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PubMed1995

A novel repetitive sequence from Mycoplasma hyopneumoniae.

We have isolated a novel repetitive DNA element from Mycoplasma hyopneumoniae strain VPP11, the entire structure of which is distinct from those of prokaryotic transposons, insertion sequences or eukaryotic retroposons reported. Southern blot hybridization experiments indicate that at least eight copies of this element locate on the M. hyopneumoniae genome. The size of this repetitive sequence is 4,193 bp which includes 270- and 272-bp direct long terminal repeats at each terminus. The internal domain of this element defines three open reading frames.

Animals↗

PubMed2001

Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea).

Mobile element dynamics in seven alleles of the chalcone synthase D locus (CHS-D) of the common morning glory (Ipomoea purpurea) are analyzed in the context of synonymous nucleotide sequence distances for CHS-D exons. By using a nucleotide sequence of CHS-D from the sister species Ipomoea nil (Japanese morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. & Iida, S. (1999) Genes Genet. Syst. 74, 141-147], it is also possible to determine the relative frequency of insertion and loss of elements within the CHS-D locus between these two species. At least four different types of transposable elements exist upstream of the coding region, or within the single intron of the CHS-D locus in I. purpurea. There are three distinct families of miniature inverted-repeat transposable elements (MITES), and some recent transpositions of Activator/Dissociation (Ac/Ds)-like elements (Tip100), of some short interspersed repetitive elements (SINEs), and of an insertion sequence (InsIpCHSD) found in the neighborhood of this locus. The data provide no compelling evidence of the transposition of the mites since the separation of I. nil and I. purpurea roughly 8 million years ago. Finally, it is shown that the number and frequency of mobile elements are highly heterogeneous among different duplicate CHS loci, suggesting that the dynamics observed at CHS-D are locus-specific.

Acyltransferases↗

PubMed1991

Positive and negative elements upstream of the meiosis-specific glucoamylase gene in Saccharomyces cerevisiae.

The SGA1 gene encoding glucoamylase is specifically expressed late in meiotic development of the yeast Saccharomyces cerevisiae. We found that accumulation of both enzyme activity and transcripts was regulated negatively by both nutritional signals and a haploid-specific negative regulator gene of meiosis. RME1, and positively by the inducer genes for meiosis, IME1 and IME2. To study the role of sequences upstream of the SGA1 gene in its expression and regulation, we generated internal deletions in the 5' non-coding region of the gene and chimeric genes with portions of the upstream sequence inserted into a reporter gene. By analyzing the expression of these genes, we have identified both a 19 bp upstream activation sequence (UAS) and a 49 bp negatively regulating element (NRE). The UAS activated transcription with no requirement for heterozygosity at the mating-type locus, but this activation was still under negative control by nutrients. The NRE showed no UAS-like activity but conferred IME2-dependent (or meiosis-specific) expression on a heterologous promoter. These results suggest that meiosis-specific expression of the SGA1 gene is established by a regulatory hierarchy including positive and negative factors, the actions of which are mediated through the two separate upstream regulatory elements, UAS and NRE, respectively. Also, that two independently acting cascades exist for the regulation of SGA1 expression: one transduces both the mating-type and nutritional signals and includes the IME2 product, which acts to relieve the repression through NRE; and another transduces only the nutritional signal independently of the above pathway and inhibits positive factors acting on UAS.

Autoradiography↗

PubMed1988

Rhizomucor miehei triglyceride lipase is synthesized as a precursor.

