Nucleotide Sequences of Human Globin Messenger Rna *

Section: Resultssupporting

confidence: 57%

Hemoglobin Cranston, an unstable variant having an elongated beta chain due to nonhomologous crossover between two normal beta chain genes.

Bunn¹,

Schmidt²,

Haney³

et al. 1975

“…Labeled complementary RNA (cRNA) was transcribed from the slow band cDNA, in the presence of [a-'2P]GTP and E. coli RNA polymerase. The cRNA was digested with T1 RNase and the digest was fractionated in two dimensions (16,17) to obtain the fingerprint pattern shown in Fig. 3C.…”

Section: Resultsmentioning

confidence: 99%

“…cDNA of low specific activity (16,17) was transcribed into 32P-labeled RNA using E. coli RNA polymerase as described (16,17). The RNA was then digested with T, ribonuclease and analyzed by the usual nucleotide sequencing technics (16,17).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Forget¹,

Housman²,

Benz³

et al. 1975

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)

“…We have shown that appropriate doses of IF markedly inhibit FLC growth both in vvo (25) and in vitro (26), as well as Hb synthesis in Me2SO-stimulated cultures (26). Virus production is also inhibited, although accumulation of viral antigens is increased (27 (32). After alkaline hydrolysis and Sephadex G-50 gel filtration, [3HIcDNA (specific activity, 107 cpm/,ug) was purified by alkaline sucrose density gradient centrifugation; fractions corresponding to 9-10 S were pooled, neutralized, ethanol-precipitated, and resuspended in water.…”

mentioning

confidence: 99%

Inhibition of transcription and translation of globin messenger RNA in dimethyl sulfoxide-stimulated Friend erythroleukemic cells treated with interferon.

Rossi¹,

Dolei²,

Cioé³

et al. 1977

The addition of appropriate doses of interferon (IF) to cultures of Friend erythroleukemic cells inhibits dimethyl sulfoxide (Me2SO)-stimulated erythroid differentiation. In this study, the synthesis of heme, hemoglobin, and globin mRNA in Me2SO-stimulated cultures, with or without IF added, was compared. Although the hemoglobin content in Me2SO+IF-treated cultures was reduced 6- to 9-fold compared to that of cultures treated with Me2SO alone, there was less than a 2-fold decrease in the amount of heme accumulated. Globin mRNA, although unchanged in size or base sequence, was reduced in content in the Me2SO+IF cultures. The level of reduction of globin mRNA was insufficient to account for the lack of globin synthesis. Thus, it appears that IF may operate on two levels--one involving the transcription of globin mRNA and the other involving its translation.