scholarly journals GATA1-Mediated Megakaryocyte Differentiation and Growth Control Can Be Uncoupled and Mapped to Different Domains in GATA1

2005 ◽  
Vol 25 (19) ◽  
pp. 8592-8606 ◽  
Author(s):  
Christiane Kuhl ◽  
Ann Atzberger ◽  
Francisco Iborra ◽  
Bernhard Nieswandt ◽  
Catherine Porcher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Dna Binding ◽  
Growth Control ◽  
Zinc Finger Transcription Factor ◽  
Naturally Occurring ◽  
Platelet Release ◽  
And Control ◽  
Megakaryocyte Progenitors ◽  
Megakaryocyte Differentiation

ABSTRACT The DNA-binding hemopoietic zinc finger transcription factor GATA1 promotes terminal megakaryocyte differentiation and restrains abnormal immature megakaryocyte expansion. How GATA1 coordinates these fundamental processes is unclear. Previous studies of synthetic and naturally occurring mutant GATA1 molecules demonstrate that DNA-binding and interaction with the essential GATA1 cofactor FOG-1 (via the N-terminal finger) are required for gene expression in terminally differentiating megakaryocytes and for platelet production. Moreover, acquired mutations deleting the N-terminal 84 amino acids are specifically detected in megakaryocytic leukemia in human Down syndrome patients. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by ectopically expressing modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte progenitors. In addition to DNA binding, distinct N-terminal regions, including residues in the first 84 amino acids, promote platelet release and restrict megakaryocyte growth. In contrast, abrogation of GATA1-FOG-1 interaction leads to loss of differentiation, but growth of blocked immature megakaryocytes is controlled. Thus, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth, and these processes can be uncoupled.

scholarly journals Regulation of rat magnocellular neurosecretory system by D-aspartate: evidence for biological role(s) of a naturally occurring free D-amino acid in mammals

2000 ◽  
Vol 167 (2) ◽  
pp. 247-252 ◽  
Author(s):  
H Wang ◽  
H Wolosker ◽  
J Pevsner ◽  
SH Snyder ◽  
DJ Selkoe
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Amino Acid ◽  
Physiological Function ◽  
Neurosecretory System ◽  
Milk Ejection ◽  
Magnocellular Neurons ◽  
Rat Hypothalamus ◽  
Naturally Occurring

Little evidence is available for the physiological function of D-amino acids in species other than bacteria. Here we demonstrate that naturally occurring freed -aspartate (D-Asp) is present in all magnocellular neurons of rat hypothalamus. The levels of this naturally occurring D-amino acid were elevated during lactation and returned to normal thereafter in the magnocellular neurosecretory system, which produces oxytocin, a hormone responsible for milk ejection during lactation. Intraperitoneal injections of D-Asp reproducibly increased oxytocin gene expression and decreased the concentration of circulating oxytocin in vivo. Similar changes were observed in the vasopressin system. These results provide evidence for the role(s) of naturally occurring free D-Asp in mammalian physiology. The findings argue against the conventional concept that only L-stereoisomers of amino acids are functional in higher species.

scholarly journals Evolution under juvenile malnutrition impacts adult metabolism and fitness

2022 ◽  
Author(s):  
Berra Erkosar ◽  
Cindy Dupuis ◽  
Fanny Cavigliasso ◽  
Loriane Savary ◽  
Hector Gallart-Ayala ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Essential Amino Acids ◽  
Standard Diet ◽  
Beta Oxidation ◽  
Purine Nucleotides ◽  
Animal Physiology ◽  
Adult Fitness ◽  
And Control ◽  
Metabolite Abundance