A Rhizomucor miehei cDNA library constructed in Escherichia coli was screened with synthetic oligonucleotides designed from knowledge of a partial amino acid sequence of the secreted triglyceride lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from this fungus. Lipase-specific recombinants were isolated and their insert sequenced. Unlike characterized bacterial and mammalian triglyceride lipases, the fungal enzyme is synthesized as a precursor, including a 70 amino acid residue propeptide between the 24 amino acid residues of the signal peptide and the 269 residues of the mature enzyme. The precursor processing mechanism, which involves cleavage between a methionine and a serine residue, is unknown. By sequence comparison with other lipases, a serine residue involved in substrate binding was identified in the fungal lipase. The sequence around this residue is well-conserved among characterized lipases. Conservation of an intron in an isolated cDNA recombinant and immunoprecipitation of in vitro synthesized R. miehei translation products indicates that the expression of the lipase gene might involve inefficient mRNA splicing.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of a cDNA encoding enolase from the filamentous fungus, Aspergillus oryzae.

A 1.6-kbp full-length cDNA for the Aspergillus oryzae enolase gene (enoA) was cloned. The sequenced insert contained a continuous open reading frame of 1314 bp encoding a protein of molecular weight 47 405. Among all enolases sequenced to-date, the deduced amino-acid sequence showed the highest homology (74.9%) with Candida albicans enolase (ENO1). Strong codon biases and multiple transcription start sites downstream from CT-blocks in the 5'-flanking region suggested strong expression. enoA mRNA was found to occupy approximately 3% (w/w) of total mRNA of A. oryzae by quantitative RT-PCR. This strong transcription was dependent on the carbon source in the medium and correlated with the growth rate of the mycelium.

Amino Acid Sequence↗

PubMed1994

The packaging signal in hepatitis B virus pregenome functions only at the 5' end.

When the hepatitis B virus (HBV) replicates, a full-size transcript of the viral genome, called pregenome RNA, is made and is selectively packaged into virus core particles. This selective encapsidation is dependent upon the presence of a specific cis-acting sequence called the packaging signal that is found at the 5' end of the pregenome RNA. Deletion analysis revealed that this packaging signal was located in a 70 bp stretch of nucleotides. Packaging defective virus genome containing the 70 bp packaging signal sequence at several locations, showed that the packaging occurs effectively only when the signal is located near the 5' end of the pregenome RNA. Packaging also occurred with a 2.4 kb viral RNA whose 5' region sequence was different from that of the pregenome RNA, but which has the packaging signal sequence inserted at the 5' region.

Base Sequence↗

PubMed1995

The genome of echovirus 11.

Echoviruses are the largest enterovirus subgroup consisting of 32 serotypes. They are common human pathogens causing, for example, meningitis, encephalitis and exanthema, but in spite of their clinical importance, relatively little is known about their biology. To illuminate the molecular characteristics of echoviruses, we have completed the genomic sequence of serotype 11. The RNA genome is 7438 nucleotides in length and it codes for a 2195 amino acid long polyprotein. When compared to other sequenced enteroviruses, echovirus 11 (EV11) shows remarkable similarity with coxsackie B viruses (CBVs) and coxsackievirus A9 (CAV9). On the basis of amino acid sequence homology in the capsid region, CAV9 is the virus most closely related to EV11. These two viruses have an apparent insertion sequence located at the C-terminus of the VP1 polypeptide. EV11, however, lacks the RGD motif found in the corresponding region of CAV9. The organization of the 5' end noncoding region resembles that of other enteroviruses, but contains a 12 nucleotides long poly-U stretch not seen in any other enterovirus sequenced to date.

Amino Acid Sequence↗

PubMed1995

Genetic organisation and evolution of Yersinia pseudotuberculosis 3,6-dideoxyhexose biosynthetic genes.

3,6-dideoxyhexose (DDH) sugars occur in some of the O antigens of Salmonella enterica and Yersinia pseudotuberculosis, but are otherwise rarely found in nature. Y. pseudotuberculosis DDH biosynthetic genes rfbS (encoding CDP-paratose synthetase) and rfbE (encoding CDP-tyvelose epimerase) were amplified and cloned, and their sequences determined. Comparisons with the equivalent genes of S. enterica show that the genetic arrangement of DDH genes is very similar; however, in Y. pseudotuberculosis there is no suggestion that paratose producing strains are derived from tyvelose-producing strains by inactivation of rfbE, which is the case in S. enterica. The previously determined DNA sequence of the rfb region of an abequose-producing strain was re-examined. It contains the remnants of an insertion sequence (IS) adjacent to a truncated and non-functional rfbE gene. This suggests that the IS was involved in recombination events contributing to O-antigen antigenic diversity in Y. pseudotuberculosis.

