DNA sequences regulating human beta globin gene expression.

Human delta globin is expressed at approximately 1-2% of the level of human beta globin in erythroid cells despite the marked homology between these two globins. To determine the DNA sequences responsible for this effect, delta and beta globin genes and fusion products of these genes constructed in vitro were transfected and expressed in HeLa cells. The results indicate that when the small intervening sequence of the beta gene (beta IVS 1) is replaced by delta IVS 1, expression of the chimeric gene is the same as that of the normal beta globin gene. By contrast, when the large intervening sequence of the beta gene (beta IVS 2) is replaced by delta IVS 2, expression of the chimeric gene is markedly reduced. These results suggest that there are signals within IVS 2 of the delta and beta genes which affect their relative expression.     

Cloning of two adult hamster globin cDNAs (alpha and beta major).

A cDNA library was prepared from poly(A) mRNA extracted from adult anemic hamster spleen erythroid cells. cDNA clones containing inserts coding for adult alpha and beta major globin chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNA and (b) nucleotide sequence analysis of the inserts and comparison to the adult globin cDNAs of mouse, rabbit and human. Availability of sequences for embryonic (Li et al. (1992) Biochim. Biophys. Acta 1130, 218-220) and adult globin cDNAs (this report) will aid in investigations of the molecular mechanisms involved in the globin ontogeny of hamsters.     

Nucleotide sequences of the mouse globin beta gene cDNAs in a wild derived new haplotype Hbb w1

Haplotypes of the beta-globin gene complex (Hbb) in laboratory mice have been defined as d, p, and s. We previously found a new haplotype w1 in wild mice collected from northwestern China. This study analyzed the nucleotide sequences of b1 and b2-globin gene cDNAs of both the p and w1 haplotypes, in comparison with those of the d haplotype. In Hbb-b1 cDNA, six base substitutions were found between the d and w1 haplotypes and also between p and w1, but none existed between d and p. In Hbb-b2 cDNA, three base substitutions were found between the d and w1 haplotypes and also between d and p, but none between p and w1. This result indicated that the Hbb gene complex of the p haplotype carries b1 ^d and b2 ^w1 genes and is probably a recombinant between d and w1 haplotypes. The hemoglobin containing the W1 phenotype showed oxygen-binding properties identical with those of the hemoglobins containing D and P phenotypes. Received: 5 January 1999 / Accepted: 5 April 1999     

Interaction between the synthesis of alpha and beta globin.

We have examined the relationship between alpha and beta globin chain syntheses by utilizing the distribution of isoleucyl residues in rabbit hemoglobin. The alpha globin chain contains three isoleucyl residues while the beta chain of certain rabbits contains no isoleucine. O-Methyl-L-threonine, an isoleucine isostere, inhibits incorporation of radiolabeled amino acids into alpha chains in rabbit reticulocytes. When alpha chain synthesis is inhibited by 50-85%, beta synthesis is stimulated by 15-50%. The excess labeled beta chains are not distinguishable from authentic beta chains by any of the following criteria: (a) carboxymethyl cellulose chromatography in sodium phosphate-urea buffers, (b) electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate, and (c) electrophoresis of methionine-containing tryptic peptides. The stimulation of beta synthesis continues after the pool of excess alpha chains has been exhausted by preincubation with O-methyl-L-threonine. The stimulation does not occur, however, when 1 mM 2-mercaptoethanol is added to the incubation medium or when the cells are excessively diluted in the incubation mixture. The rates of beta chain initiation and elongation during stimulation have been compared to the rates during normal synthesis. Although both rates are increased, the rate of elongation increases more than initiation, suggesting that initiation is the rate-limiting step in increased beta chain production. The stimulation of beta synthesis when alpha synthesis is inhibited is interpreted as resulting from relief of competition between alpha and beta mRNAs for limiting components of the protein synthetic apparatus.     

