A Festschrift for Robert H. Haynes.
Explore the source record for details and available documents.
Bibliographies as Topic↗
Biomedical subjects
M Brendel
Publications and source records attributed to M Brendel.
Formaldehyde lacks genotoxicity in formaldehyde-hyperresistant strains of the yeast Saccharomyces cerevisiae.
Formaldehyde is a weak mutagen and recombinagen in wild type strains of Saccharomyces cerevisiae. Genotoxicity of formaldehyde is influenced by the activity of the SFA yeast gene. Yeast transformants containing multiple copies of the plasmid-contained SFA gene are hyperresistant to the chemical and grow in the presence of 5 mM formaldehyde, a concentration lethal for the wild type. The SFA-encoded protein mediates the degradation of formaldehyde and its activity is most probably responsible for the low or absent genotoxic effects in formaldehyde-treated cells. Multi-copy vectors containing the SFA gene are retained in yeast transformants growing in complex media supplemented with 5 mM formaldehyde. Cells harboring such multi-copy vectors may, therefore, be propagated in large batch cultures in undefined media in the presence of formaldehyde concentrations toxic to the wild type.
Drug Resistance↗
Induction of donor-specific tolerance to rat islet allografts by intrathymic inoculation of solubilized spleen cell membrane antigens.
The development of strategies that will allow permanent survival of islet allografts without continuous host immunosuppression continues to be the most important goal in the field of pancreatic islet cell transplantation. In our study, we demonstrated that intrathymic inoculation of allogeneic spleen cell membrane antigens with a single dose of anti-lymphocyte serum induces an unresponsive state that permits survival of a subsequent pancreatic islet allograft to an extrathymic site (renal subcapsular space). This effect is donor specific and cannot be reproduced by the intravenous injection of spleen cell membrane antigens. Our results offer a potential approach for establishing donor-specific allograft acceptance in adult recipients.
Animals↗
Molecular structure of the DNA cross-link repair gene SNM1 (PSO2) of the yeast Saccharomyces cerevisiae.
A 3.2 kb yeast DNA fragment containing the DNA interstrand cross-link-specific repair gene SNM1 has been sequenced. Two genes were identified. SNM1 has an open reading frame of 1983 bp and codes for a 661 amino acid protein. Hydrophobic analysis shows that the protein is most probably not directly membrane bound. The second gene, UGX1, has an open reading frame of 573 bp coding for a polypeptide of 191 amino acid residues. The two genes are arranged head to head and share a 192 bp divergent promoter region that contains three TATAAA motives, two for the SNM1 and one for the UGX1 locus. Gene UGX1 has no apparent influence on the sensitivity of the cell to cross-linking nitrogen mustard, as its disruption in wild type does not increase sensitivity to nitrogen mustard and the presence of multiple copies of the gene fails to complement the nitrogen mustard sensitivity phenotype of snm1 disruption mutants. Northern analysis revealed that the expression of SNM1 yields an average of 0.3 copies/cell of a 2.4 kb transcript, while expression of UGX1 yields higher levels of a 0.8 kb poly(A)+ RNA.
Amino Acid Sequence↗
A ten-minute protocol for transforming Saccharomyces cerevisiae by electroporation.
We present a simplified and rapid method for the transformation of yeast cells by electroporation. Stationary cells, scraped off the agar of Petri dish cultures stored in the refrigerator for up to 6 weeks, are suspended in sorbitol buffer, spun down by gentle centrifugation, transferred into the electroporation cuvette, and immediately subjected to transformation via electroporation. Transformation efficiency of this 10-min method, which does not require the preparation of cell cultures, is about 10% of the hitherto best performing transformation procedure using cells of defined growth phase.
DNA, Fungal↗
The DNA repair gene PSO3 of Saccharomyces cerevisiae belongs to the RAD3 epistasis group.