Amino Acid Sequence↗

PubMed1986

Synthesis of mutant parathyroid hormone genes via site-specific recombination directed by crossover linkers.

A synthetic 'crossover linker' technique has been designed for gene modification. The linker has a restriction end for an initial 'cohesive end' ligation with one terminus of a linearized plasmid, a middle section carrying modified sequence information, and an 'homology-searching' sequence of 20 bp at its other end, that is homologous to a specific region in the opposite terminus of the plasmid. Inside the Escherichia coli transformation host, intramolecular recombination between the homologous ends of the resultant plasmid intermediate completes the integration of the linker. Using different crossover linkers, a human parathyroid hormone gene which had previously been cloned into plasmid pUC8 was converted to mutant coding sequences via specific base substitution, sequence deletion and sequence insertion.

Base Sequence↗

PubMed2001

Horizontal gene transfer and host specificity of beta-haemolytic streptococci: the role of a putative composite transposon containing scpB and lmb.

Beta-haemolytic streptococci are important human and animal pathogens: their genetic traits that are associated with the ability to infect human hosts remain, however, unclear. The surface protein, Lmb, mediates the adherence of Streptococcus agalactiae to human laminin. For further analysis of the corresponding gene, the adjacent genomic regions were sequenced. Lmb is localized on a putative composite transposon of 16 kb and is flanked by two copies of a novel insertion sequence element (ISSag2). It harbours the genes scpB and lmb, which are 98% identical with the respective genes of Streptococcus pyogenes. Analysis of the distribution of these genes and ISSag2 among 131 streptococcal strains revealed that all of the human isolates, but only 20% (12 of 61) of the animal isolates, contained scpB and lmb or their homologues. To investigate if the putative transposon can be mobilized, an erythromycin resistance marker was incorporated into the lmb gene of S. agalactiae. Screening for mutant strains with a regained susceptibility for erythromycin identified strains with a deletion of scpB, lmb, and one copy of ISSag2. We hypothesize that a horizontal gene transfer caused the exchange of scpB and lmb and that the ability of S. pyogenes, S. agalactiae and group C and G streptococcal strains to colonize or infect human hosts is dependent on their presence.

Adhesins, Bacterial↗

PubMed1983

Mechanisms of integration of de novo-synthesized polypeptides into membranes: signal-recognition particle is required for integration into microsomal membranes of calcium ATPase and of lens MP26 but not of cytochrome b5.

We have investigated the in vitro integration into dog pancreas microsomal membranes of three integral membrane proteins that were synthesized de novo in a wheat germ cell-free translation system: calcium ATPase of rabbit sarcoplasmic reticulum, MP26 of bovine lens fiber plasma membrane, and rat liver cytochrome b5. Biosynthetically these proteins show a common feature in that they are synthesized without a transient NH2-terminal signal sequence. Two of these proteins, ATPase and MP26, were shown to require the recently discovered signal-recognition particle (SRP) [Walter, P. & Blobel, G. (1982) Nature (London) 299, 691-698] for integration. By this criterion, therefore, they each contain at least one uncleaved signal sequence. Surprisingly, however, the uncleaved signal sequence(s) of these two proteins did not induce the characteristic SRP-mediated translation arrest that was previously shown for a cleaved signal sequence. Unlike ATPase and MP26, cytochrome b5 did not require SRP for integration into microsomal membrane. Thus, the distinction between an "insertion" sequence (specifying unassisted and opportunistic integration into any exposed membrane) and a "signal" sequence (directing integration into a specific membrane by a receptor-mediated mechanism) is a valid one. By assaying for SRP dependence, the two mechanisms of integration can now be experimentally distinguished.

