-
[
Proc Natl Acad Sci U S A,
1993]
The nematode Caenorhabditis elegans has become an organism of choice for the study of developmental processes at the genetic level. We have undertaken to develop an in vitro system to study transcription in C. elegans. As a first step we report here the cloning of the cDNA encoding the C. elegans TATA-box-binding protein (CeTBP). We used ''touchdown PCR'' to generate a specific DNA probe derived from the C-terminal region conserved in all TBP genes cloned to date. Several clones encoding an extended open reading frame were isolated from a phage lambda cDNA library. The complete amino acid sequence of CeTBP deduced from the cDNA reveals a protein of 37 kDa with an extended sequence similarity in the C-terminal region with all other TBP cDNAs sequenced so far. The N-terminal region of CeTBP (amino acids 1-153), however, does not show any homology with TBPs from other organisms. Interestingly, the N-terminal portion of the molecule contains three short direct repeats. Purified recombinant CeTBP binds specifically to the TATA box sequence, interacts with transcription factors TFIIA and TFIIB, and is able to substitute for the MID basal activity when assayed by in vitro transcription in both HeLa and C. elegans nuclear extracts. CeTBP is therefore a basal transcription factor.
-
[
EMBO J,
1995]
The nematode Caenorhabditis elegans has been a choice organism for the study of developmental regulation using classical and molecular genetic approaches. Consequently, many genetically defined pathways have been described and numerous regulatory genes have been identified. However, the biochemical and functional properties of these putative transcription factors have remained uncharacterized, partly because C.elegans cell-free transcription reactions have not been developed. Here we describe the in vitro transcriptional activation properties of two C.elegans homeodomain proteins, UNC-86 and MEC-3, in nuclear extracts derived from C.elegans embryos. Whereas the POU homeodomain protein, UNC-86, alone was able to activate transcription of the
mec-3 promoter in vitro, the LIM homeodomain protein, MEC-3, failed to bind DNA or activate transcription on its own. However, in the presence of both UNC-86 and MEC-3, we observed cooperative binding to the
mec-3 promoter and synergistic activation of transcription in vitro. Protein-protein interaction assays revealed that UNC-86 can bind directly to MEC-3, and in vitro transcription studies indicate that both proteins contain a functional activation domain. Thus, formation of a heteromeric complex containing two activation domains results in a highly potent activator. These studies provide direct functional evidence for coordinated transcriptional activation by two C.elegans DNA binding proteins that have been defined genetically as regulators of gene expression during embryogenesis.
-
[
Nature,
1999]
Activation of gene transcription in metazoans is a multistep process that is triggered by factors that recognize transcriptional enhancer sites in DNA. These factors work with co-activators to direct transcriptional initiation by the RNA polymerase II apparatus. One class of co-activator, the TAF(II) subunits of transcription factor TFIID, can serve as targets of activators and as proteins that recognize core promoter sequences necessary for transcription initiation. Transcriptional activation by enhancer-binding factors such as Sp1 requires TFIID, but the identity of other necessary cofactors has remained unknown. Here we describe a new human factor, CRSP, that is required together with the TAF(II)s for transcriptional activation by Sp1. Purification of CRSP identifies a complex of approximate relative molecular mass 700,000 (M(r) approximately 700K) that contains nine subunits with M(r) values ranging from 33K to 200K. Cloning of genes encoding CRSP subunits reveals that CRSP33 is a homologue of the yeast mediator subunit Med7, whereas CRSP150 contains a domain conserved in yeast mediator subunit Rgr1. CRSP
p200 is identical to the nuclear hormone-receptor co-activator subunit TRIP2/PBP. CRSPs 34, 77 and 130 are new proteins, but the amino terminus of CRSP70 is homologous to elongation factor TFIIS. Immunodepletion studies confirm that these subunits have an essential cofactor function. The presence of common subunits in distinct cofactor complexes suggests a combinatorial mechanism of co-activator assembly during transcriptional activation.
-
[
Zootaxa,
2022]
Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.
-
[
Nature,
1999]
Gene activation in eukaryotes is regulated by complex mechanisms in which the recruitment and assembly of the transcriptional machinery is directed by gene- and cell-type-specific DNA-binding proteins. When DNA is packaged into chromatin, the regulation of gene activation requires new classes of chromatin-targeting activity. In humans, a multisubunit cofactor functions in a chromatin-selective manner to potentiate synergistic gene activation by the transcriptional activators SREBP-1a and Sp1. Here we show that this activator-recruited cofactor (ARC) interacts directly with several different activators, including SREBP-1a, VP16 and the
p65 subunit of NF-kappaB, and strongly enhances transcription directed by these activators in vitro with chromatin-assembled DNA templates. The ARC complex consists of 16 or more subunits; some of these are novel gene products, whereas others are present in other multisubunit cofactors, such as CRSP, NAT and mammalian Mediator. Detailed analysis indicates that the ARC complex is probably identical to the nuclear hormone-receptor cofactor DRIP. Thus, ARC/DRIP is a large composite co-activator that belongs to a family of related cofactors and is targeted by different classes of activator to mediate transcriptional stimulation.
-
[
J Biol Chem,
1990]
The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.
-
[
Nat Commun,
2021]
R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes.PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.
-
[
Commun Integr Biol,
2011]
The development of bilateral symmetry during the evolution of species probably 600 million years ago brought about several important innovations: It fostered efficient locomotion, streamlining and favored the development of a central nervous system through cephalization. However, to increase their functional capacities, many organisms exhibit chirality by breaking their superficial left-right (l-r) symmetry, which manifests in the lateralization of the nervous system or the l-r asymmetry of internal organs. In most bilateria, the mechanisms that maintain consistent l-r asymmetry throughout development are poorly understood. This review highlights insights into mechanisms that couple early embryonic l-r symmetry breaking to subsequent l-r patterning in the roundworm Caenorhabditis elegans. A recently identified strategy for l-r patterning in the early C. elegans embryo is discussed, the spatial separation of midline and anteroposterior axis, which relies on a rotational cellular rearrangement and non-canonical Wnt signaling. Evidence for a general relevance of rotational/torsional rearrangements during organismal l-r patterning and for non-canonical Wnt signaling/planar cell polarity as a common signaling mechanism to maintain l-r asymmetry is presented.
-
[
J Biol Chem,
2007]
The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.
-
[
Mol Cell,
2013]
R loops are transcription byproducts that constitute athreat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation.Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonucleaseH overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.