The mutant allele pso3-1 of Saccharomyces cerevisiae confers sensitivity to treatment with UV365nm (UVA) light-activated mono- and bi-functional psoralens. When pso3-1 is combined in double mutants with selected rad and pso mutant alleles and subjected to 8-MOP + UVA treatment, epistatic interaction with regard to survival is observed with pso1, pso2, and rad3. With the same treatment the combination of pso3-1 with rad6 and rad52 leads to synergistic interaction. For the monofunctional agent 3-carbethoxypsoralen (3-CPs) the analysis of double mutants yields the same results as with the bifunctional 8-methoxypsoralen (8-MOP) with the exception of the pso1-1pso3-1 double mutant. Here we find an additive interaction, i.e., the sensitivities of both parental strains are summed in the double mutant, which indicates a different substrate specificity of the repair activity encoded by the PSO1 and PSO3 genes.
Alleles↗
Isolation and characterization of additional genes influencing resistance to various mutagens in the yeast Saccharomyces cerevisiae.
Screening of a multi-copy vector-based yeast genomic library in haploid cells of wild-type Saccharomyces cerevisiae yielded transformants hyper-resistant to various chemical mutagens. Genetical analysis of the yeast insert DNAs revealed three genes SNG1, SNQ2, and SNQ3 that confer the phenotype hyper-resistance to MNNG, to 4-NQO and triaziquone, and to mutagens 4-NQO, MNNG, and triaziquone, respectively. Integration of the gene disruption-constructs into the haploid yeast genome yielded viable null-mutants with a mutagen-sensitive phenotype. Thus, copy number of these non-essential yeast genes determines the relative resistance to certain chemical mutagens, with zero copies yielding a phenotype of mutagen sensitivity and multiple copies one of mutagen hyper-resistance, respectively.
4-Nitroquinoline-1-oxide↗
Rapid and simple isolation of DNA from agarose gels.
We present a simple method for the isolation of DNA from agarose gels that is economic, fast, and independent of electrical equipment. DNA fragments of up to 6 kb can be easily extracted within 5 min using a disposable plastic syringe and filter paper. Total extraction of DNA fragments between 10 and 20 kb in size is achieved by concentrating the DNA flushed from the gel in a DNA-binding column.
Cloning, Molecular↗
Hyper-resistance to nitrogen mustard in Saccharomyces cerevisiae is caused by defective choline transport.
The recessive hnm1 mutant allele is responsible for hyper-resistance to nitrogen mustard in Saccharomyces cerevisiae. Transformation with a single-copy HNM1 wild-type allele of such hyper-resistant mutants will restore wild-type sensitivity to nitrogen mustard. By contrast the presence of multi-copy vectors containing HNM1, in either a hyper-resistant hnm1 mutant or an HNM1 wild-type, will lead to a novel, mustard-sensitive phenotype unrelated to defects in DNA repair genes. Gene disruption of HNM1 revealed that this gene is non-essential for cells prototrophic for choline (CHO1) but lethal for cells with a cho1 genotype. Sensitivity to nitrogen mustard of wild-type HNM1, but not of hnm1 mutants, depends on the choline content of the growth medium, with cells grown in choline-free medium exhibiting the highest sensitivity. Sequencing of a 300 bp DNA fragment of HNM1 revealed the identity of this gene with the CTR locus, which is responsible for choline transport in Saccharomyces cerevisiae.
Alleles↗
The SNQ3 gene of Saccharomyces cerevisiae confers hyper-resistance to several functionally unrelated chemicals.
A multi-copy plasmid containing the SNQ3 gene confers hyper-resistance to 4-nitroquinoline-N-oxide (4NQO), Trenimon, MNNG, cycloheximide, and to sulfometuron methyl in yeast transformants. Restriction analysis, subcloning, and DNA sequencing revealed an open reading frame of 1,950 bp on the SNQ3-containing insert DNA. Gene disruption and transplacement into chromosomal DNA yielded 4NQO-sensitive null mutants which were also more sensitive than the wild-type to Trenimon, cycloheximide, sulfometuron methyl, and MNNG. Hydropathic analysis showed that the SNQ3-encoded protein is most likely not membrane-bound, while the codon bias index points to low expression of the gene.