Animals↗

PubMed2001

Intracellular generation of single-stranded DNA for chromosomal triplex formation and induced recombination.

Synthetic triple helix-forming oligodeoxyribonucleotides (TFOs) have been used to alter gene expression and to induce targeted genome modification in cells and animals. However, the efficacy of such oligodeoxyribonucleotides (ODNs) depends on efficient intracellular delivery. A novel vector system was tested for the production of single-stranded DNA (ssDNA) to serve as a TFO in mouse cells. Mouse cells carrying a substrate that can report triplex-stimulated intrachromosomal recombination were transfected with a series of ssDNA vectors, and induced recombination was assayed. Transfection with a vector set designed to generate a 34 nt G-rich ssDNA capable of triplex formation at a 30 bp polypurine target site within the reporter substrate yielded recombinants at a frequency of 196 x 10(-6), versus a background frequency of 45 x 10(-6) in mock transfected cells. No induction was seen when a vector set lacking the TFO sequence insert was tested or when the component vectors were transfected individually. Vectors engineered to express a C-rich 34 nt sequence (not expected to form triplex under physiological conditions) had no effect over background. Primer extension analyses on lysates from transfected cells confirmed the production of the intended ssDNAs. These results suggest that ssDNA molecules of a defined sequence can be generated intracellularly using a novel vector system and that such molecules are active in mediating triplex-dependent chromosomal events. The ability to produce active TFOs within cells may provide a new foundation for triplex-based gene targeting strategies.

Animals↗

PubMed2007

Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer.

Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.

Adaptation, Physiological↗

PubMed1986

Hormone-like activity of human thrombin.

Recently, we have shown that thrombin is a chemotaxin and growth-promoting agent for cells of the mononuclear phagocytic lineage. These activities are independent of thrombin's enzymatic activity. Unlike other chemotactic factors, thrombin is specific for monocytes and does not attract granulocytes. To further explore the cellular specificity we have used a human leukemia cell line HL-60 that is capable of in vitro differentiation toward either monocytes (HL-60/mono) following incubation with 1,25(OH)2D3, or granulocytes (HL-60/gran) following incubation with DMSO. In contrast to undifferentiated HL-60 cells or HL-60/gran, we find that HL-60/mono respond chemotactically to intact human alpha-thrombin, esterolytically inactive iPR2P-alpha-thrombin, and the thrombin-derived peptide CB67-129, previously shown to contain the thrombin chemotactic exosite. In addition, thrombin induces in HL-60/mono association of actin with the cytoskeleton and causes an increase in levels of free cytosolic Ca2+. These phenomena are well characterized as early events occurring concomitant with directed cell movement associated with exposure to chemotactic agents such as FMLP. Furthermore, in contrast to fibroblasts, both iPR2P-alpha-thrombin and the thrombin chemotactic peptide CB67-129 evoke dose-dependent [3H]TdR incorporation, protein synthesis, and cell replication in growth-arrested J-744 cells, a murine macrophage-like cell line. Limited tryptic digests of CB67-129 lose chemotactic activity but retain full mitogenic activity, demonstrating that as with PDGF, the sites on CB67-129 required for chemotaxis and mitogenesis are clearly dissociable. The mitogenic effects of the CB67-129 digest can be mimicked by a synthetic tetradecapeptide analogue of CB67-129 (residues 367-380) that includes the loop B insertion sequence, previously shown to be critical for thrombin's chemotactic effects. From these data, it is apparent that the loop B insertion is critical for thrombin's nonenzymic biological effects on cells, but additional sites are required for stimulation of cell movement.

Actins↗

PubMed1999

IS1631 occurrence in Bradyrhizobium japonicum highly reiterated sequence-possessing strains with high copy numbers of repeated sequences RSalpha and RSbeta.

