A Limited Number of Globin Genes in Human DNA

Abstract

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...

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

“…The RNA was trainslated in a wheat germ cell-free system, and its product was analyzed by cellulose acetate electrophoresis as described (18,19). Cellular DNA was extracted from Epstein-Barr-virus-transformed lymphocytic cell lines from normal subjects and from one of the homozygous Lepore patients (L.P.) as previously described (20).…”

“…The melting temperature of the mRNA:cDNA hybrids was obtained as previously described (25) with micrococcal nuclease to assay thermal stability of the hybrids. The hybridization of globin cDNA to cellular DNA was performed as previously described (20).…”

Quantitation of human gamma globin genes and gamma globin mRNA with purified gamma globin complementary DNA.

“…Comparable numbers of a-and /8-globin genes have been previously reported in /+-thalassemia DNA as in nonthalassemia DNA (3,4). In the studies reported here, both ,+-and g'-thalassemia DNA were used and shown to contain y-globin genes in amounts comparable to normal DNA.…”

“…Preparation of human DNA. DNA was prepared from human spleen and liver of patients with and without (8-thalassemia, as described previously (3,4). DNA from peripheral blood was prepared similarly, except that the blood sample containing white blood cells was washed three times with 10 vol of isotonic saline, frozen, and thawed before washing with 5% sucrose containing 1 mM MgCl2, 1 mM sodium phosphate (pH 6.5), 0.4% deoxycholate, and 0.8% Nonidet P40 (Shell International Ltd., London, England).…”

“…These values are consistent with genetic evidence for the presence of multiple y gene loci in human cells. The finding that the number of y-globin genes in (8-thalassemia DNA is similar to that in nonthalassemia DNA indicates that a deletion of y-globin genes cannot account for either INTRODUCTION The use of highly radioactive complementary DNA (cDNA)1 has recently permitted the measurement of the number of globin genes in a variety of mammalian species (1)(2)(3). In addition, purified a and (8 cDNAs from human cells have been used to quantitate the relative number of a-and (8-globin genes in human DNA (4,5).…”