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PubMed1992

In vitro evidence that UV-induced frameshift and substitution mutations at T tracts are the result of misalignment-mediated replication past a specific thymine dimer.

A previous study of UV-induced (254 nm) mutations in the lacI gene of Escherichia coli found that frameshift mutations accounted for about 35% of the observed mutations and that these mutations occurred predominantly at An.Tn sequences [Miller, J.H. (1985) J. Mol. Biol. 182, 48-65]. Because An.Tn sequences are hotspots for cis-syn thymine dimer formation [Brash, D.E., & Haseltine, W. A. (1982) Nature 298, 189-192], it would appear that UV-induced frameshift mutations are the result of an error during replicative bypass of a thymine dimer within such a sequence. To test the validity of such a proposal, replication experiments were carried out on templates containing cis-syn thymine dimers at each of the five possible sites of a T6 tract. The 59-mer templates were prepared by ligating oligonucleotides containing an EcoRI site to the 5'-end of decamers containing the cis-syn thymine dimer and oligonucleotides containing the primer site to the 3'-end. Primer-extension reactions were then carried out on these templates with a 3'----5' exonuclease-deficient (exo-) Klenow fragment of E. coli polymerase I and an exo-T7 polymerase (Sequenase Version 2.0). The replicative bypass products were cleaved with EcoRI to rigorously establish and quantify the presence of frameshift mutations. Both polymerases were able to bypass dimers at all sites, but only the exo-T7 polymerase led to detectable frameshifts, both -1 (approximately 30%) and -2 (approximately 5%), and only with the template containing a cyclobutane dimer at the second site from the 5'-end of the T6 tract. Sequencing of the T7 polymerase-catalyzed bypass products of all templates demonstrated that within the limits of discrimination only As were introduced opposite the dimer-containing T tracts. The only exception was for the template with the dimer at the second site which led to a readily detectable amount of a substitution mutation (approximately 30%) opposite the 5'-thymine of the T6 tract. A mechanism involving a competition between reversible misalignment and realignment steps and irreversible elongation steps is proposed to explain the origin of both the frameshift and the substitution mutations. The implications of this work to the mechanism of UV-induced frameshift and substitution mutations at T tracts in vivo are discussed.

Autoradiography↗
PubMed1992

Biologically active cymbidium ringspot virus satellite RNA in transgenic plants suppresses accumulation of DI RNA.

A full-length DNA copy of cymbidium ringspot virus (CyRSV) satellite RNA was cloned downstream of the bacteriophage T7 RNA polymerase promoter. In vitro transcripts were biologically active in plants when coinoculated with the helper virus or its RNA. Although the transcripts contained 7 or 29 extra nucleotides at the 3' end, the proper 3' terminus was restored in the satRNA progeny. Full-length cDNA clones of CyRSV satRNA under the control of the cauliflower mosaic virus 35S promoter and terminator were used to transform Nicotiana benthamiana plants. Integration of CyRSV satRNA sequence in the plant genome was tested by PCR amplification of DNA extracts from transformed plants and by detection of satRNA-related transcripts in total RNA extracts. Inoculation of transgenic plants with the helper virus induced replication of satRNA of the same size as the native molecule. Sequence analysis of the satRNA progeny showed that it was identical to natural CyRSV satRNA. Infected transgenic plants were not protected from apical necrosis and death by the presence of satRNA sequences. Rather, replication of satRNA was found to suppress accumulation of defective interfering RNA, which acts in the absence of satRNA as an attenuator of virus replication and disease.

Base Sequence↗
PubMed1989

Infectious TYMV RNA from cloned cDNA: effects in vitro and in vivo of point substitutions in the initiation codons of two extensively overlapping ORFs.

Full-length cDNA of the 6.3 kb turnip yellow mosaic virus (TYMV) genome was placed between a T7 promoter and a unique Hind III site. In vitro transcription of Hind III-linearized DNA of clone pTYMC yielded full-length RNA transcripts. In inoculations of Chinese cabbage protoplasts and plants, capped transcripts and virion RNA had similar specific infectivities and produced similar systemic symptoms. We have used the pTYMC clone in studies of the expression of two overlapping open reading frames (1.9 kb and 5.5 kb ORFs) by making mutants with alterations in the initiation codons. Evidence is presented from in vitro translations of mutant and wild type RNAs that both ORFs are expressed from TYMV RNA. A mutant in the initiation codon of the 5.5 kb ORF did not replicate in protoplasts, while mutants in the initiation codon of the 1.9 kb ORF replicated at low levels. The two groups of mutants were not able to complement each other.

