DNA Supercoiling Factor Localizes to Puffs on Polytene Chromosomes in Drosophila melanogaster

ABSTRACT DNA supercoiling factor (SCF) was first identified in silkworm as a protein that generates negative supercoils in DNA in conjunction with eukaryotic topoisomerase II. To analyze the in vivo role of the factor, we cloned a cDNA encoding Drosophila melanogaster SCF. Northern analysis revealed 1.6- and 1.8-kb mRNAs throughout development. The longer mRNA contains an open reading frame that shares homology with mouse reticulocalbin whereas the shorter one encodes a truncated version lacking the N-terminal signal peptide-like sequence. An antibody against SCF detected a 45-kDa protein in the cytoplasmic fraction and a 30-kDa protein in the nuclear fraction of embryonic extracts. Immunoprecipitation suggests that the 30-kDa protein interacts with topoisomerase II in the nucleus, and hence that it is a functional form of SCF. Immunostaining of blastoderm embryos showed that SCF is present in nuclei during interphase but is excluded from mitotic chromosomes. In larvae, the antibody stained the nuclei of several tissues including a posterior part of the salivary gland. This latter staining was associated with natural or ecdysteroid-induced puffs on polytene chromosomes. Upon heat treatment of larvae, the staining on the endogenous puffs disappeared, and strong staining appeared on heat shock puffs. These results implicate SCF in gene expression.

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A unique ribonucleoprotein complex assembles preferentially on ecdysone-responsive sites in Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5323 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5323-5330 ◽

Cited By ~ 23

Author(s):

S A Amero ◽

M J Matunis ◽

E L Matunis ◽

J W Hockensmith ◽

G Raychaudhuri ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Polytene Chromosomes ◽

Cell Extract ◽

Sequence Motifs ◽

Drosophila Cell ◽

Rnase Digestion

The protein on ecdysone puffs (PEP) is associated preferentially with active ecdysone-inducible puffs on Drosophila polytene chromosomes and contains sequence motifs characteristic of transcription factors and RNA-binding proteins (S. A. Amero, S. C. R. Elgin, and A. L. Beyer, Genes Dev. 5:188-200, 1991). PEP is associated with RNA in vivo, as demonstrated here by the sensitivity of PEP-specific chromosomal immunostaining in situ to RNase digestion and by the immunopurification of PEP in Drosophila cell extract with heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As revealed by sequential immunostaining, PEP is found on a subset of chromosomal sites bound by the HRB (heterogeneous nuclear RNA-binding) proteins, which are basic Drosophila hnRNPs. These observations lead us to suggest that a unique, PEP-containing hnRNP complex assembles preferentially on the transcripts of ecdysone-regulated genes in Drosophila melanogaster presumably to expedite the transcription and/or processing of these transcripts.

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Chromosomal distribution of the major insert in Drosophila melanogaster 28S rRNA genes

Genetics Research ◽

10.1017/s0016672300020176 ◽

1981 ◽

Vol 37 (2) ◽

pp. 209-214 ◽

Cited By ~ 35

Author(s):

W. J. Peacock ◽

R. Appels ◽

S. Endow ◽

D. Glover

Keyword(s):

Drosophila Melanogaster ◽

Polytene Chromosomes ◽

Ribosomal Gene ◽

Rrna Genes ◽

Major Type ◽

Type I ◽

28S Rrna ◽

Mitotic Chromosomes ◽

Chromosomal Distribution

SUMMARYThe major type I insert sequence for the 28S rRNA genes of Drosophila melanogaster has been mapped within the chromosomes using a probe synthesized from a cloned sequence containing the entire 5·4 kb segment. The genomic distribution was shown to be complex in that the insert sequence occurred next to many different types of sequences, in addition to occurring as an insert in the 28S rRNA genes of the X chromosome. In situ hybridization of mitotic chromosomes showed most of the insert units not contained in the ribosomal genes to be located near the ribosomal gene cluster on the X chromosome. Additional sites were detected in polytene chromosomes in region 102C, 8–12 and in the hetero-chromatin of the autosomes.

