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Biomedical subjects

D A Galloway

Publications and source records attributed to D A Galloway.

At least 109 records · Page 6Linked to original sources
PubMed1987

Characterization of the integration site of the CMV mtr in a tumor cell line.

Previous studies have shown that a 558-bp fragment of human cytomegalovirus (CMV) DNA contained within pCM4127 and designated CMV mtr can morphologically transform rodents cells in vitro. By cotransformation with pCM4127 and a plasmid conferring G418 resistance, pSV2neo, morphologically transformed NIH3T3 cell lines were isolated. Dot blot hybridization indicated that approximately 30% of the transformants retained CMV sequences. Two cell lines which retained viral DNA were chosen for further study. They were capable of anchorage independent growth and formed tumors in nude mice. Integrated viral sequences in the transformants and tumor cell lines were analyzed by Southern blotting. A bacteriophage lambda library was constructed using a tumor cell line which retained a single copy of the viral sequences, and a phage was isolated which contained the integrated plasmid and the flanking cellular sequences. A complex rearrangement between pCM4127 and pSV2neo had occurred. DNA sequence analysis showed integration of the plasmid sequences into repetitive mouse DNA and identified an adjacent mouse sequence.

Animals↗
PubMed1986

Herpes simplex virus specifies two subunits of ribonucleotide reductase encoded by 3'-coterminal transcripts.

We have previously described a transcription unit located between map coordinates 0.558 and 0.595 on the herpes simplex virus type 2 strain 333 genome which encodes two mRNAs of 5.0 and 1.2 kilobases that share a common 3' terminus, and we have determined the nucleotide sequence of a 38,000-dalton protein specified by the smaller RNA (D. A. Galloway and M. A. Swain, J. Virol. 49:724-730, 1984). The entire nucleotide sequence of the 140,000-dalton protein specified by a 3,432-base-pair open reading frame within the large mRNA is presented, as are transcriptional regulatory sequences upstream of the RNA. The 140,000-dalton protein shows strong homology with the large subunit of well-characterized ribonucleotide reductase enzymes from the mouse and from Escherichia coli and with an Epstein-Barr virus gene. The 38,000-dalton protein has been shown previously to have homology with the small subunit of these enzymes (B.-M. Sjoberg, H. Eklund, J. A. Fuchs, J. Carlson, N. M. Standart, J. V. Ruderman, S. J. Bray, and T. Hunt, FEBS Lett. 183:99-102, 1985). This is the first example of a herpesvirus transcriptional unit that encodes functionally related proteins.

Amino Acid Sequence↗
PubMed1986

Cloning and characterization of oriL2, a large palindromic DNA replication origin of herpes simplex virus type 2.

An origin of replication within the long unique sequence of herpes simplex virus type 2 designated oriL2 has been identified in a position homologous to its type 1 counterpart, oriL1, between map coordinates 0.398 and 0.413. The difficulties encountered in previous attempts to clone both oriL2 and oriL1 in an undeleted form were surmounted by minimizing the growth of the host Escherichia coli, using a recBC sbcB E. coli host, and purifying the full-length plasmid from delected forms by using a novel method which exploits the ability of a palindrome-containing plasmid to adopt a cruciform conformation, thereby decreasing its supercoiling. In a previously developed assay for functional origin activity, oriL2 was localized to a 241-base-pair ApaI-SstII fragment. DNA sequence analysis revealed a 136-base pair, almost perfect palindrome. Comparison with oriL1 showed a very high degree of conservation: the two origins differ in only 16 of the 144-base-pair oriL1 palindromic region. Most significantly, the differences between oriL1 and oriL2 mainly occur in pairs so as to generally preserve the potential for intrastrand base pairing. The central region of oriL2 is homologous with the shorter palindromic structures detected in origins located within the repetitive sequences of the short component of herpes simplex virus type 1 or 2.

Chromosome Deletion↗
PubMed1985

Characterization of the gene encoding herpes simplex virus type 2 glycoprotein C and comparison with the type 1 counterpart.