Amino Acid Sequence↗
2',3'-Dideoxycytidine preferentially inhibits in vitro growth of granulocyte-macrophage colony-forming cells from patients with chronic myeloid leukemia.
The effect of three nucleoside analogs, including 2-methyl-2'-deoxyadenosine (MDA), 5-amino-2'-deoxyuridine (ADU) and 2',3'-dideoxycytidine (DDC) on colony formation from unfractionated human bone marrow obtained from volunteers expressing parameters typical for normal cells (NBM) and from patients with the diagnosis of chronic myeloid leukemia (CMLBM) was observed. For the clonal growth of granulocyte-macrophage colony-forming cells (GM-CFC), a semisolid fibrin clot culture medium supplemented with 20% fetal bovine serum and 10% human placental conditioned medium was used. DDC has been shown to be at least a 10-fold more potent inhibitor for the growth of GM-CFC from CMLBM than from NBM. On the other hand, the effect of MDA and ADU on CMLBM did not differ markedly from the effect on NBM. These results suggest that DDC inhibits preferentially progenitor cells from CMLBM.
Bone Marrow↗
[Therapy of banal HSV lesions: molecular mechanisms of the antiviral activity of zinc sulfate].
Preparations of heparin in combination with the physiologically harmless and even beneficial zinc sulphate are available for the topical treatment of superficial mucocutaneous lesions caused by HSV (herpes simplex virus). We study the molecular mechanism of the antiviral effects of zinc ions. A concentration of 100 mumol/l Zn2+ in the culture medium reduces the virus yield in a HSV infected AGMK cell line to less than 1/1000 of the control. At this concentration zinc sulphate does not exert any major cytotoxic effects, nor does it block the synthesis of viral or cellular DNA. Free virus, however, is inactivated by 8 orders of magnitude by 15 mmol/l zinc sulphate within a few hours. The inactivated virus is limited in the glycoprotein-dependent functions adsorption and penetration. Electron micrographs show massive deposition of zinc onto virion components. The dramatic antiviral effect in vivo is therefore explained by an inhibition of virion glycoprotein functions after accumulation of zinc in the virion, presumably by binding to sulphhydryl groups of glycoprotein B.
DNA, Viral↗
The role of PSO and SNM genes in DNA repair of the yeast Saccharomyces cerevisiae.
Explore the source record for details and available documents.
DNA Repair↗
A recessive mutant allele of the HNM1 gene of Saccharomyces cerevisiae is responsible for hyper-resistance to nitrogen mustard.
A screening of haploid yeast strains for enhanced resistance to nitrogen mustard (HN2) yielded a recessive mutant allele, hnm1, that conferred hyper-resistance (HYR) to HN2. Diploids, homo- or heterozygous for the HNM1 locus, exhibit normal wild-type like resistance while homozygosity for hnm1 leads to the phenotype HYR to HN2. The hnm1 mutation could be found in yeast strains proficient or deficient in different DNA repair systems. In these mostly HN2-sensitive haploid repair-deficient mutants, hnm1 acted as a partial suppressor of HN2 sensitivity. All isolated recessive mutations conferring hyper-resistance belonged to a single complementation group. The HYR to HN2 phenotype was maximally expressed in growing cells and was associated with reduced mutability by HN2. HNM1 most probably controls uptake of HN2 which would be impaired in the hnm1 mutants.
Alleles↗
Allelism of SNQ1 and ATR1, genes of the yeast Saccharomyces cerevisiae required for controlling sensitivity to 4-nitroquinoline-N-oxide and aminotriazole.
SNQ1 gene function is required for the expression of resistance to 4NQO in wild-type yeast. The sequence of a 3.7 kb yeast DNA containing the gene SNQ1 was determined. The SNQ1 gene consists of an open reading frame of 1641 bp and encodes, according to the hydrophobicity analysis of the putative protein, a transmembrane protein of 547 amino acids. Homology searches in yeast genome databanks revealed a 100% sequence homology with gene ATR1 which controls resistance to aminotriazole in S. cerevisiae. Pre-treatment of wild-type yeast, but not of snq1-0::LEU2 disruption mutants, with sublethal doses of aminotriazole induced hyper-resistance to 4-nitroquinoline-N-oxide. Partial deletion of the nucleotide sequence coding for a putative ATP-binding site has no, or little, influence on resistance to 4NQO whereas total deletion of the region coding for this ATP-binding domain leads to 4NQO-sensitive null-mutants.
