Arrangement of the highly reiterated DNA sequences in the centric heterochromatin of Drosophila melanogaster evidence for interspersed spacer DNA

Kram, Raphaël; M, Botchan; Hearst, John E.

doi:10.1016/0022-2836(72)90323-3

Cited by 74 publications

(18 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Taken in concert with the in situ evidence of previously mentioned workers, as well as our own, and evidence provided by alternative approaches (17)(18)(19)(20)(21), the pattern of association of repeated DNA sequences and constitutive heterochromatin is becoming increasingly well established. With the coordination of preparative ultracentrifugation, and of in vitro and in situ hybridization, we should soon understand not only the extent but also the significance of the association of repeated DNA with constitutive heterochromatin.…”

supporting

confidence: 64%

Repeated DNA Sequences in the Heterochromatic Y Chromosome of Adult Drosophila melanogaster

Perreault

Kaufmann

Gay

1973

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

DNA isolated from Drosophila melanogaster adults (XX, XY, XXY, XYY) was used in DNA-DNA hybridization experiments on nitrocellulose filters. Filter-bound DNA of "high-heterochromatin" flies (those with one or more Y chromosomes) is more effective in forming hybrid duplexes than is XX DNA. The quantitative difference in hybridization efficiency is due primarily to molecules with a relatively high thermal stability (duplexes that dissociate in the temperature range 8090°). Hybridization experiments with DNA samples that have been fractionated into reiterated and unique portions show that the majority of the hybrid duplexes formed involve reiterated DNA. A small, but highly specific, interaction of unique DNA sequences has also been detected in our experiments. These data indicate that a class of repeated DNA sequences is associated with the constitutive heterochromatin, specifically with the Y chromosome of D. melanogaster. Evidence is presented that this same class of molecules, or very similar ones, is to be found in other portions of the genome, presumably in X-chromosomal or centromeric heterochromatin.Recent reports (1-4) have demonstrated a marked capacity of the centromeric regions of Drosophila chromosomes to form hybrid molecules with complementary RNA (c-RNA) transcribed from the rapidly reassociating, repetitious portion of the genome. All of these reports have used the technique of in situ hybridization, developed within the past few years by workers in several different laboratories. We wish to report the results of a series of in vitro hybridization experiments that indicate that Drosophila melanogaster DNA isolated from "high-heterochromatin flies" (i.e., those with one or more Y chromosomes) contains more of a class of repeated DNA sequences than does DNA isolated from "low-heterochromatin" XX females. We have found that the repeated DNA sequences that form hybrid duplexes in our experiments can be derived from either XY or XX flies, and are, therefore, not exclusively Y chromosomal in origin. Finally, these experiments also show that it is possible to use DNA-DNA hybridization techniques on nitrocellulose filters to distinguish between DNA sequences isolated from the unique portion of the genome of D. melanogaster females (XX) and males (XY).D. melanogaster is uniquely suited for studies of the properties of heterochromatin and euchromatin. In this organism, as in no other, we have a wealth of knowledge about the cy-

show abstract

supporting

confidence: 64%

Repeated DNA Sequences in the Heterochromatic Y Chromosome of Adult Drosophila melanogaster

Perreault

Kaufmann

Gay

1973

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…DNA, prepared from HTC cells (9) (2). Then, mixtures containing 0.4-ml portions of this cytosol were mixed with one volume of nuclear suspension and transferred to a bath at 200.…”

Section: Methodsmentioning

confidence: 99%

Role of DNA and Specific Cytoplasmic Receptors in Glucocorticoid Action

Baxter

Rousseau

Benson

et al. 1972

Proc. Natl. Acad. Sci. U.S.A.

207

View full text Add to dashboard Cite

Glucocorticoids induce tyrosine aminotransferase (EC 2.6.1.5) synthesis in cultured rat hepatoma cells. These steroids penetrate the cell membrane and bind to specific cytoplasmic receptor proteins. The resulting complex binds to the nucleus. This nuclear binding has nowlbeen studied in a cell-free preparation. The reaction appears to require a temperature-dependent modification of the steroid-receptor complex. There is a fixed number of nuclear sites that are half saturated at a complex concentration of 6 to 24 X 10-11 M. Treatment with deoxyribonuclease destroys nuclear-binding capacity. The complex also binds to purified HTC cell DNA with characteristics similar to the binding to isolated nuclei, and, as in intact cells,' receptors complexed with an anti-inducer steroid bind very poorly to DNA. These data suggest that the nuclear sites for binding steroid-receptor complexes are on the DNA. Since the extent of complex binding to purified DNA exceeds that observed with isolated nuclei, chromosomal proteins may act to restrict binding to certain reg'ions of the DNA. These studies suggest that steroid hormones stimulate the synthesis of specific proteins by affecting the transcription of structural or regulatory genes.Induction of tyrosine aminotransferase (EC 2.6.1.5) by steroid hormones in hepatoma tissue culture (HTC) cells is being studied to understand the mechanism of gene regulation in mammals (1). HTC cell cytosol contains specific steroid "receptor" proteins that appear to mediate the induction (2-5). When intact cells are exposed to inducer steroids, specifically bound hormone is predominantly found in the nuclear fraction (5). From other studies we concluded that this is due to accumulation of steroid-cytoplasmic receptor complexes in the nucleus (4). This mechanism, therefore, closely resembles that proposed for systems responsive to other steroid hormones [for review see Jensen et al. (6)]. To account for the effects of inducer, anti-inducer, and suboptimal inducer steroids, we have proposed (7) that the cytoplasmic receptor is an allosteric protein that has different conformations depending on the type of steroid with which it is complexed. In support of this idea we found that inducer-bound receptors differ from those that are uncomplexed or are bound by an anti-inducer (3, 4) and that, in intact cells, inducerreceptor complexes accumulate in the nucleus, whereas antiinducer-receptor complexes remain in the cytosol (4, 5).Thus, the biologic actions of steroids could depend on their capacity to alter the allosteric equilibrium between different conformations of the cytoplasmic receptors.In this report, some characteristics of the nuclear binding reaction, studied in a cell-free preparation, are presented.Abbreviation: HTC cells, hepatoma tissue culture cells.Inducer steroid-receptor complexes bind with high affinity to a limited number of DNA-containing nuclear sites. MATERIALS AND METHODSHTC cells, grown at 370 in suspension cultures to 2 to 10 X 105 cells/ml (8), were washed by centrifugat...

