Nucleotide sequence of the coding portion of human alpha globin messenger RNA.

J.T Wilson,L.B Wilson,V.B Reddy,C Cavallesco,P.K Ghosh,J.K Deriel,B.G Forget,S.M Weissman

doi:10.1016/s0021-9258(19)85810-6

Abstract

The nucleotide sequence of the coding portion of human alpha globin mRNA has been determined by sequence analysis using human alpha globin cDNA cloned in bacterial plasmids. The sequence was obtained by a combination of direct sequence analysis of the cloned cDNA and analysis of cDNA obtained by primer extension, using short restriction endonuclease fragments of cloned alpha cDNA that were hybridized to human globin mRNA and elongated on the mRNA template by viral reverse transcriptase. The human alpha globin mRNA has an unexpectedly high G + C base composition (64.7%), similar to that observed for rabbit globin alpha mRNA, and displays a striking bias in the use of synonym codons for various amino acids. The bias in codon usage of human alpha globin mRNA is similar, with some exceptions, to that previously observed for rabbit alpha globin mRNA as well as for human and rabbit beta globin mRNAs. A detailed restriction endonuclease map of the human alpha globin cDNA is presented.

Highlights

The nucleotide sequence of the coding portion of homologies [13]
The sequence was obtained bya and p globin mRNAs.Such a comparison is of particular combination of direct sequence analysis of the cloned interest since these mRNAs arseimilar inlength andfunction, cDNA and analysis of cDNAobtained by primer exten- are expressed in the same highly specialized cell at similar sion, using short restriction endonuclease fragments of stages of differentiation, yet derive from separate genes that cloned a cDNA that were hybridized to human globin are situated ondifferent chromosomes andmay have evolved mRNA and elongated on the mRNA template by viral somewhat independently
The end-labeled fragments were recut with a second restriction endonuclease, and the resulting products were separated by slab gel electrophoresis, identified by autoradiography, excised, eluted, and sequenced by the procedure of Maxam and Gilbert [21] or the DNA was subjected to partial digestion with snake venom phosphodiesterase followed by two-dimensional fractionation of the resulting oligonucleotides as previously described [1, 2]

Summary

76 VAL A S P ASP YET P R G

GAAGCCCACCTGG (GC)" GGTGCCGTTC GTTCTTCCACCGG TCCCCGCCGAGT 'Terminal oligonucleotidedetermined to be G andC. The suspension was repeatedly centrifuged to remove acrylamide, and the DNA was precipitated from the supernatant solution by the addition of 0.1 volume of3.0 M sodium acetate, pH 7.5, and 2 volumes of ethanol at -70". The end-labeled fragments were recut with a second restriction endonuclease, and the resulting products were separated by slab gel electrophoresis, identified by autoradiography, excised, eluted, and sequenced by the procedure of Maxam and Gilbert [21] or the DNA was subjected to partial digestion with snake venom phosphodiesterase followed by two-dimensional fractionation of the resulting oligonucleotides as previously described [1, 2]. The fractionated bands of DNA were visualized by autoradiography, excised, eluted,and precipitated with ethanol, and their sequence was determined by the chemical degradation procedure of Maxam and Gilbert [21]

RESULTS

DISCUSSION

I 4 4 3 m 3 3 7 I38 u