==================================BSR53================================== 53. I want the sequence for exon 2 in the alpha-2 coding region of human alpha globin. 1 UI - 87009038 AU - Shakin SH ; Liebhaber SA TI - Translational profiles of alpha 1-, alpha 2-, and beta-globin messenger ribonucleic acids in human reticulocytes. AB - In human reticulocytes, the critical balancing of alpha- and beta-globin synthesis may be controlled in part by differential translation of the three major adult globin messenger RNAs (mRNAs), alpha 1, alpha 2, and beta. In this study, we determined, as a parameter of translational efficiency, the relative ribosome loading of these three mRNAs. Using oligonucleotide probes specific for the alpha 1- and alpha 2-globin mRNAs, we find that these two mRNAs have identical translational profiles. Their distribution contrasts with that of beta-globin mRNA, which is present on heavier polyribosomes and is less prevalent in pre-80S messenger ribonucleoprotein fractions. The relative distribution of alpha- vs. beta-globin mRNA is consistent with more efficient beta-globin translation. In contrast, the parallel distributions of alpha 1- and alpha 2-globin mRNAs suggests they are translated with equal efficiencies. Considering the relative concentrations of the two alpha-globin mRNAs in normal reticulocytes, this result predicts a dominant role for the alpha 2-globin locus in human alpha-globin expression. MH - Base Sequence ; Gene Expression Regulation ; Globin/*GENETICS ; Human ; Polyribosomes/METABOLISM ; Reticulocytes/*METABOLISM ; RNA, Messenger/ *METABOLISM ; Support, U.S. Gov't, P.H.S. ; *Translation, Genetic SO - J Clin Invest 1986 Oct;78(4):1125-9 2 UI - 87004552 AU - Jarman AP ; Nicholls RD ; Weatherall DJ ; Clegg JB ; Higgs DR TI - Molecular characterisation of a hypervariable region downstream of the human alpha-globin gene cluster. AB - We have characterised an unusual, highly polymorphic region of DNA located 8-kb downstream of the human alpha-globin gene complex. This hypervariable region (alpha-globin 3' HVR) is composed of an array of 17-bp tandem repeats, the number of which differs considerably (70-450) from one allele to another. The sequence of the 17-bp repeats is highly conserved within and between alleles. Furthermore, this sequence identifies a core oligonucleotide [5'-GNGGGG(N)ACAG-3'] that is common to three previously characterised hypervariable regions. At reduced stringency, a probe to the 3' HVR detects a new family of multiallelic loci that will be of value in the study of human genetics. MH - Alleles ; Base Sequence ; Cloning, Molecular ; DNA Restriction Enzymes ; Evolution ; *Genes, Structural ; Globin/*GENETICS ; Human ; Nucleic Acid Hybridization ; Repetitive Sequences, Nucleic Acid ; Support, Non-U.S. Gov't ; *Variation (Genetics) SO - EMBO J 1986 Aug;5(8):1857-63 3 UI - 86289452 AU - Scharf SJ ; Horn GT ; Erlich HA TI - Direct cloning and sequence analysis of enzymatically amplified genomic sequences. AB - A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human beta-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ alpha locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5' ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA. MH - Antigens, Immune Response/*GENETICS ; Base Sequence ; Cloning, Molecular/ *METHODS ; Coliphages/*GENETICS ; DNA Polymerase I/METABOLISM ; Gene Amplification ; *Genetic Vectors ; Globin/*GENETICS ; Human ; In Vitro ; Polymorphism (Genetics) SO - Science 1986 Sep 5;233(4768):1076-8 4 UI - 86205234 AU - Morle F ; Starck J ; Godet J TI - Alpha-thalassemia due to the deletion of nucleotides -2 and -3 preceding the AUG initiation codon affects translation efficiency both in vitro and in vivo. AB - We previously hypothesized that a 2 nucleotide deletion, causing a A-greater than C change at position -3 preceding the ATG initiation codon of alpha globin gene, reduced translation efficiency of alpha globin mRNA and was responsible for a form of alpha + thalassemia displayed by an Algerian patient. We presently show that this deletion leads to a 30-45% reduction in translation efficiency of synthetic alpha globin mRNA in rabbit reticulocyte lysate. In other experiments, we constructed alpha/G gamma hybrid globin genes in which the 3' end of normal or mutated alpha globin genes downstream to the ATG initiation codon was substituted by the 3' part of a G gamma globin gene. COS cells transfected with either of these 2 hybrid genes were shown to synthesize a similar amount of alpha/G gamma hybrid mRNAs but 50% less G gamma globin when transfected with the alpha/G gamma hybrid gene carrying the deletion. These results definitively establish that the 2 nucleotide deletion reduces translation efficiency by 30-50%. This contrasts with the 93% reduction induced by a similar A-greater than C change at position -3 in the different nucleotide context preceding the ATG codon of the rat preproinsulin gene. MH - Base Sequence ; *Chromosome Deletion ; *Codon ; *Genes, Structural ; Globin/*GENETICS/ISOLATION & PURIFICATION ; Human ; Macromolecular Systems ; *Peptide Chain Initiation ; *RNA, Messenger ; Support, Non-U.S. Gov't ; Templates ; Thalassemia/*FAMILIAL & GENETIC ; Transcription, Genetic ; Translation, Genetic SO - Nucleic Acids Res 1986 Apr 25;14(8):3279-92 5 UI - 86148516 AU - Hardison RC ; Sawada I ; Cheng JF ; Shen CK ; Schmid CW TI - A previously undetected pseudogene in the human alpha globin gene cluster. AB - The sequence of the DNA between two pseudogenes in the human alpha-like globin gene cluster has been determined. Comparison of this sequence with sequences from other alpha-like globin gene clusters revealed another pseudogene, psi alpha 2, between the previously recognized pseudogenes zeta 1 and psi alpha 1. Therefore, the human alpha-like globin gene family is organized 5'-zeta 2-zeta 1-psi alpha 2-psi alpha 1-alpha 2-alpha 1-3'. The new pseudogene psi alpha 2 is very close to zeta 1, beginning only 65 base pairs 3' to the polyadenylation site of zeta 1. The first exon and the first intron of psi alpha 2 are interrupted by large inserts which are flanked by short (6 to 8 base pairs) direct repeats. The pseudogene psi alpha 2 lacks a promoter for transcription by RNA polymerase II, the first exon is highly divergent, one splice site is mutated, and five different frameshift mutations have occurred in the coding regions. Thus psi alpha 2 cannot encode a globin polypeptide. This pseudogene was not recognized in previous hybridization analyses of the human alpha-like globin gene cluster, and our discovery of it by sequence analysis suggests that divergent copies of a large number of genes may comprise a substantial fraction of the slowly renaturing DNA of mammalian genomes. MH - Base Sequence ; Comparative Study ; Genes ; Globin/*GENETICS ; Human ; Linkage (Genetics) ; Nucleic Acid Hybridization ; Sequence Homology, Nucleic Acid ; Support, U.S. Gov't, P.H.S. SO - Nucleic Acids Res 1986 Feb 25;14(4):1903-11 NP (Y)