Abdominal Visceral Fat is Associated with a BclI Restriction Fragment Length Polymorphism at the Glucocorticoid Receptor Gene Locus

Several investigations have suggested that body fat distribution is influenced by nonpathologic variations in the responsiveness to Cortisol. Genetic variations in the glucocorticoid receptor (GRL) could therefore potentially have an impact on the level of abdominal fat. A restriction fragment length polymorphism (RFLP) has previously been detected with the BelI restriction enzyme in the GRL gene identifying two alleles with fragment lengths of 4.5 and 2.3 kb. This study investigates whether abdominal fat areas measured by computerized tomography (CT) are associated with this polymorphism in 152 middle-aged men and women. The less frequent 4.5-kb allele was found to be associated with a higher abdominal visceral fat (A VF) area independently of total body fat mass (4.5/4.5 vs. 2.3/2.3 kb genotype; men: 190.7 ± 30.1 vs. 150.7 ± 33.3 cm², p=0.04; women: 132.7 ± 37.3 vs. 101.3 ± 34.5 cm², p=0.06). However, the association with AVF was seen only in subjects of the lower tertile of the percent body fat level. In these subjects, the polymorphism was found to account for 41% (p=0.003) and 35% (p=0.007), in men and women, respectively, of the total variance in AVF area. The consistent association between the GRL polymorphism detected with BelI and AVF area suggests that this gene or a locus in linkage disequilibrium with the BelI restriction site may contribute to the accumulation of AVF.     

TaqI dimorphic site in a human pancreatic phospholipase A2 (PLA2) gene localized on chromosome 12

We report the identification of a TaqI dimorphic site in the first intron of a human phospholipase A2 gene that has been localized on chromosome 12. This single two-allele polymorphism was detected by DNA blot hybridization using a full-length, 584 bp human phospholipase A2 cDNA isolated from a human lung gt10 cDNA library. Fragment lengths were detected at either 1.3 kb or 2.2 kb. The PIC value of this dimorphism was 0.369 in a random population of 353 Caucasians.     

Organization of the ribosomal ribonucleic acid genes in various wild-type strains and wild-collected strains of Neurospora

Summary The organization of the ribosomal DNA (rDNA) repcat unit in the standard wild-type strain of Neurospora crassa, 74-OR23-1A, and in 30 other wild-type strains and wild-collected strains of N. crassa, N. tetrasperma, N. sitophila, N. intermedia, and N. discreta isolated from nature, was investigated by restriction enzyme digestion of genomic DNA, and probing of the Southern-blotted DNA fragments with specific cloned pieces of the rDNA unit from 74-OR23-1A. The size of the rDNA unit in 74-OR23-1A was shown to be 9.20 kilobase pairs (kb) from blotting data, and the average for all strains was 9.11+0.21 kb; standard error=0.038; coefficient of variation (C.V.)=2.34%. These data indicate that the rDNA repeat unit size has been highly conserved among the Neurospora strains investigated. However, while all strains have a conserved HindIII site near the 5 end of the 25 S rDNA coding sequence, a polymorphism in the number and/or position of HindIII sites in the nontranscribed spacer region was found between strains. The 74-OR23-1A strain has two HindIII sites in the spacer, while others have from 0 to at least 3. This restriction site polymorphism is strain-specific and not species-specific. It was confirmed for some strains by restriction analysis of clones containing most of the rDNA repeat unit. The current restriction map of the 74-OR23-1A rDNA repeat unit is presented.     

Lack of a BglII site at the 5′ region of the PGK 1 locus: a new variant discovered in two Chibchan Amerindian groups from Costa Rica

A new 3.8-kb allele at the 5 region of the PGK 1 locus detected by the probe pSPT/PGK is reported. This variant was discovered in the Cabecar and Guaymi, two Chibchan Amerindian groups of Costa Rica. So far, a polymorphism that consists of an EcoRI/BglI (1.3-kb) variable site within an EcoRI/BglII (1.7-kb) fragment when DNA is simultaneously digested with EcoRI, BglI and BglII is known to occur in black and Caucasian populations. These two alleles were also found in the Amerindians tested. The newly described band is due to the lack of the BglII site situated 1.7 kb downstream from the EcoRI site and to the cleavage of another BglII site 2.1 kb downstream from the lacking one. This variant might be restricted to some Amerindian groups and perhaps also to Asiatic populations. Thus, it could be a useful marker in evolutive studies and for forensic applications. Moreover, the presence of a third allele in populations with Amerindian ancestry can increase the heterozygosity of the region disclosed by the pSPT/PGK probe, thus improving its application in issues dealing with X-chromosome activation ratios in females.     

