scholarly journals Overexpression of SIS2, which contains an extremely acidic region, increases the expression of SWI4, CLN1 and CLN2 in sit4 mutants.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 95-107 ◽  
Author(s):  
C J Di Como ◽  
R Bose ◽  
K T Arndt
Keyword(s):  
Growth Rate ◽  
Protein Phosphatase ◽  
Rnase A ◽  
Micrococcal Nuclease ◽  
Nuclear Fraction ◽  
High Copy Number Plasmid ◽  
Carboxyl Terminal ◽  
Rna Accumulation ◽  
Bud Initiation ◽  
Acidic Region

Abstract The Saccharomyces cerevisiae SIS2 gene was identified by its ability, when present on a high copy number plasmid, to increase dramatically the growth rate of sit4 mutants. SIT4 encodes a type 2A-related protein phosphatase that is required in late G1 for normal G1 cyclin expression and for bud initiation. Overexpression of SIS2, which contains an extremely acidic carboxyl terminal region, stimulated the rate of CLN1, CLN2, SWI4 and CLB5 expression in sit4 mutants. Also, overexpression of SIS2 in a CLN1 cln2 cln3 strain stimulated the growth rate and the rate of CLN1 and CLB5 RNA accumulation during late G1. The SIS2 protein fractionated with nuclei and was released from the nuclear fraction by treatment with either DNase I or micrococcal nuclease, but not by RNase A. This result, combined with the finding that overexpression of SIS2 is extremely to a strain containing lower than normal levels of histones H2A and H2B, suggests that SIS2 might function to stimulate transcription via an interaction with chromatin.

scholarly journals Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins.

1991 ◽  
Vol 114 (4) ◽  
pp. 623-638 ◽  
Author(s):  
M M Luke ◽  
A Sutton ◽  
K T Arndt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Phosphatase ◽  
Slow Growth ◽  
Nuclear Fraction ◽  
Yeast Protein ◽  
Rich Region ◽  
Amino Terminal ◽  
Growth Phenotype ◽  
Carboxyl Terminal

The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one-fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1.

Decondensation of Xenopus sperm chromatin using Saccharomyces cerevisiae whole-cell extractsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

2006 ◽  
Vol 84 (3-4) ◽  
pp. 451-458
Author(s):  
Troy A.A. Harkness
Keyword(s):  
Rnase A ◽  
Yeast Cells ◽  
Micrococcal Nuclease ◽  
Sperm Chromatin ◽  
Temperature Sensitive ◽  
Whole Cell ◽  
Multiple Systems ◽  
Cell Extracts ◽  
A Cell ◽  
Chromosome Decondensation

Biochemical studies using highly condensed Xenopus sperm chromatin and protein extracts prepared from multiple systems have lead to the identification of conserved proteins involved in chromosome decondensation. However, mutations to these proteins are unavailable as the systems used are not amenable to genetic studies. We took a genetic approach to isolating chromosome decondensation mutants by incubating Xenopus sperm chromatin with whole-cell extracts prepared from the Hartwell library of random temperature sensitive (ts) yeast cells. We show that decondensation of Xenopus sperm chromatin using wild type yeast extracts was rapid, ATP- and extract-dependent, and resistant to heat, N-ethylmaleimide, protease K, RNase A, and micrococcal nuclease. From 100 mutant extracts screened, we obtained one strain, referred to as rmc4, that was chromosome decondensation defective. The mutant was slow growing and exhibited germination defects. Low concentrations of rmc4 extract would eventually decondense sperm heads, and fractionation of the mutant extract produced a decondensation competent fraction, suggesting the presence of an overactive inhibitor in rmc4 cells. We performed a multicopy suppressor screen that identified PDE2, a gene encoding a protein that inhibits protein kinase A (PKA) activity. As PKA was previously shown in human cells to maintain condensed chromatin, our results suggest that PKA activity is elevated in rmc4 cells, causing a decondensation defect. Thus, our experiments reveal that yeast encodes an evolutionarily conserved chromosome decondensation activity that can be genetically manipulated.

