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PubMed2025

Establishment of reverse genetics systems for Colorado tick fever virus.

The Colorado tick fever virus (CTFV), which has 12-segmented double-stranded RNA genomes, is a pathogenic arbovirus that causes severe diseases in humans. However, little progress has been made in the analysis of replication mechanisms and pathogenicity. This virological constraint is due to the absence of a reverse genetics system for CTFV; therefore, we aimed to establish the system. Initially, the efficacy of CTFV replication was investigated in various cell lines. CTFV was found to grow in many cell types derived from different hosts and organs. Subsequently, BHK-T7 cells stably expressing T7 RNA polymerase were transfected with plasmids encoding each of the 12 CTFV gene segments, expression plasmids encoding all CTFV proteins, and a vaccinia virus RNA-capping enzyme. Following transfection, the cells were co-cultured with Vero or HeLa cells. Using this system, we rescued monoreassortants and recombinant viruses harboring peptide-tagged viral proteins. Furthermore, an improved system using Expi293F cells expressing T7 RNA polymerase was established, which enabled the generation of recombinant reporter CTFVs. In conclusion, these reverse genetics systems for CTFV will greatly contribute to the understanding of viral replication mechanisms, pathogenesis, and transmission, ultimately facilitating the development of rational treatments and candidate vaccines.

Animals↗
PubMed2005

Bacillus cereus DNA topoisomerase I and IIIalpha: purification, characterization and complementation of Escherichia coli TopoIII activity.

The Bacillus cereus genome possesses three type IA topoisomerase genes. These genes, encoding DNA topoisomerase I and IIIalpha (bcTopo I, bcTopo IIIalpha), have been cloned into T7 RNA polymerase-regulated plasmid expression vectors and the enzymes have been overexpressed, purified and characterized. The proteins exhibit similar biochemical activity to their Escherichia coli counterparts, DNA topoisomerase I and III (ecTopo I, ecTopo III). bcTopo I is capable of efficiently relaxing negatively supercoiled DNA in the presence of Mg2+ but does not possess an efficient DNA decatenation activity. bcTopo IIIalpha is an active topoisomerase that is capable of relaxing supercoiled DNA at a broad range of Mg2+ concentrations; however, its DNA relaxation activity is not as efficient as that of bcTopo I. In addition, bcTopo III is a potent DNA decatenase that resolves oriC-based plasmid replication intermediates in vitro. Interestingly, bcTopo I and bcTopo IIIalpha are both able to compensate for the loss of ecTopo III in E.coli cells that lack ecTopo I. In contrast, ecTopo I cannot substitute for ecTopo III under these conditions.

Bacillus cereus↗
PubMed1976

Genetic and physical studies of recombinant plasmids formed between an R plasmid of compatibility group FI and sex factor F of HfrH.

Recombinant plasmids between an R plasmid of the FI group (R162/3) and the sex factor F or HfrH were produced after the conjugal transfer of this R plasmid into HfrH. Three types of recombinant plasmids were identified after the mating of HfrH (R162/3) with recA and rec+ recipients. One specimen of each type (pIP218, pIP222, pIP226) was studied in this report. All three recombinant plasmids carry the same genetic information for resistance to antibiotics (CSSuT) retained from R162/3. pIP218 retained all the other properties from F of HfrH: derepression for pilus synthesis, mobilization of the chromosome for the proximally transferred HfrH genes (thr, leu, proA), interference with T7 propagation, and ability to be cured by acridine orange. pIP222 retained from F of HfrH the derepression for pilus synthesis and the same polarity of chromosome transfer (thr, leu, proA), while pIP226 retained the interference with T7 propagation and acridine orange curing. Physical studies revealed that replication control and/or recovery of F and pIP218 as covalent circles of deoxyribonucleic acid are similar, and are different from R162/3. The new plasmids are more likely the result of a substitutive recombination event than a fusion. We propose genetic maps of these recombinant plasmids, showing the unequal participation of the parental plasmids in their formation.

Acridines↗
PubMed1995

Genetic organization of a small cryptic plasmid of Helicobacter pylori.

