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PubMed2004

Cg-Rel, the first Rel/NF-kappaB homolog characterized in a mollusk, the Pacific oyster Crassostrea gigas.

We report here the identification and functional characterization of Cg-Rel, a gene encoding the Crassostrea gigas homolog of Rel/NF-kappaB transcription factors found in insects and mammals. Sequence and phylogenetic analysis showed that Cg-Rel shares the structural organization of Rel/NF-kappaB transcription factors of class II. It includes a Rel homology domain as well as a C-terminal transactivation domain (TD). Overexpression of Cg-Rel in the Drosophila S2 cell line activated the expression of a NF-kappaB-dependent reporter gene, whereas transfection with a Cg-Rel construct containing a C-terminal deletion of the TD or using a reporter gene with mutated kappaB binding sites failed to activate expression. These results suggest that Cg-Rel is a functional member of the Rel family of transcription factors, making this the sixth structurally homologous component of the Rel/NF-kappaB pathway characterized in C. gigas. Based on homology to other invertebrates' Rel/NF-kappaB cascade, the function of the oyster pathway may serve to regulate genes involved in innate defense and/or development. These findings serve to highlight a potentially important regulatory pathway to the study of oyster immunology, hence allowing comparison of the immune system in vertebrates and invertebrates, an important key issue to understand its evolution.

Animals↗
PubMed2006

Topology of splicing and snRNP biogenesis in dinoflagellate nuclei.

BACKGROUND INFORMATION: Dinoflagellates are protists that are hypothesized to have experienced a secondary loss of histones. Amongst eukaryotes, they are unique in lacking these proteins. To date, information on the mechanisms involving remodelling, transcription and splicing of their chromatin is limited. Dinoflagellate genes lack TATA boxes and downstream polyadenylation sites and particular linear arrangements. They have an alpha-amanitin-sensitive RNA polymerase, specific transcription factors and regulators, and both transcriptional and post-transcriptional regulation of gene expression. Dinoflagellates produce either polycistronic or discrete mRNAs, and have conserved snRNAs (small nuclear RNAs), indicating that their genes are spliced. RESULTS: Five representative dinoflagellate species (Amphidinium carterae, Akashiwo sanguinea, Alexandrium lusitanicum, Alexandrium fundyense and Prorocentrum micans), which show diversity in their DNA content, nuclear organization and taxonomic position, were investigated. The nuclear distribution and ultrastructural organization of splicing and snRNP (small nuclear ribonucleoprotein) biogenesis were determined by fluorescent and electron microscopy immunolabelling with Y12 sera [recognizing the sDMA (symmetrical dimethylarginine) domain of Sm and other nuclear proteins], anti-p105-PANA [proliferation-associated nuclear antigen; a marker of IGs (interchromatin granules)] and anti-DNA antibodies. In parallel, ultrastructural analysis, including cytochemical staining for RNA, phosphorylated proteins and DNA, was carried out. Splicing factors were distributed in a diffuse perichromosomal layer containing perichromatin granules and fibrils that co-localized with the decondensed peripheral DNA loops, but not with the main chromosome body. Interchromosomal domains with IGs and Cajal-like bodies were also detected. CONCLUSIONS: Dinoflagellates are rather dissimilar to other eukaryotes in their genomes, their mechanisms of gene expression and their chromosome ultrastructure. However, they share common splicing nuclear domains and snRNP biogenesis with that of other eukaryotes.

Animals↗
PubMed2001

Veterinary pharmacovigilance: between regulation and science.

Veterinary pharmacovigilance has shown a remarkable development in recent years. After presenting briefly the regulatory context, this paper considers the scope of veterinary pharmacovigilance, causality assessment of suspected adverse drug reactions (ADRs), the systems existing in different countries and the evolution for the coming years. The involvement of veterinary research and teaching institutions should certainly become more important in this cross-road activity.

Adverse Drug Reaction Reporting Systems↗
PubMed1997

Conservation of the C.elegans tra-2 3'UTR translational control.