Electron microscope analysis of mouse and rabbit globin and immunoglobulin gene sequences

Mouse and rabbit globin and immunoglobulin gene sequences, which had been synthesized in vitro from eukaryotic mRNAs and inserted into plasmids, have been examined in the electron microscope. The size of the inserted β rabbit and α and β mouse globin DNA sequences has been estimated as 620 base pairs while the size of the inserted α rabbit globin DNA sequences was found to be about 490 base pairs. Heteroduplex analysis has revealed no structural abnormalities at the insertion sites of the chimeric plasmids except in the case of a plasmid containing an immunoglobulin light chain gene sequence of about 830 bases, in which a 3 kb deletion adjacent to the insertion site was observed.     

Cloning of chicken lysozyme structural gene sequences synthesized in vitro.

Double-stranded chicken lysozyme cDNA was synthesized from an oviduct mRNA fraction enriched for lysozyme mRNA. The ds-cDNA was inserted into the BamHI site of plasmid pBR322 using chemically synthesized DNA linker molecules containing the BamHI restriction endonuclease cleavage site. After bacterial transformation, colonies carrying lysozyme DNA were identified by hybridization with highly purified lysozyme cDNA. The 555 base pairs long cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The DNA sequence of selected parts of the inserted DNA is as predicted from the amino acid sequence of prelysozyme. The sequence data allows the unambiguous location of the coding region within lysozyme mRNA.     

高原世居藏族α、β珠蛋白编码基因的克隆与测序

目的:通过对高原世居藏族α、β珠蛋白编码基因的分析,探讨藏族Hb高氧亲合力的分子机制.方法:高原现场采集健康成年男性藏族人骨髓样品,提取总RNA,通过逆转录聚合酶链反应(RT-PCR)获得人α和β珠蛋白的cDNA,与PGEM-T Easy质粒连接后,将α和β珠蛋白的cDNA转化JM109大肠杆菌中扩增培养,经酶切鉴定后测序,结果与NCBI数据库进行同源性比较.结果:藏族人α珠蛋白的cDNA与NCBI数据库登录的人cDNA序列相同,没有突变位点.一例藏族人β珠蛋白143位密码子发生了氧亲和力增高的碱基突变(CAC-＞CGC),其对应的氨基酸由His变为Arg(即Hb Abruzzo).结论:藏族人高氧亲和力变种的发现,为今后高原低氧适应相关基因的研究提供了线索.     

Cloning of soybean leghemoglobin structural gene sequences synthesized in vitro.

Double-stranded soybean leghemoglobin DNA was synthesized from leghemoglobin mRNA isolated from soybean nodules. The dsDNA was inserted into the Bam H1 site of plasmid pBR322 using the poly-dAT-joiner method. A cloned DNA fragment of one recombinant plasmid was isolated and characterized by restriction endonuclease digestion. The restriction cleavage map and the DNA sequence of a selected part of the inserted DNA are in complete accordance with the amino-acid sequence of soybean leghemoglobin.     

Cloning and amplification of rabbit alpha- and beta-globin gene sequences into Escherichia coli plasmids.

cDNA, synthesized from rabbit globin mRNA, was used in a self-priming reaction, with avian myeloblastosis virus DNA polymerase, for the synthesis of double-stranded DNA. Globin DNA ranging from about 400 to 650 base pairs was elongated with dG tails using deoxypolynucleotide transferase and was annealed to linear Escherichia coli plasmic pCR1, elongated with dC tails. Preparation of the plasmid DNA involved an enzymatic reconstruction of one EcoRI-specific site on each side of the molecule. After transformation of E. coli cells to kanamycin resistance with the hybrid molecules, bacterial clones harboring recombinant plasmids were studied for the presence of globin-specific DNA. Plasmids containing either alpha or beta rabbit globin gene sequences were obtained. There was a 4-fold excess of recombinant plasmids containing beta-globin sequences over those with alpha-globin DNA. The longest beta-globin sequences found in plasmids were about 550 to 600 pairs long, and correspond therefore to the entire beta-globin structural gene and to some of the untranslated regions. The alpha-globin sequences were 400 to 450 base pairs long. Treatment of clone pCR1betarG 19 with EcoRI endonuclease released two DNA fragments (410 and 210 base pairs) resulting from cleavage at two reconstructed external EcoRI sites and at one internal EcoRI site within the rabbit globin gene. The same treatment of pCR1alpharG 11 released one fragment. In most other recombinant plasmids studied however, no fragment was released by EcoRI digestion.     

