Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity

Mapping Intimacies ◽

10.32747/2000.7575285.bard ◽

2000 ◽

Author(s):

Gideon Grafi ◽

Brian Larkins

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Storage Protein ◽

Molecular Mechanisms ◽

S Phase ◽

Cyclin B ◽

Regulatory Subunit ◽

Maize Endosperm ◽

M Phase

The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.

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Deficiency of Bone Marrow Stromal Cx43 Expression Induces Hematopoietic Progenitor Homing Deficiency and Cell-Cycle Arrest of Hematopoietic Stem Cells and Progenitors.

Blood ◽

10.1182/blood.v108.11.349.349 ◽

2006 ◽

Vol 108 (11) ◽

pp. 349-349

Author(s):

Lina Li ◽

Cynthia A. Presley ◽

Bryan Kastl ◽

Jose A. Cancelas

Keyword(s):

Cell Cycle ◽

S Phase ◽

Chimeric Mice ◽

Wild Type ◽

Hematopoietic Progenitor ◽

Cx43 Expression ◽

Hematopoietic Stem ◽

M Phase ◽

Deficient Mice

Abstract Contact between bone marrow (BM) hematopoietic stem cells (HSC) and osteoblast/stromal (OS) cells has been shown to be critical in the regulation of hematopoiesis. However, very little is known about the regulatory mechanisms of direct cell-to-cell communication in the hematopoietic microenvironment. BM cells are directly connected through gap junctions (GJs) which consist of narrow channels between contacting cells and are composed by connexins. Connexin 43 (Cx43) is expressed by BM OS cells. Multiple osteogenic defects have been reported in human Cx43 mutations and Cx43 has been shown to be essential in controlling osteoblast functions. Due to the perinatal death of Cx43 germline null mice, an interferon-inducible, conditional genetic approach (Mx1-Cre), expressed by both hematopoietic and stromal BM cells, was used to study the role of Cx43 in stem cell function. We have previously reported that Cx43 is critical for the interaction between stroma and HSC in CAFC assays (Cancelas J.A. et al., Blood 2000) and in adult hematopoiesis after 5-fluorouracil (5-FU) administration (Presley C, et al., Cell Comm. Adh., 2005). Here, we observed that after 5-FU administration, Cx43 expression is predominantly located in the endosteum. To study the role of stroma-dependent Cx43 in hematopoiesis, we developed hematopoietic chimeras by BM transplantation of wild-type Cx43 HSC into stromal Cx43-deficient mice. Stromal Cx43 deficiency induced a severe impairment of blood cell formation during the recovery phase after 5-FU administration compared to stromal Mx1-Cre-Tg wild-type controls (Table 1), as well as a significant decrease in BM cellularity (~60% reduction) and progenitor cell content (~83% reduction). Cell cycle analysis of 5-FU-treated BM progenitors from stromal Cx43-deficient mice showed an S-phase arrest (S phase: 63.5%; G2/M phase: <1%) compared to wild-type chimeric mice (S phase: 38.6%, G2/M phase: 7.8%, p=0.01) suggesting a cell division blockade. Unlike Cx43-deficient primary mice, a differentiation arrest at the HSC compartment was observed in 5-FU-treated, stromal Cx43-deficient mice, since the content of competitive repopulating units (CRU) at 1 month, of 14-day post-5-FU BM of stromal Cx43-deficient mice was increased (27.7 ± 0.67) compared to recipients of HSC from stromal wild-type counterparts (26.5 ± 0.92 CRU, p < 0.01). Interestingly, wild-type hematopoietic progenitor homing in stromal Cx43-deficient BM was severely impaired with respect to wild-type BM (5.1% vs10.4 %, respectively, p < 0.01), while hematopoietic Cx43-deficient BM progenitors normally homed into the BM, suggesting a differential role for Cx43 in stromal and HSC. In conclusion, expression of Cx43 in osteoblasts and stromal cells appears to play a crucial role in the regulation of HSC homing in BM and hematopoietic regeneration after chemotherapy. Peripheral blood counts of WT and stromal Cx43-deficient chimeric mice after 5-FU administration (150 mg/Kg) Neutrophil counts (×10e9/L) Reticulocyte count (%) Day post-5-FU WT Cx43-deficient WT Cx43-deficient * p < 0.05 Day +8 2.89 ± 0.06 0.81 ± 0.02* 2.0 ± 0.6 3.0 ± 0.9 Day +11 9.11 ± 2.5 3.13 ± 0.8* 6.1 ± 0.6 2.7 ± 0.3* Day +14 6.22 ± 5.7 7.58 ± 8.2 7.5 ± 0.5 2.5 ± 0.5*

