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 “Sardinian”HLA-A30,B18,DR3,DQw2 haplotype constantly lacks the21-OHA andC4B genes. Is it an ancestral haplotype without duplication?

TheC4 and21-OH loci of the class III HLA have been studied by specific DNA probes and the restriction enzymeTaq I in 24 unrelated Sardinian individuals selected from completely HLA-typed families. All 24 individuals had theHLA extended haplotypeA30,Cw5,B18, BfF1,DR3,DRw52,DQw2, named “Sardinian” in the present paper because of its frquency of 15% in the Sardinian population. Eighteen of these were homozygous for the entire haplotype, and six were heterozygous at theA locus and blank (or homozygous) at all the other loci. In all completely homozygous cells and in four heterozygous cells at theA locus, the restriction fragments of the21-OHA (3.2 kb) andC4B (5.8 kb or 5.4 kb) genes were absent, and the fragments of theC4A (7.0 kb) and21-OHB (3.7 kb) genes were present. It is suggested that the “Sardinian” haplotype is an ancestral haplotype without duplication of theC4 and21-OH genes, practically always identical in its structure, also in unrelated individuals. The diversity of this haplotype in the class III region (about 30 kb less) may be at least partially responsible for its misalignment with most haplotypes, which have duplicatedC4 and21-OH genes, and therefore also for its decreased probability to recombine. This can help explain its high stability and frequency in the Sardinian population. The same conclusion can be suggested for the Caucasian extended haplotypeA1,B8,DR3 that always seems to lack theC4A and21-OHA genes.     

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.     

Novel detection of restriction fragment length polymorphisms in the human major histocompatibility complex

Cosmid genomic DNA clones have been used as hybridization probes in genomic Southern blot analysis to define restriction fragment length polymorphisms (RFLPs) in the major histocompatibility complex (MHC). Using 14 different enzymes and three overlapping cosmid clones we have detected six RFLPs in a 100 kilobase (kb) segment of DNA in the class III region extending centromeric of theTNFA gene towardHLA-DR. Four of the five RFLPs, defined using the enzymesTaqI,Rsa I,Hinc II, andHind III, and detected by the cosmid clone cosM7B, map to a 29 kb segment of DNA that includes all of the recently described G2 (BAT2) gene and a large portion of the 3 end of the G3 (BAT3) gene. The different RFLP variants were established by analyzing the DNA from three informative families and a panel of 51HLA-homozygous typing cell lines. CosM7B detectsTaq I variants of 4.3 kb, and 2.9 kb or 2.8 kb, Rsa I variants of 2.9 kb or 2.4 kb,Hinc II variants of 5.8 kb or 3.8 kb and 1.4 kb, and aHind III variant of 4.8 kb, while cosOT2 detects Taq I variants of 4.5 kb or 4 kb. The distribution of theRsa 1, Hinc II and Taq I RFLPs detected by cosM7B, and theTaq I RFLP detected with cosOT2, within the panel of cell line DNAs was assessed by Southern blotting. The 4.3 kbTaq I variant was observed in only one cell line with the extended haplotypeHLA-A29, C-, B44, SC30, DR4. The other RFLPs, however, occurred much more frequently. The 2.8 kb Taq I variant was observed in 20 % of haplotypes, the 2.9 kbRsa I variant was observed in 42% of haplotypes, and the 5.8 kbHinc I variant was observed in 12 % of haplotypes analyzed. The 4.5 kbTaq I variant detected by the overlapping cosmid cosOT2 was present in 21 % of haplotypes. Analysis of the RFLP variants with each other revealed seven different haplotypic combinations. Three of the haplotypic combinations were each subdivided into two subsets on the basis of the Nco I RFLP variant they carried at theTNF-B locus. These haplotypic combinations potentially allow differentiation among different extended haplotypes such asHLA-B8, SC01, DR3, HLA-B18, F1 C30, DR3, andHLA-B44, FC31, DR7. The RFLPs detected by the cosmid clones thus provide new tools which will be useful in the further genetic analysis of the MHC class III region.     

