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PubMed1989

The organization of the right-end early region of bacteriophage PRD1 genome.

Bacteriophage PRD1 is the only protein-primed DNA replication system known to operate in Escherichia coli. The left-genome end of PRD1 contains the early genes for the terminal protein and the DNA polymerase. These genes have been sequenced and the proteins have been produced separately. In this investigation we completed the analysis of the PRD1 early DNA regions by cloning and sequencing the right end genome containing early genes XII and XIX. We compared the structure of the right- and left-terminal regions. The genome organization of both ends was found to be rather uniform. The inverted terminal repeats, the first promoters and the first translation start codons are located almost exactly at the same distance from the genome ends. The PRD1 early gene products, P12 and P19, do not share similarities with proteins found in other protein-primed replication systems.

Amino Acid Sequence↗
PubMed1990

Identification of an insertion-sequence-like genetic element in the newly recognized human pathogen Mycoplasma incognitus.

Cloned 2.2-kb DNA (plasmid psb-2.2) of Mycoplasma incognitus, a pathogen in AIDS and non-AIDS patients [Lo et al., Am. J. Trop. Med. Hyg. 41 (1989) 364-376; 601-616], contains a 1405-bp genetic element closely resembling bacterial insertion sequence (IS) elements. This IS-like element has 29-bp terminal inverted repeats with seven mismatches, is immediately flanked by 3-bp direct repeats, and has typical stem-and-loop structures at or near both the termini. Two potential open reading frames (ORF-1 and ORF-2) encode 143 amino acids (aa) and 103 aa, respectively, in this IS-like element. Part (57 aa) of the deduced aa sequence of ORF-2 has a significant homology (43%) with the putative transposase of Escherichia coli IS3. In this study, a series of synthetic oligodeoxyribonucleotides each containing a specific sequence of a selected segment in psb-2.2, have been used as probes which reveal that the IS-like element occurs more than ten times in the genome of M. incognitus. This potentially transposable element has many characteristic features in common with bacterial IS elements.

Amino Acid Sequence↗
PubMed1991

Vaccinia virus-like early transcriptional control sequences flank an early gene in orf virus.

The purpose of this study was to map the initiation (tsp) and termination points of transcripts arising from an open reading frame (ORF3) found in the inverted terminal repeat of the orf virus genome and also, to identify probable transcriptional control sequences. Early transcripts of approx. 0.76 kb were mapped to ORF3 and found to be transcribed toward the ends of the genome. Using the S1 nuclease and primer-extension methods, the bulk of the tsp were mapped to a position 12-13 nucleotides (nt) downstream from a sequence which resembles A + T-rich vaccinia virus early promoters. The 5' ends were 81-82 nt upstream from the first ATG in ORF3. Most of 3' ends of the transcripts mapped to a region 24-32 nt downstream from a T5NT sequence found near the ORF3 stop codon. A second transcription termination point was found 25 nt downstream from another T5NT sequence located downstream and separated by 85 nt from the first. These results infer that the A + T-rich, early transcriptional control sequences found in other poxvirus genomes have been conserved in the G + C-rich genome of orf virus.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of IS402 from Pseudomonas cepacia.

IS402, a transposable gene-activating element isolated on the basis of its ability to increase expression of the Tn1 bla gene in Pseudomonas cepacia, was cloned from pTGL52 into the vector, pBluescript KS+, and its nucleotide (nt) sequence was determined. This 914-bp element had terminal inverted repeats of 17 bp with a single mismatch, and upon insertion into Tn1 generated a direct target duplication of 3 bp. Comparison of its nt sequence with the GenBank and EMBL databases indicated that IS402 is unrelated to previously described bacterial IS elements.

Base Sequence↗
PubMed1992

Sequence of ISRm4 from Rhizobium meliloti strain GR4.

ISRm4, an IS-like sequence structurally similar to Pseudomonas cepacia insertion element IS402, was identified by sequence analysis. This 933-bp element carries 17-bp putative terminal inverted repeats with five mismatches and a putative direct target duplication of 3 bp.

Base Sequence↗
PubMed1993

In-vivo-generated fusion promoters in Pseudomonas putida.

