共查询到20条相似文献,搜索用时 15 毫秒
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Drosophila polytene chromosomes were transformed into lampbrush-like structures by exposure to solutions of alkali-urea. In this process, the chromosomes shorten and widen, and the bands (chromomeres) extend laterally into loops leaving a central core between the paired homologues. The expanded polytene chromosomes are very similar in appearance to the true lampbrush chromosomes of amphibian oocytes and to ordinary chromosomes in pachytene. The denaturing effects of alkali-urea were partially counteracted by return of the treated chromosomes to Ringer solution. These observations are interpreted in terms of recent findings on protein backbones in chromosomes, and indicate that chromosomes generally may have very similar basic organization, despite differences due to species, polyteny and degree of condensation. To gain more information on the specific location of a structural gene, 125I-labelled low molecular weight (containing 5S RNA) was hybridized in situ to normal and lampbrush-like polytene chromosomes. Autoradiography showed silver grain distribution for 5S RNA consistent with hybridization primarily to the loop regions of the lampbrush chromosomes rather than the core. This provides further indirect evidence that structural genes like 5S RNA may be located on the bands (chromomeres) and not the interbands of normal polytene chromosomes. 相似文献
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An antiserum directed against a non-histone chromosomal protein was found to stain preferentially the heterochromatic chromocenter of the polytene chromosomes of the salivary glands of several Drosophila species. The highly condensed α-heterochromatin stained most prominently. The chromocenter-associated antigen probably consists of a single polypeptide of molecular weight of 38000. This protein may play a role in the specific chromatin condensation of this chromosomal region. 相似文献
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Quantitative in situ hybridization of ribosomal RNA species to polytene chromosomes of Drosophila melanogaster. 总被引:3,自引:0,他引:3
In situ hybridization of 125I-labelled 5 S and 18 + 28 S ribosomal RNAs to the salivary polytene chromosomes of Drosophila melanogaster was successfully quantitated. Although the precision of the data is low, it is possible to compare the hybridization reaction between an RNA sample and chromosomes in situ with the reaction between the same RNA sample and Drosophila DNA immobilized on nitrocellulose filters. The in situ hybrid dissociates over a narrow temperature range with a midpoint similar to the value expected for the filter hybrid. The kinetics of the in situ hybridization reaction can be fit with a single first-order rate constant that has a value from three to five times smaller than the corresponding filter hybridization reaction. Although the reaction saturates at longer times or higher RNA concentrations, the saturation value does not correspond to an RNA molecule bound to every available DNA sequence. With the acid denaturation procedure most commonly used to preserve cytological quality, only 5 to 10% of the complementary DNA in the chromosomes is available to form hybrids in situ. This hybridization efficiency is a function of how the slides are prepared and the conditions of annealing, but is approximately constant with a given procedure for both 5 S RNA and 18 + 28 S RNA over a number of different cell types with different DNA contents. The results provide further evidence that the formation of RNA-DNA hybrids is the sole basis of in situ hybridization, and show that the properties of the in situ hybrids are remarkably similar to those of filter hybrids. It is also suggested that for reliable chromosomal localization using the in situ hybridization technique, the kinetics of the reaction should be followed to ensure that the correct rate constant is obtained for the major RNA species in the sample and an impurity in the sample is not localized instead. 相似文献
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J. G. J. Bauman J. Wiegant P. Van Duijn N. H. Lubsen P. J. A. Sondermeijer W. Hennig E. Kubli 《Chromosoma》1981,84(1):1-18
Fluorochrome-labeled RNA allows the rapid detection of in situ hybrids without the need for long exposure times as in the autoradiographical hybridisation methods. Resolution is high because of the high resolving power of fluorescence microscopy. The application of a previously reported method for the hybrido-cytochemical detection of DNA sequences to polytene chromosomes of Drosophilia is described. — The specificity and sensitivity of the method are demonstrated by the hybridisation with polytene chromosomes of 1) rhodamine-labeled 5S RNA, to the 5S rRNA sites of D. melanogaster (56F) and D. hydei (23 B), 2) rhodamine-labeled RNA complementary to a plasmid containing histone genes, to the 39DE region of D. melanogaster, 3) rhodamine-labeled D. melanogaster tRNA species (Gly-3 and Arg-2), to their respective loci in D. melanogaster, 4) rhodamine-labeled RNA complementary to the insert of plasmid 232.1 containing part of a D. melanogaster heat shock gene from locus 87 C, to D. hydei heat shock locus 2-32A. In the latter instance it was possible to demonstrate the labeling of a double band which escaped unambiguous detection by autoradiography in the radioactive cytochemical hybridisation procedure because of the low topological resolution of autoradiograms. — The sensitivity of the fluorochrome-labeled RNA method is compared with the radioactive methods which use 3H- or 125I-labeled RNAs. The factors governing the sensitivity and the number of bound fluorochrome molecules to be expected are discussed.Dedicated to Professor W. Beermann in honour of his 60th birthday 相似文献
