scholarly journals A specific dimerization of rabbit β-globin messenger ribonucleic acid

1978 ◽  
Vol 175 (1) ◽  
pp. 159-169 ◽  
Author(s):  
V A Mezl ◽  
J A Hunt
Keyword(s):  
Beta Globin ◽  
Globin Mrna ◽  
Cross Hybridization ◽  
Rapid Preparation ◽  
Free System ◽  
Messenger Ribonucleic Acid ◽  
Low Salt ◽  
Alpha Globin ◽  
Two Peaks ◽  
Second Peak

Rabbit globin mRNA, when layered in low salt on 0.1 M-NaCl/sucrose gradients, separates into two peaks of material. Translation of these two RNA fractions in the wheat-germ cell-free system, hybridization against globin complementary DNA (cDNA) and cross-hybridization against cDNA species prepared from each fraction show that the first peak sedimenting at 10S is a alpha-globin mRNA and the second peak, sedimenting at approx. 15S, is beta-globin mRNA. The sedimentation rate of the beta-globin mRNA is concentration-dependent. By changing concentration and pH, it is indicated that in low-salt beta-globin mRNA adopts a conformation that leads to specific, but weak, self-dimerization during centrifugation in 0.1M-NaCl. This property permits rapid preparation of intact and relatively pure alpha- and beta-globin mRNA species.

Characterization of the major regulatory element upstream of the human alpha-globin gene cluster

1991 ◽  
Vol 11 (9) ◽  
pp. 4679-4689
Author(s):  
A P Jarman ◽  
W G Wood ◽  
J A Sharpe ◽  
G Gourdon ◽  
H Ayyub ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Globin Gene ◽  
Major Elements ◽  
Beta Globin ◽  
Globin Mrna ◽  
Expression Studies ◽  
Primary Element ◽  
Alpha Globin ◽  
Binding Sequence

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.

scholarly journals Constitutive expression of GATA-1, EPOR, alpha-globin, and gamma-globin genes in myeloid clonogenic cells from juvenile chronic myelocytic leukemia

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 323-328 ◽  
Author(s):  
E Privitera ◽  
R Schiro ◽  
D Longoni ◽  
A Ronchi ◽  
A Rambaldi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemic Cells ◽  
Globin Genes ◽  
Chronic Myelocytic Leukemia ◽  
Beta Globin ◽  
Globin Mrna ◽  
Gamma Globin ◽  
Myelocytic Leukemia ◽  
Alpha Globin

Juvenile chronic myelocytic leukemia (JCML) is a rare disorder of early childhood. Characteristic of JCML are the progressive appearance of high levels of fetal hemoglobin (HbF), reflecting a true reversion to a fetal type of erythropoiesis, and the presence of colony-forming cells able to grow in vitro spontaneously in the absence of growth factors. To better understand the relationship between the erythroid abnormalities and the leukemic process, we analyzed the expression pattern of specific genes related to erythroid differentiation--GATA-1, EPOR, alpha-globin, beta-globin, and gamma-globin genes--in JCML peripheral blood (PB) cells and in vitro-derived colonies. Northern blot analysis of PB cells from five JCML patients indicated levels of GATA-1 transcripts much higher than those usually found in other types of leukemic cells, and S1 nuclease protection assay detected significantly increased expression of gamma-globin mRNA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of single granulocyte-macrophage colony-forming unit (CFU-GM) colonies, obtained in vitro in the absence of added growth factors from four JCML patients, detected GATA-1, EPOR, and globin (alpha and gamma) transcripts in most of the colonies tested, in contrast with control CFU-GM from normal bone marrow, which were positive only for GATA-1. Single JCML colonies were tested for the presence of two different transcripts; whereas alpha- and gamma-globin genes appeared mostly coexpressed, beta-globin mRNA was detected only in a minority of the gamma-globin-positive colonies, indicating that the leukemic pattern of hemoglobin synthesis is mainly fetal. In addition, the leukemic cells occurring during blast crisis of one of our patients displayed the typical features of a stem cell leukemia (CD34+, CD19-, CD2-, myeloperoxidase-). In this sorted CD34+ population, we detected the presence of a marker chromosome, der(12)t(3;12), previously identified in bone marrow cells at diagnosis and an expression pattern superimposable to that of the JCML colonies, consistently displaying a high gamma-globin:beta-globin mRNA ratio. The expression of erythroid markers within populations of leukemic cells, both in vivo and in vitro, supports the hypothesis that abnormal JCML erythroid cells may originate from the same mutated progenitor that sustains the growth of the leukemic cells.