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The 190 kDa centrosome-associated protein of Drosophila melanogaster contains four zinc finger motifs and binds to specific sites on polytene chromosomes

Journal of Cell Science ◽

10.1242/jcs.108.11.3377 ◽

1995 ◽

Vol 108 (11) ◽

pp. 3377-3387 ◽

Cited By ~ 9

Author(s):

W.G. Whitfield ◽

M.A. Chaplin ◽

K. Oegema ◽

H. Parry ◽

D.M. Glover

Keyword(s):

Drosophila Melanogaster ◽

Zinc Finger ◽

Polytene Chromosomes ◽

Predicted Amino Acid Sequence ◽

Reading Frame ◽

Zinc Finger Motifs ◽

Drosophila Larvae ◽

Indirect Immunofluorescence Staining ◽

Cycle Dependent ◽

Cp190 Protein

Microinjection of a bacterially expressed, TRITC labelled fragment of the centrosome-associated protein CP190 of Drosophila melanogaster, into syncytial Drosophila embryos, shows it to associate with the centrosomes during mitosis, and to relocate to chromatin during interphase. Indirect immunofluorescence staining of salivary gland chromosomes of third instar Drosophila larvae, with antibodies specific to CP190, indicate that the protein is associated with a large number of loci on these interphase polytene chromosomes. The 190 kDa CP190 protein is encoded by a 4.1 kb transcript with a single, long open reading frame specifying a polypeptide of 1,096 amino acids, with a molecular mass of 120 kDa, and an isoelectric point of 4.5. The central region of the predicted amino acid sequence of the CP190 protein contains four CysX2CysX12HisX4His zinc-finger motifs which are similar to those described for several well characterised DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent associations with both the centrosome, and with specific chromosomal loci.

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Centromeric DNA cloned from functional kinetochore fragments in mitotic cells with unreplicated genomes

Journal of Cell Science ◽

10.1242/jcs.105.2.359 ◽

1993 ◽

Vol 105 (2) ◽

pp. 359-367 ◽

Cited By ~ 2

Author(s):

I.I. Ouspenski ◽

B.R. Brinkley

Keyword(s):

Topoisomerase Ii ◽

Chinese Hamster ◽

Single Copy ◽

Chromosome 1 ◽

Sequence Motifs ◽

Mitotic Chromosomes ◽

Distinctive Features ◽

Repetitive Structure ◽

Species Specific

Treatment of cells arrested in the cell cycle at the G1/S-phase boundary with 5 mM caffeine induces premature mitosis, resulting in chromosomal fragmentation and detachment of centromere-kinetochore fragments, which are subsequently attached to the mitotic spindle and segregated in anaphase. Taking advantage of this in vivo separation of the centromere, we have developed a procedure for isolation of a centromere-enriched fraction of mitotic chromatin. Using this method, we have isolated and cloned DNA from the centromere-enriched material of Chinese hamster cells. One of the clones thus obtained was characterized in detail. It contains 6 kb of centromere-associated sequence that exhibits no recognizable homology with other mammalian centromeric sequences and is devoid of any extensive repetitive structure. This sequence is present in a single copy on chromosome 1 and is species-specific. Distinctive features of the clone include the presence of several A+T-rich regions and clusters of multiple topoisomerase II consensus cleavage sites and other sequence motifs characteristic of nuclear matrix-associated regions. We hypothesize that these features might be related to the more compact packaging of centromeric chromatin in interphase nuclei and mitotic chromosomes.

