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PubMed1996

The conserved lymphokine element-0 in the IL5 promoter binds to a high mobility group-1 protein.

The conserved lymphokine elements-0 (CLE0) in the IL5 promoter is essential for the expression of IL-5. Here, we report the cloning and expression of a cDNA encoding a novel CLE0-binding protein, CLEBP-1 from a mouse Th2 clone, D10.G4.1. Interestingly, it was found that the CLEBP1 cDNA sequence was almost identical to the sequences of known high mobility group-1 (HMG1) cDNAs. When expressed as a recombinant fusion protein in Escherichia coli, CLEBP-1 was shown to bind to the IL5-CLE0 element in electrophoretic mobility-shift assays (EMSA) and southwestern blot analysis. The CLEBP-1 fusion protein cross-reacts with and-HMG-1/2 in Western blot analysis. It also binds to the CLE0 elements of IL4, GMCSF and GCSF genes. CLEBP-1 and closely related HMG-1 and HMG-2 proteins may play key roles in facilitating the expression of the lymphokine genes that contain CLE0 elements.

Animals↗
PubMed1993

Protein splicing: excision of intervening sequences at the protein level.

Protein splicing is an extraordinary post-translational reaction that removes an intact central "spacer" domain (Sp) from precursor proteins (N-Sp-C) while splicing together the N- and C-domains of the precursor, via a peptide bond, to produce a new protein (N-C). All of the available data on protein splicing fit a model in which these intervening sequences excise at the protein level via a self-splicing mechanism. Several proteins have recently been discovered that undergo protein splicing, and in two such cases, the excised spacer protein is an endonuclease. Such endonucleases are capable of conferring genetic mobility upon the intervening sequences that encodes them. These intervening sequences define a new family of mobile genetic elements that are translated yet remain phenotypically silent by excising at the protein rather than the RNA level.

Amino Acid Sequence↗
PubMedDate not supplied

Physical genome analysis of bacteria.

Pulsed-field gel electrophoresis (PFGE) is a general analytical tool to separate large DNA molecules and may therefore be applied to problems from all areas of bacteriology. The genome size of bacteria covers the range of 0.6 to 10 megabase pairs. For genome fingerprinting, the bacterial chromosome is cleaved with a restriction endonuclease that gives a resolvable and informative number of five to one hundred fragments on the PFGE gel. Restriction enzymes are chosen according to GC content, degree of methylation, and codon usage of the respective bacterial genus. Macrorestriction fingerprinting allows the identification of bacterial strains and the distinction between related and unrelated strains. If fragment patterns of several restriction digestions are quantitatively evaluated, strains can be classified according to genetic relatedness at the level of genus, species, and biovar. In particular, members of a clonal lineage can be uncovered. Hence, any problem from applied, environmental, and clinical microbiology may be addressed by PFGE restriction analysis where the spatiotemporal spread of a bacterial clone is of interest. In bacterial genomics, PFGE is employed for the top-down construction of macrorestriction maps of the chromosome which yields data about genome organization, mobile genetic elements, and the arrangement of gene loci and gene families. The genomic diversity of a bacterial species is elucidated by comparative chromosome mapping. Map positions of restriction sites and gene loci of interest serve as landmarks to assess the extent of gross chromosomal modification, namely insertions, deletions and inversions. Intra- and interspecies comparisons of genome organization provide insights into the structure and diversity of bacterial populations and the phylogeny of bacterial taxa.

Bacteria↗
PubMed1983

Molecular analysis of large transposable elements carrying the white locus of Drosophila melanogaster.