Brassica↗
PubMed1990

Cloning, nucleotide sequence, and expression of the Rhodobacter sphaeroides Y thioredoxin gene.

Synthetic oligodeoxynucleotide probes based on the known amino acid sequence of Rhodobacter sphaeroides Y thioredoxin were used to identify, clone, and sequence the structural gene. The amino acid sequence derived from the DNA sequence of the R. sphaeroides gene was identical to the known amino acid sequence of R. sphaeroides thioredoxin. An NcoI site was created by directed mutagenesis at the beginning of the thioredoxin gene, inducing in the encoded protein the replacement of serine in position 2 by alanine. The 421-base-pair NcoI-PstI restriction fragment obtained was ligated in the pKK233-2 expression vector and the resulting hybrid plasmid was used to transform Escherichia coli strains lacking functional thioredoxin. Transformants that complemented mutations in the trxA gene were identified by increased colony size on rich medium, growth on minimal medium with methionine sulfoxide, and ability to support M13 growth and T7 replication; this latter phenotype implies correct interaction between R. sphaeroides thioredoxin and the product of T7 gene 5. The presence of R. sphaeroides thioredoxin was further confirmed by enzyme assay.

Amino Acid Sequence↗
PubMed2004

Conformational model of the Holliday junction transition deduced from molecular dynamics simulations.

Homologous recombination plays a key role in the restart of stalled replication forks and in the generation of genetic diversity. During this process, two homologous DNA molecules undergo strand exchange to form a four-way DNA (Holliday) junction. In the presence of metal ions, the Holliday junction folds into the stacked-X structure that has two alternative conformers. Experiments have revealed the spontaneous transitions between these conformers, but their detailed pathways are not known. Here, we report a series of molecular dynamics simulations of the Holliday junction at physiological and elevated (400 K) temperatures. The simulations reveal new tetrahedral intermediates and suggest a schematic framework for conformer transitions. The tetrahedral intermediates bear resemblance to the junction conformation in complex with a junction-resolving enzyme, T7 endonuclease I, and indeed, one intermediate forms a stable complex with the enzyme as demonstrated in one simulation. We also describe free energy minima for various states of the Holliday junction system, which arise during conformer transitions. The results show that magnesium ions stabilize the stacked-X form and destabilize the open and tetrahedral intermediates. Overall, our study provides a detailed dynamic model of the Holliday junction undergoing a conformer transition.

Computational Biology↗
PubMed2004

Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29.

The DNA polymerase from phage phi29 is a B family polymerase that initiates replication using a protein as a primer, attaching the first nucleotide of the phage genome to the hydroxyl of a specific serine of the priming protein. The crystal structure of phi29 DNA polymerase determined at 2.2 A resolution provides explanations for its extraordinary processivity and strand displacement activities. Homology modeling suggests that downstream template DNA passes through a tunnel prior to entering the polymerase active site. This tunnel is too small to accommodate double-stranded DNA and requires the separation of template and nontemplate strands. Members of the B family of DNA polymerases that use protein primers contain two sequence insertions: one forms a domain not previously observed in polymerases, while the second resembles the specificity loop of T7 RNA polymerase. The high processivity of phi29 DNA polymerase may be explained by its topological encirclement of both the downstream template and the upstream duplex DNA.

Amino Acid Sequence↗
PubMed1995

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Animals↗
PubMed2004

A vaccinia replication system for producing recombinant hepatitis C virus.

AIM: To develop a cell culture system capable of producing high titer hepatitis C virus (HCV) stocks with recombinant vaccinia viruses as helpers. METHODS: Two plasmids were used for the generation of recombinant HCV: one containing the full-length HCV cDNA cloned between T7 promoter and T7 terminator of pOCUS-T7 vector, and the other containing the HCV polyprotein open reading frame (ORF) directly linked to a vaccinia late promoter in PSC59. These two plasmids were co-transfected into BHK21 cells, which were then infected with vTF7-3 recombinant vaccinia helper viruses. RESULTS: After 5 d of incubation, approximately 3.6X10(7) copies of HCV RNA were present per milliliter of cell culture supernatant, as detected by fluorescence quantitative RT-PCR (FQ-PCR). The yield of recombinant HCV using this cell system increased 100- to 1 000- fold compared to in vitro- transcribed HCV genomic RNA or selective subgenomic HCV RNA molecule method. CONCLUSION: This cell culture system is capable of producing high titer recombinant HCV.

