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N D Stow

Publications and source records attributed to N D Stow.

At least 55 records · Page 3Linked to original sources
PubMed1986

Identification of a varicella-zoster virus origin of DNA replication and its activation by herpes simplex virus type 1 gene products.

We have identified and characterized an origin of DNA replication in the genome of the human herpesvirus, varicella-zoster virus (VZV). This origin of replication (VZV ORIS) is located within the major inverted repeats in a position equivalent to that occupied by one of the herpes simplex virus type 1 (HSV-1) replication origins. Products encoded by both VZV and HSV-1 activate cloned copies of VZV ORIs, generating high molecular weight molecules consisting of tandem duplications of the input plasmid. The VZV ORIS region contains a tract of alternating A and T residues located at the centre of symmetry of an almost perfect palindrome of 45 bp, and the use of plasmid deletion mutants has demonstrated that this tract is an important functional element of the origin. Two sequences common to the VZV ORIS region and the regions specifying the two HSV-1 origins (ORIS, located within the TRS/IRS regions, and ORIL, located within the UL region) were identified and these may represent important recognition sites. One is an 11 bp sequence (CGTTCGCACTT), and the other is represented by the tract of alternating A and T residues. VZV does not appear to contain an origin of replication in a position equivalent to that of HSV-1 ORIL.

Animals↗
PubMed1985

Expression of an immediate early polypeptide and activation of a viral origin of DNA replication in cells containing a fragment of herpes simplex virus DNA.

A thymidine kinase cotransformation procedure has been used to introduce the sequences encoding the herpes simplex virus type 1 (HSV-1) immediate early protein, Vmw175, into permissive cells either in the presence or the absence of the adjacent origin of viral DNA replication. Cells transformed by either origin-plus or origin-minus DNA were capable of expressing functional Vmw175 as indicated by their ability to complement the growth at the nonpermissive temperature of an HSV-1 mutant, ts K, containing a temperature-sensitive lesion in the Vmw175 gene. A proportion of the virus yield from cells transformed with the origin-plus, but not the origin-minus, plasmid exhibited a ts+ phenotype. The generation of ts+ virus correlated with an amplification of input plasmid DNA sequences which occurred following superinfection, suggesting that recombination between the ts mutant and the amplified viral DNA sequences had taken place. Encapsidation of the amplified DNA sequences was also detected, suggesting that in addition to a functional origin of replication and Vmw175 gene the transformed cells also retain the viral DNA packaging signals.

Capsid↗
PubMed1985

Mutagenesis of a herpes simplex virus origin of DNA replication and its effect on viral interference.

Supercoiled plasmid molecules containing cloned copies of a DNA fragment which includes a functional herpes simplex virus type 1 origin of DNA replication were cleaved preferentially at two positions within the viral insert by nuclease S1. Plasmids with molecular linker insertions at these sites were constructed, and analysis of two representative plasmids demonstrated the presence of palindromic DNA sequences at the preferred cleavage positions. One of these palindromic sequences occurred within a 90 bp region in which the cis-acting sequences essential for viral origin function had previously been located. Insertion of a linker at this position abolished origin activity, demonstrating an essential role for sequences within the palindrome in the initiation of DNA synthesis. In transfection assays, plasmids containing a functional viral origin of DNA replication markedly interfered with the infectivity of non-defective viral DNA even in the absence of viral encapsidation signals. Inactivation of the origin greatly reduced this effect on DNA infectivity, suggesting that viral interference may be mediated by a mechanism involving the replicative machinery.

Base Sequence↗
PubMed1984

Replication of adenovirus mini-chromosomes.

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear mini-chromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.

Adenoviridae↗
PubMed1984

Analysis of DNA sequences which regulate the transcription of a herpes simplex virus immediate early gene.

The locations and functions of DNA sequences involved in transcription of the gene encoding herpes simplex virus type 1 immediate early (IE) mRNAs 4 and 5 were analyzed by use of a transient-expression assay. The region upstream of the genes encoding IE mRNAs 4 and 5 was fused to the thymidine kinase gene coding sequences, and production of enzyme or RNA was measured after transfection of plasmids into BHK cells. The effect of deletions in the upstream region was determined in the absence or presence of a virus structural component which stimulates herpes simplex virus IE transcription. Two distinct units were identified. One of these was a promoter which required not more than 69 base pairs of DNA specific for the genes encoding IE mRNAs 4 and 5 upstream from the mRNA 5' terminus. The other was a far-upstream region which mediated the response to the virion component and had an upstream boundary between nucleotides -347 and -335. An origin of DNA replication was interposed between these two units. The element TAATGAGATAC , which represents a consensus sequence present in the upstream regions of all herpes simplex type 1 IE genes, appeared to be essential for stimulation by the virion component. The activity of this element was modulated by the sequences which flank it, especially by regions having extremely high contents of guanine plus cytosine and which contain a conserved unit CCCGCCC or its complement GGGCGGG .