The gene encoding the glycoprotein C (gC) of herpes simplex virus type 1 maps to the region of the viral genome from 0.62 to 0.64. Recently, a herpes simplex virus type 2 glycoprotein previously designated gF and now designated gC was mapped to a homologous location. Analysis of the herpes simplex virus type 2 mRNA species encoded in this region revealed a major transcript of 2.5 kilobases, a 0.73-kilobase transcript (the 5' ends of which were mapped by primer extension), and several minor species, all nearly identical to the herpes simplex virus type 1 pattern. A polypeptide of ca. 60,000 daltons was identified by in vitro translation of hybrid-selected mRNA. A smaller protein of ca. 20,000 daltons was also mapped to this region. The nucleotide sequence of a 3.4-kilobase segment of DNA encompassing gC was determined, and an open reading frame of 1,440 nucleotides specifying a 480-amino acid protein with properties consistent with that of a glycoprotein was identified. Comparative DNA sequence analysis showed regions of limited homology within the coding sequences for gC and a deletion which results in 31 fewer amino acids in the gC-2 near the amino terminus of the protein. The carboxy termini of gC-1 and gC-2 are very similar, as are the 20,000-dalton proteins.

Base Sequence↗
PubMed1984

Small fragments of herpesvirus DNA with transforming activity contain insertion sequence-like structures.

A 737-base-pair fragment of herpes simplex virus type 2 DNA with morphological-transforming ability was identified by transfecting into rodent cells deleted fragments of the left-hand end of the Bgl II N fragment region (map position 0.58-0.625), which were constructed in vitro. The transforming sequences lie within the coding region for a Mr 61,000 protein, but the fragment itself does not appear to specify a viral polypeptide. Contained within the transforming fragment are sequences that can be drawn as a stem-loop structure flanked by direct repeats, similar to an insertion sequence-like element. An insertion sequence-like structure was also found in a small fragment of human cytomegalovirus DNA that has transforming activity. Possible mechanisms of herpesvirus transformation are discussed.

Animals↗
PubMed1984

HSV, CMV, and HPV in human neoplasia.

We are studying the role of sexually transmitted viruses in the development of human tumors. The persistence of herpes simplex virus, cytomegalovirus, and human papillomavirus nucleic acid sequences has been examined using cloned viral DNA sequences as probes. The relationship of the viruses to various stages in the progression of neoplasia is examined, with particular reference to the role of viral and/or cellular genes in the initiation, promotion, and maintenance of the neoplastic phenotype. The human tumors of major interest in this context are carcinomas of the cervix, vulva, and anus and Kaposi's sarcoma. The minimal fragment of HSV-2 DNA detected in cervical tumors is contained within a 656-bp sequence that can be used in transfection experiments to transform rodent cells in vitro to a malignant phenotype. However, neither this fragment nor any other is consistently retained in cervical tumors, suggesting that this viral DNA may initiate but not maintain the transformed phenotype.

Carcinoma↗
PubMed1984

Structure of the transforming region of human cytomegalovirus AD169.

The minimum size fragment of human cytomegalovirus AD169 required to initiate transformation was determined by transfection of primary rat embryo cells with deletion fragments constructed by digestion of a cloned fragment containing the transforming region (pCM4000) with exonuclease III and S1 nuclease. The results indicate that the left-hand boundary of the minimum size sequence for transformation must reside between 490 and 318 bases from the HindIII site of pCM4000. The right-hand boundary was defined by the EcoRI site which is 20 bases from the HindIII site. The nucleotide sequence of the transforming fragment of human cytomegalovirus was determined by the chemical degradation technique of Maxam and Gilbert and with the dideoxynucleoside triphosphate chain termination method. The sequence of pCM4000 comprises 2,848 base pairs and has an A X T composition of 59.5%. Reading frame analysis of the sequence indicated the longest open reading frame was 118 amino acids in length. Northern blot analysis of polyadenylated and non-polyadenylated RNA extracted from cells at immediate early and late times after infection with human cytomegalovirus indicate that one 5.0-kilobase RNA species hybridized to pCM4000 (Jahn et al., J. Virol, in press). The direction of transcription was determined by hybridization of this transcript with M13 single-stranded probes, and S1 analysis of this RNA did not detect introns.

Base Sequence↗
PubMed1984

Organization of the left-hand end of the herpes simplex virus type 2 BglII N fragment.

