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Journal ArticleDOI

Replication of DNA in larval salivary glands of Drosophila after in vivo synchronization

01 Jan 1981-Chromosoma (Chromosoma)-Vol. 82, Iss: 4, pp 505-514
TL;DR: The replication of DNA in polytene chromosomes of Drosophila seems to proceed in a regular sequence of DD-3C-1D, where 3C is the mid-replication phase and 1D is the initial phase.
Abstract: The replication of DNA in the giant chromosomes in different cells of Drosophila larval salivary glands is asynchronous. A method of in vivo synchronization of the nuclei has been successfully devised by a 5′-fluorodeoxyuridine (FdU) block-release-thymidine chase technique, and the patterns of replication sequences have been examined by 3H-thymidine autoradiography. When the larvae of Drosophila melanogaster are fed on FdU for 48 h, and the block is released thereafter, most cells are found in mid-replication phase (termed 3C). When the larvae are subjected to a chase in normal Drosophila medium (or sucrose), a series of cells arrive at 3C phase about every 8 h. When they are chased in sucrose containing thymidine, the number of cells in 3C phase rises to 70%, and then drops rapidly to 1–2% of all labelled cells. The terminal phases (3D, 2D and 1D) reach a peak between 4–8 h. At 12–14 h of chase the 3D-1D peaks decline and a third peak consisting mostly of the initial phases (DD-1C) is found at 14–16 h. The replication of DNA in polytene chromosomes of Drosophila thus seems to proceed in a regular sequence of DD-3C-1D.
Citations
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Journal ArticleDOI
TL;DR: Five different patterns in immunoperoxidase-stained monolayers of human and rodent cancer cells are defined and compared mean nuclear areas as measured by computerized planimetry indicate that the observed patterns reflect the spatial and temporal organization of DNA synthesis, which seems to be characterized by at least three successive stages of replication.
Abstract: After pulse labeling of mammalian cells in vivo or in vitro with 5-bromodeoxyuridine (BrdUrd), followed by immunostaining with a monoclonal antibody to DNA-incorporated BrdUrd, various intranuclear staining patterns are observed. These results are obtained in various labeling, fixation, denaturation, and staining conditions. We defined five different patterns in immunoperoxidase-stained monolayers of human and rodent cancer cells and compared mean nuclear areas as measured by computerized planimetry. Furthermore, we evaluated frequency distributions of these patterns in partly synchronized cell populations and correlated these results with flow cytometric DNA histograms. The results indicate that the observed patterns reflect the spatial and temporal organization of DNA synthesis, which seems to be characterized by at least three successive stages of replication. Evaluation of these patterns may have various applications in studies on cell cycle kinetics.

102 citations

Journal ArticleDOI
TL;DR: Immunochemical detection of BrdUrd can be used to determine proliferative characteristics of differentiating tissues and to obtain birth dates for actual differentiation events and to monitor DNA repair.
Abstract: This paper is a continuation of parts I (history, methods and cell kinetics) and II (clinical applications and carcinogenesis) published previously (Dolbeare, 1995Histochem. J. 27, 339, 923). Incorporation of bromodeoxyuridine (BrdUrd) into DNA is used to measure proliferation in normal, diseased and injured tissue and to follow the effect of growth factors. Immunochemical detection of BrdUrd can be used to determine proliferative characteristics of differentiating tissues and to obtain birth dates for actual differentiation events. Studies are also described in which BrdUrd is used follow the order of DNA replication in specific chromasomes, DNA replication sites in the nucleus and to monitor DNA repair. BrdUrd incorporation has been used as a tool forin situ hybridization experiments.