The Caenorhabditis elegans sex-determination gene, tra-2, is translationally regulated by two 28 nt elements (DREs) located in the 3'UTR that bind a factor called DRF. This regulation requires the laf-1 gene activity. We demonstrate that the nematode Caenorhabditis briggsae tra-2 gene and the human oncogene GLI are translationally regulated by elements that are functionally equivalent to DREs. Here, we rename the DREs to TGEs (tra-2 and GLI elements). Similarly to the C.elegans tra-2 TGEs, the C.briggsae tra-2 and GLI TGEs repress translation of a reporter transgene in a laf-1 dependent manner. Furthermore, they regulate poly(A) tail length and bind DRF. We also find that the C.elegans TGEs control translation and poly(A) tail length in C.briggsae and rodent cells. Moreover, these same organisms contain a factor that specifically associates with the C.elegans TGEs. These findings are consistent with the TGE control being present in C.briggsae and rodent cells. Three lines of evidence indicate that C.briggsae tra-2 and GLI are translationally controlled in vivo by TGEs. First, like C.elegans tra-2 TGEs, the C.briggsae tra-2 and GLI TGEs control translation and poly(A) tail lengths in C.briggsae and rodent cells, respectively. Second, the same factor in C.briggsae and mammalian cells that binds to the C.elegans tra-2 TGEs binds the C.briggsae tra-2 and GLI TGEs. Third, deletion of the GLI TGE increases GLI's ability to transform cells. These findings suggest that TGE control is conserved and regulates the expression of other mRNAs.

Amino Acid Sequence↗
PubMed1987

A single gene encoding vitellogenin in the sea urchin Strongylocentrotus purpuratus: sequence at the 5' end.

The synthesis of vitellogenin (yolk protein precursor) in the sea urchin, Strongylocentrotus purpuratus, is unique in that both males and females produce a high level of the protein. In this paper we show that this organism also is unique in possessing only a single vitellogenin gene. Like the genes that encode analogous proteins in vertebrates, the sea urchin gene is large, about 19 kb in length. The sequence surrounding the 5' end of the gene revealed several other similarities to vertebrate vitellogenin genes: the signal sequence is exceptionally short and has a sequence similar to those from frog and chick; there is a canonical TATA box at -32; and there is a sequence closely resembling the estrogen-responsive element at -207.

Amino Acid Sequence↗
PubMed1988

The left end of rat L1 (L1Rn, long interspersed repeated) DNA which is a CpG island can function as a promoter.

Here we report that the 600 bp promoter-like region at the left end of a newly isolated and characterized rat L1 DNA element can activate the prokaryotic chloramphenicol acyltransferase gene in a rat cell line. Activation only occurs when the promoter region is oriented to the transferase gene as it is to the L1 protein encoding sequences and is 75% inhibited by methylation of just 5 of the 22 CpGs present in the promoter. The G + C rich promoter contains enough CpGs to qualify it as a CpG island, but in contrast to other CpG islands, genomic L1 promoters are fully methylated in both somatic cell and sperm DNA as judged by restriction enzyme analysis. Partial demethylation of the genomic promoters by treatment with 5-azacytidine failed to produce discrete L1 transcripts. The relationship of methylation to the evolutionary history and fate of the rat L1 promoter is discussed.

Animals↗
PubMed2000

RecA-independent ectopic transposition in vivo of a bacterial group II intron.

RmInt1 is a group II intron of Sinorhizobium meliloti which was initially found within the insertion sequence ISRm2011-2. Although the RmInt1 intron-encoded protein lacks a recognizable endonuclease domain, it is able to mediate insertion of RmInt1 at an intron-specific location in intronless ISRm2011-2 recipient DNA, a phenomenon termed homing. Here we have characterized three additional insertion sites of RmInt1 in the genome of S.meliloti. Two of these sites are within IS elements closely related to ISRm2011-2, which appear to form a characteristic group within the IS630-Tc1 family. The third site is in the oxi1 gene, which encodes a putative oxide reductase. The newly identified integration sites contain conserved intron-binding site (IBS1 and IBS2) and delta' sequences (14 bp). The RNA of the intron-containing oxi1 gene is able to splice and the oxi1 site is a DNA target for RmInt1 transposition in vivo. Ectopic transposition of RmInt1 into the oxi1 gene occurs at 20-fold lower efficiency than into the homing site (ISRm2011-2) and is independent of the major RecA recombination pathway. The possibility that transposition of RmInt1 to the oxi1 site occurs by reverse splicing into DNA is discussed.