Organization of chromosomes in HeLa cells: isolation of histone-depleted nuclei and nuclear scaffolds

1980 ◽  
Vol 42 (1) ◽  
pp. 291-304
Author(s):  
K.W. Adolph
Keyword(s):  
Electron Microscopy ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nuclear Periphery ◽  
Sucrose Density Gradient ◽  
Rnase A ◽  
Micrococcal Nuclease ◽  
Order Structure ◽  
Dextran Sulphate ◽  
Thin Sections ◽  
Thin Sectioning

Histone-depleted nuclei were prepared from isolate HeLa nuclei by extracting the histones and other proteins with polyanions (dextran sulphate and heparin) or with high salt concentrations as used previously. The particles were characterized by sucrose density gradient sedimentation, thin sectioning and electron microscopy, and by polyacrylamide gel electrophoresis. The general result of the experiments is that the DNA in the histone-depleted nuclei is highly organized, and that this residual, higher-order structure is maintained by a reproducible subset of nuclear proteins, and perhaps by RNA. Furthermore, the residual proteins remain associated, in some conditions, as rapidly sedimenting structures even when the DNA is digested with nucleases. These nuclear scaffolds can resemble extracted nuclei. Histone-depleted HeLa nuclei sediment in sucrose density gradients as well defined peaks with sedimentation coefficients of around 12 000 S, when 2M NaCl is used to extract the histones, or 6 000 S, when dextran sulphate is used. The rate of sedimentation is drastically decreased by treating the particles with trypsin, and reduced to a lesser extent with RNase A. Thin sectioning and electron microscopy show that histone-depleted nuclei possess the nuclear periphery and that internal material is also present. These general features are also seen in thin sections of nuclear scaffolds, which are prepared by treating the nuclei with micrococcal nuclease of DNase I in addition to extracting the histones. Two groups of major proteins are associated with histone-depleted HeLa nuclei and the nuclear scaffolds: One group has molecular weights of 50 000-55 000 Daltons. The major species of this latter group of proteins have mobilities that are similar to the proteins of the metaphase chromosomal scaffold.

mRNA-decapping enzyme from Saccharomyces cerevisiae: purification and unique specificity for long RNA chains

1988 ◽  
Vol 8 (5) ◽  
pp. 2005-2010
Author(s):  
A Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rnase A ◽  
Micrococcal Nuclease ◽  
Mrna Decapping ◽  
Nuclease P1 ◽  
High Concentrations ◽  
Decapping Enzyme ◽  
Unique Specificity ◽  
End Group

An enzyme that hydrolyzes one PPi bond of the cap structure of mRNA, yielding m7GDP and 5'-p RNA was purified from Saccharomyces cerevisiae to a stage suitable for characterization. The specificity of the enzyme was studied, using both yeast mRNA and synthetic RNAs labeled in the cap structure. A synthetic capped RNA (540 nucleotides) was not reduced in size, while as much as 80% was decapped. Yeast mRNA treated with high concentrations of RNase A, nuclease P1, or micrococcal nuclease was inactive as a substrate. The use of synthetic capped RNAs of different sizes (50 to 540 nucleotides) as substrates showed that the larger RNA can be a better substrate by as much as 10-fold. GpppG-RNA was hydrolyzed at a rate similar to that at which 5'-triphosphate end group were not hydrolyzed.

scholarly journals Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections.

1985 ◽  
Vol 33 (5) ◽  
pp. 389-399 ◽  
Author(s):  
F J Moreno ◽  
D Hernandez-Verdun ◽  
C Masson ◽  
M Bouteille
Keyword(s):  
Silver Staining ◽  
Organizer Region ◽  
Nucleolar Organizer Region ◽  
Nucleolar Organizer ◽  
Enzymatic Digestion ◽  
Rnase A ◽  
Micrococcal Nuclease ◽  
Nucleolar Organizer Regions ◽  
Step Procedure ◽  
Ultrathin Sections

Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.

scholarly journals An RNase-sensitive particle containing Drosophila melanogaster DNA topoisomerase II.

