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Y Koltin

Publications and source records attributed to Y Koltin.

At least 37 records · Page 2Linked to original sources
PubMed1992

Neutralizing monoclonal antibodies against alpha and beta subunits of the Ustilago maydis virus encoded toxin.

The toxins secreted by Ustilago maydis are encoded by dsRNA viruses. The KP6 toxin encoded by subtype P6 consists of two polypeptides alpha and beta, which are not covalently bound. Neutralizing monoclonal antibodies (MoAbs) were raised against each subunit. Some of the anti-beta MoAbs identify different epitopes in the antigen. The MoAbs were used to affinity purify alpha and beta polypeptides from culture media and to detect the precursor of the mature toxin.

Antibodies, Monoclonal↗
PubMed1991

Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate.

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including beta-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.

Amino Acid Sequence↗
PubMed1991

Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

Amino Acid Isomerases↗
PubMed1990

Functional expression of the Candida albicans beta-tubulin gene in Saccharomyces cerevisiae.

Expression of the beta-tubulin-encoding gene (TUB2) of Candida albicans has been examined in Saccharomyces cerevisiae. Overexpression of the TUB2 gene of C. albicans, as well as that of S. cerevisiae, was found to be lethal. Chromosomal integration of the C. albicans TUB2 gene into a strain in which the native TUB2 gene had been deleted led to functional complementation. The results demonstrate that correct splicing of the two introns present in the C. albicans TUB2 gene occurs in the heterologous host strain containing this gene. Such strains are supersensitive to the tubulin-binding agent benomyl, indicating that the natural resistance of C. albicans to benomyl is not related to the structure of its beta-tubulin.

Benomyl↗
PubMed1990

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species. Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins. The best characterized of the U. maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked. In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus. They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae k1 killer toxin, also encoded by a double-strand RNA virus. Active U. maydis KP6 toxin was secreted from S. cerevisiae transformants expressing the KP6 preprotoxin. The two secreted polypeptides were not glycosylated in U. maydis, but one was glycosylated in S. cerevisiae. Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a three-amino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi. The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.

Amino Acid Sequence↗
PubMed1989

A very small viral double-stranded RNA.

UmV is a double-stranded RNA (dsRNA) virus of the corn fungal pathogen Ustilago maydis. UmV has no infectious cycle. Some UmV subtypes have viral dsRNAs encoding secreted toxins that kill sensitive cells of the same species and related species. There are three viral subtypes, P1, P4 and P6, which differ in the specificity of their secreted killer toxins. Each has three size classes of dsRNA: H (heavy), M (medium) and L (light). The L segments of UmV are unique in being derived from one end of the larger M segments. We have sequenced P1 L and placed it at the 3' end of the P1 M1 plus strand. In their overlapping regions, these dsRNAs are identical in sequence. In vitro translation of P1 M1 results in a peptide whose size is consistent with its being encoded by the non-L region of M1. P1 L is a very small dsRNA of 355 bp. It has no long open reading frames and produces no detectable in vitro translation product. The sequence of P1 L suggests that it is derived by a process unique among dsRNA viruses: replication and packaging of the 3' end fragment of a processed mRNA.

Base Sequence↗
PubMed1989

Detection of killer-independent dsRNA plasmids in Ustilago maydis by a simple and rapid method of extraction of dsRNA.

A novel method for efficient and rapid isolation of dsRNA molecules was developed. The dsRNA content of Ustilago maydis was reexamined; two distinct dsRNA classes were identified. Class I includes the dsRNA segments reported earlier for U. maydis virus systems and class II includes unencapsidated dsRNA molecules that were barely detected by the conventional extraction methods despite their high titer. Segments of the class II, some of which are reported for the first time, were further characterized; all the segments are independent of the killer system and other encapsidated dsRNA molecules. These segments are cytoplasmically transmitted and, in sharp contrast with class I-encapsidated dsRNA segments, their relative copy number decreases rapidly while entering the stationary phase.

Basidiomycota↗
PubMed1988

Enhancement of copper resistance and CupI amplification in carcinogen-treated yeast cells.