scholarly journals Analysis of the ratio of α- to β-globin and globin messenger ribonucleic acid content of fractionated rabbit erythroid bone-marrow cells

1979 ◽  
Vol 179 (3) ◽  
pp. 525-535 ◽  
Author(s):  
V A Mezl ◽  
E S Kawasaki ◽  
J A Hunt
Keyword(s):  
Bone Marrow Cells ◽  
Beta Globin ◽  
Globin Mrna ◽  
Constant Amount ◽  
Messenger Ribonucleic Acid ◽  
Mrna Ratio ◽  
Linear Reaction ◽  
Mrna Content ◽  
Cdna Hybridization ◽  
Better Than

The ratio of alpha- to beta-globin mRNA was measured by hybridization of a constant amount of highly purified alpha- or beta-globin cDNA (complementary DNA) with increasing amounts of RNA in the range up to 20% cDNA hybridization, where an essentially linear reaction is obtained. Statistical analysis indicates that the ratio of alpha- to beta-globin can be measured within a maximal error of +/- 0.3 and in most cases is better than +/- 0.15. Under these conditions there is no significant deviation from the ratio of 1.3 in the alpha- to beta-globin mRNA ratio of RNA isolated from erythroid cells rich in pronormoblasts through to reticulocytes. If the ratio of alpha- to beta-globin mRNA exceeded 1.7 or was less than 0.9 in pronormoblasts, it would be detected in these experiments. The overall globin mRNA content increases to a maximal value in the fractions rich in basophilic normoblasts of 30,000–50,000 molecules/cell. However, the accuracy of these determinations is not as great as for the ratio determinations, and no significant deviations were seen except in the cells rich in pronormoblasts, which contained less globin mRNA than the later stages.

scholarly journals The stability of human beta-globin mRNA is dependent on structural determinants positioned within its 3' untranslated region

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5314-5323 ◽  
Author(s):  
JE Russell ◽  
SA Liebhaber
Keyword(s):  
Mrna Stability ◽  
Globin Gene ◽  
Structural Features ◽  
Structural Basis ◽  
Dependent Manner ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Coding Region ◽  
Globin Mrna ◽  
Alpha Globin

Controls that act at both transcriptional and posttranscriptional levels assure that globin genes are highly expressed in developing erythroid cells. The extraordinary stabilities of alpha- and beta- globin mRNAs permit globin proteins to accumulate to substantial levels in these cells, even in the face of physiologic transcriptional silencing. Structural features that determine alpha-globin mRNA stability have recently been identified within its 3′UTR; in contrast, the structural features that determine beta-globin mRNA stability remain obscure. The current study begins to define the structural basis for beta-globin mRNA stability. Two tandem antitermination mutations are introduced into the wild-type human beta-globin gene that permit ribosomes to read into the 3′UTR of the encoded beta-globin mRNA. The readthrough beta-globin mRNA is destabilized in cultured erythroid cells, indicating that, as in human alpha-globin mRNA, an unperturbed 3′UTR is crucial to maintaining mRNA stability. Additional experiments show that the beta-globin and alpha-globin mRNA 3′UTRs provide equivalent levels of stability to a linked beta-globin mRNA coding region, suggesting a parallel in their functions. However, destabilization of the antiterminated beta-globin mRNA is independent of active translation into the 3′UTR, whereas translation into the alpha-globin mRNA 3′UTR destabilizes a linked beta-globin coding region in a translationally dependent manner. This indicates that the alpha- and beta-globin 3′UTRs may stabilize linked mRNAs through distinct mechanisms. Finally, it is shown that neither of the two mutations that, in combination, destabilize the beta-globin mRNA have any effect on beta-globin mRNA stability when present singly, suggesting potential redundancy of stabilizing elements. In sum, the current study shows that a functionally intact beta-globin mRNA 3′UTR is crucial to maintaining beta-globin mRNA stability and provides a level of stability that is functionally equivalent to, although potentially mechanistically distinct from, the previously characterized alpha- globin mRNA 3′UTR stability element.

