Protein synthesis in a cell free human reticulocyte system: ribosome function in thalassemia.

Bank, Arthur; Marks, Paul A.

doi:10.1172/jci105347

Cited by 59 publications

(13 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…It is currently believed that the basic defect in beta thalassemia is decreased synthesis of structurally normal messenger RNA (mRNA)1 for beta chains, or synthesis of a structurally and/or functionally abnormal mRNA for beta chains (8,9). There is much experimental evidence which indirectly supports this hypothesis: thalassemic ribosomes synthesize polyphenylalanine normally when primed by polyuridylic acid in a cell-free system (10); the rate of beta chain transDr. Forget is a fellow of the Medical Foundation Inc., Boston, Mass.…”

Section: Introductionmentioning

confidence: 99%

Defect in messenger RNA for human hemoglobin synthesis in beta thalassemia

Benz¹,

Forget²

1971

J. Clin. Invest.

View full text Add to dashboard Cite

A B S T R A C T Functional messenger RNA for human hemoglobin synthesis was prepared from reticulocyte lysates of patients with homozygous beta thalassemia and sickle cell anemia. The messenger RNA stimulated the synthesis of human globin chains by a cell-free system derived from Krebs mouse ascites cells. In the presence of beta thalassemia messenger RNA, the system synthesized much less beta chain than alpha chain whereas in the presence of sickle cell anemia messenger RNA, nearly equal amounts of alpha and beta chains were synthesized. The beta/alpha synthetic ratios obtained in the cell-free system were similar to those obtained by incubating intact beta thalassemia and sickle cell anemia reticulocytes in the presence of radioactive leucine. The experiments provide direct evidence of a defect in messenger RNA for beta chains as a cause for the decreased synthesis of beta chains observed in beta thalassemia.

show abstract

Section: Introductionmentioning

confidence: 99%

Defect in messenger RNA for human hemoglobin synthesis in beta thalassemia

Benz¹,

Forget²

1971

J. Clin. Invest.

View full text Add to dashboard Cite

show abstract

“…In a thalassemia, a-chain synthesis is specifically diminished (7,8). Assays with cell-free protein-synthesizing systems have shown that mRNAs from 3 thalassemia cells reflect a decrease in /-chain mRNA activity (9)(10)(11)(12). A similar decrease in a globin mRNA activity has been demonstrated in a thalassemia (Hemoglobin H) cells (13).…”

mentioning

confidence: 99%

Decreased Globin Messenger RNA in Thalassemia Detected by Molecular Hybridization

Kacian

Gambino

Dow

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

101

View full text Add to dashboard Cite

In previous studies of patients with /3 thalassemia, mRNA extracted from reticulocytes in peripheral blood when added to cell-free systems reproduces the deficient /-chain synthesis characteristic of intact cells. The present studies with specific probes for a and /3 mRNA were designed to decide whether the decreased /3 mRNA activity is due to the presence of abnormal or reduced /3 globin mRNA in these cells. Purified a and /3 complementary DNAs (cDNAs) have been synthesized with RNA-instructed DNA polymerase; a and /3 mRNAs isolated from heavy (,3-producing) and light (a-producing) polyribosomes of rabbit reticulocytes were used as templates. Each of the cDNAs is more than 80% pure by the criterion of biological activity. The a cDNA labeled with [32P]dCTP and the 3 cDNA labeled with [3H]dCTP have been added simultaniously to reaction mixtures containing various concentrations of mRNA from thalassemic and nonthalassemic subjects. The extent and rate of hybridization were determined, permitting a comparison of relative a and / mRNA content in the same annealing mixture. In six nonthalassemic patients, relatively equal amounts of hybridizable a and / mRNA appear to be present.

show abstract

“…Our results indicate that in the a thalassemia syndrome of Hb H disease the a/fl mRNA ratio is 1: 6, whereas in homozygous fl thalassemia the a/f mRNA ratio is 10 Preparation of Human and Rabbit Globin mRNA. Rabbit globin mRNA was obtained as described (27).…”

mentioning

confidence: 74%

“…Studies on various aspects of Iolypeptide chain initiation and translation in homozygous f thalassemia have revealed no abnormalities (10)(11)(12)(13)(14)(15). However, when globin mRNA isolated from fi thalassemia reticulocytes is added to a heterologous cell-free system, much less human fi chain is synthesized than a chain (16)(17)(18).…”

mentioning

confidence: 99%

“…The amount of labeled DNA hybridized was determined by diluting the reaction mixture into 2 ml of a solution containing 0.1 M sodium acetate (pH 4.5), 1 mM ZnSO4, 10 Ag/ml of calf-thymus DNA (heat-denatured), and sufficient Aspergillus oryzae S1 nuclease to digest denatured 3H-labeled DNA of bacteriophage P22 so that less than 5% of its cpm remained acid-precipitable, while no more than 5% acid-soluble counts were released from the native (double-stranded) phage DNA; incubation was for 30 min at 45°. The remaining nuclease-resistant trichloroacetic acidprecipitable counts (less the counts in a similarly processed blank containing cDNA but no RNA) were taken to represent the specific hybrid; percent hybridization was calculated in reference to the acid-precipitable counts in a similarly incubated but nondigested blank reaction mixture containing cDNA but no RNA.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Quantitative Deficiency of Chain-Specific Globin Messenger Ribonucleic Acids in the Thalassemia Syndromes

Housman

Forget²,

Skoultchi³

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

114

View full text Add to dashboard Cite

A hybridization assay procedure was devised that makes possible quantitation of the ratio of mRNA of alpha to mRNA of beta globin chains in an RNA sample. The assay uses the radioactive synthetic DNA copies obtained by incubation of RNA-dependent DNA polymerase of avian myeloblastosis virus with rabbit globin mRNA that is 80-90%4 enriched in mRNA specific for synthesis of alpha or beta globin chains. The rabbit alpha-chain mRNA is obtained from the postribosomal supernatant of rabbit reticulocyte lysates; the rabbit beta-chain mRNA is obtained from the largest polysomes of rabbit reticulocytes treated with L-O-methylthreonine. Sufficient homology exists between rabbit and human globin chains and globin mRNAs that the synthetic DNA copies of chainspecific rabbit globin mRNA hybridize with human globin mRNA. Applied to the study of globin mRNA isolated from reticulocytes of humans with alpha and beta thalassemia, the technique revealed marked quantitative deficiency of alpha-chain mRNA relative to beta-chain mRNA in alpha thalassemia and similar deficiency of beta-chain mRNA relative to alpha-chain mRNA in beta thalassemia. The thalassemia' syndromes are therefore characterized by true quantitative deficiency of the mRNA specific for the affected globin chain.

show abstract

Protein synthesis in a cell free human reticulocyte system: ribosome function in thalassemia.

Cited by 59 publications

References 23 publications

Defect in messenger RNA for human hemoglobin synthesis in beta thalassemia

Defect in messenger RNA for human hemoglobin synthesis in beta thalassemia

Decreased Globin Messenger RNA in Thalassemia Detected by Molecular Hybridization

Quantitative Deficiency of Chain-Specific Globin Messenger Ribonucleic Acids in the Thalassemia Syndromes

Contact Info

Product

Resources

About