Academic literature on the topic 'Molecular biology, Hematopoiesis, Gene regulation'
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Journal articles on the topic "Molecular biology, Hematopoiesis, Gene regulation"
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Stellrecht, C. M., G. Fraizer, C. Selvanayagam, L. Y. Chao, A. Lee, and G. F. Saunders. "Transcriptional regulation of a hematopoietic proteoglycan core protein gene during hematopoiesis." Journal of Biological Chemistry 268, no. 6 (February 1993): 4078–84. http://dx.doi.org/10.1016/s0021-9258(18)53582-1.
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de Rooij, Laura P. M. H., Derek C. H. Chan, Ava Keyvani Chahi, and Kristin J. Hope. "Post-transcriptional regulation in hematopoiesis: RNA binding proteins take control." Biochemistry and Cell Biology 97, no. 1 (February 2019): 10–20. http://dx.doi.org/10.1139/bcb-2017-0310.
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Normal hematopoiesis is sustained through a carefully orchestrated balance between hematopoietic stem cell (HSC) self-renewal and differentiation. The functional importance of this axis is underscored by the severity of disease phenotypes initiated by abnormal HSC function, including myelodysplastic syndromes and hematopoietic malignancies. Major advances in the understanding of transcriptional regulation of primitive hematopoietic cells have been achieved; however, the post-transcriptional regulatory layer that may impinge on their behavior remains underexplored by comparison. Key players at this level include RNA-binding proteins (RBPs), which execute precise and highly coordinated control of gene expression through modulation of RNA properties that include its splicing, polyadenylation, localization, degradation, or translation. With the recent identification of RBPs having essential roles in regulating proliferation and cell fate decisions in other systems, there has been an increasing appreciation of the importance of post-transcriptional control at the stem cell level. Here we discuss our current understanding of RBP-driven post-transcriptional regulation in HSCs, its implications for normal, perturbed, and malignant hematopoiesis, and the most recent technological innovations aimed at RBP–RNA network characterization at the systems level. Emerging evidence highlights RBP-driven control as an underappreciated feature of primitive hematopoiesis, the greater understanding of which has important clinical implications.
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Choi, Kyunghee. "Hemangioblast development and regulation." Biochemistry and Cell Biology 76, no. 6 (December 1, 1998): 947–56. http://dx.doi.org/10.1139/o99-007.
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Hematopoietic and endothelial cell lineages are the first to mature from mesoderm in the developing embryo. However, little is known about the molecular and (or) cellular events leading to hematopoietic commitment. The recent applications of technology utilizing gene targeted mice and the employment of many available in vitro systems have facilitated our understanding of hematopoietic establishment in the developing embryo. It is becoming clear that embryonic hematopoiesis occurs both in the extra-embryonic yolk sac and within the embryo proper in the mouse. The existence of the long pursued hemangioblast, a common progenitor of hematopoietic and endothelial cells, is now formally demonstrated. Based on this new information, many studies are being conducted to understand hematopoietic commitment events from mesoderm. In this review, we will first discuss the establishment of the hematopoietic system with special emphasis on the most primitive hematopoietic committed cells, the hemangioblast. We will then discuss mesoderm-inducing factors and their possible role in hematopoietic lineage commitment.Key words: hematopoietic commitment, hemangioblast, in vitro embryonic stem cell differentiation.
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Gandillet, Arnaud, Alicia G. Serrano, Stella Pearson, Michael Lie-A-Ling, Georges Lacaud, and Valerie Kouskoff. "Sox7-sustained expression alters the balance between proliferation and differentiation of hematopoietic progenitors at the onset of blood specification." Blood 114, no. 23 (November 26, 2009): 4813–22. http://dx.doi.org/10.1182/blood-2009-06-226290.
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Abstract The molecular mechanisms that regulate the balance between proliferation and differentiation of precursors at the onset of hematopoiesis specification are poorly understood. By using a global gene expression profiling approach during the course of embryonic stem cell differentiation, we identified Sox7 as a potential candidate gene involved in the regulation of blood lineage formation from the mesoderm germ layer. In the present study, we show that Sox7 is transiently expressed in mesodermal precursors as they undergo specification to the hematopoietic program. Sox7 knockdown in vitro significantly decreases the formation of both primitive erythroid and definitive hematopoietic progenitors as well as endothelial progenitors. In contrast, Sox7-sustained expression in the earliest committed hematopoietic precursors promotes the maintenance of their multipotent and self-renewing status. Removal of this differentiation block driven by Sox7-enforced expression leads to the efficient differentiation of hematopoietic progenitors to all erythroid and myeloid lineages. This study identifies Sox7 as a novel and important player in the molecular regulation of the first committed blood precursors. Furthermore, our data demonstrate that the mere sustained expression of Sox7 is sufficient to completely alter the balance between proliferation and differentiation at the onset of hematopoiesis.
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Guo, Fukun, Wei Liu, Kankana Chava, Jose Cancelas, George Thomas, Sara C. Kozma, and Yi Zheng. "Role of mTOR in Hematopoiesis and Hematopoietic Stem Cell Regulation." Blood 114, no. 22 (November 20, 2009): 1490. http://dx.doi.org/10.1182/blood.v114.22.1490.1490.
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Abstract Abstract 1490 Poster Board I-513 The mammalian target of rapamycin (mTOR) integrates nutrients, growth factors, and cellular energy status to control protein synthesis that determines cell growth and metabolism. It is also known that mTOR plays an essential role in cell survival by regulating Akt/PKB signaling. By using the inhibitor rapamycin, mTOR has previously been suggested to regulate proliferation of megakaryocyte progenitors and late stage of megakaryocyte differentiation without a general impact on normal hematopoiesis or hematopoietic stem cell (HSC) function. Due to limitations of rapamycin and the early lethality of conventional mTOR gene targeted mice, the physiological role of mTOR in blood development remains undefined. In this study, we have utilized an inducible conditional mTOR knockout mouse model by crossbreeding mTORflox/flox mice with Mx-Cre mice that allow interferon-induced mTOR deletion in the bone marrow following a transplantation and polyI:C induction protocol, in an effort to determine the genetic role of mTOR in hematopoiesis. Depletion of mTOR drastically affected hematopoiesis in a blood cell autonomous manner in Mx-Cre;mTORflox/flox bone marrow transplant recipients: the mice showed marked reduction in BM cellularity and in the numbers of myeloid and lymphoid lineage cells, erythrocytes, and platelets in peripheral blood, bone marrow, and thymus, leading to bone marrow failure, blood cell exhaustion and lethality. In vitro colony-forming activities by bone marrow or spleen progenitors were completely abolished in the absence of mTOR. Interestingly, the number and frequency of HSCs in bone marrow (Lin−Sca-1+c-Kit+) increased transiently while the number of early progenitors (CMP, GMP, MEP, CLP) detected by cell surface markers remained unchanged or only mildly affected in the mutant mice within 14 days after polyI:C treatment. Concomitantly, mTOR deletion led to a massive egress of HSCs from bone marrow to distal organs including spleen (∼60-fold increase). Transplantation of mTOR−/− bone marrow cells into NOD-SCID mice or competitive transplantation of mTOR−/− bone marrow cells into BoyJ mice further demonstrated that mTOR deficiency caused a complete failure in HSC engraftment and repopulation. Surprisingly, at the cellular level these phenotypes are associated with increased proliferation of HSCs in vivo and in vitro by 60% and 2.5-fold, respectively, as assessed by 5-bromodeoxyuridine incorporation assays whereas the cell survival index appears to be unaffected. Moreover, mTOR−/− HSCs and progenitor cells displayed impaired adhesion to fibronectin CH296 fragment (∼30% decrease) and migration toward SDF-1α gradients (∼30% decrease). At the molecular level, gene chip microarray analysis of mTOR−/− HSCs revealed that the cell cycle regulators myb, wee1, FANCD2, and FANCE were significantly downregulated while Rb and E2F5 were upregulated, the survival/apoptosis regulators MCL1 and BCL2L1 were upregulated, and the actin cytoskeleton and cell extracellular matrix adhesion regulators Arp2/3 complex subunit 5, paxillin, laminin α5, integrin β3, and myosin light chain 6B were upregulated. Further, immunoblotting analysis of isolated Lin− cells showed that SCF-stimulated activation of translational regulators S6K and 4E-BP and survival regulator Akt were abolished upon mTOR deletion. Taken together, these data suggest that mTOR is a critical regulator of HSC quiescence, self-renewal, and engraftment through the regulation of cell cycle, survival and actin cytoskeleton signals, and is essential in multiple stages of hematopoiesis. Disclosures Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.