Animals↗
PubMed1983

Fragments from both termini of the herpes simplex virus type 1 genome contain signals required for the encapsidation of viral DNA.

A 535 base pair DNA fragment which maps entirely within the IRS/TRS regions of the herpes simplex virus type 1 (HSV-1) genome and contains all the cis-acting signals necessary for it to function as an origin of viral DNA replication has previously been identified (N.D. Stow and E.C. McMonagle, Virology, in press). When BHK cells were transfected with circular plasmid molecules containing cloned copies of this DNA fragment, and superinfected with wt HSV-1 as helper, amplification of the input plasmid was detected. Two observations indicated that the amplified DNA was not packaged into virus particles. Firstly, when the transfected cells were disrupted the amplified DNA was susceptible to digestion by added DNase, and secondly, it was not possible to further propagate the DNA when virus from the cells was passaged. Fragments from the joint region and from both termini of the viral genome were inserted into origin-containing plasmids and the resulting constructs analysed. In all cases the inserted fragment allowed the amplified DNA to be further passaged, and a proportion to become resistant to digestion with DNase. These observations suggest that signals required for the encapsidation of HSV-1 DNA are located within DNA sequences shared by the inserted fragments and therefore lie within the reiterated 'a' sequence of the viral genome.

Animals↗
PubMed1983

Characterization of the TRS/IRS origin of DNA replication of herpes simplex virus type 1.

In previous experiments, an origin of viral DNA replication was localized within a 995-bp DNA fragment that mapped entirely within the TRS/IRS repeated region of the herpes simplex virus type 1 genome (N. D. Stow, EMBO J. 1, 863-867, 1982). In this paper, this origin is now shown to reside within a 535-bp segment subcloned from the above fragment. Deletions extending various distances into each end of the 535-bp segment were generated using nuclease BAL 31, and the resulting plasmids screened for their ability to replicate in cells superinfected with wild-type HSV-1 helper virus. This analysis indicated that the cis-acting sequences essential for DNA replication were present within a 90-bp region, and a 100-bp viral DNA fragment containing all the signals necessary for origin function was identified. The origin lies within an untranscribed region located between the 5'-ends of two divergently transcribed immediate-early mRNAs. A prominant feature of the origin region is an almost perfect palindromic sequence 45 bp long containing 18 consecutive A or T residues at its center.

Base Sequence↗
PubMed1982

The infectivity of adenovirus genomes lacking DNA sequences from their left-hand termini.

Deletions extending various distances into the left-hand terminal DNA sequences of the adenovirus type 2 (Ad2) genome were generated in a plasmid containing a cloned fragment spanning from 0 to 4.9 map units. The altered Ad2 DNA sequences were introduced into viral genomes by ligating a plasmid-derived fragment, which included the sequences extending to 3.8 map units, to the 3.8-100 map unit fragment generated by XbaI cleavage of the DNA of the Ad5 variant, d1309 (N. Jones and T.Shenk, Cell 17 683-689, 1979). The infectivity of the ligation products was studied by transfection of line 293 cells. Genomes lacking 11, 40, or 51 nucleotides from their left-hand termini, or containing an additional 18dG residues linked to this position were infectious, and analysis of the progeny virus genomes demonstrated that the structure of these modified termini had been restored to normal. In contrast, genomes from which the first 160 base pairs (bp), including the entire 102 bp left hand inverted terminal repeat (ITR), had been removed were non-infectious. The results indicate that the ITRs present at the opposite ends of transfecting DNA molecules are able to interact in vivo, and enable the production of viable viruses containing corrected left-hand terminal sequences. Possible mechanisms for this interaction are discussed.

Adenoviruses, Human↗
PubMed1982

Localization of an origin of DNA replication within the TRS/IRS repeated region of the herpes simplex virus type 1 genome.

