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Using in situ nucleic acid hybridizations, the genes that code for 28, 18 and 5S rRNA have been localized in the polytene chromosomes of Drosophila tumiditarsus. The 5S genes are found at a single site near the centromere of the second chromosome, whereas the 28 and 18S genes are found at the nucleolar organizer region of the dot chromosome. The dot chromosome has been previously described as alpha-heterochromatic. However, our cytochemical and autoradiographic results do not support such a conclusion. The autoradiographic results reveal that the dot chromosome is transcriptionally active and is not late-replicating, as is expected of alpha-heterochromatin. Further, the dot chromosomes possess none of the usual staining characteristics of heterochromatin except for its lack of polytene bands. Using rRNA-DNA filter hybridizations, we find that the rDNA of D. tumiditarsus salivary glands is under-replicated. This is the first species of Drosophila where the rDNA in not found on the sex chromosomes, and is the first report of an under-replicated autosomal locus which is not located in heterochromatic blocks.
Using in situ nucleic acid hybridizations, the genes that code for 28, 18 and 5S rRNA have been localized in the polytene chromosomes of Drosophila tumiditarsus. The 5S genes are found at a single site near the centromere of the second chromosome, whereas the 28 and 18S genes are found at the nucleolar organizer region of the dot chromosome. The dot chromosome has been previously described as alpha-heterochromatic. However, our cytochemical and autoradiographic results do not support such a conclusion. The autoradiographic results reveal that the dot chromosome is transcriptionally active and is not late-replicating, as is expected of alpha-heterochromatin. Further, the dot chromosomes possess none of the usual staining characteristics of heterochromatin except for its lack of polytene bands. Using rRNA-DNA filter hybridizations, we find that the rDNA of D. tumiditarsus salivary glands is under-replicated. This is the first species of Drosophila where the rDNA in not found on the sex chromosomes, and is the first report of an under-replicated autosomal locus which is not located in heterochromatic blocks.
The distribution of fast, intermediate and slow renaturing fractions of Rhynchosciara americana DNA was examined in the polytene salivary gland chromosomes by in situ hybridization. Heterochromatic areas readily hybridized but hybrid formation in the euchromatin depended more on the repetitiveness of the RNA probe.
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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