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The presence of heterogeneous RNA of high molecular weight has been demonstrated on the giant chromosomes, in the nuclear sap and in the cytoplasm of the salivary glands in Chironomus tentans. The kinetic properties of this heterogeneous RNA have also been outlined in some detail. — Salivary glands were incubated for different time intervals (20, 45 and 180 min) in haemolymph, supplemented with tritiated cytidine and uridine. The different cellular components were isolated by micromanipulation and RNA extracted with an SDS-pronase solution and analysed with electrophoresis in agarose. — Heterogeneous, high molecular weight RNA with a peak around 35 S was saturated with label on chromosome I, II and III in 45 min, although the synthetic capacity was unchanged during at least 180 min incubation. This indicated a complete turnover of heterogeneous RNA on the chromosomes in less than 45 min. The turnover time in the giant puffs (the so called Balbiani rings) on the fourth chromosome, was even shorter and estimated to less than 30 min. No shift in the electrophoretic pattern of this heterogeneous RNA was found to occur on the chromosomes during long incubation times or during actinomycin D experiments. These labelling characteristics of heterogeneous RNA on the chromosomes indicate that all the different molecules in the heterogeneous RNA have a similar and rapid turnover. A conversion to smaller, stable molecules was excluded. — Heterogeneous RNA of a distribution corresponding to that on the chromosomes was found in the nuclear sap and also in the cytoplasm. The activity in both these cellular compartments increased between 45 and 180 min incubation. The distribution pattern for high molecular weight RNA was in all experiments similar on the chromosomes, in the nuclear sap and in the cytoplasm. It appears that at least a considerable part of the high molecular weight RNA leaves the chromosomes to enter the nuclear sap and lateron to some extent the cytoplasm in this high molecular form. Stable molecules of smaller size (6–15 S) did not appear during 180 min incubation. The data indicate, however, also a substantial breakdown of heterogeneous RNA to acid soluble products during this time. 相似文献
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Identification of left-handed Z-DNA by indirect immunofluorescence in polytene chromosomes of Chironomus thummi thummi 总被引:8,自引:0,他引:8
F. Lemeunier C. Derbin B. Malfoy M. Leng E. Taillandier 《Experimental cell research》1982,141(2):508-513
In this work, antibodies against Z-DNA were used to stain polytene chromosomes of Chironomus thummi thummi. By indirect immunofluorescence we report the first identification of left-handed conformation of DNA in a band region. The Chironomus pattern also contrasts with the general staining observed in Drosophila. In Chironomus the antibodies to Z-DNA bind to one interband region of the chromosome II and two bands regions of the chromosome IV. 相似文献
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The incorporation of 3H-uridine in different regions of polytene chromosomes in live cells of the Drosophila melanogaster salivary glands was compared with the incorporation of 3H-UTP in the same regions under the incubation of cytological preparations of these chromosomes with the E. coli RNA polymerase. The label distribution by regions was compared with the DNA content in them. Individual regions of chromosomes differ by 3H-uridine incorporation in live cells to a much greater extent than by 3H-UTP incorporation in vitro under the incubation with a non-homologous enzyme. RNA synthesis in an exogenous enzyme depends on the DNA content in different chromosome regions to a much greater extent than RNA synthesis in vivo. The correlation of label distribution after 3H-uridine incorporation in live cells and after RNA synthesis in vitro on the preparations by the bacterial RNA polymerase is, correspondingly, very low. This enzyme forms, however, RNA's on puffs 2-3 times more actively than on the same regions in non-puffing state but this difference is dozens of times greater in live cells. RNA synthesis in vitro is, thus, non-specific and does not correspond practically to the intensity of RNA synthesis on the same chromosome regions in live cells. At the same time, as in live cells, the E. coli enzyme synthesizes twice more RNA on the single X-chromosome of males (1X2A) than on each of X-chromosomes of diploid (2X2A) and triploid (3X3A) females or superfemales (3X2A), whereas in intersexes (2X3A) X-chromosomes display intermediate template activity. Thus, RNA synthesis by a heterologous enzyme in vitro does not differ by this index from the synthesis in live cells. It is suggested that differences in the template activity of X-chromosomes in vitro depending on the sex index (X : A) are due to different degree of DNP condensation in these chromosomes. In spite of differences in the degree of condensation, the male X-chromosome binds on the fixed preparation approximately the same amount of thymus histone F1 carrying fluorochrome as each of two female X-chromosomes. Hence, there is no sharp difference between the male and female X-chromosomes by the number and length of DNA regions accessible for interaction with exogenous proteins. On the basis of the data obtained, a hypothesis about two levels and, respectively, two mechanisms of control gene activity in animal chromosomes is considered. The first mechanism is, supposedly, based on decondensation of DNP appears to result in that the same proteins-regulators in the same amount activate corresponding genes in X-chromosome in males twice more strongly than in females. 相似文献
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Carlos Quijano Pavel Tomancak Jesus Lopez-Marti Mikita Suyama Peer Bork Marco Milan David Torrents Miguel Manzanares 《Genome biology》2009,9(12):R176
Background
The physical organization and chromosomal localization of genes within genomes is known to play an important role in their function. Most genes arise by duplication and move along the genome by random shuffling of DNA segments. Higher order structuring of the genome occurs in eukaryotes, where groups of physically linked genes are co-expressed. However, the contribution of gene duplication to gene order has not been analyzed in detail, as it is believed that co-expression due to recent duplicates would obscure other domains of co-expression. 相似文献13.
