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     

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.     

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.     

Unstable and stable CAD gene amplification: importance of flanking sequences and nuclear environment in gene amplification.

We analyzed the amplification of the CAD gene in independently isolated N-(phosphonacetyl)-L-aspartate-resistant clones derived from single parental clones in two mouse cell lines. We report for the first time that the CAD gene is amplified unstably in mouse cells, that the degree of instability varies greatly between clones, and that minute chromosomes and highly unstable chromosomelike structures contain the amplified sequences. These data are most consistent with the idea that the amplified unit in each clone consists of different flanking DNA and that such differences engender amplified sequences with unequal stability. We also introduced the mouse chromosome containing the CAD gene into hamster cells by microcell-mediated chromosome transfer to determine whether the propensity for unstable extrachromosomal amplification of the mouse CAD gene would prevail in the hamster cell nuclear environment. We report that the mouse CAD gene was amplified stably in expanded chromosomal regions in each of seven hybrids that were analyzed. This observation is consistent with the idea that the nuclear environment influences whether mutants containing intra- or extrachromosomally amplified sequences will be isolated.     

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.     

Size heterogeneity of polyadenylate sequences in mouse globin messenger RNA

Heterogeneity in the length of the poly(A) region has been demonstrated in mouse α and β-globin messenger RNAs. This finding is based on the initial observation that only 30% of the globin mRNA purified by oligo(dT)-cellulose affinity chromatography binds to Millipore filters under conditions where other poly(A)-containing mRNAs have been shown to bind, and the subsequent finding that the bound and non-bound fractions contain different size classes of poly(A). The poly(A) size was determined by polyacrylamide gel electrophoresis of the T₁ and pancreatic RNAase-resistant fragments. The unbound mRNA fraction gives a fragment 35 to 45 adenine nucleotides long, while the bound mRNA contains two fragments with average lengths of 55 to 65 and 75 to 120 nucleotides.The heterogeneity of the poly(A) region is present in both α and β-globin mRNAs as both Millipore-bound and unbound RNA fractions directed the synthesis of comparable amounts of mouse α and β-globin chains.Change in the distribution of the various size classes of poly(A) was analyzed by Millipore binding assays after various times of labeling in vivo. The percentage of labeled mRNA bound to Millipore filters decreased with time, suggesting either a shortening of the poly(A) region or differential synthesis of mRNAs containing shorter poly(A) at earlier stages in erythropoeisis.     

Differential expression of mouse beta/goat beta c, mouse beta/goat beta F, and mouse beta/goat epsilon II hybrid globin genes in murine erythroleukemia cells.

We assembled three hybrid beta-globin genes by fusing the mouse beta-major promoter and initial transcribed region to one of three goat beta-like globin gene bodies: beta c (preadult), beta F (fetal), or epsilon II (embryonic). Thymidine kinase (tk)-deficient murine erythroleukemia (MEL) cells were cotransformed with one of these constructs and a separate plasmid bearing the tk gene. Half of the 24 cell lines containing either the mouse beta/goat beta c or mouse beta/goat beta F genes expressed the transferred genes at significant levels; in many cases the hybrid genes were, like the endogenous beta-globin genes, inducible with dimethyl sulfoxide. We obtained 13 cell lines containing the mouse beta/goat epsilon II hybrid gene, 6 of which were cotransfected with a mouse beta/human beta fusion gene known to function in MEL cells. In contrast to the results with the other fusion genes, the mouse beta/goat epsilon II hybrid was very poorly expressed: in two separate experiments, 0 of 13 and 2 of 13 lines showed significant mouse beta/goat epsilon II RNA levels after induction. In all these lines the endogenous mouse beta and cotransfected mouse beta/human beta genes were expressed. As an initial test of possible reasons for the inactivity of the mouse beta/goat epsilon II hybrid, we recloned this fusion gene into a tk-bearing plasmid, adjacent to the tk gene. Of 12 cell lines transformed with this plasmid, 11 produced mouse beta/goat epsilon II RNA; in 6 cases the expression was both strong and dimethyl sulfoxide inducible.