Fast solid support detection of PCR amplified viral DNA sequences using radioiodinated or hapten labelled primers

We have cloned and propagated in prokaryotic vectors the viral DNA sequences that are integrated in a variety of cells transformed by adenovirus 2 or SV40. Analysis of the clones reveals that the viral DNA sequences sometimes are arranged in a simple fashion, collinear with the viral genome; in other cell lines there are complex arrangements of viral sequences in which tracts of the viral genome are inverted with respect to each other. In several cases the nucleotide sequences at the joints between cell and viral sequences have been determined: usually there is a sharp transition between cellular and viral DNAs. The viral sequences are integrated at different locations within the genomes of different cell lines; likewise there is no specific site on the viral genomes at which integration occurs. Sometimes the viral sequences are integrated within repetitive cellular DNA, and sometimes within unique sequences. In some cases there is evidence that the viral sequences along with the flanking cell DNA have been amplified after integration. The sequences that flank the viral insertion in the line of SV40-transformed rat cells known as 14B have been used as probes to isolate, from untransformed rat cells, clones that carry the region of the chromosome in which integration occurred. Analysis of the structure of these clones by restriction endonuclease digestion and heteroduplex formation shows that a rearrangement of cellular sequences has occurred, presumably as a consequence of integration.

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Recombination in SV40-infected cells: Viral DNA sequences at sites of circularization of transfecting linear DNA

Virology ◽

10.1016/0042-6822(81)90506-7 ◽

1981 ◽

Vol 109 (2) ◽

pp. 353-365 ◽

Cited By ~ 8

Author(s):

Mary Woodworth-Gutai

Keyword(s):

Dna Sequences ◽

Viral Dna ◽

Linear Dna ◽

Infected Cells

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Cloning full-length dengue type 4 viral DNA sequences: Analysis of genes coding for structural proteins

Virology ◽

10.1016/0042-6822(86)90169-8 ◽

1986 ◽

Vol 155 (1) ◽

pp. 77-88 ◽

Cited By ~ 105

Author(s):

Bangti Zhao ◽

Erich Mackow ◽

Alicia Buckler-White ◽

Lewis Markoff ◽

Robert M. Chanock ◽

...

Keyword(s):

Dna Sequences ◽

Full Length ◽

Structural Proteins ◽

Viral Dna ◽

Sequences Analysis

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Transcriptional control signals of a herpes simplex virus type 1 late (gamma 2) gene lie within bases -34 to +124 relative to the 5' terminus of the mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.6.11.3652-3666.1986 ◽

1986 ◽

Vol 6 (11) ◽

pp. 3652-3666

Author(s):

F L Homa ◽

T M Otal ◽

J C Glorioso ◽

M Levine

Keyword(s):

Herpes Simplex ◽

Dna Sequences ◽

Viral Dna ◽

Regulated Expression ◽

Base Pair Fragment ◽

Simplex Virus ◽

Pair Fragment ◽

Gc Gene ◽

Hsv 1

The cis-acting DNA sequences required for regulated expression of a herpes simplex virus type 1 (HSV-1) late (gamma 2) gene were studied by using viruses containing specific deletions in the 5' transcribed noncoding and upstream regions of the HSV-1 glycoprotein C (gC) gene, a model gamma 2 gene. Nine mutant viruses which had variable 5' and 3' deletions within bases -569 to +124 relative to the 5' terminus of the gC mRNA were isolated. The mutants were isolated by a simple in situ hybridization screening procedure not requiring any prior selective pressure for or against expression of the gC gene. Analysis of RNA extracted from cells infected with individual mutants showed that the DNA sequences required for regulated expression of this gamma 2 gene lay within bases -34 to +124. This 158-base-pair fragment was sufficient to confer accurate and quantitative expression of gC mRNA and to maintain the stringent requirement on viral DNA replication for expression of this gene. Moreover, it was found that sequences located between -34 and +14 contained signals essential for expression of gC. To determine whether the -34 to +124 sequences would function as a gamma 2 promoter when moved to another region of the HSV-1 genome, the 158-base-pair fragment was substituted for the normal thymidine kinase promoter-regulatory sequences in the thymidine-kinase gene locus. Transcription of this chimeric gene was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis. The only recognizable consensus sequence upstream of the transcription initiation site for this gene was the TATAAA sequence at -30.