Polymorphism ofTcrb andTcrg genes in Biozzi mice: Segregation analysis of a newTcrg haplotype with antibody responsiveness

Tcrb andTcrg gene polymorphism was investigated in high (H) and low (L) responder Biozzi mice from selection I, II, and GS by Southern blot analysis with appropriateV andC probes. No polymorphism of theTcrb haplotype was detected between H and L mice in all selections which were all found to be of the BALB/c type. The H-I and H-II g genotype was of BALB/c and DBA/2 type, respectively. In contrast, a newTcrg haplotype shared by L-I and L-II mice was identified and characterized by C1, 2, 3, C4, V1, 2, 3, V5, and V6 restriction fragment length polymorphisms (RFLPs).Tcrg genotypes were not fixed in the GS selection and two additional new haplotypes were identified in two L-GS mice. An attempt was made to correlate the L-Ig genotype with the low responder status by analyzingg haplotypes among highest and lowest responder (H-1 x L-I)F₂ hybrids immunized with sheep red blood cells (SRBC). No correlation was found in this segregation study, whereas a highly significant one was established with theH-2 haplotype, a locus already known to participate in the genetic control of H-I/L-I difference. The lack of correlation between SRBC response and theTcrg genotype was consistent with the heterogenousg haplotypes found in mice of the GS selection. Together, the present results suggest that H and L mice have the sameTcrab potential repertoire and that T-cell receptor (Tcr) genes cannot be considered as immune response genes in this model. Our results also indicate that the F₂ segregation analysis, given a polymorphic gene, is suitable for an investigation of its immune response functions.     

Newly characterized genetic polymorphism of uropepsinogen group A (PGA) using both isoelectric focusing and immunoblotting

Summary Genetic polymorphism of uropepsinogen group A (PGA) was characterized in human urine using a technique involving both polyacrylamide gel isoelectric focusing and immunoblotting with an anti-PGA antibody. PGA was clearly separable into five fractions, termed I to V in order of decreasing anodal mobility. The most slowly migrating fraction V was composed of F (fast) and/or S (slow) band(s). The population frequencies of the three patterns of fraction V (F, FS, and S) and family studies indicated that PGA V is controlled by a pair of alleles, PGA V ^* F and PGA V ^* S, at a single autosomal locus, and that both are codominant. The frequencies of the genes are 0.07 for PGA V ^* F and 0.93 for PGA V ^* S.     

Multiple restriction fragment length polymorphisms associated with the Vc determinant of the MN blood group-related chimpanzee V-A-B-D system

Twelve restriction fragment length polymorphisms (RFLPs) were detected in common chimpanzee using two restriction enzymes (HindIII andMspI) and four DNA probes to the coding regions of the human glycophorin A (GPA) and glycophorin B (GPB) genes and their 3-untranslated regions. Seven RFLPs correlated with red cell expression of the V^c determinant of the MN blood group-related V-A-B-D system and five RFLPs correlated with nonexpression of this antigen. Animals heterozygous for theV allele that encodes the V^c determinant had all 12 polymorphic restriction fragments and appeared to show reduced intensity of probe hybridization to these fragments, consistent with the presence of aV and a non-V allele. No RFLPs were detected withEcoRI,SstI, orBamHI, in spite of the relatively large segment of DNA (at least 20 kb) involved in the polymorphisms. The RFLPs were chimpanzee specific and were not found in man, gorilla, orangutan, or gibbon. Multiple RFLPs distinguishing primate species are rare and may be useful markers for molecular evolution.This work was supported in part by National Institutes of Health Grants AM 33463 and CA 33000.     

Evolutionary relationship ofHLA-DRB genes inferred from intron sequences

The major histocompatibility complex (Mhc) consists of class I and class II genes. In the humanMhc (HLA) class II genes, nineDRB loci have been identified. To elucidate the origin of these duplicated loci and allelic divergences at the most polymorphicDRBI locus, introns 4 and 5 as well as the 3′ untranslated region (altogether approximately 1,000 base pairs) of sevenHLA-DRB loci, threeHLA-DRBI alleles, and nine nonhuman primateDRB genes were examined. It is shown that there were two major diversification events inHLA-DRB genes, each involving gene duplications and allelic divergences. Approximately 50 million years (my) ago,DRBI ^*04 and an ancestor of theDRB1 ^*03 cluster (DRBI ^*03, DRBI^*15, andDRB3) diverged from each other andDRB5, DRB7, DRB8, and an ancestor of theDRB2 cluster (DRB2, DRB4, andDRB6) arose by gene duplication. Later, about 25 my ago,DRBI ^*15 diverged fromDRBI*03, andDRB3 was duplicated fromDRBI ^*03. Then, some 20 my ago, the lineage leading to theDRB2 cluster produced two new loci,DRB4 andDRB6. TheDRBI ^*03 andDRBI ^*04 allelic lineages are extraordinarily old and have persisted longer than some duplicated genes. The orthologous relationships ofDRB genes between human and Old World monkeys are apparent, but those between Catarrhini and New World monkeys are equivocal because of a rather rapid expansion and contraction of primateDRB genes by duplication and deletion. Correspondence to: Y. Satta     

