A second polymorphic dinucleotide repeat in the 5′ flanking region of the human IL10 gene

Received: 1 July 1996 / Revised: 10 July 1996     

Characterization of genetic markers in the 5′ flanking region of the apo A1 gene

Genetic variation of apoA1/C3/A4 is associated with hyperlipidaemia and coronary heart disease. We report the polymerase chain reaction (PCR) conditions for determining three polymorphic sites in the 5 flanking region of apoA1 using DNA prepared from small aliquots of whole blood. These polymorphisms identify six haplotypes that will be of value in genetic studies.     

DNA polymorphism in the 5′ flanking region of the human carbonic anhydrase II gene on chromosome 8

Summary A restriction-fragment-length polymorphism (RFLP) is described which is associated with the human carbonic anhydrase II gene (CA2) that codes for one of the three genetically distinct carbonic anhydrase isozymes, CA I, CA II, and CA III. The isolated DNA was cleaved with several restriction enzymes and subjected to Southern blot hybridization analysis using a DNA probe containing the 5 end of the human CA II gene. A two allele RFLP which was detected with the restriction endonuclease, Taq I, is expressed phenotypically on Southern blots as either a 5.4 kilobase (kb) fragment or as 4.0 and 1.4 kb fragments. These fragments result from the presence or absence of a Taq I recognition site in the 5 flanking region approximately 1.0kb from the initiation codon of the CA II gene. Segregation analysis showed that the alleles are inherited in a Mendelian fashion, with a frequency of 50%.     

An EcoRI RFLP in the 5′ region of the human NF1 gene

Von Recklinghausen neurofibromatosis or type l neurofibromatosis (NF1), is one of the most common autosomal dominant disorders. NF1 is characterized by neurofibromas, café-au-lait spots and Lisch nodules of the iris. The NF1 gene is located in 17q11.2. The restriction fragment length polymorphism reported here will be useful in linkage analysis in NF1 families.     

Tissue-specific binding of nuclear factors to the 5′-flanking region of rat gene for calcium-binding protein regucalcin

The existence of nuclear factors which bind to the 5-flanking region of calcium-binding protein regucalcin gene in rats was investigated. We previously reported that rat regucalcin mRNA is expressed in a highly tissue-specific manner; the mRNA was mainly present in the liver but only slightly in the kidney. When the nuclear proteins extracted from the liver and kidney of rats were used in the gel mobility shift assays, a protein-DNA complex was uniquely formed with the DNA fragment containing the upstream region from the first exon of rat regucalcin gene. On the other hand, this complex was not found by using the nuclear extracts from rat brain, spleen, and heart. The nuclear proteins of these extracts, however, could specifically bind to the DNA fragment containing the first exon region of rat regucalcin gene, although Northern blot analysis did not show detectable amount of regucalcin mRNA levels in rat brain, spleen, and heart. The present study demonstrates that the existence of nuclear protein components which bind to the regucalcin gene. These identified components may be involved in the tissue-specific regulation of regucalcin gene expression.     

Differential binding of rat pituitary-specific nuclear factors to the 5′-flanking region of pituitary and placental members of the human growth hormone gene family

Summary Placental chorionic somatomammotropin (hCS-A or B) and growth hormone variant (hGH-V) are members of the human growth hormone family, and are related by structure and function to pituitary growth hormone (hGH-N). However, while the hGH-N gene is expressed specifically in the anterior pituitary, hGH-V and hCS are produced in the placenta. Hybrid hGH-N, hGH-V and hCS-A genes containing 5-flanking sequences, including the endogenous promoter, are preferentially expressed in rat pituitary tumor (GC) cells, after gene transfer. Since interaction with a pituitary-specific protein (Pit 1) is required for efficient hGH-N as well as rat growth hormone (rGH) gene expression in GC cells, binding of pituitary proteins to the hGH-V and hCS-A promoter sequences was investigated. Rat Pit 1 binds at two locations on the hGH-N gene, a distal (–140/–107) and proximal site (–97/–66), in a similar manner to that observed with the rGH gene. By contrast, efficient Pit 1 binding was seen only to the distal site of the hGH-V gene and the proximal site of the hCS-A gene. Although binding of a protein to the distal hCS-A sequences was observed, the site of interaction was truncated (–140/–116), not pituitary-specific, and was more consistent with the binding of Sp1. These data indicate that rat Pit 1 binds to the placental hGH-V and hCS-A genes and correlates with their promoter activity in GC cells after gene transfer. However, the data also indicate that rat Pit 1 binds to human and rat pituitary growth hormone in a similar manner (two sites of interaction) and that the pattern of binding is distinct from the placental members of the hGH gene family. These data indicate that human Pit 1, unlike the rat equivalent, might distinguish these genes functionally (tissue-specifically) as well as structurally.     

