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D A Sinclair

Publications and source records attributed to D A Sinclair.

At least 19 recordsLinked to original sources
PubMed2005

A genetic and molecular profile of third chromosome centric heterochromatin in Drosophila melanogaster.

In this review, we combine the results of our published and unpublished work with the published results of other laboratories to provide an updated map of the centromeric heterochromatin of chromosome 3 in Drosophila melanogaster. To date, we can identify more than 20 genes (defined DNA sequences with well-characterized functions and (or) defined genetic complementation groups), including at least 16 essential loci. With the ongoing emergence of data from genetic, cytological, and genome sequencing studies, we anticipate continued, substantial progress towards understanding the function, structure, and evolution of centric heterochromatin.

Animals↗
PubMed2001

Recombination-mediated lengthening of terminal telomeric repeats requires the Sgs1 DNA helicase.

The Saccharomyces cerevisiae SGS1 gene encodes a RecQ-like DNA helicase, human homologues of which are implicated in the genetic instability disorders, Bloom syndrome (BS), Rothmund-Thomson syndrome (RTS), and Werner syndrome (WS). Telomerase-negative yeast cells can recover from senescence via two recombinational telomere elongation pathways. The "type I" pathway generates telomeres with large blocks of telomeric and subtelomeric sequences and short terminal repeat tracts. The "type II" pathway generates telomeres with extremely long heterogeneous terminal repeat tracts, reminiscent of the long telomeres observed in telomerase-deficient human tumors and tumor-derived cell lines. Here, we report that telomerase-negative (est2) yeast cells lacking SGS1 senesced more rapidly, experienced a higher rate of telomere erosion, and were delayed in the generation of survivors. The est2 sgs1 survivors that were generated grew poorly, arrested in G(2)/M and possessed exclusively type I telomeres, implying that SGS1 is critical for the type II pathway. The mouse WS gene suppressed the slow growth and G(2)/M arrest phenotype of est2 sgs1 survivors, arguing that the telomeric function of SGS1 is conserved. Reintroduction of SGS1 into est2 sgs1 survivors restored growth rate and extended terminal tracts by approximately 300 bp. Both phenotypes were absolutely dependent on Sgs1 helicase activity. Introduction of an sgs1 carboxyl-terminal truncation allele with helicase activity restored growth rate without extending telomeres in most cases, demonstrating that type II telomeres are not necessary for normal growth in the absence of telomerase.

Animals↗
PubMed2001

Essential genes in proximal 3L heterochromatin of Drosophila melanogaster.

We have further characterized essential loci within the centric heterochromatin of the left arm of chromosome 3 (3L) of Drosophila melanogaster, using EMS, radiation and P element mutagenesis. We failed to find any new essential genes, a result that suggests a lower-than-average gene density in this region. Mutations affecting expression of the most proximal gene [lethal 1, l1 or l(3)80Fj] act as dominant suppressors of Polycomb (Pc), behavior which is consistent with a putative trithorax group (trx-G) gene. The third gene to the left of the centromere [lethal 3, l3 or l(3)80Fh] is likely to correspond to verthandi (vtd), a known trx-G gene that plays a role in the regulation of hedgehog (hh) expression and signalling. The intervening gene [lethal 2, l2 or l(3)80Fi] is required throughout development, and mutant alleles have interesting phenotypes; in various allelic combinations that survive, we observe fertility, bristle, wing, eye and cuticle defects.

Animals↗
PubMed2000

Cloning and characterization of a new theta-class glutathione-S-transferase (GST) gene, gst-3, from Drosophila melanogaster.

We report here on the cloning and characterization of a new theta-class glutathione-S-transferase (GST) gene, gst-3, from Drosophila melanogaster. Its sequence is distinct from previously characterized Drosophila GST genes, and Southern blotting shows no other closely related genes in the genome. In-situ hybridization localizes the gene to chromosome 2 (55D), near gst-2 (53F), and clearly separate from the gst-D cluster at 87B. The gene is intronless and appears to possess conventional 5' TATA, Cap and 3' polyadenylation signals. A single transcript, approximately 1kb in size, appears to be expressed at high levels in all developmental stages examined. When this gene is overexpressed using various upstream GAL4 driver systems, no striking phenotypes are observed; however, we detect bristle morphology defects in some progeny. The gst-3 gene does not appear to be essential, based upon our observation that mutant flies homozygous for an EP element insertion 5' to the TATA box produce little or no detectable gst-3 mRNA; these flies are viable and fertile at 25 and 29 degrees C. Nevertheless, the gst-3 gene appears to be evolutionarily conserved in other Drosophila species, suggesting that it may be functionally important.

