scholarly journals Erythroid Krüppel-Like Factor Directly Activates the Basic Krüppel-Like Factor Gene in Erythroid Cells

2007 ◽  
Vol 27 (7) ◽  
pp. 2777-2790 ◽  
Author(s):  
Alister P. W. Funnell ◽  
Christopher A. Maloney ◽  
Lucinda J. Thompson ◽  
Janelle Keys ◽  
Michael Tallack ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Chromatin Immunoprecipitation ◽  
Erythroid Cells ◽  
Factor Gene ◽  
Upstream Promoter ◽  
Murine Embryos ◽  
Inhibitor Of Protein Synthesis ◽  
Kinetics Of ◽  
In Erythroid Cells

ABSTRACT The Sp/Krüppel-like factor (Sp/Klf) family is comprised of around 25 zinc finger transcription factors that recognize CACCC boxes and GC-rich elements. We have investigated basic Krüppel-like factor (Bklf/Klf3) and show that in erythroid tissues its expression is highly dependent on another family member, erythroid Krüppel-like factor (Eklf/Klf1). We observe that Bklf mRNA is significantly reduced in erythroid tissues from Eklf-null murine embryos. We find that Bklf is driven primarily by two promoters, a ubiquitously active GC-rich upstream promoter, 1a, and an erythroid downstream promoter, 1b. Transcripts from the two promoters encode identical proteins. Interestingly, both the ubiquitous and the erythroid promoter are dependent on Eklf in erythroid cells. Eklf also activates both promoters in transient assays. Experiments utilizing an inducible form of Eklf demonstrate activation of the endogenous Bklf gene in the presence of an inhibitor of protein synthesis. The kinetics of activation are also consistent with Bklf being a direct Eklf target. Chromatin immunoprecipitation assays confirm that Eklf associates with both Bklf promoters. Eklf is typically an activator of transcription, whereas Bklf is noted as a repressor. Our results support the hypothesis that feedback cross-regulation occurs within the Sp/Klf family in vivo.

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

1994 ◽  
Vol 92 (4) ◽  
pp. 585-594 ◽  
Author(s):  
T. J. Bouma ◽  
R. De Visser ◽  
J. H. J. A. Janssen ◽  
M. J. De Kock ◽  
P H. Van Leeuwen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Energy Requirements ◽  
Inhibitor Of Protein Synthesis

An in vitro bioassay for a mulluscan gonadotropin

1975 ◽  
Vol 62 (2) ◽  
pp. 433-446
Author(s):  
M. J. Wells ◽  
R. K. O'Dor ◽  
S. K. Buckley
Keyword(s):  
Protein Synthesis ◽  
High Rate ◽  
Follicle Cells ◽  
Octopus Vulgaris ◽  
Amino Acid Incorporation ◽  
Gland Extract ◽  
Acid Incorporation ◽  
Kinetics Of

1. Protein synthesis occurs at a high rate in the ovaries of maturing Octopus vulgaris and can be measured from the incorporation of [14C]leucine in vivo and in isolated groups of eggs in vitro. 2. Removal of the optic glands in vivo 1--3 days prior to testing markedly reduces amino acid incorporation in vivo or in vitro. After 5 days in vivo incorporation stops. 3. The rate of incorporation in vitro is increased by the addition of optic gland extract. 4. Analysis of the kinetics of leucine uptake and incorporation in vitro indicates that the hormone has an effect on the inward transport of leucine which is independent of its action on protein synthesis. 5. Electron-microscope studies of the follicle cells and ova show that the former are the site of protein synthesis. 6. Changes in either uptake or incorporation into protein by the follicle cells can be used as a qualitative biolobical assay for the optic gland hormone. Uptake is very easy to measure but incorporation is the more sensitive parameter. Either is potentially suitable as a quantitative assay for this and perhaps also for other molluscan gonadotropins.

scholarly journals A human beta-globin gene fused to the human beta-globin locus control region is expressed at high levels in erythroid cells of mice engrafted with retrovirus-transduced hematopoietic stem cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1384-1392 ◽  
Author(s):  
I Plavec ◽  
T Papayannopoulou ◽  
C Maury ◽  
F Meyer
Keyword(s):  
Bone Marrow Cells ◽  
Globin Gene ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Beta Globin Gene ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
In Erythroid Cells