A 2.9-kb cryptic plasmid of Helicobacter pylori (Hp), pHel1, was isolated and the complete nucleotide (nt) sequence was determined. An open reading frame (ORF1) was identified encoding a putative polypeptide of 63,709 Da, the existence and correct size of which was confirmed by T7 promoter expression analysis. The ORF1 sequence showed strong amino-acid sequence identity to a recently identified putative ORF1 protein of a cryptic Hp plasmid, pHPM180, and significant homologies to putative Rep proteins of Campylobacter coli (RepB) and Pediococcus halophilus (RepA), and was therefore designated RepA. A functional role of RepA in replication of pHel1 was demonstrated by the fact that only pHel1 plasmid derivatives with an intact repA gene were able to autonomously replicate in Hp. Upstream of repA, a 22-bp sequence was recognized which was tandemly repeated four and a half times, a feature typical for many replication origins (ori) and commonly termed a DNA iteron. Analysis of the repA upstream region by primer extension identified a transcription start point for the repA mRNA, but did not correspond to known consensus promoter sequences. Southern hybridizations using pHel1 as a probe under stringent conditions revealed that homologous sequences to pHel1 were present in nearly all plasmid-carrying Hp strains, but not in a plasmid-carrying Helicobacter felis strain, suggesting that this type of replicon is predominantly found in the Hp species.

Amino Acid Sequence↗
PubMed2004

Complete genomic sequence of the virulent Salmonella bacteriophage SP6.

We report the complete genome sequence of enterobacteriophage SP6, which infects Salmonella enterica serovar Typhimurium. The genome contains 43,769 bp, including a 174-bp direct terminal repeat. The gene content and organization clearly place SP6 in the coliphage T7 group of phages, but there is approximately 5 kb at the right end of the genome that is not present in other members of the group, and the homologues of T7 genes 1.3 through 3 appear to have undergone an unusual reorganization. Sequence analysis identified 10 putative promoters for the SP6-encoded RNA polymerase and seven putative rho-independent terminators. The terminator following the gene encoding the major capsid subunit has a termination efficiency of about 50% with the SP6-encoded RNA polymerase. Phylogenetic analysis of phages related to SP6 provided clear evidence for horizontal exchange of sequences in the ancestry of these phages and clearly demarcated exchange boundaries; one of the recombination joints lies within the coding region for a phage exonuclease. Bioinformatic analysis of the SP6 sequence strongly suggested that DNA replication occurs in large part through a bidirectional mechanism, possibly with circular intermediates.

Amino Acid Sequence↗
PubMed1989

Characterization of the binding specificity of two anticruciform DNA monoclonal antibodies.

Two monoclonal antibodies (2D3 and 4B4) have been raised against a stable cruciform DNA structure containing the 27-base pair palindrome of the SV40 origin of replication on one strand and an unrelated 26-base pair palindrome on the complementary strand (pRGM 21 x pRGM 29) and have been shown to recognize conformational determinants specific to cruciform DNA structures (Frappier, L., Price, G.B., Martin, R. G., and Zannis-Hadjopoulos, M. (1987) J. Mol. Biol. 193, 751-758). To define the region(s) of the cruciform that is recognized by these antibodies, we examined the ability of 2D3 and 4B4 to protect the single-stranded tips of the loops or the four-way junctions at the base of the stem of stable cruciform molecules against cleavage by mung bean nuclease or T7 endonuclease 3, respectively. Both antibodies were found to protect two of the four elbow-like structures at the base of the cruciform from T7 endonuclease 3 cleavage, but not the tips of the cruciform arms from mung bean nuclease cleavage. Also, predigestion of the cruciform with mung bean nuclease did not affect the binding of either antibody. In addition, 2D3 bound to a cruciform and a T-shaped structure involving the palindromic sequence at the cloning site of pUC7, which is completely unrelated in sequence to the palindrome of pRGM 21 x pRGM 29, and protected the base of these stem-loop structures against cleavage by T4 endonuclease VII. These results indicate that 2D3 and 4B4 bind at or near the base of the cruciform molecules and that, at least for 2D3, binding is independent of DNA sequence.

Antibodies, Monoclonal↗
PubMed1995

In vitro transcripts of a full-length cDNA of a naturally deleted RNA2 of barley mild mosaic virus (BaMMV) replicate in BaMMV-infected plants.