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Gu, Yi, Michael C. Byrne, Nivanka C. Paranavitana, Bruce Aronow, Jamie E. Siefring, Maria D'Souza, Heidi F. Horton, Lawrence A. Quilliam, and David A. Williams. "Rac2, a Hematopoiesis-Specific Rho GTPase, Specifically Regulates Mast Cell Protease Gene Expression in Bone Marrow-Derived Mast Cells." Molecular and Cellular Biology 22, no. 21 (November 1, 2002): 7645–57. http://dx.doi.org/10.1128/mcb.22.21.7645-7657.2002.
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ABSTRACT Rho family GTPases activate intracellular kinase cascades to modulate transcription of multiple genes. Previous studies have examined the roles of the ubiquitously expressed Rho GTPase, Rac1, in regulation of gene expression in cell lines and implicated NF-κB, serum response factor, and kinase signaling pathways in this regulation. To understand the role of the closely related but hematopoiesis-specific Rho GTPase, Rac2, in regulation of gene transcription, we compared the gene expression profiles between wild-type and Rac2−/− bone marrow-derived mast cells. Our data demonstrate remarkable specificity in the regulation of gene expression by Rac2 versus Rac1. Microarray analysis demonstrated that expression of 38 known genes was significantly altered in Rac2−/− mast cells after cytokine stimulation compared with those in wild-type cells. Of these, the expression of the mouse mast cell protease 7 (MMCP-7) gene in wild-type cells was highly induced at the transcriptional level after stimulation with stem cell factor (SCF). In spite of compensatorily increased expression of Rac1 in Rac2-deficient cells, SCF-induced MMCP-7 transcription did not occur. Surprisingly, the loss of MMCP-7 induction was not due to decreased activation of NF-κB, a transcription factor postulated to lie downstream of Rac1 and known to play a critical role in hematopoietic cell differentiation and proliferation. However, the activities of c-Jun N-terminal kinases (JNKs) were markedly decreased in Rac2−/− mast cells. Our results suggest that cytokine-stimulated activation of MMCP-7 gene transcription is selectively regulated by a Rac2-dependent JNK signaling pathway in primary mast cells and imply a remarkable specificity in the regulation of transcriptional activity by these two highly related Rho GTPases.
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Liao, Eric C., Nikolaus S. Trede, David Ransom, Augustin Zapata, Mark Kieran, and Leonard I. Zon. "Non-cell autonomous requirement for thebloodlessgene in primitive hematopoiesis of zebrafish." Development 129, no. 3 (February 1, 2002): 649–59. http://dx.doi.org/10.1242/dev.129.3.649.
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Vertebrate hematopoiesis occurs in two distinct phases, primitive (embryonic) and definitive (adult). Genes that are required specifically for the definitive program, or for both phases of hematopoiesis, have been described. However, a specific regulator of primitive hematopoiesis has yet to be reported. The zebrafish bloodless (bls) mutation causes absence of embryonic erythrocytes in a dominant but incompletely penetrant manner. Primitive macrophages appear to develop normally in bls mutants. Although the thymic epithelium forms normally in bls mutants, lymphoid precursors are absent. Nonetheless, the bloodless mutants can progress through embryogenesis, where red cells begin to accumulate after 5 days post-fertilization (dpf). Lymphocytes also begin to populate the thymic organs by 7.5 dpf. Expression analysis of hematopoietic genes suggests that formation of primitive hematopoietic precursors is deficient in bls mutants and those few blood precursors that are specified fail to differentiate and undergo apoptosis. Overexpression of scl, but not bmp4 or gata1, can lead to partial rescue of embryonic blood cells in bls. Cell transplantation experiments show that cells derived from bls mutant donors can differentiate into blood cells in a wild-type host, but wild-type donor cells fail to form blood in the mutant host. These observations demonstrate that the bls gene product is uniquely required in a non-cell autonomous manner for primitive hematopoiesis, potentially acting via regulation of scl.
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Contreras, Jorge R., Thilini Fernando, Tiffany M. Tran, Matteo Zampini, Norma Iris Rodriguez-Malave, Jayanth Kumar Palanichamy, Jasmine Gajeton, et al. "Molecular Characterization of Long Non-Coding RNA CASC15 in Leukemogenesis." Blood 128, no. 22 (December 2, 2016): 5103. http://dx.doi.org/10.1182/blood.v128.22.5103.5103.
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Abstract High throughput transcriptome sequencing has uncovered a previously uncharacterized layer of gene regulation by long non-coding RNAs (lncRNAs). LncRNAs are characterized by capped, polyadenylated, and spliced transcripts that lack an open reading frame. Despite the similarities in their genetic organization, they play variety of roles at the cellular level, including regulation of transcription and translation, leading to alterations in gene expression. One of these functions is the regulation of expression of chromosomally adjacent genes. Here, we examined the function of the lncRNA CASC15 that was originally discovered as being dysregulated in in ETV6-RUNX1-translocated B-acute lymphoblastic leukemia. Enforced expression of CASC15 in hematopoietic stem and progenitor cells led to a myeloid bias in development with an overall decrease in engraftment and colony formation. Conversely, using a CRISPR-based approach, CASC15 deletion skewed hematopoietic cell progenitors towards a B cell fate. CASC15 was also demonstrated to regulate cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown in cell lines were enrichment for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. To further characterize this, we queried a functional relationship between YY1 and CASC15. Interestingly, we found that YY1 interacts with CASC15, and that CASC15 enhanced YY1-mediated transcription at the SOX4 promoter. Together these studies represent some of the first functional characterizations of lncRNAs in leukemia and highlight the importance of non-coding regulatory mechanisms in malignant hematopoiesis. Disclosures No relevant conflicts of interest to declare.
