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PubMed1995

The mitochondrial DNA of the amoeboid protozoon, Acanthamoeba castellanii: complete sequence, gene content and genome organization.

In phylogenetic trees based on comparison of nuclear small subunit rRNA sequences, Acanthamoeba castellanii (an amoeboid protozoon) is positioned near the base of the radiation leading to the animals, fungi and plants. However, the specific affiliation of this protist with the major multicellular lineages of eukaryotes is currently uncertain. To further explore the evolutionary position of A. castellanii, we have determined the complete primary sequence of its mitochondrial genome. We find that the circular mtDNA (41,591 bp; 70.6% A+T) encodes two rRNAs (small subunit and large subunit), 16 tRNAs and 33 proteins (17 subunits of the respiratory chain and 16 ribosomal proteins). As well, this genome contains eight open reading frames (ORFs) larger than 60 codons and of undefined function. Two of these ORFs (orf124 and orf142) have homologs in other mtDNAs ("orf25" and "orfB", respectively), three are unique to A. castellanii mtDNA (orf83, orf115 and orf349), and three are intronic ORFs. Among notable features of A. castellanii mtDNA are the following: (1) Genes and ORFs are all encoded on the same strand and are tightly packed, with only 6.8% of the total sequence not having an evident coding function and intergenic spacer sequences ranging from only 1 to 616 bp (average 64 bp). Ten pairs of protein-coding genes overlap by up to 38 bp and two subunits of cytochrome oxidase (COX1 and COX2) are specified by a single continuous ORF. (2) Only three introns, all group I and each containing a free-standing ORF, are present; these are localized in the 3'-half of the large subunit rRNA gene. (3) The genome encodes fewer than the minimal number of tRNA species required to support mitochondrial protein synthesis, suggesting that additional tRNAs are imported from the cytosol into A. castellanii mitochondria. Of the 16 tRNAs specified by A. castellanii mtDNA (one with an 8-nucleotide anticodon loop), 13 have been shown or are predicted to undergo a novel form of RNA editing within the acceptor stem. (4) A modified genetic code is used in which UGA specifies tryptophan. (5) Repeated sequences and obvious small sequence motifs that might represent regulatory elements are absent. In overall size, gene content and organizational pattern, A. castellanii mtDNA most closely resembles the mtDNA of the chlorophycean alga Prototheca wickerhamii (55,326 bp; 74.2% A+T), but is quite different in these respects from the mtDNA of Chlamydomonas reinhardtii (15,758 bp; 54.8% A+T), another chlorophycean alga, as well from characterized animal and fungal mitochondrial genomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acanthamoeba↗
PubMed1987

[Optimization of the gene expression for human alpha F- and beta 1-interferons in Escherichia coli cells].

The basic results of the studies on expression of the genes of human alpha F- and beta 1-interferons in E. coli cells are presented. To synthesize the fibroblast interferon, the respective fragment of the human chromosome was cloned, the complete nucleotide sequence of the structural moiety of mature beta-interferon was determined and the genes of "hybrid (interferon-like) proteins" and "hybrid sites of ribosome binding" were constructed with control of the beta-interferon gene by the prokaryotic regulatory areas. Synthesis of beta-interferon was achieved (1.10(7)-5.10(7) IU per 1 l of the bacterial culture) with the use of the tryptophan operon promoter. A new procedure for optimization of allogenic genetic information in E. coli cells: constructing of "hybrid operons with partially overlapping genes" or artificial "overlappons" was developed following the example of the alpha F-interferon gene cloned in the Laboratory headed by E. D. Sverdlov at the Institute of Bioorganic Chemistry of the USSR Academy of Sciences. The use of this procedure enabled production of up to 5.10(7) IU/l of alpha F-interferon under the control of the lacUV5-promoter. On the basis of the newly constructed vector molecules expression of the genes of alpha F- and beta 1-interferons was amplified with the "overlappon" procedure.

Chromosome Mapping↗
PubMed1996

Characterization of component-I gene of botulinum C2 toxin and PCR detection of its gene in clostridial species.

Botulinum C2 toxin is composed of two nonlinked protein components, component-I (light chain) and component-II (heavy chain). It is produced by Clostridium botulinum types C and D, and is thought to play a lethal pathogenic role. These biological activities of C2 toxin may be due to the ADP-ribosylation of non-muscle actin by component-I of the toxin. We were able to isolate two overlapping gene fragments encoding component-I from the chromosomal DNA of Clostridium botulinum type C strain (C)-203U28, and determine the complete nucleotide sequence of component-I gene. The gene for component-I, bc21, consists of one open reading frame (ORF) encoding 431 amino acid residues (1293 nucleotides) without signaling peptide sequence. The molecular mass calculated from the deduced amino acid sequence was 49400.37 Da. Mono-ADP-ribosyltransferase activity was demonstrated in the lysate from E. coli transformed by the recombinant plasmid, pGEM-C2 encompassing whole component-I gene with its own promoter.