Amino Acid Sequence↗
PubMed2000

Essential genes in autosomal heterochromatin of Drosophila melanogaster.

We are taking two approaches to understanding the structure, function and regulation of essential genes within Drosophila heterochromatin. In the first, we have undertaken a genetic and molecular characterization of essential genes within proximal 3L heterochromatin. The expression of such 'resident' genes within a heterochromatic environment is paradoxical and poorly understood, given that the same environment can inactivate euchromatic sequences (position effect variegation, or PEV). A second approach involves the study of the local chromosomal environment of heterochromatic (het) genes, as assayed both biochemically, and via the effects of genetic modifiers of PEV, the latter being putative components important for het gene expression. Our results to date suggest that the three most proximal genes in 3L heterochromatin have key roles in development, and indicate strong effects of combinations of genetic modifiers of PEV on het gene expression.

Animals↗
PubMed1999

Yeast aging research: recent advances and medical relevance.

The molecular mechanisms of aging are most fully understood for the budding yeast Saccharomyces cerevisiae. Recent advances in our understanding of aging in this organism have enabled researchers to answer some fundamental questions about the aging process. Is aging due to a multitude of 'mechanisms' or can there be a key few? Can we design single-gene mutations that will prolong life? Can we prolong life whilst maintaining health and fecundity? The various contributing factors to yeast longevity, uncovered thus far, fall into three classes: DNA metabolism, heterochromatin, and metabolic activity. However, these separate classes may actually represent different aspects of the same aging mechanism based on genome stability. This review examines the recent advances in our understanding of yeast aging and discusses their relevance, if any, to the human condition.

Aging↗
PubMed1999

MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks.

The yeast Sir2/3/4p complex is found in abundance at telomeres, where it participates in the formation of silent heterochromatin and telomere maintenance. Here, we show that Sir3p is released from telomeres in response to DNA double-strand breaks (DSBs), binds to DSBs, and mediates their repair, independent of cell mating type. Sir3p relocalization is S phase specific and, importantly, requires the DNA damage checkpoint genes MEC1 and RAD9. MEC1 is a homolog of ATM, mutations in which cause ataxia telangiectasia (A-T), a disease characterized by various neurologic and immunologic abnormalities, a predisposition for cancer, and a cellular defect in repair of DSBs. This novel mode by which preformed DNA repair machinery is mobilized by DNA damage sensors may have implications for human diseases resulting from defective DSB repair.

Ataxia Telangiectasia↗
PubMed1999

Control of expression of one-carbon metabolism genes of Saccharomyces cerevisiae is mediated by a tetrahydrofolate-responsive protein binding to a glycine regulatory region including a core 5'-CTTCTT-3' motif.

Expression of yeast genes involved in one-carbon metabolism is controlled by glycine, by L-methionine, and by nitrogen sources. Here we report a novel control element containing a core CTTCTT motif mediating the glycine response, demonstrating that a protein binds this element, that binding is modulated by tetrahydrofolate, and that folate is required for the in vivo glycine response. In an heterologous CYC1 promoter the region needed for the glycine response of GCV2 (encoding the P-subunit of glycine decarboxylase) mediated repression that was relieved by glycine. It was also responsible for L-methionine control but not nitrogen repression. GCV1 and GCV3 have an homologous region in their promoters. The GCV1 region conferred a glycine response on an heterologous promoter acting as a repressor or activator depending on promoter context. A protein was identified that bound to the glycine regulatory regions of GCV1 and GCV2 only if the CTTCTT motif was intact. This protein protected a 17-base pair CATCN7CTTCTT region of GCV2 that is conserved between GCV1 and GCV2. Protein binding was increased by tetrahydrofolate, and use of a fol1 deletion mutant indicated the involvement of a folate in the in vivo glycine response. Tetrahydrofolate or a derivative may act as a ligand for the transcription factor controlling expression of one-carbon metabolism genes.

Amino Acid Oxidoreductases↗
PubMed1999

The Additional sex combs gene of Drosophila is required for activation and repression of homeotic loci, and interacts specifically with Polycomb and super sex combs.

