Biomedical subjects

D E Gottschling

Publications and source records attributed to D E Gottschling.

28 records · Page 2Linked to original sources

PubMed1994

Overcoming telomeric silencing: a trans-activator competes to establish gene expression in a cell cycle-dependent way.

Genes located near telomeres in yeast are subject to position-effect variegation. To better understand the mechanism of this variegation, we investigated how a telomeric URA3 gene switches from a silent to an expressed state. We found that silencing of a telomeric URA3 gene was attributable to the elimination of its basal transcription. The reversal of that silencing was dependent on the presence of PPR1, the trans-activator protein of URA3. Maximum expression of URA3 required a higher concentration of PPR1 when URA3 was telomeric compared with when it was at a nontelomeric location. The ability of PPR1 to overcome silencing varied at different points in the cell cycle. In cells arrested in G2/metaphase, PPR1 was able to activate transcription of a telomeric URA3, but in cells arrested in G0, G1, or early S phase it was not. In comparison, a nontelomeric URA3 could be activated by PPR1 at all times. We conclude that once established, telomeric silent chromatin is a relatively stable structure, making a gene recalcitrant to activation. Following the disassembly of silent chromatin during DNA replication, competition of assembly ensues between components of telomeric chromatin, to establish a silent state, and the trans-activator, to establish gene expression. These results help explain the stochastic nature of phenotypic switching in variegated gene expression.

Base Sequence↗

PubMed1993

Silent domains are assembled continuously from the telomere and are defined by promoter distance and strength, and by SIR3 dosage.

In Saccharomyces cerevisiae, telomeres repress transcription of genes located nearby. This region-specific gene inactivation is thought to involve the packaging of telomeric domains into silent chromatin. To gain insight into the mechanism of telomeric silencing, a genetic assay to examine the spread of silencing along the distal right arm of chromosome V was developed. The frequency of silencing a telomere-adjacent URA3 gene decreased with increasing distance of the gene's promoter from the telomere, irrespective of transcriptional orientation. The distance over which telomeric silencing of URA3 was observed was extended by weakening the gene's promoter--specifically, by deleting PPR1, the trans-activator of URA3. The silent telomeric domain was extended even farther by increasing the gene dosage of SIR3. These results suggest that a gene's promoter is a key determinant in controlling silencing on that gene and that SIR3 is a crucial component of the silent chromatin domain that initiates at the telomere and is assembled inwardly along the yeast chromosome. Finally, silencing is not observed on the centromeric side of an actively transcribed telomeric gene, demonstrating that the repressed telomeric domain is propagated continuously along the DNA. Taken together, these data reflect the complex and dynamic organization of eukaryotic genomes into functionally distinct regions.

Chromosomes, Fungal↗

PubMed1992

Telomere-proximal DNA in Saccharomyces cerevisiae is refractory to methyltransferase activity in vivo.

Genes located near telomeres in Saccharomyces cerevisiae undergo position-effect variegation; their transcription is subject to reversible but mitotically heritable repression. This position effect and the finding that telomeric DNA is late replicating suggest that yeast telomeres exist in a heterochromatin-like state. Mutations in genes that suppress the telomeric position effect suggest that a special chromatin structure exists near chromosomal termini. Thus transcriptional repression may be explained by the inability of DNA binding proteins to access the DNA near telomeres. To test this hypothesis, the Escherichia coli Dam DNA methyltransferase, which modifies the sequence GATC, was introduced into S. cerevisiae cells. DNA sequences near the telomere were highly refractive to Dam methylation but were modified when located at positions more internal on the chromosome. Telomeric sequences were accessible to methyltransferase activity in strains that contained a mutation that suppressed the telomeric position effect. These data support the model that sequence-specific DNA binding proteins are excluded from telomere-proximal sequences in vivo and show that expression of DNA methyltransferase activity may serve as a useful tool for mapping chromosomal structural domains in vivo.

Chromatin↗

PubMed1992

Saccharomyces telomeres assume a non-nucleosomal chromatin structure.

The chromatin structures of the telomeric and subtelomeric regions on chromosomal DNA molecules in Saccharomyces cerevisiae were analyzed using micrococcal nuclease and DNAse I. The subtelomeric repeats X and Y' were assembled in nucleosomes. However, the terminal tracts of C1-3A repeats were protein protected in a particle larger than a nucleosome herein called a telosome. The proximal boundary of the telosome was a DNase I hypersensitive site. This boundary between the telosome and adjacent nucleosomes was completely accessible to Escherichia coli dam methylase when this enzyme was expressed in yeast, whereas a site 250 bp internal to the telomeric repeats was relatively inaccessible. Telosomes could be cleaved from chromosome ends with nuclease and solubilized as protein-DNA complexes. Immunoprecipitation of chromosomal telosomes with antiserum to the RAP1 protein indicated that RAP1 was one component of isolated telosomes. Thus, the termini of chromosomal DNA molecules in yeast are assembled in a non-nucleosomal structure encompassing the entire terminal C1-3A tract. This structure is separated from adjacent nucleosomes by a region of DNA that is highly accessible to enzymes.

