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M J Leibowitz

Publications and source records attributed to M J Leibowitz.

At least 37 records · Page 2Linked to original sources
PubMed1993

Genetics of heat-curability of killer virus of yeast.

The cytoplasmically inherited M double-stranded (ds) RNA genome segment of killer virus of Saccharomyces cerevisiae is heat-curable in some yeast strains but not in others. Temperature sensitivity is conferred on both M1 and M2 dsRNA satellite virus segments by the L-A-HN allele of the killer helper virus genome, but not by the L-A-H allele. Both diploidy and mating type heterozygosity of the host cell are also correlated with increased virus curability.

Base Sequence↗
PubMed1993

Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA.

On the basis of the reported enhanced antisense activity of polylysine-oligonucleotide conjugates, a synthetic 12-mer oligodeoxyribonucleotide has been coupled at its 5' terminus to a series of positively charged (delta-ornithine)n cysteine peptides. Binding between the nucleic acid-peptide conjugate and its complementary DNA target sequence was detected by the impact of complexation on the melting temperature (Tm). It was found that the Tm for the nucleic acid-peptide gradually increased with increasing net charge on the conjugated peptide. Site-directed cleavage with RNase H demonstrates that the peptide-modified oligomer also hybridizes with its RNA target sequence. Increased affinity for target mRNA with net charge was shown by a cell-free translation arrest assay.

Base Sequence↗
PubMed1992

Design of peptide-acridine mimics of ribonuclease activity.

A series of peptide-acridine conjugates was designed and synthesized, based on three features of the proposed catalytic mechanism of RNase A: 2'-proton abstraction by His-12, proton donation to the leaving 5'-oxygen by His-119, and stabilization of the pentacoordinated phosphorous transition state by Lys-41. The substrate binding capability of RNase A was mimicked by the intercalator, acridine. Lysine served as a linker between acridine and the catalytic tripeptide. Cleavage of target RNA was monitored by agarose gel electrophoresis and by gel-permeation chromatography. The carboxyl-amidated conjugates HGHK(Acr)-NH2, HPHK(Acr)-NH2, and GGHK(Acr)-NH2 (where Acr indicates 2-methyl-9-acridinemethylene) all had similar hydrolytic activity. The catalytic mechanism most likely involved only the abstraction of the 2'-proton and stabilization of the transition state. These RNase mimics utilized rRNA and single-stranded RNA but not double-stranded RNA and tRNA as substrates.

Acridines↗
PubMed1992

Sequence and variability of the 5.8S and 26S rRNA genes of Pneumocystis carinii.

The sequence of the coding region of the rRNA operon of rat-derived Pneumocystis carinii has been completed, including the genes for 5.8S and 26S rRNA. These genes show homology to the rRNA genes of yeast, and an apparent group I self-splicing intron is present in the 26S rRNA gene. Like a similar intron in the 16S rRNA gene, this intron is in a phylogenetically conserved region. Variation in the 26S rRNA sequence was noted between P. carinii organisms isolated from two different sources.

Base Sequence↗
PubMed1992

Yeast killer virus transcription initiation in vitro.

Killer virions isolated from infected Saccharomyces cerevisiae cells contain an RNA polymerase activity which catalyzes the transcription in vitro of positive polarity RNAs from the L-A and M double-stranded RNA genomic segments of the virus. The RNA polymerase can initiate transcription in vitro with gamma-thio-GTP, whose thiophosphate group is found on the 5' terminus of transcripts. Transcripts produced in vitro by the virion-associated RNA polymerase in the presence of 7mGpppG are significantly more active as translational templates than are transcripts produced in its absence. However, unlike Escherichia coli RNA polymerase transcripts from viral cDNA made in the presence of 7mGpppG, transcripts produced by viral RNA polymerase in the presence of 7mGpppG fail to bind to antibody against 7mG.

DNA-Directed RNA Polymerases↗
PubMed1991

Coupling of killer virus transcription with translation in yeast cell-free extracts.