Plasmid pEST1463 carrying the promoterless pheBA operon was cloned into Pseudomonas putida PaW85, and phenol-utilizing colonies were isolated on minimal plates containing phenol as the only carbon and energy source. In these clones, chromosomally located Tn4652 was transposed upstream from the coding sequencing of pheA (encoding phenol monooxygenase). Sequence analysis together with mapping of the transcription start point of the pheBA operon in the recombinant plasmids revealed that fusions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated functional promoters under selective pressure in P. putida cells. These promoter sequences show similarity to the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. In three of the six fusion promoters studied, the generation combined two distinct events: transposition of Tn4652 into DNA containing potential -10 sequences and point mutations in these sequences. These mutations made the -10 sequences more like the sigma 70 promoter consensus sequences.

Base Sequence↗
PubMed1994

Characterization of IS1201, an insertion sequence isolated from Lactobacillus helveticus.

IS1201, a 1387-bp insertion sequence isolated from Lactobacillus helveticus, was identified by its nucleotide (nt) sequence. It carries a single open reading frame encoding a 369-amino-acid protein, which shares homology with transposases found in a class of related IS, including ISRm3 from Rhizobium meliloti, IS256 from Staphylococcus aureus, IS6120 from Mycobacterium smegmatis, IS1081 from M. bovis, IST2 from Thiobacillus ferroxidans and IS406 from Pseudomonas cepacia. IS1201 has terminal inverted repeats of 24 bp in length and a target site duplication of 8 bp. Its copy number ranges from 3 to about 16 per L. helveticus genome. No homology was found between the nt sequence of IS1201 and those of the other bacterial IS from the same class. These results, together with previous observations [de los Reyes-Gavilán et al., Appl. Environ. Microbiol., 58 (1992) 3429-3432], confirm that IS1201 can be used as a specific DNA probe for the identification of L. helveticus strains.

Amino Acid Sequence↗
PubMed1994

Characterization of IS1203, an insertion sequence in Escherichia coli O111:H-.

The complete nucleotide (nt) sequence of IS1203 from Escherichia coli O111:H- strain PH has been determined. IS1203 is 1312-nt long, with imperfect 26-bp terminal inverted repeats. The two major ORFs in IS1203 encode polypeptides of 12.7 and 33.7 kDa, the latter being the putative transposase. IS1203 is closely related to IS629 of Shigella sonnei and IS3411 of E. coli. At least twelve copies of IS1203 were found in the genome of E. coli O111:H- strain PH.

Amino Acid Sequence↗
PubMed1994

Construction of Tn917ac1, a transposon useful for mutagenesis and cloning of Bacillus subtilis genes.

A Tn917 derivative was constructed for the purposes of mutagenesis and cloning of Bacillus subtilis genes. This transposon, Tn917ac1 (4.6 kb), consisted of terminal inverted repeats of Tn917, the res sequence, a ColE1 origin of replication (ori) and two drug-resistance genes. The plasmid carrying this transposon, named pD917, contained the erm-tnpR-tnpA gene cluster of Tn917 and a temperature-sensitive ori of pE194. For the purpose of mutagenesis, transposition of Tn917ac1 was induced by culturing strains harboring pD917 in a medium containing a low concentration of erythromycin. Cells with a Tn917ac1 insertion in the chromosome were selected on agar containing chloramphenicol after heat treatment to eliminate the plasmidic form of pD917. DNA fragments adjacent to Tn917ac1 could be cloned by restriction digestion of the chromosomal DNA and by transforming the self-ligated restriction fragments into Escherichia coli. Sequence analysis revealed that Tn917ac1 was integrated into the chromosome of B. subtilis by transposition in a recE strain and by transposition or integration of pD917 in a wild-type strain. Tn917ac1 has been demonstrated to be useful for mutating and cloning of the genes involved in the biosynthesis of fengycin in B. subtilis F29-3.

Bacillus subtilis↗
PubMed1995

The insertion sequence element ISRm2011-2 belongs to the IS630-Tc1 family of transposable elements and is abundant in Rhizobium meliloti.

The insertion sequence (IS) element ISRm2011-2 of Rhizobium meliloti (Rm) is characterized by 19-bp imperfect terminal inverted repeats (three mismatches) and a size of 1053 bp. Upon transposition, ISRm2011-2 generates a putative target duplication of 2 bp. ISRm2011-2 carries two major overlapping open reading frames (ORFA and B) with a coding capacity of 135 and 201 amino acids (aa), respectively. A potential translational frameshifting window (5'-AAAAAAAG) is located in the overlapping region of both ORFs. The putative fusion product of both proteins, which probably represents the mature transposase, has a predicted molecular mass of 35.8 kDa and a pI of 10.5. Comparison of the deduced aa sequence of ORFA with database entries revealed homology to putative transposases of some IS elements of the IS3 family, as well as to eukaryotic transcription factors. The protein encoded by ORFB shows homology to transposases (Tps) of the recently proposed IS630-Tc1 family which includes Tps of both prokaryotic and eukaryotic transposable elements. Analyses of the distribution of ISRm2011-2 in natural Rm populations showed that this IS element is abundant in Rm strains.