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He, XY, VP Antao, D. Basila, JC Marx, and BR Davis. "Isolation and molecular characterization of the human CD34 gene." Blood 79, no. 9 (May 1, 1992): 2296–302. http://dx.doi.org/10.1182/blood.v79.9.2296.2296.
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Abstract The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5′ termini of mRNAs by 5′-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5′ and 3′ untranslated regions, amino acid coding sequences, and 5′ flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.
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He, XY, VP Antao, D. Basila, JC Marx, and BR Davis. "Isolation and molecular characterization of the human CD34 gene." Blood 79, no. 9 (May 1, 1992): 2296–302. http://dx.doi.org/10.1182/blood.v79.9.2296.bloodjournal7992296.
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The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5′ termini of mRNAs by 5′-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5′ and 3′ untranslated regions, amino acid coding sequences, and 5′ flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.
Dissertations / Theses on the topic "Molecular biology, Hematopoiesis, Gene regulation"
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CANTU', CLAUDIO. "The Sox6 transcription factor: its role in human and murine erythroid differentiation and mechanisms for its regulation." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2009. http://hdl.handle.net/10281/8374.
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To identify new genes functionally involved in erythropoiesis during development and maturation, we analysed by DNA microarray three cell populations of different maturity during mouse fetal liver development (E11.5 - E13.5). Among genes whose expression increases in parallel with erythroid maturation there is Sox6, a member of the Sry–related transcription factors family.
Sox6 is known to play a major role in erythropoiesis: its ablation in mouse causes a strong relative increase of the expression of embryonic (εy) versus other non-alpha globin genes in late gestation, and a high number of circulating nucleated and misshapen erythrocytes.
To study the role of Sox6 in human erythropoiesis, we overexpressed it by lentiviral vector transduction both in the K562 erythroleukemic cell line and in human primary CD34+ cord blood cells .
Sox6 induces significant differentiation in both models as shown by morphological and FACS analysis. Moreover, several erythroid specific transcripts are greatly increased , i.e. mRNAs for enzymes controlling the heme-biosynthetic pathway, for transcription factors and for all globins (although the ratio between epsilon and gamma-globin is decreased), suggesting that Sox6 is a general positive regulator of erythroid genes expression.
Despite their erithroleukemic origin, K562 overexpressing Sox6 grow at a very low rate when compared with control cells, and die in culture within about ten days after transduction.
Sox6 overexpression causes in fact a strong increase of SOCS3 (suppressor of cytokine-signaling) transcript, which is known to block Jak2 signalling.
Since erythroblasts undergo cell-cycle withdrawal during terminal differentiation,
our working hypothesis is that Sox6 may induce the erythroid terminal maturation program through a block in the cell-cycle progression, possibly by acting as a repressor of the Jak signalling pathway.
We then used the Sox6 consensus from the εy-globin promoter to perform a bioinformatic genome-wide search for similar evolutionarily conserved motifs, and we found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in-vitro and in-vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells. Moreover, the binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by a strong downregulation of the endogenous Sox6 transcript.
Finally, we demonstrated that Sox6 expression, in human erythroid cultures and in mouse bone marrow cell populations, peaks at the erythroblast stage and decreases along with erythroid differentiation. Together these observations suggest that the negative Sox6 autoregulation mediated by the double Sox6 binding site within its own promoter, might be relevant to control the Sox6 transcriptional downregulation observed in late erythroid maturation
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Martin, Richard. "Regulation of SCL expression and function in hematopoiesis." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85582.
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The development of the hematopoietic system occurs in two waves: a first wave of primitive erythropoiesis, which consists in the production of a single lineage, primitive erythrocytes, and a second wave of definitive hematopoiesis, which describes the generation of many specialized blood cell types from common hematopoietic stem cells. Whereas definitive hematopoiesis is fairly well understood, involves signals from the environment and the expression of lineage-specific transcription factors, the molecular mechanisms regulating primitive erythropoiesis remain to be defined. The aim of this thesis was to clarify the roles of the Stem Cell Leukemia (SCL) gene and Vascular Endothelial Growth Factor (VEGF) during primitive and definitive hematopoiesis. Although gene targeting experiments indicate essential roles for VEGF/Flk-1 signaling and SCL at the onset of hematopoiesis, their exact functions remain elusive. This work has revealed that different thresholds of VEGF are required for the migration of hematopoietic precursors from mesoderm to sites of hematopoiesis and for their subsequent expansion. Furthermore, it shows that SCL, a basic helix-loop-helix transcription factor, acts downstream of VEGF signaling to ensure the survival of primitive erythrocytes. During definitive hematopoiesis, conditional knock-out experiments establish a non-redundant role for SCL during erythroid and megakaryocytic differentiation. Yet, it remains unclear whether SCL is essential for commitment to these lineages. Results presented in this thesis suggest that SCL is not involved in commitment to these pathways, but rather acts to consolidate and expand the erythroid and megakaryocytic compartments, following lineage choice. Finally, despite the central role for SCL during hematopoietic development, the mechanisms regulating its tissue specific expression remain unknown. This work provides molecular and functional evidence that demonstrate that the homeodomain-Taken together, this work has elucidated molecular mechanisms which underlie cell fate decisions. It describes how the activity of a master regulator of erythroid differentiation, SCL, is regulated both by signals from the environment and at the transcriptional level, through combinatorial interactions between lineage-specific transcription factors.
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Xu, Yong Zhong. "Molecular mechanisms of regulation of SLC11A1 gene expression." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121167.
