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PubMed2001

A family of Tc1-like transposons from the genomes of fishes and frogs: evidence for horizontal transmission.

Tc1-like transposons are very widely distributed within the genomes of animal species. They consist of an inverted repeat sequence flanking a transposase gene with homology to the mobile DNA element, Tc1 of the nematode Caenorhabditis elegans. These elements seem particularly to infest the genomes of fish and amphibian species where they can account for 1% of the total genome. However, all vertebrate Tc1-like elements isolated so far are non-functional in that they contain multiple frameshifts within their transposase coding regions. Here I describe a Tc1-like transposon (PPTN) from the genome of a marine flatfish species (Pleuronectes platessa) which bears conserved inverted repeats flanking an apparently intact transposase gene. Closely related, although degenerate, Tc1-like transposons were also isolated from the genomes of Atlantic salmon (SSTN, Salmo salar) and frog (RTTN, Rana temporaria). Consensual nucleic acid sequences were derived by comparing several individual isolates from each species and conceptual amino acid sequences were thence derived for their transposases. Phylogenetic analysis of these sequences with previously isolated Tc1-like transposases shows that the elements from plaice, salmon and frog comprise a new subfamily of Tc1-like transposons. Each member is distinct in that it is not found in the genomes of the other species tested. Plaice genomes contain about 300 copies of PPTN, salmon 1200 copies of SSTN and frog genomes about 500 copies of RTTN. The presence of these closely related elements in the genomes of fish and frog species, representing evolutionary lines, which diverged more than 400 million years ago, is not consistent with a vertical transmission model for their distributions.

Amino Acid Sequence↗
PubMed2002

Specific binding of a protein to a novel DNA element in the cyanobacterial small heat-shock protein gene.

Previously, it was shown that transcription of the small heat-shock protein gene, hspA, from the thermophilic cyanobacterium Synechococcus vulcanus is transiently heat-inducible at a vegetative promoter that lacks any known regulatory DNA elements. A novel regulatory mechanism that suppresses the expression of hspA under non-heat-shock condition has been postulated. In this study, it is demonstrated that a protein(s) in the extract of unstressed cells of the thermophilic cyanobacterium Thermosynechococcus elongatus, a cyanobacterium closely related to S. vulcanus, specifically binds to a 5'-untranslated region of the hspA gene. An AT-rich imperfect inverted-repeat sequence (ACAAgcAAA-TTTagTTGT) as a target for a putative DNA-binding protein has been identified. The DNA-binding activity in the cell as well as in the cell extract was lost much more quickly at a heat-shock temperature than a normal growth temperature. In a cell, the activity was restored within 45 min after a heat-shock by the heat-induced synthesis and stabilization of a DNA-binding protein. It is proposed that the inverted repeat is a specific target for a DNA-binding protein and that it plays a role in the regulation of the cyanobacterial hspA gene expression.

5' Untranslated Regions↗
PubMed1982

Partial restriction map of Marek's disease virus DNA.

A partial restriction map of Marek's disease virus (MDV) DNA was constructed by digestion with endonucleases BamHI, Bg/I and SmaI and by blotting hybridization. The data suggest that there is a terminal heterogeneous sequence at least on one end of the MDV DNA molecule. The data did not reveal four different orientations of the terminal fragments of MDV DNA molecules despite the observation that MDV DNA contains inverted repeat sequences as also present in Herpes simplex virus (HSV) DNA molecules (Cebrian et al., 1981). Terminal deletion of MDV DNA, SalI-H and I, was found in high passage number preparations.

Animals↗
PubMed2003

Characterization of the cryptic plasmid pBGR1 from Bartonella grahamii and construction of a versatile Escherichia coli-Bartonella spp. shuttle cloning vector.