Amino Acid Sequence↗
PubMed1996

Cloning and characterization of an IS-like element present in the genome of Brevibacterium lactofermentum ATCC 13869.

A repetitive DNA element of the Gram+ Brevibacterium lactofermentum (Bl), cloned by a modification of the subtractive hybridization method, contained a 1.4-kb IS-like element, IS13869, which included an open reading frame (ORF) inside a perfect 26-bp terminal inverted repeat (TIR). An 8-bp direct repeat (DR) was found outside each TIR. The ORF encoded a deduced protein of 436 amino acids (49 380 Da) with extensive similarity to other known transposases of insertion elements of Mycobacterium smegmatis (IS1096). Pseudomonas sp. (tpnA) and Corynebacterium glutamicum (IS31831). Distinct patterns were observed in different strains of Bl by hybridization with a probe internal to IS13869: four copies of IS13869 occurred in the wild type (wt) and R31 strains, but only three of them were observed in a recA derivative of the wt. Analysis by pulsed-field gel electrophoresis suggested that at least one copy of IS13869 had changed its position inside the chromosome during the lineage of a Bl derivative.

Bacterial Proteins↗
PubMed1996

Computer analyses reveal a hobo-like element in the nematode Caenorhabditis elegans, which presents a conserved transposase domain common with the Tc1-Mariner transposon family.

The present report describes the use of computer analyses to reveal a hobo-like element in the genome of Caenorhabditis elegans. This hobo-like sequence is 3039 bp long, contains two inverted terminal repeats of 25-27 bp and probably does not encoded a functional transposase. Sequence comparisons suggest that each transposase of hobo elements probably has a D(D/S)E motif. Thus the transposases of the hAT superfamily of transposons appear to be close to the other transposases and intregrases.

Amino Acid Sequence↗
PubMed1996

Transposon-like sequences at the TOX2 locus of the plant-pathogenic fungus Cochliobolus carbonum.

The ascomycete fungus Cochliobolus carbonum race 1 is pathogenic on certain genotypes of maize due to the production of HC-toxin, a host-specific cyclic peptide. HC-toxin production is controlled, at least in part, by a duplicated 22-kb region of DNA that is found only in toxin-producing isolates of the fungus. This 22-kb region of DNA is flanked by a repetitive element. We have sequenced the element and found an interrupted reading frame that would encode a product similar to transposases from the fungal transposons Fot1 of Fusarium oxysporum and Pot2 of Magnaporthe grisea. The individual element cloned from C. carbonum is likely to function neither in cis nor trans, as it had a nonsense mutation in frame and several substitutions in its terminal inverted repeats. However, similar elements in the C. carbonum genome may be active, as the putative transposase-encoding region hybridized to mRNA of the size predicted by the reading frame. The element was found in varying copy number in the genomes of all Cochliobolus spp. examined, giving a distinct fingerprint in each species and race tested. The sequence similarity of the C. carbonum repetitive element to other fungal transposons, along with its presence in multiple copies per genome, strongly suggest that the C. carbonum repetitive element is a member of the Fot1 family of fungal transposons.

Amino Acid Sequence↗
PubMed1992

Rapid identification and differentiation of the vaccine strain Rac H from EHV 1 field isolates using a non-radioactive DNA probe.

A method for rapid differentiation between the EHV 1 live vaccine strain Rac H and field isolates is described. Total DNA was isolated from virus-infected small scale cell cultures. DNA fragments digested with restriction endonuclease BamHI were separated, transferred and immobilized on filter membranes. A Digoxigenin-labeled probe derived from EHV 1 was used for hybridization. This probe hybridized specifically to sequences of the inverted terminal repeat region which in case of Rac H include a deletion of 0.8 kb. By comparing the different migration patterns after blot hybridization it could be shown that in 65 isolates from cases of abortion the live vaccine strain Rac H was not involved.

Abortion, Veterinary↗
PubMed2005

Persistent liver expression of murine apoA-l using vectors based on adeno-associated viral vectors serotypes 5 and 1.