Carcinogen-induced amplification at the CupI locus, coding for a metallothionein protein, was studied in the yeast Saccharomyces cerevisiae. Exposure of cells from three different haploid strains, 4939, DBY746 and 320, to chemical carcinogens such as N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), ethylmethanesulfonate (EMS) and 4-nitroquinoline-N-oxide (4NQO) enhanced the frequency of copper-resistant colonies up to several hundred fold. Copper-resistant clones obtained from strains DBY746 and 320, which contain more than one copy of the CupI locus, displayed a four- to eightfold amplification of the CupI sequences. In these clones the amplified CupI sequences were organized in a tandem array. Carcinogen treatment of strain 4939 in which only one copy of the CupI gene is present produced resistant colonies without CupI amplification. The possible use of the yeast system to study gene duplication and amplification is discussed.

Carcinogens↗
PubMed1988

Transcription and in vitro translation of the dsRNA virus isolated from Rhizoctonia solani.

A segmented double-stranded dsRNA virus has been isolated from virulent strains of Rhizoctonia solani. The dsRNA genome has mol. wts. of 1.45 and 1.32 X 10(6). Two full-size transcripts with mol. wts. of 0.74 and 0.66 X 10(6) (2.2 kb and 2 kb, respectively) were synthesized by the virus-associated RNA-dependent RNA polymerase and resolved by denaturing polyacrylamide gel electrophoresis. The transcripts cross-hybridized to the viral dsRNA isolated from a number of strains. The transcripts did not hybridize with the genomic DNA. An unencapsidated species of dsRNA with mol. wt. of 1.6 X 10(6) did not hybridize with the viral transcripts. No cross-hybridization between the two viral dsRNA segments was obtained. The viral-encoded proteins were studied by in vitro translation using the rabbit reticulocyte lysate system. The transcripts served as mRNA for the synthesis of the major capsid protein of 55 kD, and a number of other products. The viral coat protein was immunoprecipitated with antibodies against purified virus particles. Partial proteolysis of the major in vitro product and the authentic capsid protein using Staphylococcus aureus V8 protease produced similar peptide patterns. Denatured viral dsRNA also directed the synthesis of proteins identical to those translated from the transcripts in vitro.

Animals↗
PubMed1988

Isolation and characterization of a beta-tubulin gene from Candida albicans.

We report the isolation and nucleotide sequence determination of a beta-tubulin gene (TUB2) from the pathogenic dimorphic fungus Candida albicans. Nucleotide sequence analysis revealed that TUB2 encodes a protein of 449 amino acids (aa) with considerable sequence homology to beta-tubulins isolated from other fungal species. The nucleotide sequence of the C. albicans gene is 70% homologous to that of the Saccharomyces cerevisiae gene. The coding region for the C. albicans beta-tubulin gene is interrupted by two introns. The first intron occurs after the 4th aa and the second intron occurs after the 13th aa. A comparison with other fungal beta-tubulin genes indicates that the intron locations are highly conserved. Codon usage in the C. albicans TUB2 gene is nonrandom, as has been observed for other fungal beta-tubulin genes. The C. albicans TUB2 gene is transcribed to yield a 1.8-kb mRNA species. On the basis of genomic Southern-blot analysis, we conclude that C. albicans most likely possesses a single beta-tubulin gene.

Amino Acid Sequence↗
PubMed1988

Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes.

By using orthogonal-field alternating gel electrophoresis (OFAGE), field-inversion gel electrophoresis (FIGE), and contour-clamped homogeneous field gel electrophoresis (CHEF), we have clearly resolved 11 chromosomal bands from various Candida albicans strains. OFAGE resolves the smaller chromosomes better, while FIGE, which under our conditions causes the chromosomes to run in the reverse order of OFAGE, is more effective in separating the larger chromosomes. CHEF separates all chromosomes under some conditions, but these conditions do not often resolve homologs. The strains examined are highly polymorphic for chromosome size. Fourteen cloned Candida genes, isolated on the basis of conferral of new properties to or complementation of auxotrophic deficiencies in Saccharomyces cerevisiae, and three sequences of unknown function have been hybridized to Southern transfers of CHEF, FIGE, and OFAGE gels. Four sets of resolvable bands have been shown to be homologous chromosomes. On the basis of these data, we suggest that C. albicans has seven chromosomes. Genes have been assigned to the seven chromosomes. Two chromosomes identified genetically have been located on the electrophoretic karyotype.

Candida albicans↗
PubMed1987

Virus-encoded toxin of Ustilago maydis: two polypeptides are essential for activity.