Differential expression of alpha- and beta-globin genes in erythroleukemic cell lines

1990 ◽  
Vol 10 (7) ◽  
pp. 3591-3595
Author(s):  
N Beru ◽  
P B Maples ◽  
O Hermine ◽  
E Goldwasser
Keyword(s):  
Cell Lines ◽  
Globin Gene ◽  
Restriction Enzymes ◽  
Erythroid Differentiation ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Alpha Globin ◽  
Erythroleukemic Cell

The IW32, NN10, and IW201 cell lines are erythroleukemic cell lines isolated from the spleens of mice infected with the Friend virus. IW32 and NN10 cells can be induced toward erythroid differentiation and hemoglobin synthesis by hemin or butyrate. Both cell lines contain some mature alpha- and beta-globin mRNA before induction, and addition of the inducers greatly increases the amount of globin message. Unlike IW32 and NN10 cells, IW201 cells are only partially inducible. Uninduced 201 cells contain a small amount of alpha-globin mRNA but no detectable beta-globin message. After induction, the cells contain markedly increased amounts of alpha-globin mRNA but still do not express the beta-globin gene. Southern blot analysis with 10 restriction enzymes shows that the restriction map of the beta-globin gene in IW201 cells is indistinguishable from that in IW32 and NN10 cells.

scholarly journals Endogenous read-through of a UGA termination codon in a Saccharomyces cerevisiae cell-free system: evidence for involvement of both a mitochondrial and a nuclear tRNA.

1982 ◽  
Vol 2 (5) ◽  
pp. 490-497 ◽  
Author(s):  
M F Tuite ◽  
C S McLaughlin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Deae Cellulose ◽  
In Vitro Translation ◽  
Translation System ◽  
Beta Globin ◽  
Mitochondrial Trna ◽  
Globin Mrna ◽  
Free System ◽  
Cell Free System

Globin mRNA, translated in a Saccharomyces cerevisiae cell-free protein synthesizing system prepared from a [psi+ rho+] strain, primarily directed the synthesis of alpha- and beta-globin. A third globin mRNA-specific polypeptide was also synthesized, representing approximately 10% of the total translation products. This polypeptide (beta') was synthesized by translational read-through of the beta- globin mRNA UGA terminator and was mediated primarily by an endogenous tRNA coded for by the mitochondria. This mitochondrial tRNA, when charged, could be preferentially bound, in high salt, to benzoylated DEAE-cellulose, a characteristic of a tRNATrp. The synthesis of beta- mediated by this mitochondrial tRNATrp was significantly reduced when the translation system was prepared from an isogenic [psi-] strain. Evidence for a nuclear-coded tRNA, also able to suppress the beta-globin mRNA UGA terminator in [psi+] but not [psi-] lysates, was also obtained. The presence of these endogenous UGA suppressor activities in the yeast cell-free system should allow successful in vitro translation of mitochondrial mRNAs.

scholarly journals Differential expression of alpha- and beta-globin genes in erythroleukemic cell lines.

