scholarly journals The cell cycle program of polypeptide labeling in Chlamydomonas reinhardtii.

1977 ◽  
Vol 72 (2) ◽  
pp. 223-241 ◽  
Author(s):  
S H Howell ◽  
J W Posakony ◽  
K R Hill
Keyword(s):  
Cell Cycle ◽  
Chlamydomonas Reinhardtii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Generation ◽  
Temperature Sensitive ◽  
Dark Phase ◽  
Restrictive Temperature ◽  
One Dimensional ◽  
A Cell

The cell cycle program of polypeptide labeling in syndhronous cultures of wild-type Chlamydomonas reinhardtii was analyzed by pulse-labeling cells with 35SO4 = or [3H]arginine at different cell cycle stages. Nearly 100 labeled membrane and soluble polypeptides were resolved and studied using one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The labeling experiments produced the following results. (a) Total 35SO4 = and [3H]arginine incorporation rates varied independently throughout the cell cycle. 35SO4 = incorporation was highest in the mid-light phase, while [3H]arginine incorporation peaked in the dark phase just before cell division. (b) The relative labeling rate for 20 of 100 polypeptides showed significant fluctuations (3-12 fold) during the cell cycle. The remaining polypeptides were labeled at a rate commensurate with total 35SO4 = or [3H]arginine incorporation. The polypeptides that showed significant fluctuations in relative labeling rates served as markers to identify cell cycle stages. (c) The effects of illumination conditions on the apparent cell cycle stage-specific labeling of polypeptides were tested. Shifting light-grown asynchronous cells to the dark had an immediate and pronounced effect on the pattern of polypeptide labeling, but shifting dark-phase syndhronous cells to the light had little effect. The apparent cell cycle variations in the labeling of ribulose 1,5-biphosphate (RUBP)-carboxylase were strongly influenced by illumination effects. (d) Pulse-chase experiments with light-grown asynchronous cells revealed little turnover or inter-conversion of labeled polypeptides within one cell generation, meaning that major polypeptides, whether labeled in a stage-specific manner or not, do not appear transiently in the cell cycle of actively dividing, light-grown cells. The cell cycle program of labeling was used to analyze effects of a temperature-sensitive cycle blocked (cb) mutant. A synchronous culture of ts10001 was shifted to restrictive temperature before its block point to prevent it from dividing. The mutant continued its cell cycle program of polypeptide labeling for over a cell generation, despite its inability to divide.

scholarly journals Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽  
1993 ◽  
Vol 134 (1) ◽  
pp. 63-80 ◽  
Author(s):  
T A Weinert ◽  
L H Hartwell
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Control ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
A Cell ◽  
G2 Phase ◽  
Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

scholarly journals CONDITIONAL MUTANTS IN CHLAMYDOMONAS REINHARDTII BLOCKED IN THE VEGETATIVE CELL CYCLE

1973 ◽  
Vol 57 (3) ◽  
pp. 760-772 ◽  
Author(s):  
Stephen H. Howell ◽  
Jay A. Naliboff
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Chlamydomonas Reinhardtii ◽  
Simple Technique ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
General Applicability ◽  
Cell Cycles ◽  
Point Analysis ◽  
A Cell

Conditional "cycle-blocked" (cb) mutants of Chlamydomonas reinhardtii have been detected and isolated. These mutants exhibit normal vegetative growth at permissive temperature but are unable to complete a cell cycle (or a specified number of cell cycles) at restrictive temperature. A simple technique has been devised to determine the cell cycle stage in each mutant when the defective gene product, which ultimately affects cell division, completes its function. This stage is called the "block point", and is determined by scoring the residual cell division in an exponentially growing population after shift to temperature restrictive conditions. In the cb mutants isolated so far, block points representing many stages throughout the cell cycle have been found. Two categories of cb mutants are described here: one set which prevents the subsequent cell division when the cell encounters the block point after a shift to restrictive temperature, and another set which permits an additional round of cell division after the block point is encountered. The general applicability of block point analysis to other cell systems is presented.

