scholarly journals Decision letter: A cell cycle-independent, conditional gene inactivation strategy for differentially tagging wild-type and mutant cells

2017 ◽  
Keyword(s):  
Cell Cycle ◽  
Gene Inactivation ◽  
Wild Type ◽  
A Cell ◽  
Mutant Cells

scholarly journals A cell cycle-independent, conditional gene inactivation strategy for differentially tagging wild-type and mutant cells

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sonal Nagarkar-Jaiswal ◽  
Sathiya N Manivannan ◽  
Zhongyuan Zuo ◽  
Hugo J Bellen
Keyword(s):  
Cell Cycle ◽  
Gene Inactivation ◽  
Inversion Method ◽  
Wild Type ◽  
Flip Flop ◽  
Temporal Cues ◽  
A Cell ◽  
Novel Method ◽  
Mutant Cells

Here, we describe a novel method based on intronic MiMIC insertions described in Nagarkar-Jaiswal et al. (2015) to perform conditional gene inactivation in Drosophila. Mosaic analysis in Drosophila cannot be easily performed in post-mitotic cells. We therefore, therefore, developed Flip-Flop, a flippase-dependent in vivo cassette-inversion method that marks wild-type cells with the endogenous EGFP-tagged protein, whereas mutant cells are marked with mCherry upon inversion. We document the ease and usefulness of this strategy in differential tagging of wild-type and mutant cells in mosaics. We use this approach to phenotypically characterize the loss of SNF4Aγ, encoding the γ subunit of the AMP Kinase complex. The Flip-Flop method is efficient and reliable, and permits conditional gene inactivation based on both spatial and temporal cues, in a cell cycle-, and developmental stage-independent fashion, creating a platform for systematic screens of gene function in developing and adult flies with unprecedented detail.

Yeast dom34 Mutants Are Defective in Multiple Developmental Pathways and Exhibit Decreased Levels of Polyribosomes

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 45-56
Author(s):  
Luther Davis ◽  
JoAnne Engebrecht
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Heterologous Expression ◽  
Protein Translation ◽  
Developmental Pathways ◽  
G1 Phase ◽  
Growth Defect ◽  
Wild Type ◽  
Drosophila Homolog ◽  
Mutant Cells

Abstract The DOM34 gene of Saccharomyces cerevisiae is similar togenes found in diverse eukaryotes and archaebacteria. Analysis of dom34 strains shows that progression through the G1 phase of the cell cycle is delayed, mutant cells enter meiosis aberrantly, and their ability to form pseudohyphae is significantly diminished. RPS30A, which encodes ribosomal protein S30, was identified in a screen for high-copy suppressors of the dom34Δ growth defect. dom34Δ mutants display an altered polyribosome profile that is rescued by expression of RPS30A. Taken together, these data indicate that Dom34p functions in protein translation to promote G1 progression and differentiation. A Drosophila homolog of Dom34p, pelota, is required for the proper coordination of meiosis and spermatogenesis. Heterologous expression of pelota in dom34Δ mutants restores wild-type growth and differentiation, suggesting conservation of function between the eukaryotic members of the gene family.

The SAC3 gene encodes a nuclear protein required for normal progression of mitosis

1996 ◽  
Vol 109 (6) ◽  
pp. 1575-1583
Author(s):  
A. Bauer ◽  
R. Kolling
Keyword(s):  
Cell Cycle ◽  
Actin Cytoskeleton ◽  
Nuclear Protein ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Cell Cycle Delay ◽  
A Cell ◽  
Single Nucleus ◽  
Microtubule Function ◽  
Mitotic Defect