Amino Acid Sequence↗
PubMed1986

Infection of a Chlorella-like alga with the virus PBCV-1: transcriptional studies.

Infection of the unicellular, eukaryotic Chlorella-like green alga NC64A by the large dsDNA containing virus PBCV-1 immediately reduced host RNA synthesis. Chloroplast rRNAs, but not cytosolic rRNAs, were degraded following viral infection. Northern blot analysis utilizing four cloned fragments of PBCV-1 DNA as probes, which represent about 12% of the viral genome, revealed several properties of PBCV-1 transcription: A few viral transcripts were detected within 5 min after infection. Each PBCV-1 DNA clone hybridized to both early and late transcripts which implies that early and late genes are dispersed throughout the viral genome. The transition from early to late transcription occurred between 40 and 60 min after infection coincident with the onset of viral DNA synthesis. Three of the four DNA clones hybridized to transcripts which additively were larger than the corresponding DNA probe. This could reflect RNA processing, presence of overlapping genes, or transcription from both DNA strands. A few, but not all, early transcripts were synthesized in the presence of cycloheximide. This suggests that the virus either carries in its own RNA polymerase or uses a host RNA polymerase for very early viral transcription and that synthesis of additional, later transcripts depends on translation of an early gene product(s).

Chlorella↗
PubMed2000

IS1414, an Escherichia coli insertion sequence with a heat-stable enterotoxin gene embedded in a transposase-like gene.

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic E. coli (ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of astA nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that astA is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the -1 reading frame in reference to the toxin gene. In vitro expression of the predicted M(r)- approximately 46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS285 from Yersinia pestis, IS1356 from Burkholderia cepacia, and ISRm3 from Rhizobium meliloti. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS1414. Preliminary experiments to show IS1414 translocation were unsuccessful. Overlapping genes of the type suggested by the IS1414 core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.

Alleles↗
PubMed2006

Functional genome and proteome analyses of cutaneous autoimmune diseases.

The use of functional genomics and proteomics technologies has dramatically increased through recent years with a special emphasis on cancer biology. However, a series of more recent reports has also addressed inflammatory diseases. These included studies on different autoimmune diseases, such as rheumatoid arthritis, lupus erythematosus, and systemic sclerosis. Gene and protein expression profiles from these studies have emphasized the role of cytokines, chemokines, and apoptosis-related molecules for the pathogenesis of autoimmune diseases. Much less is known about gene and protein patterns of these diseases in dermatology. Here we provide an overview on current knowledge about genomics and proteomics analyses of cutaneous autoimmune diseases. These diseases include psoriasis, lupus erythematosus, systemic sclerosis, vitiligo, and alopecia areata. The presented findings not only provide deeper insights into the pathogenesis of each individual disease but also show overlapping gene patterns suggestive for common pathogenic mechanisms. However, many open questions remain to be resolved since data about local gene expression pattern in affected tissues are still scarce.

Animals↗
PubMed1993

Characterization of the gene coding for the Rickettsia prowazekii DNA primase analogue.

The gene (dnaG) coding for DNA primase in the obligate intracellular parasitic bacterium, Rickettsia prowazekii, has been isolated and characterized. An open reading frame (ORF) of 1848 bp capable of encoding 616 amino acids (aa) is located 18 bp upstream from the gene coding for the major sigma factor of R. prowazekii, sigma 73. Based on aa sequence comparisons of DNA primase from R. prowazekii, Escherichia coli, Salmonella typhimurium and Bacillus subtilis, we propose that R. prowazekii dnaG begins 69 bp into the ORF and encodes 593 aa with a calculated M(r) of 68,683. An upstream ORF overlaps 66 of the first 69 bp of the larger R. prowazekii dnaG ORF, suggesting either an overlapping gene structure or the generation of the smaller protein product of 593 aa. Predicted aa sequence of R. prowazekii primase compared to E. coli, S. typhimurium and B. subtilis primases reveals 30.5%, 30.5% and 29.7% aa identity, respectively. The R. prowazekii dnaG gene failed to complement an E. coli dnaG temperature sensitive mutation perhaps due to poor expression of the gene or inability to function properly in E. coli. The gene organization of an ORF followed by DNA primase (dnaG) and then the major sigma factor (rpoD) is consistent with the major macromolecular synthesis operons of E. coli, S. typhimurium and B. subtilis.