Juveniles are often first to suffer from nutrient shortage, and juvenile undernutrition is likely an important force of natural selection shaping animal physiology, with consequences potentially extending into adulthood. We combined RNAseq, targeted metabolomics and genomics to study the consequences of experimental evolution under juvenile undernutrition for metabolism of reproductively active adult females of Drosophila melanogaster. Compared to six Control populations maintained on standard diet, six Selected populations evolved for over 230 generations on a nutrient-poor larval diet showed major changes in adult gene expression and metabolite abundance. In particular, Selected flies were relatively deficient in essential amino acids and purine nucleotides, but showed overabundance of several non-essential amino-acids involved in purine synthesis and overexpression of multiple enzymes catalyzing this pathway. Selected flies also accumulated medium-chain acylcarnitines suggestive of congestion in beta-oxidation, possibly linked to deficiency of electron transporters. Some aspects of the metabolic profile of Selected flies resembled that of flies subject to starvation. Furthermore, differences between Selected and Control populations in adult gene expression were in general positively correlated with differences in larval expression, consistent with pleiotropy in gene regulation between the life stages. Finally, Selected flies were less fit in terms of fecundity than Controls even when both were raised under the conditions under which the Selected populations evolved. These results suggest that evolutionary adaptation to juvenile undernutrition has large pleiotropic consequences for adult metabolism, and that they are costly rather than adaptive for adult fitness.

The Carboxy-Terminal Portion of the Aflatoxin Pathway Regulatory Protein AFLR of Aspergillus parasiticus ActivatesGAL1::lacZ Gene Expression inSaccharomyces cerevisiae

1999 ◽  
Vol 65 (6) ◽  
pp. 2508-2512 ◽  
Author(s):  
Perng-Kuang Chang ◽  
Jiujiang Yu ◽  
Deepak Bhatnagar ◽  
Thomas E. Cleveland
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Dna Binding ◽  
Aspergillus Parasiticus ◽  
Regulatory Protein ◽  
Lacz Gene ◽  
Coding Region ◽  
Coding Regions ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal

ABSTRACT AFLR, a DNA-binding protein of 444 amino acids, transactivates the expression of aflatoxin biosynthesis genes in Aspergillus parasiticus and Aspergillus flavus, as well as the sterigmatocystin synthesis genes in Aspergillus nidulans. We show here by fusion of various aflR coding regions to the GAL4 DNA-binding coding region that the AFLR carboxyl terminus contained a region that activatedGAL1::lacZ gene expression inSaccharomyces cerevisiae and that the AFLR internal region was required for the activation activity. Compared to the AFLR carboxy-terminal fusion protein (AFLRC), a mutant AFLRC retained approximately 75% of the activation activity after deletion of three acidic amino acids, Asp365, Glu366, and Glu367, in a previously identified acidic stretch. Removal of the carboxy-terminal amino acid, Glu444, did not affect the activation activity. Substitutions of acidic Glu423, Asp439, or Asp436/Asp439 with basic amino acids, Lys and His, resulted in 10- to 15-fold-lower activation activities. Strikingly, the Asp436His mutation abolished the activation activity. Substitutions of basic His428 and His442 with acidic Asp resulted in 20 and 40% decreases in the activation activities, respectively. Simultaneous substitutions of Arg427, Arg429, and Arg431 with Leu also significantly decreased the activation activity; the decrease was approximately 50-fold. Results suggest that the AFLR carboxy-terminal region is involved in transcription activation and that total acidity in this region is not a major determinant of AFLR’s activation ability inS. cerevisiae.

173 THE EFFECT OF AMINO ACIDS AND HYALURONAN ON BOVINE EMBRYO IN VITRO DEVELOPMENT AND GENE EXPRESSION PATTERN

2006 ◽  
Vol 18 (2) ◽  
pp. 194 ◽  
Author(s):  
A. T. Palasz ◽  
J. Beltrán Breña ◽  
P. De la Fuente ◽  
M. F. Martinez ◽  
A. Gutiérrez-Adán
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Embryo Development ◽  
Control Group ◽  
Bovine Embryo ◽  
In Vitro Development ◽  
Glut 1 ◽  
Vitro Development ◽  
And Control

