Control of Haemoglobin Synthesis: a Difference in the Size of the Polysomes making α and β Chains

Hunt, Rosanna; Hunter, A. R.; Munro, Alan

doi:10.1038/220481a0

Cited by 79 publications

(31 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This finding is consistent with various studies (18)(19)(20)(21)(22) which indicate that /-mRNA is initiated-at a faster rate than a-mRNA and that there must be an excess of a-mRNA in order to achieve equal synthesis of a and /3 globin chains in reticulocytes.…”

Section: Resultssupporting

confidence: 81%

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Forget¹,

Housman²,

Benz³

et al. 1975

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

View full text Add to dashboard Cite

Human globin messenger RNA, purified by oligo(dT)-cellulose column chromatography, is reproducibly separated into two bands by polyacrylamide gel electrophoresis in the presence of 99% formamide. The more rapidly migrating (fast) band is somewhat more abundant than the slow band in normal (nonthalassemic) total reticulocyte globin messenger RNA. In a-thalassemic (Hb H disease) messenger RNA, the slow band is 6.5 times more abundant than the fast band, whereas in 0-thalassemic messenger RNA the fast band is three times more abundant than, a second band, which has a slightly greater mobility than the slow band of normal and a-thalassemic RNA. The RNA bands of nonthalassemic globin messenger RNA were eluted from the gel and efficiently transcribed into DNA copies by use of the RNA-dependent DNA polymerase of avian myeloblastosis virus. Hybridization of these copy DNAs to fast and slow band RNAs and to nonfractionated normal, a-thalassemic, and fl-thalassemic messenger RNAs revealed that the eluted fast band RNA contains predominantly a-chain specific sequences, whereas the eluted slow band RNA contains predominantly d-chain specific sequences. Nucleotide sequence analysis of 32P-labeled-RNA transcribed from the slow band copy DNA also indicated that the slow band RNA is,B messenger RNA.The ability to separate biochemically the messenger RNAs (mRNAs) for the a and ,B globin chains of human hemoglobin would greatly facilitate detailed studies of the normal human globin genes and of the precise molecular basis of the thalassemia syndromes. The a-and ,B-thalassemias are inherited disorders of human hemoglobin synthesis which are characterized by absent or decreased synthesis of a and # globin chains, respectively (1). This defect in hemoglobin synthesis has been shown, by molecular hybridization studies, to be associated with absent or decreased quantities of a or , globin chain mRNA (2-4).Globin mRNA migrates as a single broad 9-lOS RNA band during electrophoresis in polyacrylamide gels in aqueous media (5-8). However, in the presence of 98-99% formamide, polyacrylamide gel electrophoresis separates the globin mRNA into two discrete bands (8-10, 23, 24 and translated in cell-free protein synthesizing systems (8, 10, 24); the more rapid (fast) band stimulates the synthesis predominantly of a globin chains, whereas the slow band stimulates the synthesis predominantly of , globin chains.We report here the fractionation of human globin mRNA by polyacrylamide gel electrophoresis in formamide, and the characterization of its separated components by molecular hybridization and nucleotide sequencing technics utilizing DNA copies of the eluted RNA components, synthesized by means of the RNA-dependent DNA polymerase of avian myeloblastosis virus. MATERIALS AND METHODSMaterials. Most materials for RNA isolation, polyacrylamide gel electrophoresis, complementary DNA (cDNA) synthesis, and RNA-cDNA hybridization are the same as previously described (2,(11)(12)(13)

show abstract

Section: Resultssupporting

confidence: 81%

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Forget¹,

Housman²,

Benz³

et al. 1975

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

View full text Add to dashboard Cite

show abstract

“…These modifications of standard methods permitted adequate recovery of human polysomes. It was then possible to show that human, as well as rabbit (4,15), nascent beta chains are associated with heavier polysomes than are alpha chains; presumably, translation of human-beta messengers is initiated at a more rapid rate than alpha, as is the case in rabbits (15 The patient with homozygous beta thalassemia chosen for this study produced sufficient peripheral blood reticulocytes and hemoglobin A. Gamma-chain synthesis was only 15% of the alpha in peripheral blood cells. These necessary bio- logical qualities, together with the availability of [I5S]methionine of very high specific activity, led to successful labeling of the thalassemic polysomes and the detection on them of the normal distribution of nascent beta-and alpha-chains.…”

Section: Discussionmentioning

confidence: 99%

“…Measurement of the polysome sizes associated with the alphaand beta-chain message in human reticulocytes demanded certain modifications of the methods used for similar analyses in rabbit reticulocytes (4 The cells were then divided into two aliquots. A 0.5-ml sample of a rabbit-reticulocyte lysate (9) was added to one of the aliquots.…”

Section: Methodsmentioning

confidence: 99%

Beta Thalassemia and Translation of Globin Messenger RNA

Nathan

Lodish

Kan³

et al. 1971

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

View full text Add to dashboard Cite

To define the quality and relative quantity of beta and alpha messenger RNA in human nonthalassemic and thalassemic reticulocytes, intact cells were incubated with [35S]methionine. The relative amounts of beta-and alpha-nascent chains on polysomes of different sizes were measured by tryptic digestion of pooled polysomes and by determination of the specific activities of beta and alpha peptides that contain methionine. Betachain synthesis predominated on heavy polysomes in nonthalassemic, as well as in thalassemic cells. Since beta chains in thalassemia are made on normal-size polyribosomes, we conclude that the defect in thalassemia does not involve reduction in the rate of initiation of translation due to the production of an abnormal beta message. Such would lead to beta-chain synthesis on very small polysomes. We therefore suggest that the decreased production of beta-globin chains results from a decreased amount of functional beta-globin messenger RNA.

show abstract

“…It is worth bearing in mind that in a situation where initiation frequency was already decreased fivefold, and thus protein output would be fivefold reduced with the repressed mRNA located in very small polysomes, superimposing an approximately fourfold decrease in elongation rate would result in little additional reduction in reporter protein output, even though the target mRNA would move into larger polysomes. For a fuller discussion of the relationship between protein output rates, polysome size, initiation frequency, and elongation rate, under steady-state conditions likely to pertain in transfected cell assays, the reader is referred to Hunt et al (1968Hunt et al ( , 1969. ]…”

Section: Conclusion That Can Be Drawn From the In Vitro Recapitulatimentioning

confidence: 99%

MicroRNAs repress translation of m⁷Gppp-capped target mRNAs in vitro by inhibiting initiation and promoting deadenylation

Standart¹,

Jackson²

2007

Genes Dev.

118

View full text Add to dashboard Cite

Control of Haemoglobin Synthesis: a Difference in the Size of the Polysomes making α and β Chains

Cited by 79 publications

References 9 publications

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Beta Thalassemia and Translation of Globin Messenger RNA

MicroRNAs repress translation of m⁷Gppp-capped target mRNAs in vitro by inhibiting initiation and promoting deadenylation

Contact Info

Product

Resources

About

Control of Haemoglobin Synthesis: a Difference in the Size of the Polysomes making α and β Chains

Cited by 79 publications

References 9 publications

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Beta Thalassemia and Translation of Globin Messenger RNA

MicroRNAs repress translation of m7Gppp-capped target mRNAs in vitro by inhibiting initiation and promoting deadenylation

Contact Info

Product

Resources

About

MicroRNAs repress translation of m⁷Gppp-capped target mRNAs in vitro by inhibiting initiation and promoting deadenylation