We report herein the isolation and molecular characterization of pBGR1, the first native plasmid isolated from the genus Bartonella. Cloning and sequencing revealed a 2725-base pair (bp) cryptic plasmid comprising two open reading frames of considerable length, which were designated rep and mob. The regions containing rep and mob are separated by 140-bp inverted repeat sequences and display a difference in G + C content from one another. A 1435-bp SacI-BclI fragment containing the rep gene is sufficient to mediate replication in the species Bartonella henselae and Bartonella tribocorum, while this replicon does not appear to be functional in Escherichia coli. The Rep protein of 190 amino acids (aa) shares homology to putative replication proteins of cryptic plasmids of Gram-negative origin, which form a subgroup of the rolling-circle replication proteins of the pSN2 plasmid superfamily of Gram-positive bacteria. The Mob protein of 333 aa is related to mobilization proteins of several cryptic plasmids and is associated with a conserved recombination site A. The tra functions of RP4 can mobilize pBGR1 derivatives in a mob-dependent manner. Mobilizable pBGR1-based E. coli-Bartonella spp. shuttle vectors were constructed and were shown to be maintained in B. tribocorum during in vivo passage in a rat model in the absence of antibiotic selection. The small size and stability of these shuttle cloning vectors should render them particularly valuable for genetic studies in Bartonella spp.

Adenosine Triphosphatases↗
PubMed1985

The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences.

We have studied the interaction of purified FLP protein with restriction fragments from the substrate 2mu circle DNA of yeast. We find that FLP protects about 50 bp of DNA from nonspecific nuclease digestion. The protected site consists of two 13 bp inverted repeat sequences separated by an 8 bp spacer region. A third 13 bp element is also protected by binding of the FLP protein. We demonstrate that FLP introduces single- and double-strand breaks into the substrate DNA. This site-specific cleavage occurs at the margins of the spacer region, generating 8 bp 5' protruding ends with 5'-OH and 3'-protein-bound termini. Binding to mutant sites and half-sites demonstrates that the third symmetry element is not important for binding and cleavage by the FLP protein. The integrity of the core region is important for the cleavage activity of FLP.

Base Composition↗
PubMed1980

Comparison of foldback sequences of herpes simplex virus types 1 and 2 DNA.

The DNAs of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) were separately denatured and allowed to renature briefly. The intrastand foldback structures that resulted from base pairing of inverted repeated sequences on otherwise single-stranded (ss) DNA were visualized in the electron microscope. The two genomes were found to contain similar size classes of small duplex stem DNA sequences. However, HSV-2 DNA appeared to possess an additional, larger size class of foldback structures not found on HSV-1 DNA. Both HSV DNAs were found to contain stem-plus-loop structures; the larger stem-plus-loop structures of the two genomes had similar stem lengths but dissimilar loop lengths. Thus, a comparison of the genomes of HSV-1 and HSV-2 showed that they possessed similar size classes of foldback sequences.

Base Sequence↗
PubMed1987

DNA replication-blocking properties of adducts formed by aflatoxin B1-2,3-dichloride and aflatoxin B1-2,3-oxide.

The carcinogen aflatoxin B1 (AFB1), upon activation to a hypothesized AFB1-2,3-oxide (AFB1-oxide), reacts with DNA guanines. Aflatoxin B1-2,3-dichloride (AFB1-Cl2) was originally synthesized as an electronic analog for the putative AFB1-oxide, which has never been isolated due to presumed reactivity. We have previously shown that AFB1-oxide reacts with base-paired DNA guanines in a sequence-specific manner, as revealed by an alkali-degradation analysis. On the basis of a replication-block analysis, we have shown that AFB1-Cl2 reacts with single-stranded DNA preferentially at inverted repeat sequences, which were suggested to be capable of forming intrastrand base-paired structures. Here, we present data to show the following. Both AFB1-oxide and AFB1-Cl2 react with guanines in double-stranded DNA to induce similar sequence-specific, alkali-labile sites. Reactivity with partial DNA duplexes as well as the use of single-strand specific chemical probes directly demonstrates that AFB1-Cl2, like AFB1-oxide, prefers base-paired guanines over non-base-paired guanines. DNA replication block patterns induced by AFB1-oxide are essentially similar to those induced by AFB1-Cl2. Unexpectedly, and unlike other tested DNA lesions, Mn2+ does not appear to affect the template blocking properties of the adduct formed by AFB1-Cl2 or AFB1-oxide. The sites for replication stoppage as well as the lack of a Mn2+ effect on adducted templates have implications for the mechanisms of mutagenesis by activated AFB1.