Plasma levels of high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-l (apoA-l) are inversely related to risk for coronary heart disease. Overexpression of apoA-l inhibits atherosclerosis in animal models. A method of stably expressing apoA-l using somatic gene transfer would be of interest. Pseudotyped adeno-associated virus (AAV) vectors comprised of inverted terminal repeats from AAV serotype 2 have been used for liver-directed gene transfers. We hypothesized that liver-directed gene transfer of apoA-l using vectors based on AAV serotypes 1 and 5 would result in higher-level, prolonged expression of apoA-l and increased HDL-C. To test this hypothesis we injected apoA-l-/- mice via the tail vein with either AAV2, AAV1 or AAV5 vectors encoding the murine apoA-l cDNA driven by the liver-specific thyroxine binding globulin promoter. Plasma levels of murine apoA-l and HDL-C were highest in mice injected with the AAV1-based vector and lowest in mice injected with the AAV2-based vector. Expression of apoA-l was stable up to 1 year after vector injection. These results indicate that AAV5 and AAV1 are more effective vectors for achieving higher levels of stable transgene expression of apoA-l after liver-directed gene transfer than AAV2. Furthermore, AAV1-based vectors generate higher apoA-l levels than AAV5-based vectors. It is possible that the levels of expression achieved using these vectors will be therapeutic in preventing atherosclerosis.

Animals↗
PubMed2006

A hybrid vector for ligand-directed tumor targeting and molecular imaging.

Merging tumor targeting and molecular-genetic imaging into an integrated platform is limited by lack of strategies to enable systemic yet ligand-directed delivery and imaging of specific transgenes. Many eukaryotic viruses serve for transgene delivery but require elimination of native tropism for mammalian cells; in contrast, prokaryotic viruses can be adapted to bind to mammalian receptors but are otherwise poor vehicles. Here we introduce a system containing cis-elements from adeno-associated virus (AAV) and single-stranded bacteriophage. Our AAV/phage (AAVP) prototype targets an integrin. We show that AAVP provides superior tumor transduction over phage and that incorporation of inverted terminal repeats is associated with improved fate of the delivered transgene. Moreover, we show that the temporal dynamics and spatial heterogeneity of gene expression mediated by targeted AAVP can be monitored by positron emission tomography. This new class of targeted hybrid viral particles will enable a wide range of applications in biology and medicine.

Animals↗
PubMed2004

The Bacillus thuringiensis phage GIL01 encodes two enzymes with peptidoglycan hydrolase activity.

Bacteriophage GIL01, infecting Bacillus thuringiensis serovar israelensis, possesses a linear dsDNA genome of 14,931 bp with proteins attached to its 5' extremities and terminal inverted repeats at both ends. Viral particles are sensitive to organic solvents, suggesting that a lipid membrane is present in the capsid. All these characteristics are reminiscent of those found in members of the Tectiviridae family. Sequence analysis of GIL01 revealed the presence of two open reading frames (ORF25 and ORF30) encoding potential lytic enzymes, which were cloned and overexpressed in Escherichia coli. The muralytic activity of these two proteins, designated Mur1 and Mur2 respectively, was confirmed in situ using renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing bacterial cell wall preparations. While Mur2 degrading activity is limited to B. thuringiensis israelensis, Mur1 has a broader cleavage spectrum.

Amino Acid Sequence↗
PubMed2004

Guest, a transposable element belonging to the Tc1/mariner superfamily is an ancient invader of Neurospora genomes.

Guest is a transposable element of the Tc1/mariner superfamily with 30-40bp terminal inverted repeats and a TA dinucleotide target site duplication. Guest was originally discovered in the St. Lawrence 74A laboratory strain of the filamentous fungus Neurospora crassa. In this report, Guest iterations subcloned from a cosmid library of the Oakridge 74A strain were used to design PCR primers that permitted the detection of Guest in wild isolates of N. crassa. Guest is present in N. crassa as multiple copies ranging between 100bp and 2.4kb and is present in the mating type locus of several Neurospora species. Bioinformatic analysis of the entire N. crassa genome (Oakridge 74A strain) detected 48 Guest iterations. All iterations appeared to have been inactivated either by repeat-induced point mutation or sequence deletion, with the majority being remnants less than 400bp in length. The possible involvement of Guest in the evolution of the variable region that flanks the mating type idiomorphs in several Neurospora species is discussed.

Amino Acid Sequence↗