Restriction site polymorphisms in the pig β-globin gene cluster

A restriction fragment length polymorphism was detected in pig DNA digested with Hind III restriction endo nuclease and probed with rabbit β₁-globin gene. Eight different phenotypes were observed and for six of them family data demonstrated that they are determined by three alleles. As this polymorphism is not found with four other restriction endo nucleases (Bam HI, Eco RI, Kpn I, and Pst I), single point mutations are proposed to explain the observed differences.     

Restriction fragment length polymorphism of the human beta-globin gene cluster: analysis of a beta-thalassemic family in Taiwan

We have analysed seven polymorphic restriction sites of the human beta-globin gene cluster of six members of a Chinese family with a beta +-thalassemic sibling. The seven polymorphic sites analysed are the HincII site at the 5'-end of the epsilon-globin gene, the HindIII sites in the two gamma-globin genes, two HincII sites within and at the 3'-end of the psi beta 1 pseudogene, the AvaII site in the beta-globin gene and the BamHI site located at the 3' side of the beta-globin gene. The beta thal chromosome has been identified to have a haplotype of +----++ with respect to these seven polymorphic sites. This is also the most predominant haplotype associated with beta +-thalassemia in Mediterranean and Chinese populations (Chen et al., 1984; Orkin et al., 1982). Of the seven sites analysed in this family, four will be useful in prenatal diagnosis of beta-thalassemia in subsequent pregnancies in the family.     

A silent deletion in the beta-globin gene cluster.

A survey of the gamma-globin gene region of over 1000 normal individuals revealed a novel 2.5 kb deletion which removes the 5' end of the A gamma-globin gene. Unusually, this deletion in the beta-globin gene cluster is not associated with increased fetal haemoglobin production. Sequence analysis of the deletion endpoints revealed no significant homology at the breakpoint and failed to support a role for a proposed recombination hotspot in IVS-2 in the generation of this illegitimate recombination event. The existence of small "silent" deletions in the beta-globin gene cluster emphasizes the importance of deletion size in altering expression of the fetal globin genes.     

Nonuniform recombination within the human beta-globin gene cluster.

Population genetic analysis of 15 restriction site polymorphisms demonstrates nonuniform recombination within the human beta-globin gene cluster. These DNA polymorphisms show two clusters of high nonrandom associations, one 5' and another 3' to the beta-globin structural gene, with no significant linkage disequilibrium between the two clusters. The 5'- and 3'-association clusters are 34.6 kilobases (kb) and 19.4 kb long, respectively, and are separated by 9.1 kb of DNA immediately 5' to the beta-globin gene. For each of these three DNA regions, we have observed a relationship between nonrandom associations and physical distance between the polymorphisms. However, this relationship differed for each of these regions. On the assumption that the effective population size (Ne) is 5,000-50,000, we estimate the total recombination rate to be 0.0017%-0.0002% in the 5' cluster, 0.0931%-0.0093% in the 3' cluster, and 0.2912%-0.0219% in the 9.1-kb region between them. The beta cluster thus shows nonuniformity in recombination. Moreover, the recombination rate in the 9.1-kb DNA segment is 3-30 times greater than expected and is thus a hot spot for meiotic recombination.     

Detection of a novel DNA polymorphism in the beta-globin gene cluster

Analysis of DNA from the beta-globin gene cluster in an Albanian family identified a novel RsaI site approximately 550 base pairs 5' to the beta-globin gene. In this family, two chromosomes carrying otherwise identical beta-globin haplotypes were found to differ at the RsaI site. Population screening demonstrated the presence and absence of the site in DNA from individuals of northern European, Mediterranean, Middle Eastern, Southeast Asian, African, and Asian Indian descent, indicating that this site is a DNA polymorphism common in many ethnic groups. The polymorphism is also present in DNA from individuals carrying different beta-globin alleles. Additional nucleotide sequence changes identified in an RsaI (+) genomic clone in the region immediately 3' to the RsaI site suggest a mechanism for the randomization of the site with respect to haplotype.     

The beta-globin gene cluster distribution revisited-patterns in Native American populations

New frequencies for the beta-globin gene cluster haplotypes are presented for the Aché (N = 82 individuals), Guarani (N = 76), and Kaingang (N = 54), three Native South American populations that live in an area between parallels 20 degrees S and 30 degrees S not covered by previous studies at this locus. The haplotype frequencies obtained for the three populations are within the interval observed for 28 other Native American populations. The Aché show much less haplotypes (five) than the other two populations (9-10), the haplotype prevalences being more similar to those of the Guarani than to the Kaingang. The Native American total heterozygosity was about half (0.41) that obtained for the African populations (0.71), but was not much different from those obtained for other continents. A geographical pattern was disclosed in South America by mapping the frequencies of the most common haplotype (haplotype 2), and by means of spatial correlation analysis. The analysis of molecular variance (AMOVA) and pairwise F(ST) data suggest three distinct sectors for the genetic landscape of Native South America: the Andes, the Center/Southeast region, and the Amazon.     