Animals↗
PubMed1980

[Protection of foreign DNA against host-controlled restriction in bacterial cells. II. Protection of pSF2124 plasmid by the gene function of bacteriophages T3 and T7].

When restriction-active Escherichia coli cells (R+P1m+P1) are transformed with the pSF2124 plasmid, a common vector in experimental gene transfer, the efficiency of transformation (e.o.t.) is lowered by 2 orders of magnitude compared with restriction-negative (r-P1m-P1 or r-P1m+P1) recipient cells due to restriction of the pSF2124 DNA by endoR.EcoP1. Preinfection of r+P1m+P1 cells with UV-inactivated ocr+ phages (T3, T7, T3sam-) still able to express their early genes protects the plasmid DNA against restriction by endoR.EcoP1: The e.o.t. of r+P1m+P1 recipient cells with pSF2124 attains the same high value as that of r-m- cells. The specific role of the ocr+ gene function was demonstrated by the use of ocr- mutants (T3/R7, T7/D111): Preinfection with such phage mutants does not increase the e.o.t. of r+P1m+P1 cells. An unspecific e.o.t. alteration of restriction-negative (r-m-) recipient cells by ocr+ or ocr- phages was excluded. The ocr+ gene function can be exploited to protect pSF2124 against DNA restriction. The recipient cells survive the process of phage preinfection and transformation and stably replicate themselves as well as the plasmid DNA.

DNA Restriction Enzymes↗
PubMed1989

The essential 65-kilodalton DNA-binding protein of herpes simplex virus stimulates the virus-encoded DNA polymerase.

The 65-kilodalton DNA-binding protein (65KDBP) of herpes simplex virus type 1 (HSV-1), the product of the UL42 gene, is required for DNA replication both in vitro and in vivo, yet its actual function is unknown. By two independent methods, it was shown that the 65KDBP stimulates the activity of the HSV-1-encoded DNA polymerase (Pol). When Pol, purified from HSV-1-infected cells, was separated from the 65KDBP, much of its activity was lost. However, addition of the 65KDBP, purified from infected cells, stimulated the activity of Pol 4- to 10-fold. The ability of a monoclonal antibody to the 65KDBP to remove the Pol-stimulating activity from preparations of the 65KDBP confirmed that the activity was not due to a trace contaminant. Furthermore, the 65KDBP did not stimulate the activity of other DNA polymerases derived from T4, T7, or Escherichia coli. The 65KDBP gene transcribed in vitro from cloned DNA and translated in vitro in rabbit reticulocyte lysates also was capable of stimulating the product of the pol gene when the RNAs were cotranslated. The product of a mutant 65KDBP gene missing the carboxy-terminal 28 amino acids exhibited wild-type levels of Pol stimulation, while the products of two large deletion mutants of the gene could not stimulate Pol activity. These experiments suggest that the 65KDBP may be an accessory protein for the HSV-1 Pol.

Animals↗
PubMed2006

A novel, topologically constrained DNA molecule containing a double Holliday junction: design, synthesis, and initial biochemical characterization.

The double Holliday junction (dHJ) is a central intermediate to homologous recombination, but biochemical analysis of the metabolism of this structure has been hindered by the lack of a substrate that adequately replicates the endogenous structure. We have synthesized a novel dHJ substrate that consists of two small, double stranded DNA circles conjoined by two Holliday junctions (HJs). Its biochemical synthesis is based on the production of two pairs of single stranded circles from phagemids, followed by their sequential annealing with reverse gyrase. The sequence between the two HJs is identical on both strands, allowing the HJs to migrate without the generation of unpaired regions of DNA, whereas the distance between the HJs is on the order of gene conversion tracts thus far measured in Drosophila and mouse model systems. The structure of this substrate also provides similar topological constraint as would occur in an endogenous dHJ. Digestion of the dHJ substrate by T7 endonuclease I resolves the substrate into crossover and non-crossover products, as predicted by the Szostak model of double strand break repair. This substrate will greatly facilitate the examination of the mechanism of resolution of double Holliday junctions.

Animals↗
PubMed2000

Functional hydrogen-bonding map of the minor groove binding tracks of six DNA polymerases.