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Cell Cycle Dysregulation by Human Cytomegalovirus: Influence of the Cell Cycle Phase at the Time of Infection and Effects on Cyclin Transcription

Journal of Virology ◽

10.1128/jvi.72.5.3729-3741.1998 ◽

1998 ◽

Vol 72 (5) ◽

pp. 3729-3741 ◽

Cited By ~ 135

Author(s):

Bryan S. Salvant ◽

Elizabeth A. Fortunato ◽

Deborah H. Spector

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Cell Cycle Progression ◽

Hcmv Infection ◽

Cyclin A ◽

S Phase ◽

Cyclin B ◽

Cycle Progression ◽

Cycle Arrest ◽

Time Of Infection

ABSTRACT Human cytomegalovirus (HCMV) infection inhibits cell cycle progression and alters the expression of cyclins E, A, and B (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697–6704, 1995). In this study, we examined cell cycle progression, cyclin gene expression, and early viral events when the infection was initiated at different points in the cell cycle (G0, G1, and S). In all cases, infection led to cell cycle arrest. Cells infected in G0 or G1phase also showed a complete or partial absence, respectively, of cellular DNA synthesis at a time when DNA synthesis occurred in the corresponding mock-infected cells. In contrast, when cells were infected near or during S phase, many cells were able to pass through S phase and undergo mitosis prior to cell cycle arrest. S-phase infection also produced a delay in the appearance of the viral cytopathic effect and in the synthesis of immediate-early and early proteins. Labeling of cells with bromodeoxyuridine immediately prior to HCMV infection in S phase revealed that viral protein expression occurred primarily in cells which were not engaged in DNA synthesis at the time of infection. The viral-mediated induction of cyclin E, maintenance of cyclin-B protein levels, and inhibitory effects on the accumulation of cyclin A were not significantly affected when infection occurred during different phases of the cell cycle (G0, G1, and S). However, there was a delay in the observed inhibition of cyclin A in cells infected during S phase. This finding was in accord with the pattern of cell cycle progression and delay in viral gene expression associated with S-phase infection. Analysis of the mRNA revealed that the effects of the virus on cyclin E and cyclin A, but not on cyclin B, were primarily at the transcriptional level.

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Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.4888 ◽

1999 ◽

Vol 19 (7) ◽

pp. 4888-4896 ◽

Cited By ~ 76

Author(s):

Guy Oshiro ◽

Julia C. Owens ◽

Yiqun Shellman ◽

Robert A. Sclafani ◽

Joachim J. Li

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Replication ◽

Kinase Activity ◽

Cell Cycle Control ◽

S Phase ◽

Regulatory Subunit ◽

Anaphase Promoting Complex ◽

Initiation Of Dna Replication

ABSTRACT In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G1 phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G1 cells. This G1-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G1 phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.

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SLC4A11 and MFSD3 Gene Expression Changes in Deoxynivalenol Treated IPEC-J2 Cells

Frontiers in Genetics ◽

10.3389/fgene.2021.697883 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yafei Xu ◽

Xiaolei Chen ◽

Luchen Yu ◽

Yi Wang ◽

Haifei Wang ◽

...

Keyword(s):

Cell Cycle ◽

Dna Methylation ◽

Drug Targets ◽

Molecular Mechanisms ◽

The Novel ◽

Animal Cells ◽

Cytotoxic Effects ◽

M Phase ◽

Detection Of Biomarkers

Deoxynivalenol (DON) caused serious cytotoxicity for animal cells. However, genes involved in regulating DON toxicity and the underlying molecular mechanisms remain largely unknown. This study explored the role of SLC4A11 and MFSD3 in alleviating DON toxicity and analyzed the DNA methylation changes of these two genes. Viability and cell cycle analysis showed that DON exposure decreased the IPEC-J2 viability (P < 0.01), blocked the cell cycle in the G2/M phase (P < 0.01), and increased the rate of apoptosis (P < 0.05). Expression of the SLC4A11 and MFSD3 genes was significantly downregulated upon DON exposure (P < 0.01). Overexpression of SLC4A11 and MFSD3 can enhance the cell viability (P < 0.01). DNA methylation assays indicated that promoter methylation of SLC4A11 (mC-1 and mC-23) and MFSD3 (mC-1 and mC-12) were significantly higher compared with those in the controls and correlated negatively with mRNA expression (P < 0.05). Further analysis showed that mC-1 of SLC4A11 and MFSD3 was located in transcription factor binding sites for NF-1 and Sp1. Our findings revealed the novel biological functions of porcine SLC4A11 and MFSD3 genes in regulating the cytotoxic effects induced by DON, and may contribute to the detection of biomarkers and drug targets for predicting and eliminating the potential toxicity of DON.