Proteins binding to the 5‘—flanking regualtory elements of the human β—globin gene

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5‘-flanking regulatory elements of human β-globin gene,two negative control regions(NCR1,-610to-490 bp;NCR2,-338,to-233bp),was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5‘-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in art for the silence of β-glolbin gene in the embryonic and fetal stages.     

Characterization of the rat carbonic anhydrase II gene structure: sequence analysis of the 5′ flanking region and 3′ UTR

The rat carbonic anhydrase II gene was characterized and found to be approximately 15.5 kb in length and to contain 7 exons and 6 introns. All intron/exon junction and branch point sequences conform to consensus sequences, and the overall rat CA II genomic structure appears to be conserved upon comparison with mouse, human, and chicken CA II genes. The putative cis-acting elements within the analyzed 1014 bp 5′ flanking region include: TATA box, 4 Sp1 binding sites, 2 AP2 sites and putative tissue-specific β-globin-like repeat elements. A CpG island of approximately 800 bp was identified that begins about 600 bp upstream of exon 1 and extends about 200 bp into intron 1. In the 3′ UTR, two polyadenylation signals (AATAAA) are present, the second of which is believed to be utilized. Northern blot analysis reveals that the 1.7 kb rat CA II mRNA is abundantly expressed in adult male brain and kidney, while negligible amounts are detected in heart and liver.     

Restriction fragment length polymorphism in the 3′ flanking region of the rabbit β1-globin gene

By Southern blot analysis, a restriction fragment length polymorphism in the 3' flanking region of the rabbit beta 1-globin gene was detected. Two alleles, characterized by 9.7- and 12.4-kb BamHI fragments and by 15.3- and 18.0-kb HindIII fragments, have been detected in a small population of White New Zealand rabbits. The long allele is the most frequent (about 70%). The simultaneous changes in the restriction patterns of the two endonucleases and the constant distance between BamHI and HindIII sites in short and long fragments suggest the possibility that the two alleles arise from a rearrangement phenomenon involving a DNA segment 2.7 kb long. In addition, the presence of the two alleles in individuals genetically unrelated to the White New Zealand breed suggests that this polymorphism is widespread.     

DNA polymorphisms in the 5′-flanking region of the HLA-DQA1 gene

The HLA-DQA1 gene exhibits haplotype-specific restriction fragment polymorphisms due to DNA rearrangements. We found that some of these polymorphisms extend into the 5 flanking region of the gene and are distinct from other HLA-DQA1 related DNA polymorphisms so far reported. Sequencing of genomic DNA subclones derived from the 5 flanking region of HLA-DQA1 showed the presence, in a DR4 haplotype, of two repetitive elements of the Alu family, oriented in opposite directions and bracketing an approximately 3 kilobase region immediately adjacent to the promoter of the gene. When DNAs extracted from several cell lines were analyzed by genomic hybridization using single-copy probes relative to these intervening sequences, polymorphisms were observed. No structural alterations of the gene immediately outside the DNA portion delimited by the two Alu elements were observed, thus suggesting that polymorphisms of the 5 end of HLA-DQA1 may be limited to the intervening region between the two Alu repeats. The latter includes upstream regulatory elements controlling the expression of the genes. The possibility that the structure of the DNA in this region may influence the regulation of HLA-DQA1 gene expression in different haplotypes is discussed.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M72411. Address correspondence and offprint requests to: J. Guardiola.     

