In patients with betao thalassaemia from Ferrara, beta globin mRNA sequences are either absent or structurally abnormal while in betao thalassaemia in Catania, beta globin mRNA sequences are present. In deltabeta thalassaemia there is a deletion of beta-like globin genes, while in betao Catania DNA, no beta globin gene deletion is detectable.
IPurified a and 63 globin complementary DNAs (cDNAs) have been separated from total radioactively labeled human globin cDNA using mRNA purified' from liver of a hydrops fetalis (a thalassemia). The P cDNA hybridizes to the hydrops fetalis mRNA while the a eDNA remains single-stranded. The purified a and iB cDNAs were assayed for their purity by their hybridization to mRNA prepared from reticulocytes of nonthalas.semia, a thalassemia, and I thalassemia subjects. The results indicate that the separated cDNAs are selective in hybridization to a or j3 globin mRNAs, respectively. The previously reported deficiency of globin mRNA in thalassemia cells has been confirmed with these purified cDNAs.The purified a and # cDNAs were' hybridized to cellular DNA to determine the relative number of a-and i3-like genes in non-thalassemia, B+ thalassemia, and hydrops fetalis (a thalassemia) DNA. The a cDNA hybridized to hydrops fetalis liver DNA to a much lower extent than B cDNA, confirming the previously reported deletion of a globin genes in hydrops fetalis. By contrast, both the a and j5 eDNA probes hybridized to the same extent to spleen DNA from non-thalassemia and from ,B + thalassemia patients. Between two and five globin genes in non-thalassemia and .B + thalassemia DNA hybridize to is cDNA and one to five to a cDNA. These studies indicate that in s+ thalassemia, there is no detectable deletion in g3 globin genes. The genetic defect in , + thalassemia appears to be due to either repression of transcription of j3 globin genes or abnormal processing of B globin mRNA.Using radioactively labeled complementary DNA (cDNA) as a probe, we have recently reported that there are less than 10 copies of globin genes present per haploid genome in human DNA (1). In these studies, #+ thalassemia spleen DNA was found to have a complement of globin genes similar to that of non-thalassemia DNA when cDNA containing both a and , sequences were used. We report here the measurement of the relative numbers of a-and ,8-like globin genes in human DNA using purified human a and gB cDNA. Hydrops fetalis (a thalassemia) mRNA which contains no a mRNA has been used to separate a and j3 cDNA from total normal human cDNA (2). Hybridization of total globin cDNA (a plus P cDNA) with hydrops fetalis mRNA leads to # cDNA ,3 mRNA hybrids, whereas a cDNA remains single stranded. The single stranded and double stranded species were separated using hydroxylapatite chromatography (1), and g3 mRNA removed from j3 mRNA -, cDNA hybrids by alkaline hydrolysis. The specificity of each of the purified a and 8 cDNAs has been determined by hybridization to globin mRNA from normal, #+ thalassemia, and a thalassemia cells.Previous studies using cell-free systems (3-7) and hybridization to cDNA (8, 9) have shown that mRNA from a thalassemia cells contains decreased a mRNA, and mRNA from 16 thalassemia cells decreased amounts of # mRNA. In the present experiments, hybridization of the purified a and P cDNA probes to 5 thalassemia mRNA shows a 10-fold excess of a co...
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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