Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated chromosomes of Drosophila melanogaster

1997 ◽  
Vol 110 (17) ◽  
pp. 1979-1988 ◽  
Author(s):  
H. Bousbaa ◽  
L. Correia ◽  
G.J. Gorbsky ◽  
C.E. Sunkel
Keyword(s):  
Drosophila Melanogaster ◽  
Metaphase Plate ◽  
Cell Types ◽  
Early Prophase ◽  
Mitotic Chromosomes ◽  
Microtubule Inhibitors ◽  
Phosphatase Inhibitor ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Late Telophase

The progression of cells from metaphase to anaphase is thought to be regulated by a checkpoint that delays entry into anaphase until all chromosomes reach a stable bi-polar attachment at the metaphase plate. Previous work has suggested that the 3F3/2 kinetochore phosphoepitopes are involved in this checkpoint system. We show that the 3F3/2 centromere phosphoepitopes are present in Kc cells, third instar larval neuroblasts and isolated chromosomes of Drosophila melanogaster. In tissue culture cells and neuroblasts isolated from third instar larvae, centromere labelling is detected from early prophase to the metaphase-anaphase transition but absent once cells center anaphase. During anaphase, the antibody stains the spindle mid zone and during telophase the midbody is labelled until cells separate. In both cell types, the 3F3/2 antibody stains the centrosome from prophase to late telophase. The 3F3/2 staining is retained in Kc cells and third instar larval neuroblasts arrested at the prometaphase state with microtubule inhibitors. Also, two mitotic mutants that show abnormal spindle morphology retain the centromere labelling in a metaphase-like configuration, suggesting that they activate the metaphase-anaphase checkpoint. Finally, mitotic chromosomes isolated in the presence of a phosphatase inhibitor show phosphoepitopes at the primary constriction on the surface of each chromatid, however, chromosomes isolated in the absence of a phosphatase inhibitor do not. Incubation of these chromosomes with ATP causes the rephosphorylation of the phosphoepitopes at the centromere.

Sulfhydryl bond formation is a prerequisite for proper cycling of centrosomes and chromosomes in mammalian tissue culture cells

1993 ◽  
Vol 51 ◽  
pp. 342-343
Author(s):  
Heide Schatten ◽  
Neidhard Paweletz ◽  
Ron Balczon
Keyword(s):  
Tissue Culture ◽  
Cell Cycle Progression ◽  
Group Formation ◽  
Sulfhydryl Group ◽  
Mammalian Tissue ◽  
Mitotic Chromosomes ◽  
Microtubule Network ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Transmission Electron

To study the role of sulfhydryl group formation during cell cycle progression, mammalian tissue culture cells (PTK2) were exposed to 100¼M 2-mercaptoethanol for 2 to 6 h during their exponential phase of growth. The effects of 2-mercaptoethanol on centrosomes, chromosomes, microtubules, membranes and intermediate filaments were analyzed by transmission electron microscopy (TEM) and by immunofluorescence microscopy (IFM) methods using a human autoimmune antibody directed against centrosomes (SPJ), and a mouse monoclonal antibody directed against tubulin (E7). Chromosomes were affected most by this treatment: premature chromosome condensation was detected in interphase nuclei, and the structure in mitotic chromosomes was altered compared to control cells. This would support previous findings in dividing sea urchin cells in which chromosomes are arrested at metaphase while the centrosome splitting cycle continues. It might also support findings that certairt-sulfhydryl-blocking agents block cyclin destruction. The organization of the microtubule network was scattered probably due to a looser organization of centrosomal material at the interphase centers and at the mitotic poles.

scholarly journals DNA replication of histone gene repeats in Drosophila melanogaster tissue culture cells: multiple initiation sites and replication pause sites.

