Immunofluorescent characterization of DNA�RNA hybrids on polytene chromosomes of Trichosia pubescens (Diptera, Sciaridae)
| Autor: | José Mariano Amabis, O. Leoncini, Francisco Lara, W. Büsen, B D Stollar |
|---|---|
| Rok vydání: | 1982 |
| Předmět: |
Polytene chromosome
medicine.diagnostic_test Diptera Endoribonuclease Fluorescent Antibody Technique Nucleic Acid Hybridization RNA DNA Biology Immunofluorescence Molecular biology Chromosomes Salivary Glands Uridine Staining chemistry.chemical_compound chemistry Transcription (biology) Larva Genetics medicine Animals Genetics (clinical) |
| Zdroj: | Chromosoma. 87:247-262 |
| ISSN: | 1432-0886 0009-5915 |
| DOI: | 10.1007/bf00327628 |
| Popis: | We have studied the distribution of DNA X RNA hybrids on polytene chromosomes with the aid of a goat antibody against DNA X RNA hybrids using the immunofluorescence technique. Fixed polytene chromosomes of the sciarid Trichosia pubescens (Diptera) show distinct, stage-specific labelling patterns throughout larval development. Controls for the staining procedure - including preincubation with hybrid-specific endoribonuclease H - prove that DNA X RNA hybrids are present on fixed chromosomes. They are revealed only under mild fixation conditions which do not efficiently immobilize all chromosomal proteins, indicating that some proteins have to be removed to make the antigens accessible to antibody. Certain fixation conditions may also cause local denaturation of chromosomal DNA, and some hybrids may possibly form during specimen preparation. After incorporation of radioactive uridine, a combination of phase contrast, fluorescent, and autoradiographic images of one and the same chromosomal preparation demonstrates that hybrid fluorescence is confined to transcriptionally active regions. Two puff classes can be distinguished. The first binds antibody and includes most RNA puffs and all DNA puffs so far studied; the second, comprising some RNA puffs, does not show bright fluorescence in spite of the fact that RNA synthesis is high as revealed by 3H-uridine incorporation. DNA X RNA hybrids are not found at DNA puff sites during the DNA amplification period; these sites contain detectable hybrids only when transcription is taking place. - Combination of the fluorescent technique with its excellent resolution and autoradiography should be helpful in studying detailed topological aspects of transcriptionally active chromosomal regions. |
| Databáze: | OpenAIRE |
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