Plasmid formation in Streptomyces: excision and integration of the SLP1 replicon at a specific chromosomal site

Summary We present data showing that the SLP1 plasmids found in Streptomyces lividans after mating with S. coelicolor strain A3(2) orginate as deletion mutants of a 17 kb segment of the S. coelicolor chromosome. Excision of the entire 17 kb segment yields a transiently existing plasmid containing a site for integration into the chromosome of recipient SLP1^- S. lividans strains at a unique locus that corresponds to the original chromosomal location of SLP1 in S. coelicolor. The deletion mutants of SLP1 lack the attachment site and/or other regions required for its integration, and thus persist in the recipient as autonomously replicating plasmids. Plasmids that contain the complete 17 kb sequence of the chromosomally integrated SLP1 segment were constructed in vitro by circularization of restriction endonuclease-generated fragements of chromosomal DNA carrying a tandemly-duplicated integrant of SLP1. Transformation of an SLP1^- S. lividans strain with such plasmids results in chromosomal integration of the SLP1 sequence at the same site at which it is integrated in S. lividans cells that acquire the sequence by mating with S. coelicolor. A model for the site-specific excision and integration of SLP1 is presented.     

Conservation of the central MHC genome: PFGE mapping and RFLP analysis of complement,HSP70, and TNF genes in the goat

A degree of conservation of the genes located between class II and class I [central major histocompatibility complex (MHC) genes] is apparent among mammalian species including primates and the mouse. Few others have been analyzed. The caprine MHC is of particular interest, since it has recently been observed that susceptibility to a lentivirus-induced polyarthritis (caprine arthritis) segregates with serologically defined MHC class I antigens. This arthritis resembles, in a number of respects, rheumatoid arthritis in man. Human cDNA probes were used to examine the caprine central MHC and class I and II genes by restriction fragment length polymorphism (RFLP) and by pulsed field gel electrophoresis (PFGE) in order to define the polymorphism and linkage of central MHC genes to class I and class II genes. An outbred population of dairy goats (Saanen, British Alpine, Anglo Nubian, and Toggenberg) was examined for class I and class II RFLPs. Both regions were found to be highly polymorphic. The number of fragments hybridizing to an HLA-B7 probe after Eco RI, Bam HI, Bgl II, or Hind III digestion suggests there may be 10–13 class I genes. The degree of polymorphism was comparable to that reported in the mouse. Limited polymorphism was found in the central MHC genes. The caprine C4 and CYP21 genes were duplicated and demonstrated RFLP with Bam HI, Hind III, Eco RV, and Taq I. An infrequent Taq I C2 polymorphism was found. PFGE revealed substantial conservation of both the order and linkage of the central MHC genes when compared with mous and man. C4, C2, CYP21, HSP70, and tumor necrosis factor (TNF) genes are all located within 800 kilobase (kb) of the class I loci. Distant from the class I region, the C4, C2, and CYP21 genes are linked on a short genomic segment (180 kb Not I and 190 kb Pvu I fragments). HSP70 cohybridizes with the complement genes on a 380 kb Mlu I fragment. Linkage of HSP70, TNF, and class I genes was found on a single Not I fragment (610 kb). TNF and class I cohybridize on Pvu I (730 kb) and Not I (610 kb) fragments. Conservation of a similar central MHC genomic structure across species argues for functional interaction between the central MHC genes. We postulate selection for these central MHC genes through their role as non antigen-specific regulators of immune response.     

Identification of TaqI polymorphism in the mitochondrial acetoacetyl-CoA thiolase gene and familial analysis of 3-ketothiolase deficiency

We analyzed the mitochondrial acetoacetyl-CoA thiolase gene (T2) by Southern blotting. Fifteen unrelated healthy individuals and members of five families with 3-ketothiolase deficiency (3KTD) were analyzed. We found a TaqI polymorphism, the heterozygosity of which was calculated to be 0.5 among healthy Japanese individuals. This restriction fragment length polymorphism (RFLP) proved to be useful for detecting 3KTD patients and its obligatory carriers, at the DNA level and in two out of five 3KTD families. This polymorphism was found to be generated by the presence/ absence of a TaqI site in intron 9 of the T2 gene. With in vitro amplification of the genomic region around the TaqI site, this RFLP can be detected within 2 days.     