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Solute carrier family 11 member 1 protein (SLC11A1), also known as natural resistance-associated macrophage protein 1 (NRAMP1), plays an important role in the host immune defense and inflammatory response. It is a highly conserved transmembrane protein which transports divalent metal cations in a proton-dependent manner. It regulates iron homeostasis in macrophages and exerts pleiotropic effects on macrophage activation. In mice, natural resistance or susceptibility to a range of intracellular pathogens is controlled by the Slc11a1 gene. In human, genetic polymorphisms of the SLC11A1 gene have been shown to be associated with a susceptibility to a variety of infectious and autoimmune diseases. The expression of the SLC11A1 gene is strictly regulated during myeloid differentiation. The human promyelocytic leukemia cell lines such as HL-60 and U937 cells are useful models to study the regulation of SLC11A1 gene expression during experimentally induced granulocytic, monocytic, or macrophage-like differentiation. Herein, we demonstrated that during the PMA-induced differentiation of HL-60 cells and human monocytes toward macrophages, β-actin translocates from the cytoplasm to the nucleus where it is associated with RNA polymerase II and binds to the promoter of the SLC11A1 gene. β-actin knockdown inhibits the SLC11A1 promoter-driven transcription, and neutralization of nuclear actin by in vivo microinjection of antibodies against β-actin into nuclei significantly blocks the expression of SLC11A1 mRNA. Further studies revealed that an AP-1-like element present in the proximal region of the SLC11A1 gene promoter is essential for PMA-induced transcriptional activation of this gene. β-actin, as a subunit of the SWI/SNF complex, and another subunit BRG1 are associated with the transcription factor ATF-3 and are recruited to the AP-1 like element in an ATF-3-dependant manner . ATF-3 cooperates with BRG1 and β-actin to activate the SLC11A1 promoter. Furthermore, a proximal (GT/AC)n repeat (t(gt)5ac(gt)5ac(gt)9g) region adjacent to the AP-1-like element is converted into a Z-DNA structure in response to PMA treatment, and BRG1 is involved in this process. Our results suggest that recruitment of the SWI/SNF complex initiates Z-DNA formation and subsequently helps to transactivate the SLC11A1 gene. Previous studies have shown that SLC11A1 is extensively glycosylated and phosphorylated, and is localized at the membrane of late endosomes/lysosomes in macrophages. The present study revealed that SLC11A1 is tyrosine-phosphorylated during the differentiation of U937 cells into macrophages induced by PMA. Using the kinase inhibitor PP2 and RNA interference experiments, we demonstrated that Src family kinases including c-Src are required for the tyrosine phosphorylation of SLC11A1 protein. In vitro phosphorylation assays showed that SLC11A1 is a direct substrate for active c-Src kinase. Furthermore, tyrosine 15 is identified as the tyrosine phosphorylation site by Src family kinases and phosphorylation of tyrosine 15 modulates SLC11A1-mediated nitric oxide production. We also showed that the Src family kinases including c-Src are also involved in lysosomal targeting of SLC11A1. These results suggest an important role of Src family kinases in subcellular localization and function of SLC11A1 in macrophages. Overall, our studies contributed important information on the regulation of expression of SLC11A1 in macrophages and its role in regulation of macrophage functions.La protéine « Solute carrier family 11 member 1 » (SLC11A1), aussi connue sous le nom « natural resistance associated macrophage protein 1 » (NRAMP1), jour un rôle important dans la défense immunitaire et réponse inflammatoire de l'hôte. C'est une protéine transmembranaire hautement conservée qui transporte des cations divalents métalliques d'une manière dépendent des protons. Elle régularise l'homéostasie du fer dans les macrophages et a des effets pléiotropiques sur l'activation de ces cellules. Chez les souris, le gène Slc11a1 contrôle la resistance naturelle ou la susceptibilité aux pathogènes intracellulaires. Chez les humains, les polymorphismes génétiques de SLC11A1 sont associés à une susceptibilité à une variété de maladies infectieuses ou auto-immunitaires. L'expression du gène SLC11A1 est strictement régularisée pendant la différenciation myéloïde. Les lignées cellulaires humaines dérivées de la leucémie aiguë promyélocytaire, telles que la HL-60 et l'U937, sont des modèles utiles pour étudier le contrôle de l'expression du gène SLC11A1 pendant la différenciation de type granulocytaire, monocytaire ou de macrophage induite expérimentalement. Ici, nous avons démontré que durant la différenciation induite par PMA des cellules HL-60 et des monocytes humains aux macrophages, β-actine passe du cytoplasme au noyau où il s'associe avec l'ARN polymérase II et se fixe sure le promoteur du gène SLC11A1. Le « knock-down » de la β-actine inhibe la transcription menée par le promoteur de gène SLC11A1. Dans le noyau, l'expression de l'ARN de SLC11A1 est bloqué significativement en neutralisant l'actine par la microinjection in vivo des anticorps contre β-actine. Autres études ont démontré qu'un élément « semblable à AP-1 » est présent dans la région proximale du promoteur de SLC11A1 et celui-ci est essentiel pour l'activation transcriptionnelle de ce gène induite par PMA. β-actine, étant une sous-unité du complexe SWI/SNF, et la sous-unité BRG1 sont associés avec le facteur de transcription ATF-3. Ensemble, elles sont recrutées à l'élément semblable à AP-1 en une manière qui dépendante sur ATF-3. ATF-3 coopère avec BRG1 et β-actine pour activer le promoteur de SLC11A1. De plus, la région du répète proximale (GT/AC)n [t(gt)5ac(gt)5ac(gt)9g] adjacent au élément semblable à AP-1 est convertit en une structure de Z-ADN en réponse au traitement de PMA, un processus dans lequel BRG1 est impliqué. Nos résultats suggèrent que le recrutement du complexe SWI/SNF amorce la formation de Z-ADN et aide à transactiver le gène SLC11A1. Des études précédentes ont démontrés que SLC11A1 est extensivement glycosylée et phosphorylée, et que cette protéine se trouve chez les macrophages dans les membranes des endosomes tardifs ou des lysosomes. L'étude présentée ici a révélé que SLC11A1 est phosphorylée sur les tyrosines pendant la différenciation des cellules U937 aux macrophages par PMA. En utilisant l'inhibiteur de kinase PP2 et des essais d'interférence d'ARN, nous avons démontré que les kinases de la famille Src, incluant c-Src, sont requises pour la phosphorylation de tyrosine de la protéine SLC11A1. Les essais in vitro de phosphorylation ont montré que SCL11A1 est un substrat direct pour la kinase active c-Src. De plus, la tyrosine 15 a été identifiée comme étant le site de phosphorylation de kinases de la famille Src. La phosphorylation de tyrosine 15 fait moduler la production de l'oxyde nitrique de laquelle SLC11A1 fait parti. Nous avons aussi montré que les kinases de la famille Src ont un rôle important dans la localisation subcellulaire et dans le fonctionnement de SLC11A1 dans les macrophages. Globalement, nos études ont contribué d'information importante sur la régularisation de l'expression de SCL11A1 dans les macrophages et son rôle dans le fonctionnement des macrophages.
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Sutherland, Leslie C. "Transcriptional regulation of the murine PGK-1 gene." Thesis, University of Ottawa (Canada), 1994. http://hdl.handle.net/10393/10111.
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The gene encoding the glycolytic enzyme phosphoglycerate kinase is transcriptionally regulated at two levels. Expression of enzyme is related to the glycolytic activity of the cell, and is highest in transcriptionally active cells. Expression is also regulated by X chromosome inactivation, as the somatically expressed Pgk-1 gene is X-linked. The role of 5$\sp\prime$-flanking cis-acting DNA elements and trans-acting factors in the regulation of Pgk-1 expression was examined. The murine Pgk-1 gene contains an upstream activator sequence (UAS) in its 5$\sp\prime$-flanking region. This region was found to be responsible for elevating transcription levels at least ten-fold above basal Pgk-1 promoter levels in P19 embryonal carcinoma (EC) cells. Part of this activity was attributed to the R2 protein binding site, first identified by DNase 1 footprinting techniques. Mutation of the middle region of R2 resulted in a 5-fold reduction in expression of a Pgk-1 driven construct in stable transfection experiments into P19 cells. It was also determined that the R2 site was not important for transcription in P19 cells induced to differentiate with retinoic acid (RA). In undifferentiated P19 cells, another UAS protein binding site, R1, was identified by band shift analysis. R1 could not be detected by footprint analysis, suggesting that the affinity of binding at R1 was lower than at R2. The mutation of the R2 site did not abolish protein binding, which led to the hypothesis that multiple factors were binding the DNA at R2. R1 was also hypothesized to interact with multiple factors. However, fractionation of the P19 nuclear extract and use in band shift studies against the R1 DNA resulted in a single fraction with binding activity, suggesting a single R1 DNA binding protein and a non-DNA binding component. This non-DNA binding component at R1 was found to be tissue- or species-specific. Southwestern analysis in conjunction with fractionation experiments suggested that one of the R2 DNA binding proteins was approximately 70 kD and that the R1 DNA binding protein was 120 kD. Treatment of the P19 cells with RA led to a reduction in gene expression. Two days after exposure to the drug, the contribution to expression from the UAS was reduced by 50%, and four days after exposure the UAS no longer contributed to gene expression. Protein binding to the UAS was also altered after RA-treatment. A new site of protein interaction was detected in the distal region of the UAS, at R3, and binding at R1 was altered. There was, therefore, a correlation between protein interaction within the UAS and gene expression during differentiation. The results presented in this thesis demonstrate that the regulation of PGK-1 occurs, at least in part, at the level of gene expression, and that the UAS has an important role to play in regulating expression levels of the gene during differentiation. The results also suggest that the transcriptional stimulatory activity of the UAS depends on higher-order interactions between multiple low affinity DNA binding proteins which change upon differentiation.