Base Sequence↗
PubMed1998

The regulatory particle of the Saccharomyces cerevisiae proteasome.

The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.

Amino Acid Sequence↗
PubMed2006

The chromatin regulatory code: beyond a histone code.

In this commentary on the contribution by Arndt Benecke in this issue, I discuss why the notion of "chromatin code" introduced and elaborated in this paper is to be preferred to that of "histone code". Speaking of a code as regards nucleosome conformation and histone tail post-translational modifications only makes sense within the chromatin fiber, where their physico-chemical features can be translated into regulatory programs at the genome level, by means of a complex, multi-level interplay with the fiber architecture and dynamics settled in the course of Evolution. In particular, this chromatin code presumably exploits allosteric transitions of the chromatin fiber. The chromatin structure dependence of its translation suggests two alternative modes of transcription initiation regulation, also proposed in the paper by A. Benecke in this issue for interpreting strikingly bimodal micro-array data.

Chromatin↗
PubMed2005

Vestige: maximum likelihood phylogenetic footprinting.

BACKGROUND: Phylogenetic footprinting is the identification of functional regions of DNA by their evolutionary conservation. This is achieved by comparing orthologous regions from multiple species and identifying the DNA regions that have diverged less than neutral DNA. Vestige is a phylogenetic footprinting package built on the PyEvolve toolkit that uses probabilistic molecular evolutionary modelling to represent aspects of sequence evolution, including the conventional divergence measure employed by other footprinting approaches. In addition to measuring the divergence, Vestige allows the expansion of the definition of a phylogenetic footprint to include variation in the distribution of any molecular evolutionary processes. This is achieved by displaying the distribution of model parameters that represent partitions of molecular evolutionary substitutions. Examination of the spatial incidence of these effects across regions of the genome can identify DNA segments that differ in the nature of the evolutionary process. RESULTS: Vestige was applied to a reference dataset of the SCL locus from four species and provided clear identification of the known conserved regions in this dataset. To demonstrate the flexibility to use diverse models of molecular evolution and dissect the nature of the evolutionary process Vestige was used to footprint the Ka/Ks ratio in primate BRCA1 with a codon model of evolution. Two regions of putative adaptive evolution were identified illustrating the ability of Vestige to represent the spatial distribution of distinct molecular evolutionary processes. CONCLUSION: Vestige provides a flexible, open platform for phylogenetic footprinting. Underpinned by the PyEvolve toolkit, Vestige provides a framework for visualising the signatures of evolutionary processes across the genome of numerous organisms simultaneously. By exploiting the maximum-likelihood statistical framework, the complex interplay between mutational processes, DNA repair and selection can be evaluated both spatially (along a sequence alignment) and temporally (for each branch of the tree) providing visual indicators to the attributes and functions of DNA sequences.

Algorithms↗
PubMed2002

Characterisation of the GH gene cluster in a new-world monkey, the marmoset (Callithrix jacchus).

In most mammals pituitary GH is encoded by a single gene with no close relatives. However, in man the GH gene has been shown to be one of a cluster of five closely related genes, four of which are expressed in the placenta. Rhesus monkey also expresses at least five closely related GH-like genes, although the genomic organisation of these has not been fully reported. Here we describe the cloning and characterisation of GH-like genes in a new-world monkey, the marmoset (Callithrix jacchus). This species possesses a cluster of eight GH-like 'genes'. The gene at the 5' end of this cluster encodes pituitary GH and is similar to that encoding human GH. Five of the eight marmoset 'genes' are probably pseudogenes, since they include mutations which would prevent normal expression, including stop codons and small insertions/deletions that would change the reading frame. In one case a large part of a gene is deleted, and in another a large insertion is introduced into an exon. The remaining two marmoset genes are potentially expressible, as proteins with sequences substantially different (at 25-30% of all residues) from that of marmoset GH itself; whether and in which tissue(s) such expression actually occurs is not yet known. None of the marmoset genes is clearly equivalent to any of the human GH-like genes expressed in the placenta, and this and phylogenetic analysis suggest that the duplications that gave rise to the marmoset GH gene cluster occurred independently of those that gave rise to the corresponding cluster in man. Although it includes more 'genes', the marmoset cluster extends over a shorter region of chromosomal DNA (about 35 kb) than does the human GH gene cluster (about 50 kb).

