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

Housman, David E.; Forget, Bernard G.; Skoultchi, Arthur I.; Benz, Edward J.

doi:10.1073/pnas.70.6.1809

Cited by 114 publications

(60 citation statements)

References 33 publications

(39 reference statements)

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“…In the erythroid cells of patients with (t thalassemia, there is a decreased amount of ( mRNA present, as determined by biologic activity assay in cell-free systems (21)(22)(23) and by molecular hybridization (16,24). The underlying gene defect responsible for reduced (3 globin mRNA synthesis may be due to (1) deletion of ( globin genes, (2) (3,6, and y mRNAs will be necessary to quanti- tate more precisely the number of each of the globin genes in the genomes of patients with and without thalassemia.…”

Section: Resultsmentioning

confidence: 99%

A Limited Number of Globin Genes in Human DNA

Gambino

Kacian

O'Donnell

et al. 1974

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

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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...

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Section: Resultsmentioning

confidence: 99%

A Limited Number of Globin Genes in Human DNA

Gambino

Kacian

O'Donnell

et al. 1974

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

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“…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%

“…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%

See 1 more Smart Citation

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.

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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)

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“…Housman [8], has demonstrated by hybridization studies that cr-and @-globin mRNA prepared from polysomes are in a I : I ratio and we have made the simplest assumption that one of the bands is (Y messenger RNA, the other fl messenger RNA.…”

Section: Polyacrylamide Gel Elektrophoresis and In Vitromentioning

confidence: 99%

Molecular weights of separated rabbit α‐ and β‐globin messenger RNAs

1974

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Several authors [l-3] have shown that translation of the 'supernatant' messenger RNA from rabbit reticulocytes in vitro results in an excess of a-globin over @-globin chains. We have previously demonstrated that rabbit reticulocyte polysomal messenger RNA, which codes for roughly equal amounts of the two globin chains in vitro, exhibits two major zones in polyacrylamide gel electrophoresis in formamide. The molecular weight of the more rapidly migrating RNA species was estimated to be 202 000 and that of the more slowly migrating RNA species was estimated to be 227 000 f4] i Since rabbit a-globin chains contain five fewer amino acids than rabbit /I-globin chains, we suggested that the smaller RNA may represent the a+. globin messenger and the larger RNA the @globin messenger. But the size difference (25 000) is considerably more than necessary (4800) to code for the additions number of amino acids. We present evidence in this report that the 'supernatant' messenger, which we confirm by translation in vitro is enriched by a factor of 2-3 as compared with polysomal messenger in cu-globin messenger activity, dispiays one major zone in polyac~l~ide gel electrophoresis in formamide. The mobility of this RNA is identical to that of the smaller RNA species in polysomal messenger. The mol. wt. of rabbit o-globin messenger RNA is therefore 202 000. Conversely, the larger polysomal RNA species must represent /I-globin messenger, and its mol. wt. is therefore 227 000. Methods Preparation of post-ribosomal 'supernatant' messenger RhfARibosomes, including native subunits, were removed from a rabbit reticulocyte lysate by 553 000 g hr centrifugation. The supernatant was carefully removed and the RNA extracted using the method of Lee et al [S] . The RNA was washed twice with 70% ethanol, dried in vacua and the polyadenylated RNA isolated by oligo dT-cellulose chromatography [6] . This RNA was concentrated lo-fold by lyoph~isation and precipitated with 2 vol of ethanol. After overnight storage at -2O'C the RNA was recovered by low speed centrifugation, washed twice with 70% ethanol, dried in vacua and dissolved in water or formamide. Protein synthesis in vitroPre-incubated Krebs II ascites S30 was prepared as described by Mathews and Korner [7]. 200 ~1 incubation mixtures contained: 80 ~1 ascites S30,20 mM Tris-HCl buffer, pH 7.5,80 mM KCl, 3 mM magnesium acetate, 1 mM dithiothreitol, 10 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, 1 mM ATP, 0.2 mM GTP, 100 PM of all natur~ly occurring L-amino acids except those added in radioactive form, 2.0 /.&i

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

Cited by 114 publications

References 33 publications

A Limited Number of Globin Genes in Human DNA

A Limited Number of Globin Genes in Human DNA

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

Molecular weights of separated rabbit α‐ and β‐globin messenger RNAs

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