From Bradyrhizobium japonicum highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSalpha and RSbeta (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845-1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of B. japonicum differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of B. japonicum contained many copies of IS1631, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of B. japonicum were examined. Consequently, IS1631 occurrence was well correlated with B. japonicum HRS strains possessing high copy numbers of the repeated sequence RSalpha or RSbeta. DNA sequence analysis indicated that IS1631 is 2,712 bp long. In addition, IS1631 belongs to the IS21 family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS21, and its terminal inverted repeat sequences with multiple short repeats.

Amino Acid Sequence↗

PubMed2002

A Drosophila full-length cDNA resource.

BACKGROUND: A collection of sequenced full-length cDNAs is an important resource both for functional genomics studies and for the determination of the intron-exon structure of genes. Providing this resource to the Drosophila melanogaster research community has been a long-term goal of the Berkeley Drosophila Genome Project. We have previously described the Drosophila Gene Collection (DGC), a set of putative full-length cDNAs that was produced by generating and analyzing over 250,000 expressed sequence tags (ESTs) derived from a variety of tissues and developmental stages. RESULTS: We have generated high-quality full-insert sequence for 8,921 clones in the DGC. We compared the sequence of these clones to the annotated Release 3 genomic sequence, and identified more than 5,300 cDNAs that contain a complete and accurate protein-coding sequence. This corresponds to at least one splice form for 40% of the predicted D. melanogaster genes. We also identified potential new cases of RNA editing. CONCLUSIONS: We show that comparison of cDNA sequences to a high-quality annotated genomic sequence is an effective approach to identifying and eliminating defective clones from a cDNA collection and ensure its utility for experimentation. Clones were eliminated either because they carry single nucleotide discrepancies, which most probably result from reverse transcriptase errors, or because they are truncated and contain only part of the protein-coding sequence.

Amino Acid Sequence↗

PubMed2001

Canine COL1A2 mutation resulting in C-terminal truncation of pro-alpha2(I) and severe osteogenesis imperfecta.

RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-alpha2(I) suggested comigration with the similarly sized pro-alpha2(I) derived from the mutant allele. Furthermore, a-chains were overhydroxylated and the ratio of alpha1(I):alpha2(I) was 3.2:1, consistent with the presence of alpha1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-alpha2(I) C-propeptide and confirmed a diagnosis of OI.

Amino Acid Sequence↗

PubMed1995

[The mode of infection of tuberculosis--analysis using molecular epidemiologic methods].

In the 1950's, evidence showed that INH-resistant Mycobacterium tuberculosis organisms were attenuated in both virulence and pathogenicity in animals, and it was postulated that these bacilli might also be attenuated in their virulence to humans. Subsequent studies, however, indicated that all INH-resistant strains should not be considered as being attenuated in their virulence to humans. The general conclusion was that patients excreting INH-resistant organisms are somewhat less infectious to their contacts than patients excreting INH-susceptible organisms. Insertion sequences are suitable tools for the diagnosis and epidemiology of tuberculosis because of the highly variable copy number and variability of insertion sites in the chromosome. This variability allows the subtyping of M. tuberculosis strains by restriction fragment length polymorphism (RFLP) analysis. Patients with the same RFLP pattern constitute an epidemiologically linked cluster. Clustering indicates recent infection and rapid progression to clinical illness. Nearly one thirds of new cases of tuberculosis in San Francisco are the result of recent infection. In Thailand, tuberculosis has now emerged as the most common opportunistic disease associated with HIV infection. Cluster analysis showed the risk of progression to active tuberculosis among individuals infected with HIV. Ther have been numerous outbreaks of multidrug-resistant tuberculosis in the United States. Most of such outbreaks have primarily involved persons infected with HIV, who are thought to have been exposed to the strains in medical or correctional facilities.

AIDS-Related Opportunistic Infections↗