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The Drosophila melanogaster dodecasatellite sequence is closely linked to the centromere and can form connections between sister chromatids during mitosis

Journal of Cell Science ◽

10.1242/jcs.105.1.41 ◽

1993 ◽

Vol 105 (1) ◽

pp. 41-50 ◽

Cited By ~ 4

Author(s):

M. Carmena ◽

J.P. Abad ◽

A. Villasante ◽

C. Gonzalez

Keyword(s):

Cell Cycle ◽

Drosophila Melanogaster ◽

In Situ Hybridisation ◽

Polytene Chromosomes ◽

Colchicine Treatment ◽

Dependent Manner ◽

Fluorescence In Situ Hybridisation ◽

Mitotic Chromosomes ◽

Sister Chromatids ◽

Diploid Cells

We have used fluorescence in situ hybridisation to wild-type and rearranged mitotic chromosomes to map the Drosophila melanogaster dodecasatellite sequence. It is located at a unique site, within the pericentric heterochromatin of the right arm of the third chromosome, closely linked to the primary constriction. In polytene chromosomes, dodecasatellite is found as one or a few dots in the central region of the chromocentre. In untreated diploid cells, dodecasatellite sequences are found as one or two dots throughout the cell cycle. This distribution can be altered in a cell cycle-dependent manner in two ways. Firstly, in interphase cells, hypotonic shock promotes the decondensation of the genomic region containing this satellite, resulting in a string-like structure. Secondly, some of the precociously separated sister chromatids produced by colchicine treatment show dodecasatellite within the intervening space connecting the main dodecasatellite signals of each chromatid. The distribution of dodecasatellite seems to be rather constant between individuals of the same species, as indicated by the lack of any detectable variations in its pattern amongst individuals from six geographically distant strains of D. melanogaster. On the other hand, the distribution of dodecasatellite shows a remarkable degree of variation amongst closely related species of the melanogaster subgroup ranging from a non-detectable signal in Drosophila yakuba and Drosophila teissieri, to staining in the X, second and third chromososomes of Drosophila mauritiana.

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Topoisomerase II Inhibitors Influence Simian Virus 40 Chromatin Structure in vivo Accompanied with Inhibition of Replication, Transcription and Changes in DNA Supercoiling

Oncology ◽

10.1159/000226543 ◽

1988 ◽

Vol 45 (2) ◽

pp. 107-116 ◽

Cited By ~ 3

Author(s):

Waldemar Waldeck ◽

Hanswalter Zentgraf ◽

Frank Rösl

Keyword(s):

Chromatin Structure ◽

Topoisomerase Ii ◽

Simian Virus 40 ◽

Dna Supercoiling ◽

Simian Virus ◽

Topoisomerase Ii Inhibitors

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Arginine-Rich Regions Mediate the RNA Binding and Regulatory Activities of the Protein Encoded by the Drosophila melanogaster suppressor of sable Gene

Molecular and Cellular Biology ◽

10.1128/mcb.20.21.8198-8208.2000 ◽

2000 ◽

Vol 20 (21) ◽

pp. 8198-8208 ◽

Cited By ~ 7

Author(s):

Michael A. Turnage ◽

Paul Brewer-Jensen ◽

Wen-Li Bai ◽

Lillie L. Searles

Keyword(s):

Drosophila Melanogaster ◽

Rna Binding ◽

Consensus Sequence ◽

Polytene Chromosomes ◽

Ectopic Expression ◽

Binding Activity ◽

Suppressor Of Sable ◽

Rna Accumulation

ABSTRACT The Drosophila melanogaster suppressor of sable gene,su(s), encodes a novel, 150-kDa nuclear RNA binding protein, SU(S), that negatively regulates RNA accumulation from mutant alleles of other genes that have transposon insertions in the 5′ transcribed region. In this study, we delineated the RNA binding domain of SU(S) and evaluated its relevance to SU(S) function in vivo. As a result, we have defined two arginine-rich motifs (ARM1 and ARM2) that mediate the RNA binding activity of SU(S). ARM1 is required for in vitro high-affinity binding of SU(S) to small RNAs that were previously isolated by SELEX (binding site selection assay) and that contain a common consensus sequence. ARM1 is also required for the association of SU(S) with larval polytene chromosomes in vivo. ARM2 promotes binding of SU(S) to SELEX RNAs that lack the consensus sequence and apparently is neither necessary nor sufficient for the stable polytene chromosome association of SU(S). Use of the GAL4/UAS system to drive ectopic expression of su(s) cDNA transgenes revealed two previously unknown properties of SU(S). First, overexpression of SU(S) is lethal. Second, SU(S) negatively regulates expression of su(s)intronless cDNA transgenes, and the ARMs are required for this effect. Considering these and previous results, we propose that SU(S) binds to the 5′ region of nascent transcripts and inhibits RNA production in a manner that can be overcome by splicing complex assembly.

