A method is described for localizing DNA sequences hybridized in situ to Drosophila polytene chromosomes. This procedure utilizes a biotin-labeled analog of TTP that can be incorporated enzymatically into DNA probes by nick-translation. After hybridization in situ, the biotin molecules in the probe serve as antigens which bind affinity-purified rabbit antibiotin antibodies. The site of hybridization is then detected either fluorimetrically, by using fluorescein-labeled goat anti-rabbit IgG, or cytochemically, by using an anti-rabbit IgG antibody conjugated to horseradish peroxidase. When combined with Giemsa staining, the immunoperoxidase detection method provides a permanent record that is suitable for detailed cytogenetic analysis. This immunological approach offers four advantages over conventional autoradiographic procedures for detecting in situ hybrids: (i) the time required to determine the site of hybridization is decreased markedly; (ii) biotin-labeled probes are chemically stable and give -reproducible results for many months; (iii) biotin-labeled probes appear to produce less background noise than do radiolabeled probes; and (iv) the resolving power is equal to and often greater than that achieved autoradiographically.In situ hybridization, initially developed by Gall and Pardue (1) and John et at (2), has proven to be a valuable method for determining the cellular or chromosomal location of hybridized nucleic acids (3)(4)(5)(6)(7)(8)(9)(10). Standard in situ hybridization protocols use radiolabeled RNA or DNA probes and autoradiographic methods of detection or quantification. By using probes of high specific activity under conditions such that hybridization "networks" are formed (8-12), it is now possible to localize unique sequences in mammalian chromosome spreads after autoradiographic exposures of 5-22 days (9, 10). However, the inherent drawbacks of radiolabeled probes-notably chemical lability due to radiolytic decomposition, concern for personnel safety, and disposal problems-make it desirable to have sensitive methods for detecting polynucleotide sequences that do not rely on the use of radioisotopes, especially for routine applications in clinical medicine. Several
We have developed a novel method for high resolution mapping of specific DNA sequences after in situ hybridization. DNA probes, labeled with biotin-nucleotides in conventional nick-translation reactions, are hybridized to cytological preparations and detected with affinity- purified rabbit antibiotin antibodies followed by antibodies to rabbit IgG that are conjugated to fluorescent or enzymatic reagents. Using peroxidase labeled anti-rabbit IgG, we are able to detect and localize specific sequences at both the light and electron microscopic levels. Initial studies were done with repeated DNA sequences previously mapped by light microscope autoradiography to assess the fidelity and resolution of this method. An analysis using biotin-labeled mouse satellite DNA is presented here.
In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70% ethanol vapor; hybridization sites are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Interphase nuclei present in some of the metaphase chromosome preparations also show defined patterns of satellite DNA labeling which suggests that satellite-containing regions are associated with each other during interphase. The sensitivity of this method is estimated to be at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of colloidal gold particles (~20 nm in diameter). Gold particles bind specifically both directly over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructural localization of DNA sequences in chromosomes and chromatin.In situ hybridization has become a classical method for mapping DNA sequences in cytological preparations and it has been used extensively to map repeated genes in polytene and metaphase chromosomes at the light microscope (LM) level (see reference 1 for review). Recent refinements, which enhance the efficiency of hybridization and the sensitivity of hybrid detection, have made it possible to identify and localize specific cellular or viral DNA and RNA sequences even when present in low copy number (2-12). Furthermore, by using high molecular weight radioactive-probe networks (3), or by including dextran sulfate in the hybridization mixture (13, 14), unique sequences have been mapped to metaphase chromosomes after autoradiographic exposure of 5-22 d.A few attempts have been made to extend in situ hybridization to the electron microscope (EM) level. However, most of these involved hybridization to tissue sections, tissue blocks, or whole cells (see reference 15 for review); no general method is available for hybridization to whole mount metaphase chromosomes or chromatin spreads. Such a method would (a) allow gene mapping in systems where the metaphase chromosomes are very small (e.g., Drosophila, avian microchromosomes,...
Murine 3T3L1 preadipocytes transformed by avian sarcoma virus were unable to differentiate in response to insulin or dexamethasone plus 1-methyl-3-isobutylxanthine, both potent inducers of differentiation of the nontransformed 3T3L1 parental line. Conditioned medium from transformed cells contained a relatively heat-stable factor(s) which inhibited the differentiation of untransformed parental 3T3L1 cells but did not induce any changes in their morphology. A protease-sensitive mitogen was also detected in the medium. The relationship between the two activities remains to be elucidated.
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