The RNA2 of a German isolate of the bipartite barley mild mosaic bymovirus (BaMMV-ASL1) is 3524 nucleotides long excluding the 3'-terminal poly(A) tail. The isolate was propagated by mechanical inoculation for several years. Electrophoretic comparison of viral nucleic acids during this period revealed that a spontaneous reduction in the length of the RNA2 occurred, resulting in the isolate BaMMV-ASL1a. The deleted RNA2 of BaMMV lacked a fragment that was 630 nucleotides long. The deletion occurred in the 3' half of the single open reading frame (ORF) found in RNA2; this ORF encodes a polyprotein that has a molecular mass of 98 kDa, which is assumed to be processed autocatalytically into proteins of 25 kDa and 73 kDa. A full-length cDNA of the deleted RNA2 was synthesized and cloned under the control of the phage T7 promoter. In vitro transcripts of the BaMMV-ASL1a clone replicated in barley plants after co-inoculation with a wild-type-like isolate of BaMMV. The deletions in RNA2 of BaMMV-ASL1a and those of a number of other isolates that were examined were found to affect a domain of the putative 73 kDa protein that is obviously not essential for replication but may be important for the transmission of BaMMV by its natural vector Polymyxa graminis.

Base Sequence↗
PubMed1974

Suppression of a mutation in gene 3 of bacteriophage T7 (T7 endonuclease I) by mutations in phage and host polynucleotide ligase.

Bacteriophage T7 bearing amber mutations in both gene 1.3 (T7 DNA ligase) and gene 3 (T7 endonuclease I) are viable when grown in suppressor-negative, ligase-negative hosts. This is evidenced by a high plating efficiency and a large burst size compared to the single mutants. These findings may be explained by a limited destruction of cellular DNA by the double mutant.

Carbon Radioisotopes↗
PubMed1992

Genetic analysis of the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin.

Gene 5 protein of bacteriophage T7 is a nonprocessive DNA polymerase. During infection of Escherichia coli, T7 annexes the host protein thioredoxin for use as a processivity factor for T7 DNA polymerase. We describe here a genetic method to investigate the interaction between T7 gene 5 protein and E. coli thioredoxin. The strategy is to use thioredoxin mutants that are unable to support the growth of wild-type T7 phage to select for T7 revertant phage that suppress the defect in thioredoxin. A thioredoxin mutation that replaces glycine at position 74 with aspartic acid fails to support the growth of wild-type T7. This mutation is suppressed by six different mutations within T7 gene 5, each of which results in a single amino acid substitution within gene 5 protein. Three of the suppressor mutations are located within the putative polymerization domain of gene 5 protein, and three are located within the putative 3'-to-5' exonucleolytic domain. Each suppressor mutation alone is necessary and sufficient to confer the revertant phenotype.

DNA Replication↗
PubMed1993

Suicidal nucleotide sequences for DNA polymerization.

Studying the activity of T7 DNA polymerase (Sequenase) on open circular DNAs, we observed virtually complete termination within potential triplex-forming sequences. Mutations destroying the triplex potential of the sequences prevented termination, while compensatory mutations restoring triplex potential restored it. We hypothesize that strand displacement during DNA polymerization of double-helical templates brings three DNA strands (duplex DNA downstream of the polymerase plus a displaced overhang) into close proximity, provoking triplex formation, which in turn prevents further DNA synthesis. Supporting this idea, we found that Sequenase is unable to propagate through short triple-helical stretches within single-stranded DNA templates. Thus, DNA polymerase, by inducing triplex formation at specific sequences in front of the replication fork, causes self-termination. Possible biological implications of such 'conformational suicide' are discussed. Our data also provide a novel way to target DNA polymerases at specific sequences using triplex-forming oligonucleotides.

Base Sequence↗
PubMed1992

Purified MotA protein binds the -30 region of a bacteriophage T4 middle-mode promoter and activates transcription in vitro.