Amino Acid Sequence↗
PubMed1996

The aroA gene of Campylobacter jejuni.

The gene for 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (aroA) cloned from Campylobacter jejuni (Cj) strain 81116 was identified by complementation of an Escherichia coli (Ec) auxotrophic aroA mutant. The Cj aroA gene has been sequenced. It encodes an enzyme of 428 amino acids (aa), that is homologous to other bacterial EPSP synthases, especially that of Bacillus subtilis with which it has a 39% aa identity. The transcriptional start point was mapped. It is present in an upstream open reading frame (ORF) that has a strong homology to the gene encoding phenylalanine tRNA synthetase (pheS). Downstream from aroA another ORF is present which is homologous to the lytB gene of Ec. The stop codon of the aroA gene overlaps the start codon of lytB.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1988

Retinoic acid and thyroid hormone induce gene expression through a common responsive element.

Studies of steroid receptors have led to the identification of a superfamily of ligand-inducible regulatory proteins that includes receptors for thyroid hormones and retinoic acid. This family of receptors regulates gene expression through binding to short cis-acting sequences referred to as hormone-response elements. Identification of a functional retinoic acid responsive element is crucial to our understanding of the mechanisms by which retinoic acid receptors activate gene expression and regulate cell differentiation. One impediment to such a study is the absence of any identified gene whose transcription is directly dependent on the receptor-hormone complex. Because the DNA-binding domains of the retinoic acid and thyroid hormone receptors are highly related (62% identical in their amino acid sequences), we have investigated the possibility that the retinoic acid receptor could activate gene expression through a thyroid hormone response element. We now report that a human retinoic acid receptor expressed from cloned complementary DNA or the endogenous retinoic acid receptor present in F9 teratocarcinoma cells can activate gene expression from promoters fused to a natural or synthetic thyroid hormone response element. The product translated in vitro from the human retinoic acid receptor cDNA can bind to a thyroid hormone response element with high affinity. The unexpected implication of these findings is that retinoic acid and thyroid hormones, acting through their respective receptors, could control overlapping gene networks involved in the regulation of vertebrate morphogenesis and homeostasis.

Binding Sites↗
PubMed2004

Sp17 gene expression in myeloma cells is regulated by promoter methylation.

The mechanisms underlying sperm protein 17 (Sp17) gene expression in myeloma cells remained unclear. Using reverse transcription-polymerase chain reaction (RT-PCR), Sp17 transcripts were detected in ARK-B, ARP-1, RPMI-8226 and KMS-11 but not in H929, IM-9, MM1-R and U266 cells. Using a panel of primer pairs in methylation-sensitive PCR to amplify overlapping gene segments, our screening studies showed that the HpaII sites at -359 and -350 are involved in the regulation of Sp17 gene expression. To confirm the differences in methylation status between Sp17-positive and Sp17-negative cell lines, KMS-11 cells (Sp17-positive) and IM-9 cells (Sp17-negative) were subjected to the more accurate method of bisulphite conversion. KMS-11 cells were more hypomethylated at these HpaII sites of exon 1 compared to IM-9 cells, indicating the association of hypomethylated promoter with Sp17 gene expression. In addition, the level of methylation at other CpG sites within the promoter sequence was also higher in IM-9 than KMS-11. Exon 1 was cloned into a reporter vector, pCAT*3 Enhancer. Chloramphenicol acetyl transferase (CAT) activity was restored in cells transfected with the recombinant plasmid, indicating the promoter function of exon 1. Exposure of Sp17-negative cell lines to the hypomethylating agent, 5-azacytidine, resulted in the upregulation of Sp17 gene expression. Our results therefore provide evidence for the regulation of Sp17 gene expression by promoter methylation.

Antigens, Surface↗
PubMed1989

Characterization of the Escherichia coli protein-export gene secB.

The Escherichia coli secB gene product is required for normal export of envelope proteins out of the cell cytoplasm. In this report, we present the identification and nucleotide sequence of the secB coding sequence. The secB structural gene overlaps almost completely with a predicted open reading frame (ORF) that is encoded on the opposite strand. To establish the identity of the secB ORF, we characterized a secB mutation that caused total loss of secB function, based upon its phenotype. This mutation resulted from a nucleotide change that caused an ochre mutation in one ORF (the secB gene) and a silent (no amino acid change) codon change in the opposite ORF.