Abstract Retroviral-mediated gene transfer of human beta-globin provides a model system for the development of somatic gene therapy for hemoglobinopathies. Previous work has shown that mice receiving a transplant of bone marrow cells infected with a retroviral vector containing the human beta-globin gene can express human beta-globin specifically in erythroid cells; however, the level of expression of the transduced globin gene was low (1% to 2% per gene copy as compared with that of the endogenous mouse beta-globin gene). We report here the construction of a recombinant retrovirus vector encoding a human beta- globin gene fused to the 4 major regulatory elements of the human beta- globin locus control region (LCR). The LCR cassette increases the level of expression of the globin gene in murine erythroleukemia cells by 10- fold. To study the level of expression in vivo, mouse bone marrow cells were infected with virus-producing cells and the transduced cells were injected into lethally irradiated recipients. In the majority of provirus-containing mice (up to 75%), expression of human beta-globin in peripheral blood was detected at least 3 to 6 months after transplantation. Twelve animals representative of the level of expression of the transduced gene in blood (0.04% to 3.2% of the endogenous mouse beta-globin RNA) were selected for further analysis. A range of 0.4% to 12% of circulating erythrocytes stained positive for human beta-globin protein. Based on these values, the level of expression of the transduced gene per cell was estimated to be 10% to 39% of the endogenous mouse beta-globin gene. These data demonstrate that fusion of the LCR to the beta-globin gene in a retroviral vector increases the level of beta-globin expression in murine erythroleukemia cells and suggest that high-level expression can be obtained in erythroid cells in vivo after transduction into hematopoietic stem cells.

Ferritin Heavy Chain, a Repressor of the Human β-Globin Gene That Binds a Conserved Cagtgc Motif, Is Bound to the Repressed β-Globin Promoter In Vivo in K562 Cells and Specifically Binds an Analogous Site in the Mouse βMajor-Globin Promoter.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1223-1223
Author(s):  
Robert H. Broyles ◽  
Visar Belegu ◽  
Charles A. Stewart ◽  
Quentin N. Pye ◽  
Austin C. Roth ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Chromatin Immunoprecipitation ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cells ◽  
Ferritin Heavy Chain ◽  
Polyclonal Antisera ◽  
Globin Promoter ◽  
F1 Generation

Abstract Ferritin heavy chain (FH), an embryonically-expressed protein in the erythroid lineage, localizes to the nucleus and represses the human adult β-globin promoter in transient expression assays (Broyles et al., PNAS98: 9145, 2001). Recently, we have performed chromatin immunoprecipitation (ChIP) assays with cross-linked chromatin of K562 cells in which the β-globin gene is repressed, using anti-FH polyclonal antisera. These results strongly indicate that FH occupies the repression site (a CAGTGC motif) in vivo. Binding to this -150 site has been previously demonstrated to be required for β-promoter repression in co-transfections. EMSA assays (competitive gel shifts) have revealed that the mouse βMajor-globin promoter has an analogous CAGTGN motif at -160 bp from the cap site that competes specifically with the human CAGTGC site for FH binding. The mouse βMinor-globin promoter lacks the -150/-160 CAGTGN motif and, therefore, the FH binding site. Thus, a human FH transgenic mouse, in which the FH gene is driven by a truncated β-promoter lacking the CAGTGN motif, should express human FH in definitive erythroid cells where the FH would be predicted to repress βMajor-globin but not βMinor-globin. Such a mouse would be predicted to survive but be born with a mild β-thalassemia due to the decreased βMajor/βMinor ratio in its definitive erythroid cells. Preliminary results from the litters of F1 generation FH-tg mice indicate that such is indeed the case, i.e., that human FH functions as a βMajor-globin repressor in vivo.

Adenine Reversal of Ethionine-Induced Inhibition of Protein Synthesis: A Comparison with Computer-Generated Data

1973 ◽  
Vol 51 (10) ◽  
pp. 1428-1432
Author(s):  
R. Kisilevsky ◽  
G. Matheson
Keyword(s):  
Protein Synthesis ◽  
Female Rats ◽  
Reversal Effect ◽  
In Vivo Labeling ◽  
Inhibition Of Protein Synthesis ◽  
Reduced Rate ◽  
Kinetics Of

In vivo labeling kinetics of (a) nascent peptides on liver polysomes, and (b) proteins of the 100 000 × g supernatant have been determined for ethionine-intoxicated female rats given adenine, to reverse the effect of ethionine. When compared to computer-generated data the results indicate that the reversal effect of adenine is not simply through the increase in the previously reduced rate of initiation. In addition to increasing the rate of initiation, adenine, soon after its administration, reduces the rate of elongation. The rate of elongation subsequently returns to normal levels.