Aflatoxin B1↗
PubMed1987

Isolation and characterization of a cDNA clone encoding testis protamine Z1 from the dog-fish Scylliorhinus caniculus.

A clone containing a 445-bp cDNA insert was isolated from a cDNA library synthesized from dog-fish testes mRNA. The nucleotide sequence was determined and corresponded to a 50-amino-acid protein. The known five-amino-acid N-terminal sequence corresponded exactly to our deduced amino acid sequence. After in vitro transcription of this cDNA using SP6 RNA polymerase, the translated polypeptide comigrated with the Z1 scylliorhinine marker. Analysis of the cDNA 3' flanking region of our Scylliorhinus protamine Z1 revealed an inverted repeat sequence, an ACAA motif and a CAGGAAAGA box known as regulatory signals for transcription termination in histone genes. In addition, sequences homologous to the simian virus (SV 40) and polyoma virus core enhancer elements were identified in the 5' and 3' flanking regions.

Amino Acid Sequence↗
PubMed1990

Human gastric (H+ + K+)-ATPase gene. Similarity to (Na+ + K+)-ATPase genes in exon/intron organization but difference in control region.

The human gastric (H+ + K+)-ATPase gene (15 kilobases) was cloned, and its nucleotide sequence was determined. The gene has 22 exons and codes a protein of 1,035 residues including the initiator methionine (Mr = 114,047). A conserved lysine-rich sequence with inserted glycine residues was found near the amino terminus of the enzyme. The phosphorylation site and pyridoxal 5'-phosphate- and fluorescein isothiocyanate-binding residues found in the rat and pig enzymes are also conserved in the human enzyme. The positions of introns in the human (H+ + K+)-ATPase gene are essentially the same as those in the human (Na+ + K+)-ATPase alpha and alpha III subunits; but the first introns of the two enzymes are difficult to align, and unlike in the (Na+ + K+)-ATPase gene, the sixth exon in the (H+ + K+)-ATPase gene is not separated by an intron. Furthermore, the ninth intron is located two bases upstream of the position for the corresponding intron of the (Na+ + K+)-ATPase alpha III subunit. The similarity in organization of these two ATPase genes and the homology in the primary structures of their proteins (approximately 60%) suggest that these two genes were derived from a common ancestral gene. However, the 5'-flanking regions of the genes for (H+ + K+)-ATPase and the (Na+ + K+)-ATPase alpha (+) subunit show no apparent sequence homology, indicating that their transcriptions are regulated differently. The control region of the fast-twitch sarcoplasmic reticulum Ca2(+)-ATPase gene also showed no sequence homology to that of (H+ + K+)-ATPase. The 5'-flanking region of the (H+ + K+)-ATPase gene contains potential binding sites for RNA polymerase II and various transcriptional regulation factors and several direct and inverted repeat sequences which may be important for specific and controlled expression of the gene in gastric parietal cells. There are two polyadenylation signals in the 3'-flanking region of the (H+ + K+)-ATPase gene, but the sequence of this region shows no homology to those of the corresponding regions of the genes for the (Na+ + K+)-ATPase alpha and alpha III subunits.

Adenosine Triphosphatases↗
PubMed2000

Identification of the gene encoding BmpB, a 30 kDa outer envelope lipoprotein of Brachyspira (Serpulina) hyodysenteriae, and immunogenicity of recombinant BmpB in mice and pigs.