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Bakopanos, Evangelos. "Regulation of the 3-adrenergic receptor gene expression." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38148.
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The beta3-adrenergic receptor (beta3-AR) represents a very attractive target for the development of anti-obesity drugs. Selective agonists have been developed based on the rodent beta3-AR and successfully used to treat obesity and Type 2 diabetes in these animals. In humans, however, these particular compounds appear to be less effective and not as specific for the beta3-AR as they are in rodents. An additional problem is the low level of expression of these receptors in human adipose, which may ultimately limit the clinical use of stronger more specific agonists. The objective of this research was to examine the regulation of the beta3-AR gene expression. In order to characterize functionally the cis-acting elements necessary for 3-AR proximal promoter activity, a series of luciferase reporter constructs containing various portions of the mouse 5'-flanking region of the gene, spanning from -1.4 kb to -0.126 kb relative to the translation start site (since there is no unique transcription start site), were generated and their transcriptional activity was evaluated by transient transfection experiments. We identified a 13 by element located between -0.208 kb to -0.196 kb that is essential for basal and constitutive proximal promoter activity. The beta3-AR mRNA has a rapid turnover and is readily expressed in the absence of apparent stimuli, in cells cultured in medium with charcoal-stripped serum. Out of several possible regulators we focused on glucocorticoids and thiazolidinediones, the former because it was a potentially important negative regulator, the latter because it could possibly stimulate the expression. In HIB-1B brown adipocytes, glucocorticoids appear to have two opposing effects on beta3-AR gene expression: they rapidly and directly inhibit transcription but also induce a rapidly turned-over protein that stimulates transcription of the gene. The level of beta3-AR expression in response to glucocorticoids may ultimately depend on the relative magnitu
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Bales, Mark. "Molecular regulation of gene expression in anterior mesendoderm of vertebrates." Diss., The University of Arizona, 2002. http://hdl.handle.net/10150/280000.
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The signaling pathways that regulate the fate of cells located in the anterior lateral (AL) region of the vertebrate embryo are not well understood. Mesodermal cells in this region will assume the heart muscle phenotype while adjacent endoderm gives rise to foregut tissues such as the liver. The AL endoderm supplies key signaling molecules to promote the survival and differentiation of the precardiac mesoderm. These AL endoderm factors are know to up-regulate transcription factors, such as Nkx2--5, that regulate cardiac genes. However, little is known about how the AL endoderm is patterned and the exact mechanism by which the cardiac transcription factors function within the mesoderm. Therefore, two projects were pursued to understand the developmental pathways that promote early heart development. One project looks at defining the mechanism by which the Nkx2 homeobox genes regulate cardiac gene expression in mouse embryonic stem cells. Unfortunately, this project was plagued with difficulties. Mouse ES cells were used as a model system to study cardiac differentiation. However, these cells were found to contain a potent genome protection mechanism that prevented the stable integration of transcription factors. This phenomenon is addressed and discussed within the thesis. The second project defines the role of retinoids in patterning the AL endoderm. In this study, the homeobox gene cHex, a gene required for hepatocyte development, was used as an AL endoderm marker. It was found that retinoids act directly on the cHex gene promoter to reduce its activity and restrict its expression domain to the AL endoderm.
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Argentin, Stefania. "Transcriptional regulation of the rat atrial natriuretic factor gene." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74556.
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Atrial natriuretic factor (ANF), a 28 amino acid peptide hormone, is the major secretory product of the heart. Because of its diuretic, natriuretic and vasodilating activities, this peptide may be involved in the maintenance of proper fluid and electrolyte balance and blood pressure control. In order to study the transcriptional regulation of ANF, we have isolated the rat ANF gene and we have established a system of cardiocytes in primary cell culture for studies on the hormonal, tissue-specific and developmental regulation of the ANF gene. Using this in vitro system, as have demonstrated that thyroid hormone increases ANF mRNA levels about 2- to 4-fold in atrial and ventricular cells in primary cardiocyte cell cultures, respectively. Similarly, glucocorticoids augment by about 3-fold both atrial and ventricular ANF mRNA levels in cardiac cells in culture. Glucocorticoids exert this effect at the transcriptional level probably via the binding of glucocorticoid receptor to a DNA element in the distal 5$ sp prime$-flanking sequences of the gene as suggested by DNA-mediated transfection studies in cardiocyte cultures. In order to better understand the mechanisms governing the cardiac-specific as well as developmental expression of the ANF gene, we have analyzed ANF promoter sequences by transient transfection studies in primary cardiocyte cultures. Our data show that the ANF promoter is active only in cells of cardiac origin. Moreover, up to $-$1.6 kb of 5$ sp prime$ upstream sequences are necessary for full expression of the ANF gene in cardiac cells. Within these sequences, two particular elements, a proximal and a distal, are necessary for full ANF transcriptional activity. The proximal element can confer cardiac specificity to an otherwise non tissue-specific heterologous promoter. Further upstream sequences, between $-$2.5 and $-$1.6 kb appear to be implicated in the developmental control of ANF gene expression, as assessed by differential activity in 1 and 4 d
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Lefebvre, Tania. "Role of USP4 in the regulation of gene expression." Thesis, University of Ottawa (Canada), 2007. http://hdl.handle.net/10393/27471.
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USP4 is a deubiquitinating enzyme whose levels have been shown to be elevated in certain human lung tumors. USP4 is thought to possess oncogenic properties due to its ability to promote tumors in nude mice assays. The lack of an overall effect on ubiquitin levels in overexpression studies has led to the hypothesis that USP4 may act on a few select substrates to edit their ubiquitination status. Although the structure/function relationship is more documented, the physiological substrates and role in vivo are not. In order to elucidate the mechanism by which USP4 could potentially exert its tumorigenic effect, an RNA knockdown approach was undertaken. The effect of changes in USP4 levels was investigated to determine if USP4 plays a role in transcription or mRNA stability. The data suggest that USP4 does not affect levels of mRNAs containing an ARE sequence in NIH-3T3 or Cos-7 cells. Although USP4 was not shown to have an mRNA stabilizing effect, USP4 was found to bind CBP in vivo using an immunoprecipitation experiment and to exert an effect on basal transcription levels. This data suggests that USP4 levels may affect global transcription, perhaps through binding with the transcriptional co-activator CBP.
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Garnier, France. "Study of transcription regulation of the gene mdr1." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=56986.