The SAC3 gene of Saccharomyces serevisiae has been implicated in actin function by genetic experiments showing that a temperature sensitive mutation in the essential actin gene (actl-1) can be suppressed by mutations in SAC3. An involvement of SAC3 in actin function is further suggested by the observation that the actin cytoskeleton is altered in SAC3 mutants. Our fractionation experiments, however, point to a nuclear localization of Sac3p. On sucrose density gradients Sac3p co-fractionated with the nuclear organelle markers examined. Furthermore, Sac3p was enriched 10-fold in a nuclei preparation along with the nuclear protein Nop1p. In this report we further show that SAC3 function is required for normal progression of mitosis. SAC3 mutants showed a higher fraction of large-budded cells in culture, indicative of a cell cycle delay. The predominant population among the large-budded sac3 cells were cells with a single nucleus at the bud-neck and a short intranuclear spindle. This suggests that a cell cycle delay occurs in mitosis prior to anaphase. The observation that SAC3 mutants lose chromosomes with higher frequency than wild-type is another indication for a mitotic defect in SAC3 mutants. We further noticed that SAC3 mutants are more resistant against the microtubule destabilizing drug benomyl. This finding suggests that SAC3 is involved, directly or indirectly, in microtubule function. In summary, our data indicate that SAC3 is involved in a process which affects both the actin cytoskeleton and mitosis.

Growth in cell length in the fission yeast Schizosaccharomyces pombe

1985 ◽  
Vol 75 (1) ◽  
pp. 357-376 ◽  
Author(s):  
J.M. Mitchison ◽  
P. Nurse
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Size ◽  
Single Cells ◽  
Temperature Shift ◽  
Time Lapse ◽  
Cell Length ◽  
Wild Type ◽  
Cycle Growth ◽  
A Cell ◽  
Mutant Cells

The cylindrical cells of Schizosaccharomyces pombe grow in length by extension at the ends and not the middle. At the beginning of the cell cycle, growth is restricted to the ‘old end’, which existed in the previous cycle. Later on, the ‘new end’, formed from the septum, starts to grow at a point in the cycle that we have called NETO (‘new end take-off’). Fluorescence microscopy on cells stained with Calcofluor has been used to study NETO in size mutants, in blocked cdc mutants and with different growth temperatures and media. In wild-type cells (strain 972) NETO happens at 0.34 of the cycle with a cell length of 9.5 microns. With size mutants that are smaller at division, NETO takes place at the same size (9.0-9.5 microns) but this is not achieved until later in the cycle. Another control operates in larger size mutants since NETO occurs at the same stage of the cycle (about 0.32) as in wild type but at a larger cell size. This control is probably a requirement to have completed an event in early G2, since most cdc mutant cells blocked before this point in the cycle do not show NETO whereas most of those blocked in late G2 do show it. We conclude that NETO only happens if: (1) the cell length is greater than a critical value of 9.0-9.5 microns; and (2) the cell has traversed the first 0.3-0.35 of the cycle and passed early G2. NETO is delayed in poor media, in which cell size is also reduced. Temperature has little effect on NETO under steady-state conditions, but there is a transient delay for some hours after a temperature shift. NETO is later in another wild-type strain, 132. Time-lapse photomicrography was used to follow the rates of length growth in single cells. Wild-type cells showed two linear segments during the first 75% of the cycle. There was a rate-change point (RCP), coincident with NETO, where the rate of total length extension increased by 35%. This increase was not due simply to the start of new-end growth, since old-end growth slowed down in some cells at the RCP. cdc 11.123 is a mutant in which septation and division is blocked at 35 degrees C but nuclear division continues.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Disease-causing mutation in α-actinin-4 promotes podocyte detachment through maladaptation to periodic stretch

2018 ◽  
Vol 115 (7) ◽  
pp. 1517-1522 ◽  
Author(s):  
Di Feng ◽  
Jacob Notbohm ◽  
Ava Benjamin ◽  
Shijie He ◽  
Minxian Wang ◽  
...  
Keyword(s):  
Direct Consequence ◽  
Adhesion Assay ◽  
Actin Network ◽  
Wild Type ◽  
Spatially Correlated ◽  
Mechanical Responses ◽  
Podocyte Detachment ◽  
A Cell ◽  
Cross Links ◽  
Mutant Cells