Amino Acid Sequence↗
PubMed1997

Homeoboxes in sea anemones (Cnidaria:Anthozoa): a PCR-based survey of Nematostella vectensis and Metridium senile.

Homeobox genes belong to a phylogenetically widespread family of regulatory genes that play important roles in pattern formation and cell-fate specification in several model systems (e.g., Drosophila, mouse, and C. elegans). Although the evolution of many classes of homeobox genes predates the diversification of the Bilateria, comparatively little is known about homeobox genes in outgroups to the Bilateria, such as the Cnidaria. We used the polymerase chain reaction to recover 12 partial homeoboxes from 2 species of sea anemones, Metridium senile and Nematostella vectensis (phylum Cnidaria; class Anthozoa). These homeoboxes appear to represent 9 distinct, mutually paralogous homeobox genes, 5 of which belong to previously identified cnidarian homeobox classes, and 4 of which appear to represent previously unidentified classes. The evolutionary relationships between the homeodomains of sea anemones and of bilaterian animals were assessed through database searches and phylogenetic analyses. As many as 5 of the anemone homeoboxes may belong to the Hox class, which suggests that the Hox gene complement of cnidarians is larger than previously expected. Homologs of the even-skipped gene of Drosophila were also identified in both Metridium and Nematostella.

Amino Acid Sequence↗
PubMed1975

[Heat resistance of 2 types of protoplasmic movement in Physarum polycephalum plasmodia].

The thermostabilities of the "unordered" and shuttle protoplasmic streamings in myxomycete Physarum polycephalum plasmodia was studied. A comparison of these thermostabilities has revealed that the cessation of the former streaming occurs at temperatures higher than those required for arresting the shuttle streaming. The difference between the two types of protoplasmic streamings is better seen in the rate of repair of protoplasmic streaming halted by a 10 minutes heating at 38-41 degrees C. For example, the unordered streaming is restored 2 minutes after heating plasmodia at 39 degrees for 10 min., while the shuttle streaming is resumed in 24 minutes. It is supposed that the two protoplasmic streamings are independent to an appreciable extent, and that the shuttle streaming, being more complex and coordinated, has appeared in the evolution at later stages than the unordered one. The higher heat sensitivity of the shuttle streaming substantiates a view of the lower stability to injury in regulatory mechanisms if compared to the stability of motile mechanisms.

Acclimatization↗
PubMed1992

[The role of mobile genetic elements (MGE) in microevolution].

The MGEs of Drosophila and other objects contain open reading frames (ORFs) encoding transposition enzymes, and "motifs" similar to functional sites: promoters, enhancers, heat shock regulatory sites, those of reception of different stress external signals and hormones, recombination sites, etc. In other words, MGE play a role of "movable cassettes of regulatory elements" in the genomes. The patterns of genome MGE localization are the important components of polygenic systems of character expression, the subjects of variation and evolution. The summed up density of MGE localization along the genome has probably the upper value corresponding to approx. 1 MGE copy per gene. The transpositional variability of MGE patterns could be random, non-random, self-related and inducible. The MGE patterns are important subjects of microevolution and reconstruction of trees of the pattern similarity is an effective method for its description. The patterns could be changed by selection of limited quantitative characters. The stress induction (temperature, treatment, dysgenic cross, etc.) stimulated the MGE transpositions and excisions, mass in population and multiple in individuals. The temperature induction acts probably through the system of response to heat shock treatment. The totality of MGE patterns make up the genomic system capable of quick reorganizations after stress external and genomic influences. The stress external influences are often correlated with passing of the population through the "bottle-neck" stage. The rate of transpositions has an upper limiting border, so named "boundary of regulation error catastrophe", that corresponds to approx. 1 transposition per genome, per generation. After the stress induction of transpositions this boundary could be exceeded, the state of the population norm becoming disrupted. The changes in MGE patterns are also supposed to accompany the changes of characters of isolation, i.e. to accompany the speciation.

