The nucleotide sequences of the untranslated 5' regions of human alpha- and beta-globin mRNAs.

Abstract

The complete sequences of the untranslated 5' regions of human a-and ftlobin mRNAs were determined by sequence analysis of full-length cDNAs. The single-stranded cDNAs were digested with the restriction endonuclease Hae Ill, and the two 3'-terminal fragments of 75 (5)(6)(7)(8)(9)(10)(11)(12). In the human, the ,Bglobin mRNA from the initiation codon AUG to the 3'-poly(A) end has been sequenced, and portions of the translated region of the a-globin mRNA and the complete untranslated 3' end are known (10, 13-16). The primary structure of the untranslated 5' region of mRNA is of great interest because it may play a role in ribosomal binding and the initiation of protein synthesis. Lockard and RajBhandary (17) determined the sequence of the 5' termini of the separated rabbit a-and ,B-globin mRNAs by direct RNA sequence analysis. By analyzing the cDNAs synthesized with a primer complementary to the region of the initiation codon, Baralle (11,12) sequenced the complete untranslated 5' regions of both rabbit a-and ,B-globin mRNAs.We report here the complete sequences of the untranslated 5' region of the human a-and,B-globin mRNAs. We utilized the ability of the restriction enzyme Hae III to cleave singlestranded DNA at G-G-C-C sequences (18,19). Full-length single-stranded a-and ,B-globin cDNAs were digested with Hae III and the two 3'-terminal fragments corresponding to the 5' termini of the a-and f3-mRNAs were identified and isolated.The sequences of these two fragments were then determined according to the method of Maxam and Gilbert (4) by labeling either at their 3' ends with ribonucleoside [32P]diphosphate with terminal deoxyribonucleotidyl transferase, or at their 5' ends with 32P by using polynucleotide kinase.MATERIALS AND METHODS Source of Human Globin mRNA. Blood was obtained from a patient with sickle cell disease, one with hemoglobin H disease, and one with homozygous 3 thalassemia. RNA was prepared from these samples and the poly(A)-rich RNA was isolated by two passages over oligo(dT)-cellulose as described (20).Preparation of Globin cDNAs. Single-stranded globin cDNAs were prepared according to the method of Verma et al. (21). Nonradioactive dATP, dGTP, and TTP at 400,M each and radioactive dCTP at 100 MM were used to maximize the synthesis of full-length cDNAs as shown by Efstratiadis et al. (22). The radioactive precursors used for the synthesis of internally labeled cDNA in the different experiments were [3H]dCTP (New England Nuclear, 23 Ci/mmol) diluted to a specific activity of 2.3 Ci/mmol and [a-32P]dCTP (New England Nuclear, 110-115 Ci/mmol) diluted to a specific activity of 25 or 0.1 Ci/mmol to yield high-or low-specific activity 32p products, respectively.Digestion of cDNA with Restriction Enzyme. cDNA was digested with the restriction endonuclease Hae III (New England Biolabs), 200 units/Mg of DNA, in 6 mM Tris-HCI, pH 7.4/6 mM MgCl2/6 mM NaCl/6 mM 2-mercaptoethanol for 16 hr at 37°. To analyze the restriction fragments, digested high-specific activity [32P]cDNA was separated by ...

A new method for the size estimation of the RNA genome segments of influenza virus

“…Chang et al (29) obtained high yields of full length cDNA from human r-globin mRNA, using an oligo dT primer with reverse transcriptase.…”

Hemoglobin Long Island is caused by a single mutation (adenine to cytosine) resulting in a failure to cleave amino-terminal methionine.

“…By using an 8% gel we were also able to define the sequence of 56 bases upstream from the adenine-to-cytosine transversion. In both the control and the propositus' reticulocyte RNA these sequences were normal (21,22). These results indicate that two separate species of 8-globin mRNA are present in the Patient A C G T propositus heterozygous for Hb Long Island, one with CAC coding for histidine in the second B-globin codon position and the other with a CCC codon corresponding to proline.…”

“…The analysis of the mRNA sequence of the /3-globin mutant, Hb Long Island, confirmed the histidineto-proline substitution (1) resulting from an adenine-tocytosine transversion. The RNA sequence immediately adjacent to the AUG initiation codon was normal, as was the more upstream region of the untranslated portion of 83-globin mRNA (21). Although the mutant protein consisted of two separate amino acid abnormalities (1), no other nucleic acid mutation was found.…”

Insertion of synthetic copies of human globin genes into bacterial plasmids

“…The globin genes are one of the best characterized eucaryotic gene systems available to study problems of gene regulation and the process of differentiation. Extensive structural information is already available for the human a globin chain mRNA and partial information is available for the human a chain mRNA (2)(3)(4)(5)(6). There are limitations in perforrfing extensive structural analyses of isolated mRNAs both because of possible heterogeneity (polymorphism) between multiple gene loci for the same globin chain (7), and because of the difficulty in obtaining large quantities of pure mRNA.…”

“…Digestions with Eco RI were carried out at 37°C in 100 mM Tris-HCl, pH 7.5, 5 mM MgC12, and 50 mM NaCl. Digestions with MboII were performed at 37°C in 6 mM Tris-HCl, pH 7.5, 6 mM MgC12, 6 mM NaCl, and 6 mM a mercaptoethanol. The number of enzyme units used and the time of digestion varied with different batches of enzyme.…”