Full textAbstract:
In order to characterize cis-acting sequences and trans-acting factors important in regulating the expression of the mouse mdr1 gene, in vitro DNAsel footprinting experiments were carried out on a mdr1 promoter segment between positions $-$245 and +84, using nuclear protein extracts prepared from cell lines expressing different endogenous amounts of mdr1 mRNAs. Three footprinted sequences were detected on the non-coding strand of the $-$245 to +84 mdr1 promoter fragment (between -77 to -56, between -46 to -24, and between +5 and +15) with nuclear extracts from mdr1 expressing cells (CMT-93, LTA, and Y-1 cells). In addition, a specific footprinted sequence ($-$14 to +5) was detected on both strands only with nuclear extracts from the mdr1 non-expressing cell line (RAG cells) suggesting the presence and binding of a putative negative regulatory factor in these cells. However, replacement of this sequence in the mdr1 basal promoter ($-$93 to +84) by a heterologous, although similar positioned SV40 sequence did not restore promoter activity in RAG cells. The basal mdr1 promoter was further characterized in bidirectional deletion mutants, in order to identify cis-acting elements important for general transcriptional regulation. These studies further localized the mdr1 basal promoter between positions $-$74 and +84, and also suggested the presence of possible positive and negative cis-acting sequence elements modulating the activity of this basal promoter.
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Canaff, Lucie. "Extracellular calcium-sensing receptor : studies of gene expression and regulation." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85136.
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The calcium-sensing receptor (CaSR), expressed in parathyroid, thyroid and kidney, is essential for maintenance of calcium homeostasis. Extracellular calcium (Ca2+o) affects several hepatic functions including bile secretion, metabolic activity, regeneration, and the response to xenobiotics. We have demonstrated the presence, in hepatocytes, of a functional CaSR. Western blot analysis using a specific CaSR antibody showed staining in both whole liver and hepatocyte extracts. Immunohistochemistry and in situ hybridization of rat liver sections showed expression of CaSR protein and mRNA by a subset of hepatocytes. CaSR agonists, gadolinium (Gd3+; 0.5-3.0 mM) and spermine (1.25-20 mM), in the absence of Ca2+o, elicited dose-related increases in intracellular calcium (Ca2+i) in isolated rat hepatocytes loaded with Fura 2-AM and this response was abrogated when IP3-sensitive calcium pools had been depleted by pretreatment with either thapsigargin or phenylephrine. Addition of the deschloro-phenylalkylamine compound, NPS-R467, but not the S enantiomer, NPS-S467, increased the sensitivity of the Ca 2+i mobilization response to 1.25 mM spermine. Bile flow ceased after Ca2+o withdrawal and its recovery was enhanced by spermine in isolated perfused liver preparations. Ca2+ and Gd3+ increased bile flow and NPS-R467 but not the S compound enhanced the response to a sub maximal Ca2+ concentration. Thus, the data demonstrate that rat hepatocytes harbor a CaSR capable of mobilizing Ca2+i from IP3-sensitive stores and that activation of the CaSR stimulates bile flow.The human CASR gene contains at least 7 exons and spans more than 100 kilobases. Little is known about the 5' flanking region and transcriptional regulatory sequences that control expression of the CASR in specific cells. The human CASR gene has two promoters (P1 and P2) yielding alternative transcripts containing either exon 1A or exon 1B 5'-untranslated region sequences that splice to exon 2 some 242 bp before the ATG translation start site. We have cloned the CASR promoter and transcriptional start sites were identified in parathyroid gland and in human thyroid C-cell (TT) cells; that for promoter P1 lies 27-bp downstream of a TATA box, whereas that for promoter P2, which lacks a TATA box, lies in a GC-rich region.
Books on the topic "Molecular biology, Hematopoiesis, Gene regulation"
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G, Abraham Nader, and Symposium on the Molecular Biology of Hematopoiesis (11th 1998 : Bormio, Italy), eds. Molecular biology of hematopoiesis 6. New York: Kluwer Academic/Plenum Publishers, 1999.
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S, Sohal R., Birnbaum Linda S, and Cutler Richard G, eds. Molecular biology of aging: Gene stability and gene expression. New York: Raven Press, 1985.
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P, Vanden Heuvel John, and Peters Jeffrey M, eds. Regulation of gene expression: Molecular mechanisms. Totowa, N.J: Humana Press, 2006.
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1928-, Watson James D., ed. Molecular biology of the gene. 5th ed. San Francisco, California: Pearson/Benjamin Cummings, 2004.
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1928-, Watson James D., ed. Molecular biology of the gene. 4th ed. Menlo Park, California: Benjamin/Cummings, 1987.
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1928-, Watson James D., ed. Molecular biology of the gene. 4th ed. Menlo Park, California: Benjamin/Cummings Pub. Co., 1988.
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1928-, Watson James D., ed. Molecular biology of the gene. 6th ed. San Francisco: Pearson/Benjamin Cummings, 2008.
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1928-, Watson James D., ed. Molecular biology of the gene. 5th ed. San Francisco: Pearson/Benjamin Cummings, 2004.
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1928-, Watson James D., ed. Molecular biology of the gene. 5th ed. Delhi: Pearson Education, 2004.
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Collado-Vides, Julio, and Ralf Hofestädt. Gene regulation and metabolism: Postgenomic computational approaches. Cambridge, Mass: MIT Press, 2002.
Find full textBook chapters on the topic "Molecular biology, Hematopoiesis, Gene regulation"
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Kobayashi, Ichizo. "Homologous Gene Targeting For Human Gene Therapy." In Molecular Biology of Hematopoiesis 5, 345–49. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4613-0391-6_42.
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Bina, Minou. "Gene Regulation." In Methods in Molecular Biology, 1–11. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-284-1_1.
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Feder, J. N., D. M. Penny, A. Irrinki, G. A. Mintier, J. A. Lebron, C. N. Gross, L. Lee, et al. "The Hereditary Hemochromatosis Gene and Iron Homeostasis." In Molecular Biology of Hematopoiesis 6, 353–62. Boston, MA: Springer US, 1999. http://dx.doi.org/10.1007/978-1-4615-4797-6_43.
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Imagawa, Shigehiko, Masayuki Yamamoto, and Yasusada Miura. "Gata Transcription Factors Negatively Regulate Erythropoietin Gene Expression." In Molecular Biology of Hematopoiesis 5, 501–13. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4613-0391-6_61.
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Rameshwar, Pranela, and Pedro Gascón. "Neural Regulation of Hematopoiesis by the Tachykinins." In Molecular Biology of Hematopoiesis 5, 463–70. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4613-0391-6_56.
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Shibahara, Shigeki, Kazuhisa Takeda, Shoji Okinaga, Miki Yoshizawa, Kazuhiro Takahashi, and Hiroyoshi Fujita. "Transcriptional Control of the Human Heme Oxygenase-1 Gene by Stress." In Molecular Biology of Hematopoiesis 5, 439–44. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4613-0391-6_53.
