Relative numbers of human globin genes assayed with purified alpha and beta complementary human DNA.

Abstract

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...

Defects in DNA and globin messenger RNA in homozygotes for hemoglobin Lepore.

“…The results also provide insights into the molecular defect in thalassemia at the gene level. We and others have previously shown that in p8-and 80-thalassemia, structural f-globin genes are present in normal amounts (5,10). It is postulated by us and others (32,33) that regulatory DNA sequences in the linked y-8-,3-gene sequences are responsible for the expression of these genes.…”

“…A role is postulated for the DNA sequences between the y-, 8-, and ,8-genes in limiting y-globin gene expression in the B3-thalassemias. We and others (6,7,11,12) have previously shown that extensive deletion of 8-globin genes, and probably 8-genes as well, in 8,8-thalassemia and hereditary persistence of fetal Hb is associated with greater y-globin gene expression, in contrast to ,0-and 8+-thalassemia in which no deletion of 8-and /8-structural genes is detectable (5,10). Similarly, in the Lepore homozygote the presence of a fusion-gene product including the N-terminal end of 8-globin suggests that DNA sequences between y-and 8-genes are intact and may account for the (36) and rabbit 8-globin genes (37); in the mouse it has been shown that these intervening sequences are transcribed into nuclear globin RNA precursors (38), although they are not present in mature mouse globin mRNA.…”

“…736 mal human cells and those of patients with different forms of thalassemia (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). We report here the analysis of Lepore-globin mRNA and genomic DNA in three patients homozygous for hemoglobin (Hb) Lepore.…”

Hemoglobin Indianapolis (beta 112[G14] arginine). An unstable beta-chain variant producing the phenotype of severe beta-thalassemia.

“…Most often, there are decreased amounts of the specific globin messenger RNA which serves as a template for the synthesis of the affected globin chain (4)(5)(6). The messenger RNA deficit results from either globin gene deletions (7)(8)(9)(10) or transcriptional or post-transcriptional impairment of the expression of globin genes (11)(12)(13). Analysis of the hemoglobins synthesized in patients with thalassemia has not shown abnormalities in the primary structure of the globin chains (1)(2)(3).…”

Trimodality in the proportion of hemoglobin G Philadelphia in heterozygotes: evidence for heterogeneity in the number of human alpha chain loci.

“…Implications for the a-thalassemia syndromes Recent studies (4,(23)(24)(25) indicate that a-thalassemia may be caused by at least partial deletion of the a-chain structural genes. The varying degrees of severity in the a-thalassemia syndromes are believed to reflect the number of a-chain genes deleted.…”