrial mutagen. In mammalian cells, some neoplastic changes occur at the mitochondrial level. Analogies between yeast and mammalian mitochondria suggest that agents which increase petite mutagenesis in yeast may have some carcinogenic potential. Although some types of petite inducer may have potential as antitumour drugs, those which are very effective antimitochondrial agents appear to be too toxic for therapeutic use. A process comparable to early stages in petite mutagensis occurs in human degenerative diseases and it seems possible that a consequence of exposure to petite mutagens could be an increase in the rate of degenerative diseases or of the aging process.

Animals↗
PubMed1991

Sequence and localization of the gene encoding yeast phosphoglycerate mutase.

In this study we report on the complete nucleotide sequence of the yeast phosphoglycerate mutase gene (GPM1) and its essential 5' and 3' non-coding regions. The transcriptional start points were determined by S1-mapping and sequencing of a cDNA clone. Several sequences identified as important for transcriptional regulation in yeast promoters are present upstream of the transcription start point. 3' to the coding region we sequenced a composite repetitive element which, apparently, originated from a recombination between a delta- and a tau-element. Finally, we mapped the GPM1 gene 13 cM distal to fas1 on chromosome XI.

Amino Acid Sequence↗
PubMed1990

Antimutagenesis in yeast by sodium chloride, potassium chloride, and sodium saccharin.

Aqueous salt solutions containing NaCl, KCl, MgCl2, Na2SO4, CaCl2, NH4Cl, or sodium saccharin are mutagenic in yeast when logarithmic growth of cells is interrupted by exposure to a 0.5-2.0 M salt solution. Stationary-phase cells are not mutated by this treatment. When placed in an enriched medium with the salt, the stationary-phase cells grow after a prolonged lag period. The compounds tested (NaCl, KCl, and sodium saccharin), under conditions in which growth in medium can take place, exhibit an antimutagenic response as measured by the compartmentalization test. The antimutagenic action of salt solutions in yeast is concentration-dependent. Unlike the mutagenic action of these compounds, which approximates an osmolality-dependent response, the antimutagenic action seems to be correlated with toxicity as measured by growth rate reduction at increasing concentrations of the compounds. For example, sodium saccharin and NaCl exhibit almost identical osmolalities; however, 0.3 M sodium saccharin reduces the growth rate much more than does 0.3 M NaCl. At these same molar concentrations, the spontaneous mutation rate for histidine prototrophy is, for the control, 6.2 x 10(-8) mutations/cell/-generation, 3.5 x 10(-8) with 0.3 M NaCl, and 1.7 x 10(-8) with 0.3 M sodium saccharin.

Depression, Chemical↗
PubMed1989

The phosphofructokinase genes of yeast evolved from two duplication events.

Yeast phosphofructokinase (PFK) is an octameric enzyme composed of four alpha-subunits and four beta-subunits, encoded by the genes PFK1 and PFK2, respectively. PFK1 was mapped 23 cM distal to ADE3 on chromosome VII, and PFK2 30 cM proximal to RNA1 on chromosome XIII. The entire nucleotide sequences for the two genes were obtained by sequencing both DNA strands. Only one major open reading frame was found for each gene. They encode 987 aa for PFK1 (Mr 107,984) and 959 aa for PFK2 (Mr 104,589). Both genes show a biased codon usage. The deduced amino acid sequences showed: (i) 20% homology between the N- and the C-terminal halves of each subunit, (ii) 55% homology between the two subunits, and (iii) significant homologies to the PFK sequences from human and rabbit muscle (42%), Escherichia coli (34%), and Bacillus (36%). These data support the view that two gene duplication events occurred in the evolution of the yeast PFK genes. The first duplication event took place soon after the separation of prokaryotic and eukaryotic lineage and the second in Saccharomyces later in the phylogeny. Functional domains in the yeast subunits were deduced by comparison to the rabbit muscle enzyme.

Amino Acid Sequence↗
PubMed1989

Scope of action of the immunoglobulin mutator system.

The authors have developed a method to measure the rate of spontaneous mutations taking place in IgH, the gene encoding the immunoglobulin heavy chain. When an amber chain-termination codon mutates to a sense codon, translation of the polypeptide chain will be completed, and mutant cells producing the heavy chain can be detected with a fluorescent labelled antibody. The protocol used is the compartmentalization test which minimizes any effect of selection. In subclones of the pre-B lymphocyte line 18-81, the spontaneous mutation rate in the part of IgH encoding the variable region is somewhat greater than 10(-5) mutations per base pair per generation. This supports the hypothesis that hypermutation is not dependent on cell stimulation by an antigen. In a hybrid between a cell of this line and a myeloma (which represents the terminal stage of the B-cell lineage), the mutation rate was too low to be determined by this test, less than 10(-9). When the same loss to gain procedure system was used with an opal chain-terminating codon in the part of IgH encoding the constant region (C mu), a high rate of reversion by deletion was found. Long (more than one exon) and short (less than one exon) deletions occurred at rates of 1.7 x 10(-5) and 1.4 x 10(-7) per generation, respectively. It is thought that the high rate of deletion is not related to somatic hypermutation but rather to DNA rearrangement during the heavy-chain class switch, which is occurring in these pre-B cell lines. The point mutation rate was too low to be detected above the background of deletion mutants, less than 5 x 10(-8). The immunoglobulin mutator system works weakly, if at all, on two other, nonimmunoglobulin, genes tested: B2m (beta 2 microglobulin) and the gene for ouabain resistance.

Antibody Diversity↗