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Summary Three TaqI restriction fragment length polymorphisms (RFLP) detected by the central portion of von Willebrand factor cDNA, which recognizes the true gene and in addition pseudogenic sequences, were characterized and mapped. Small cDNA fragments which hybridized with DNA from families with von Willebrand disease were used. Two of the RFLP, recognized by 1.7 and 0.45 kb cDNA fragments, are not in linkage either with von Willebrand disease or with RFLP located in the von Willebrand factor (vWF) gene, which indicates their pseudogenic location. These markers located in 22q11, near to the bcr gene, provide new tools for the study of several somatic and constitutional alterations affecting this chromosomal region. The third RFLP is recognized by a cDNA fragment corresponding to the N-terminal portion of mature vWF and is localized in the true gene. Since significant linkage disequilibrium with other informative RFLP is not present, this marker contributes to the definition of family haplotypes associated with von Willebrand disease.

The synthesis of α and β-globin have been determined in normal and thalassemic erythroid cells, incubated at temperatures decreasing from 37 to 10°C. Synthesis of β-globin by thalassemic reticulocytes decreased with temperature clearly less than α and β-globin synthesis by normal erythroid cells, and also less than α-globin synthesis by thalassemic reticulocytes. Consequently the excess of α-globin synthesis, typical of β-thalassemia, disappeared when cells where incubated at temperatures of approximately 10°C. The normalization of β-globin synthesis, obtained in our experimental conditions, clearly suggests that the defect of β-globin synthesis, characteristic of β-thalassemia, does not result from a reduction in amount of β-globin messenger, but rather from a reduction of the rate of β-globin mBNA translation.

The intracellular behaviour of the excess newly synthesized α-globin has been studied by incubating peripheral blood and bone marrow samples from 9 homozygous β-thalassemic subjects with labeled amino acids. The globin chains present in the cytoplasm and in association with the stromal material have been separated by column chromatography, and the incorporation of radioactivity has been determined for each globin at various incubation times. The results obtained show that the excess of α-globin is removed from the cell cytoplasm and becomes associated with the stromal material.

The authors have investigated the possibility that the Ferrara type of β-thalassemia, in which β-globulin synthesis is absent, may be due to a nonsense mutation on the gene for β-globin. This hypothesis has been tested by trying to detect the presence of newly synthesized β-globin fragments in the reticulocytes of homozygous β-thalassemic subjects. The red blood cells were incubated with [3H]leucine. The whole hemolysate was hydrolyzed with trypsin and several β-chain peptides were isolated by column chromatography and assayed for radioactivity. No incorporation of radioactive leucine could be detected in the various β-globin peptides isolated and examined. The hypothesis that a nonsense mutation is responsible for the absence of β-globin synthesis in the Ferrara β-thalassemia is not supported by these findings.

The kinetics of incorporation of 3H-labeled amino acids into α, β, and γ globin has been determined, following incubation of intact reticulocytes, in stroma-free hemolysates and in purified Hb F and Hb A from homozygous β-thalassemic subjects. The labeled globins present in the cytoplasm as well as in purified Hb F and Hb A at various incubation times, were separated by CM-cellulose chromatography, and the ratios of [3H]α to [3H]counterpart globins were determined. The results obtained show that in the initial stages of incubation, the ratios of [3H]α to [3H]-counterpart globins are higher than 1 in the total lysate and in purified hemoglobins; these ratios, after long periods of incubation, decrease to values below 1. These findings suggest that the excess of newly synthesized α molecules flows very rapidly from the polyribosomes to the hemoglobins, and then, more slowly, to a “stromal”α pool. A model is presented, showing the most likely mechanism for the overall process.