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Nanmoku, Koji, Yasuyoshi Miyao, Masakazu Tamura, Kazuyoshi Tamura, Tadanori Yoshimatsu, Keiji Shimizu, and Kazuhiro Ikenaka. "Gene Transfer to the Nervous System Using High-Titer Retroviral Vector." In Molecular Biology of Hematopoiesis 6, 299–305. Boston, MA: Springer US, 1999. http://dx.doi.org/10.1007/978-1-4615-4797-6_37.
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Shimada, Takashi, and Koichi Miyake. "Targeted Gene Transfer into CD4 Positive Cells by HIV-Based Retroviral Vectors." In Molecular Biology of Hematopoiesis 5, 323–29. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4613-0391-6_39.
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Gruissem, Wilhelm. "Regulation of Plastid Gene Expression." In Plant Molecular Biology, 361–70. Berlin, Heidelberg: Springer Berlin Heidelberg, 1994. http://dx.doi.org/10.1007/978-3-642-78852-9_34.
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Kusy, Sophie, and Christopher H. Contag. "Reporter Gene Technologies for Imaging Cell Fates in Hematopoiesis." In Methods in Molecular Biology, 1–22. New York, NY: Springer New York, 2013. http://dx.doi.org/10.1007/978-1-4614-9437-9_1.
Full textConference papers on the topic "Molecular biology, Hematopoiesis, Gene regulation"
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"Molecular diagnostics of hearing loss due to mutations in the SLC26A4 gene in indigenous peoples of Southern Siberia (Russia)." In Bioinformatics of Genome Regulation and Structure/ Systems Biology. institute of cytology and genetics siberian branch of the russian academy of science, Novosibirsk State University, 2020. http://dx.doi.org/10.18699/bgrs/sb-2020-262.
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"Identification of a novel small RNA encoded in the mouse urokinase receptor uPAR gene (Plaur) and its molecular targets." In Bioinformatics of Genome Regulation and Structure/Systems Biology (BGRS/SB-2022) :. Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences, 2022. http://dx.doi.org/10.18699/sbb-2022-559.
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"Revealing the molecular basis of interactions of COVID-19 with hyperglycemia and diabetic complications based on the bioinformatics analysis of the gene networks." In Bioinformatics of Genome Regulation and Structure/Systems Biology (BGRS/SB-2022) :. Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences, 2022. http://dx.doi.org/10.18699/sbb-2022-520.
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LeDuc, Philip. "Linking Molecular to Cellular Biomechanics With Nano- and Micro-Technology." In ASME 2003 International Mechanical Engineering Congress and Exposition. ASMEDC, 2003. http://dx.doi.org/10.1115/imece2003-43987.
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The link between mechanics and biochemistry has been implicated in a myriad of scientific and medical problem, from orthopedics and cardiovascular medicine, to cell motility and division, to signal transduction and gene expression. Most of these studies have been focused on organ-level issues, yet cellular and molecular level research has become essential over the last decade in this field thanks to the revolutionary developments in genetics, molecular biology, fabrication processes, and biotechnology. Developing the link between molecular and cellular biomechanics through subcellular studies can help uncover the complex interactions requisite for understanding higher order macroscopic behavior. Here, we will explore the link between molecular and cellular research through novel systems of nano- and micro-technology. In this, I will discuss novel technologies that we have developed and are utilizing, which include magnetic needles, three-dimension cell stretching systems, and microfluidics to examine the link between mechanics and biochemistry (including structural regulation through the cytoskeleton). By combining these novel approaches between engineering and biology, this multidisciplinary research can make a tremendous impact on the studies of human health and diseases through advances in fields such as proteomics, tissue engineering, and medical diagnostics.
Reports on the topic "Molecular biology, Hematopoiesis, Gene regulation"
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Hirschberg, Joseph, and Gloria A. Moore. Molecular Analysis of Carotenoid Biosynthesis in Plants: Characterizing the Genes Psy, Pds and CrtL-e. United States Department of Agriculture, August 1993. http://dx.doi.org/10.32747/1993.7568744.bard.
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In this research we have studied the molecular biology of carotenoid biosynthesis in tomato. The investigations focused on the genes Pds and Psy, encoding desaturase and phytoene synthase, respectively, which are key enzymes in the biosynthetic pathway of lycopene and b-carotene. In addition, we have investigated the genes for lycopene cyclase. We have cloned from tomato and characterized the cDNA of CrtL-e, which encodes the lycopene e-cyclase, and analyzed its expression during fruit development. The results establish a paradigm for the regulation of carotenoid pigment biosynthesis during the ripening process of fruits. It is concluded that transcriptional regulation of genes that encode carotenoid-biosynthesis enzymes is the major mechanism that governs specific pigment accumulation. During the ripening of tomato fruits transcription of the genes encoding the enzymes phytoene synthase and phytoene desaturase is up-regulated, while the transcription of the genes for both lycopene cyclases decreases and thus the conversion of lycopene to subsequent carotenoids is inhibited. These findings support the working hypothesis of the molecular approach to manipulating carotenogenesis by altering gene expression in transgenic plants, and offer obvious strategies to future application in agriculture. The molecular and physiological knowledge on carotenogenesis gained in this project, suggest a concept for manipulating gene expression that will alter carotenoid composition in fruits and flowers.
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Shaw, John, Arieh Rosner, Thomas Pirone, Benjamin Raccah, and Yehezkiel Antignus. The Role of Specific Viral Genes and Gene Products in Potyviral Pathogenicity, Host Range and Aphid Transmission. United States Department of Agriculture, August 1992. http://dx.doi.org/10.32747/1992.7561070.bard.
Full textAbstract:
In this research we have studied the molecular biology of carotenoid biosynthesis in tomato. The investigations focused on the genes Pds and Psy, encoding desaturase and phytoene synthase, respectively, which are key enzymes in the biosynthetic pathway of lycopene and b-carotene. In addition, we have investigated the genes for lycopene cyclase. We have cloned from tomato and characterized the cDNA of CrtL-e, which encodes the lycopene e-cyclase, and analyzed its expression during fruit development. The results establish a paradigm for the regulation of carotenoid pigment biosynthesis during the ripening process of fruits. It is concluded that transcriptional regulation of genes that encode carotenoid-biosynthesis enzymes is the major mechanism that governs specific pigment accumulation. During the ripening of tomato fruits transcription of the genes encoding the enzymes phytoene synthase and phytoene desaturase is up-regulated, while the transcription of the genes for both lycopene cyclases decreases and thus the conversion of lycopene to subsequent carotenoids is inhibited. These findings support the working hypothesis of the molecular approach to manipulating carotenogenesis by altering gene expression in transgenic plants, and offer obvious strategies to future application in agriculture. The molecular and physiological knowledge on carotenogenesis gained in this project, suggest a concept for manipulating gene expression that will alter carotenoid composition in fruits and flowers.
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Horwitz, Benjamin A., and Barbara Gillian Turgeon. Fungal Iron Acquisition, Oxidative Stress and Virulence in the Cochliobolus-maize Interaction. United States Department of Agriculture, March 2012. http://dx.doi.org/10.32747/2012.7709885.bard.