The protein products of Polycomb group (PcG) and trithorax group (trxG) genes are required for the maintenance of the transcriptionally repressed and active states, respectively, of the homeotic genes. Mutations in PcG genes produce gain-of-function (posterior) homeotic transformations, while mutations in trxG genes produce loss-of-function (anterior) homeotic transformations. Double mutant combinations between a PcG gene and a trxG gene suppress the homeotic transformations seen with either mutation alone, suggesting that PcG and trxG genes act antagonistically. The PcG gene Additional sex combs (Asx) is interesting because one mutant allele, AsxP1, causes both anterior and posterior homeotic transformations. AsxP1 and other Asx mutations were crossed to mutations in the PcG gene Polycomb (Pc) and the trxG gene trithorax (trx). Asx alleles enhance both PcG and trxG homeotic transformations, showing that Asx is required for both the activation and the repression of homeotic loci. Asx also shows strong allele-specific interactions with the PcG genes Pc and super sex combs (sxc). Together, these data indicate that there are functional interactions between Asx, Pc and sxc in vivo. ASX may interact with a PcG complex containing PC and SXC and mediate activation versus repression at target loci, perhaps by interacting directly with the TRX protein.

Alleles↗
PubMed1999

Genomic imprinting and position-effect variegation in Drosophila melanogaster.

Genomic imprinting is a phenomenon in which the expression of a gene or chromosomal region depends on the sex of the individual transmitting it. The term imprinting was first coined to describe parent-specific chromosome behavior in the dipteran insect Sciara and has since been described in many organisms, including other insects, plants, fish, and mammals. In this article we describe a mini-X chromosome in Drosophila melanogaster that shows genomic imprinting of at least three closely linked genes. The imprinting of these genes is observed as mosaic silencing when the genes are transmitted by the male parent, in contrast to essentially wild-type expression when the same genes are maternally transmitted. We show that the imprint is due to the sex of the parent rather than to a conventional maternal effect, differential mitotic instability of the mini-X chromosome, or an allele-specific effect. Finally, we have examined the effects of classical modifiers of position-effect variegation on the maintenance and the establishment of the imprint. Factors that modify position-effect variegation alter the somatic expression but not the establishment of the imprint. This suggests that chromatin structure is important in maintenance of the imprint, but a separate mechanism may be responsible for its initiation.

Alleles↗
PubMed1999

A genetic and molecular characterization of the garnet gene of Drosophila melanogaster.

The garnet gene was one of the first genes to be identified in Drosophila melanogaster. Mutations in the garnet gene affect both of the biochemically distinct types of pigments in the eye and disrupt pigmentation of other organs. As an initial step in the analysis of this gene, we have analyzed the pigmentation defects in several of the garnet alleles. We have also cloned the gene and examined its expression in various tissues and at different stages of development. The garnet gene is expressed throughout development and in all tissues examined. Structurally related sequences can be detected in a variety of other eukaryotes. The predicted protein sequence of the garnet product resembles clathrin and nonclathrin adaptin proteins and is highly similar to the delta subunit of the newly isolated mammalian AP-3 adaptin complex, which is associated with the trans-Golgi network and endosomes. This suggests that garnet encodes a protein that acts in the intracellular sorting and trafficking of vesicles from the trans-Golgi network to endosomes, and related specialized organelles such as the pigment granule. This finding provides an explanation for the phenotype of garnet mutations and predicts that other Drosophila eye-colour genes will be a rich resource for the genetic dissection of intracellular vesicle transport.

Adaptor Protein Complex 3↗
PubMed1998

Characterization of a Drosophila homologue of the 160-kDa subunit of the cleavage and polyadenylation specificity factor CPSF.

Processing of the 3' end of mRNA precursors depends on several proteins. The multisubunit cleavage and polyadenylation specificity factor (CPSF) is required for cleavage of the mRNA precursor as well as polyadenylation. CPSF interacts with the cleavage stimulatory factor complex (CstF), and this interaction increases the specificity of binding. Following cleavage downstream of the AAUAAA site, CPSF and poly(A) polymerase (PAP) are required for efficient polyadenylation. Recently, it has been shown that 160-kDa subunit of CPSF interacts directly with the 77-kDa subunit of CstF, which is homologous to the product encoded by the Drosophila gene su(f), and with PAP. Here we report the cloning and characterization of a Drosophila homologue of CPSF-160. The 1329-amino acid dCPSF protein exhibits about 45% and 20% sequence identity, respectively, to its mammalian and yeast counterparts over its entire length. We show that the CPSF homologue is expressed throughout development and that CPSF is essential for viability. Mutations in the cpsf gene did not alter the phenotype of homozygous su(f) mutations, suggesting that, for most genes, processing of 3' termini is not sensitive to small changes in cpsf and su(f) dosage.

Amino Acid Sequence↗
PubMed1998

Molecular mechanisms of yeast aging.