Restriction polymorphisms of the ceruloplasmin gene on chromosome 3

Using a probe isolated from a human liver cDNA library, polymorphisms were observed in the human ceruloplasmin gene with the enzymes PstI and MspI. The PstI polymorphism was frequent (allele frequencies, 0.46 and 0.54) whereas the polymorphisms found with MspI were rare.     

The duck beta-globin gene cluster contains a single enhancer element

An erythroid-specific enhancer was previously identified in the 3'-flanking region of the beta adult gene in chicken and duck, by transfection into AEV transformed chicken erythroblasts. Here we show that the duck enhancer is equally active in erythroid human K562 cells, presenting an embryonic/fetal program of globin gene expression. Furthermore, no other enhancer was found within the 20 kb of DNA including four beta-like globin genes as well as a 1.5 kb upstream and a 3 kb downstream sequence.     

Recent insertion of an alu sequence in the beta-globin gene cluster of the gorilla

We present the nucleotide sequence of a new Alu family member that lies between the delta- and beta-globin genes in gorilla DNA. The sequence exhibits 91% similarity with a consensus sequence of the Alu family. It is flanked by a perfect repetition of a 16-nucleotide target sequence and terminates with 24 adenylic residues. As this sequence is absent at this locus in other primate DNAs, its insertion occurred less than 8 million years ago, thus supporting the idea that Alu sequences are still mobile elements in the hominoid genome.     

Characteristic beta-globin gene cluster haplotypes of Evenkis and Oroqens in north China

Haplotype frequencies of the beta-globin gene cluster were estimated for 114 Evenkis and 81 Oroqens from northeast China, and their characteristics were compared with those in Japanese, Koreans, and three Colombian Amerindian groups of South America (Wayuu, Kamsa, and Inga tribes). A major 5' subhaplotype (5' to the delta-globin gene) was + - - - - in Evenkis, whereas + - - - -, - + + - +, and - + - + + were the major subhaplotypes in Oroqens. One possible candidate for an ancestral 5' subhaplotype, - - - - -, was found in one Evenki (0.5%) and three Oroqen chromosomes (2.0%). They were observed as heterozygous forms for + ---- and -----. Major haplotypes were +-----+, + -----+-, and + - - - - + + in Evenkis, whereas they were +-----+,-++-+-+, +----+-, and -+-++-+ in Oroqens. The lowest Nei's genetic distance values of Evenkis or Oroqens based on the 5' subhaplotype frequency distributions were observed in relation to the Wayuu or Koreans, respectively, but those of Evenkis and Oroqens based on the haplotype frequency distributions were found in relation to Koreans.     

Restriction fragment length polymorphisms at the growth hormone gene in pigs

Pigs from four Danish and two Swedish populations were examined for restriction fragment length polymorphism (RFLP) at the growth hormone (GH) gene. Polymorphism was detected with the restriction enzymes DraI and TaqI. A comparison of the Danish populations showed significant differences among their allelic frequencies.     

Interleukin-1 gene cluster polymorphisms in cerebral infarction

Interleukin-1 (IL-1) has pleiotropic actions in the central nervous system. During the past decade, a growing corpus of evidence has indicated an important role of this cytokine in the development of brain damage following cerebral ischaemia. The expression of IL-1 in the brain is dramatically increased during the early and chronic stage of infarction. The IL-1 gene cluster on chromosome 2ql4 contains three related genes (IL-1alpha, IL-1beta, and IL-1 receptor antagonist, IL-1ra) located within a 430-kb region. Polymorphisms in the genes encoding IL-1alpha, IL-1beta, and IL-1ra have been associated with several inflammatory diseases. Therefore we hypothesized that these cytokines might be good candidates for cerebral infarction (CI). We ascertained these genotypes in 363 CI patients and 640 controls matched for age and gender. A significant increase was found for the IL-1alpha (-889) allele 2 carriers in CI patients compared with controls (chi2 = 5.633, P = 0.018, odds ratio (OR) = 1.5). Furthermore, the IL-1alpha (-889) allele 2 carriers increased the relative risk for CI in the subjects without the IL-1ra allele 2 (chi2 = 7.989, P = 0.005, OR = 1.7). There was no significant association between IL-1beta (+3,953) polymorphism and CI. These results suggest that IL-1alpha-889 and IL-1ra polymorphisms are effective in the development of CI in Koreans.     