We have previously shown that bovine embryos cultured in SOFaa (BME + MEM amino acids) culture medium with hyaluronan (HA) + BSA are of better quality (Guti�rrez-Ad�n et al. 2005 Reprod. Fertil. Dev. 17, 219). Our objective was to examine the effect of essential (BME) or non-essential (MEM) amino acids with or without HA (MAP-5; Bioniche, Inc., Belleville, Ontario, Canada) on bovine embryo in vitro development and mRNA transcription of five developmentally important genes; apoptosis (Bax), growth factor (IGF-II), glucose (Glut-1) and fructose (Glut-5) transport and metabolism, and cell to cell adhesion (Cx-43). A total of 1474 presumptive zygotes (5 replicates) were initially cultured in 40 �L drops in the following groups: Group 1, control, SOFaa; Group 2, SOF-1 (MEM only); and Group 3, SOF-2 (BME only). On Day 4 (~96 h post-insemination (pi) the number of zygotes that had developed to d8 cells was recorded and 10 �L of SOF-1 and SOF-2, each with 2.5 mg/mL HA, was added to half of the embryos from Groups 2 and 3, respectively; the other half of Groups 2 and 3 and control group received 10 �L of corresponding medium without HA. Embryos were cultured under paraffin oil at 39�C and 5% CO2 in humidified air. Cleavage rates were recorded on Day 2 and the number of blastocysts on Days 7, 8, and 9. Five blastocysts from each replicate from each treatment were frozen for determination of gene expression patterns later. Cleavage rates and embryo development 96 h pi were compared among groups by chi-square analysis. The effects of HA and medium on blastocyst rates were analyzed by logistic regression and the data on mRNA expression by one-way repeated-measures ANOVA. Cleavage rates were 81.1% in SOFaa and 79.3% in SOF-1 (P = 0.48) and different from those in the SOF-2 group (72.4%; P < 0.02). The proportion of embryos that developed to d8 cells at Day 4 was higher in the control (46.7%) and SOF-1 (46.8%) groups than in the SOF-2 group (32.6%). The number of blastocysts that developed in SOFaa (37.0%), SOF-1 (37.7%), and SOF-1 + HA (37.8%) were higher (P < 0.001) than those in SOF-2 (19.6%) and SOF-2 + HA (21.8%). The level of expression of Glut-5 was not different among the groups. However, SOF-2 was the only group that had significantly lower expression of Glut-5, Igf II, and Cx43, and higher expression of BAX (P < 0.05) as compared to the control group and the SOF-1 groups with or without HA. Addition of HA to SOF-2 medium increased expression of Glut-1 and Igf II and decreased expression of BAX as compared to the SOF-1 only and control groups and the SOF-2 groups with or without HA (P < 0.05). The level of expression of Cx43 was higher in the control than in four remaining groups, and lower in the SOF-2 than in the SOF-1 group (P < 0.05). Addition of HA increased expression of Cx43 in both SOF-1 and SOF-2 groups but this level of expression was lower than in the control group; the level in the SOF-2 + HA group was lower (P < 0.05) than in the SOF-1 + HA group. We conclude that, within our protocol, MEM amino acids only stimulate embryo development to the blastocyst stage and the addition of HA to the SOF-MEM and SOF-BME media on Day 4 of culture improved embryo quality.

scholarly journals The N-terminal 96 residues of MCM1, a regulator of cell type-specific genes in Saccharomyces cerevisiae, are sufficient for DNA binding, transcription activation, and interaction with alpha 1.

1992 ◽  
Vol 12 (8) ◽  
pp. 3563-3572 ◽  
Author(s):  
L Bruhn ◽  
J J Hwang-Shum ◽  
G F Sprague
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Binding ◽  
Transcription Activation ◽  
Reporter Genes ◽  
Cell Type ◽  
Cell Type Specific