A gene encoding a 30kDa outer envelope protein of the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, was cloned and expressed in Escherichia coli strain XLOLR. Five phagemids containing DNA inserts encoding the protein were established and one clone (pSHA) was sequenced. An 816bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site (AGGAG), and putative -10 (TATAAT) and -35 (TTGAAA) promoter regions upstream from the ATG start of the ORF. A 12bp inverted repeat sequence, possibly serving as a transcription terminator, was identified downstream from the TAA stop codon. Analysis of the amino acid sequence identified a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. Further analysis of the amino acid usage of this lipoprotein, designated BmpB, showed its possible outer membrane localisation. Comparison of the gene encoding the lipoprotein, bmpB, with GenBank nucleotide sequences showed that it has homology with the gene (plp3) encoding Plp3, an outer membrane lipoprotein of Pasteurella haemolytica (54% identity in 735bp). Comparison of the deduced amino acid sequence with the SWISS-PROT amino acid database revealed greatest homology with the outer membrane lipoproteins (Plp1, 2, 3) of P. haemolytica (34% identity in 242 aa, 37% identity in 250 aa, and 39% identity in 272 aa, respectively), and lipoproteins (rcsF and lipoprotein-28) of E. coli (40% identity in 267 aa and 36% identity in 263 aa, respectively). Three of the recombinant E. coli clones (pSHA, pSHD, and pSHE) were formalinised and used to immunise mice. A bacterin preparation of one recombinant E. coli clone (pSHA) was used to immunise pigs. Sera from these mice and pigs recognised the 30kDa lipoprotein in outer membrane preparations of B. hyodysenteriae, indicating the immunogenicity of recombinant BmpB. Sera from pigs naturally infected with B. hyodysenteriae also reacted with recombinant BmpB expressed in E. coli.

Amino Acid Sequence↗
PubMed2004

Transcription factor YB-1 mediates DNA polymerase alpha gene expression.

Y-box protein-1 involvement in cyclin A and B1 gene regulation has recently been demonstrated. A more generalized role of this protein for cell replication is hypothesized as numerous regulatory sequences of cell cycle-related genes contain putative binding sites. In the present study the DNA polymerase alpha (DPA) gene is identified as another YB-1-responsive gene with a Y-box and 3' inverted repeat sequence, designated DPA RE-1, in the serum-responsive promoter region. Overexpressed YB-1 concentration-dependently trans-activated DPA gene expression in reporter assays and Southwestern blotting as well as DNA binding analyses revealed binding of distinct endogenous proteins to the RE-1 with molecular sizes of 26, 32 and 52 kDa. Among these, YB-1 binding was confirmed using recombinant as well as endogenous proteins, with preferential single-stranded DNA binding. Early serum growth response in mesangial cells was accompanied by a nuclear YB-1 shift and nucleocomplex formation at the RE-1. Fine mapping of the DPA RE-1 sequence unraveled a dependence on co-factors for trans-regulation with gene activation in the context of a heterologous SV40 promoter but suppression in the context of the abbreviated homologous promoter sequence. A YB-1 knock down resulted in decreased DPA transcription rates and abrogated the serum-dependent induction of DPA transcription. These results link YB-1 with serum responsiveness of DPA gene expression and provide insight into the required sequence and protein binding context.

Acetylcysteine↗
PubMed1993

Comparative DNA sequence features in two long Escherichia coli contigs.

The recent sequencing of two relatively long (approximately 100 kb) contigs of E.coli presents unique opportunities for investigating heterogeneity and genomic organization of the E.coli chromosome. We have evaluated a number of common and contrasting sequence features in the two new contigs with comparisons to all available E.coli sequences (> 1.6 Mb). Our analyses include assessments of: (i) counts and distributions of restriction sites, special oligonucleotides (e.g., Chi sites, Dam and Dcm methylase targets), and other marker arrays; (ii) significant distant and close direct and inverted repeat sequences; (iii) sequence similarities between the long contigs and other E.coli sequences; (iv) characterization and identification of rare and frequent oligonucleotides; (v) compositional biases in short oligonucleotides; and (vi) position-dependent fluctuations in sequence composition. The two contigs reveal a number of distinctive features, including: a cluster of five repeat/dyad elements with very regular spacings resembling a transcription attenuator in one of the contigs; REP elements, ERICs, and other long repeats; distinction of the Chi sequence as the most frequent oligonucleotide; regions of clustering, overdispersion, and regularity of certain restriction sites and short palindromes; and comparative domains of inhomogeneities in the two long contigs. These and other features are discussed in relation to the organization of the E.coli chromosome.