An assay has been developed and used to locate an origin of DNA replication on the herpes simplex virus type 1 (HSV-1) genome. Baby hamster kidney cells were transfected with circular plasmid molecules containing cloned copies of HSV-1 DNA fragments, and helper functions were provided by superinfection with wild-type HSV-1. The presence of an HSV-1 origin of replication within a plasmid enabled amplification of the vector DNA sequences, which was detected by the incorporation of [32P]orthophosphate. By screening various HSV-1 DNA fragments it was possible to identify a 995-bp fragment that maps entirely within the reiterated sequences flanking the short unique region of the viral genome and contains all the cis-acting signals necessary to function as an origin of viral DNA replication. The products of plasmid replication were shown to be high mol. wt. DNA molecules consisting of tandem duplications of the complete plasmid, suggesting that replication was occurring by a rolling-circle mechanism.

Animals↗
PubMed1982

The nucleotide sequence of mRNA for the Mr 19 000 glycoprotein from early gene block III of adenovirus 2.

The cytoplasmic poly(A)RNA from early stages of infection of HeLa cells by adenovirus 2 (Ad2), was used to synthesize cDNA. The resulting cDNA segments were inserted at the PstI site of the plasmid pBR322 after dG/dC tailing. The clones containing sequences corresponding to early region III transcripts were identified by hybridization against the SmaI-C fragment (76.5 to 91.9 map units) of the Ad2 genome. The clone pE22 which contained a copy of the mRNA coding for the Mr 19 000 glycoprotein was characterized in detail. A comparison with the genomic sequences allowed the identification of the splice between the x and y leaders. The nucleotide sequences CCGGTG and CAGTTT were found at the donor and acceptor sites of the splice junction, respectively. The coding region consisted of a continuous stretch of 159 amino acids with a hydrophobic N-terminus and two possible glycosylation sites. The triplet ATG was encountered twice in phase 3 before the actual site of initiation of translation, which was in phase 2. The 3'-untranslated region was 500 nucleotides long and contained an open, translational reading frame for a Mr 11 000 protein, following a potential initiator ATG. The sequence ATTAAA was observed 17 bp before the poly(A) tail.

Adenoviruses, Human↗
PubMed1981

Cloning of a DNA fragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directed mutagenesis.

The HpaI E fragment (0-4.5 map units) of adenovirus type 2 (Ad2) DNA was cloned in the plasmid vector pBR322. Excision of the viral insert with PstI and XbaI generated a fragment which comigrated with Ad2 XbaI-E (0-3.8 map units), and this fragment was ligated to the 3.8-100 fragment generated by XbaI cleavage of the DNA of the Ad5 mutant, dl309 (N. Jones and T. Shenk, Cell 17:683-689, 1979). Transfection with the ligation products resulted in the production of progeny virus which was able to replicate on both HeLa and line 293 cells, demonstrating the biological activity of the sequences rescued from the plasmid. Small deletions were introduced around the SmaI site (map position 2.8) within the cloned viral insert, and the altered DNA sequences were reintroduced into progeny virus as described above. The mutant viruses grew well on line 293 cells but plaqued with greatly reduced efficiency on HeLa cells, exhibiting a host range phenotype similar to previously described mutants with lesions located within this region of the genome. When plasmid-derived left-end fragments containing pBR322 DNA sequences to the left of map position 0 were ligated to the 3.8-100 fragment of dl309 DNA, the infectivity of the ligation products was not reduced. However, all progeny viruses examined yielded normal-size restriction enzyme fragments from their left-hand ends, indicating that the bulk of the pBR322 DNA sequences are removed either prior to or as a consequence of the replication of the transfecting DNA molecules.

Adenoviruses, Human↗
PubMed1979

Physical mapping of paar mutations of herpes simplex virus type 1 and type 2 by intertypic marker rescue.

Mutations (paar) in herpes simplex virus (HSV) which confer resistance to phosphonoacetic acid involve genes associated with virus-induced DNA polymerase activity. Two mutants of HSV (HSV-1 tsH and HSV-2 ts6) produce a thermolabile DNA polymerase activity. In this study, the ts lesions present in these mutants and those present in two independent phosphonoacetic acid-resistant mutants of HSV-1 and HSV-2 (paar-1 and paar-2) have been physically mapped by restriction endonuclease analysis of recombinants produced between HSV-1 and HSV-2 by intertypic marker rescue. All four mutations mapped within a 3.3-kilobase pair region around map unit 40. The accuracy of the method is reflected by the mapping results for tsH and paar-2, which were found to lie in the same 1.3-kilobase pair region. paar-1 was found to lie to the right of ts6. Virus-induced DNA polymerase is thought to have a molecular weight of 150,000, necessitating a gene with a coding capacity of 4.6 kilobase pairs. The four mutations mapped in this study all lie within a region smaller than this, but the results do not yet prove that all four lesions reside in this or any single gene.

