S-fl-thalassemia had a ratio of (3 mRNA to ac mRNA of 0.75 while two subjects with homozygous 13-thalassemia had severe deficiencies of (3 mRNA. Conversely, a patient with a-thalassemia (Hb H disease) had a ratio of (3 mRNA to ax mRNA on reticulocyte polyribosomes of 6. These data provide further evidence of a quantitative deficiency of chain-specific globin mRNA in patients with the thalassemia syndromes.The thalassemia syndromes are an inherited group of hemolytic, hypochromic anemias in which the synthesis of a specific hemoglobin chain is impaired (1-3). Thus, in j#-thalassemia, which occurs in various ethnic groups (4), the synthesis of normal (3 chains is deficient relative to that of a chains. In contrast, a-thalassemia, one form of which is Hb H disease, is characterized by a deficiency of a-chain synthesis (5). Since genetic analyses have placed the f3-thalassemia locus or loci close to the fl-chain structural locus (6-8), recent work on the underlying defect in the thalassemias has focused on the globin mRNAs.In the (3-and a-thalassemias, assays of globin mRNA in cell-free translating systems have demonstrated a relative deficiency in the activity of (3 mRNA and a mRNA, respectively, commensurate with the deficiency in globin-chain synthesis in these disorders (9-12). RNA-DNA hybridization studies have suggested that the mRNA defect in these conditions is a quantitative one (13,14), and that in #°-thalassemia, characterized by an absence of fl-chain synthesis, the de-
METHODSThe methods used for reticulocyte lysis and polyribosome isolation have been described (19,20). In some experiments polyribosomes were treated with EDTA, and the RNA was fractionated on a 10-30% linear sucrose gradient (21). The 14S region containing 10S RNA was collected; the RNA was extracted with phenol and precipitated with ethanol (21).This RNA was then subjected to oligo(dT)-cellulose chromatography to isolate globin mRNA (22,23). In other experiments, the polyribosomes were directly extracted two to five times with phenol-choloroform-isoamyl alcohol (50: 50:1) (23). TKhe RNA was 'precipitated in ethanol and was applied to an oligo(dT)-cellulose column. If the quantity of RNA was greater than 2 mg, the RNA eluted in 0.5 M KCI was reapplied to the column, the two poly(A)-rich RNA fractions were then pooled, and these fractions were then chromatographed a third time. RNA (4-8 /Ag) was subjected to formamide electrophoresis on 7.5% polyacrylamide gels as described (24, 16, 17); the gels were stained with and scanned at 600 nm. The quantity of an RNA was estimated from the area under the recorded curve. After electrophoresis, the two 10S RNA bands were observed in similar proportions no matter which isolation procedure was used, but an improved yield of mRNA was obtained by omitting the sucrose gradient step.
567
