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R Sternglanz

Publications and source records attributed to R Sternglanz.

10 recordsLinked to original sources
PubMed1992

Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease.

An assay was developed that detected DNA cruciform cutting endonuclease activity in crude extracts of Saccharomyces cerevisiae. A collection of temperature-sensitive strains was screened using this assay, and a mutant lacking the activity was found. The mutation leading to the enzymatic defect was mapped to the left arm of chromosome XI within 3 cM of the centromere. Cloning of the gene for this endonuclease was achieved by chromosome walking from the nearby PUT3 locus. The gene, called CCE1 (cruciform cutting endonuclease), was sequenced and found to have an open reading frame encoding a 41 kDa protein. The amino acid sequence of this eukaryotic endonuclease shows homology neither to its prokaryotic counterparts nor to other proteins in available databases. A cce1 null mutant has no obvious growth defect, and despite the ability of the CCE1 enzyme to cleave Holliday junction analogs, the mutant shows no defect in meiotic or mitotic recombination. A second cruciform cutting activity was detected in extracts from a cce1 null mutant, indicating that yeast has at least two such enzymes. The only phenotype observed for cce1 mutants is a higher than normal frequency of appearance of petite cells, suggesting that the CCE1 protein is important for the maintenance of mitochondrial DNA.

Amino Acid Sequence↗
PubMed1992

Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation.

Previous work has shown that rRNA synthesis is strongly inhibited in yeast top1-top2 double mutants. Here, we show that inactivation of yeast topoisomerases can have paradoxical effects on transcription by RNA polymerase I. For example, transcription of ribosomal minigenes on extrachromosomal plasmids is greatly stimulated in top1-top2 cells while accumulation of full-length endogenous rRNA is strongly inhibited. We present evidence for a mechanism that can partly account for these opposing effects on transcription. On the one hand, transcription initiation can be stimulated owing to an accumulation of negative superhelicity because polymerase I prefers to initiate on negatively supercoiled templates. Conversely, synthesis of full-length rRNA is inhibited owing to the fact that chain elongation requires a DNA relaxing activity.

Cloning, Molecular↗
PubMed1991

The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest.

We describe a method that detects proteins capable of interacting with a known protein and that results in the immediate availability of the cloned genes for these interacting proteins. Plasmids are constructed to encode two hybrid proteins. One hybrid consists of the DNA-binding domain of the yeast transcriptional activator protein GAL4 fused to the known protein; the other hybrid consists of the GAL4 activation domain fused to protein sequences encoded by a library of yeast genomic DNA fragments. Interaction between the known protein and a protein encoded by one of the library plasmids leads to transcriptional activation of a reporter gene containing a binding site for GAL4. We used this method with the yeast SIR4 protein, which is involved in the transcriptional repression of yeast mating type information. (i) We used the two-hybrid system to demonstrate that SIR4 can form homodimers. (ii) A small domain consisting of the C terminus of SIR4 was shown to be sufficient to mediate this interaction. (iii) We screened a library to detect hybrid proteins that could interact with the SIR4 C-terminal domain and identified SIR4 from this library. This approach could be readily extended to mammalian proteins by the construction of appropriate cDNA libraries in the activation domain plasmid.

Cloning, Molecular↗
PubMed1991

Nucleosome fractionation by mercury affinity chromatography. Contrasting distribution of transcriptionally active DNA sequences and acetylated histones in nucleosome fractions of wild-type yeast cells and cells expressing a histone H3 gene altered to encode a cysteine 110 residue.

A technique for the separation of transcriptionally active and inactive nucleosomes by mercury affinity chromatography has been applied to study the nucleosomal distribution of DNA sequences from the GAL1, ACT1, HIS4, MAT alpha, and HMRa genes of yeast. In mammalian cells, the method has been shown to separate active from inactive nucleosomes and to fractionate the active nucleosomes into two classes, one retained on the mercury column because of salt-labile associations with certain thiol-reactive non-histone proteins, and the other bound by covalent linkage of the cysteine 110 thiol groups of histone H3 molecules to the mercurated support. The first class of nucleosomes is elutable in 0.5 M NaCl; the second is displaced by 10 mM dithiothreitol (DTT) (Walker, J., Chen, T. A., Sterner, R., Berger, M., Winston, F., and Allfrey, V.G. (1990) J. Biol. Chem. 265, 5736-5746). We show that, in wild-type yeast cells, in which histone H3 lacks cysteinyl residues, very little DNA and a negligible complement of nucleosomes appear in the DTT-eluate, confirming the requirement for the H3-thiols in the mercury-binding reaction. Moreover, the DTT-eluted fraction is seriously deficient in the actively transcribed GAL1, ACT1, HIS4, and MAT alpha DNA sequences. Site-directed mutagenesis was employed to create an H3 gene containing a cysteine codon in place of the alanine codon at position 110 of the yeast H3 amino acid sequence. A strain was constructed containing the mutant histone H3 gene instead of the normal H3 gene. Subsequent fractionations of the mutant nucleosomes by mercury-affinity chromatography revealed a characteristic nucleosome peak in the DTT-eluted fraction. Its content of transcribed GAL1, ACT1, and HIS4 DNA sequences was 20- to 500-fold higher than that of the corresponding DTT-eluted fraction of wild-type yeast. Although this result is in accord with the finding that, in mammalian cells, the thiol groups of histone H3 become accessible when nucleosomes "unfold" during transcription, we find that nucleosomes containing the GAL1 DNA sequences of the yeast H3-mutant also bind to the mercury column when that gene is not being expressed. We conclude that many yeast nucleosomes are maintained in a "primed," potentially active state, possibly due to the very high constitutive levels of acetylation of the core histones. However, the nucleosomes of the HMRa gene, which is not expressed in a MAT alpha yeast strain, are virtually absent from the DTT-eluted nucleosome fractions of the H3-mutant cells, indicating that prolonged silencing of the gene is accompanied by compaction and loss of H3-thiol reactivity of its nucleosomes.