A large transposable element (TE) comprising the white-apricot and roughest genes has been found to transpose to well over a hundred sites scattered over the Drosophila genome. We report the cloning of the essential parts of several TEs. TE98 and TE28 sequences were cloned by ;walking' along the chromosome from the previously cloned heatshock genes. The ends of the TEs are characterized by dispersed repetitive elements belonging to the foldback (FB) family. FB elements are also associated with two independently isolated transposable elements originating from the white locus, Tp w-1 and Tp w. The strong correlation between FB elements and large composite transposons suggests that a pair of these elements can mobilize large intermediary DNA segments. One particular FB family member, FB-NOF, is associated with TE28, the white-crimson (w) mutant, the w-derived Tp w-1 and probably also with Tp w. A unique sequence located close to the white end of TE28 was used to clone the borders of TE77 and the surrounding sequences in the bithorax region, indicating that the TE can be used as a probe for gene isolation. Some evolutionary implications of the large composite transposons are discussed.

Journal Article↗
PubMed1991

Chloroplast ribosomal intron of Chlamydomonas reinhardtii: in vitro self-splicing, DNA endonuclease activity and in vivo mobility.

All chloroplast 23S ribosomal RNA genes of the unicellular alga Chlamydomonas reinhardtii contain an 888 bp group I intron with an internal open reading frame (ORF). A precursor RNA encompassing the intron with its 5' and 3' flanking sequences was shown to self-splice both during in vitro transcription and upon incubation of the isolated pre-RNA under self-splicing conditions. Expression of the internal ORF in Escherichia coli in the presence of a plasmid containing a cDNA corresponding to the intronless form of the 23S rRNA gene resulted in specific cleavage of the cDNA at or close to the exon junction sequence. To test whether this ORF-encoded double-strand DNA endonuclease is involved in intron mobility in vivo, the same ribosomal cDNA was stably integrated into the C. reinhardtii chloroplast genome using particle gun mediated transformation. All the transformants with the cDNA integrated at the expected site in the chloroplast genome had the intron precisely inserted at the artificial exon junction site. These experiments demonstrate that the chloroplast ribosomal intron of C. reinhardtii behaves as a ribozyme in vitro and also as a mobile genetic element in vivo provided a target site is present.

Animals↗
PubMed2002

Insertional mutagenesis in yeasts using T-DNA from Agrobacterium tumefaciens.

Insertional mutagenesis is a powerful tool for the isolation of novel mutations. The gene delivery system of the bacterium Agrobacterium tumefaciens, which mediates transfer not only to plants but also to yeasts and fungi, could be exploited to generate collections of yeasts containing insertional mutations if there were no bias towards particular integration sites, as is the case in plants. To test this, we have analysed a small collection of Saccharomyces cerevisiae strains with T-DNA copies integrated in the S. cerevisiae genome. The position of 54 of these T-DNAs was determined. The T-DNA showed no clear preference for certain DNA sequences or genomic regions. We have isolated insertions in the coding regions of the genes YGR125w, YDR250c, YGR141w, YGR045c, YPL017c, YGR040w, YDL052c, YJL148w, YCL033c, YFL061w, YJR033c, YDR175c and YLR309c confirming that these genes are non-essential for S. cerevisiae haploid growth on minimal medium. Given the advantages of T-DNA, we propose its use as an ideal mobile DNA element for insertional mutagenesis in yeasts.

Agrobacterium tumefaciens↗
PubMed2003

Ty3/gypsy-like retrotransposons in Candida albicans and Candida dubliniensis: Tca3 and Tcd3.

Ty3/gypsy retrotransposons are a widespread group of eukaryote mobile genetic elements. They are similar in structure to, and may be ancestors of, the vertebrate retroviruses. Here we describe the first Ty3/gypsy retrotransposons from the pathogenic yeasts Candida albicans and Candida dubliniensis, which we refer to as Tca3 and Tcd3, respectively. Tca3 was first identified in a variety of strains as an element lacking a large part of its coding region. Comparative analyses between C. albicans and C. dubliniensis allowed us to identify the closely related full-length Tcd3 element, and, subsequently, the full-length Tca3 elements. The full-length versions of Tca3 and Tcd3 are broadly similar in structure to other Ty3/gypsy elements, but have several features of special interest, e.g. both elements appear to have a novel mechanism for priming minus-strand DNA synthesis, probably involving conserved secondary structures adjacent to the 5' LTRs. Also, while closely related to each other, the two elements appear to be fairly distantly related to other known Ty3/gypsy-like elements. Finally, the occurrence of the internally deleted forms of Tca3 in many strains raises interesting questions concerning the evolution of these transposable elements in Candida and the evolution of Candida itself. The sequences reported in this paper have been assigned GenBank Accession Nos AF499463, AF499464 and AF510498.