Chromatin↗

PubMed1991

Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae.

Genes placed near telomeres in S. cerevisiae succumb to position-effect variegation. SIR2, SIR3, SIR4, NAT1, ARD1, and HHF2 (histone H4) were identified as modifiers of the position effect at telomeres, since transcriptional repression near telomeres was no longer observed when any of the modifier genes were mutated. These genes, in addition to SIR1, have previously been shown to repress transcription at the silent mating loci, HML and HMR. However, there were differences between transcriptional silencing at telomeres and the HM loci, as demonstrated by suppressor analysis and the lack of involvement of SIR1 in telomeric silencing. These findings provide insights into telomeric structure and function that are likely to apply to many eukaryotes. In addition, the distinctions between telomeres and the HM loci suggest a hierarchy of chromosomal silencing in S. cerevisiae.

Chromosomes, Fungal↗

PubMed1990

Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription.

S. cerevisiae chromosomes end with the telomeric repeat (TG1-3)n. When any of four Pol II genes was placed immediately adjacent to the telomeric repeats, expression of the gene was reversibly repressed as demonstrated by phenotype and mRNA analyses. For example, cells bearing a telomere-linked copy of ADE2 produced predominantly red colonies (a phenotype characteristic of ade2- cells) containing white sectors (characteristic of ADE2+ cells). Repression was due to proximity to the telomere itself since an 81 bp tract of (TG1-3)n positioned downstream of URA3 when URA3 was approximately 20 kb from the end of chromosome VII did not alter expression of the gene. However, this internal tract of (TG1-3)n could spontaneously become telomeric, in which case expression of the URA3 gene was repressed. These data demonstrate that yeast telomeres exert a position effect on the transcription of nearby genes, an effect that is under epigenetic control.

Chromosome Mapping↗

PubMed1986

Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear DNA.

The macronuclear DNA in the ciliated protozoan O. nova consists of integral of 10(7) gene-sized DNA molecules, all of which terminate with 20 bp of C4A4 repeats followed by a 3' (G4T4)2 single-stranded tail. Two immunologically distinct proteins of 55 and 26 kd, which are tenaciously, but noncovalently associated with Oxytricha macronuclear DNA termini, have been purified. These proteins protect DNA termini from degradation by the exonuclease Bal31. They also facilitate retention of natural and synthetic telomeric DNAs onto nitrocellulose. The Oxytricha proteins are not simply C4A4-binding proteins. Rather, their efficient binding requires both the 3' single-stranded (G4T4)2 tail and the adjacent duplex region. Thus, these proteins require both the sequence and the structure of natural DNA termini for efficient binding. As such they represent the first described example of telomeric-specific proteins.

Animals↗

PubMed1984

Chromatin structure of the molecular ends of Oxytricha macronuclear DNA: phased nucleosomes and a telomeric complex.

Oxytricha macronuclear DNA exists as approximately 24 X 10(6) gene-sized molecules terminating with a C4A4 repeat. DNA-protein interactions at the ends of bulk macronuclear molecules were probed with micrococcal nuclease and methidiumpropyl-EDTA X Fe(II) (MPE X Fe[II]). The ends were indirectly labeled by hybridizing with (C4A4)2. Alternatively, a novel method using MPE X FE(II) as a probe and directly labeling the 3' ends with terminal transferase was implemented. A terminal complex involving approximately 100 bp with nucleosomes phased inward from the complex was found to be characteristic of most or all of the ends. Analysis of two specific genes confirmed the pattern and showed that the special structure was on both ends of each molecule. We conclude that a DNA-protein complex involving 100 bp and terminating with the C4A4 repeat can be sufficient to provide the fundamental functions of telomeres, allowing linear DNA replication and conferring stability of linear DNA.

Animals↗

PubMed1983

Different nucleosome spacing in transcribed and non-transcribed regions of the ribosomal RNA gene in Tetrahymena thermophila.

The chromatin structure of the palindromic macronuclear ribosomal RNA genes of Tetrahymena thermophila was probed with micrococcal nuclease. Independent of the state of transcriptional activity, the transcribed region had a shorter nucleosome repeat (184 +/- 3 base pairs) than the non-transcribed central spacer or bulk chromatin (both 200 base pairs). The transcribed region displayed an increased sensitivity to micrococcal nuclease in rapidly growing cells, which suggested an altered chromatin structure during transcription. At early stages of nuclease digestion, the central spacer appeared to be in a highly structured nucleosomal array. Based on the differences in nucleosome repeat distance and sensitivity to nuclease, we conclude that quite different chromatin structures are maintained in two adjacent regions of the Tetrahymena ribosomal RNA gene. The DNA of the non-transcribed terminal spacer was found to contain sequences which are highly susceptible to micrococcal nuclease, precluding any conclusions about nucleosome structure in this region.

Animals↗

PubMed1982

Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena.

In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid. Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized. When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing. The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons. We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions. We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds. The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.

Animals↗