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PubMed2004

Genome-wide identification and mapping of variable sequences in the genomes of Burkholderia mallei and Burkholderia pseudomallei.

Burkholderia mallei and Burkholderia pseudomallei, closely related Gram-negative bacteria, are the causative agents of such serious infectious diseases of humans and animals as glanders and melioidosis, respectively. Despite numerous studies of these pathogens, the detailed mechanisms of their pathogenesis is still poorly understood. One of the serious obstacles to revealing factors responsible for pathogenicity lies in the considerable natural variability of B. pseudomallei and B. mallei, which is also a challenge to development of rapid and efficient diagnostic tools facilitating unambiguous identification of the infectious agents. To gain a deeper insight into B. mallei and B. pseudomallei interspecies divergence and intraspecies polymorphism, we compared the genomes of B. mallei C-5 and B. pseudomallei C-141 strains using a subtractive hybridization technique. A library of DNA fragments specific for B. mallei C-5 and absent from B. pseudomallei C-141 was obtained and analyzed. Some of the differential sequences detected were also not found in the recently sequenced genome of B. pseudomallei K96243. However, a multitude of B. mallei C-5 sequences absent from the B. pseudomallei C-141 genome were detected in the genome of B. pseudomallei K96243. On the other hand, some sequences identified as constituents of the B. mallei C-5 genome were not found in the genome of B. mallei ATCC 23344. Some of the differential DNA fragments displayed similarity to different mobile elements that have not yet been described for B. mallei, whereas the others matched fragments of various prophages, or, when translated into protein sequences, components of active transport systems and different enzymes. A substantial proportion of the differential clones had no database matches either at the nucleotide or amino acid sequence level. The results suggest great genome-wide intra- and interspecies variability of B. mallei and B. pseudomallei. The differences identified may be useful as molecular signatures for identification of B. mallei strains.

Animals↗
PubMed1997

Mutations in the carboxy-terminal part of IS30 transposase affect the formation and dissolution of (IS30)2 dimer.

The transposase of IS30 catalyses different transpositional rearrangements via the dimer (IS30)2 intermediate structure. Mutation analysis provides evidence that the C-terminal part of IS30 transposase is required for the formation and dissolution of (IS30)2 dimer. C-terminal mutants are also defective in transpositional fusion; however, this deficiency can be 'suppressed' by addition of the final product of site-specific dimerisation, the core (IS30)2 intermediate structure. The transposase part studied shows significant homologies in three highly conserved regions to proteins of IS30-related mobile elements.

Amino Acid Sequence↗
PubMed1998

The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae.

The currently available yeast mitochondrial DNA (mtDNA) sequence is incomplete, contains many errors and is derived from several polymorphic strains. Here, we report that the mtDNA sequence of the strain used for nuclear genome sequencing assembles into a circular map of 85,779 bp which includes 10 kb of new sequence. We give a list of seven small hypothetical open reading frames (ORFs). Hot spots of point mutations are found in exons near the insertion sites of optional mobile group I intron-related sequences. Our data suggest that shuffling of mobile elements plays an important role in the remodelling of the yeast mitochondrial genome.

Base Composition↗
PubMed2002

LINE drive. retrotransposition and genome instability.

The LINE-1 (L1) retrotransposon, the most important human mobile element, shapes the genome in many ways. Now two groups provide evidence that L1 retrotransposition is associated with large genomic deletions and inversions in transformed cells. If these events occur at a similar frequency in vivo, they have had a substantial effect on human genome evolution.

Animals↗
PubMed1997

Identification and status of design improvements to the NASA Shuttle EMU for International Space Station application.

To meet the significant increase in EVA demand to support assembly and operations of the International Space Station (ISS), NASA and industry have improved the current Shuttle Extravehicular Mobility Unit (EMU), or "space suit", configuration to meet the unique and specific requirements of an orbital-based system. The current Shuttle EMU was designed to be maintained and serviced on the ground between frequent Shuttle flights. ISS will require the EMUs to meet increased EVAs out of the Shuttle Orbiter and to remain on orbit for up to 180 days without need for regular return to Earth for scheduled maintenance or refurbishment. Ongoing Shuttle EMU improvements have increased reliability, operational life and performance while minimizing ground and on-orbit maintenance cost and expendable inventory. Modifications to both the anthropomorphic mobility elements of the Space Suit Assembly (SSA) as well as to the Primary Life Support System (PLSS) are identified and discussed. This paper also addresses the status of on-going Shuttle EMU improvements and summarizes the approach for increasing interoperability of the U.S. and Russian space suits to be utilized aboard the ISS.