Restriction fragment length polymorphism of the major histocompatibility complex of the pig

Human HLA cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the SLA major histocompatibility complex in swine. Cellular genomic DNA from 19 SLA homozygous pigs representing 13 different haplotypes was digested with restriction endonucleases Eco RI, Hind III, or Bam H1, separated by electrophoresis, and transferred onto diazobenzyloxymethyl paper by the Southern blot technique. The blots were probed with ³²P-labeled class I or beta-DR class II cDNA. Depending on the haplotypes and the endonucleases used, seven to ten restriction fragments hybridized with the class I probe, and five to seven with the beta-DR probe. Their sizes ranged from 3.4 to 22 kilobase-pairs. Few bands were common to all 13 haplotypes. With all but one haplotype, identical autoradiogram patterns were obtained from unrelated, but phenotypically SLA-identical pigs, suggesting that most of the RFLP revealed were controlled by the SLA region. Further polymorphism was found in a group of seven unrelated pigs which typed serologically as SLA A15 CI B18 homozygotes but could be divided into two subgroups, with five animals in one subgroup and two in the other, when the genomic DNA was hybridized with the class I probe. When the class 11 beta-DR probe was tested on the same seven pigs, another subdivision was seen, and this correlated with MLR data. These results demonstrate that HLA class I and class II probes can be used to identify certain well-established SLA haplotypes and to identify subclasses within at least one SLA haplotype.Abbreviations MHC major histocompatibility complex - MLR mixed lymphocyte reaction - kbp kilobase pair(s) - RFLP restriction fragment length polymorphism     

DNA polymorphisms and linkage relationship of the human complement componentC6,C7, andC9 genes

In this report we describe the linkage between genes encoding human complement componentsC6,C7, andC9. Polymorphisms have been described at the DNA level for theC7 andC9 genes. We have studied 20 individuals by Southern blot analysis with fourC6 cDNA subclones to detect restriction fragment length polymorphisms (RFLPs). We have found a Taq I polymorphism defined by two alleles of 8.0 (C6 H) and 6.0 (C6 L) kilobases (kb). RFLP segregation for theC6, C7, andC9 loci in informative families allowed us to estimate the maximum Lod scores at a recombination fraction of =0.0 (C6–C7), =0.0 (C7–C9), and =0.0 (C6–C9). Significant linkage disequilibrium was found betweenC6 andC7 and betweenC7 andC9 loci in directly determined haplotypes of unrelated parents. Data from this study show that the genes encoding the human terminal complement componentsC6, C7, andC9 define a cluster in the short arm of chromosome 5. We propose that the clusters involving theC8A andC8B and theC6, C7, andC9 genes be referred to as MACI and MACH, respectively.     

Human immunoglobulin variable region genes: A new VH sequence used to detect polymorphism

A human heavy chain variable region subgroup III pseudogene (HV3.3) was isolated, characterized, and sequenced. When HV3. was hybridized to Southern blots of human DNA, two potentially informative polymorphic bands, resulting from 2.7 kb Hind III (HH2.7) and 7.3 kb Eco RI(HE7.3) fragments, were detected with frequencies of 0.553 and 0.606, respectively. These polymorphic bands showed Mendelian segregation in families and appeared to be in tight linkage disequilibrium with each other ( ₁ ² =24.91, P<0.001). Evidence from sibling-pair data indicated linkage of the Hind III polymorphic band to constant region allotypic and restriction fragment length polymorphism markers. Bands representing alternative forms of the polymorphic restriction sites were not detected for either HH2.7 or HE7.3. This indicates either that the alternative fragments comigrate with homologous fragments resulting from conserved restriction sites, or that the polymorphism is due to a gene duplication or deletion. No band segregating with HH2.7 was found in separate digests using eight other enzymes. Although this indicates that a major deletion is unlikely, it does not exclude the possibility of a gene deletion or duplication affecting the intergenic region(s) of one or more homologous genes. Whatever the precise explanation, these findings support the hypothesis that there is polymorphic variation of V _H gene repertoires in man.Previous address     