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BK virus-transformed inbred hamster brain cells: status of viral DNA in subclones

Molecular and Cellular Biology ◽

10.1128/mcb.2.7.837-844.1982 ◽

1982 ◽

Vol 2 (7) ◽

pp. 837-844

Author(s):

M M Pater ◽

A Pater ◽

G di Mayorca ◽

E Beth ◽

G Giraldo

Keyword(s):

Cell Line ◽

Dna Sequences ◽

Copy Number ◽

Tumor Antigens ◽

Blot Hybridization ◽

Virus Production ◽

Bk Virus ◽

Free Form ◽

Brain Cells ◽

Viral Dna

We have recently reported that viral DNA sequences in inbred LSH hamster brain cells transformed by the GS variant of BK virus (LSH-BR-BK) are present predominantly in a free form (Beth et al., J. Virol. 40:276-284, 1981). In this report, we confirm that the presence of viral DNA sequences in these cells is not due to virus production, since viral capsid proteins were not detected by immunoprecipitation. Furthermore, we examined the status of viral DNA in 15 subclones of this cell line and detected free and integrated viral DNA sequences in only 5 of the subclones. The other 10 subclones contained exclusively integrated viral DNA sequences, as shown by the blot hybridization of high-molecular-weight cell DNA which was uncleaved or digested with HincII, for which there are no sites in viral DNA. The arrangement of viral DNA in these clones was further analyzed by cleavage of cellular DNA with HpaII and HindIII. Mitomycin (0.03 microgram/ml) treatment of subclones containing only integrated sequences resulted in the appearance of free viral DNA sequences in some of these cells. This result supports the postulation that free viral DNA in LSH-BR-BK cells is made up of excision products of observed tandemly repeated integrated sequences. In addition to the large T- and small t-antigens, LSH-BR-BK and all of its 15 subclones contained two antigen species which were larger than large T and one species which was smaller than small t. The number of tumor antigens in the LSH- BR-BK cell line and its subclones with a large copy number in a free form was not more than in the subclones with low copy number and integrated DNA. This suggests that free viral DNA is not a template for tumor antigen production in transformed cells.

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Retroviral DNA Integration

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.63.4.836-843.1999 ◽

1999 ◽

Vol 63 (4) ◽

pp. 836-843 ◽

Cited By ~ 106

Author(s):

Patrick Hindmarsh ◽

Jonathan Leis

Keyword(s):

Dna Sequences ◽

Host Genome ◽

Single Site ◽

Enzyme Complex ◽

Enzymatic Process ◽

Long Terminal Repeats ◽

Host Proteins ◽

Viral Dna ◽

Dna Integration ◽

Terminal Repeats

SUMMARY DNA integration is a unique enzymatic process shared by all retroviruses and retrotransposons. During integration, double-stranded linear viral DNA is inserted into the host genome in a process catalyzed by the virus-encoded integrase (IN). The mechanism involves a series of nucleophillic attacks, the first of which removes the terminal 2 bases from the 3′ ends of the long terminal repeats and of the second which inserts the viral DNA into the host genome. IN specifically recognizes the DNA sequences at the termini of the viral DNA, juxtaposing both ends in an enzyme complex that inserts the viral DNA into a single site in a concerted manner. Small duplications of the host DNA, characteristic of the viral IN, are found at the sites of insertion. At least two host proteins, HMG-I(Y) and BAF, have been shown to increase the efficiency of the integration reaction.

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Tumorigenic poxviruses: Analysis of viral DNA sequences implicated in the tumorigenicity of shope fibroma virus and malignant rabbit virus

Virology ◽

10.1016/0042-6822(86)90134-0 ◽

1986 ◽

Vol 152 (2) ◽

pp. 308-321 ◽

Cited By ~ 30

Author(s):

C. Upton ◽

G. McFadden

Keyword(s):

Dna Sequences ◽

Viral Dna

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Detection of viral DNA and RNA by in situ hybridization.

Journal of Histochemistry & Cytochemistry ◽

10.1177/34.1.3001177 ◽

1986 ◽

Vol 34 (1) ◽

pp. 33-38 ◽

Cited By ~ 36

Author(s):

J K McDougall ◽

D Myerson ◽

A M Beckmann

Keyword(s):

Dna Sequences ◽

Viral Dna ◽

Rna Sequences ◽

Hpv Dna ◽

Dna And Rna ◽

Common Warts ◽

Anogenital Region ◽

Simplex Virus ◽

Human Viruses

Using cloned restriction endonuclease fragments of Herpes simplex virus (HSV), human papillomavirus (HPV), and cytomegalovirus (CMV) DNA as probes, viral DNA and RNA sequences have been detected in human tissues. The probes were labeled either with a radioactive isotope, for subsequent detection by autoradiography, or with biotin. This latter technique has been successfully used to visualize HPV DNA in tissues that have been fixed in formalin and embedded in paraffin, and is therefore of value in retrospective studies of histological specimens. HPV DNA was detected under non-stringent conditions (Tm = -42 degrees C) with heterologous probes in plantar and common warts, laryngeal papillomas, and anogenital condylomas. The specific type of HPV was established using stringent hybridization conditions (Tm = - 17 degrees C). Results from these and from malignant tissues show the distribution and localization of HSV and HPV RNA and DNA sequences in malignancies of squamous cell origin in the anogenital region. Both HSV and HPV DNA sequences have occasionally been detected in the same tumor, providing a further impetus to test the hypothesis that an initiator-promoter relationship might involve these common human viruses in the development of some tumors.

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