Analysis of variation in the human beta-globin gene cluster using a novel DHPLC technique

We have implemented a technique combining allele-specific PCR (AS-PCR) and denaturing high-performance liquid chromatography (DHPLC) to identify new polymorphic variants within an intergenic region in the beta-globin cluster. This technique is applicable to the detection of new variants in genomic regions where variation is apportioned into distinct classes of haplotype. Duplexes for DHPLC analysis were created by denaturation and re-annealing of a mixture of two AS-PCR products of known and unknown sequence from the same haplotypic class, permitting detection of new haplotypes in each class. A 454bp fragment 3.5kb 5' to the human delta-globin gene, which may have a gene regulatory function, was analysed in 840 chromosomes from a global sampling of human populations using this method. Two divergent haplotypes were found to predominate in all populations studied, possibly as a result of balancing selection.     

ELISA detection of restriction site polymorphisms in the pig ryanodine receptor locus

We compare two strategies for ELISA detection of restriction site polymorphisms (EDRSP) that are suitable for high-throughput genotyping of the pig ryanodine receptor point mutation (RYR1 ^hal ). In both procedures, target DNA is amplified by PCR with one primer that is 5′ biotinylated and a second primer that is 5′ fluoresceinylated. PCR products are captured in duplicate wells on a streptavidin-coated, 96-well plate. The duplicates may be treated in two ways. In a single restriction enzyme assay, one duplicate is exposed to a restriction enzyme that cuts one allele specifically, and the second duplicate is exposed to no restriction enzyme. In a dual restriction enzyme assay, the second replicate is exposed to a second restriction enzyme that cuts the alternate allele specifically. Thereafter, the two procedures are similar; anti-fluorescein antibodies conjugated to peroxidase are allowed to bind to the fluoresceinylated ends, the plate is washed, and a substrate is converted to a colored end product. The ratio of the absorbances in the two wells is used to classify subjects by genotype. When the dual restriction enzyme assay is run, three genotype groups are easily distinguishable. When the single restriction enzyme assay is run, heterozygotes generate values that may overlap with those of the homozygotes that are not cut by the restriction enzyme. Dual restriction enzyme assays are more accurate than single restriction enzyme assays; however, single restriction enzyme assays are sufficient for identifying pigs that carry RYR1 ^hal . Received: 30 December 1997 / Accepted: 20 April 1998     

Function of the upstream hypersensitive sites of the chicken beta-globin gene cluster in mice.

We have shown previously that the chicken beta A-globin gene, with its 3' enhancer, is expressed in a copy number-dependent manner in transgenic mice. The expression level was low but increased approximately 6-fold upon inclusion of 11 kb of upstream DNA containing four DNase I hypersensitive sites. To study the effect of the individual upstream hypersensitive sites on transgene expression, we produced lines of mice in which the individual upstream sites were linked to the beta A gene and enhancer. RNA levels were measured in blood from adult animals. With each of these four constructs, the level of transgene RNA per DNA copy varied over a > 20-fold range. These data suggest that addition of a hypersensitive site to the beta A-globin/enhancer region abrogates its position independent expression. The average beta A-globin expression per copy in the lines carrying an upstream site was comparable with that in lines without an upstream site. Thus, no single upstream hypersensitive site accounts for the higher level of beta A-globin expression seen in mice containing the complete upstream region. We had shown previously that control of the chicken beta-globin cluster is distributed between at least two regions, the beta A/epsilon enhancer and the upstream region. Our current results suggest that the control mediated by the upstream DNA is itself distributed and is not due to a single hypersensitive site.     

The hemopexin gene maps to the same location as the beta-globin gene cluster on human chromosome 11

Using human hemopexin cDNA clones isolated from lambda gt11 cDNA library as probes, we have carried out Southern blot analysis of a series of human-Chinese hamster somatic cell hybrids containing different combinations of human chromosomes. Synteny analysis revealed 100% concordance between the hemopexin gene and human chromosome 11. In situ hybridization of 3H-labeled hemopexin cDNA to metaphase chromosomes prepared from human lymphocytes further localized the gene to the region p15.4-p15.5, the same location as the beta-globin gene cluster.     

Restriction site polymorphism in the phosphoglycerate kinase gene on the X chromosome

Summary Using a human phosphoglycerate kinase (PGK) cDNA probe, we have identified a common Pst I restriction site polymorphism on the human X chromosome in all ethnic groups studied. the polymorphic Pst I site was absent in 40.4% of 94 X chromosomes of unrelated subjects. Heterozygous females can only be detected by the combined use of a Pst I digest and a Xba I+Pst I digest due to the presence of autosomal and X-linked bands of the same size in simple Pst I digests. Since 48% of females are heterozygotes for the Pst I polymorphism, this site can serve as a useful genetic marker on the long arm of X chromosome in man.