In situ hybridization using a biotinylated probe and streptavidin-alkaline phosphatase was applied to the detection of hepatitis B virus(HBV)-DNA in formaim-fixed and paraffin-embedded liver-biopsied tissues from 24 patients with chronic type B liver diseases (all carriers of serum HBsAg, and the results were compared with those of the immunohistochemical detection of HBsAg and HBcAg.The specificity of in situ hybridization reactions was confirmed by negative staining of the control tests. HBV-DNA was detected in the sections of 8 cases with serum HBeAg and one case without serum HBeAg, and was located predominantly in the cytoplasm of hepatocytes showing various staining patterns such as diffuse, regional or peripheral.The lobular or pseudolobular distribution of HBV-DNA in the liver tissues was divided into scattered, clustered and diffuse type. The scattered type was observed predominantly in the sections of cases with active liver cirrhosis.As regards HBsAg and HBcAg in the liver tissues, HBsAg was detected in the sections of 18 cases with or without serum HBeAg; 8 were detectable for HBV-DNA by in situ hybridization and 10 were not detectable. HBcAg was detected in the sections of 9 cases all with serum HBeAg; 6 were detectable for HBV-DNA by in situ hybridization and 3 were not detectable. The sections of 5 out of the 6 detectable cases for HBV-DNA revealed the cytoplasmic HBcAg in hepatocytes.These findings suggested that HBV-DNA in hepatocytes detected by in situ hybridization correlated with the presence of serum HBeAg and cytoplasmic HBcAg rather than nuclear HBcAg in hepatocytes in chronic type B liver diseases.Examinations of hepatitis B (HB) surface antigen (HBsAg) and core antigen (HBcAg) in liver tissues as well as serum HBeAg, anti-HBe antibody and anti-HBc antibody have provided useful information for diagnosis and prognosis in chronic type B liver diseases. Of these antigens or antibodies, HBcAg in hepatocytes and serum HBeAg are considered to be markers of active replication of the HB virus (HBV). Recently, HBV-DNA, as a direct marker of HBV-replication in chronic type B liver diseases, has been detected in serum or liver tissues by nucleic acid hybridizations including in situ hybridization (1-5, 7, 8, 10, 11, 14, 17, 18, 20, 23). The technique of in situ hybridization has allowed for identification of viral infected cells and observa-489

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

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