scholarly journals Dominantly Transmitted β-Thalassemia Arising From the Production of Several Aberrant mRNA Species and One Abnormal Peptide

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 685-690 ◽  
Author(s):  
Paula Faustino ◽  
Leonor Osório-Almeida ◽  
Luı́sa Romão ◽  
José Barbot ◽  
Berta Fernandes ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Globin Gene ◽  
Dominant Mode ◽  
In Vitro Translation ◽  
Exon 2 ◽  
Mrna Species ◽  
Moderate Anemia ◽  
Cryptic Splice Sites ◽  
Extension Analysis

Abstract We describe a dominantly inherited β-thalassemia intermedia phenotype observed in a five-generation Portuguese family. Carriers are characterized by moderate anemia, hypochromia, microcytosis, elevated hemoglobin (Hb)A2 and HbF levels, splenomegaly, hepatomegaly, and inclusion bodies in pheripheral red blood cells after splenectomy. The molecular basis of this condition is a small deletion within the 5′ consensus splicing sequence of the second intron of the β-globin gene, IVS-II-4,5 (-AG). Reticulocyte RNA studies performed by reverse transcription-polymerase chain reaction (RT-PCR) and primer extension analysis showed three abnormally processed transcripts, which, upon sequencing, were shown to correspond to (1) skipping of exon 2, and (2) activation of two cryptic splice sites (between codons 59/60, and at IVS-II-47). In vitro translation studies of these patients' reticulocyte RNA have shown that at least one of these aberrant mRNA species is translated into an abnormally elongated peptide whose cytotoxic properties could, in part, be causing the atypical dominant mode of inheritance observed in this family. We suggest that this elongated β chain is unable to combine with an α-globin chain to form a functional Hb molecule. Its degradation would, then, exhaust the proteolytic defense mechanism of the erythroid precursors, leading to inefficient proteolysis of the free α chains in excess.

scholarly journals Dominantly Transmitted β-Thalassemia Arising From the Production of Several Aberrant mRNA Species and One Abnormal Peptide

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 685-690 ◽  
Author(s):  
Paula Faustino ◽  
Leonor Osório-Almeida ◽  
Luı́sa Romão ◽  
José Barbot ◽  
Berta Fernandes ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Globin Gene ◽  
Dominant Mode ◽  
In Vitro Translation ◽  
Exon 2 ◽  
Mrna Species ◽  
Moderate Anemia ◽  
Cryptic Splice Sites ◽  
Extension Analysis

We describe a dominantly inherited β-thalassemia intermedia phenotype observed in a five-generation Portuguese family. Carriers are characterized by moderate anemia, hypochromia, microcytosis, elevated hemoglobin (Hb)A2 and HbF levels, splenomegaly, hepatomegaly, and inclusion bodies in pheripheral red blood cells after splenectomy. The molecular basis of this condition is a small deletion within the 5′ consensus splicing sequence of the second intron of the β-globin gene, IVS-II-4,5 (-AG). Reticulocyte RNA studies performed by reverse transcription-polymerase chain reaction (RT-PCR) and primer extension analysis showed three abnormally processed transcripts, which, upon sequencing, were shown to correspond to (1) skipping of exon 2, and (2) activation of two cryptic splice sites (between codons 59/60, and at IVS-II-47). In vitro translation studies of these patients' reticulocyte RNA have shown that at least one of these aberrant mRNA species is translated into an abnormally elongated peptide whose cytotoxic properties could, in part, be causing the atypical dominant mode of inheritance observed in this family. We suggest that this elongated β chain is unable to combine with an α-globin chain to form a functional Hb molecule. Its degradation would, then, exhaust the proteolytic defense mechanism of the erythroid precursors, leading to inefficient proteolysis of the free α chains in excess.

In vitro translation of mRNA species from cells infected with Machupo virus

1987 ◽  
Vol 92 (3-4) ◽  
pp. 315-319
Author(s):  
I. S. Lukashevich ◽  
T. A. Stelmakh ◽  
E. P. Stchesljenok ◽  
T. V. Shkolina
Keyword(s):  
In Vitro Translation ◽  
Mrna Species ◽  
Machupo Virus

scholarly journals Inclusion body beta-thalassemia trait in a Swiss family is caused by an abnormal hemoglobin (Geneva) with an altered and extended beta chain carboxy-terminus due to a modification in codon beta 114

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 801-805 ◽  
Author(s):  
P Beris ◽  
PA Miescher ◽  
JC Diaz-Chico ◽  
IS Han ◽  
A Kutlar ◽  
...  
Keyword(s):  
Inclusion Body ◽  
Gradient Centrifugation ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Red Cell ◽  
Beta Chain ◽  
Inclusion Body Formation ◽  
Moderate Anemia ◽  
Abnormal Hemoglobin