Environment, Controlled↗
PubMed2004

Isolation of Salmonella enterica serotype choleraesuis resistant to ceftriaxone and ciprofloxacin.

Salmonella enterica serotype choleraesuis (S choleraesuis) usually causes systemic infections in man that need antimicrobial treatment. We isolated a strain of S choleraesuis that was resistant to ceftriaxone and ciprofloxacin from a patient with sepsis. Ciprofloxacin resistance was associated with mutations in gyrA and parC, whereas the ampC gene (bla(CMY-2)), responsible for ceftriaxone resistance, was carried by a transposon-like mobile element. This element was found inserted into finQ of a potentially transmissible 140 kb plasmid, with an 8 bp direct repeat flanking the junction regions. The appearance of this resistant S choleraesuis is a serious threat to public health, and thus constant surveillance is warranted.

Ceftriaxone↗
PubMed2001

Analysis of human Per4.

The molecular mechanism of the circadian pacemaker depends on the oscillatory expression of clock gene constituents. The Drosophila period gene is central to the clock mechanism in these animals. Three homologs of this gene identified in mice (mPer1-3) and humans (hPer1-3) display rhythmic expression and are important for normal clock function. Recently, analysis of the draft sequence of the human genome has revealed the presence of a fourth Per gene family member. Surprisingly, the deduced hPer4 cDNA has no open reading frame encoding a full-length PER-like protein. This sequence is characterized by numerous deletions, insertions, frame shifts and base pair changes, and its genomic structure is devoid of introns. The presence of an MER-2 mobile element fossil within the Per4 locus predicted that this gene would also be present in non-human primates. Rhesus monkey Per4 displays similar sequence anomalies and is 92.8% identical to hPer4. Sequence comparisons indicate that Per4 originated from a Per3 predecessor and that it is relatively new to the Period gene family. We conclude that hPer4 and RmPer4 are pseudogenes and descended from the retrotransposition of an ancestral Per3 gene.

Amino Acid Sequence↗
PubMed1997

Developmental expression analysis of the 1731 retrotransposon reveals an enhancement of Gag-Pol frameshifting in males of Drosophila melanogaster.

Extensive analyses of Drosophila melanogaster retrotransposon transcriptions in cultured cells or during development have been reported, but little is known about their translation during the development of the fly. Analysis of the translational products of the 1731 Drosophila melanogaster retrotransposon in Kc Drosophila cultured cells has been reported, showing the existence of primary products (Gag and Pol) and of processed polypeptides of various sizes. Study of 1731 retrotransposon expression at both levels of transcription and translation during the development of Drosophila melanogaster, is presented. 1731 transcripts were detected by in situ hybridization and 1731 proteins were detected by immunostaining and immunoblotting in embryos and in adult gonads. 1731 transcripts and proteins were detected in the mesoderm and central nervous system during embryonic development, in nurse cells and follicle cells in adult ovaries and in primary spermatocytes in adult testes. Moreover, Western blot analysis of the 1731 proteins with anti-Gag or anti-Pol antibodies in gonads revealed that the 1731 mRNA could be translated differentially according to the expressing tissue: essentially, ovarian translation and/or processing of 1731 products is different from that operating in testes, where the Gag-Pol fusion polyprotein is the most prominent product. Our results indicate that expression of the 1731 mobile element is regulated not only at the transcriptional level but also at the translational level, and that this regulation is different in the two sexes.

Animals↗
PubMed1998

Constitutive and barbital-induced expression of the Cyp6a2 allele of a high producer strain of CYP6A2 in the genetic background of a low producer strain.