MCM1 performs several functions necessary for its role in regulating cell type-specific gene expression in the yeast Saccharomyces cerevisiae: DNA binding, transcription activation, and interaction with coregulatory proteins such as alpha 1. We analyzed a set of MCM1 deletion derivatives using in vivo reporter gene assays and in vitro DNA-binding studies to determine which regions of MCM1 are important for its various activities. We also analyzed a set of LexA-MCM1 hybrids to examine the ability of different segments of MCM1 to activate transcription independent of MCM1's DNA-binding function. The first third of MCM1 [MCM1(1-96)], which includes an 80-residue segment homologous to the mammalian serum response factor, was sufficient for high-affinity DNA binding, for activation of reporter gene expression, and for interaction with alpha 1 in vitro and in vivo. However, the ability of MCM1(1-96) to activate transcription and to interact with alpha 1 was somewhat reduced compared with wild-type MCM1 [MCM1(1-286)]. Optimal interaction with alpha 1 required residues 99 to 117, in which 18 of 19 amino acids are acidic in character. Optimal transcription activation required a segment from residues 188 to 286, in which 50% of the amino acids are glutamine. Deletion of this segment from MCM1 reduced expression of reporter genes by about twofold. Moreover, LexA-MCM1 hybrids containing this segment were able to activate expression of reporter genes that rely on LexA binding sites as potential upstream activation sequences. Thus, glutamine-rich regions may contribute to the activation function of yeast transcription activators, as has been suggested for glutamine-rich mammalian proteins such as Sp1.

scholarly journals GATA1-Mediated Megakaryocyte Differentiation and Growth Control Can Be Uncoupled and Mapped to Different Domains in GATA1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 827-827
Author(s):  
Christiane Kuhl ◽  
Ann Atzberger ◽  
Francisco Iborra ◽  
Catherine Porcher ◽  
Paresh Vyas
Keyword(s):  
Amino Acids ◽  
Zinc Finger ◽  
Short Form ◽  
Growth Control ◽  
Fetal Life ◽  
Erythroid Progenitor ◽  
Exon 2 ◽  
N Terminus ◽  
Platelet Release

Abstract The zinc finger transcription factor GATA1 plays a critical non-redundant role in megakaryopoiesis in mice and human patients with both germline and acquired GATA1 mutations. Germline deletion of the mouse regulatory element, HSI (ΔneoΔHS mice), specifically reduces megakaryocyte GATA1 expression to 5% of wild-type levels. ΔneoΔHS mice are viable but have defective megakaryocyte maturation and growth control. Mutant megakaryocytes fail to shed proplatelets in vitro and the platelet count is ~5 fold lower in mutant mice. Moreover, mutant megakaryocyte precursors hyperproliferate in vivo and in vitro. Here, we show there is a specific ~10-fold increase in abnormal CD41+ FcγRlowc-kit+CD9+ megakaryocyte-erythroid progenitor cells in fetal liver. These progenitors form abnormally large colonies in vitro and are unable to form proplatelets. In contrast, earlier myeloid progenitors (the common myeloid progenitor CMP) were present at expected frequencies and differentiated to all non-megakaryocyte myeloid lineages normally. Consistent with this GATA1 expression was selectively reduced in the abnormal progenitors and immature megakaryocyte precursors. We then used these GATA1-deficient primary megakaryocyte progenitors as a cellular substrate to define the amino acid domains within GATA1 required to facilitate proplatelet release and retard immature megakaryocyte growth. Previous data suggested that the bi-functional GATA1 zinc fingers could bind both DNA and interact with protein partners. The C-terminal zinc finger is essential for binding to all GATA sites whereas the N-terminal finger helps binding to a subset of GATA sites and mediates interaction with the critical GATA1 cofactor, FOG-1. Finally, the N-terminal 84 amino acids of GATA1 are important for megakaryopoiesis. Neonates and children with Down Syndrome (DS) can develop megakaryocyte leukaemia acquire mutations in GATA1 exon 2 in fetal life that result in the production of a short form of GATA1 protein (GATA1s) lacking the N-terminal 84 amino acids. In this study, we have systematically dissected GATA1 domains required for platelet release and control of megakaryocyte growth by using retroviral-mediated gene transfer to ectopically express modified GATA1 molecules in primary GATA1-deficient fetal megakaryocyte CD41 + FcγRlowc-kit+CD9+progenitors. In addition to the GATA1-FOG-1 interaction and C-terminal zinc finger, two domains within the first N-terminal 110 amino acids are required for platelet release. To restrict megakaryocyte growth, two domains are required; the C-terminal zinc finger and residues in the N-terminus between amino acids 54–110. Importantly, interaction with FOG-l is not required to restrain megakaryocyte growth. Furthermore, we showed that chicken GATA1 was able to rescue both, platelet release and growth control. As only a few residues are conserved in the N-terminus between mouse and chicken GATA1, this potentially pinpoints critical functional residues in the N-terminus. In summary, distinct GATA1 domains regulate terminal megakaryocyte gene expression leading to platelet release and restrain megakaryocyte growth. Analysis of distinct mutations in the N-terminus and a modified version of GATA1 that is crippled in its interaction with FOG-1 shows platelet production and megakaryocyte growth can be uncoupled. Finally, these results have implications for how GATA1s may function in megakaryocytic leukemia in DS.