Abstract We have analyzed the sequence of the beta globin gene of a chromosome that is linked to the occurrence of an inclusion body beta-thalassemia characterized in the heterozygote by moderate anemia, severe red cell abnormalities, splenomegaly, inclusion body formation, elevated Hb A2 levels, and an increased in vitro alpha/beta chain synthetic ratio. The data indicate a change in codon 114 from CTG (Leu) to -GG that resulted in a frameshift and the presumed synthesis of an abnormal beta chain that is 156 residues long with a completely different C-terminal amino acid sequence. The change in codon 114 gives a -GGGCCC- sequence that creates a new ApaI site; the resulting 2.6-kilobase fragment has been observed in all subjects with this thalassemia condition. Protein structural analyses failed to demonstrate any trace of the abnormal beta chain, even in reticulocytes and nucleated red cells that were isolated by density gradient centrifugation. The inclusion bodies appear to contain mainly normal alpha chains. It is assumed that the structure of the beta-Geneva chain prevents it from combining with normal alpha chains; this results in a rapid breakdown of the abnormal protein during the early stages of red cell maturation and an accumulation of free alpha chains.

scholarly journals Locus assignment of two alpha-globin structural mutants from the Caribbean basin: alpha Fort de France (alpha 45 Arg) and alpha Spanish Town (alpha 27 Val)

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 833-835
Author(s):  
FE Cash ◽  
N Monplaisir ◽  
M Goossens ◽  
SA Liebhaber
Keyword(s):  
Dominant Role ◽  
Globin Gene ◽  
In Vitro Translation ◽  
Globin Mrna ◽  
Alpha Globin ◽  
Structural Mutations ◽  
The Caribbean ◽  
Spanish Town

Abstract Two alpha-globin structural mutants were mapped to their encoding loci by in vitro translation of hybrid-selected alpha 1- and alpha 2-globin mRNA. The more highly expressed mutant, alpha Spanish Town (alpha 27Val), is encoded at the alpha 2 locus and the less expressed mutant, alpha Fort de France (alpha 45Arg), is encoded at the alpha 1 locus. These results further define the distribution of alpha-globin structural mutations within the alpha-globin gene cluster and substantiate the dominant role of the alpha 2-globin locus in alpha- globin expression.

scholarly journals Locus assignment of two alpha-globin structural mutants from the Caribbean basin: alpha Fort de France (alpha 45 Arg) and alpha Spanish Town (alpha 27 Val)

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 833-835
Author(s):  
FE Cash ◽  
N Monplaisir ◽  
M Goossens ◽  
SA Liebhaber
Keyword(s):  
Dominant Role ◽  
Globin Gene ◽  
In Vitro Translation ◽  
Globin Mrna ◽  
Alpha Globin ◽  
Structural Mutations ◽  
The Caribbean ◽  
Spanish Town

Two alpha-globin structural mutants were mapped to their encoding loci by in vitro translation of hybrid-selected alpha 1- and alpha 2-globin mRNA. The more highly expressed mutant, alpha Spanish Town (alpha 27Val), is encoded at the alpha 2 locus and the less expressed mutant, alpha Fort de France (alpha 45Arg), is encoded at the alpha 1 locus. These results further define the distribution of alpha-globin structural mutations within the alpha-globin gene cluster and substantiate the dominant role of the alpha 2-globin locus in alpha- globin expression.

Translational Control of the Hematopoiesis-Specific RhoH Gene Is Mediated by Upstream Open Reading Frames.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4190-4190
Author(s):  
Sébastien Lahousse ◽  
Claude Denis ◽  
Jean-Pierre Kerckaert ◽  
Sylvie Galiègue-Zouitina
Keyword(s):  
Protein Synthesis ◽  
Translational Control ◽  
Molecular Mechanisms ◽  
Chromosomal Translocation ◽  
Bound State ◽  
Inhibitory Effect ◽  
Cellular Growth ◽  
In Vitro Translation ◽  
Exon 2