Recent studies have identified amino acid side chains forming several hydrogen bonds in the DNA minor groove as potentially important in polymerase replication of DNA. Few studies have probed these interactions on the DNA itself. Using non-hydrogen-bonding nucleoside isosteres, we have now studied effects in both primer and template strands with several polymerases to investigate the general importance of these interactions. All six polymerases show differences in the H-bonding effects in the minor groove. Two broad classes of activity are seen, with a first group of DNA polymerases (KF(-), Taq, and HIV-RT) that efficiently extends nonpolar base pairs containing nucleoside Q (9-methyl-1H-imidazo[4,5-b]pyridine) but not the analogue Z (4-methylbenzimidazole), implicating a specific minor groove interaction at the first extension site. A second group of polymerases (Pol alpha, Pol beta, and T7(-)) fails to extend all non-H-bonding base pairs, indicating that these enzymes may need minor groove hydrogen bonds at both minor groove sites or that they are especially sensitive to noncanonical DNA structure or stability. All DNA polymerases examined use energetically important minor groove interactions to probe newly synthesized base pairs before extending them. The positions of these interactions vary among the enzymes, and only a subset of the interactions identified structurally appears to be functionally important. In addition, polymerases appear to be differently sensitive to small changes in base pair geometry.

Animals↗
PubMed2001

Vaccinia virus-free recovery of vesicular stomatitis virus.

The advent of reverse-genetics represents a powerful new approach to elucidate aspects of negative-sense RNA virus replication. The reverse-genetics system established previously for vesicular stomatitis virus (VSV) required four plasmids encoding the nucleoprotein (N), phosphoprotein (P), polymerase (L), and the full-length, anti-genomic RNA. Transcription to yield the antigenomic RNA as well as the N, P, and L, mRNAs was initiated by bacteriophage T7 polymerase expressed from a recombinant Vaccinia virus. In this report, we describe the successful recovery of infectious VSV in the absence of Vaccinia virus. The N, P, and L genes of VSV were inserted downstream of both the T7 promoter and an internal ribosomal entry site (IRES element). T7 polymerase was expressed constitutively from BSR-T7/5 cells. RTPCR was used to confirm that the recovered VSV was derived from transfected DNA. Virion protein profile, CPE in tissue culture, and virus titer of the recombinant VSV were indistinguishable from those of parental VSV. Thus, the need for Vaccinia virus is eliminated with this system, making it an attractive, alternative approach for the recovery of infectious VSV from DNA.

Animals↗
PubMed1996

Mutations within the 5' nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells.

Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell culture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in cultured African green monkey kidney (BS-C-1) cells but not in fetal rhesus monkey kidney (FRhK-4) cells (S. P. Day, P. Murphy, E. A. Brown, and S. M. Lemon, J. Virol. 66: 6533-6540, 1992). To determine whether these mutations enhance cap-independent translation directed by the HAV internal ribosomal entry site (IRES), we compared the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses in two stably transformed cell lines (BT7-H and FRhK-T7) which constitutively express cytoplasmic bacteriophage T7 RNA polymerase and which are derived from BS-C-1 and FRhK-4 cells, respectively. Translational activity was assessed by monitoring expression of a reporter protein, chloramphenicol acetyltransferase (CAT), following transfection with plasmid DNAs containing bicistronic T7 transcriptional units of the form luciferase-5'NTR-CAT. In both cell types, transcripts containing the 5'NTR of HM175/P16 expressed CAT at levels that were 50- to 100-fold lower than transcripts containing the IRES elements of Sabin type 1 poliovirus or encephalomyocarditis virus, confirming the low activity of the HAV IRES. However, in BT7-H cells, transcripts containing the 5'NTR of wild-type virus. This translational enhancement was due to additive effects of a UU deletion at nucleotides 203 and 204 and a U-to-G substitution at nucleotide 687 of HM175/P16. These mutations did not enhance translation in FRhK-T7 or Huh-T7 cells (a T7 polymerase-expressing cell line derived from human hepatoblastoma cells) or in vitro in rabbit reticulocyte lysates. These results demonstrate that mutations in the 5'NTR of a cell culture-adapted HAV enhance viral replication by facilitating cap-independent translation in a cell-type-specific fashion and support the concept that picornaviral host range is determined in part by differences in cellular translation initiation factors.

Animals↗
PubMed1988

Site-specific binding of viral plus single-stranded RNA to replicase-containing open virus-like particles of yeast.