Amino Acid Sequence↗
PubMed2000

Regulation of HGF/SF gene expression in MRC-5 cells by N-acetylcysteine.

The effect of N-acetylcysteine (NAC) on levels of hepatocyte growth factor/scatter factor (HGF/SF) gene transcripts was investigated in the human lung embryonic fibroblast cell line, MRC-5. NAC increased expression of HGF/SF mRNA, in a dose- and time-dependent fashion, by a mechanism independent of glutathione synthesis but sensitive to oxidant stress induced by H(2)O(2). Using actinomycin D to block RNA synthesis, it was observed that NAC had no effect on the stability of the HGF/SF mRNA transcripts. NAC increased HGF/SF promoter activity in cells transiently transfected with chloramphenicol acetyltransferase (CAT) reporter genes driven by HGF/SF gene 5'-flanking sequences. Primer extension analysis demonstrated that NAC enhanced the expression of HGF/SF mRNA transcribed from the main transcription initiation site. Although the 5' flanking region of the HGF/SF gene contains a sequence at -1019 to -1011 with homology to the NF-kappaB response element, electrophoretic mobility shift assay demonstrated that this site did not bind nuclear factors in MRC-5 cells in the presence or absence of NAC. In contrast to the effect on HGF/SF mRNA, NAC did not increase HGF/SF protein production by MRC-5 cells.

Acetylcysteine↗
PubMed1998

Distribution of the fungal transposon Restless: full-length and truncated copies in closely related strains.

The fungal transposon Restless of Tolypocladium inflatum ATCC 34921 is a member of the hAT family of mobile DNA elements. In order to study the distribution of this transposon we have looked at 13 fungal strains, most of which are taxonomically related to strain ATCC 34921. Three strains, which show identical banding patterns in a comparative RAPD analysis with strain ATCC 34921, similarly carry multiple copies of Restless. In addition, 1 T. inflatum strain and 2 Beauveria nivea strains contain only a few or even single copies of the transposon. Inverse PCR and DNA sequencing analysis revealed that 1 strain contains a nonmobile truncated version of the element, while the other one harbors a full-length transposon copy which was named Restless-2. The presence of a single transposon copy of Restless in a defined Beauveria strain indicates recent acquisition of this transposon, since class II transposons usually occur in several copies per haploid genome. Notably, the corresponding genomic location is not occupied by a transposon copy in strain ATCC 34921.

Base Sequence↗
PubMed2001

Selective recognition of pyrimidine motif triplexes by a protein encoded by the bacterial transposon Tn7.

The bacterial transposon Tn7 is distinguished among mobile genetic elements by its targeting abilities. Recently, we reported that Tn7 is able to selectively insert adjacent to triple-helical DNA. The binding of TnsC, a Tn7-encoded protein, to the triplex DNA target leads to the specific transposition of Tn7 adjacent to both inter- and intramolecular pyrimidine motif triplexes. Here, we further probe how Tn7 targets triplex DNA. We report that TnsC discriminates between different types of triplexes, showing binding preference for pyrimidine but not for purine motif intermolecular triplex DNA. The binding preferences of TnsC and the Tn7 insertion profiles were obtained using psoralenated, triplex- forming oligonucleotides annealed to plasmid DNAs. Although the presence of psoralen is not required for targeting nor is it alone able to attract TnsC, we show that the location of psoralen within the pyrimidine motif triplex does alter the position of Tn7 insertion relative to the triplex. Comparison between the triplex-targeting pathway and the highly site-specific targeting pathway mediated by the binding of the Tn7-encoded protein, TnsD, to the unique site attTn7, suggests that similar structural features within each target DNA are recognized by TnsC, leading to site-specific transposition. This work demonstrates that a prokaryotic protein involved in the targeting and regulation of Tn7 translocation, TnsC, can selectively recognize pyrimidine motif triplexes.