The levels of one or more cytochrome P450 (CYP) enzymes and the respective mRNAs are found to be higher in insecticide-resistant insects than in susceptible insects. To understand better how insects regulate the levels of CYPs, we examined the expression of the Cyp6a2 gene in various strains of Drosophila melanogaster. We also took a transgenic approach to understand the molecular mechanisms that are involved in strain variation of Cyp6a2 expression. RNA blot analysis showed that the constitutive expression of Cyp6a2 varies from strain to strain; the level of CYP6A2 mRNA is barely detectable in the underproducer ry506 strain, whereas it is very high in the overproducer 91-R and MHIII-D23 strains. The long terminal repeat (LTR) of mobile element 17.6 that is found in the 3' untranslated region (UTR) of the Cyp6a2 gene of some strains does not appear to have any role on the steady-state CYP6A2 mRNA level. We also found that the Cyp6a2 gene is inducible by barbital in 91-R, ry506 as well as 91-C, which carries an LTR insertion. To examine the genetic background of the underproducer ry506 strain with respect to Cyp6a2 expression, we transformed the ry506 strain with the Cyp6a2 allele of the overproducer 91-R strain (Cyp6a2-91 R) and measured the constitutive and barbital-induced expression of the Cyp6a2-91 R transgene in the transformed flies. The Cyp6a2-91 R transgene carrying 129 bp of DNA upstream of the ATG codon did not show any constitutive or barbital-induced expression in the ry506 host genome. However, transgenes with 1331 and 985 bp upstream DNA showed similar levels of constitutive expression that were higher than that of the endogenous Cyp6a2 gene of the ry506 host strain, but lower than the expression of the same gene in the 91-R strain. Both these transgenes, with 1331 and 985 bp upstream DNA, also showed induction with 0.1 M barbital. DNA sequence analysis revealed that in both 91-R and ry506, the upstream DNA between +1 and -985 bp contains a distal and a proximal group of three potential barbie boxes, i.e. cis-elements that are thought to be involved in barbiturate-mediated induction of CYP genes. Except for four bases located near the distal cluster of barbie boxes and two other bases, the base sequence of the upstream DNA is identical in ry506 and 91-R strains. These results suggest that the underproducer ry506 strain has the trans-regulatory factors to support constitutive and induced expression of the Cyp6a2-91 R allele carrying DNA between -129 and -1331 bp regions. Possible reasons for low constitutive expression of the endogenous Cyp6a2 gene and moderate level of expression of the Cyp6a2-91 R allele in the ry506 genetic background are discussed.

Alleles↗
PubMed2003

Uncovering genomic differences in human pathogenic Yersinia enterocolitica.

To map out genomic differences between highly pathogenic Yersinia enterocolitica WA-314C biogroup 1B, serotype O:8 strain and low-pathogenic Y. enterocolitica Y-108C biogroup 4, serotype O:3 strain we have applied a method of suppression subtractive hybridization (SSH). In total, 428 WA-314-specific and 83 Y-108-specific sequences were uncovered by SSH. Among them were DNA fragments with similarity to known genes from several groups: (1) genes involved in O-antigen biosynthesis, (2) host-specific restriction-modification systems, (3) systems of iron and heme acquisition and storage, (4) flagellar biogenesis genes, (5) putative virulence factors, (6) drug resistance genes, and (7) mobile elements. Mapped out genomic differences may be applied in identification and development of novel therapeutic strategies for the treatment of enteropathogenic Yersinia.

Animals↗
PubMed1993

The retron: a bacterial retroelement required for the synthesis of msDNA.

'Retrons' are bacterial retroelements responsible for the synthesis of msDNA, a hybrid nucleic acid consisting of a single-stranded DNA that is branched out from an internal guanosine of an RNA molecule via a 2',5'-phosphodiester linkage. Retrons are found in a minor population of various bacterial species and are extensively diverse. Two important questions now demanding attention are whether retrons are mobile elements and why are they so diverse?

Base Sequence↗
PubMed1996

Transposition: mobile introns get into line.

Group II introns encode highly structured, frequently self-splicing RNAs; they are also mobile genetic elements. This mobility has been found to involve DNA-primed reverse transcription, with similarities to retrotransposition and telomere maintenance.

Base Sequence↗
PubMed1997

Origins of the mobile gene cassettes found in integrons.

Many of the acquired antibiotic resistance genes found in enterobacteria and pseudomonads are part of small mobile elements known as gene cassettes, and other genes are also likely to be found in cassettes. The origins of the genes and the recombination sites that make up cassettes are not known, but recent analyses of available data suggest that cassettes may be ancient structures, and some hypotheses for how they are formed can now be examined.

DNA Transposable Elements↗
PubMed1998

Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons.