Chromosome Mapping↗
PubMed1978

Physical mapping of herpes simplex virus type 1 mutations by marker rescue.

A generally applicable technique which permits the rescue of selected genetic markers from fragments of herpes simplex virus DNA is described. Baby hamster kidney cells infected at the nonpermissive temperature with intact DNA from temperature-sensitive mutants or with fragmented wild-type DNA produce no, or little, infectious progeny. Coinfection results in an increased yield of virus, demonstrating the rescue of genetic information from the DNA fragments. This progeny virus consists of both wild-type and temperature-sensitive virus, demonstrating that both recombination and complementation can occur in coinfected cells. Rescue experiments using isolated fragments produced with various restriction endonucleases have enabled us to locate five temperature-sensitive mutations on the herpes simplex virus type 1 physical map. An adaptation of the technique has allowed the physical mapping of a mutation which affects the herpes simplex virus type 1 pyrimidine deoxyribonucleoside kinase gene. Comparison of the genetic and physical maps for these mutants reveals several anomalies which are discussed.

Cell Line↗
PubMed1978

Physical mapping of herpes simplex virus-induced polypeptides.

Analysis of the polypeptides induced by 29 herpes simplex virus type 1/type 2 intertypic recombinants and correlation of the data with the crossover points in the recombinant DNAs have enabled the map positions of many polypeptides to be deduced. These include 25 polypeptides which label with [35S]methionine, 11 which label with [32P]orthophosphate, and 4 which label with [14C]glucosamine. Together with the data of Preston et al. (J. Virol., in press) on the mapping of five immediate-early polypeptides, the results show that representatives of four groups of proteins--immediate-early, late, phosphorylated, and glycosylated--map in both long and short regions. The functional organization of the herpes simplex virus genome does not therefore restrict any of these four groups to either the long or the short region.

Cell Line↗
PubMed1978

Physical mapping of herpes simplex virus-coded functions and polypeptides by marker rescue and analysis of HSV-1/HSV-2 intertypic recombinants.

A number of temperature-sensitive (ts) mutants and one pyrimidine deoxyribonucleoside kinase-deficient mutant of herpes simplex virus (HSV), have been located on the physical map of the genome by means of marker rescue experiments and by the analysis of the crossover points in intertypic recombinants between HSV types 1 and 2. The physical map is compared to the genetic map and certain anomalies identified. Analysis of infected-cell polypeptides specified by intertypic recombinants has allowed tentative map co-ordinates to be assigned to the structural genes (or genes which cause post-translational modification) for many of the polypeptides. Immediate-early, phosphorylated, glycosylated and structural as well as non-structural polypeptides have been analysed in this way and it can be concluded that there is no restriction of any of these groups of polypeptides to either the long or the short regions of the genome. One of the recombinants, 2853, is at least partially "frozen" in one orientation of the long region. This orientation is also the one which exhibits a minimum number of crossovers in three other recombinants.

Chromosome Mapping↗
PubMed1976

Phage and defective phage of strains of Myxococcus.

1. Phage-like particles were found in the supernatants of cultures of strains of Myxococcus xanthus, M. virescens and M. fulvus. The largest number of such particles was associated with M. virescens V2. Most of the particles were similar in morphology to the virulent Myxococcus phage, MX-1. 2. Several new phages were isolated from soil and animal droppings. A new phage was isolated from cultures of M. virescens V2. All resembled phage MX-1 in morphology and were related to phage MX-1 serologically. One of these phage, om, was characterized by fractionation of its proteins by SDS-polyacrylamide gel electrophoresis and by analysis of restriction fragments of its DNA. The very close relatedness with MX-1 was confirmed by these techniques. Phage om, was found to exist in a state of pseudolysogeny with strains of M. virescens and M. fulvus. 3. Two types of bacteriocin-like activity were found associated with Myxococcus strains. In one case, the activity was extracted from chloroformkilled or from sonicated cells. In the second case it was associated with extracellular material. Strains of Salmonella and Cytophaga were found to be good indicators for this latter activity. These strains were found to be killed by phage MX-1. 4. The significance of these data for origin of the phages of myxococci are discussed and it is proposed that MX-1 and the newly isolated phages may be virulent mutants of a family of lysogenic phages.

Bacteriocins↗