Sabra C. Goff

Globin Chain Electrophoresis: a New Approach to the Determination of the Gγ/Aγ Ratio in Fetal Haemoglobin and to Studies of Globin Synthesis

Summary. Separation of globin chains by electrophoresis provides a simple and rapid method for the determination of the Gγ/Aγ ratio in human fetal haemoglobin, and of biosynthetic rates of the globin chains. Whole haemolysates were analysed by electrophoresis on polyacrylamide gels in urea, acetic acid and Triton X‐100. Electrophoresis of haemolysates from newborn infants led to four bands: Aγ, Gγ, β and α. The identity of these bands was indicated by examination of haemoglobins of known globin chain composition. In 15 samples, the %Gγ was similar by Triton gels and by amino acid analysis of the γCB‐3 peptide. Some mutant globin chains were also separable with the electrophoretic technique. Triton gel electrophoresis provides rapid analysis of very small amounts of haemoglobin, and permits examination of globin chain composition as well as globin synthetic ratios.

Molecular characterization of seven beta-thalassemia mutations in Asian Indians.

To characterize systematically the mutations which produce beta‐thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta‐globin gene cluster of 44 beta‐thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta‐gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5′ to the beta‐globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5′ to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5′ and 3′ to it, suggesting that it lies within a region of relative sequence randomization.

The duplicated human α-globin genes: Their relative expression as measured by RNA analysis

Abstract The human α-globin genes are duplicated and encode identical polypeptides. Recently we detected in cloned genomic DNAs characteristic sequence differences between the 3′ untranslated regions of the 5′ (α2) and 3′ (α1) genes, not previously recognized by direct analysis of mRNA and cDNA transcripts. Based on these untranslated region differences, we have now used S1 nuclease mapping of RNA to detect and quantitate the two predicted α-mRNA species. With this assay we have examined the relative expression of the α-globin genes during normal development and in α-thalassemia syndromes. In normal adult reticulocytes, α2 RNA is slightly more abundant than the α1 species (ratio 60:40). This relative abundance of the a RNAs was consistently observed in fetal blood and liver RNA samples from 10 weeks of gestation to birth. In both deletion and nondeletion forms of α thalassemia, only the α1 RNA species was present in erythroid RNA. These studies resolve prior conflicts regarding the heterogeneity of α RNA and establish the normal pattern of relative α-gene expression during development independent of protein variants. RNA analysis also permits for the first time identification of the mutant genes in nondeletion forms of a thalassemia.

Heterogeneity of DNA deletion in gamma delta beta-thalassemia.

By restriction endonuclease mapping, gene cloning, and DNA sequencing we have determined the region of DNA that is deleted in a family with gamma delta beta-thalassemia. The deletion removes the linked epsilon, gamma-, and delta-globin structural genes and terminates within the coding portion of the beta-globin gene. Since the extent of DNA deletion in this family differs from that reported in another family, we conclude that gamma delta beta-thalassemia is heterogeneous at the molecular level.

Electrophoretic separation of human embryonic globin demonstrates "α-thalassemia" in human leukemia cell line K562

Abstract Human embryonic, fetal, and adult globin chains ( ζ, e, A γ, G γ, β, α ) can be separated by electrophoresis on Triton Acid urea gels. K562, a human leukemia cell line, was induced with hemin, labelled with [ 3 H]-leucine, and globin synthesis analyzed. All globins except β were produced. e > ζ; G γ: A γ=70:30; non-α:α=>2:1 . Thus, hemin-induced K562 synthesized embryonic and fetal globin chains, and had globin synthetic imbalance, with “α-thalassemia.”

Transient expression of human adenosine deaminase cDNAs: identification of a nonfunctional clone resulting from a single amino acid substitution.

Human adenosine deaminase (ADA) is an important purine catabolic enzyme which irreversibly deaminates adenosine and deoxyadenosine. Severe genetic deficiency of ADA leads to an immunological deficiency state in which T-lymphoid cells are selectively destroyed by the accumulation of toxic levels of deoxyadenosine and deoxy-ATP. In preparation for transfer of ADA sequences into a variety of cell types, we explored expression of ADA cDNAs transfected into cultured cells within a simian virus 40-based expression vector. After transfection into monkey kidney (COS) cells, ADA cDNA encompassing the entire coding region of the protein generated human ADA activity. An unexpected finding, however, was the identification of a cDNA clone that failed to produce either human enzyme activity or immunoreactive ADA protein. As this pattern is typical of many naturally occurring mutant ADA alleles, we characterized the molecular defect in this clone. DNA sequence analysis revealed a single nucleotide substitution in amino acid position 50 (glycine-valine). Northern blotting with a unique 17-mer oligonucleotide demonstrated the absence of the mutant sequence in the mRNA from which the cDNA library giving rise to the mutant cDNA was constructed. Therefore, the substitution in the variant cDNA was created during cloning. These data define one critical region of the human ADA protein molecule and suggest a convenient strategy for characterization of the phenotypes associated with naturally occurring mutant alleles.

Chemical Evidence for the Separation of Gy and AY Globin Chains by Polyacrylamide Gel Electrophoresis in Urea and Triton X-100

Abstract Polyacrylamide gel electrophoresis in urea and Triton X-100 of a hemolysate from human fetal red blood cells produces four major protein bands: α, β, and 2 γ globin chains. We have verified that the latter two are the Gγ and Aγ globin chains which have respectively glycine or alanine at position 136. After incorporation of either [3H] alanine or [3H] glycine into newly synthesized globin each y chain was isolated by preparative electrophoresis. The chains were cleaved with cyanogen bromide at methionines 55 and 133, then subjected to automated sequencing, and the residues from each sequencer turn counted. Glycine incorporation was detected for the third turn (position 136) of the Gγ chain and alanine for the Aγ Substantial metabolic conversion of [3H] glycine to serine and proline was also noted.

748 RELATIVE EXPRESSION OF THE α-GLOBIN GENES IN MAN DURING DEVELOPMENT

The structural genes for α-globin in man are duplicated. They encode identical polypeptides, but differ in sequence in the 3′-untranslated region of the gene. Because this segment is found in all mature mRNAs, detection of this sequence difference in mRNA would provide a means of assessing relative expression of the two α-genes independent of identifiable protein variants. We have devised a sensitive, quantitative assay for the two predicted α-mRNA species based on this principle. Using this approach we have investigated whether the relative expression of the α-genes changes during the transition from fetal to adult erythropoiesis in utero. Normally the mRNA derived from the more 5′ gene (α2) predominates slightly relative to that from the more 3′ (α1) gene: ratio 60/40. In fetal blood of 17-18 weeks gestation and newborn blood samples this ratio is preserved. During hepatic hematopoiesis (10-16 weeks gestation) the α-mRNAs are present in this proportion as well. Therefore, during the transition from γ to β-globin expression, the relative expression of the α-genes is unaltered. Furthermore, the relative expression of the α-genes is similar in hepatic and bone marrow phases of erythropoiesis.