We have determined the complete nucleotide sequence surrounding the region coding for a 38,000-dalton protein of herpes simplex virus type 2 strain 333 that is encoded between map coordinates 0.58 to 0.595 on the viral genome. The sequence data have revealed an open translational reading frame of 1,011 nucleotides encoding a protein of 337 amino acids. Upstream of those sequences, transcriptional regulatory signals could be identified that overlap another open reading frame of 489 nucleotides, which presumably encodes the carboxy-terminal 163 amino acids of a 140,000-dalton protein. Downstream of the gene encoding the 38,000-dalton protein a polyadenylation signal was found as well as one further along on the other strand near a termination codon that presumably punctuates the gene specifying a 61,000-dalton protein. The DNA sequence data were compared with the amended sequence of the herpes simplex virus type 1 strain KOS 40,000-dalton protein (K. G. Draper, R. F. Frink, and E. K. Wagner, J. Virol. 43:1123-1128, 1982) and with the 5' end of the same gene from herpes simplex virus type 1 strain 17 (J. McLaughlan and J. B. Clements, J. Gen. Virol. 64:997-1006, 1983) to assess the degree of inter- and intratypic variation. The comparison of the sequences has provided a basis for the type specifity of this viral protein and for different mechanisms giving rise to the diversity between herpes simplex virus types 1 and 2.

Amino Acid Sequence↗
PubMed1983

The oncogenic potential of herpes simplex viruses: evidence for a 'hit-and-run' mechanism.

Experiments to determine the mechanism of transformation of herpes simplex virus (HSV) have identified fragments of viral DNA which are able to initiate transformation. No set of viral genes seems to be consistently retained or expressed in the transformed cells or in human cervical tumours, suggesting that viral DNA is not needed to maintain the transformed phenotype. In fact there is no conclusive evidence that initiation of neoplasia is mediated by a viral protein. Here we revisit the 'hit-and-run' hypothesis and its implications for HSV-induced tumorigenicity.

Animals↗
PubMed1983

Nucleotide sequence of the herpes simplex virus type 2 thymidine kinase gene.

We have determined the complete nucleotide sequence of the thymidine kinase gene of herpes simplex virus (HSV) type 2 strain 333. The sequence of the thymidine kinase gene exhibits an open translational reading frame of 1,128 nucleotides encoding a protein of 376 amino acids. The DNA sequence was compared with that of the HSV type 1 thymidine kinase gene from strain MP (S. L. McKnight, Nucleic Acids Res. 8:5949-5964, 1980) and from strain CL 101 (M. J. Wagner, J. A. Sharp, and W. C. Summers, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445, 1981) to assess the extent of intra- and intertypic variation for one viral gene. The nucleotides encoding the structural gene varied 1.7% between the two HSV type 1 strains and 19% between HSV type 1 and HSV type 2, which translated to differences in the amino acid sequence of the two proteins of 1.9 and 27%, respectively. The DNA encoding the 5' regulatory sequences appeared to be more conserved than the DNA coding for the structural gene, and the DNA at the 3' end of the gene was the least homologous.

Base Sequence↗
PubMed1982

Herpesvirus-specific RNA and protein in carcinoma of the uterine cervix.

Cloned probes of herpes simplex virus type 2 DNA were used in cytological hybridization experiments to detect herpesvirus RNA transcripts in the neoplastic cells of tumors of the uterine cervix. Virus-specific RNA was shown to represent transcription of limited regions of the genome, of which one is known to code for a DNA-binding protein that can be found by immunoperoxidase staining in the neoplastic cells of these tumors and has also been detected in cells transformed in vitro by this virus.

Antigens, Viral↗
PubMed1982

Limited transcription of the herpes simplex virus genome when latent in human sensory ganglia.

Human paravertebral ganglia were examined for the presence of specific sequences of viral RNA by in situ cytological hybridization, using as probes individual fragments of herpes simplex virus type 2 DNA. Ganglia were obtained at autopsy from 40 patients, and 14 were positive for herpes simplex virus RNA. Transcripts from the left-hand 30% of the viral genome were detected in all of the herpes simplex virus-positive cases. RNA homologous to other sequences from the L component were present less frequently, and no RNA from the S component was detected. These results argue that specific transcription of the herpes simplex virus genome occurs in latently infected human ganglion cells.