Amino Acid Sequence↗
PubMed2002

Activation of prophage eib genes for immunoglobulin-binding proteins by genes from the IbrAB genetic island of Escherichia coli ECOR-9.

Four distinct Escherichia coli immunoglobulin-binding (eib) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in E. coli reference (ECOR) strain ECOR-9. Each eib gene was transferred to test E. coli strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. Supporting this hypothesis, we cloned from ECOR-9 overlapping genes, ibrA and ibrB (designation is derived from "immunoglobulin-binding regulator"), which together activated eib expression in the derived lysogens. The proteins encoded by ibrA and ibrB are very similar to uncharacterized proteins encoded by genes of Salmonella enterica serovar Typhi and E. coli O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933). The genomic segment containing ibrA and ibrB has been designated the IbrAB island. It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes. The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the E. coli K-12 genome. Homology to IbrAB was found in certain other ECOR strains, including the other five eib-positive strains and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion of ibrAB revealed that the other eib-positive strains diverge by </=0.1% from ECOR-9, whereas eib-negative ECOR-47 diverges by 16%.

Chromosome Mapping↗
PubMed2002

Genetic and physiological data implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia.

A map of 191 single-nucleotide polymorphism (SNPs) was built across a 5-Mb segment from chromosome 13q34 that has been genetically linked to schizophrenia. DNA from 213 schizophrenic patients and 241 normal individuals from Canada were genotyped with this marker set. Two 1,400- and 65-kb regions contained markers associated with the disease. Two markers from the 65-kb region were also found to be associated to schizophrenia in a Russian sample. Two overlapping genes G72 and G30 transcribed in brain were experimentally annotated in this 65-kb region. Transfection experiments point to the existence of a 153-aa protein coded by the G72 gene. This protein is rapidly evolving in primates, is localized to endoplasmic reticulum/Golgi in transfected cells, is able to form multimers and specifically binds to carbohydrates. Yeast two-hybrid experiments with the G72 protein identified the enzyme d-amino acid oxidase (DAAO) as an interacting partner. DAAO is expressed in human brain where it oxidizes d-serine, a potent activator of N-methyl-D-aspartate type glutamate receptor. The interaction between G72 and DAAO was confirmed in vitro and resulted in activation of DAAO. Four SNP markers from DAAO were found to be associated with schizophrenia in the Canadian samples. Logistic regression revealed genetic interaction between associated SNPs in vicinity of two genes. The association of both DAAO and a new gene G72 from 13q34 with schizophrenia together with activation of DAAO activity by a G72 protein product points to the involvement of this N-methyl-d-aspartate receptor regulation pathway in schizophrenia.

Amino Acid Sequence↗
PubMed1987

The bacteriophage Mu com gene appears to specify a translation factor required for mom gene expression.

Expression of the bacteriophage Mu mom gene is subject to a variety of regulatory controls. Both the host Dam DNA-adenine methylase and the phage Mu C protein are required for mom gene transcription. In addition, the Mu com gene product is required for production of the mom protein. Because the com and mom genes overlap on the same mRNA transcript (with com being located proximal to the 5' end), it is likely that Com function is exerted after transcription initiation. To study the role of Com, two segments of Mu were cloned in both orientations (+ and -) into the HindIII site of the galactokinase expression vector, pKG1800; the HindIII site is located between the galK structural gene and its promoter. In (+) plasmids, the Mu DNA inserts were transcribed from the gal promoter in the same orientation as in the phage genome; (-) plasmids had the Mu DNA inserted in the reverse orientation. Each Mu insert contained the same segment of the mom gene from the 3' terminus, but differed in the extent of com gene included at the 5' terminus; one contained a truncated com gene and the other a complete com gene, as well as upstream Mu regulatory sequences. The results are summarized as follows: (1) both (-) plasmids produced only about 10% as much galactokinase activity following fucose induction as the parental vector, pKG1800; (2) plasmid pGTVH(+), with an intact com gene produced about 30% as much galactokinase as pKG1800; (3) plasmid pMTVH(+), with a truncated com gene, produced only about 10% as much enzyme as pKG1800.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage mu↗
PubMed1990

Bacillus subtilis ada operon encodes two DNA alkyltransferases.

