Human immunoglobulin variable region genes: V κ polymorphisms suggest variation in V-gene repertoires

The present study characterizes four potentially informative polymorphic bands of 5.2, 2.3, 1.9, and 1.2 kb, detected by Southern blot hybridization of Eco RI digests of human DNA using HK101/80 (an immunoglobulin V I probe). These restriction fragment length polymorphisms (RFLP) show Mendelian segregation and they are linked to each other and to Km(1), the allotypic marker on the kappa constant region. There is strong linkage disequilibrium between the 2.3 and 1.2 kb polymorphisms. A 0.7 kb Pst I polymorphic band and a 2.9 kb Sac I polymorphic band were also identified; the latter may reflect a region of tandem repeats in the V region. No bands representing the alternative forms of any of the polymorphic restriction sites were identified. This implies either that genes are missing from the V repertoire or that such bands are hidden because of comigration of fragments due to conservation of restriction sites. Evidence for comigration of fragments was obtained from independent V clones and suggests that dark bands on Southern blots of Pst I digests must often represent several superimposed genes which have conserved restriction sites. The demonstration of RFLP within the V region provides circumstantial evidence for polymorphic variation in the repertoire of V structural genes. The RFLP reported here should be useful as genetic markers in future studies on the immune response and disease susceptibility in man.     

Molecular genotyping of the HLA-DQ α gene region

Restriction fragment analysis has been applied to genomic DNA extracted from human tumor cell lines. Polymorphic restriction fragments encompassing the HLA-DQ gene were observed upon digestion with Bgl II, Eco RI, and Hind III. Analysis of these polymorphic fragments (or allogenotopes) showed that for each restriction enzyme a series of three differently sized allogenotopes existed. Clusters of cosegregating allogenotopes belonging to the different allelic series defined three different allogenotypes. Each allogenotype exhibited a distinctive restriction map generated by digestion with five restriction enzymes. Comparison of these restriction maps showed that generation of the polymorphisms observed at the HLA-DQ region in these sets of cell lines is not caused by a single event. In some B- and T-lymphoma cell lines a fourth allogenotype was found. The restriction site map of genomic DNA from these cell lines suggested that the latter distribution of restriction enzyme sites was most probably generated by recombination between two of the previously observed allogenotypes at a crossover site(s) adjacent to the HLA-DQ gene.     

Cloning of sporulation gene spoIVC in Bacillus subtilis

Summary Sporulation gene spoIVC of Bacillus subtilis was cloned by the prophage transformation method in temperate phage 105. The specialized transducing phage, 105spoIVC-1, restored the sporulation of the asporogenous mutant of B. subtilis strain 1S47 (spoIVC133). Transformation experiments showed that the spoIVC gene resides on a 7.3 kb HindIII restriction fragment. Subsequent analysis of the 7.3 kb HindIII fragment with restriction endonuclease EcoRI showed that the spoIVC gene resides on a 3.6 kb EcoRI fragment within the 7.3 kb fragment. The 3.6 kb fragment was recloned into the unique EcoRI site of plasmid pUB110 and deletion derivatives having a deletion within the 3.6 kb insert were constructed. The plasmid carrying the entire spoIVC gene restored the sporulation of strain HU1214 (spoIVC133, recE4) at a frequency of 10⁷ spores/ml, and reduced the sporulation of strain HU1018 (spo ⁺, recE4) to 10⁷ spores/ml.     

Restriction endonuclease analysis of human globin genes in cellular DNA

Using samples of human cellular DNA digested with restriction endonucleases Eco RI, Hind III, Hinc II, Bam HI, Alu I, or Hae III, we were able to localize globin gene fragments separated by agarose gel electrophoresis. The fragments were transferred to nitro-cellulose filters and identified by hybridization to [³²P] cDNA for total adult globin mRNA. The α-globin gene fragments were specifically identified by their presence in normal controls and absence in DNA from homozygous α-thalassemia, a genetic disorder due to deletion of α-globin genes. In addition, the patterns with Hind III indicate a 4.1 kb distance between the centers of the normal duplicated α-globin gene loci.     

Physical map of Aspergillus nidulans mitochondrial genes coding for ribosomal RNA: an intervening sequence in the large rRNA cistron

Summary A detailed map of the 32 kb mitochondrial genome of Aspergillus nidulans has been obtained by locating the cleavage sites for restriction endonucleases Pst I, Bam H I, Hha I, Pvu II, Hpa II and Hae III relative to the previously determined sites for Eco R I, Hind II and Hind III. The genes for the small and large ribosomal subunit RNAs were mapped by gel transfer hybridization of in vitro labelled rRNA to restriction fragments of mitochondrial DNA and its cloned Eco R I fragment E3, and by electron microscopy of RNA/DNA hybrids.The gene for the large rRNA (2.9 kb) is interrupted by a 1.8 kb insert, and the main segment of this gene (2.4 kb) is separated from the small rRNA gene (1.4 kb) by a spacer sequence of 2.8 kb length.This rRNA gene organization is very similar to that of the two-times larger mitochondrial genome of Neurospora crassa, except that in A. nidulans the spacer and intervening sequences are considerably shorter.     