show abstract

“…Recently Kram et al (1972) BUOYANT DENSITY (g/cm 3) FIGUR• 6 Buoyant density in CsCI of DNA isolated from the 13,000 g, crude mitoehondrial pellet of unfertilized D. melanogaster eggs. M. lysodeikticus DNA was used as a density standard, neutral p = 1.731 g/cm 3 and alkaline p = 1.789 g/cm 3. a.…”

Section: Melanogastermentioning

confidence: 99%

ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Polan

Friedman

Gall

et al. 1973

The Journal of Cell Biology

View full text Add to dashboard Cite

A B S T R A C TMitochondrial DNA (MtDNA) with a neutral buoyant density of 1.681 g/cm a has been isolated from unfertilized eggs of Drosophila mdanogaster. This DNA is a circular molecule with an average length of 5.3 /~m; it reassociates with a low Coil~2 after denaturation, and in alkaline isopycnic centrifugation it separates into strands differing in density by 0.005 g/cm 3 MtDNA isolated from purified mitochondria of unfertilized eggs or from total larval DNA melts with three distinct thermal transitions. The three melting temperature values suggest that the molecule may have three regions differing in average base composition. DNA isolated from unfertilized eggs of D. melanogaster contains approximately equal amounts of MtDNA and another DNA with a buoyant density of 1.697 g/cm a, slightly less dense than main peak DNA. The possibility that the heavier DNA fraction consists of amplified ribosomal DNA was excluded by hybridization experiments, but otherwise nothing is known of its origin or function. I N T R O D U C T I O NMitochondrial DNA (MtDNA) 1 from several higher organisms renatures rapidly (Corneo et al., 1966;Borst and Ruttenberg, 1966;Nass, 1966;Kroon et al., 1966; Dawid and Wolstenholme, 1968), is circular (Van Bruggen et al., 1966;Sinclair and Stevens, 1966; Wolstenholmc and Dawid, 1967), and shows strand separation in alkaline isopycnic centrifugation (Dawid and Wolstenholme, 1967; Leftter et al., 1970;Corneo et al., 1968). We report here the isolation of Mt-DNA from Drosophila melanogaster and its charac1Abbreviations used in this paper: EBr, ethidium bromide; EDTA, ethylenediaminetetraacetate; MtDNA, mitochondrial DNA; rDNA, the DNA coding for ribosomal RNA; rRNA, ribosomal RNA; SSC, 0.15 M NaCI, 0.015 M Na-citrate, pH 7.0; T,,, melting temperature.terization by isopycnic centrifugation, thermal denaturation, and electron microscopy.MtDNA from D. melanogaster is a circular molecule, renatures rapidly, and separates into two strands in alkaline CsC1. However, it has unusual thermal denaturation properties. MtDNA from Rana pipiens, Xenopus laevis (Dawid, 1965), and sheep heart shows only one transition on thermal denaturation. Leffier et al. (1970) have reported a biphasic melting curve for rat liver MtDNA and correlated the transitions with sequential denaturation of nicked and intact circles. Renatured rat liver MtDNA melted with a single transition. Wolstenholme et al. (1972) have observed the same phenomenon and, using the electron microscope, have identified at least three specific regions of rat liver MtDNA which melt 1°-2°C below the rest of the circle. We have ob-$80

show abstract

Arrangement of the highly reiterated DNA sequences in the centric heterochromatin of Drosophila melanogaster evidence for interspersed spacer DNA

Cited by 74 publications

References 31 publications

Repeated DNA Sequences in the Heterochromatic Y Chromosome of Adult Drosophila melanogaster

Repeated DNA Sequences in the Heterochromatic Y Chromosome of Adult Drosophila melanogaster

Role of DNA and Specific Cytoplasmic Receptors in Glucocorticoid Action

ISOLATION AND CHARACTERIZATION OF MITOCHONDRIAL DNA FROM DROSOPHILA MELANOGASTER

Contact Info

Product

Resources

About