Sequence analysis of the 5′-untranslated region of the human H1 histamine receptor-encoding gene

A clone containing the H₁ histamine receptor (H₁HR)-encoding gene was isolated from a human genomic DNA library. The 5′-UTR of the H₁HR gene reported here differs upstream from bp −142 from that reported previously [Fukui et al., Biochem. Biophys. Res. Comm. 201 (1994) 894–901]. PCR amplification utilizing primer pairs derived from the 5′-UTR reported herein amplified a DNA fragment of the expected size from human genomic DNA whereas 5′-UTR primers derived from the Fukui et al. sequence did not yield a PCR product. The 5′-UTR of H₁HR contains potential TATA and CCAAT boxes, a CACCC sequence, potential GREs and other DNA-binding motifs.     

Polymorphism of pentanucleotide repeats in the 5′ flanking region of glutathione S-transferase (GST) π gene

The upstream sequence of the glutathione S-transferase gene contains pentanucleotide (ATAAA) repeats. Analysis of the region using polymerase chain reaction indicated that the repeat sequence was polymorphic and segregation of the polymorphic alleles was codominant heredity. Heterozygosity of the new VNTR was 0.818 in healthy Japanese and 0.794 in American whites. Allelic frequencies among healthy controls and alcoholics as well as other diseases were not significantly different.     

The length polymorphism in the 5′ flanking region of the human β-globin gene with denaturing gradient gel electrophoresis in a Japanese population

Summary The ATTTT repeat polymorphism located approximately 1,400 base pairs (bp) upstream from the -globin structural gene was analyzed by denaturing gradient gel electrophoresis (DGGE) of RNA: DNA duplexes. A study of 81 unrelated Japanese from Hiroshima revealed a sequence heteromorphism in this site. The alleles with five and six repeats of the ATTTT unit, which have been reported, were found in polymorphic proportions. Two unreported alleles were also detected, the first, in two persons, characterized by seven repeats and the other, in a single person, having an A-to-G nucleotide substitution in the fifth repeat.     

Dinucleotide repeat in the 3′ flanking region provides a clue to the molecular evolution of the Duffy gene

The Duffy blood group system consists of three alleles, FYA, FYB, and FY. To study the molecular evolution of the three alleles, we established the polymorphism of a dinucleotide (GT) repeat sequence (designated FyGT/ C) in the 3′ flanking region of the Duffy gene, and studied the relationship between FyGT/C and Duffy polymorphism in Japanese, people of African origin, and chimpanzee. By single-strand conformation polymorphism and sequence analysis, five and two alleles were identified in Japanese and Africans, respectively. In 110 random Japanese, the FyGT/C genotypes observed were in agreement with Hardy-Weinberg law. From the sequence of the chimpanzee Duffy gene, including both flanking regions, FYB was identified as the ancestral gene of the human alleles. The FyGT/C sequences associated with the FY allele of Africans were distinct from those of Duffy positives, whereas the FYB and FYA alleles shared common FyGT/C sequences. Thus, it is suggested that the first split took place between the FYB and FY alleles, and the second between the FYB and FYA alleles. Received: 25 July 1996 / Revised: 10 October 1996     

Crystal structure of the N-terminal region of human Topoisomerase IIβ binding protein 1

Human DNA Topoisomerase IIβ binding protein 1 (TopBP1) is a modulating protein that plays an essential role in the response to DNA damage. The N-terminal region of TopBP1, which contains predicted BRCA1-carboxy terminal (BRCT) domains 1 and 2, binds to Rad9, a component of the cell cycle checkpoint clamp Rad9-Hus1-Rad1 complex. Here, we report the crystal structure of the TopBP1 N-terminal region (residues 1-290) at 2.4 Å resolution. Interestingly, in addition to the predicted tandem BRCT1-2 repeats (residues 103-284), residues 7-98 form a previously unreported BRCT domain (here, BRCT0). In contrast to both BRCT1 and BRCT2, which possess the conventional phosphopeptide binding residues within a surface pocket, the corresponding pocket in BRCT0 is largely hydrophobic. Structural comparisons together with peptide binding studies indicate that the tandem BRCT1-2 domains are the binding region for phosphorylated Ser387 in Rad9.     

Polymorphism of the Hinf I restriction site located 1 Kb 5′ to the human β-globin gene

Summary Mapping of the DNA from 14 Mediterranean subjects indicates a genetic variation in an Hinf I recognition site located 1 kilobase 5 to the -globin gene. This Hinf I site was found associated with eight -thalassemic genes and 11 normal genes, and hence is not specifically linked to -thalassemia.