1993 ◽  
Vol 13 (7) ◽  
pp. 4098-4106 ◽  
Author(s):  
T Shinomiya ◽  
S Ina
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Dna Replication ◽  
Present Report ◽  
Polymerase Chain Reaction Method ◽  
Mapping Method ◽  
Histone Gene ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Replication Intermediates

We showed previously that DNA replication initiates at multiple sites in the 5-kb histone gene repeating unit in early embryos of Drosophila melanogaster. The present report shows evidence that replication in the same chromosomal region initiates at multiple sites in tissue culture cells as well. First, we analyzed replication intermediates by the two-dimensional gel electrophoretic replicon mapping method and detected bubble-form replication intermediates for all fragments restricted at different sites in the repeating unit. Second, we analyzed bromodeoxyuridine-labeled nascent strands amplified by the polymerase chain reaction method and detected little differences in the size distribution of nascent strands specific to six short segments located at different sites in the repeating unit. These results strongly suggest that DNA replication initiates at multiple sites located within the repeating unit. We also found several replication pause sites located at 5' upstream regions of some histone genes.

Heterogeneous nuclear ribonucleoprotein complexes and proteins in Drosophila melanogaster

1992 ◽  
Vol 12 (2) ◽  
pp. 847-855
Author(s):  
G Raychaudhuri ◽  
S R Haynes ◽  
A L Beyer
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Rna Sequences ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Culture Cells ◽  
Ribonucleoprotein Complexes ◽  
Tissue Culture Cells ◽  
Hnrnp Proteins ◽  
Nuclear Ribonucleoprotein ◽  
Basic Region

Pre-mRNAs cotranscriptionally associate with a small group of proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. We have previously identified two genes in Drosophila melanogaster, Hrb98DE and Hrb87F (i.e., genes at 98DE and 87F encoding putative hnRNA binding proteins), which encode five protein species homologous to the mammalian A-B hnRNP proteins. The studies presented herein show that antibodies against the RNP domains of Hrb98DE reacted with 10 to 15 distinct spots of 38 to 40 kDa in the basic region of two-dimensional gels. These nuclear proteins bound single-stranded nucleic acids and were extracted from Drosophila tissue culture cells as 40 to 80S hnRNP complexes in association with 300 to 800 nucleotide fragments of RNA. The peak of poly(A)+ RNA sequences was coincident with the peak of HRB proteins in sucrose gradients, strongly suggesting that the HRB complexes identified are Drosophila hnRNP complexes. The repertoire of HRB proteins did not change significantly during embryogenesis and was similar to that observed in Drosophila tissue culture cells. Analyses with peptide-specific antisera demonstrated that the major proteins in the hnRNP complex were encoded by the two genes previously identified. Although the Drosophila HRB proteins are only approximately 60% identical throughout the RNP domains to the mammalian A-B hnRNP proteins, features of the basic pre-mRNA packaging mechanism appear to be highly conserved between D. melanogaster and mammals.

p75, a polypeptide component of karyoskeletal protein-enriched fractions associated with transcriptionally active loci of Drosophila melanogaster polytene chromosomes

1988 ◽  
Vol 8 (5) ◽  
pp. 1877-1886
Author(s):  
B M Benton ◽  
S Berrios ◽  
P A Fisher
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Transcriptional Regulation ◽  
Salivary Gland ◽  
Indirect Immunofluorescence ◽  
Polytene Chromosomes ◽  
Nuclear Interior ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Larval Salivary Gland

A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed.

scholarly journals Targeted Integration of Single-Copy Transgenes in Drosophila melanogaster Tissue-Culture Cells Using Recombination-Mediated Cassette Exchange

Genetics ◽  
2015 ◽  
Vol 201 (4) ◽  
pp. 1319-1328 ◽  
Author(s):  
Sathiya N. Manivannan ◽  
Thomas L. Jacobsen ◽  
Peter Lyon ◽  
Bhavani Selvaraj ◽  
Peter Halpin ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Drosophila Melanogaster ◽  
Single Copy ◽  
Culture Cells ◽  
Cassette Exchange ◽  
Tissue Culture Cells ◽  
Targeted Integration

scholarly journals Effect of heat shock on ribosome synthesis in Drosophila melanogaster.