Cells of Ustilago maydis containing double-stranded RNA viruses secrete a virus-encoded toxin to which other cells of the same species and related species are sensitive. Mutants affected in the expression of the KP6 toxin were characterized, and all were viral mutants. A temperature-sensitive nonkiller mutant indicated that the toxin consists of two polypeptides, 12.5K and 10K, that are essential for the toxic activity. The temperature-sensitive nonkiller mutant was affected in the expression of the 10K polypeptide, and its toxic activity was restored by the addition of the 10K polypeptide to its secreted inactive toxin. These results led to the reexamination of other mutants that were known to complement in vitro. Each was found to secrete one of the two polypeptides. Here we show for the first time that P6 toxin consists of two polypeptides that do not interact in solution, but both are essential for the toxic effect. Studies on the interaction between the two polypeptides indicated that there are no covalent or hydrogen bonds between the polypeptides. Toxin activity is not affected by the presence of 0.3 M NaCl in the toxin preparations and in the medium, suggesting that no electrostatic forces are involved in this interaction. Also, the two polypeptides do not share common antigenic determinants. The activity of the two polypeptides appears to be dependent on a sequential interaction with the target cell, and it is the 10K polypeptide that initiates the toxic effect. The similarity of the U. maydis virus-encoded toxin to that of Saccharomyces cerevisiae is discussed.

Basidiomycota↗
PubMed1985

Isolation of genes from Candida albicans by complementation in Saccharomyces cerevisiae.

A genomic library of the asexual pathogenic yeast Candida albicans was constructed in the S. cerevisiae vector YEp13. The library contains a representation of the entire genome with a probability of 99%. The expression of the genes of C. albicans in S. cerevisiae was examined and two mutations his3-1 and trp1-289 of S. cerevisiae were complemented by the cloned genes of C. albicans. The hybridization data indicates that the plasmids complementing the mutations of S. cerevisiae contain sequences from C. albicans.

Candida albicans↗
PubMed1984

RNA polymerase activity in virions from Ustilago maydis.

RNA polymerase activity is associated with the double-stranded RNA virions of Ustilago maydis. The reaction products of the polymerase activity are single-stranded RNA molecules. The RNA molecules synthesized are homologous to the three classes of double-stranded RNA molecules that typify the viral genome. The single-stranded RNA synthesized is released from the virions. The molecular weight of the single-stranded RNA transcripts is about half the size of the double-stranded RNA segments, and thus, it appears that in the in vitro reaction, full-length transcripts can be obtained.

Basidiomycota↗
PubMed1984

Six groups of double-stranded RNA mycoviruses.

Six groups of double-stranded (ds) RNA mycoviruses have been proposed. The main characteristics which define a group are described, and the properties of members and probable members of each group are tabulated. Possibilities for organization of the groups into families, genera and species are discussed. The classification scheme could ultimately accommodate the majority of the well-characterized dsRNA mycoviruses.

Fungi↗
PubMed1983

Two Ustilago maydis viral dsRNAs of different size code for the same product.

UmV is a double-stranded RNA (dsRNA) virus of the corn fungus Ustilago maydis. There are three viral subtypes, P1, P4 and P6, which differ in the specificity of their secreted killer toxins. Each has three size classes of dsRNAs: H (heavy), M (medium), and L (light). We find that, unique among dsRNA viruses, two segments of different size code for the same product--the toxin resistance factor. The smaller dsRNA (L) is homologous to one end of the larger (M), and may have arisen by replication and packaging of a sub-genomic mRNA. We have also compared all the UmV dsRNAs with each other and with the dsRNAs of the similar yeast virus (ScV) by Northern gel and by 3' sequence analysis. Like those of ScV, many of the UmV dsRNAs have one 3' terminus with the sequence UUUUUCAOH or UUUUUCGOH. The H and L dsRNAs of similar size in different viral subtypes are generally related in sequence. The UmV H dsRNAs of different size are not detectably related in sequence.

Base Sequence↗
PubMed1983

Mutator activity in Schizophyllum commune.

A strain with an elevated level of spontaneous mutations and an especially high rate of reversion at a specific locus (pab-) was identified. The mutator trait is recessive. UV sensitivity and the absence of a UV-specific endonucleolytic activity were associated with the enhancement of the mutation rate in mutator strains. The endonuclease associated with the regulation of the mutation rate also acted on single-stranded DNA. The molecular weight of this enzyme is about 38000 daltons.

Agaricales↗