Base Sequence↗
PubMed1983

The interactions of enzyme and chemical probes with inverted repeats in supercoiled DNA.

In negatively supercoiled DNA molecules some inverted repeat sequences adopt a perturbed conformation which is characterised by the following properties. They are centrally hypersensitive to single-strand-specific nucleases such as S1, and to a much lower extent the flanking regions may also be sensitive. They are also hypersensitive to modification by bromoacetaldehyde, particularly in their flanking region. They may be resistant to endonucleolysis by restriction enzymes and are cleaved (resolved) by a T4 resolving enzyme. All these properties can only be consistently explained by a model in which the inverted repeat adopts a cruciform structure. This property has been shown to depend sharply on a superhelix density, and the transition to nuclease sensitivity is accompanied by a marked alteration in the overall molecular geometry as judged by frictional properties. The probable dynamics of these structures are discussed.

Base Sequence↗
PubMed1988

Determinants for the DNase I-hypersensitive chromatin structure 5' to a human HSP70 gene.

A DNase I hypersensitive site was detected in chromatin formed over a human hsp70 gene segment after amplification in COS7 cells. Deletion mutant analysis was used to evaluate the sequence requirements for this chromatin structure. Determinants sufficient to form the hypersensitive site are contained in a 280 base-pair sequence corresponding approximately to the region that is hypersensitive. Deletion of sequences from either end of this region resulted in reduced hypersensitivity, suggesting that multiple genetic elements contribute to the formation of this chromatin structure. As has been reported for other heat shock genes, the hypersensitive chromatin structure is present prior to heat treatment and does not change in intensity or position after heat shock, in spite of the fact that hsp70 gene expression is completely dependent on heat induction. Sequence requirements for hypersensitivity were generally similar to those for heat-induced gene expression when mutant plasmids were tested at low copy number (e.g. in HeLa cells or in COS cells without amplification); however, deletion of sequences between -223 and -162 with respect to the start of transcription abolished the hypersensitive site but had no effect on gene expression. A barrier to exonuclease III digestion was detected within this region (near an imperfect inverted repeat sequence centered at position -202), suggesting that proteins are tightly bound to the DNA at this location.

Animals↗
PubMed1988

Resolution of model Holliday junctions by yeast endonuclease is dependent upon homologous DNA sequences.

Holliday junctions, in which two double-stranded DNA molecules are linked by single-stranded crossovers, are thought to be central intermediates in genetic recombination. We report here the in vitro specificity of a yeast endonuclease for structures analogous to Holliday junctions. Plasmids that extrude inverted repeat sequences into cruciform junctions are cleaved by the introduction of nicks into strands of like polarity, approximately 4-8 nucleotides from the base of the junction. In all cases, cleavage occurs within homologous sequences, and with precise symmetry across the junction. In contrast, a junction containing four arms of unrelated sequence is cleaved asymmetrically. The dependence upon homology for symmetrical cleavage is not found with T4 endonuclease VII, which cleaves branched structures in vitro. Holliday junction resolution appears to occur in a concerted manner by the introduction of nicks into two homologous DNA helices held in alignment.

Base Sequence↗
PubMed1994

Homology between Borrelia burgdorferi OspC and members of the family of Borrelia hermsii variable major proteins.