Gene amplification in methotrexate-resistant mouse cells. II. Rearrangement and amplification of non-dihydrofolate reductase gene sequences accompany chromosomal changes

Total amplified DNA in methotrexate-resistant mouse lymphoma EL4 cells and mouse melanoma PG19 cells has been characterized in two ways. Metaphase spreads show the presence of additional chromosome forms that are either “homogeneously staining” chromosomes or “double minute” and ring chromosomes. Gel electrophoresis of restriction enzyme-digested nuclear DNA shows the presence of amplified sequences, the pattern of which is unique in each of five cell lines. We conclude that extensive DNA rearrangement has taken place during amplification.     

Absence from the mouse genome of DNA sequences related to the globin gene family.

Radioactively labelled DNA, complementary to mouse α and β globin messenger RNA, was annealed with unlabelled mouse embryo DNA under conditions of both optimum and lowered stringency. Although an increase in saturation of unlabelled DNA fragments with complementary DNA molecules is produced by lowered stringency, the values obtained are within the range expected for the known globin chains.It is concluded that within the limits of our experimental conditions, there does not exist a large family of DNA sequences related to the globin genes.     

Rabbit globin pseudogene psi beta 2 is a hybrid of delta- and beta-globin gene sequences

The evolutionary history of the rabbit globin pseudogene psi beta 2 was studied by completing its nucleotide sequence and aligning the sequence with that of the rabbit adult globin gene beta 1 and the human minor adult globin gene delta. The 5' flanking region and exon 1 of psi beta 2 were most similar to rabbit beta 1, but the large intervening sequence and the 3' untranslated region were most similar to human delta. Intron 1 and exon 2 were equally similar to both delta and beta 1. This pattern indicates that psi beta 2 was originally a delta-like gene that acquired the 5' portion of gene beta 1 by intrachromosomal gene conversion. The presence of a delta-globin gene sequence in both rabbits and humans shows that it is an ancient gene, predating the mammalian radiation that occurred over 85 Myr ago. Delta has shown a pronounced tendency to be altered in its 5' end during the course of mammalian evolution. Quantitative divergence analysis shows that the ancestor to rabbit psi beta 2 was active until 20-30 Myr ago, during which time the lagomorph beta-globin gene family apparently functioned without a pseudogene.      

Isolation and identification of separated messenger RNAs for rabbit alpha and beta globin.

Rabbit globin messenger RNA was separated into two species by polyacrylamide gel electrophoresis in formamide. The two species were isolated from the gel and assayed for messenger activity in the ascites cell-free system. The product of the cell-free system was analysed by column chromatography and by finger-printing. The RNA species with the lower mobility (mol. wt 227,000) codes mainly for β globin, whilst the RNA with the higher mobility (mol. wt 202,000) codes mainly for α globin. Fingerprint analysis of DNA copies of the separated mRNA species may be distinguished, and suggest that the polynucleotide sequences are of homogeneity comparable to the messenger activity. We conclude therefore that a physical separation of the two messenger species has been obtained.     

Intergenic DNA sequences flanking the pseudo alpha globin genes of human and chimpanzee.

We have determined the sequence of 2400 base pairs upstream from the human pseudo alpha globin (psi alpha) gene, and for comparison, 1100 base pairs of DNA within and upstream from the chimpanzee psi alpha gene. The region upstream from the promoter of the psi alpha gene shows no significant homology to the intergenic regions of the adult alpha 2 and alpha 1 globin genes. The chimpanzee gene has a coding defect in common with the human psi alpha gene, showing that the product of this gene, if any, was inactivated before the divergence of human and chimpanzee. However the chimpanzee gene contains a normal ATG initiation codon in contrast to the human gene which has GTG as the initiation codon. The psi alpha genes of both human and chimpanzee are flanked by the same Alu family member. The structure and position of this repeat have not been altered since the divergence of human and chimpanzee, and it is at least as well conserved as its immediate flanking sequence. Comparing human and chimpanzee, the 300 bp Alu repeat has accumulated only two base substitutions and one length mutation; the adjacent 300 bp flanking region has accumulated five base substitutions and twelve length mutations.