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%.     

Repeating units of xenopus laevis oocyte-type 5S DNA are heterogeneous in length

The restriction enzymes Hind III and Hae III cleave Xenopus laevis 5S DNA at one and three sites, respectively, in each repeating unit of approximately 700 base pairs. The cleavage sites for both enzymes have been located within the repeating unit by denaturation mapping of the restriction fragments. The Hind III products and one of the Hae III fragments are variable in length, indicating heterogeneity in the length of the repeating unit in 5S DNA. This length heterogeneity is confined to the major A + T-rich spacer region. Repeating units differ from each other by discrete quanta of approximately 15 base pairs. The A + T-rich spacer has been shown to consist largely of tandem subrepeats of just this size (Brownlee, Cartwright, and Brown, 1974). We suggest that the repeat-length heterogeneity is due to variable numbers of these subrepeats in the spacer regions of the major repeating units.     

DNA sequences homologous to the T DNA region of Agrobacterium tumefaciens are present in diverse Rhizobium species

Summary DNA sequences homologous to the T DNA region of the octopine-type Ti plasmid from Agrobacterium tumefaciens are present in different Rhizobium species. Plasmid DNA from each of two R. leguminosarum, two R. meliloti, and four slow-growing Rhizobium strains examined contain restriction endonuclease fragments that hybridize with the T DNA region, or with DNA sequences at or near the adjacent Ti plasmid transfer (ra) region. Four different BamHI fragments that contain homology to the T DNA region were cloned from R. leguminosarum 300 plasmid DNA. Cloned fragments of 5.9 kb and 10.3 kb hybridize to each other and are homologous to sequences which map at the right boundary region (EcoRI fragment 24) of the core T DNA. Ti plasmid sequences homologous to those present in cloned fragments of 10.9 kb and 2.0 kb map in adjacent fragments near the tra genes, approximately 10 kb to the right of the core T DNA.     

Genomic organization of a polymorphic duplicated region centromeric of HLA-B

The region between tumor necrosis factor (TNF) and HLA-B in the central major histocompatibility complex (MHC) is polymorphic and associated with several autoimmune diseases. The polymorphisms are haplospecific or haplotypic and retained within the same MHC ancestral haplotype (AH). We have cloned this region from four AHs into bacteriophage and found that a highly polymorphic region in the TNF-HLA-B interval is duplicated. Clones from this region isolated from three MHC AHs show two populations. The regions, designated CL1 and CL2, have different sizes of Bam HI fragments carrying the duplicated sequences. These fragments correspond to those seen after Bam HI restriction fragment length polymorphism (RFLP) analysis of genomic DNA from the same cell lines. Pulsed field gel electrophoresis analysis shows that both CL1 and CL2 are in the central MHC and are about 16 kilobases apart. DNA cloning and RFLP analysis demostrate that the Cl region is highly polymorphic but retained within an MHC AH. Polymorphism and duplication are common characteristics of the genes found in the MHC and therefore the CL sequences have the potential to be interesting in this respect.     

Inheritance of a mutant histocompatibility gene and a new mammary tumor virus genome in the B6.KH-84 mouse strain

The potential association between integration or deletion of mouse mammary tumor virus (MMTV) retroviral sequences and the appearance of non-H-2 histocompatibility (H) antigen mutations was investigated. Genomic blots from inbred strains carrying 22 loss, gain-loss, and gain mutations on the BALB/c and C57BL/6 backgrounds were hybridized with probes homologous to the long terminal repeat (LTR) and envelope (env) regions of MMTV. Twenty-one mutants were identical in restriction patterns to the respective background strains with all tested restriction enzymes and both probes. However, genomic blots of one gain mutant, B6.C-KH-84, exhibited restriction fragments which were not exhibited by either of the parental strains, C57BL/6 or BALB/c. An additional 5.5 kb Eco RI fragment was observed with the env probe and additional 9.2 kb and 5.5 kb fragments were observed with the LTR probe. These observations were substantiated by hybridization of these two probes with genomic blots generated with additional restriction enzymes. Assuming that the new provirus contains a single, internal Eco RI site as has been observed for other MMTV proviral sequences, it is presumed that the new provirus includes both 5 and 3 LTRs in addition to the env region. Based on the unique sizes of the observed restriction fragments relative to other identified MMTV proviral sequences, this provirus has been designated Mtv-22. The potential role of Mtv-22 in the genesis of the gained histocompatibility antigen in B6.C-KH-84 is discussed.On leave of absence from Istituto Nazionale per to Studio e la Cura dei Tumori, Milano, Italy     