Labelled chromosomal RNA of the dipteran Chironomus tentans was studied with respect to its migration properties during electrophoresis in agarose. The RNA was isolated from polytene chromosomes which had been microdissected from fixed salivary glands and obtained free from nucleoli and nuclear sap. Labelled material migrates as 4–5 S RNA and as polydisperse material in a range where the lower limit corresponds to 10–15 S, the upper limit to 80–90 S RNA and the maximum in the distribution to 30–40 S RNA. The data indicate that the latter fractions are formed by unbroken, single-stranded RNA molecules, partly of very high molecular weights. It is shown in a number of tests that the distribution is not a consequence of formation of complexes or aggregates between RNA molecules on one hand and DNA, proteins or other RNA molecules on the other. 相似文献
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Modifications in the synthesis of salivary gland RNA were induced by treatments with 10 /ml cycloheximide (CHM) on 4th instar larvae of Chironomus pallidivitattus. After 3, 6 and 24 h CHM treatment, RNA was labeled in vitro, by incubating the salivary glands in a medium containing H3-uridine. The electrophoretical analyses corresponding to the 3 and 6 h treatment showed a stimulation of the non-ribosomal components of the newly synthesized RNA, while preribosomal RNA synthesis appeared depressed. This fact was also confirmed at cytological level, since autoradiograms made after 3 h of CHM treatment showed a reduced H3-uridine label over the nucleolus and an increase of diffuse labeling over the chromosomes. Longer treatments (24 h) causes a considerable inhibition of the synthesis of all RNA species. The role played by protein synthesis inhibition in the aforementioned effects is discussed. — Some of the morphological implications of CHM treatment, such as modifications of the nucleolar structure (nucleolar segregation) are also reported. The use of a squash technique based on glutaraldehyde fixation of the salivary glands, considerably facilitates such studies. 相似文献
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Cynthia K. Warner Deborah T. Watts Victoria Finnerty 《Molecular & general genetics : MGG》1980,180(2):449-453
Summary A reliable method for visualizing the Drosophila enzyme pyridoxal oxidase in polyacrylamide gels is described. Antiserum to pyridoxal oxidase has been produced and used in quantitative immunoelectrophoresis to determine the relative amounts of pyridoxal oxidase cross reacting material (CRM) in several mutants including lpo, lxd, ma-l and cin. The lpo variant did not have CRM for PO, thus further supporting the idea that it represents a structural gene for pyridoxal oxidase in Drosophila. CRM for PO was found in ma-l and lxd indicating that their effects upon the enzyme are probably post-translational. No CRM for PO could be found in the cin mutants.This work was supported by PHS grant GM 23736 to V. Finnerty 相似文献
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Drosophila salivary chromosomes have been isolated at neutral pH and physiological ionic strength. They display only background level binding of antibodies against Z-DNA. Following exposure to the commonly used fixative 45% acetic acid all of the polytene chromosomes, X and autosomes, show a massive increase in anti-Z-DNA antibody binding. The enhancement from background to intense fluorescence occurs whether the chromosomes are stabilised by two orders of magnitude lower concentration of formaldehyde than that used to minimise protein extraction in classical acid squash preparations, or by physiological concentrations of spermine and spermidine. Nicking of acetic acid-treated chromosomes by DNase I dramatically reduces their Z-DNA immunoreactivity. The histones and non-histones extracted by 45% acetic acid from unfixed and formaldehyde-fixed Drosophila chromatin have been analysed. Exposure of isolated salivary chromosomes to the non-protein-extracting fixative 95% ethanol also enhances Z-DNA immunoreactivity. All of these phenomena must be taken into account in the search for the Z-DNA conformation in cells by cytological techniques. 相似文献
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We have investigated the blood cell types present in Drosophila at postembryonic stages and have analysed their modifications during development and under immune conditions. The anterior lobes of the larval hematopoietic organ or lymph gland contain numerous active secretory cells, plasmatocytes, few crystal cells, and a number of undifferentiated prohemocytes. The posterior lobes contain essentially prohemocytes. The blood cell population in larval hemolymph differs and consists mainly of plasmatocytes which are phagocytes, and of a low percentage of crystal cells which reportedly play a role in humoral melanisation. We show that the cells in the lymph gland can differentiate into a given blood cell lineage when solicited. Under normal nonimmune conditions, we observe a massive differentiation into active macrophages at the onset of metamorphosis in all lobes. Simultaneously, circulating plasmatocytes modify their adhesion and phagocytic properties to become pupal macrophages. All phagocytic cells participate in metamorphosis by ingesting doomed larval tissues. The most dramatic effect on larval hematopoiesis was observed following infestation by a parasitoid wasp. Cells within all lymph gland lobes, including prohemocytes from posterior lobes, massively differentiate into a new cell type specifically devoted to encapsulation, the lamellocyte. 相似文献