1988 ◽  
Vol 8 (1) ◽  
pp. 91-95 ◽  
Author(s):  
J Bell ◽  
L Neilson ◽  
M Pellegrini
Keyword(s):  
Tissue Culture ◽  
Protein Synthesis ◽  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Ribosomal Protein Synthesis ◽  
Correct Processing

In Drosophila tissue culture cells, the synthesis of ribosomal proteins was inhibited by a 1-h 37 degrees C heat shock. Ribosomal protein synthesis was repressed to a greater extent than that of most other proteins synthesized by these cells at 25 degrees C. After a 1-h heat shock, when the cells were returned to 25 degrees C, the ribosomal proteins were much slower than most other 25 degrees C proteins to return to pre-heat shock levels of synthesis. Relative to one another, all the ribosomal proteins were inhibited and later recovered to normal levels of synthesis at the same rate and to the same extent. Unlike the ribosomal proteins, the precursor to the large rRNAs was continually synthesized during heat shock, although at a slightly reduced level, but was not processed. It was rapidly degraded, with a half-life of approximately 16 min. Pre-heat shock levels of synthesis, stability, and correct processing were restored only when ribosomal protein synthesis returned to at least 50% of that seen in non-heat-shocked cells.

The dynamic localisation of the Drosophila APC/C: evidence for the existence of multiple complexes that perform distinct functions and are differentially localised

2002 ◽  
Vol 115 (14) ◽  
pp. 2847-2856 ◽  
Author(s):  
Jun-yong Huang ◽  
Jordan W. Raff
Keyword(s):  
Fusion Proteins ◽  
Cyclin B ◽  
Subcellular Localisation ◽  
Anaphase Promoting Complex ◽  
Mitotic Spindles ◽  
Mitotic Chromosomes ◽  
Nuclear Envelope Breakdown ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Drosophila Cells

In Drosophila cells, the destruction of cyclin B is spatially regulated. In cellularised embryos, cyclin B is initially degraded on the mitotic spindle and is then degraded in the cytoplasm. In syncytial embryos,only the spindle-associated cyclin B is degraded at the end of mitosis. The anaphase promoting complex/cyclosome (APC/C) targets cyclin B for destruction,but its subcellular localisation remains controversial. We constructed GFP fusions of two core APC/C subunits, Cdc16 and Cdc27. These fusion proteins were incorporated into the endogenous APC/C and were largely localised in the cytoplasm during interphase in living syncytial embryos. Both fusion proteins rapidly accumulated in the nucleus prior to nuclear envelope breakdown but only weakly associated with mitotic spindles throughout mitosis. Thus, the global activation of a spatially restricted APC/C cannot explain the spatially regulated destruction of cyclin B. Instead, different subpopulations of the APC/C must be activated at different times to degrade cyclin B. Surprisingly,we noticed that GFP-Cdc27 associated with mitotic chromosomes, whereas GFP-Cdc16 did not. Moreover, reducing the levels of Cdc16 or Cdc27 by >90%in tissue culture cells led to a transient mitotic arrest that was both biochemically and morphologically distinct. Taken together, our results raise the intriguing possibility that there could be multiple forms of the APC/C that are differentially localised and perform distinct functions.

A deletion of the 3' end of the Drosophila melanogaster hsp70 gene increases stability of mutant mRNA during recovery from heat shock

1985 ◽  
Vol 5 (12) ◽  
pp. 3397-3402
Author(s):  
A A Simcox ◽  
C M Cheney ◽  
E P Hoffman ◽  
A Shearn
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Protein Gene ◽  
Hsp70 Mrna ◽  
X Ray ◽  
Culture Cells ◽  
Hsp70 Protein ◽  
Tissue Culture Cells ◽  
High Level ◽  
Hsp40 Protein

hsp40, an X-ray-induced deletion mutant of the major Drosophila melanogaster heat shock protein gene hsp70, was shown to be incorrectly regulated at the translational level. hsp40 protein synthesis persisted at a high level after the release from heat shock, whereas hsp70 protein production was rapidly repressed. This result was observed both in flies heterozygous for the hsp40 gene and in tissue culture cells transfected with the truncated gene. Analysis of the transcription of the hsp40 gene indicated that its mRNA, unlike hsp70 mRNA, was not actively destabilized after a return to control temperatures, permitting prolonged production of the mutant protein.

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