X double-stranded RNA is a deletion mutant of L-A double-stranded RNA and is encapsidated in viral particles by the L-A-encoded major coat protein. X double-stranded RNA has all the cis sites necessary to be transcribed, encapsidated, and replicated. We have cloned X double-stranded RNA and sequenced it. The complete X double-stranded RNA sequence deduced indicates that the first 25 bases of the X plus-strand 5' end originated from the 5' end of the L-A plus strand and that most, if not all, of the rest comes from the 3' end of the L-A plus strand. The X plus strand made by X double-stranded RNA-containing virus-like particles binds specifically to empty open virus-like particles and is converted by these particles to X double-stranded RNA. RNA transcripts of the X complementary DNA clones and deletion derivatives thereof were made in vitro by T7 and T3 RNA polymerases and tested for specific binding to the virus-like particles. The results suggest that the binding is due to the sequence UUUGGCCAGG, 370 bases upstream from the X plus-strand 3' end. This sequence is also present in the M1 plus strand 140 bases from its 3' end.

Amino Acid Sequence↗
PubMed1990

Studies of coronavirus DI RNA replication using in vitro constructed DI cDNA clones.

Sequence analysis of an intracellular defective-interfering (DI) RNA, DIssE, of mouse hepatitis virus (MHV) revealed that it is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the 5'-end, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the 3'-end of the parental MHV genome. DIssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. A system was developed for generating DI RNAs to study the mechanism of MHV RNA replication. A cDNA copy of DIssE RNA was placed downstream of T7 RNA polymerase promoter to generate DI RNAs capable of extremely efficient replication in the presence of a helper virus. We demonstrated that, in the DI RNA-transfected cells, the leader sequence of these DI RNAs was switched to that of the helper virus during one round of replication. This high-frequency leader sequence exchange was not observed if a nine-nucleotide stretch at the junction between the leader and the remaining DI sequence was deleted. This observation suggests that a free leader RNA is utilized for the replication of MHV RNA.

Animals↗
PubMed1992

Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural RNA.

The in vitro fidelity of reverse transcriptase from human immunodeficiency virus type I (HIV-1 RT) upon copying an RNA template was measured using the phi Xam 16 reversion assay. A phi X174 sequence harboring the amber 16 codon was cloned into a transcription vector. RNA obtained from transcription by bacteriophage T7 RNA polymerase was used as a template for RNA-directed DNA synthesis by HIV-1 RT. An imbalance of dNTP concentrations during the reverse transcription step served to distinguish between errors that arose from the transcription step and errors from reverse transcription. The frequency of dGTP.U mismatches was determined to be 1/360, while dGTP.rA mismatches formed at a rate of 1/4600. These are 20-fold and sevenfold higher, respectively, than the error rates determined for the same sequence with a DNA template. Due to a high background of errors in the RNA template originating from the transcription step only upper limits for the frequency of three other mismatches can be given. The data indicate that the reverse transcription step of the HIV-1 replication cycle contributes significantly to the generation of mutant viruses.

Base Sequence↗
PubMed1996

Intracellular membrane proliferation in E. coli induced by foot-and-mouth disease virus 3A gene products.

During picornavirus infection replication of genomic RNA occurs in membrane-associated ribonucleoprotein complexes. These replication complexes contain different nonstructural viral proteins with mostly unknown function. To examine the function of nonstructural picornaviral proteins in more detail, cDNA of foot-and-mouth-disease virus (FMDV) strain O1 Lausanne was cloned into lambda ZAP II, and different parts of the P3-coding sequence were expressed in E. coli by the T7 polymerase system. Expression products constituted (a) fusion proteins composed of N-terminal leader peptide of bacteriophage T7 phi 10 protein fused to FMDV P3-sequences of different lengths, (b) translation products of authentic P3-region genes, and (c) carboxy-terminally truncated 3A proteins. Expression products were characterized by NaDodSO4-polyacrylamide gel electrophoresis, immunoblotting, as well as electron and immunoelectron microscopy. We show here that in the T7 polymerase system a high level of expression of 3A-containing peptides is achieved in E. coli. Remarkably, the expression of 3A-derived proteins induced a dramatic intracellular membrane proliferation in E. coli cells, similar to the vesicle induction observed in FMDV-infected cells. By immunoelectron microscopy, 3A-reactive material was found associated with these membranes. We hypothesize that the FMDV 3A protein is instrumental in eliciting intracellular membrane proliferation in infected cells as a prerequisite for viral RNA replication.

Aphthovirus↗