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

Abstract

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)

Molecular analysis of the beta-thalassemia phenotype associated with inheritance of hemoglobin E (alpha 2 beta2(26)Glu leads to Lys).

“…The assay does not permit individual quantitation of I3A_mRNA relative to 8E_ mRNA. In contrast, we have repeatedly shown (13)(14)(15)(16) that the hybridization conditions employed reproducibly detect 83-mRNA without cross-hybridization to y-mRNA. RNA from both patients exhibited a striking decrease in the total /3 globin mRNA content (i.e., /3A + JJE/a ratio).…”

“…Both exhibit typical thalassemic facies, growth retardation, and erythroid hyperplasia. Patients with Hb S-,8-thalassemia and "classical" /3-thalassemia who were studied have been described previously (13)(14)(15)(16). In addition, the father ofpatient 1, a heterozygote for Hb E, was studied (Table I).…”

“…Globin chains synthesized in cell-free translation systems primed with reticulocyte RNA were analyzed by carboxymethylcellulose chromatography after addition of authentic globin chain markers, as described in the preceding section. ,3/a mRNA ratios assessed by molecular hybridization were obtained by saturation hybridization analysis, comparing the slopes obtained from titration of equal amounts of a-and /8-cDNA by increasing amounts of each RNA sample, as described elsewhere (14)(15)(16). However, hybridization reactions were conducted under conditions which we have previously shown (14)(15)(16) to prohibit cross-hybridization among /8-, 8-, e-, and -y-mRNA and cDNA sequences.…”

“…,3/a mRNA ratios assessed by molecular hybridization were obtained by saturation hybridization analysis, comparing the slopes obtained from titration of equal amounts of a-and /8-cDNA by increasing amounts of each RNA sample, as described elsewhere (14)(15)(16). However, hybridization reactions were conducted under conditions which we have previously shown (14)(15)(16) to prohibit cross-hybridization among /8-, 8-, e-, and -y-mRNA and cDNA sequences. The molecular hybridization analyses do not distinguish between ,A_ and 3E_mRNA, since these mRNA most likely differ only in one or a few base pairs.…”

“…RNA was desalted by centrifugation through a Sephadex G-25 (coarse) syringe column (22), and used directly for further analysis without purification of globin mRNA fractions. Cell-free translations in wheat germ extracts were performed utilizing methods described in detail elsewhere, as was molecular hybridization analysis, utilizing radioactive, purified a-and ,8-globin cDNA hybridization probes (13)(14)(15)(16)(17)19). Globin chains synthesized in cell-free translation systems primed with reticulocyte RNA were analyzed by carboxymethylcellulose chromatography after addition of authentic globin chain markers, as described in the preceding section.…”