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Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae.

Molecular Biology of the Cell ◽

10.1091/mbc.8.8.1587 ◽

1997 ◽

Vol 8 (8) ◽

pp. 1587-1601 ◽

Cited By ~ 49

Author(s):

M R Young ◽

B K Tye

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Synthesis ◽

S Phase ◽

Specific Cell ◽

Replication Origins ◽

Proliferating Cells ◽

M Phase ◽

Mcm Proteins ◽

And Function

The Mcm2-7 proteins are a family of conserved proteins whose functions are essential for the initiation of DNA synthesis in all eukaryotes. These patients are constitutively present in high abundance in actively proliferating cells. In Saccharomyces cerevisiae, the intracellular concentrations of Mcms are between 100 and 500 times the number of replication origins. However, these proteins are limiting for the initiation of DNA synthesis at replication origins. Our studies indicate that only a small fraction of Mcm2 and Mcm3 tightly associates with chromatin, from late M phase to the beginning of the S phase. The rest of the Mcm2 and Mcm3 proteins are disturbed to both the cytoplasm and nucleoplasm in relatively constant levels throughout the cell cycle. We also show that S. cerevisiae Mcm3 is a phosphoprotein that exists in multiple isoforms and that distinct isoforms of Mcm2 and Mcm3 can be detected at specific stages of the cell cycle. These results suggest that the localization and function of the Mcm proteins are regulated by posttranslational phosphorylation in a manner that is consistent with a role for the Mcm proteins in restricting DNA replication to once per cell cycle.

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The role of cdc2 and other genes in meiosis in Schizosaccharomyces pombe.

Genetics ◽

10.1093/genetics/140.4.1235 ◽

1995 ◽

Vol 140 (4) ◽

pp. 1235-1245 ◽

Cited By ~ 4

Author(s):

Y Iino ◽

Y Hiramine ◽

M Yamamoto

Keyword(s):

Dna Synthesis ◽

Schizosaccharomyces Pombe ◽

Nuclear Division ◽

Large Subunit ◽

S Phase ◽

Dependent Manner ◽

M Phase ◽

Cdc2 Gene ◽

Meiosis I

Abstract The requirement of the cdc2, cdc13 and cdc25 genes for meiosis in Schizosaccharomyces pombe was investigated using three different conditions to induce meiosis. These genes were known to be required for meiosis II. cdc13 and cdc25 are essential for meiosis I. The cdc2 gene, which is required for the initiation of both mitotic S-phase and M-phase, is essential for premeiotic DNA synthesis and meiosis II. The requirement of cdc2 for meiosis I was unclear. This contrasts with Saccharomyces cerevisiae, where CDC28, the homolog of cdc2, is required for meiosis I but not for premeiotic DNA synthesis. Expression of cdc13 and cdc25 was induced after premeiotic DNA synthesis, reaching a sharp peak before the first nuclear division. Expression of cdc22, encoding the large subunit of ribonucleotide reductase, was also induced but the peak was before premeiotic DNA synthesis. The induction of cdc13 and cdc25 was largely dependent on DNA synthesis and the function of the mei4 gene. The mei4 gene itself was also induced in a DNA synthesis-dependent manner. The chain of gene expression activating cdc25 may be important as part of the mechanism that ensures the dependency of nuclear division on DNA replication during meiosis.

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LncRNA RP1-85F18.6 affects osteoblast cells by regulating the cell cycle

Open Life Sciences ◽

10.1515/biol-2020-0090 ◽

2020 ◽

Vol 15 (1) ◽

pp. 951-958

Author(s):

