Protocol of electron microscope in situ nucleic acid hybridization for the exclusive detection of double-stranded DNA sequences in cells containing large amounts of homologous single-stranded DNA and RNA sequences: Application to adenovirus type 5 infected Hela cells

1993 ◽  
Vol 25 (1) ◽  
pp. 2-11 ◽  
Author(s):  
Francine Puvion-Dutilleul
Keyword(s):  
Electron Microscope ◽  
Nucleic Acid ◽  
Dna Sequences ◽  
Adenovirus Type ◽  
Nucleic Acid Hybridization ◽  
Rna Sequences ◽  
Dna And Rna ◽  
Double Stranded Dna ◽  
Adenovirus Type 5

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.

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.

scholarly journals CHROMOSOMAL LOCALIZATION OF REPETITIVE DNA IN THE NEWT, TRITURUS

1972 ◽  
Vol 54 (3) ◽  
pp. 580-591 ◽  
Author(s):  
Giuseppina Barsacchi ◽  
Joseph G. Gall
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Repetitive Dna ◽  
Ribosomal Dna ◽  
Dna Sequences ◽  
Chromosomal Localization ◽  
Nucleic Acid Hybridization ◽  
Repetitive Dna Sequences ◽  
Lampbrush Chromosomes

The repetitive DNA sequences of the newt, Triturus viridescens, have been studied by nucleic acid hybridization procedures. Complementary RNA was synthesized enzymatically from unfractionated newt DNA. This RNA hybridized strongly to the centromeric regions of both somatic and lampbrush chromosomes It also bound to other loci scattered along the lengths of the chromosomes The amplified ribosomal DNA in the multiple oocyte nucleoli was demonstrated by in situ hybridization

In situ nucleic acid hybridization

1989 ◽  
Vol 34 (3) ◽  
pp. 187-192 ◽  
Author(s):  
William R. Freeman ◽  
Clayton A. Wiley
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Hybridization

scholarly journals Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2

2020 ◽  
Vol 48 (7) ◽  
pp. 3869-3887 ◽  
Author(s):  
Linlin Hou ◽  
Yuanjie Wei ◽  
Yingying Lin ◽  
Xiwei Wang ◽  
Yiwei Lai ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
High Mobility ◽  
Binding Motif ◽  
Dna And Rna ◽  
Somatic Cell Reprogramming ◽  
Double Stranded Dna

Abstract Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.

scholarly journals Genetic Identification of Adenovirus Type 5 Genes That Influence Viral Spread

2006 ◽  
Vol 80 (4) ◽  
pp. 2000-2012 ◽  
Author(s):  
T. Subramanian ◽  
S. Vijayalingam ◽  
G. Chinnadurai
Keyword(s):  
Dna Sequences ◽  
Control Cell ◽  
Cancer Cell Line ◽  
Adenovirus Type ◽  
Viral Proteins ◽  
Genetic Identification ◽  
Large Plaque ◽  
Viral Spread ◽  
Leader Protein ◽  
Adenovirus Type 5

ABSTRACT The mechanisms that control cell-to-cell spread of human adenoviruses (Ad) are not well understood. Two early viral proteins, E1B-19K and E3-ADP, appear to have opposing effects since viral mutants that are individually deficient in E1B-19K produce large plaques (G. Chinnadurai, Cell 33:759-766, 1983), while mutants deficient in E3-ADP produce small plaques (A. E. Tollefson et al., J. Virol. 70:2296-2306, 1996) on infected cell monolayers. We have used a genetic strategy to identify different viral genes that influence adenovirus type 5 (Ad5) spread in an epithelial cancer cell line. An Ad5 mutant (dl327; lacking most of the E3 region) with the restricted-spread (small-plaque) phenotype was randomly mutagenized with UV, and 27 large-plaque (lp) mutants were isolated. A combination of analyses of viral proteins and genomic DNA sequences have indicated that 23 mutants contained lesions in the E1B region affecting either 19K or both 19K and 55K proteins. Four other lp mutants contained lesions in early regions E1A and E4, in the early L1 region that codes for the i-leader protein, and in late regions that code for the viral structural proteins, penton base, and fiber. Our results suggest that the requirement of E3-ADP for Ad spread could be readily compensated for by abrogation of the functions of E1B-19K and provide genetic evidence that these two viral proteins influence viral spread in opposing manners. In addition to E1B and E3 proteins, other early and late proteins that regulate viral replication and infectivity also influence lateral viral spread. Our studies have identified novel mutations that could be exploited in designing efficient oncolytic Ad vectors.

scholarly journals In Situ Loading and Delivery of Short Single- and Double-Stranded DNA by Supramolecular Organic Frameworks

CCS Chemistry ◽  
2019 ◽  
Vol 1 (2) ◽  
pp. 156-165 ◽  
Author(s):  
Bo Yang ◽  
Xiao-Dan Zhang ◽  
Jian Li ◽  
Jia Tian ◽  
Yi-Peng Wu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Dna Sequences ◽  
Laser Scanning ◽  
High Stability ◽  
Intracellular Delivery ◽  
Water Soluble ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nucleic Acid Delivery

Short DNA represents an important class of biomacromolecules that are widely applied in gene therapy, editing, and modulation. However, the development of simple and reliable methods for their intracellular delivery remains a challenge. Herein, we describe that seven water-soluble, homogeneous supramolecular organic frameworks (SOFs) with a well-defined pore size and high stability in water that can accomplish in situ inclusion of single-stranded (ss) and double-stranded (ds) DNA (21, 23, and 58 nt) and effective intracellular delivery (including two noncancerous and six cancerous cell lines). Fluorescence quenching experiments for single and double end-labeled ss- and ds-DNA support that the DNA sequences can be completely enveloped by the SOFs. Confocal laser scanning microscopy and flow cytometry reveal that five of the SOFs exhibit excellent delivery efficiencies that, in most of the studied cases, outperform the commercial standard Lipo2000, even at low SOF–nucleic acid ratios. In addition to high delivery efficiencies, the water-soluble, self-assembled SOF carriers have a variety of advantages, including convenient preparation, high stability, and in situ DNA inclusion, which are all critical for practical applications in nucleic acid delivery.

Sign in / Sign up

Export Citation Format

Share Document