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R H Morse

Publications and source records attributed to R H Morse.

At least 19 recordsLinked to original sources
PubMed1993

Nucleosome disruption by transcription factor binding in yeast.

Studies in vivo and in vitro have shown that the packaging of DNA into chromatin can affect gene expression. Here, binding of the yeast transcriptional activator GAL4 to DNA in chromatin has been investigated in vivo with a yeast episome. A positioned nucleosome that is present in cells grown in glucose and contains a single GAL4 binding site is disrupted by GAL4 binding in galactose. GAL4 can also bind to DNA in chromatin when the carboxyl-terminal activation domain of GAL4 is either masked by GAL80 or is absent. These results show that a transcription factor can bind to its site in vivo in what would appear to be a repressive chromatin structure.

Base Sequence↗
PubMed1992

Transcribed chromatin.

In eukaryotes, DNA that is transcribed is packaged first into nucleosomes and then into chromatin fibres. How does transcription proceed through chromatin? Studies of transcription through nucleosomes in vitro suggest that the intracellular environment may provide factors which alleviate the inhibitory effect that nucleosomes have on transcription, possibly via positive supercoiling induced by the migrating polymerase. Stable changes in nucleosome structure have been correlated with transcriptionally active chromatin, but the precise mechanism by which RNA polymerase transcribes through nucleosomal DNA remains unknown.

Animals↗
PubMed1992

A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo.

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.

Base Sequence↗
PubMed1991

Topoisomer heterogeneity of plasmid chromatin in living cells.

Previous investigations of topoisomer distributions of simian virus 40 (SV40) DNA from monkey cells have revealed that these circular mini-chromosomes, like relaxed, naked, closed circular DNA, exist as a Gaussian distribution of topoisomers. I have extended this comparison by measuring topoisomer distributions for a variety of plasmid episomes that are stably propagated in cells of the yeast Saccharomyces cerevisiae. The breadth of the topoisomer distributions for plasmid chromatin, including SV40, is approximately constant when normalized for DNA length, as is the breadth of distribution for naked DNA. However, the distributions for plasmid chromatin are substantially broader than those for the corresponding relaxed, naked DNAs. The breath is constant for plasmids differing in transcriptional activity, and varies only slightly between synchronized and unsynchronized populations of yeast cells, suggesting that variation in plasmid linking number with transcription or replication does not account for the observed heterogeneity in linking number. Topoisomer heterogeneity for plasmid chromatin in vivo may be due to heterogeneity in the number of nucleosomes on each plasmid, which could reflect either the nature of the assembly process or the dynamics of nucleosomes within the cell.

Cell Cycle↗
PubMed1990

The transcription complex of the Xenopus somatic 5 S RNA gene. A functional analysis of protein-DNA interactions outside of the internal control region.

We have defined protein-DNA interactions associated with the transcription complex of a Xenopus somatic 5 S RNA gene under efficient in vitro transcription conditions. Transcription factor IIIA, the internal control region of the 5 S RNA gene and specific DNA sequences 5' and 3' of the internal control region are all involved in the formation of a multiprotein complex. Specific protein-DNA interactions outside of the 5 S RNA gene itself, revealed by DNase I footprinting, have no apparent role in the transcription process. Sequences within the 5 S RNA gene, 5' of the internal control region, are not essential for transcription by RNA polymerase III in vitro, but do contribute to its efficiency.

Animals↗
PubMed1990

Effect of transcription of yeast chromatin on DNA topology in vivo.

Coding regions of transcribed and non-transcribed genes typically differ in chromatin structure. However, it is not known what kind of alterations in nucleosome or chromatin structure these differences reflect. To determine whether changes in nucleosome topology accompany transcription, we introduced into yeast a multicopy plasmid bearing the gene coding for the heat shock protein HSP26. The plasmid-borne gene is assembled into chromatin, and is induced by heat shock in the same manner as the endogenous HSP26 gene. A small change in linking number in the HSP26 plasmid accompanies heat shock. This change is consistent with that previously reported for thermal untwisting of DNA in yeast chromatin, and is equivalent in magnitude to that observed in control plasmids which lack heat-shock response elements. These data indicate that no stable alteration in nucleosome topology accompanies transcription of the heat shock gene. Moreover, the kinetics of the observed changes in linking number indicate that topoisomerase relaxes the thermally induced torsional stress in 1-5 min. We conclude that if alterations in nucleosome topology accompany polymerase passage, recovery must take place within this time period.