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Our project focused on genes for high affinity iron acquisition in Cochliobolus heterostrophus, a necrotrophic pathogen of maize, and their intertwined relationship to oxidative stress status and virulence of the fungus on the host. An intriguing question was why mutants lacking the nonribosomal peptide synthetase (NRPS) gene (NPS6) responsible for synthesis of the extracellular siderophore, coprogen, are sensitive to oxidative stress. Our overall objective was to understand the mechanistic connection between iron stress and oxidative stress as related to virulence of a plant pathogen to its host. The first objective was to examine the interface where small molecule peptide and reactive oxygen species (ROS) mechanisms overlap. The second objective was to determine if the molecular explanation for common function is common signal transduction pathways. These pathways, built around sensor kinases, response regulators, and transcription factors may link sequestering of iron, production of antioxidants, resistance to oxidative stress, and virulence. We tested these hypotheses by genetic manipulation of the pathogen, virulence assays on the host plant, and by following the expression of key fungal genes. An addition to the original program, made in the first year, was to develop, for fungi, a genetically encoded indicator of redox state based on the commercially available Gfp-based probe pHyper, designed for animal cell biology. We implemented several tools including a genetically encoded indicator of redox state, a procedure to grow iron-depleted plants, and constructed a number of new mutants in regulatory genes. Lack of the major Fe acquisition pathways results in an almost completely avirulent phenotype, showing how critical Fe acquisition is for the pathogen to cause disease. Mutants in conserved signaling pathways have normal ability to regulate NPS6 in response to Fe levels, as do mutants in Lae1 and Vel1, two master regulators of gene expression. Vel1 mutants are sensitive to oxidative stress, and the reason may be underexpression of a catalase gene. In nps6 mutants, CAT3 is also underexpressed, perhaps explaining the sensitivity to oxidative stress. We constructed a deletion mutant for the Fe sensor-regulator SreA and found that it is required for down regulation of NPS6 under Fe-replete conditions. Lack of SreA, though, did not make the fungus over-sensitive to ROS, though the mutant had a slow growth rate. This suggests that overproduction of siderophore under Fe-replete conditions is not very damaging. On the other hand, increasing Fe levels protected nps6 mutants from inhibition by ROS, implying that Fe-catalyzed Fenton reactions are not the main factor in its sensitivity to ROS. We have made some progress in understanding why siderophore mutants are sensitive to oxidative stress, and in doing so, defined some novel regulatory relationships. Catalase genes, which are not directly related to siderophore biosynthesis, are underexpressed in nps6 mutants, suggesting that the siderophore product (with or without bound Fe) may act as a signal. Siderophores, therefore, could be a target for intervention in the field, either by supplying an incorrect signal or blocking a signal normally provided during infection. We already know that nps6 mutants cause smaller lesions and have difficulty establishing invasive growth in the host. Lae1 and Vel1 are the first factors shown to regulate both super virulence conferred by T-toxin, and basic pathogenicity, due to unknown factors. The mutants are also altered in oxidative stress responses, key to success in the infection court, asexual and sexual development, essential for fungal dissemination in the field, aerial hyphal growth, and pigment biosynthesis, essential for survival in the field. Mutants in genes encoding NADPH oxidase (Nox) are compromised in development and virulence. Indeed the triple mutant, which should lack all Nox activity, was nearly avirulent. Again, gene expression experiments provided us with initial evidence that superoxide produced by the fungus may be most important as a signal. Blocking oxidant production by the pathogen may be a way to protect the plant host, in interactions with necrotrophs such as C. heterostrophus which seem to thrive in an oxidant environment.
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Shani, Uri, Lynn Dudley, Alon Ben-Gal, Menachem Moshelion, and Yajun Wu. Root Conductance, Root-soil Interface Water Potential, Water and Ion Channel Function, and Tissue Expression Profile as Affected by Environmental Conditions. United States Department of Agriculture, October 2007. http://dx.doi.org/10.32747/2007.7592119.bard.
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Constraints on water resources and the environment necessitate more efficient use of water. The key to efficient management is an understanding of the physical and physiological processes occurring in the soil-root hydraulic continuum.While both soil and plant leaf water potentials are well understood, modeled and measured, the root-soil interface where actual uptake processes occur has not been sufficiently studied. The water potential at the root-soil interface (yᵣₒₒₜ), determined by environmental conditions and by soil and plant hydraulic properties, serves as a boundary value in soil and plant uptake equations. In this work, we propose to 1) refine and implement a method for measuring yᵣₒₒₜ; 2) measure yᵣₒₒₜ, water uptake and root hydraulic conductivity for wild type tomato and Arabidopsis under varied q, K⁺, Na⁺ and Cl⁻ levels in the root zone; 3) verify the role of MIPs and ion channels response to q, K⁺ and Na⁺ levels in Arabidopsis and tomato; 4) study the relationships between yᵣₒₒₜ and root hydraulic conductivity for various crops representing important botanical and agricultural species, under conditions of varying soil types, water contents and salinity; and 5) integrate the above to water uptake term(s) to be implemented in models. We have made significant progress toward establishing the efficacy of the emittensiometer and on the molecular biology studies. We have added an additional method for measuring ψᵣₒₒₜ. High-frequency water application through the water source while the plant emerges and becomes established encourages roots to develop towards and into the water source itself. The yᵣₒₒₜ and yₛₒᵢₗ values reflected wetting and drying processes in the rhizosphere and in the bulk soil. Thus, yᵣₒₒₜ can be manipulated by changing irrigation level and frequency. An important and surprising finding resulting from the current research is the obtained yᵣₒₒₜ value. The yᵣₒₒₜ measured using the three different methods: emittensiometer, micro-tensiometer and MRI imaging in both sunflower, tomato and corn plants fell in the same range and were higher by one to three orders of magnitude from the values of -600 to -15,000 cm suggested in the literature. We have added additional information on the regulation of aquaporins and transporters at the transcript and protein levels, particularly under stress. Our preliminary results show that overexpression of one aquaporin gene in tomato dramatically increases its transpiration level (unpublished results). Based on this information, we started screening mutants for other aquaporin genes. During the feasibility testing year, we identified homozygous mutants for eight aquaporin genes, including six mutants for five of the PIP2 genes. Including the homozygous mutants directly available at the ABRC seed stock center, we now have mutants for 11 of the 19 aquaporin genes of interest. Currently, we are screening mutants for other aquaporin genes and ion transporter genes. Understanding plant water uptake under stress is essential for the further advancement of molecular plant stress tolerance work as well as for efficient use of water in agriculture. Virtually all of Israel’s agriculture and about 40% of US agriculture is made possible by irrigation. Both countries face increasing risk of water shortages as urban requirements grow. Both countries will have to find methods of protecting the soil resource while conserving water resources—goals that appear to be in direct conflict. The climate-plant-soil-water system is nonlinear with many feedback mechanisms. Conceptual plant uptake and growth models and mechanism-based computer-simulation models will be valuable tools in developing irrigation regimes and methods that maximize the efficiency of agricultural water. This proposal will contribute to the development of these models by providing critical information on water extraction by the plant that will result in improved predictions of both water requirements and crop yields. Plant water use and plant response to environmental conditions cannot possibly be understood by using the tools and language of a single scientific discipline. This proposal links the disciplines of soil physics and soil physical chemistry with plant physiology and molecular biology in order to correctly treat and understand the soil-plant interface in terms of integrated comprehension. Results from the project will contribute to a mechanistic understanding of the SPAC and will inspire continued multidisciplinary research.