1994 ◽  
Vol 126 (6) ◽  
pp. 1331-1340 ◽  
Author(s):  
V H Meller ◽  
M McConnell ◽  
P A Fisher
Keyword(s):  
Rna Polymerase Ii ◽  
Topoisomerase Ii ◽  
Sedimentation Coefficient ◽  
Rnase A ◽  
Micrococcal Nuclease ◽  
Nuclear Antigen ◽  
Nuclear Pore ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Topo Ii

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).

scholarly journals FCP1, the RAP74-Interacting Subunit of a Human Protein Phosphatase That Dephosphorylates the Carboxyl-terminal Domain of RNA Polymerase IIO

1998 ◽  
Vol 273 (42) ◽  
pp. 27593-27601 ◽  
Author(s):  
Jacques Archambault ◽  
Guohua Pan ◽  
Grace K. Dahmus ◽  
Mireille Cartier ◽  
Nick Marshall ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Protein Phosphatase ◽  
Human Protein ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal

Nuclear distribution of Drosophila DNA topoisomerase II is sensitive to both RNase and DNase

1995 ◽  
Vol 108 (4) ◽  
pp. 1651-1657 ◽  
Author(s):  
V.H. Meller ◽  
P.A. Fisher
Keyword(s):  
Topoisomerase Ii ◽  
Immunoblot Analysis ◽  
Dnase I ◽  
Rnase A ◽  
Drosophila Embryo ◽  
Micrococcal Nuclease ◽  
Cell Fractionation ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Nuclear Distribution

The nuclear distribution of Drosophila DNA topoisomerase II was determined by immunoblot analysis after nuclease digestion and cell fractionation. About 60% of DNA topoisomerase II could be removed from nuclei by RNase A, about 70% by DNase I, and about 90% by incubation with both enzymes together or with micrococcal nuclease. Nuclease treatment of nuclei did not affect the distribution of lamins Dm1 and Dm2 or other nuclear proteins similarly. Nuclease-mediated solubilization of DNA topoisomerase II from Drosophila nuclei was also dependent on NaCl concentration. Solubilization was not efficient below 100 mM NaCl. Sucrose velocity gradient ultracentrifugation demonstrated that DNA topoisomerase II solubilized from nuclei by either RNase A or DNase I migrated at about 9 S, as expected for the homodimer. Results of chemical crosslinking supported this observation. We conclude that DNA topoisomerase II has both RNA- and DNA-dependent anchorages in Drosophila embryo nuclei.

The effect of plant density on tiller growth and morphology in barley

1972 ◽  
Vol 78 (2) ◽  
pp. 281-288 ◽  
Author(s):  
E. J. M. Kirby ◽  
D. G. Faris
Keyword(s):  
Growth Rate ◽  
Final Stage ◽  
Plant Density ◽  
Practical Importance ◽  
Leaf Sheath ◽  
Second Phase ◽  
Endogenous Gibberellin ◽  
Competition For Light ◽  
Bud Initiation ◽  
Tiller Bud

SUMMARYA detailed study was made of the tillering of barley plants grown at densities which ranged from 50 to 1600 plants m–2. Throughout the season the initiation of tiller buds and their growth in length and leaf number were followed, and measurements were made of leaf sheath and lamina length and lamina width. A proportion of tillers died and their positions and times of death are given.Three phases of tillering were recognized. The first, tiller bud initiation, was little affected directly by density. During the second phase, buds did not grow or did grow and emerge from the subtending leaf sheath. If they grew, there was no difference in growth rate of tillers at different densities. At the higher densities fewer buds developed, and the morphology of those tillers which did grow was affected by density. This suggests that the growth of the tiller bud may be controlled by levels of endogenous gibberellin, while in the final stage, growth after emergence from the subtending leaf sheath, competition for light appears to be the factor which determines whether a tiller survives to produce an ear. The data are discussed in relation to the practical importance of tillering.

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