The cytoplasmically inherited killer virus of Saccharomyces cerevisiae expresses its dsRNA genome via apparently uncapped viral transcripts produced in the cytoplasm of infected cells. Virions of this naturally temperature-sensitive virus can be added to cell-free translational extracts of uninfected yeast cells resulting in a reaction in which viral transcription and translation are coupled at 15 degrees C in vitro. In this reaction nucleotides are incorporated into full-length transcripts of the M and L-A dsRNA segments, with lower levels of incorporation into genomic RNA. In addition, incorporation of nucleotides is observed into a smaller RNA species showing no sequence relatedness to M or L-A.

Base Sequence↗
PubMed1987

Nucleotide phosphotransferase, nucleotide kinase and inorganic pyrophosphatase activities of killer virions of yeast.

The intracellular killer virions of yeast co-purify with an RNA polymerase activity which catalyzes the synthesis of full-length transcripts of the two viral genomic double-stranded RNA segments. This polymerase utilizes ribonucleoside diphosphates or triphosphates as substrates. The virions have other associated nucleotide-metabolizing enzyme activities, including nucleoside diphosphate kinase, adenosine monophosphate kinase, and nucleoside triphosphate phosphotransferase, an activity which catalyzes the exchange of gamma-phosphate from any ribonucleoside triphosphate with any ribonucleoside or deoxyribonucleoside triphosphate. The purified virions also contain an inorganic pyrophosphatase activity. These enzymes may allow the virus to utilize nucleotide pools distinct from those utilized in host cell transcription.

Adenosine Diphosphate↗
PubMed1987

Killing of Saccharomyces cerevisiae by the lysosomotropic detergent N-dodecylimidazole.

The lysosomotropic detergent N-dodecylimidazole (C12-Im) has previously been found to kill mammalian cells by concentrating in lysosomes, followed by lysosomal disruption and release of cytotoxic enzymes into the cytoplasm. The action of C12-Im on Saccharomyces cerevisiae is described in this report. C12-Im prevented growth of colonies when present in 1% yeast extract-2% Bacto-Peptone-2% glucose plates at concentrations of 5 micrograms/ml or above, or when present in a soft agar overlay at 20 micrograms/ml. Treatment of cells suspended in glucose-containing buffer (pH 8.0, 37 degrees C) with C12-Im (6 micrograms/ml) caused greater than 95% cell death within 6 min. Dependence of killing on C12-Im concentration was sigmoidal, suggesting a cooperative mode of action. Killing was pH dependent, being much more effective at pH 8.0 than at pH 5.0. Ammonium sulfate and imidazole protected against killing if added before, but not after, the addition of C12-Im. Sensitivity to C12-Im was strongly growth dependent: the cells were most sensitive at early to mid-logarithmic phase of growth and became progressively less sensitive during progression through late logarithmic and stationary phase. Vacuolar disruption by C12-Im was demonstrated by using cells loaded with lucifer yellow CH or fluoresceinated dextran in their vacuoles; vacuoles of logarithmically growing cells were more sensitive than those of stationary-phase cells. These results suggest that vacuolar disruption by C12-Im may underlie its cytotoxic effects.

Ammonium Sulfate↗
PubMed1986

The internal polyadenylate tract of yeast killer virus M1 double-stranded RNA is variable in length.

The 1.8-kbp M1 double-stranded (ds) RNA from type 1 killer strains of Saccharomyces cerevisiae contains an internal 200-bp adenine- and uracil-rich region. We have previously demonstrated that this region consists primarily of adenine residues on the plus strand of M1 dsRNA and on the full-length, in vitro synthesized (+) transcript (denoted m) of M1 dsRNA, neither of which contains 3'-terminal polyadenylate. We now show that there is variability in the length of the polyadenylate tracts of m transcripts synthesized in vitro by virions purified from either of the K1 diploid killer strains A364A X S7 or A364A X 1384. This variability reflects size differences seen in the corresponding M1 dsRNA genomes which, along with other data presented, localizes the variability in the length of M1 dsRNA to the adenine- and uracil-rich region.

Molecular Weight↗
PubMed1986

Sequence of the M1-2 region of killer virus double-stranded RNA.

A full-length complementary DNA (cDNA) copy of the M1-2 region of the double-stranded genome of the yeast killer virus was synthesized by reverse transcription, utilizing the m in vitro transcript as template and synthetic primers for both strands. The sequence lacks any long open reading frames (ORFs). The internal portion of the M1-2 region includes the sequence that is linked to the subterminal 229 bases of the M1-1 homologous region in the S3 defective-interfering mutant of killer virus double-stranded RNA (dsRNA). Thus, the probable site at which the deletion occurred in S3 has been identified.