scholarly journals Amino Acids in the Basic Domain of Epstein-Barr Virus ZEBRA Protein Play Distinct Roles in DNA Binding, Activation of Early Lytic Gene Expression, and Promotion of Viral DNA Replication

2006 ◽  
Vol 80 (18) ◽  
pp. 9115-9133 ◽  
Author(s):  
Lee Heston ◽  
Ayman El-Guindy ◽  
Jill Countryman ◽  
Charles Dela Cruz ◽  
Henri-Jacques Delecluse ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Dna Replication ◽  
Dna Binding ◽  
Lytic Cycle ◽  
Late Gene Expression ◽  
Lytic Gene ◽  
Basic Domain ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The ZEBRA protein of Epstein-Barr virus (EBV) drives the viral lytic cycle cascade. The capacity of ZEBRA to recognize specific DNA sequences resides in amino acids 178 to 194, a region in which 9 of 17 residues are either lysine or arginine. To define the basic domain residues essential for activity, a series of 46 single-amino-acid-substitution mutants were examined for their ability to bind ZIIIB DNA, a high-affinity ZEBRA binding site, and for their capacity to activate early and late EBV lytic cycle gene expression. DNA binding was obligatory for the protein to activate the lytic cascade. Nineteen mutants that failed to bind DNA were unable to disrupt latency. A single acidic replacement of a basic amino acid destroyed DNA binding and the biologic activity of the protein. Four mutants that bound weakly to DNA were defective at stimulating the expression of Rta, the essential first target of ZEBRA in lytic cycle activation. Four amino acids, R183, A185, C189, and R190, are likely to contact ZIIIB DNA specifically, since alanine or valine substitutions at these positions drastically weakened or eliminated DNA binding. Twenty-three mutants were proficient in binding to ZIIIB DNA. Some DNA binding-proficient mutants were refractory to supershift by BZ-1 monoclonal antibody (epitope amino acids 214 to 230), likely as the result of the increased solubility of the mutants. Mutants competent to bind DNA could be separated into four functional groups: the wild-type group (eight mutants), a group defective at activating Rta (five mutants, all with mutations at the S186 site), a group defective at activating EA-D (three mutants with the R179A, S186T, and K192A mutations), and a group specifically defective at activating late gene expression (seven mutants). Three late mutants, with a Y180A, Y180E, or K188A mutation, were defective at stimulating EBV DNA replication. This catalogue of point mutants reveals that basic domain amino acids play distinct functions in binding to DNA, in activating Rta, in stimulating early lytic gene expression, and in promoting viral DNA replication and viral late gene expression. These results are discussed in relationship to the recently solved crystal structure of ZEBRA bound to an AP-1 site.