98 citations

Journal ArticleDOI
TL;DR: The sequence of activation of the hormone-induced puffs conforms to a cascade pattern, and the nucleoli, Balbiani rings, and DNA puffs are specific types of active regions in the polytene chromosomes.
Abstract: Publisher Summary The interphase polytene chromosome is morphologically differentiated into tightly compacted (the chromomeric) and decompacted (the interchomomeric) regions, as the chromatid from telomere to telomere has the appearance of a string with threaded beads, the chromomeres. During the activation of transcription of genes located within chromomeres, the constituent material of a chromomere loosens and a large swelling, the puff, is formed. Study of the sequence of the appearance of puffs in the course of development and other experiments have led to the concept that hormones are involved in regulating puff activity. Thus, the sequence of activation of the hormone-induced puffs conforms to a cascade pattern. The nucleoli, Balbiani rings, and DNA puffs are specific types of active regions in the polytene chromosomes. Their morphology differs to a large extent from that of the usual puffs. The nucleolar organizer contains a gene block, organized in a complex manner and encoding ribosomal RNA. Balbiani rings are composed of numerous repetitive elements, and they encode RNA molecules of a giant size. The amplification of DNA takes place in the DNA puffs.

52 citations

Journal ArticleDOI
TL;DR: Observations on autoradiographic labelling of partially lysed polytene chromosomes provide evidence for a lack of temporal and spatial agreement in the activation of origin points in homologous regions of the lateralPolytene strands; these observations also suggest local variations in levels of polyteny within a chromosome.
Abstract: It is widely known that the bulk of the pericentromeric heterochromatin (α-heterochromatin) does not replicate during polytenization in Drosophila. However, a recent DNA-Feulgen cytophotometric study (Dennhofer 1982a) has claimed equal polytenization of all heterochromatin regions. To re-examine this issue, the amount of Hoechst 33258-bright heterochromatin in non-polytene and polytene nuclei in salivary glands and Malpighian tubules of late third instar larvae of D. nasuta has been compared by cytofluorometry. Since the amount of Hoechst 33258-bright heterochromatin is similar in non-polytene and polytene nuclei in spite of the latter having an enormously high euchromatin DNA content, it is concluded that the α-heterochromatin does not replicate during polytenization. The present results further indicate that in the polytene nuclei of Malpighian tubules the α-heterochromatin remains at the 2C level whereas in salivary gland polytene nuclei it varies between the 2C and 4C levels.

31 citations

Journal ArticleDOI
TL;DR: In this article, DNA fiber autoradiography of highly polytenized nuclei in salivary glands of Drosophila nasuta larvae reveals two distinct types of active replicons: type I replicons are longer (mean size = 64 gin), have a very high rate of fork migration (average rate = 0.95 gm/min) and generally occur in large arrays often extending over several thousand tim.
Abstract: DNA fibre autoradiography of highly polytenized nuclei in salivary glands of Drosophila nasuta larvae reveals two distinct types of active replicons. Type I replicons are longer (mean size= 64 gin), have a very high rate of fork migration (average rate = 0.95 gm/min) and generally occur in large arrays often extending over several thousand tim. In contrast, the type II replicons are smaller (mean size = 20 gm), slow replicating (average rate = 0.07 gin/rain) and occur in short arrays containing only a few closely spaced active replicons. Evidence is presented that type I replicons are active in the early S and type II in the late S. Observa- tions on autoradiographic labelling of partially lysed poly- tene chromosomes provide evidence for a lack of temporal and spatial agreement in the activation of origin points in homologous regions of the lateral polytene strands; these observations also suggest local variations in levels of poly- teny within a chromosome. On the basis of this and other available information on replication in polytene chromo- somes the possible roles of the two replicon types in the generation of the different 3H-thymidine labelling patterns of polytene chromosomes are discussed.

24 citations

References
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Journal ArticleDOI
01 May 1972-Genetics
TL;DR: It is postulated that a chromomere is one cistron within which much of the DNA is regulatory in function.
Abstract: An average size chromomere of the polytene X chromosome of Drosophila melanogaster contains enough DNA in each haploid equivalent strand to code for 30 genes, each 1,000 nucleotides long. We have attempted to learn about the organization of chromosomes by asking how many functional units can be localized within a chromomere. This was done by 1) recovery of mutants representative of every cistron in the 3A2-3C2 region; 2) the characterization of the function of each mutant type and grouping by complementation tests; 3) the determination of the genetic and cytological position of each cistron by recombination and deletion mapping. The data clearly show one functional group per chromomere. It is postulated that a chromomere is one cistron within which much of the DNA is regulatory in function.