Resistance of gram-negative organisms to antibiotics such as beta-lactams, aminoglycosides, trimethoprim and chloramphenicol is caused by many different acquired genes, and a substantial proportion of these are part of small mobile elements known as gene cassettes. A gene cassette consists of the gene and a downstream sequence, known as a 59-base element (59-be), that acts as a specific recombination site. Gene cassettes can move into or out of a specific receptor site (attl site) in a companion element called an integron, and integration or excision of the cassettes is catalysed by a site-specific recombinase (Intl) that is encoded by the integron. At present count there are 40 different cassette-associated resistance genes and three distinct classes of integron, each encoding a distinct Intl integrase. The same cassettes are found in all three classes of integron, indicating that cassettes can move freely between different integrons. Integrons belonging to class I often contain a further antibiotic resistance gene, sull, conferring resistance to sulphonamides. The sull gene is found in a conserved region (3'-CS) that is not present in all members of this class. Class I integrons of the sull type are most prevalent in clinical isolates and have been found in many different organisms. Even though most of them are defective transposon derivatives, having lost at least one of the transposition genes, they are none the less translocatable and consequently found in many different locations. The transposon Tn7 is the best known representative of class 2 integrons, and Tn7 and relatives are also found in many different species.

Journal Article↗
PubMed1998

The structure and evolution of angiosperm nuclear genomes.

Despite several decades of investigation, the organization of angiosperm genomes remained largely unknown until very recently. Data describing the sequence composition of large segments of genomes, covering hundreds of kilobases of contiguous sequence, have only become available in the past two years. Recent results indicate commonalities in the characteristics of many plant genomes, including in the structure of chromosomal components like telomeres and centromeres, and in the order and content of genes. Major differences between angiosperms have been associated mainly with repetitive DNAs, both gene families and mobile elements. Intriguing new studies have begun to characterize the dynamic three-dimensional structures of chromosomes and chromatin, and the relationship between genome structure and co-ordinated gene function.

Chromosomes↗
PubMed2003

Long-chain fatty acyl-CoA esters induce lipase activation in the absence of a water-lipid interface.

In most lipases a mobile element or lid domain covers the catalytic site of the enzyme and the lid opening event, which usually proceed at a lipid-water interface, is required to form the catalytically competent lipase. We report here a noticeable increase in activity of two fungal lipases assayed in aqueous solution in absence of any interface when adding submicellar concentrations of amphipathic physiological molecules like long-chain acyl-CoAs. The catalytic activity was dramatically dependent on the acyl chain length of the amphiphile and could be related with a lid-opening process. Our data support that lipase activation can be triggered in the absence of a well-defined interface, and stresses the notion that other non-aggregated amphipathic constituents of the local microenvironment can act as putative regulators of lipase activity.

Acyl Coenzyme A↗
PubMed1991

Nucleolus organizer-suppressed position-effect variegation in Drosophila melanogaster.

The white locus is inactivated in a cell-by-cell variegated pattern when juxtaposed with the proximal or distal parts of the nucleolus organizer region (NO) by X chromosome inversion. Recombinants for two such inversions, wm51b and wm4, were obtained and randomized for genetic background. White locus activity was much higher in the wm4 recombinant duplicated for most of the NO and much lower in the wm51b recombinant deficient for it. Although there may be other molecular differences between the heterochromatic regions of the recombinants, the most obvious is the dosage of NO. Suppression of a NO region-evoked variegated phenotype by additional NO doses is discussed in relation to four different classes of models for position-effect variegation (PEV): chromatin structure, nuclear geometry, incomplete transposition of mobile elements, and heterochromatin promoter-driven transcription. A corollary of the structural model is functional subdivision of heterochromatin, which would enable the use of PEV as a tool for its study.

Animals↗
PubMed1993

The use of retrotransposons as markers for mapping genes responsible for fitness differences between related Drosophila melanogaster strains.

Hitch-hiking of dispersed mobile elements serving as molecular markers was used as a new tool for mapping quantitative trait loci in Drosophila melanogaster. Two Drosophila strains with high fitness (HA) were backcrossed repeatedly to a closely related strain with low fitness (LA) to initiate experimental populations with expected HA gene frequencies of 1/32. The frequencies of 19 insertion sites of the retrotransposons mdg1 and copia were analyzed after 11 to 17 generations. Frequencies of sites from the HA line increased substantially in the pericentromeric region, indicating that one or more loci responsible for the fitness difference between the strains were located there. A maximum likelihood (ML) procedure was applied to estimate selection coefficients associated with the markers, and this indicated a broad, strongly selected region of the chromosome. At least one additional locus was localized in the middle of the 2L arm. Possible applications of this method are discussed.

Animals↗