Base Sequence↗
PubMed1986

Translation of homologous and heterologous messenger RNAs in a yeast cell-free system.

A stable mRNA-dependent cell-free translation system from Saccharomyces cerevisiae, prepared by a modification of the method of Hofbauer et al. [Eur. J. Biochem. 122 (1982) 199-203] was active in translation of exogenous homologous and heterologous mRNAs. Optimal translational activity required the addition of polyamines and yeast tRNA. The m transcript of the M segment of double-stranded RNA, synthesized in vitro using the killer virus-associated RNA polymerase, directed the synthesis of preprotoxin polypeptide (M-p32), which was immunologically identified using antitoxin antibody. Sindbis virus capsid protein and rabbit globin were also translated from their mRNAs. Translation was inhibited by puromycin, sparsomycin and anisomycin. Analogues of the 5'-terminal caps present on most eukaryotic mRNA molecules inhibited translation of added mRNAs, including capped mRNAs and the uncapped killer virus mRNA.

Cell-Free System↗
PubMed1986

5S RNA and tRNA-like molecules are associated with killer virus dsRNA of yeast.

A population of oligonucleotides co-purified with the dsRNA genomic segments of the killer virus of Saccharomyces cerevisiae during electrophoresis through agarose gels. These smaller RNA molecules must be separated from the viral genome in order to determine the structure of the dsRNA molecules. Sequence analysis of these isolated oligonucleotides showed that the population contained tRNA-like molecules, as well as 5S RNA, which are presumably encoded by the host cell genome.

Base Sequence↗
PubMed1985

Structure and expression of the M2 genomic segment of a type 2 killer virus of yeast.

The M2 double-stranded (ds) RNA species encodes toxin and resistance functions in Saccharomyces cerevisiae strains with the K2 killer specificity. RNA sequence analysis reveals the presence of a large open reading frame on the larger heat-cleavage product of M2 dsRNA, which is translated in vitro to yield a 28 kd polypeptide as a major product. The postulated translation initiator AUG triplet is located within a stem and loop structure near the 5' terminus of the positive strand, which also contains plausible 18S and 5.8S ribosomal RNA binding sites. These features may serve to regulate the translation of the K2 toxin precursor. The M1 (from type 1 yeast killers) and M2 dsRNA species lack extensive sequence homology, although specific features are shared, which may represent structural elements required for gene expression and replication.

Amino Acid Sequence↗
PubMed1985

On the mechanism of exclusion of M2 double-stranded RNA by L-A-E double-stranded RNA in Saccharomyces cerevisiae.

L-A-E double-stranded RNA (dsRNA), when introduced into cells carrying L-A-H and M2 dsRNAs, does not eliminate the L-A-H dsRNA, but (i) L-A-E does lower the copy number of L-A-H dramatically and (ii) L-A-E eliminates M2 dsRNA from the cell. That these two effects of L-A-E are related is shown by the fact that mutants of a strain carrying L-A-H and M2 selected for their resistance to exclusion of M2 by L-A-E [effect (ii)] have an altered L-A-H whose copy number is not lowered by L-A-E [effect (i)]. Although the L-A in K1 strains (L-A-HN in all cases examined) differs significantly both genetically and physically from the L-A in the K2 strain studied (L-A-H), the L-A-HN from the K1 strains can maintain M2 dsRNA, and the L-A-H from the K2 strains can maintain M1 dsRNA.

Chromosomes↗
PubMed1985

[1,4]Benzoxazine-2,3-diones as antiallergic agents.

The synthesis of a series of [1,4]benzoxazine-2,3-diones and a new class of compounds, benzobisoxazinetetrones, is described. These compounds were evaluated for their effect in the rat mast cell (RMC) test passively sensitized in vitro with rat antiovalbumin serum and for their effect in inhibitory passive cutaneous anaphylaxis (PCA) in the rat. Some of these compounds are of the same potency level as disodium cromglycate in the RMC test and some are effective orally in PCA.

Animals↗