The life cycle of many organisms involves a progressive decline in fitness and fecundity with age, and yeast is no exception. Many theories have been proposed to explain the mortality of yeast cells, including the increase in cell size and accumulation of bud scars on the cell surface. None of these has survived closed scrutiny. However, recent discoveries might have validated one aging model in which the triggering of a molecular aging clock results in the replication and accumulation of a senescence factor that eventually overwhelms old cells.

DNA, Fungal↗
PubMed1998

Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster.

Polycomb group (PcG) genes of Drosophila are negative regulators of homeotic gene expression required for maintenance of determination. Sequence similarity between Polycomb and Su(var)205 led to the suggestion that PcG genes and modifiers of position-effect variegation (PEV) might function analogously in the establishment of chromatin structure. If PcG proteins participate directly in the same process that leads to PEV, PcG mutations should suppress PEV. We show that mutations in E(Pc), an unusual member of the PcG, suppress PEV of four variegating rearrangements: In(l)wm4, B(SV), T(2;3)Sb(V) and In(2R)bw(VDe2). Using reversion of a Pelement insertion, deficiency mapping, and recombination mapping as criteria, homeotic effects and suppression of PEV associated with E(Pc) co-map. Asx is an enhancer of PEV, whereas nine other PcG loci do not affect PEV. These results support the conclusion that there are fewer similarities between PcG genes and modifiers of PEV than previously supposed. However, E(Pc) appears to be an important link between the two groups. We discuss why Asx might act as an enhancer of PEV.

Animals↗
PubMed1998

The enhancer of polycomb gene of Drosophila encodes a chromatin protein conserved in yeast and mammals.

The Polycomb group of genes in Drosophila are homeotic switch gene regulators that maintain homeotic gene repression through a possible chromatin regulatory mechanism. The Enhancer of Polycomb (E(Pc)) gene of Drosophila is an unusual member of the Polycomb group. Most PcG genes have homeotic phenotypes and are required for repression of homeotic loci, but mutations in E(Pc) exhibit no homeotic transformations and have only a very weak effect on expression of Abd-B. However, mutations in E(Pc) are strong enhancers of mutations in many Polycomb group genes and are also strong suppressors of position-effect variegation, suggesting that E(Pc) may have a wider role in chromatin formation or gene regulation than other Polycomb group genes. E(Pc) was cloned by transposon tagging, and encodes a novel 2023 amino acid protein with regions enriched in glutamine, alanine and asparagine. E(Pc) is expressed ubiquitously in Drosophila embryogenesis. E(Pc) is a chromatin protein, binding to polytene chromosomes at about 100 sites, including the Antennapedia but not the Bithorax complex, 29% of which are shared with Polycomb-binding sites. Surprisingly, E(Pc) was not detected in the heterochromatic chromocenter. This result suggests that E(Pc) has a functional rather than structural role in heterochromatin formation and argues against the heterochromatin model for PcG function. Using homology cloning techniques, we identified a mouse homologue of E(Pc), termed Epc1, a yeast protein that we name EPL1, and as well as additional ESTs from Caenorhabditis elegans, mice and humans. Epc1 shares a long, highly conserved domain in its amino terminus with E(Pc) that is also conserved in yeast, C. elegans and humans. The occurrence of E(Pc) across such divergent species is unusual for both PcG proteins and for suppressors of position-effect variegation, and suggests that E(Pc) has an important role in the regulation of chromatin structure in eukaryotes.

Amino Acid Sequence↗
PubMed1998

The Additional sex combs gene of Drosophila encodes a chromatin protein that binds to shared and unique Polycomb group sites on polytene chromosomes.

The Additional sex combs (Asx) gene of Drosophila is a member of the Polycomb group of genes, which are required for maintenance of stable repression of homeotic and other loci. Asx is unusual among the Polycomb group because: (1) one Asx allele exhibits both anterior and posterior transformations; (2) Asx mutations enhance anterior transformations of trx mutations; (3) Asx mutations exhibit segmentation phenotypes in addition to homeotic phenotypes; (4) Asx is an Enhancer of position-effect variegation and (5) Asx displays tissue-specific derepression of target genes. Asx was cloned by transposon tagging and encodes a protein of 1668 amino acids containing an unusual cysteine cluster at the carboxy terminus. The protein is ubiquitously expressed during development. We show that Asx is required in the central nervous system to regulate Ultrabithorax. ASX binds to multiple sites on polytene chromosomes, 70% of which overlap those of Polycomb, polyhomeotic and Polycomblike, and 30% of which are unique. The differences in target site recognition may account for some of the differences in Asx phenotypes relative to other members of the Polycomb group.

Amino Acid Sequence↗
PubMed1998

The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

Amino Acid Sequence↗