Acetylation↗
PubMed1991

The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants.

The SIR1 gene product of Saccharomyces cerevisiae is one of several proteins involved in repressing transcription of the silent mating-type genes. Strains with mutations in the genes coding for these proteins are defective in mating due to derepression of the silent loci. We have found that overexpression of the SIR1 gene suppresses the mating defects of several of these mutants, including nat1 and ard1 mutants (the products of these two genes are responsible for N-terminal acetylation of a subset of yeast proteins), certain sir3 mutants, and a histone H4 mutant. The SIR1 gene has been sequenced and found to contain an open reading frame coding for a 678-amino-acid protein.

Amino Acid Sequence↗
PubMed1979

Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli.

A new Escherichia coli deoxyribonucleic acid (DNA) ligase mutant has been identified among a collection of temperature-sensitive DNA replication mutants isolated recently (Sevastopoulos, Wehr, and Glaser, Proc. Natl. Acad. Sci. U.S.A. 74:3485-3489, 1977). At the nonpermissive temperature DNA synthesis in the mutant stops rapidly, the DNA is degraded to acid-soluble material, and cell death ensures. This suggests that the mutant may be among the most ligase-deficient strains yet characterized.

Chromosome Mapping↗
PubMed1978

Ionization and divalent cation dissociation constants of nalidixic and oxolinic acids.

The ionization constants and some divalent cation dissociation constants of nalidixic and oxolinic acids, both specific inhibitors of bacterial DNA replication, have been determined. The carboxylic pKa' values are 6.1 and 6.9 at 25 degrees for nalidixic and oxolinic acids, respectively. These values indicate that intramolecular hydrogen-bonding stabilizes the un-ionized form of these compounds in aqueous solution. Both compounds bind divalent cations; the divalent cation dissociation constants for oxolinic acid are somewhat smaller that those for nalidixic acid. We suggest that both compounds may act by forming a complex in situ with a divalent cation in a metalloprotein involved in DNA replication. The evidence that both drugs inhibit at the same target site is briefly reviewed.

Cations, Divalent↗
PubMed1977

Recent excitement in the DNA replication problem.

It is now possible to reproduce most of the reactions involved in DNA replication using prokaryotic enzymes in vitro. Such systems have revealed that DNA replication is a complex process depending on a relatively large number of proteins, and that nucleoside triphosphate hydrolysis energy is used at several discrete steps. Much of the complexity of DNA replication may arise from the need for extreme copying fidelity.

Adenosine Triphosphate↗
PubMed1976

Evidence that both growing DNA chains at a replication fork are synthesized discontinuously.

Escherichia coli, Bacillus subtilis, and T7-infected E. coli have been labeled with short pulses of [3H] thymidine, and the labeled DNA has been examined by sedimentation in alkaline sucrose. In all three systems, the great majority of the DNA labeled by a short pulse is found in the form of small DNA chains of 10S, the so-called Okazaki pieces. The B. subtilis and T7 nascent DNA fragments hybridize with equal efficiency to the separated strands of B. subtilis and T7 DNA, respectively. The results suggest that both growing DNA chains at a given replication fork are synthesized discontinuously in the case of E. coli, B. subtilis, and T7. We have found that the method used to terminate the pulse affects the size distribution of the labeled DNA; some methods allow joining of nascent DNA fragments after termination of the pulse. Previous reports of discontinuous DNA synthesis on only one growing DNA chain and continuous synthesis on the other DNA chain are probably due to preferential joining of Okazaki pieces on the DNA chain growing in the overall 5' leads to 3' direction.

Bacillus subtilis↗