scholarly journals Detection of viral DNA and RNA by in situ hybridization.

1986 ◽  
Vol 34 (1) ◽  
pp. 33-38 ◽  
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.

High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

1995 ◽  
Vol 53 ◽  
pp. 752-753
Author(s):  
B.A. Hamkalo ◽  
S. Narayanswami ◽  
A.P. Kausch
Keyword(s):  
Nucleic Acid ◽  
Interphase Nucleus ◽  
Genome Mapping ◽  
Precise Location ◽  
Electron Microscope Level ◽  
Rna Sequences ◽  
Fundamental Interest ◽  
Whole Cells ◽  
Dna And Rna

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

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

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.

scholarly journals DNA-mediated gene transfer without carrier DNA.

1981 ◽  
Vol 91 (1) ◽  
pp. 153-156 ◽  
Author(s):  
K M Huttner ◽  
J A Barbosa ◽  
G A Scangos ◽  
D D Pratcheva ◽  
F H Ruddle
Keyword(s):  
Gene Transfer ◽  
Dna Sequences ◽  
Kinase Gene ◽  
Whole Cell ◽  
Cell Carrier ◽  
Wide Range ◽  
Dna Restriction ◽  
Simplex Virus

DNA-mediated gene transfer is a procedure which uses purified DNA to introduce new genetic elements into cells in culture. The standard DNA-mediated gene transfer procedure involves the use of whole cell DNA as carrier DNA for the transfer. We have modified the standard DNA-mediated gene transfer procedure to transfer the Herpes simplex virus type 1 thymidine kinase gene (TK) into TK- murine recipient cells in the absence of whole cell carrier DNA. The majority (8/10) of carrier-free transformant lines expressed the TK+ phenotype stably, in sharp contrast to our results with carrier-containing DNA-mediated gene transfer. There was a wide range in donor DNA content among independent transformants. Further analysis on one transformant line using DNA restriction digests and in situ hybridization provided evidence that, in the absence of whole cell carrier DNA, multiple donor DNA sequences became integrated at a single chromosomal site.

Localization of histone gene transcripts in newt lampbrush chromosomes by in situ hybridization

1977 ◽  
Vol 27 (1) ◽  
pp. 57-79
Author(s):  
R.W. Old ◽  
G.H. Callan ◽  
K.W. Gross
Keyword(s):  
Dna Sequences ◽  
Histone Gene ◽  
Lampbrush Chromosomes ◽  
Rna Sequences ◽  
Psammechinus Miliaris ◽  
Lateral Loop ◽  
Gene Transcripts ◽  
Label Distribution ◽  
Chromosome I

Denatured 3H-labelled DNAs containing histone gene sequences originating from the echinoderms Psammechinus miliaris, Echimus esculentus and Strongylocentrotus purpuratus have been in situ hybridized to RNA transcripts on newt lampbrush chromosomes. Autoradiographs of the hybridized lampbrush preparations show labelling restricted to four or fewer lateral loop pairs all lying within the heteromorphic regions of chromosome I, also one or two loop pairs on chromosome VI, one loop pair on chromosome X and one loop pair on chromosome XI. For oocytes from a single newt, coincident label distribution is found with DNA's of diverse echinoderm origin; however different newts show some specific individual diversity in label distribution, including heterozygosity in the case of loops on bivalents VI and X. The more conspicuously labelled loops, particularly those on chromosome I, show a pattern of labelling which is explicable if the newt histone DNA sequences are confined to short intercepts of lateral loop axis. Transcription is initiated prior to the histone DNA sequences, proceeds through the histone DNA sequences, and beyond, and the histone RNA sequences are cut from the transcripts before the termination of transcription.

scholarly journals The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters

2015 ◽  
Vol 89 (15) ◽  
pp. 7506-7520 ◽  
Author(s):  
Irene Lo Cigno ◽  
Marco De Andrea ◽  
Cinzia Borgogna ◽  
Silvia Albertini ◽  
Manuela M. Landini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Herpes Simplex ◽  
Viral Gene Expression ◽  
Restriction Factor ◽  
Viral Gene ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Hpv Dna ◽  
U2os Cells ◽  
Simplex Virus

ABSTRACTThe human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) infection by inhibiting both viral-DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict human papillomavirus 18 (HPV18) replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used, in which the IFI16 protein was knocked down through the use of small interfering RNA (siRNA) technology and overexpressed following transduction with the adenovirus IFI16 (AdVIFI16) vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral-load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral-gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinized HPV DNA through epigenetic modifications and plays a broad surveillance role against viral DNA in the nucleus that is not restricted to herpesviruses.IMPORTANCEIntrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as a sensor of foreign DNA and an antiviral restriction factor, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral-gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS cells transfected with HPV18 minicircles, suggesting that it is not cell type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating the protein in the epigenetic regulation of HPV gene expression and replication.

scholarly journals A toolkit for studying Varroa genomics and transcriptomics: Preservation, extraction, and sequencing library preparation

2020 ◽  
Author(s):  
Nonno Hasegawa ◽  
Maeva Techer ◽  
Alexander S. Mikheyev
Keyword(s):  
Honey Bee ◽  
Dna Sequences ◽  
Room Temperature ◽  
Post Treatment ◽  
Population Declines ◽  
Rna Sequences ◽  
Deformed Wing Virus ◽  
Rna Integrity ◽  
Dna And Rna ◽  
Sequencing Library

AbstractBackgroundThe honey bee parasite, Varroa destructor, is a leading cause of honey bee population declines. In addition to being an obligate ectoparasitic mite, Varroa carries several viruses that infect honey bees and act as the proximal cause of colony collapses. Nevertheless, until recently, studies of Varroa have been limited by the paucity of genomic tools. Lab- and field-based methods exploiting such methods are still nascent. This study developed a set of methods for preserving Varroa DNA and RNA from the field to the lab and processing them into sequencing libraries. We performed preservation experiments in which Varroa mites were immersed in TRIzol, RNAlater, and absolute ethanol for preservation periods up to 21 days post-treatment to assess DNA and RNA integrity.ResultsFor both DNA and RNA, mites preserved in TRIzol and RNAlater at room temperature degraded within 10 days post-treatment. Mites preserved in ethanol at room temperature and 4°C remained intact through 21 days. Varroa mite DNA and RNA libraries were created and sequenced for ethanol preserved samples, 15 and 21 days post-treatment. All DNA sequences mapped to the V. destructor genome at above 95% on average, while RNA sequences mapped to V. destructor, but also sometimes to high levels of the deformed-wing virus and to various organisms.ConclusionEthanolic preservation of field-collected mites is inexpensive and simple, and allows them to be shipped and processed successfully in the lab for a wide variety of sequencing applications. It appears to preserve RNA from both Varroa and at least some of the viruses it vectors.

scholarly journals 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.

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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