Synthesis of the Borrelia burgdorferi outer surface protein C (OspC) is quite variable. We have cloned and sequenced the ospC gene from B. burgdorferi isolate CA-11.2A, a clone in which ospC expression varies. The 5' flanking region of the gene contains at least two consensus promoter regions, as well as two large overlapping inverted repeats. Sequence comparison to other OspC proteins indicated that the CA-11.2A OspC is as closely related to OspC from two different genospecies of Lyme disease spirochetes as it is to OspC from the prototype B. burgdorferi strain, B31. Comparisons of the OspC amino acid (aa) sequence with those in aa sequence databases revealed partial identity with the variable major proteins Vmp3 and Vmp24 of B. hermsii, a causative agent of tick-borne relapsing fever. An ospC probe hybridized to B. hermsii restriction fragments and linear plasmids that also were recognized by the vmp3 and vmp24 probes. OspC and these Vmp appear to be related, but their synthesis is regulated differently in the two species of spirochetes. This represents a fascinating example of the evolution of the number, position, regulation and perhaps function of homologous genes in two related pathogens. These parameters may relate to characteristic properties of the pathogens and their separate tick vectors.

Amino Acid Sequence↗
PubMed2000

Heat shock proteins and inflammatory acne vulgaris: molecular cloning, overexpression and purification of a propionibacterium acnes GroEL and DnaK homologue.

Propionibacterium acnes is associated with inflammatory acne. The genes encoding two putative mediators of inflammation, the heat shock proteins GroEL and DnaK, were cloned from this organism and sequenced. groEL and dnaK encode proteins of 56.8 and 66.4 kDa, respectively, which show a high degree of homology (>75% similarity) to the GroEL and DnaK proteins of mycobacteria and streptomycetes. The promoter regions of both genes contain inverted repeat sequences believed to be involved in the transcriptional regulation of heat shock genes. Recombinant P. acnes GroEL and DnaK were overexpressed in Escherichia coli with C-terminal histidine tags. The recombinant proteins were purified from E. coli by metal affinity chromatography. These proteins will now be used in immunological investigations to determine their role in inflammatory acne.

Acne Vulgaris↗
PubMed1997

DNA sequences and their binding proteins required for Sertoli cell-specific transcription of the rat androgen-binding protein gene.

The rat androgen-binding protein (ABP) gene is transcriptionally regulated from two promoters: the P1 promoter regulates expression of transcripts starting at exon 1, whereas P(A) regulates transcripts containing exon A. The P1 promoter directs cell-specific gene regulation of ABP secreted by Sertoli cells. In this study, the Sertoli cell-regulatory sequences of P1 were further examined using a luciferase reporter system with three cell lines, including a Sertoli cell line (MSC-1) that expresses the ABP gene. Deletion mapping experiments determined that the sequences required for full activity in MSC-1 cells were included within 619 bp of the start site and identified several regions that demonstrated increased luciferase activity: the -583 bp to -564 bp, -503 bp to -484 bp, and -114 bp to -65 regions. The activities contributed by each region were much higher (up to 120-fold) in MSC-1 cells than in MA10 Leydig or NIH3T3 fibroblast cells. Nuclear-binding proteins and their binding sequences were identified using several molecular biology techniques. Complexes formed by nuclear proteins of MSC-1, MA10, and NIH3T3 cells, which bind specifically to the -114 to -65-bp region, were identified using gel retardation assays. Furthermore, the inverted repeat sequence in this region, 5'-AGGGTCAGTGTCCCT-3' was identified by deoxyribonuclease (DNase) I footprinting. The regulatory element contained within the -503 to -484-bp region was identified by scanning mutagenesis, but no protein was found that bound to this sequence by gel retardation or DNase I protection assays. This element is characterized by the core sequence, 5'-GGAGGC-3'. The third regulatory region (residues -583 to -564) bound a protein complex that retarded mobility of the free DNA probe in a gel shift assay. Using several techniques, the binding sequence was identified as 5'-TTCATAGTATCCATTAAAC-3'. In summary, these data have identified several transcriptional regulatory sequences and their binding proteins, which appear to play a role in the Sertoli cell-specific expression of the ABP gene.

Androgen-Binding Protein↗