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Characterization of the Rhesus Cytomegalovirus US28 Locus

Journal of Virology ◽

10.1128/jvi.77.19.10404-10413.2003 ◽

2003 ◽

Vol 77 (19) ◽

pp. 10404-10413 ◽

Cited By ~ 32

Author(s):

M. E. T. Penfold ◽

T. L. Schmidt ◽

D. J. Dairaghi ◽

P. A. Barry ◽

T. J. Schall

Keyword(s):

Viral Entry ◽

Intracellular Signaling ◽

Northern Analysis ◽

Reading Frame ◽

Reverse Transcription Pcr ◽

Wide Range ◽

Infected Cells ◽

G Protein Coupled

ABSTRACT Human cytomegalovirus (CMV) US28 (and the related open reading frame [ORF] US27) are G-protein-coupled receptor homologs believed to play a role in viral pathogenesis. In vitro, US28 has been shown to bind and internalize ligands, as well as activate intracellular signaling in response to certain chemokines, and to initiate the migration of smooth muscle cells to chemokine gradients. To assess the role of US28 in vivo, we examined the rhesus model and sequenced and characterized the rhesus CMV US28 locus. We found that rhesus CMV carries five tandem homologs of US28, all widely divergent from US28 and from each other. By reverse transcription-PCR and Northern analysis, we demonstrated expression of these ORFs in infected cells. With stable cell lines expressing these ORFs, we analyzed the homolog's binding and signaling characteristics across a wide range of chemokines and found one (RhUS28.5) to have a ligand binding profile similar to that of US28. In addition, we localized US28 and the rhesus CMV homolog RhUS28.5 to the envelope of infectious virions, suggesting a role in viral entry or cell tropism.

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The Drosophila salivary gland chromocenter contains highly polytenized subdomains of mitotic heterochromatin.

Genetics ◽

10.1093/genetics/139.2.659 ◽

1995 ◽

Vol 139 (2) ◽

pp. 659-670 ◽

Cited By ~ 1

Author(s):

P Zhang ◽

A C Spradling

Keyword(s):

Salivary Gland ◽

Dna Sequences ◽

Polytene Chromosomes ◽

Centromeric Heterochromatin ◽

Mitotic Chromosomes ◽

Satellite Dnas ◽

Central Mass ◽

P Elements

Abstract Peri-centromeric regions of Drosophila melanogaster chromosomes appear heterochromatic in mitotic cells and become greatly underrepresented in giant polytene chromosomes, where they aggregate into a central mass called the chromocenter. We used P elements inserted at sites dispersed throughout much of the mitotic heterochromatin to analyze the fate of 31 individual sites during polytenization. Analysis of DNA sequences flanking many of these elements revealed that middle repetitive or unique sequence DNAs frequently are interspersed with satellite DNAs in mitotic heterochromatin. All nine Y chromosome sites tested were underrepresented > 20-fold on Southern blots of polytene DNA and were rarely or never detected by in situ hybridization to salivary gland chromosomes. In contrast, nine tested insertions in autosomal centromeric heterochromatin were represented fully in salivary gland DNA, despite the fact that at least six were located proximal to known blocks of satellite DNA. The inserted sequences formed diverse, site-specific morphologies in the chromocenter of salivary gland chromosomes, suggesting that domains dispersed at multiple sites in the centromeric heterochromatin of mitotic chromosomes contribute to polytene beta-heterochromatin. We suggest that regions containing heterochromatic genes are organized into dispersed chromatin configurations that are important for their function in vivo.

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