Defect in messenger RNA for human hemoglobin synthesis in beta thalassemia

The number of globin genes in human cells was determined by hybridizing DNA from human spleens to 3H-labeled DNA complementary to human globin mRNA. Assuming the rates of reannealing of complementary DNA and cellular DNA are similar, the extent of hybridization of complementary DNA at various ratios of cellular DNA to complementary DNA indicate that there are fewer than 10 globin gene copies per haploid human genome. An alternative analysis of the data, which introduces no assumptions concerning the relative rates of reaction of complementary DNA and cellular DNA, indicates fewer than 20 globin gene copies are present. DNA isolated from the spleen of a patient with ( + thalassemia contained a number of globin gene copies similar to that of normal DNA.The mechanisms regulating human hemoglobin synthesis at the gene level are poorly understood. Since both transcriptional and translational controls may be involved, it is of importance to determine the number of globin genes in man. The availability of highly radioactive DNA (cDNA) complementary to globin mRNA (1-3) provides a useful tool for this purpose. Recent reports of the use of mouse liver and duck reticulocytes indicate that there are fewer than 10 copies of the globin sequences present per haploid genome (4-6). In addition, the number of globin genes present in duck reticulocytes and liver cells has been found to be similar (4). The experiments reported here were undertaken to measure the number of globin genes present in human genomes, and to compare the number of genes in the cells of patients with B+ thalassemia and without thalassemia. Human cDNA was used to probe DNA from human spleens for the number of globin gene copies. The results are consistent with genetic evidence, and indicate that fewer than 20 globin genes are present in normal haploid human genomes. In addition, there is no detectable difference in the number of globin genes present in DNA from the cells of a patient with (3+ thalassemia. METHODSPreparation and Characterization of Human DNA. Human DNA was isolated from individual spleens of patients with and without thalassemia obtained when splenectomy was indicated for treatment of the patient. The spleens were collected within 1 hr after surgery, cut into small pieces, frozen immediately in liquid nitrogen, and stored at -70°. The spleens were ground to a coarse powder in a mortar cooled with dry ice, and the DNA was isolated by the following procedure modified from those reported (7, 8): Frozen spleen powder (10-50 g) was suspended at 40 in 5% sucrose buffer containing 1 mM MgCl2 and 1 mM NaH2PO4 (pH 6.5). The cells were broken by 25 strokes in a tight-fitting Dounce homogenizer. The nuclei were separated by centrifugation at 800 X g for 10 min, washed once with the 5% sucrose solution, and taken up in 10-20 volumes of 10 mM Tris (pH 8.3), 0.15 M NaCl, 5 mM EDTA, 1% sodium dodecyl sulfate, 1.0 M NaClO4.After addition of an equal volume of CHCl3-isoamyl alcohol (24: 1), the mixture was shaken for 30 min at room temperature. The aq...

Section: Resultsmentioning

confidence: 99%

A Limited Number of Globin Genes in Human DNA

Gambino

Kacian

O'Donnell

et al. 1974

“…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) …”

Section: Methodsmentioning

confidence: 99%

“…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) (15), with the modifications described by Gould and Hamlyn (9). Formamide was deionized by mixing for 4 hr with 40 g/liter of Rexyn I-300® (Fisher Scientific Co.), previously dried by washing with absolute ethanol, then ether.…”

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)

“…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). The opposite is observed in the a thalassemia syndrome of Hb H disease (19,20 there exists in these conditions a normal amount of a structurally and functionally abnormal mRNA.…”

mentioning

confidence: 99%

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

Housman

Forget²,

Skoultchi³

et al. 1973

Self Cite

114

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.