Solution Structure of the B-Myb DNA-Binding Domain:  A Possible Role for Conformational Instability of the Protein in DNA Binding and Control of Gene Expression†,‡

Biochemistry ◽  
1998 ◽  
Vol 37 (27) ◽  
pp. 9619-9629 ◽  
Author(s):  
Pauline B. McIntosh ◽  
Tom A. Frenkiel ◽  
Ute Wollborn ◽  
John E. McCormick ◽  
Karl-Heinz Klempnauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Solution Structure ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Control Of Gene Expression ◽  
And Control

The N-terminal 96 residues of MCM1, a regulator of cell type-specific genes in Saccharomyces cerevisiae, are sufficient for DNA binding, transcription activation, and interaction with alpha 1

1992 ◽  
Vol 12 (8) ◽  
pp. 3563-3572
Author(s):  
L Bruhn ◽  
J J Hwang-Shum ◽  
G F Sprague
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Binding ◽  
Transcription Activation ◽  
Reporter Genes ◽  
Cell Type ◽  
Cell Type Specific

MCM1 performs several functions necessary for its role in regulating cell type-specific gene expression in the yeast Saccharomyces cerevisiae: DNA binding, transcription activation, and interaction with coregulatory proteins such as alpha 1. We analyzed a set of MCM1 deletion derivatives using in vivo reporter gene assays and in vitro DNA-binding studies to determine which regions of MCM1 are important for its various activities. We also analyzed a set of LexA-MCM1 hybrids to examine the ability of different segments of MCM1 to activate transcription independent of MCM1's DNA-binding function. The first third of MCM1 [MCM1(1-96)], which includes an 80-residue segment homologous to the mammalian serum response factor, was sufficient for high-affinity DNA binding, for activation of reporter gene expression, and for interaction with alpha 1 in vitro and in vivo. However, the ability of MCM1(1-96) to activate transcription and to interact with alpha 1 was somewhat reduced compared with wild-type MCM1 [MCM1(1-286)]. Optimal interaction with alpha 1 required residues 99 to 117, in which 18 of 19 amino acids are acidic in character. Optimal transcription activation required a segment from residues 188 to 286, in which 50% of the amino acids are glutamine. Deletion of this segment from MCM1 reduced expression of reporter genes by about twofold. Moreover, LexA-MCM1 hybrids containing this segment were able to activate expression of reporter genes that rely on LexA binding sites as potential upstream activation sequences. Thus, glutamine-rich regions may contribute to the activation function of yeast transcription activators, as has been suggested for glutamine-rich mammalian proteins such as Sp1.

Microstructural investigation of surface roughness in MBE-grown AlAs

1995 ◽  
Vol 53 ◽  
pp. 454-455
Author(s):  
R. Rajesh ◽  
R. Droopad ◽  
C. H. Kuo ◽  
R. W. Carpenter ◽  
G. N. Maracas
Keyword(s):  
Thin Film ◽  
Real Time ◽  
Growth Control ◽  
Native Oxide ◽  
Effusion Cell ◽  
Surface Oxide Layer ◽  
Microstructural Investigation ◽  
Mbe Growth ◽  
Film Substrate ◽  
And Control

Knowledge of material pseudodielectric functions at MBE growth temperatures is essential for achieving in-situ, real time growth control. This allows us to accurately monitor and control thicknesses of the layers during growth. Undesired effusion cell temperature fluctuations during growth can thus be compensated for in real-time by spectroscopic ellipsometry. The accuracy in determining pseudodielectric functions is increased if one does not require applying a structure model to correct for the presence of an unknown surface layer such as a native oxide. Performing these measurements in an MBE reactor on as-grown material gives us this advantage. Thus, a simple three phase model (vacuum/thin film/substrate) can be used to obtain thin film data without uncertainties arising from a surface oxide layer of unknown composition and temperature dependence.In this study, we obtain the pseudodielectric functions of MBE-grown AlAs from growth temperature (650°C) to room temperature (30°C). The profile of the wavelength-dependent function from the ellipsometry data indicated a rough surface after growth of 0.5 μm of AlAs at a substrate temperature of 600°C, which is typical for MBE-growth of GaAs.

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