Duplication of exons 13, 14 and 15 of the LDL-receptor gene in a patient with heterozygous familial hypercholesterolemia

Summary During a survey of Italian patients with familial hypercholesterolemia (FH), we identified an FH heterozygous patient with a gross rearrangement of the low density lipoprotein (LDL) receptor gene. Southern blot analysis of the proband's DNA digested with restriction enzymes PvuII, BamHI, BglII and XbaI and hybridization with cDNA probes complementary to the 3 end of the gene revealed the presence of abnormal fragments that were approximately 7 kb larger than their normal counterparts. DNA digestion with other enzymes (EcoRV, NcoI, KpnI and StuI) and hybridization with probes complementary to exons 13–17 generated normal fragments and an abnormal fragment of 6.3–6.8 kb. These results are consistent with the presence of an insertion of approximately 7 kb caused by a duplication of exons 13, 14 and 15. This is a novel mutation that is most probably the result of an unequal crossing-over between repetitive sequences located in intron 12 and intron 15. This novel mutation has been designated FH_{Bologna 2}.     

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.     

Polymorphism of the T-cell receptor gamma variable and constant region genes in a Chinese population

Summary The human T-cell receptor gamma gene region spans 160 kb genomic DNA. Restriction fragment length polymorphisms (RFLPs) have been previously documented for the constant region (TRGC) genes, the joining (TRGJ) segments and the variable (TRGV) genes. We have recently defined the alleles of the T-cell receptor gamma V, J and C genes and we have described seven haplotypes of the V gamma subgroup I genes characterized either by RFLPs or by deletion or insertion of V gamma genes. The number of VI genes may vary from 7 to 10 per haploid genome, the 9-gene haplotype being the most frequent. Allelic fragments can unambiguously characterize the TRGC2 gene with duplication or triplication of the exon 2. These alleles and haplotypes have been analyzed in four different populations (French, Lebanese, Tunisian and Black African). In this paper, we compare these allele and haplotype frequencies with those found in a Chinese population and we describe new TRGV allelic restriction fragments found only in the Chinese samples. These results and the previous data demonstrate the flexibility of the human T cell receptor gamma locus and the importance of unequal crossing-overs in the evolution of that locus. Moreover, they underline the importance of studying these polymorphisms in population genetics.     

The binding of exogenous DNA fragments to bovine spermatozoa

Abstract

The ability of mature, freeze‐thawed bovine sperm to bind exogenous end‐labelled or oligo‐labelled λ Hind III DNA restriction fragments was examined. Following 30 min. incubation of bovine sperm with P³² end‐labelled λ Hind III DNA and five washes with medium, approximately 5.8 ng DNA were bound to 10⁷ sperm. Agarose gel autoradiography revealed that all of the λ Hind III DNA bands were present following sperm washes except for the smaller 0.5 Kb and 0.125 Kb bands. Incubation of sperm with ³H oligo‐labelled λ Hind III DNA gave a much higher level of binding (138 ng/10⁷ sperm) than that found with end‐labelled DNA. This binding was entirely eliminated by DNase I. The separation of live and dead sperm fractions on Percoll gradients revealed that more oligo‐labelled λ Hind III DNA was found to be associated with the dead sperm fraction (31.2 ng/10⁷ sperm) rather than the live sperm fraction (2.7 ng/10⁷ sperm). Analysis of supravital stained, light microscopic autoradiographs confirmed that oligo‐labelled λ Hind III DNA bound to dead sperm in the post‐acrosomal region of the sperm head although other minor distribution patterns were observed.     

Map location of the Escherichia coli origin of replication.

Summary Working with restriction fragments obtained directly from the Escherichia coli K12 chromosome, the EcoRI-HindIII restriction map of the section of the chromosome containing the replication origin has been extended by 14 kilobase pairs (kb) to cover 56kb. Within this newly mapped portion, the liv and rrnC cistrons have been identified by (1) hybridization of individual restriction fragmenents to the ilv-transducing phage dilv5 and (2) a comparison of the restriction map of this region with the EcoRI map of dilv5 and the HindIII map of the plasmid pJC110, a ColE1-ilv hybrid. The replication origin is located approximately 30 kb from the ilvE gene and 20 kb from the rrnC 16S rRNA cistron. This places the origin near 82.7 min on the genetic map, close to uncA.     