By prophage transformation and subcloning, we have obtained Bacillus subtilis DNA fragments that could complement the hypersensitivity of ada (adaptive response deficient) mutants to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The nucleotide sequence contained two open reading frames that were assigned to the genes adaA and adaB, encoding methylphosphotriester-DNA methyltransferase and O6-methylguanine-DNA methyltransferase, respectively. These two genes overlap by 11 bp and comprise a small operon. The 1.6 Kb transcripts derived from the operon were detected in ada+ cells cultured in the presence of MNNG but not in control ada+ cells. From analysis of the syntheses of DNA alkyltransferases in the ada mutant cells harboring the plasmid carrying the complete or partial fragment, we conclude that the adaA gene product functions as a transcriptional activator of the ada operon, while the adaB gene product specializes in repair of mutagenic O6-methylguanine residues. Comparison with Escherichia coli ada operon showed that the two genes correspond to portions of the E. coli ada gene, implicating gene fusion or splitting as the origin of the difference in the organizations of the genes.

Alkyl and Aryl Transferases↗
PubMed1996

Expression of a novel member of estrogen response element-binding nuclear receptors is restricted to the early stages of chorion formation during mouse embryogenesis.

Members of the nuclear hormone receptor gene family of transcription factors have been shown to be expressed in characteristic patterns during mouse organogenesis and postnatal development. Using an RT-PCR based screening assay, we have identified nuclear receptors expressed in embryonal carcinoma stem cells. One of the cDNAs characterized, mERR-2, was found to be expressed exclusively during a narrow developmental window in trophoblast progenitor cells between days 6.5 and 7.5 post coitum (p.c.). From 8.5 days p.c. and onwards, the mERR-2 gene activity evaded detection as analysed by in situ hybridization. We also show that the mERR-2 gene product and the estrogen receptor share a common target DNA-sequence recognition specificity unique among members of the gene family. Furthermore, efficient homodimerization and DNA-binding of the orphan receptor mERR-2 was found to be dependent on interaction with the heat shock protein 90, a molecular chaperone hitherto recognized to interact only with the steroid hormone receptor subgroup of nuclear receptors. Based on our results we suggest that the mouse orphan receptor mERR-2 has the potential to regulate overlapping gene networks with the estrogen receptor and may participate in signal transduction pathways during a short developmental period coinciding with the formation of the chorion.

Amino Acid Sequence↗
PubMed2002

Expression of tylM genes during tylosin production: phantom promoters and enigmatic translational coupling motifs.

In the genome of Streptomyces fradiae, the three tylM genes are codirectional with the upstream gene, tylGV. Although the introduction of transcriptional blocks into the tylM genes revealed that they are normally cotranscribed, expression of tylMI still persisted (albeit at a very low level) when either of the upstream genes, tylMII or tylMIII, was disrupted. Such expression apparently resulted from transcriptional initiation at spurious sites that probably contribute insignificantly, if at all, to promote activity in the wild type. Prior to the onset of tylosin production, tylMIII is transcribed independently of tylGV from an authentic promoter buried within tylGV. This latter observation is interesting given that the TGA stop codon of tylGV overlaps the GTG start codon of tylMIII. Evidently, terminally overlapping genes are not always translationally coupled.

Anti-Bacterial Agents↗
PubMed2025

Strong but diffuse genetic divergence underlies differentiation in an incipient species of marine stickleback.

Understanding how lineages proceed along the "speciation continuum" and how species boundaries are maintained over time remain central questions in evolutionary biology. Populations early in the speciation process can give us detailed insight into the reproductive barriers that first initiate speciation. In this study, we explore the nature of genomic divergence between two sympatric marine stickleback ecotypes from Atlantic Canada, "whites" and "commons". Males of each ecotype exhibit distinct nuptial colorations, nesting habits, and parental care strategies. Using population genomic analyses of SNPs and copy number variants (CNVs; deletions and duplications) we show that whites and commons consistently form distinct populations. We uncover genomic differentiation in the white ecotype characteristic of an incipient species, showing extremely low genome-wide differentiation (FST) and very recent divergence (~1 kya). Demographic analysis detected very low levels of ongoing gene flow between populations. Our results and prior genomic studies suggest that reproductive isolation is being maintained between ecotypes despite recent evidence that hybridization in nature does occur. Contrary to other systems, we found many small, but dispersed regions of high differentiation throughout the genome rather than explicitly within chromosomal inversions or the sex chromosomes. On chromosomes VII and XVI, we identified CNVs overlapping genes enriched for olfaction, which may play a role in differences in reproductive strategies between ecotypes. Ultimately, our results demonstrate that genome-wide rather than localized differences can underlie the early stages of divergence, and that this pattern is corroborated by both SNPs and CNVs.

Copy Number Variation↗