1990 ◽  
Vol 10 (7) ◽  
pp. 3591-3595 ◽  
Author(s):  
N Beru ◽  
P B Maples ◽  
O Hermine ◽  
E Goldwasser
Keyword(s):  
Cell Lines ◽  
Globin Gene ◽  
Restriction Enzymes ◽  
Erythroid Differentiation ◽  
Globin Genes ◽  
Beta Globin ◽  
Globin Mrna ◽  
Beta Globin Gene ◽  
Alpha Globin ◽  
Erythroleukemic Cell

The IW32, NN10, and IW201 cell lines are erythroleukemic cell lines isolated from the spleens of mice infected with the Friend virus. IW32 and NN10 cells can be induced toward erythroid differentiation and hemoglobin synthesis by hemin or butyrate. Both cell lines contain some mature alpha- and beta-globin mRNA before induction, and addition of the inducers greatly increases the amount of globin message. Unlike IW32 and NN10 cells, IW201 cells are only partially inducible. Uninduced 201 cells contain a small amount of alpha-globin mRNA but no detectable beta-globin message. After induction, the cells contain markedly increased amounts of alpha-globin mRNA but still do not express the beta-globin gene. Southern blot analysis with 10 restriction enzymes shows that the restriction map of the beta-globin gene in IW201 cells is indistinguishable from that in IW32 and NN10 cells.

scholarly journals Characterization of the major regulatory element upstream of the human alpha-globin gene cluster.

1991 ◽  
Vol 11 (9) ◽  
pp. 4679-4689 ◽  
Author(s):  
A P Jarman ◽  
W G Wood ◽  
J A Sharpe ◽  
G Gourdon ◽  
H Ayyub ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Globin Gene ◽  
Major Elements ◽  
Beta Globin ◽  
Globin Mrna ◽  
Expression Studies ◽  
Primary Element ◽  
Alpha Globin ◽  
Binding Sequence

The major positive regulatory activity of the human alpha-globin gene complex has been localized to an element associated with a strong erythroid-specific DNase I hypersensitive site (HS -40) located 40 kb upstream of the zeta 2-globin mRNA cap site. Footprint and gel shift analyses of the element have demonstrated the presence of four binding sites for the nuclear factor GATA-1 and two sites corresponding to the AP-1 consensus binding sequence. This region resembles one of the major elements of the beta-globin locus control region in its constitution and characteristics; this together with evidence from expression studies suggests that HS -40 is a primary element controlling alpha-globin gene expression.

Endogenous read-through of a UGA termination codon in a Saccharomyces cerevisiae cell-free system: evidence for involvement of both a mitochondrial and a nuclear tRNA

1982 ◽  
Vol 2 (5) ◽  
pp. 490-497
Author(s):  
M F Tuite ◽  
C S McLaughlin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Deae Cellulose ◽  
In Vitro Translation ◽  
Translation System ◽  
Beta Globin ◽  
Mitochondrial Trna ◽  
Globin Mrna ◽  
Free System ◽  
Cell Free System

Globin mRNA, translated in a Saccharomyces cerevisiae cell-free protein synthesizing system prepared from a [psi+ rho+] strain, primarily directed the synthesis of alpha- and beta-globin. A third globin mRNA-specific polypeptide was also synthesized, representing approximately 10% of the total translation products. This polypeptide (beta') was synthesized by translational read-through of the beta- globin mRNA UGA terminator and was mediated primarily by an endogenous tRNA coded for by the mitochondria. This mitochondrial tRNA, when charged, could be preferentially bound, in high salt, to benzoylated DEAE-cellulose, a characteristic of a tRNATrp. The synthesis of beta- mediated by this mitochondrial tRNATrp was significantly reduced when the translation system was prepared from an isogenic [psi-] strain. Evidence for a nuclear-coded tRNA, also able to suppress the beta-globin mRNA UGA terminator in [psi+] but not [psi-] lysates, was also obtained. The presence of these endogenous UGA suppressor activities in the yeast cell-free system should allow successful in vitro translation of mitochondrial mRNAs.

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