Animals↗
PubMed1996

A proposal for using modified site-specific recombination systems for making insertions into a chosen chromosomal site in vivo based on the analysis of lambda phage integration.

A proposal is presented here to use modified mobile genetic elements for making chromosomal insertions in vivo, into any desired chromosomal site. The idea lies in replacing the sequences which direct specificity of integration of those elements with other DNA sequences, homologous to the desired target chromosomal site. The idea is analysed on the basis of a lambda phage integration system. A design for a universal system for making chromosomal insertions into any site of choice is proposed.

Bacteriophages↗
PubMed2001

Genetic organization of the she pathogenicity island in Shigella flexneri 2a.

In this study we report the complete nucleotide sequence and genetic organization of the she pathogenicity island (PAI) of Shigella flexneri 2a strain YSH6000T. The 46 603 bp she PAI is situated adjacent to the 3' terminus of the pheV tRNA gene and includes an imperfect direct repeat of the 3'-terminal 22 bp of the pheV gene at the right boundary of the PAI. The she PAI carries a bacteriophage P4-like integrase gene within the pheV -proximal boundary of the PAI, intact and truncated mobile genetic elements, plasmid-related sequences, open reading frames exhibiting high sequence similarity to those found on the locus of enterocyte effacement (LEE) PAI of enterohemorrhagic Escherichia coli (EHEC), and the SHI-2 PAI of S. flexneri and several other open reading frames of unknown function. The she PAI also encodes two autotransporter proteins, including SigA, a cytopathic protease that contributes to intestinal fluid accumulation and Pic, a protease with mucinase, and hemagglutinin activities. In addition, an open reading frame (orf) termed sap, has high sequence similarity to the gene encoding Antigen 43, a surface-located autotransporter protein of E. coli. The ShET1 enterotoxin genes, associated predominantly with S. flexneri 2a strains, are also located on the she PAI.

Bacterial Proteins↗
PubMed1994

Characterization of pPT23B, the plasmid involved in syringolide production by Pseudomonas syringae pv. tomato PT23.

Avirulence gene D (avrD) in strain PT23 of Pseudomonas syringae pv. tomato (Pst) specifies the production of syringolides, which are elicitors of plant defense reactions. An 83-kb indigenous plasmid (pPT23B) that carries avrD has been mapped and characterized and a putative par region was identified. pPT23B contains a large amount of DNA that is repeated in other native plasmids in PT23. A putative mobile insertion element that occurs on plasmid pPT23A as well as on the chromosome was also identified in strain PT23. New broad-host-range expression vectors that functioned in Pst were constructed for overexpression of the cloned avrD gene and high-level production of the syringolides. Introduction of an avrD overexpression plasmid into PT23 or plasmid-cured strains led to identical syringolide peaks on HPLC with no new peaks observed. These results suggested that neither pPT23B nor other indigenous plasmids in Pst carry additional genes required for syringolide production or metabolism. Pst strains lacking pPT23B were not impaired in virulence on tomato plants.

Bacterial Toxins↗
PubMed1995

The staphylococcal insertion sequence IS257 is active.

The plasmid pJ3356 confers high-level mupirocin resistance on a strain of Staphylococcus aureus isolated from a hospital. The plasmid also carries two copies of IS257. Recombination of an IS257-containing plasmid conferring erythromycin resistance, pOX7-IS, into either of the IS257s of pJ3356 has been observed. The co-integration of pJ3356 and a small plasmid, pOX7, is also reported and involves duplication of one of the IS257s from pJ3356 together with 8 bp of pOX7 at the site of integration. Thus IS257 has been shown to be an active mobile genetic element.