Jiangtao Song ◽

Wenrong Song ◽

Lei Zhang

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Cyclin A ◽

S Phase ◽

Cyclin B ◽

Regulatory Proteins ◽

Osteoblast Cell ◽

Osteoblast Cells ◽

M Phase ◽

Cell Cycle Regulatory Proteins

AbstractA lncRNA RP1-85F18.6 was reported to affect cell growth by regulating the cell cycle. Here we tested whether it affects the proliferation of osteoblast cells by regulating the cell cycle. We determined the expression of RP1-85F18.6 in two osteoblast cell lines hFOB and HOB by qPCR. Then we knocked down or overexpressed RP1-85F18.6 in hFOB and tested the alteration of viability, cell cycle, and cell cycle regulatory proteins. Results showed that both hFOB and HOB expressed RP1-85F18.6. The knockdown of RP1-85F18.6 decreased the viability of hFOB, while the overexpression of it increased the viability. Higher expression of RP1-85F18.6 results in higher cell viability. The knockdown of RP1-85F18.6 caused an increase in the S phase cells and a decrease in the G2/M phase cells. The overexpression of RP1-85F18.6 caused a decrease in the S phase cells and an increase in the G2/M phase cells. The knockdown of RP1-85F18.6 decreased cyclin A, cdk1, E2F, cyclin B, p53, and p21, whereas the overexpression of RP1-85F18.6 increased cyclin A, cdk1, E2F, cyclin B, p53, and p21. This study demonstrated that RP1-85F18.6 is expressed in osteoblast cell lines hFOB and HOB. RP1-85F18.6 affects the proliferation of osteoblasts by regulating the cell cycle.

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Canonical and Alternative Pathways in Cyclin-Dependent Kinase 1/Cyclin B Inactivation upon M-Phase Exit in Xenopus laevis Cell-Free Extracts

Enzyme Research ◽

10.4061/2011/523420 ◽

2011 ◽

Vol 2011 ◽

pp. 1-8

Author(s):

Jacek Z. Kubiak ◽

Mohammed El Dika

Keyword(s):

Cell Cycle ◽

Xenopus Laevis ◽

Cell Cycle Progression ◽

Cyclin B ◽

Cyclin Dependent Kinase ◽

Cycle Progression ◽

Alternative Pathways ◽

Global View ◽

M Phase

Cyclin-Dependent Kinase 1 (CDK1) is the major M-phase kinase known also as the M-phase Promoting Factor or MPF. Studies performed during the last decade have shown many details of how CDK1 is regulated and also how it regulates the cell cycle progression. Xenopus laevis cell-free extracts were widely used to elucidate the details and to obtain a global view of the role of CDK1 in M-phase control. CDK1 inactivation upon M-phase exit is a primordial process leading to the M-phase/interphase transition during the cell cycle. Here we discuss two closely related aspects of CDK1 regulation in Xenopus laevis cell-free extracts: firstly, how CDK1 becomes inactivated and secondly, how other actors, like kinases and phosphatases network and/or specific inhibitors, cooperate with CDK1 inactivation to assure timely exit from the M-phase.

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When RAD52 Allows Mitosis to Accept Unscheduled DNA Synthesis

Cancers ◽

10.3390/cancers12010026 ◽

2019 ◽

Vol 12 (1) ◽

pp. 26 ◽

Cited By ~ 2

Author(s):

Camille Franchet ◽

Jean-Sébastien Hoffmann

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Fragile Site ◽

Repetitive Sequences ◽

Eukaryotic Cell ◽

S Phase ◽

Replication Forks ◽

Polymerase Eta ◽

Dna Polymerase Eta

Faithful duplication of the human genome during the S phase of cell cycle and accurate segregation of sister chromatids in mitosis are essential for the maintenance of chromosome stability from one generation of cells to the next. Cells that are copying their DNA in preparation for division can suffer from ‘replication stress’ (RS) due to various external or endogenous impediments that slow or stall replication forks. RS is a major cause of pathologies including cancer, premature ageing and other disorders associated with genomic instability. It particularly affects genomic loci where progression of replication forks is intrinsically slow or problematic, such as common fragile site (CFS), telomeres, and repetitive sequences. Although the eukaryotic cell cycle is conventionally thought of as several separate steps, each of which must be completed before the next one is initiated, it is now accepted that incompletely replicated chromosomal domains generated in S phase upon RS at these genomic loci can result in late DNA synthesis in G2/M. In 2013, during investigations into the mechanism by which the specialized DNA polymerase eta (Pol η) contributes to the replication and stability of CFS, we unveiled that indeed some DNA synthesis was still occurring in early mitosis at these loci. This surprising observation of mitotic DNA synthesis that differs fundamentally from canonical semi-conservative DNA replication in S-phase has been then confirmed, called “MiDAS”and believed to counteract potentially lethal chromosome mis-segregation and non-disjunction. While other contributions in this Special Issue of Cancers focus on the role of RAS52RAD52 during MiDAS, this review emphases on the discovery of MiDAS and its molecular effectors.

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