Animals↗
PubMed1990

Molecular genetics of corticosteroid action.

The recent isolation and characterization of steroid receptor coding sequences has revolutionized the field of steroid hormone action. These studies have revealed that steroid receptors are members of a much larger nuclear receptor "super family." The ligand and DNA binding domains have been shown to be molecular components that functionally interact to transform the steroid-receptor complex into a highly specific gene regulator that induces or represses the expression of cell-specific target genes. Molecular genetic approaches have been used to study structure-function relationships of several steroid receptor proteins, the most extensive analysis has been that of the glucocorticoid receptor. Several breakthroughs in the study of steroid hormone action include the construction of novel chimeric steroid receptor proteins, functional expression of steroid receptors in yeast, and the development of sensitive cloning techniques designed to isolate low abundance, hormonally regulated transcripts.

Animals↗
PubMed1998

Transgenic mice expressing a functional human photopigment.

PURPOSE: Changes in retinal photopigments represent a fundamental step in the evolution of visual systems, in that addition of new pigment types or alterations in the spectral absorption properties of existing pigments modify visual capacities and thus open new visual worlds. To provide a tool that would allow direct examination of the changes caused by the presence of novel photopigments, this study was designed to determine whether a gene encoding a human cone photopigment introduced into the mouse genome would be expressed in a cone-specific manner and would support phototransduction. METHODS: Mice transgenic for the human long wavelength-sensitive (L) photopigment were generated by microinjection of fertilized mouse eggs. RNA expression in different tissues was monitored by reverse transcription-polymerase chain reaction analysis. Photopigment protein was localized in retinal cross sections and wholemounts by antibody staining. Light transduction of the cone photopigments was assessed by flicker photometric electroretinography (ERG). RESULTS: The human transgene was expressed specifically in the mouse cones in quantities comparable to those of the mouse middle wavelength-sensitive (M) pigment gene. Immunocytochemical analysis showed that the human L pigment was abundantly synthesized in most mouse cones, was translocated to the outer segments, and caused no detectable cone degeneration. Electroretinographic spectral sensitivity analysis showed that the human L pigment was efficient in eliciting an electrical response. The degree of expression of the transgene in the two founders correlated well with the spectral responsivity of the ERG. CONCLUSIONS: The human L photopigment transduces light efficiently in mouse cones, implying that all protein domains necessary for efficient interaction with intracellular transport and signal transduction machineries in mouse cones have been conserved through evolution. The expression of the human L photopigment gene in both classes of cone of the mouse retina indicates that the transgene did not have the regulatory elements necessary for restricting its expression to mouse M cones or that such elements are not recognized in mouse UV-sensitive cones.

Animals↗
PubMed1988

Evolution of the autosomal chorion locus in Drosophila. I. General organization of the locus and sequence comparisons of genes s15 and s19 in evolutionary distant species.

We have isolated clones corresponding to the autosomal chorion locus of Drosophila melanogaster, from two distantly (D. virilis and D. grimshawi) and one closely (D. subobscura) related species. In all the species the locus is unique within the genome and encompasses the same four chorion genes and an adjacent nonchorion gene, in the same order. In all species the locus specifically amplifies in the ovary, as in D. melanogaster. We present the nucleotide sequences of DNA segments that total 8.3 kb in length and include gene s15-1 from D. subobscura, D. virilis, and D. grimshawi as well as gene s19-1 from D. subobscura and D. grimshawi. They show clearly nonuniform rates of divergence, both within and outside the limits of the genes. Highlighted by a background of extensive sequence divergence elsewhere in the extragenic region, highly conserved elements are observed in the 5' flanking DNA and might represent regulatory elements.

Amino Acid Sequence↗