Base Sequence↗
PubMed2002

A 50-kb plasmid rich in mobile gene sequences isolated from a marine micrococcus.

A 50,709-bp cryptic plasmid isolated from a marine Micrococcus has been sequenced and found to contain a number of putative mobile genetic elements. The coding regions for 11 putative transposases comprise approximately 17% of the total plasmid sequence. The majority of these transposases are located within a 13-kb cluster which includes a 1553-bp direct repeat consisting of a duplicated pair of transposase genes. The remaining putative ORFs showed similarity to a variety of proteins, the most notable being spider silk.

Base Composition↗
PubMed1993

A new middle repetitive sequence of Nicotiana plumbaginifolia genome.

A middle repetitive sequence NPR18 was isolated from Nicotiana plumbaginifolia nuclear genome [8]. Sequences homologous to the repeat are dispersed through genomes of several Nicotiana species. Computer-assisted data analysis of NPR18 primary sequence reveals several features attributed to mobile genetic elements: an AT content higher than average for nuclear DNA of genus Nicotiana plants; a number of direct and inverted repeats. Some of the repeats displayed homology to the terminal and subterminal repeats of Ac/Ds-like plant elements.

Base Sequence↗
PubMed1991

Nerve cells of Drosophila Notch mutant are differentiated inside amphibian brain: a new approach for the analysis of genetic control of nerve cell differentiation.

Fragments of the neural primordium of a new Notch mutant of Drosophila melanogaster produced in our laboratory were transplanted into the neural tube of embryos of 4 amphibian species (caudate and ecaudate) immediately after completion of neurulation. The grafts were identified by using a light microscope, scanning electron miscroscope, and in situ hybridization with mobile genetic elements of Drosophila and fluorescent dyes as markers. As has been shown, Drosophila nerve cells survive and differentiate inside the neural tube of amphibian embryos. The grafts increase in size by twentyfold and the cell proliferation zones are retained during the period of six months. Differentiated cells of the graft formed axon-dendritic contacts with recipient cells and penetrated into the organisms' brain structures. The effect of Drosophila transplants proved to be different for caudate and ecaudate amphibians. The presence of the graft accelerated the development of Xenopus laevis and it also affected their behavior. This approach can be very useful for the study of genetic basis of development and behavior.

Animals↗
PubMed1991

Molecular cloning and analysis of forked locus in Drosophila ananassae.

Drosophila ananassae, in spite of its unique genetic characters including high mutability and high frequency of male recombination has been little studied at the molecular level. Since the species is very similar to D. melanogaster, it is natural to expect that the high spontaneous mutation rate and male recombination may be caused by inserted mobile DNA elements, as in D. melanogaster. The present study was designed to determine whether or not most spontaneous mutations of the forked locus of D. ananassae are caused by insertion sequences as is found in D. melanogaster. I cloned the forked locus of D. ananassae, using forked DNA from D. melanogaster as a probe and investigated the molecular structure and transcription of the gene by Southern and Northern analyses. The results suggest that the restriction map and transcriptional patterns of the forked locus of D. ananassae are similar to those of D. melanogaster, while the spontaneous mutations available in D. ananassae are induced quite differently from those that have been described in D. melanogaster; in four (f;cd, f10-14, f49 and f86) out of eight forked alleles, neither insertions nor deletions were detected around the forked coding region. Forked transcripts are expressed in a pattern which is very similar to that of D. melanogaster and were all of normal size in these mutants. The other four mutants (f10-3, f9-10, f83i and f79b) had insertion sequences upstream of the forked coding region, while transcripts of the forked gene were of normal size. Hence, none of the eight mutations studied appear to affect the structure of the forked transcripts.

Animals↗