THE MELANOGENIC RESPONSE TO TESTOSTERONE IN SCROTAL EPIDERMIS: EFFECTS ON TYROSINASE ACTIVITY AND PROTEIN SYNTHESIS

1976 ◽  
Vol 81 (2) ◽  
pp. 435-448 ◽  
Author(s):  
Michael J. Wilson ◽  
Eugene Spaziani
Keyword(s):  
Protein Synthesis ◽  
Specific Protein ◽  
Tyrosinase Activity ◽  
Melanin Synthesis ◽  
Testosterone Treatment ◽  
Pre Treatment ◽  
Inhibitor Of Protein Synthesis ◽  
Rate Limiting

ABSTRACT Pigmentation of the scrotum of the black-pelted rat, as expressed through melanocyte melanogenic activity, is controlled by androgens. Castration decreased in vitro incorporation of [14C] tyrosine into melanin. Testosterone pre-treatment for 4 days increased melanin radioactivity over castrate controls; the increment in vitro was prevented by an inhibitor of protein synthesis (cycloheximide) added to the incubation. However, cycloheximide only partially blocked melanin synthesis when added to tissue from animals hormone treated for 6 days in vivo, and was ineffective in tissue from intacts. Bulk protein synthesis in vitro (incorporation of [14C] tyrosine or -leucine) was not affected by castration or testosterone treatment but was uniformly inhibited by cycloheximide. The data suggest that new synthesis of specific protein in vitro was necessary for initial hormone-stimulation of melanogenesis, but with longer exposure to hormone sufficient protein was pre-synthetized in vivo to permit melanogenesis during incubation with the inhibitor. Radioautographs of epidermis incubated with [14C] tyrosine showed grains concentrated over macromolecular aggregates in melanocytes, a pattern not altered by cycloheximide. Though available for incorporation into general tissue protein. [14C] tyrosine was apparently incorporated preferentially into melanin by melanocytes. DOPA (3,4-dihydroxyphenylalanine) added to incubations in cofactor amounts did not affect decreased melanin synthesis after castration and appears, therefore, not to be rate limiting in that decrease. Tissue uptake of free [14C] tyrosine or — leucine during incubation was lower than normal in castrate epidermis; uptake was elevated by testosterone treatment. Concentrations appeared sufficient in all preparations to suggest that availability is not rate limiting for synthesis of melanin or protein; however, a possible influence on amino acid permeability for melanocytes remains undetermined. Tyrosinase activity was present in both particulate and cytosol fractions of epidermis but decreased significantly after castration only in the cytosol. Testosterone increased particulate activity after 4 days and soluble activity after 9 days of treatment. These and findings above are consistent with a model that tyrosinase is synthesized and incorporated into melanosome structure within 4 days testosterone treatment; with longer treatment synthesis may then exceed that required for melanosome assembly and tyrosinase appears in the soluble milieu.

scholarly journals Heme-regulated eIF2α kinase in erythropoiesis and hemoglobinopathies

Blood ◽  
2019 ◽  
Vol 134 (20) ◽  
pp. 1697-1707 ◽  
Author(s):  
Jane-Jane Chen ◽  
Shuping Zhang
Keyword(s):  
Protein Synthesis ◽  
Stress Response ◽  
Initiation Factor ◽  
Integrated Stress Response ◽  
Iron Availability ◽  
Eukaryotic Initiation Factor ◽  
Erythroid Cells ◽  
Eif2α Kinase ◽  
In Erythroid Cells

Chen and Zhang review the role of eukaryotic initiation factor 2α (eIF2α) in regulating the balance between protein synthesis and iron availability as part of the integrated stress response in erythroid cells.

The use of an inhibitor of protein synthesis to investigate the roles of ecdysteroids and sex-determination genes on the expression of the genes encoding the Drosophila yolk proteins

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 931-941
Author(s):  
M. Bownes ◽  
A. Scott ◽  
M. Blair
Keyword(s):  
Protein Synthesis ◽  
Sex Determination ◽  
Fat Body ◽  
Yolk Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Yolk Protein Genes ◽  
Fat Bodies ◽  
Inhibitor Of Protein Synthesis

The three yolk-protein genes of Drosophila are normally expressed only in adult female fat bodies and ovaries. 20-hydroxyecdysone can affect the transcription of these genes in males and females, as can mutations in the sex-determining genes tra, tra-2, ix and dsx. We have asked a number of basic questions about how these genes are regulated, using an inhibitor of protein synthesis (cycloheximide), labelling RNA in vivo, a temperature-sensitive sex-determination mutant (tra-2ts1), and 20-hydroxyecdysone. We have found that the yolk-protein genes are continuously transcribed in the fat bodies of adult females and that maintenance of this transcription requires protein synthesis. Hormone induction in males is also inhibited by cycloheximide, suggesting that the products of other genes are essential both for 20-hydroxyecdysone to be able to switch on the genes, and for their continuous transcription in the female fat body. The products of the tra-2 gene are also required for continuous transcription of the yolk-protein genes, suggesting that the pathway inhibited by the cycloheximide is that of the sex-determination hierarchy. 20-hydroxyecdysone can override the sex-determination system and induce yolk protein synthesis in normal males and tra-2ts reared and maintained at the restrictive temperature.

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