Chromatin↗
PubMed1990

Clinical patterns of failure following stereotactic interstitial irradiation for malignant gliomas.

The vast majority of patients treated for malignant gliomas with surgery, conventional radiation therapy, and systemic chemotherapy recur within 2 cm of their original disease site as documented by CT scanning. We have analyzed the clinical patterns of failure in patients treated with stereotactic interstitial irradiation (brachytherapy) for malignant gliomas in order to determine if this modality has altered the recurrence pattern in this disease. Between December 1985 and December 1989, 53 patients with malignant glioma were treated with stereotactic interstitial irradiation using temporary high activity iodine-125. Thirty-three patients were treated as part of a primary treatment protocol that included 5940 cGy external beam prior to implantation. Twenty patients were treated at time of recurrence. The median dose of radiation given at implantation was 5040 cGy for the primary lesions and 5450 cGy for the recurrent lesions. Twenty-two patients have suffered relapse as documented by clinical and radiographic studies. The predominant patterns of failure in these 22 patients were in the margins of the implant volume (8) and distant sites (10) within the CNS (distant ipsilateral or contralateral hemisphere, spinal axis) or extraneural. Thus, marginal and distant recurrences accounted for 82% of the relapses in our patients. We conclude stereotactic interstitial irradiation has changed the recurrence pattern in patients with malignant glioma with true local recurrence no longer being the predominant pattern of failure as is seen with conventional therapy.

Adolescent↗
PubMed1989

Nucleosomes inhibit both transcriptional initiation and elongation by RNA polymerase III in vitro.

To examine the effect of nucleosomes on in vitro transcription, purified chicken erythrocyte core histones and plasmid DNA bearing the Xenopus 5S RNA gene were assembled into nucleosomes and used as templates for transcription in a Xenopus oocyte nuclear extract. Plasmids having a nucleosome incorporating a specific region of the gene were selected by treating the reconstituted molecules with restriction endonucleases. In this way, it was shown that a nucleosome on or close to the internal control region of the 5S RNA gene inhibits transcription. Furthermore, experiments with 5S maxigenes showed that RNA polymerase III, in contrast to SP6 RNA polymerase, will not transcribe through a nucleosome in vitro.

Animals↗
PubMed1987

Yeast nucleosomes allow thermal untwisting of DNA.

Thermal untwisting of DNA is suppressed in vitro in nucleosomes formed with chicken or monkey histones. In contrast, results obtained for the 2 micron plasmid in Saccharomyces cerevisiae are consistent with only 30% of the DNA being constrained from thermal untwisting in vivo. In this paper, we examine thermal untwisting of several plasmids in yeast cells, nuclei, and nuclear extracts. All show the same quantitative degree of thermal untwisting, indicating that this phenomenon is independent of DNA sequence. Highly purified yeast plasmid chromatin also shows a large degree of thermal untwisting, whereas circular chromatin reconstituted using chicken histones is restrained from thermal untwisting in yeast nuclear extracts. Thus, the difference in thermal untwisting between yeast chromatin and that assembled with chicken histones is most likely due to differences in the constituent histone proteins.

Animals↗
PubMed1986

Effect of trypsinization and histone H5 addition on DNA twist and topology in reconstituted minichromosomes.

Free DNA in solution exhibits an untwisting of the double helix with increasing temperature. We have shown previously that when DNA is reconstituted with histones to form nucleosome core particles, both the core DNA and the adjacent linker DNA are constrained from thermal untwisting. The origin of this constraint is unknown. Here we examine the effect of two modifications of nucleosome structure on the constraint against thermal untwisting, and also on DNA topology. In one experiment, we removed the highly positively charged histone amino and carboxy termini by trypsinization. Alternatively, we added histone H5, a histone H1 variant from chick erythrocytes. Neither of these modifications had any major effect on DNA topology or twist in the nucleosome.

Animals↗
PubMed1985

Nucleosome core particles suppress the thermal untwisting of core DNA and adjacent linker DNA.

Covalently closed circular DNA is known to undergo a temperature-dependent change in helical twist. We have reconstituted nucleosome core particles onto closed circular DNA and measured the thermal untwisting of the DNA as a function of nucleosome density. The results demonstrate that the DNA associated with the nucleosome core particle does not alter its twist when the temperature is varied between 4 degrees C and 37 degrees C, and that the length of DNA prevented from thermal untwisting includes the linker as well as the core DNA.

Animals↗