α-Actinin-4 (ACTN4) bundles and cross-links actin filaments to confer mechanical resilience to the reconstituted actin network. How this resilience is built and dynamically regulated in the podocyte, and the cause of its failure in ACTN4 mutation-associated focal segmental glomerulosclerosis (FSGS), remains poorly defined. Using primary podocytes isolated from wild-type (WT) and FSGS-causing point mutant Actn4 knockin mice, we report responses to periodic stretch. While WT cells largely maintained their F-actin cytoskeleton and contraction, mutant cells developed extensive and irrecoverable reductions in these same properties. This difference was attributable to both actin material changes and a more spatially correlated intracellular stress in mutant cells. When stretched cells were further challenged using a cell adhesion assay, mutant cells were more likely to detach. Together, these data suggest a mechanism for mutant podocyte dysfunction and loss in FSGS—it is a direct consequence of mechanical responses of a cytoskeleton that is brittle.

Exploratory analysis of the effect of taselisib on downstream pathway modulation and correlation with tumor response in ER-positive/HER2-negative early-stage breast cancer from the LORELEI trial.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 1050-1050 ◽  
Author(s):  
Paolo Nuciforo ◽  
Dominik Hlauschek ◽  
Cristina Saura ◽  
Evandro de Azambuja ◽  
Roberta Fasani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Early Stage ◽  
Objective Response ◽  
Wild Type ◽  
Cycle Arrest ◽  
Er Positive ◽  
A Cell ◽  
Tumor Biopsies ◽  
Clinical Trial Information

1050 Background: Taselisib (T) is an oral, potent, selective inhibitor of Class I PI3-kinase with enhanced activity against PIK3CA mutant cancer cells. Results from the LORELEI trial have demonstrated a significant improvement in ORR (objective response rate) by centrally assessed magnetic resonance imaging in all randomized patients as well as in the PIK3CA mutant (MT) cohort treated with neoadjuvant T plus letrozole (L) compared to placebo (P) plus L. Here we present the results of exploratory analyses of selected pathway-related phosphoproteins. Methods: Baseline (BL) and week3 (W3) tumor biopsies were obtained from 334 patients enrolled in the trial. Phosphoproteins (pAKT, pPRAS40 and pS6) were analyzed by IHC. BL levels as well as changes from BL to W3 were correlated with response assessed either by ORR or cell cycle arrest (Ki67 at W3 < 2.7%). Results: In the overall population, BL phosphoproteins levels were similar between the T and P arms. Higher pAKT (p < 0.001) and pPRAS40 (p = 0.004) levels were observed in MT vs wild-type (WT), whereas the opposite result was found for pS6 (p = 0.03). Treatment-induced absolute changes of phosphoproteins adjusted for BL levels were not significantly different between the T and P arms in the overall population, except for pPRAS40 with higher decrease in the T arm (p = 0.014). After stratification for PIK3CA genotype, a significantly greater decrease in expression levels was observed for pPRAS40 (p < 0.001) and pS6 (p = 0.020) in MT tumors treated with T. The treatment effects were not significantly different in the WT population. A trend for an association between decrease in pS6 levels at W3 and improved ORR was observed in the MT (p = 0.08) and T (p = 0.09) subgroups. The magnitude of pS6 suppression at W3 was higher in tumors achieving a cell cycle arrest in the MT/T subgroup (biserial correlation = -0.473). Conclusions: Exploratory analyses of phosphoproteins showed bioactivity of taselisib as indicated by downstream pathway suppression. Translational research aiming to integrate these results with additional exploratory biomarkers data is currently ongoing. Clinical trial information: NCT02273973.

scholarly journals Altered cell cycle kinetics, gene expression, and G1 restriction point regulation in Rb-deficient fibroblasts.