343 citations

Journal ArticleDOI
TL;DR: A complete reconstruction of the synaptonemal complex in 12 pachytene nuclei from one wild-type germarium has permitted the following observations as discussed by the authors : 1) Drosophila melanogaster bivalents exhibit a chromocentral arrangement; the pericentric heterochromatin of all bivalent lies in one region of the nucleus, the chromocenter.
Abstract: Complete reconstruction of the synaptonemal complex in 12 pachytene (defined here as that stage in which the synaptonemal complex is continuous throughout the bivalents) nuclei from one wild-type germarium has permitted the following observations. 1) Drosophila melanogaster bivalents at pachytene exhibit a chromocentral arrangement; the pericentric heterochromatin of all bivalents lies in one region of the nucleus, the chromocenter. Telomeric ends do not appear to abutt the nuclear envelope. 2) Synaptonemal complex is present in the pericentric heterochromatin; however, it is morphologically distinct from that present in the euchromatic portion of thesynaptonemal complex of the bivalent arms is greatest at early pachytene; the synaptonemal complex then becomes progressively shorter. Minimum length is approximately one-half of the maximum. 4) Decrease in length of synaptonemal complex is accompanied by an increase in thickness. Reconstruction of 20 pachytene nuclei from an additional 8 germaria suggests that these observations are typical. Correlations between these cytological observations and genetic observations (e.g., patterns of crossing-over) are discussed.

269 citations

Journal ArticleDOI
TL;DR: Indications were obtained that during puff formation acidic protein accumulation precedes the onset of RNA synthesis, and release of newly synthesized RNA from puffs in which RNA synthesis was inhibited by actinomycin D at a stage of maximal activity.
Abstract: In order to separate some of the factors involved in the formation of puffs the antibiotic actinomycin D was applied at different stages of puff activity. Puffs were induced by temperature shocks or eodysone. Inhibition of RNA synthesis with actinomycin D before application of a puff inducing stimulus prevents neither the appearance of the stimulus specific puffs nor the accumulation of acidic proteins in the puff regions. The puffs attained under these conditions approximately 1/3 of the size normally produced by the stimulus. Indications were obtained that during puff formation acidic protein accumulation precedes the onset of RNA synthesis. Synthesis and storage of newly synthesized RNA within the puff region was studied on the basis of grain distribution in uridine-H3 autoradiographs after various incubation periods. RNA synthesis appears to be restricted to a particular area of the puff region. After a 3 min temperature shock following injection of uridine-H3 silver grains are located only over a particular area of the newly formed puff. The same area becomes labeled during a 1 min pulse of uridine-H3 applied at a stage of maximum puff development. Longer periods of incubation result in a random distribution of the grains over the whole puff region. Grain counts on different areas of experimentally induced puffs and on the same areas at a stage of puff regression indicate that the newly synthesized RNA becomes transferred from the area where it was synthesized and is stored for a certain period within the puff region. Complete release of newly synthesized RNA from puffs in which RNA synthesis was inhibited by actinomycin D at a stage of maximal activity is accomplished within 30 to 35 min.

156 citations

Book ChapterDOI
01 Jan 1964

114 citations


"Replication of DNA in larval saliva..." refers background in this paper

  • ...In some cells the chromosomes are labelled very diffusely on the interbands and puffs, in some they are continuously labelled and in others label may be highly localized, discontinuous and restricted to thick bands, proximal heterochromatin and often telomeres (Plaut et al., 1964; Rodman, 1968 ; Lakhotia and Mukherjee, 1970; Arcos-Teran, 1972; Kalisch and H/igele, 1973; H/igele and Kalisch, 1974; Chatterjee and Mukherjee, 1975; Chatterjee and Mukherjee, 1977)....

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Journal Article
01 Jan 1969-Genetics

114 citations