4-Nitroquinoline-1-oxide↗
The mechanism of the antiherpetic activity of zinc sulphate.
The molecular mechanism of the effects of zinc ions against herpes simplex virus (HSV) infection was investigated. Zinc sulphate (100 microM) in the culture medium of an HSV-infected African green monkey kidney cell line did not block viral DNA synthesis and, at this concentration, only moderate cytotoxic effects were observed in uninfected cells. Nevertheless, virus yields were reduced to less than 1% of the control. Thus the long standing hypothesis that zinc might block multiplication of HSV by selective intranuclear inhibition of the viral DNA polymerase apparently has lost its validity. Inhibition of virus growth in the absence of severe cytotoxicity must therefore result from other effects of ZnSO4. Free virus is inactivated by 15 mM-ZnSO4 within a few hours of its addition. The inactivated virus is defective in the glycoprotein-dependent functions of penetration and, to some extent, adsorption. Electron micrographs show massive deposition of zinc onto virion components. In a virion, transmembrane transport of zinc ions is not expected and the established antiviral effect is therefore explained by an inhibition of virion glycoprotein function after non-specific accumulation of zinc into many virion membrane components.
Adsorption↗
Impact of culture and cryopreservation on MHC class II antigen expression in canine and porcine islets.
Canine and porcine islets were either cultured for 10 days at 37 degrees C or cryopreserved. The effect of these treatments on MHC class II antigen expression was examined by indirect immunofluorescence test using the class II monoclonal antibody 2MC3. Within untreated canine islets exclusively round shaped cells as leukocytes and monocyte/macrophage like cells with dendritic branches were positive with 2MC3. Porcine islets additionally exhibited strong immunofluorescence of the vascular endothelium. Tissue culture significantly reduced the class II antigen expression in both species. The majority of the untreated canine islets had between 5 and 19 2MC3 positive cells. Cell culture reduced the number of class II positive cells to a maximum of 3 and even 84.9% of the islets were completely negative for class II antigens. Porcine islets showed a total class II antigen loss of the vascular endothelium, whereas leukocytes and monocytes/macrophages remained class II antigen positive. Cryopreservation did not have clear-cut effects on the MHC expression in both species.
Animals↗
Molecular cloning of SNM1, a yeast gene responsible for a specific step in the repair of cross-linked DNA.
We have isolated yeast gene SNM1 via complementation of sensitivity towards bi- and tri-functional alkylating agents in haploid and diploid yeast DNA repair-deficient snm1-1 mutants. Four independent clones of plasmid DNA containing the SNM1 locus were isolated after transformation with a YEp24-based yeast gene bank. Subcloned SNM1-containing DNA showed (i) complementation of the repair-deficiency phenotype caused by either one of the two different mutant alleles snm1-1 and snm1-2ts; (ii) complementation in haploid and diploid yeast snm1-1 mutants by either single or multiple copies of the SNM1 locus; and (iii) that the SNM1 gene is at most 2.4 kb in size. Expression of SNM1 on the smallest subclone, however, was under the control of the GAL1 promotor. Gene size and direction of transcription was further verified by mutagenesis of SNM1 by Tn10-LUK transposon insertion. Five plasmids containing Tn10-LUK insertions at different sites of the SNM1-containing DNA were able to disrupt the function of genomic SNM1 after gene transplacement. Correct integration of the disrupted SNM1::Tn10-LUK at the genomic site of SNM1 was verified via tetrad analysis of the sporulated diploid obtained after mating of the SNM1::Tn10-LUK transformant to a haploid strain containing the URA3 SNM1 wild-type alleles. The size of the poly(A)+ RNA transcript of the SNM1 gene is 1.1 kb as determined by Northern analysis.
Blotting, Northern↗