Ganglia↗
PubMed1982

Identification of proteins encoded by a fragment of herpes simplex virus type 2 DNA that has transforming activity.

Cloned BglII fragment N (map units 0.58 to 0.625) of herpes simplex virus type 2 DNA has been shown to transform rodent cells to an oncogenic phenotype (Galloway and McDougall, J. Virol. 38: 749-760, 1981). RNA homologous to this fragment directs the synthesis of five polypeptides in a cell-free translation system. The approximate molecular weights of these proteins are 140,000, 61,000, 56,000, 35,000, and 23,500. The 35,000-dalton protein is the major species late in infection and is the only species detected before the onset of viral DNA replication. The arrangement of the sequences encoding these proteins along the herpes simplex virus type 2 genome was determined by hybridization of the RNA to cloned PstI fragment of BglII-N and to single-stranded DNA segments cloned into M13mp7. Both the hybridization experiments and immunoprecipitation with monoclonal antibodies suggested that the 140,000- and 35,000-dalton proteins are at least partially colinear and share antigenic determinants.

Animals↗
PubMed1982

Transformation of NIH 3T3 cells with cloned fragments of human cytomegalovirus strain AD169.

NIH 3T3 cells were transfected with restriction endonuclease and cloned human cytomegalovirus DNA fragments to identify the transforming region(s). Cleavage of human cytomegalovirus strain AD169 DNA with XbaI and HindIII left a transforming region intact whereas EcoRI inactivated this function. Transfection of cells with cosmids containing human cytomegalovirus DNA spanning the entire genome resulted in transformation by one cosmid, pCM1058, with the AD169 HindIII DNA fragments E, R, T, and a'. Cells were selected for their growth in 1.2% methylcellulose. The clones isolated had a significant replating efficiency and were oncogenic in BALB/c nu/nu mice. Transfection of cosmids and plasmids containing subsets of the viral sequences in pCM1058 identified a common region possessed by all of the transforming recombinant molecules. This region was in the HindIII E fragment with the left boundary defined by the EcoRI d-R junction and the right boundary defined by the HindIII E-T junction. Further mapping and transfection experiments determined that the transforming region was contained without a 2.9-kilobase fragment between map units 0.123 and 0.14 on the prototype molecule of the AD169 strain.

Animals↗
PubMed1981

Transformation of rodent cells by a cloned DNA fragment of herpes simplex virus type 2.

Transformation of rodent cells with isolated restriction endonuclease fragments of herpes simplex virus type 2 DNA identified a region of the genome located between map positions 0.58 and 0.62. These sequences were cloned into pBR322, and the recombinant plasmid was used to transform primary rat embryo cells and NIH 3T3 cells. The transformants were selected for their ability to form dense foci on a monolayer or to form colonies in semisolid medium. In contrast to the parental rat or mouse cells, cell lines transformed with the cloned herpes simplex virus type 2 fragment grow to high saturation densities, replicate in medium containing 1% serum, form colonies in dilute methylcellulose, show reduced levels of fibronectin, and are tumorigenic in nude mice and in their syngeneic hosts. Southern blot hybridizations have detected sequences homologous to the viral fragment in high-molecular-weight DNA from the transformed cell lines that are not present in DNA from normal rodents. In all cases, the plasmid DNA was present in less than one copy per cell, and the patterns of viral sequences changed with passage of the cell line in vivo.

Animals↗
PubMed1980

Cloning of Herpes simplex virus 2 DNA fragments in a plasmid vector.

DNA isolated from virions of Herpes simplex type 2 (HSV-2) strain 333 was digested with various restriction enzymes and joined to the EK2 plasmid vector pBR322. The viral DNA sequences present in the hybrids were analyzed by restriction enzyme mapping and hybridization to fragments of HSV-2 DNA. The collection of recombinant molecules represents approx. 75% of the HSV-2-genome. In most cases, the structure of the recombinants seemed identical to the organization of authentic fragments of HSV-2 DNA, however, a few hybrids contained rearrangements of viral and plasmid sequences.

Cloning, Molecular↗