Abstract The hematopoiesis-specific RhoH gene was identified in our laboratory, as a BCL6 partner gene (1), in follicular B-non Hodgkin’s lymphoma associated with the recurring t(3;4) chromosomal translocation (2). The RhoH gene encodes a Rho small G protein supposed to be a Rac antagonist (3). The RhoH protein is always in a GTP-bound state (i.e. constitutively active), which suggests a strongly regulated synthesis. This work is aimed at elucidating molecular mechanisms that may regulate this RhoH protein synthesis. The RhoH gene contains one coding exon only (exon 2) preceded by six uncoding exons, and the RhoH transcripts exhibit an important 5′ UTR heterogeneity, especially in the B-lymphoid lineage. This heterogeneity both reflects (i) an use of multiple transcription start sites and (ii) alternative splicing events of some 5′ uncoding exons (4). The present study is devoted to assess the functional relevance of these 5′ uncoding exons in the RhoH protein synthesis. Some of them (exons 1a, 1b and X4) contain an upstream ORF (uORF) sequence. Ten percent of eukaryotic mRNAs, 2/3 of which are involved in the control of cellular growth and differentiation, contain such uORFs within their 5′-UTR sequence. These uORFs are involved in the translational control of these genes (5). RhoH thus provides an additional opportunity to assess the role of uORFs in the translation of a cellular mRNA. Moreover, uORFs have never been implicated in the regulation of any small G family member gene. The data show that the uORFs sequences from the RhoH gene repress the translation of a downstream reporter gene, in transfected hematopoietic cell lines (erythroid, B- and T- lymphoid). The initiation codon from 1a-, 1b-, and X4- uORFs is strongly involved in this phenomenon, as the inhibitory effect was suppressed by an uAUG invalidation. Targeted mutations (premature Stop, silent or mis-sens) within these uORFs sequences were functionally analyzed in the same reporter LUC system. The results led us to distinguish two uORF-mediated translational regulatory mechanisms: (i) an “uAUG dependent / uORF-peptide dependent” mechanism for the 1a- and 1b- uORFs sequences, and (ii) an “uAUG dependent / uORF-peptide independent” mechanism for the X4- uORF. The RhoH transcripts exhibit a 5′ UTR heterogeneity, which might modulate the protein synthesis. Three main RhoH transcripts are expressed in the different hematopoietic lineages: “1a-X4-2”, “1a-1b-X4-2” and “1b-X4-2” (4), therefore the effects of the 1a-, 1b-, and X4- uORFs were investigated in combination (1a+X4; 1a+1b+X4; 1b+X4), as in the RhoH transcripts. The results convinced us to propose an uORF-mediated translational regulatory mechanism, which is based on a combinatory effect (synergy or antagonism) of three uORFs. This mechanism is called: “translational lock-unlock” model of the RhoH gene. In vitro translation experiments aimed at validating this model will be presented.

Inclusion body beta-thalassemia trait in a Swiss family is caused by an abnormal hemoglobin (Geneva) with an altered and extended beta chain carboxy-terminus due to a modification in codon beta 114

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 801-805 ◽  
Author(s):  
P Beris ◽  
PA Miescher ◽  
JC Diaz-Chico ◽  
IS Han ◽  
A Kutlar ◽  
...  
Keyword(s):  
Inclusion Body ◽  
Gradient Centrifugation ◽  
Globin Gene ◽  
Beta Thalassemia ◽  
Red Cell ◽  
Beta Chain ◽  
Inclusion Body Formation ◽  
Moderate Anemia ◽  
Abnormal Hemoglobin

We have analyzed the sequence of the beta globin gene of a chromosome that is linked to the occurrence of an inclusion body beta-thalassemia characterized in the heterozygote by moderate anemia, severe red cell abnormalities, splenomegaly, inclusion body formation, elevated Hb A2 levels, and an increased in vitro alpha/beta chain synthetic ratio. The data indicate a change in codon 114 from CTG (Leu) to -GG that resulted in a frameshift and the presumed synthesis of an abnormal beta chain that is 156 residues long with a completely different C-terminal amino acid sequence. The change in codon 114 gives a -GGGCCC- sequence that creates a new ApaI site; the resulting 2.6-kilobase fragment has been observed in all subjects with this thalassemia condition. Protein structural analyses failed to demonstrate any trace of the abnormal beta chain, even in reticulocytes and nucleated red cells that were isolated by density gradient centrifugation. The inclusion bodies appear to contain mainly normal alpha chains. It is assumed that the structure of the beta-Geneva chain prevents it from combining with normal alpha chains; this results in a rapid breakdown of the abnormal protein during the early stages of red cell maturation and an accumulation of free alpha chains.

scholarly journals Molecular basis for alpha-thalassemia associated with the structural mutant hemoglobin Suan-Dok (alpha 2 109leu----arg) [published erratum appears in Blood 1991 Mar 15;77(6):1404]

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2630-2636 ◽  
Author(s):  
I Weiss ◽  
FE Cash ◽  
MB Coleman ◽  
A Pressley ◽  
JG Adams ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Globin Gene ◽  
Mrna Level ◽  
Early Time ◽  
In Vitro Translation ◽  
Globin Mrna ◽  
Time Points ◽  
Alpha Thalassemia ◽  
Alpha Globin

Abstract Hemoglobin (Hb) Suan-Dok (alpha 109Arg) is a rare alpha-globin structural mutation that is linked to an alpha-thalassemia (alpha-thal) determinant. When inherited in trans to an alpha-thal-1 mutation (-), it results in Hb H disease associated with low levels (9%) of the Suan- Dok Hb. The nature of the thalassemic defect associated with the alpha SD mutation has been investigated by structural and functional studies. Sequence analysis of the cloned Suan-Dok allele showed a missense mutation (T----G) at codon 109 in an otherwise normal alpha 2-globin gene. When the alpha 2SD-globin gene was introduced into mouse erythroleukemia cells, the steady state alpha-globin messenger RNA (mRNA) level was equivalent to the alpha A-globin gene control. Although in vitro translation of a synthetic alpha 2SD-globin mRNA generated levels of alpha globin equivalent to alpha 2A-globin mRNA at early time points, the ratio of alpha SD to alpha A globin decreased markedly at later time points. These data suggest that the thalassemic defect associated with the Suan-Dok mutation results from a significant instability of the alpha SD globin.

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