Molecular cytochemistry of nucleic acids by gene technology and in situ hybridization and the advances in its application.

Abstract

Molecular cytochemistry of nucleic acids by gene technology and the advances in its application are reviewed with special reference to in situ hybridization . Studies on the in situ hybridization of repetitive and unique DNA sequences , messenger RNA, and viral genomes are outlined.A brief survey of the methods employed for labeling DNA and RNA probes with high specific radioactivity or nonradioactive markers and sensitive detection systems are given . The data of our own studies on the detection of Y chromosome by in situ hybridization with biotin-labeled DNA probes, molecular cloning of proviral DNA of a retrovirus produced in a human lymphoblastoid cell line, and in situ detection of gene expression of the molecularly cloned proviral DNA in transfected cells are also presented. The gene expression was detected in several percent of the transfected cells by indirect immunoperoxidase staining of viral proteins as well as by in situ hybridization of viral RNA with a [32P]-labeled probe and with a biotin-labeled probe.Recent advances in molecular biology of nucleic acids, especially the development of DNA cloning and sequencing technologies have brought revolutionary advances in biology and medical science. By these techniques, specific genes or DNA sequences are cloned, the base sequences determined or even altered, and then the DNA sections reinserted into cells to examine their structure and function in vivo. It became easy to obtain various DNA or RNA probes and to label with high specific radioactivity or nonradioactive markers for detecting specific genes and their expressions in cells and tissues. Double stranded DNA is dissociated into two single strands by heating or alkaline treatment (DNA denaturation), and is readily reformed into double stranded DNA by gradual cooling or neutralization (DNA annealing, renaturation, or hybridization). Similar hybridization reaction occurs between any two single-stranded DNA or RNA chains irrespective of their sources, provided they have a complementary nucleotide sequence (nucleic acid hybridization). Thus, specific DNA or RNA sequences are detected by the molecular hybridization with labeled DNA or RNA probes either in solution, on membrane filters(2, 23, 70), or in situ in histological and cytological preparations (18, 33) ( Table 1).The nucleic acid hybridization techniques have been extensively applied for the detection of specific DNA sequence (Southern blotting) (70) or RNA sequence (Northern blotting) (2) separated by gel electrophoresis and blotted onto nitrocellulose or nylon membrane filters. The hybridization is also made to nucleic acids spotted directly onto membrane filters (dot blot or spot hybridization) (6). These methods 151

Detection of hepatitis B virus-DNA in liver tissues in chronic type B liver diseases and its relevancy to viral antigens. A study by in situ hybridization using a biotinylated probe and streptavidin-alkaline phosphatase.

“…Generally, radiolabeled probes and autoradiography have been utilized for nucleic acid hybridizations including in situ hybridization (19). The use of radiolabel- ed probes, despite the high sensitivity, is restricted to special laboratories because of safety problems.…”

“…In addition, autoradiographic detection of the probes is time-consuming (23). Recently, non-radioactive probes and their colorimetric detection systems have been developed (9,15,19) and demonstrated to be available for in situ hybridization (6,16,19,23). There have been reports on the detection of HBV-DNA by in situ hybridization using a biotinylated probe in which avidin-biotin-peroxidase complex (ABC) system was used for the colorimetric detection of the probe (1, 14, 18, 20).…”

“…tion of the cellular distribution of viral genomes (6,8,19,23). There have been several reports on the detection of HBV-DNA by in situ hybridization using radiolabeled or biotinylated probes in HBV-infected liver tissues (1-3, 7, 10, 11, 14, 17, 18, 20).…”

In situ Hybridization for the Demonstration of Bovine Leukemia Virus Transcripts in Lymphosarcoma Cells Using Biotinylated Probes

“…The molecular hybridization technique provides a means for detection of specific proviral genome inside the target cells' DNA (5, 15,17). The recently developed in situ hybridization technique is applicable in detecting specific DNA or RNA targets at cellular level on tissue sections (7,13). For detection of the in situ localization of DNA or RNA, cDNAs labelled with radio-isotopes have been used as probes and the sites of DNA or RNA are visualized with radioautographic techniques (7,11).…”

“…More recently, in order to obtain better resolution and to minimize the cumbersome procedures associated with the handling of radioactive compounds, non-radioactive probes have been introduced (1, 12). By use of these non-radioactive probes, the criteria of morphological preservation of cells and tissues become more stringent (13). BLV m R N A has been detected in lymphocytes from BLV-infected sheep using radioactively labelled probes (1 I), but non-radioactively labelled probes have never been used thus far.…”