The bacteriophage T4-encoded MotA protein is critical for transcription from T4 middle-mode promoters. However, a direct interaction of this protein with a middle-mode promoter has not previously been demonstrated. We have cloned the motA gene and overexpressed the gene product using the T7 expression system. A simple procedure was then developed to purify the MotA protein to homogeneity. Using the purified protein we have demonstrated that MotA protein binds to the -30 region of the middle-mode promoter PuvsY. This promoter has previously been shown to be a necessary component of a T4 replication origin, and thus MotA is also a T4 origin-binding protein. Modified RNA polymerase purified from T4-infected cells was used to establish middle-mode transcription in vitro. Transcription from PuvsY was markedly enhanced by the addition of MotA protein, whether or not the template contained the cytosine modifications characteristic of T4 DNA. However, transcription from PuvsY was apparently independent of the MotA protein when unmodified RNA polymerase from uninfected cells was used.

Base Sequence↗
PubMed2005

Dual role of the lymphocytic choriomeningitis virus intergenic region in transcription termination and virus propagation.

Each genome segment of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), encodes two genes in ambisense orientation, separated by an intergenic region (IGR). The 3' ends of subgenomic viral mRNAs have been mapped to a stem-loop structure within the IGR, suggesting structure-dependent transcription termination. We have studied the role of the LCMV IGR by using a minigenome (MG) rescue system based on RNA analogues of the short genome segment. An ambisense MG coding for chloramphenicol acetyltransferase (CAT) and green fluorescent protein reporter genes instead of the nucleoprotein and glycoprotein open reading frames, respectively, served as a template for synthesis of full-length anti-MG (aMG) replicate and subgenomic size mRNA for reporter gene expression. An analogous MG without IGR was amplified by the virus polymerase with equal efficiency, but subgenomic mRNA was undetectable. Reporter gene expression from IGR-deficient aMG CAT-sense RNA of genomic length was approximately 5-fold less efficient than that from subgenomic CAT mRNA derived from an IGR-containing MG, but at least 100-fold more efficient than that from a T7 RNA polymerase transcript with the same sequence. Therefore, in the absence of IGR-mediated transcription termination, a fraction of full-length aMG RNA appears to behave as bona fide mRNA. Unexpectedly, MGs without IGR were dramatically impaired in their ability to passage reporter gene activity via infectious virus-like particles. These data suggest that the LCMV IGR serves individual functions in transcription termination for enhanced gene expression and in the virus assembly and/or budding, which are required for the efficient propagation of LCMV infectivity.

Chloramphenicol O-Acetyltransferase↗
PubMed1990

Endoribonucleolytic cleavage of RNA: oligodeoxynucleotide hybrids by the ribonuclease H activity of HIV-1 reverse transcriptase.

Endoribonucleolytic cleavage by the ribonuclease H activity associated with HIV-1 reverse transcriptase was observed in vitro using substrates consisting of synthetic oligodeoxynucleotides hybridized to a 345 nucleotide T7 RNA polymerase transcript derived from the gag region of HIV-1. This observation suggests that a possible mechanism of action of antisense oligonucleotides in the inhibition of viral replication and expression may involve the selective "suicidal" ribonucleolytic cleavage of viral RNA by reverse transcriptase at the site of hybridization of the oligonucleotide.

Base Sequence↗
PubMed2006

A novel processive mechanism for DNA synthesis revealed by structure, modeling and mutagenesis of the accessory subunit of human mitochondrial DNA polymerase.

Mitochondrial DNA polymerase (pol gamma) is the sole DNA polymerase responsible for replication and repair of animal mitochondrial DNA. Here, we address the molecular mechanism by which the human holoenzyme achieves high processivity in nucleotide polymerization. We have determined the crystal structure of human pol gamma-beta, the accessory subunit that binds with high affinity to the catalytic core, pol gamma-alpha, to stimulate its activity and enhance holoenzyme processivity. We find that human pol gamma-beta shares a high level of structural similarity to class IIa aminoacyl tRNA synthetases, and forms a dimer in the crystal. A human pol gamma/DNA complex model was developed using the structures of the pol gamma-beta dimer and the bacteriophage T7 DNA polymerase ternary complex, which suggests multiple regions of subunit interaction between pol gamma-beta and the human catalytic core that allow it to encircle the newly synthesized double-stranded DNA, and thereby enhance DNA binding affinity and holoenzyme processivity. Biochemical properties of a novel set of human pol gamma-beta mutants are explained by and test the model, and elucidate the role of the accessory subunit as a novel type of processivity factor in stimulating pol gamma activity and in enhancing processivity.