Strong associations between RFLP and protein polymorphisms for CD46

Human CD46 (membrane cofactor protein) is a cell surface glycoprotein with cofactor activity for the factor I mediated cleavage of components C3b and C4b. Using a CD46 cDNA clone, three restriction enzymes give simple two allele restriction fragment length polymorphisms (RFLPs) in samples of over 300 Caucasians. For Pvu II, P1 with a 16.5 kilobase (kb) fragment and P2 with 14.8 kb + 1.9 kb fragments have frequencies of .40 and .60. For Hin dIII, H1 with a 4.3 kb fragment and H2 with a 2.3 kb fragment have similar frequencies. For Bgl. II, B1 with a 10 kb fragment and B2 with 8.3 kb + 1.8 kb fragments have frequencies of 0.08 and 0.92. There is strong linkage disequilibrium between these polymorphic sites. Designating haplotypes by Hin dIII, Pvu II, Bgl II alleles, there are two common haplotypes P2, H2, B2 and P1, H1, B2, expected at frequencies of .6 and .32, one less common haplotype P1, H1, B1 expected at a frequency .08. The two major protein isoforms of CD46, as detected on peripheral blood lymphocytes by western blot, of M _r 66 000 () and 56 000 () are determined by differential splicing in production of the mRNA. A strong association between protein isoform and RFLP haplotypes in 30 unrelated subjects suggests that the splicing preference site is in linkage disequilibrium with the RFLPs. The results are consistent with haplotypes P2, H2, B2 and P1, H1, B1 producing predominantly ; P1, H1, B2 producing predominantly in about 72% of cases and in 28% of cases. Address correspondence and offprint requests to: A. Wilton, at the present address.     

Duplications and deletions of Vh genes in inbred strains of mice

evolution of variable region (Vh) gene family copy number and polymorphism was investigated by the analysis of the immunoglobulin heavy chain variable region (Igh-V) locus in 74 inbred strains and substrains of mice. Several strains were found to have slight differences from Igh-V haplotypes previously identified, usually of a single Vh gene family. These results indicate that the evolution of copy number in the mouse Igh-V locus proceeds largely by the accumulation of incremental changes, reflecting the clustered organization of the mouse Igh-V locus. We have found no evidence of very large or frequent duplication or deletion events indicative of rapid expansion or contraction processes. The existence of one or more particularly large Vh gene families most likely reflects random copy number variation, rather than selection for the amplification of their members. The identification of strains with recombinant Vh gene arrays demonstrates that recombination, both within and between haplotypes, appears to be the predominant mechanism generating the high restriction fragment length polymorphism in the Igh-V locus.Abbreviations used in this paper Igh-V immunoglobulin heavy chain variable region locus - Vh heavy chain variable region gene - Dh heavy chain diversity region gene - V immunoglobulin kappa light chain variable region gene - V T-cell receptor beta chain variable region gene     

Comparison between the organization of nuclear ribosomal DNA unit of Euglena gracilis Z and var. Bacillaris

Summary We have characterized the nuclear rDNA unit of Euglena gracilis var. bacillaris and compared it to that of the Z strain. We have localized restriction sites for Eco R1, Sal 1, Sma 1, Hind III, Bam H1 and Bgl II on this unit as well as the coding region for 20 S and 25 S rRNAs. For both strains, results suggest an homogeneity of the 11.6 kbp rDNA units. Comparison between strains shows differences characterized by two additional Sal 1 sites in bacillaris and the likely methylation of one Sma 1 site in Z. Both differences are localized in a non-coding region of the rDNA unit. Analyses of 18 Euglena strains from various origins confirm these differences and allow easy recognition of bacillaris and Z type strains.Abbreviations kb kilo base - kpb kilo base pair - plasmids pRH 59 and pRH 57 contain a Hind III-HInd III nuclear DNA fragment from W₃BUL of 5.9 and 5.7 kbp respectively, pRB 48 and pRB 35 contain a Bam H1-Bam H1 nuclear DNA fragment from wild-type Z of 4.8 and 3.5 kbp respectively - SDS sodium dodecyl sulfate - UV ultra-violet     

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     

A study of the HLA-D region in patients with classic Kaposi's sarcoma

The HLA-D region in nine Sardinian patients with classic Kaposi's sarcoma was studied with two restriction enzymes, Eco RI and Eco RV, and two cDNA probes, DR and DQ. A total of 41 polymorphic restriction fragments were identified. One, an 11.5 kb Eco RV DQ fragment, was present in three of the patients but in none of the controls; a second, an 8.0 kb Eco RV DR fragment, was present in six patients and all the controls. No single fragment was identified which was significantly over or under-represented in either group.