Sexual development in a homothallic fission yeast: synthesis of readiness proteins resolved by gel electrophoresis

1982 ◽  
Vol 60 (6) ◽  
pp. 693-704 ◽  
Author(s):  
G. B. Calleja ◽  
Byron F. Johnson ◽  
Teena Walker
Keyword(s):  
Sexual Development ◽  
Gel Electrophoresis ◽  
Catabolite Repression ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Low Concentrations ◽  
Coomassie Brilliant ◽  
Temperature Aging

Sexual development of a homothallic strain of Schizosaccharomyces pombe was monitored by radiolabelling and sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis. Of more than 60 bands detected by Coomassie brilliant blue and by autoradiography, about 30 bands synthesized during development were discrete enough for experimental analysis.About a dozen bands are preferentially vegetative, another dozen preferentially developmental. However, vegetative bands as a group are also synthesized during development. Their synthesis is relatively unaffected by low concentrations of cycloheximide or by chloramphenicol and is not temperature sensitive at 37 °C nor catabolite repressible. Only band 40 (ca. 40 000 daltons) seems to be exclusively vegetative.The synthesis of developmental bands 13, 18, 24, 30, and α, all of which first appear during late-log phase, is catabolite repressible. Developmental band 51 is also synthesized throughout the vegetative phase. The synthesis of bands 24, 30, 51, and α is temperature sensitive at 37 °C during the development, but that of band 18 is not. The synthesis of band 13 during development is not temperature sensitive, but its earlier synthesis during late-log phase is. The synthesis of all these six developmental bands is immediately inhibited by cycloheximide, but not by chloramphenicol. Their appearance as a group of radioactive bands is greatly diminished in cultures grown in cycloheximide, in chloramphenicol, or in ethidium bromide.Developmental bands 13, 18, 24, and 30 may be called readiness proteins. They first appear prior to the earliest morphological signs of sexual activity. Their developmental synthesis is inhibited by conditions mat inhibit sexual development. Such inhibitory conditions include anaerobiosis, restrictive temperature, aging in stationary phase, the presence of inhibitors of cytoplasmic protein synthesis and of mitochondrial function, and catabolite repression. Readiness proteins may be regulating the switch from vegetative metabolism.

Plasma membrane associated proteins and glycoproteins of a temperature-sensitive hamster cell line (ts 3)

1978 ◽  
Vol 56 (11) ◽  
pp. 1061-1068
Author(s):  
Vijai N. Nigam ◽  
Carlos A. Brailovsky
Keyword(s):  
Plasma Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Phenotype ◽  
Temperature Sensitive ◽  
Major Significance ◽  
Sds Page ◽  
C Distribution ◽  
Associated Proteins ◽  
A Cell

Hamster ts 3 cells grown at 32 and at 39 °C were compared for (a) cell phenotype and growth rates, (b) sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) patterns of plasma membrane polypeptides, and (c) distribution of radioactivity in SDS–PAGE of plasma membrane glycopeptides metabolically labeled with [3H]glucosamine and [3H]fucose. It was observed that ts 3 cells growing at 39 °C and having a normal phenotype contained increased amounts of a glycopeptide of about 250 000 daltons and a polypeptide of about 200 000 daltons, as compared with ts 3 cells grown at 32 °C and having a transformed phenotype. No other alterations of major significance were observed.

scholarly journals A connection between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase.