Amino Acid Sequence↗
PubMed1979

A novel bacteriophage defence mechanism: the anti-restriction protein.

Bacteriophage T3 and T7 protect their DNA from restriction by producing, as the earliest detectable phage functions, anti-restriction proteins. Although the two phage proteins differ in their chromatographic and antigenic properties, they act by the same mechanism: the anti-restriction proteins inhibit E. coli K12 restriction endonuclease by direct interaction.

Coliphages↗
PubMed1998

Sequence analysis of a functional polymerase (L) gene of bovine respiratory syncytial virus: determination of minimal trans-acting requirements for RNA replication.

The complete nucleotide sequence of a functional clone of the large polymerase (L) gene of bovine respiratory syncytial virus (BRSV) strain A51908 was determined by analysis of cloned cDNAs obtained from genomic and mRNAs. The BRSV L gene is 6573 nt in length and the derived polypeptide has 2162 aa. Alignment of the sequences of the BRSV L gene, and its encoded protein, with sequences of the L gene and protein of human respiratory syncytial virus strain A2 showed 77% identity at the nucleotide level and 84% identity at the amino acid level. By comparison, the L gene and protein of avian pneumovirus showed only 50% identity at the nucleotide level and 64% identity at the amino acid level. A minigenome was constructed to encode a BRSV vRNA analogue containing the gene for chloramphenicol acetyltransferase (CAT) under the control of putative BRSV transcription motifs and flanked by the BRSV genomic termini. Transfection of plasmids encoding the BRSV minigenome, nucleocapsid protein (N), phosphoprotein (P) and L protein, each under the control of T7 promoter, into cells infected with a vaccinia virus recombinant expressing the T7 RNA polymerase gave rise to CAT activity and progeny with the minigenome. This result indicates that the N, P and L proteins are necessary and sufficient for transcription and replication of the BRSV minigenome and are functional. Further, inclusion of small amounts of the M2 protein along with the N, P and L proteins greatly augmented minigenome transcription.

Amino Acid Sequence↗
PubMed2001

Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010.

Unwinding of double-stranded DNA into single-stranded intermediates required for various fundamental life processes is catalyzed by helicases, a family of mono-, di- or hexameric motor proteins fueled by nucleoside triphosphate hydrolysis. The three-dimensional crystal structure of the hexameric helicase RepA encoded by plasmid RSF1010 has been determined by X-ray diffraction at 2.4 A resolution. The hexamer shows an annular structure with 6-fold rotational symmetry and a approximately 17 A wide central hole, suggesting that single-stranded DNA may be threaded during unwinding. Homologs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and the helicase domain of the bacteriophage T7 gp4 protein. In a modeled complex, ATP molecules are located at the subunit interfaces and clearly define adenine-binding and ATPase catalytic sites formed by amino acid residues located on adjacent monomers; most remarkable is the "arginine finger" Arg207 contributing to the active site in the adjacent monomer. This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrolysis and helicase activity of RepA. The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published asymmetric structure of the bacteriophage T7 gp4 helicase domain.

Adenosine Triphosphatases↗
PubMed1975

Determination of the sequences of 18 nucleotides from the 5'-end of the 1-strand and 15 nucleotides from the 5'-end of the r-strand of T7 DNA.

The sequences of 18 nucleotides from the 5'-end of the 1-strand and 15 nucleotides from the 5'-end of the r-strand of T7 bacteriophage DNA have been determined to be pT-C-T-C-A-C-A-G-T-G-T-A-C-G-T-C-C-C (1-strand) and pA-G-G-G-A-C-A-C-A-G-C-G-C-T-C (r-strand). The 5'-termini of whole DNA or separated strands were kinased using polynucleotide kinase and (gamma-32-P) rATP. The DNA was partially digested with pancreatic DNase and the fragments were separated by two dimensional electrophoresis and homochromatography. To complete the sequence, snake venom phosphodiesterase digestions of these fragments were carried out. The relationship of these sequences to the proposed cleavage of concatemeric DNA during DNA replication is discussed.

Adenosine Triphosphate↗