1996 ◽  
Vol 16 (5) ◽  
pp. 2402-2407 ◽  
Author(s):  
R E Herrera ◽  
V P Sah ◽  
B O Williams ◽  
T P Mäkelä ◽  
R A Weinberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin E ◽  
Growth Cycle ◽  
Wild Type ◽  
Restriction Point ◽  
Protein Levels ◽  
Selective Growth Advantage ◽  
Molecular Events ◽  
Rb Gene ◽  
Mutant Cells

Fibroblasts prepared from retinoblastoma (Rb) gene-negative mouse embryos exhibit a shorter G1 phase of the growth cycle and smaller size than wild-type cells. In addition, the mutant cells are no longer inhibited by low levels of cycloheximide at any point in G1 but do remain sensitive to serum withdrawal until late in G1. Certain cell cycle-regulated genes showed no temporal or quantitative differences in expression. In contrast, cyclin E expression in Rb-deficient cells is deregulated in two ways. Cyclin E mRNA is generally derepressed in mutant cells and reaches peak levels about 6 h earlier in G1 than in wild-type cells. Moreover, cyclin E protein levels are higher in the Rb-/- cells than would be predicted from the levels of its mRNA. Thus, the selective growth advantage conferred by Rb gene deletion during tumorigenesis may be explained in part by changes in the regulation of cyclin E. In addition, the mechanisms defining the restriction point of late G1 may consist of at least two molecular events, one cycloheximide sensitive and pRb dependent and the other serum sensitive and pRb independent.

p53 functions as a cell cycle control protein in osteosarcomas

1990 ◽  
Vol 10 (11) ◽  
pp. 5772-5781
Author(s):  
L Diller ◽  
J Kassel ◽  
C E Nelson ◽  
M A Gryka ◽  
G Litwak ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Control ◽  
P53 Gene ◽  
Tumor Formation ◽  
Wild Type ◽  
Gene Encoding ◽  
Wide Range ◽  
A Cell ◽  
Wild Type P53

Mutations in the p53 gene have been associated with a wide range of human tumors, including osteosarcomas. Although it has been shown that wild-type p53 can block the ability of E1a and ras to cotransform primary rodent cells, it is poorly understood why inactivation of the p53 gene is important for tumor formation. We show that overexpression of the gene encoding wild-type p53 blocks the growth of osteosarcoma cells. The growth arrest was determined to be due to an inability of the transfected cells to progress into S phase. This suggests that the role of the p53 gene as an antioncogene may be in controlling the cell cycle in a fashion analogous to the check-point control genes in Saccharomyces cerevisiae.

Fission yeast Nda1 and Nda4, MCM homologs required for DNA replication, are constitutive nuclear proteins

1996 ◽  
Vol 109 (2) ◽  
pp. 319-326 ◽  
Author(s):  
N. Okishio ◽  
Y. Adachi ◽  
M. Yanagida
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Affinity Column ◽  
Wild Type ◽  
M Phase ◽  
A Cell ◽  
Mcm Proteins ◽  
Cycle Dependent

The nda1+ and nda4+ genes of the fission yeast Schizosaccharomyces pombe encode proteins similar to budding yeast MCM2 and MCM5/CDC46, respectively, which are required for the early stages of DNA replication. The budding yeast Mcm proteins display cell-cycle dependent localization. They are present in the nucleus specifically from late M phase until the beginning of S phase, so that they were suggested to be components of a replication licensing factor, a positive factor for the onset of replication, which is thought to be inactivated after use, thus restricting replication to only once in a cell cycle. In the present study, we raised antibodies against Nda1 or Nda4 and identified 115 kDa and 80 kDa proteins, respectively. Their immunolocalization was examined in wild-type cells and in various cell-cycle mutants. Both Nda1 and Nda4 proteins remained primarily in the nucleus throughout the cell cycle. In mutants arrested in G1, S, and G2 phases, these proteins were also enriched in the nucleus. These results indicate that the dramatic change in subcellular localization as seen in budding yeast is not essential in fission yeast for the functions of Nda1 and Nda4 proteins to be executed. The histidine-tagged nda1+ gene was constructed and integrated into the chromosome to replace the wild-type nda1+ gene. The resulting His-tagged Nda1 protein was adsorbed to the Ni-affinity column, and co-eluted with the untagged Nda4 protein, suggesting that they formed a complex.

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