Authentic beta-globin mRNA sequences in homozygous betaO-thalassemia.

Abstract

In a patient with homozygous PO-thalassemia in whom studies of reticulocyte hemoglobin synthesis showed no fl-globin chain synthesis in vivo and in vitro, molecular hybridization studies revealed RNA sequences complementary to -globin cDNA. The fact that these sequences were authentic P-globin mRNA was shown by fingerprint analysis of T1 ribonuclease-digested mRNA and by sequencing of oligonucleotides unique to P-globin mRNA. The Blood used as a nonthalassemic control was obtained from a patient with sickle cell anemia. Blood from a fetus that died from homozygous a-thalassemia was used as a source of ymRNA because it contained predominantly y-and a small amount of ,B-globin chain sequences but no a-globin sequences.Preparation of RNA and DNA. Peripheral blood was washed three times with isotonic saline and the erythrocytes were lysed with the Orskov reaction according to the method of Boyer et al. (16). With this technique, only erythrocytes were lysed and 98% of the leukocytes remained intact. The latter were saved for the DNA preparation as described (17). Ribonucleoproteins from the erythrocytes lysate were precipifated at pH 5, and the RNA was extracted with phenol, chloroform, and isoamyl alcohol (12). Approximately 15 A260 units of total RNA were obtained per 50 ml of blood. (One unit is the amount of material that when dissolved in 1 ml of solvent gives an absorbance of 1 with a path length of 1 cm.) The RNA was twice passed through oligo(dT)-cellulose columns (18) and the poly(A)-containing RNA eluted with low-salt buffer was separated by centrifugation on a 15-30% (wt/vol) sucrose density gradient. Fractions between the 8S and 12S positions were pooled.To prepare mRNA rich in fl-globin sequences, we separated the sickle cell mRNA by electrophoresis on 5% polyacrylamide gels in 98% formamide (19). The slowly migrating band in the 10S region containing more ,B than a sequences was isolated and the RNA was eluted from the gel, extracted with phenol and purified on oligo(dT)-cellulose (20).Hybridization Analysis. Radioactive DNAs complementary to a-and f,-globin mRNA were synthesized according to the methods of Verma et al. (21), as described (17). a-and fl-globin sequences in the mRNA were measured by hybridization with a-and f-globin cDNAs that were labeled with [a-32P]dCTP (specific activity 120 Ci-mmolh') and [3H]dCTP (specific activity 23 Ci-mmolh'), respectively. The ,B-cDNA was a tran-

Molecular basis for nondeletion alpha-thalassemia in American blacks. Alpha 2(116GAG----UAG).

“…This is contrary to previous studies in f3-thalassemia which indicate that nonsense mutations result in lowered levels of mRNA (48,49). Our measurements of a2:al mRNA ratios in the members of this kindred with normal a-globin gene maps (aa/aa) and single a-globin gene deletions (-a3.7/aa) gave results identical to those for individuals with the same genotype but lacking the nondeletion defect (42).…”

Molecular basis of the D variant phenotypes DNU and DII allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein

“…Total RNA was isolated from peripheral blood using the ammonium chloride-bicarbonate lysis method (Temple et al, 1977). RT-PCR was performed using cDNA templates synthesized as described (Sambrook et al, 1989) using oligo d(T)20 to prime synthesis.…”

beta zero thalassemia in Sardinia is caused by a nonsense mutation.

“…In some individuals, (-globin mRNA synthesis is absent; in others, a variable quantity is demonstrable in the reticulocyte (19). Previously we investigated the lesion in the mRNA from a Chinese patient affected by (30 thalassemia where (3-globin mRNA was detectable (20,21), and found the defect to be a nonsense mutation that caused premature termination of the ,8-globin chain (22). In this study we investigated thalassemia on the island of Sardinia (23), where (0 thalassemia, the predominant molecular form, is characterized by low levels of (8-globin mRNA.…”

“…Previous studies (23). We prepared poly (A+) RNA from 20 patients and cellular DNA from 10 patients, as previously described (21,24). RNA from 20 patients and DNA from five patients was quantitated by solution hybridization with cDNA enriched in a-and 18-globin sequences as previously described (20,24).…”