Summary
We have used the powerful methodology of DNA enzymatic amplification in order to assign human α-globin structural mutants to one of the two highly homologous α-globin genes. Selectively amplified α1 and α2-globin cDNAs were dot-blotted and further hybridized to synthetic oligonucleotides encompassing either the normal or the mutated sequences. The generated signals corresponded specifically to one of the two α-globin genes. Using this approach the α-globin structural mutants J-Buda and G-Pest were found to be encoded by the α2 and the α1-globin genes, respectively. Furthermore, the exact nucleotide changes were determined. We propose this technique to serve as a simple and definitive method for assigning α-globin structural mutants.
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References
Baine RM, Rucknagel DL, Dublin PA Jr, Adams JG III (1976) Trimodality in the proportion of hemoglobin G-Philadelphia in heterozygotes: evidence for heterogeneity in the number of human α-chain loci. Proc Natl Acad Sci USA 73:3633–3636
Beltz GA, Jacobs KA, Eickbush TH, Cherbas PT, Kafatos FC (1983) Isolation of multigene families and determination of homologies by filter hybridization methods. Methods Enzymol 100:266–285
Brimhall B, Duerst M, Hollan SR, Stenzel P, Szelenyi J, Jones RT (1974) Structural characterization of hemoglobins J-Buda [α61 (E10) Lys→Asn] and G-Pest [α74 (EF3) Asp→Asn]. Bichim Biophys Acta 336:344–360
Brown NA, Kafatos FC (1988) Functional cDNA libraries from Drosophila embryos. J Mol Biol 203:425–432
Budowle B, Eberhardt P (1986) Ultra thin-layer polyacrylamide gel isoelectric focusing for the identification of hemoglobin variants. Hemoglobin 10:161–172
Chehab FF, Doherty M, Cai S, Kan YW, Cooper S, Rubin EM (1987) Detection of sickle cell anaemia and thalassaemias. Nature 329:293–295
Deisseroth A, Nienhuis A, Turner P, Velez R, Anderson WF, Ruuddle F, Lawrence J, Creagan R, Kucherlapati R (1976) Localization of the human α-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay. Cell 12:205–218
Embury SH, Scharf SJ, Saiki RK, Gholson MA, Golbus M, Arnheim N, Erlich HA (1987) Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis. N Engl J Med 316:656–660
Foldi J, Cohen-Solal M, Valentin C, Blouquit N, Hollan S, Rosa J (1980) The human α-globin gene. The protein products of the duplicated genes are identical. Eur J Biochem 109:463–470
Hollan SR, Szelenyi JG, Brimhall B, Duerst M, Jones RT, Koler RD, Stocklen Z (1972) Multiple alpha chain loci for human haemoglobins: Hb J-Buda and Hb G-Pest. Nature 235:47–50
International Hemoglobin Information Center (1986) Variants of the α-chain. Hemoglobin 10:261–327
Kropp GL, Fucharoen S, Embury S (1989) Selective enzymatic amplification of α2-globin DNA for detection of the hemoglobin Constant Spring mutation. Blood 73:1987–1992
Lauer J, Shen C-KJ, Maniatis T (1980) The chromosomal arrangement of human α-like globin genes: sequence homology and α-globin gene deletions. Cell 20:119–130
Liddell J, Brown D, Beale D, Lehmann H, Huntsman RG (1964) A new hemoglobin-Ja Oxford found during a survey of an English population. Nature 204:269–270
Liebhaber SA, Cash FE (1985) Locus assignment of α-globin structural mutations by hybrid-selected translation. J Clin Invest 75:64–70
Liebhaber SA, Goossens M, Kan YM (1981) Homology and concerted evolution at the α1 and α2 loci of human α-globin. Nature 290:26–29
Liebhaber SA, Rappaport EF, Cash FE, Ballas SK, Schwartz E, Surrey S (1984) Hemoglobin I mutation encoded at both α-globin loci on the same chromosome. Science 226:1449–1451
Liebhaber SA, Cash EF, Main DM (1985) Compensatory increase in α1-globin gene expression in individuals heterozygous for the α-thalassemia-2 deletion. J Clin Invest 76:1057–1064
Liebhaber SA, Cash FE, Ballas SK (1986) Human α-globin gene expression. The dominant role of the α2-locus in mRNA and protein synthesis. J Biol Chem 261:15327–15333
Lodish H, Desalu O (1973) Regulation of synthesis of non-globin proteins in cell-free extracts of rabbit reticulocytes. J Biol Chem 248:3520–3527
Maniatis T, Fritsch EF, Sambrook J (eds) (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Marks J, Shaw J-P, Shen C-KJ (1986) Sequence organization and genomic complexity of primate θ1 globin gene, a novel α-globin-like gene. Nature 321:785–788
Michelson AM, Orkin SH (1983) Boundaries of gene conversion within the duplicated human α-globin genes. J Biol Chem 258:15245–15254
Old JM, Clegg JB, Weatherall DJ, Booth PB (1978) Hemoglobin J Tongariki is associated with α-thalassemia. Nature 273:319–320
Proudfoot NJ, Gill A, Maniatis T (1982) The structure of the human zeta-globin gene and a closely linked, nearly identical pseudogene. Cell 31:553–563
Rovera G, Magarian C, Borun TW (1978) Resolution of hemoglobin subunits by electrophoresis in acid-urea polyacrylamide gels containing Triton X-100. Anal Biochem 85:506–518
Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HAA (1986) Analysis of enzymatically amplified β-globin and HLA-DQα DNA with allele-specific oligonucleotide probes. Nature 324:163–166
Saiki RK, Chang C-A, Levenson CH, Warren TC, Boehm CD, Kazazian HH, Erlich HA (1988) Diagnosis of sickle cell anemia and β-thalassemia with enzymatically amplified DNA and non-radioactive allele-specific oligonucleotide probes. N Engl J Med 319:537–541
Sancar GB, Tatis B, Cedeno M, Reider RF (1980) Proportion of hemoglobin G-Philadelphia (α263Asn-Lys β2) in heterozygotes is determined by α-globin gene deletions. Proc Natl Acad Sci USA 77:6874–6878
Szelenyi JG, Horanyi M, Foldi J, Hollan SR (1983) The unequal expression of human α-globin genes at the protein level. Biomed Biochim Acta 42:187–191
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Mamalaki, A., Horanyi, M., Szelenyi, J. et al. Locus assignment of human α-globin structural mutants by selective enzymatic amplification of α1 and α2-globin cDNAs. Hum Genet 85, 509–512 (1990). https://doi.org/10.1007/BF00194226
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DOI: https://doi.org/10.1007/BF00194226