1996 ◽  
Vol 7 (7) ◽  
pp. 1083-1094 ◽  
Author(s):  
K Lundgren ◽  
S Allan ◽  
S Urushiyama ◽  
T Tani ◽  
Y Ohshima ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Growth Arrest ◽  
Mrna Splicing ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Yeast Gene ◽  
Temperature Sensitive Mutants ◽  
Cycle Arrest ◽  
A Cell

The fission-yeast gene cdc28+ was originally identified in a screen for temperature-sensitive mutants that exhibit a cell-division cycle arrest and was found to be required for mitosis. We undertook a study of this gene to understand more fully the general requirements for entry into mitosis. Cells carrying the conditional lethal cdc28-P8 mutation divide once and arrest in G2 after being shifted to the restrictive temperature. We cloned the cdc28+ gene by complementation of the temperature-sensitive growth arrest in cdc28-P8. DNA sequence analysis indicated that cdc28+ encodes a member of the DEAH-box family of putative RNA-dependent ATPases or helicases. The Cdc28 protein is most similar to the Prp2, Prp16, and Prp22 proteins from budding yeast, which are required for the splicing of mRNA precursors. Consistent with this similarity, the cdc28-P8 mutant accumulates unspliced precursors at the restrictive temperature. Independently, we isolated a temperature-sensitive pre-mRNA splicing mutant prp8-1 that exhibits a cell-cycle phenotype identical to that of cdc28-P8. We have shown that cdc28 and prp8 are allelic. These results suggest a connection between pre-mRNA splicing and progression through the cell cycle.

scholarly journals A Yeast taf17 Mutant Requires the Swi6 Transcriptional Activator for Viability and Shows Defects in Cell Cycle-Regulated Transcription

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1561-1576
Author(s):  
Neil Macpherson ◽  
Vivien Measday ◽  
Lynda Moore ◽  
Brenda Andrews
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Essential Gene ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Synthetic Lethal ◽  
Cycle Arrest ◽  
A Cell ◽  
Allelism Tests ◽  
Complementation Groups

Abstract In Saccharomyces cerevisiae, the Swi6 protein is a component of two transcription factors, SBF and MBF, that promote expression of a large group of genes in the late G1 phase of the cell cycle. Although SBF is required for cell viability, SWI6 is not an essential gene. We performed a synthetic lethal screen to identify genes required for viability in the absence of SWI6 and identified 10 complementation groups of swi6-dependent lethal mutants, designated SLM1 through SLM10. We were most interested in mutants showing a cell cycle arrest phenotype; both slm7-1 swi6Δ and slm8-1 swi6Δ double mutants accumulated as large, unbudded cells with increased 1N DNA content and showed a temperature-sensitive growth arrest in the presence of Swi6. Analysis of the transcript levels of cell cycle-regulated genes in slm7-1 SWI6 mutant strains at the permissive temperature revealed defects in regulation of a subset of cyclin-encoding genes. Complementation and allelism tests showed that SLM7 is allelic with the TAF17 gene, which encodes a histone-like component of the general transcription factor TFIID and the SAGA histone acetyltransferase complex. Sequencing showed that the slm7-1 allele of TAF17 is predicted to encode a version of Taf17 that is truncated within a highly conserved region. The cell cycle and transcriptional defects caused by taf17slm7-1 are consistent with the role of TAFIIs as modulators of transcriptional activation and may reflect a role for TAF17 in regulating activation by SBF and MBF.

scholarly journals Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degPMutants and Induces the Cpx and ςE Extracytoplasmic Stress Responses

2001 ◽  
Vol 183 (18) ◽  
pp. 5230-5238 ◽  
Author(s):  
Geoffrey R. Langen ◽  
Jill R. Harper ◽  
Thomas J. Silhavy ◽  
S. Peter Howard
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Stress Responses ◽  
Elevated Temperatures ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Phospholipase A ◽  
Temperature Sensitive ◽  
Outer Membrane Phospholipase A ◽  
Temperature Sensitive Phenotype

ABSTRACT DegP is a periplasmic protease that is a member of both the ςE and Cpx extracytoplasmic stress regulons ofEscherichia coli and is essential for viability at temperatures above 42°C. [U-14C]acetate labeling experiments demonstrated that phospholipids were degraded indegP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, β-lactamase, and β-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degPcells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and ςE regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype ofdegP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and ςE regulons rather than by inactivating the phospholipase per se.

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