A new polymorphism in the gene for the dopamine D2 receptor

A new polymorphism has been identified in the gene for the dopamine D₂ receptor. The polymorphism was found by single stranded conformational polymorphism (SSCP) analysis. Sequencing revealed an insertion at a BsoF1 restriction site. This polymorphism in the 3- untranslated region was found at a frequency of 0.07 in Centre d'Etude du Polymorphisme Humain (CEPH) parents.     

Detection of a NotI polymorphism with the pmetH probe by pulsed-field gel electrophoresis.

The pmetH probe, tightly linked to the locus for cystic fibrosis (CF), detected a NotI polymorphism with allele sizes of 1,000 and 550 kb that could be separated by pulsed-field gel electrophoresis. Both alleles were found on chromosomes bearing either the normal or the CF allele. Preliminary data showed that this polymorphism was not in strong linkage disequilibrium with the CF locus. Preliminary large-scale restriction mapping of the fragment showed that the NotI polymorphic site is 200-370 kb distant from the MET locus; thus it defines a new polymorphic locus in the CF region. This locus is of potential interest for studying CF families that show recombinants between CF and its tightly linked probes, in order to increase the precision of the genetic map of the region.     

The Location of a Disease-Associated Polymorphism and Genomic Structure of the Human 52-kDa Ro/SSA Locus (SSA1)

Sera from approximately 30% of patients with systemic lupus erythematosus (SLE) contain high titers of autoantibodies that bind to the 52-kDa Ro/SSA protein. We previously detected polymorphisms in the 52-kDa Ro/SSA gene (SSA1) with restriction enzymes, one of which is strongly associated with the presence of SLE (P < 0.0005) in African Americans. A higher disease frequency and more severe forms of the disease are commonly noted among these female patients. To determine the location and nature of this polymorphism, we obtained two clones that span 8.5 kb of the 52-kDa Ro/SSA locus including its upstream regulatory region. Six exons were identified, and their nucleotide sequences plus adjacent noncoding regions were determined. No differences were found between these exons and the coding region of one of the reported cDNAs. The disease-associated polymorphic site suggested by a restriction enzyme map and confirmed by DNA amplification and nucleotide sequencing was present upstream of exon 1. This polymorphism may be a genetic marker for a disease-related variation in the coding region for the protein or in the upstream regulatory region of this gene. Although this RFLP is present in Japanese, it is not associated with lupus in this race.     

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%.     

Restriction fragment length polymorphism of the human T cell receptor alpha gene

Previous studies have demonstrated restriction fragment length polymorphisms (RFLP) in the vicinity of the alpha and beta genes of the human T-cell receptor. In the course of experiments designed to discover additional polymorphic restriction sites, we found a new RFLP of the T-cell alpha gene recognized by the restriction enzyme Taq I. The site was localized to the interval between the most 3 joining (J) exon and the most 5 constant (C) region exon, about 7 kb distant from the previously described Bgl II polymorphic site which mapped to the vicinity of the 3 untranslated exon. With the use of these two polymorphic markers, four Ti-alpha alleles could be identified, allowing unambiguous assignment of all Ti-alpha genes in some families. These markers may be useful in identifying possible immune response genes or disease predisposition genes associated with the genes of the T-cell receptor for antigen.Abbreviations used in this paper RFLP restriction fragment length polymorphism - Ti-alpha alpha gene of the T-cell receptor for antigen     

Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/kanamycin resistance determinant

Summary Streptococcal plasmid pGB301 is an in vivo rearranged plasmid with interesting properties and potential for the molecular cloning of genes in streptococci. Transformation of S. sanguis (Challis) with the group B streptococcal plasmid pIP501 (29.7 kb) gave rise to the deletion derivative pGB301 (9.8 kb, copy number 10) which retained the multiple resistance phenotype of its ancestor (inducible MLS-resistance, chloramphenicol resistance). Among the eight restriction endonucleases used to physically map pGB301 were four that cleaved the plasmid at single sites yielding either sticky (HpaII, KpnI) or bluntends (HpaI, HaeIII/BspRI). Passenger DNA derived from larger streptococcal plasmids (pSF351C61, 69.5 kb; pIP800, 71 kb) was successfully inserted into the HpaII site and, by blunt-end cloning, into the HaeIII/BspRI site. The gentamicin/kanamycin resistance gene of pIP800 was expressed by recombinant plasmids carrying the insert in either orientation. Insertion of passenger DNA into the HaeIII/BspRI site (but not the HpaII site) caused instability of adjacent pGB301 sequences which were frequently deleted, thereby removing the chloramphenicol resistance phenotype. The vector pGB301 has a remarkable capacity for passenger DNA (inserts up to 7 kb) and the property of instability and loss of a resistance phenotype following insertion of passenger DNA into the HaeIII/BspRI site should facilitate the identification of cloned segments of DNA when using this plasmid in molecular cloning experiments.     

Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla

The ribosomal DNA repeat units of two closely related species of the genus Fraxinus, F. excelsior and F. oxyphylla, were characterized. The physical maps were constructed from DNA digested with BamHI, EcoRI, EcoRV and SacI, and hybridized with three heterologous probes. The presence or the absence of an EcoRV restriction site in the 18s RNA gene characterizes two ribosomal DNA unit types found in both species and which coexist in all individuals. A third unit type appeared unique to all individuals of F. oxyphylla. It carries an EcoRI site in the intergenic spacer. Each type of unit displayed length variations. The rDNA unit length of F. excelsior and F. oxyphylla was determined with EcoRV restriction. It varied between 11kb and 14.5kb in F. excelsior and between 11.8kb to 13.8kb in F. oxyphylla. Using SacI restriction, at least ten spacer length variants were observed in F. excelsior, for which a detailed analysis was conducted. Each individual carries 2–4 length variants which vary by a 0.3-kb step multiple. This length variation was assigned to the intergenic spacer. By using the entire rDNA unit of flax as probe in combination with EcoRI restriction, each species can be unambiguously discriminated. The species-specific banding pattern was used to compare trees from a zone of sympatry between the two species. In some cases, a conflicting classification was obtained from morphological analysis and the use of the species-specific rDNA polymorphism. Implications for the genetic management of both species are discussed.     

The polymorphism of the plasma inter-α-trypsin inhibitor (ITI) and its relationship to the heavy chain H1 subunit gene (ITIH1) at 3p211-212

We investigated the ITI protein polymorphism in linkage analysis, usingDraI andSstI as restriction fragment length polymorphism (RFLP) markers for the ITIH1 gene. Isoelectric focusing (IEF) classification from 76 individual plasma samples and RFLP analysis from the corresponding DNA preparations disclosed linkage disequilibrium between the phenotypic IEF patterns of the two common ITI alleles, ITI^*1 and ITI^*2, and the diallelic DNA polymorphisms of two ITIH1 RFLPs, represented byDraI 4.0 kb andDraI 2.4 + 1.6 kb, and bySstI 6.7 kb andSstI 6.0 + 0.7 kb, for the ITI 1 and ITI 2 IEF phenotypes, respectively, and byDraI 4.0/2.4 + 1.6 kb andSstI 6.7/6.0 + 0.7 kb for the heterozygous ITI 1–2 IEF phenotype. Linked segregation between either of the RFLPs and the polymorphic ITI plasma protein locus has been established in nine informative family pedigrees. The less frequent allele in Europeans, ITI^*3, is not represented by a further allelic restriction fragment in either RFLP. The significant linkage disequilibrium observed in this genetic study indicates that the ITI locus, with the alleles ITI^*1 and ITI^*2, must be close to, or reside within, the ITIH1 gene. The diallelic ITI protein polymorphism therefore provides an informative phenotypic marker system for chromosome 3p211-212.     

Detection of frequent BglII polymorphism by polymerase chain reaction and TaqI restriction fragment length polymorphism for 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase at the human HSDβ3 locus (1p11–p13)

Summary Analysis of amplified polymerase chain reaction products of 575 bp from the fourth exon of the human type I 3-hydroxysteroid dehydrogenase/⁵-⁴ isomerase gene at locus HSD3 1p11–p13, reveals a frequent two-allele polymorphism at codon Leu³³⁸ due to a silent substitution of T by C, thus creating a BglII site leading to 371- and 204-bp fragments. Southern blot analysis of BglII-digested DNA from 57 individuals using a genomic probe detects two allelic fragments of 5.3kb and 0.77 kb, respectively, while two allelic fragments of 3.7 kb and 3.4 kb are obtained in TaqI digests with multiple constant bands, as also observed with BglII digests.