CYP21/C4 gene organisation in Italian 21-hydroxylase deficiency families

Summary A total of 33 Italian 21-hydroxylase (21-OH) deficiency families were investigated using a combination of short and long range restriction mapping of the CYP21/C4 gene cluster. The analyses revealed that large-scale length polymorphism in this gene cluster strictly conformed to a compound variable number of tandem repeats (VNTR) plus insertion system with between one and four CYP21 + C4 units and seven BssHII restriction fragment length polymorphisms (RFLPs) (75kb, 80kb, 105kb, 110kb, 135kb, 140kb and 180kb). A total of 9/66 disease haplotypes, but only 1/61 nondisease haplotypes, showed evidence of gene addition by exhibiting three or more CYP21 + C4 repeat units. Of these, two were identified in one 21-OH deficiency patient who has a total of eight CYP21 + C4 units, being homozygous for the HLA haplotype DR2 DQ2 B5 A28. This haplotype carries four CYP21 + C4 units, three of which contain CYP21A-like genes and one of which contains a CYP21B-like gene that presumably carries a pathological point mutation. Of the other gene addition haplotypes associated with 21-OH deficiency, four show three CYP21 + C4 units flanked by HLA-DR1 and HLA-B14 markers. Although such haplotypes have commonly been associated with non-classical 21-OH deficiency, three examples in the present study are unexpectedly found in two salt-wasting patients, who are respectively homozygous or heterozygous for this haplotype. Only 7/66 disease haplotypes showed evidence of a CYP21B gene deletion.     

Transcription and diversity of immunoglobulin lambda chain variable genes in the rat

The DRB region of the human major histocompatibility complex displays length polymorphism: Five major haplotypes differing in the number and type of genes they contain have been identified, each at appreciable frequency. In an attempt to determine whether this haplotype polymorphism, like the allelic polymorphism, predates the divergence of humansfrom great apes, we have worked out the organization of the DRB region of the chimpanzee Hugo using a combination of chromosome walking, pulsed-field gel electrophoresis, and sequencing. Hugo is a DRB homozygote whose single DRB haplotype is some 440 kilobases (kb) long and contains five genes. At least one and possibly two of these are pseudogenes, while three are presumably active genes. The genes are designated DRB ^* A0201, DRB2 ^* 0101, DRB3 ^* 0201, DRB6 ^* 0105, and DRB5 ^* 0301, and are arranged in this order on the chromosome. The DRB2 and DRB3 genes are separated by approximately 250 kb of sequence that does not seem to contain any additional DRB genes. The DRB ^* A0201 gene is related to the DRB1 gene of the human DR2 haplotype; the DRB2 ^* 0101 and DRB3 ^* 0201 genes are related to the DRB2 and DRB3 genes of the human DR3 haplotype, respectively; the DRB6 ^* 0105 and DRB5 ^* 0301 genes are related to the DRBVI and DRB5 genes of the human DR2 haplotype, respectively. Thus the Hugo haplotype appears to correspond to the entire human DR2 haplotype, into which a region representing a portion of the human DR3 haplotype has been inserted. Since other chimpanzees have their DRB regions organized in different ways, we conclude that, first, the chimpanzee DRB region, like the human DRB region, displays length polymorphism; second, some chimpanzee DRB haplotypes are longer than the longest known human DRB haplotypes; third, in some chimpanzee haplotypes at least, the DRB genes occur in combinations different from those of the human haplotypes; fourth, and most importantly, certain DRB gene combinations have been conserved in the evolution of chimpanzees and humans from their common ancestors. These data thus provide evidence that not only allelic but also haplotype polymorphism can be passed on from one species to another in a given evolutionary lineage.     

The histocompatibility-2 system in wild mice

The I-region gene products of 29 wild-derivedH-2 haplotypes on a B10 background (B10.W congenic lines) were typed with alloantisera which detect 17 inbred I-region antigens. Five new I-region antigens were defined by expanding the inbred line panel ofH-2 haplotypes to includeH-2 ^u , H-2^v, andH-2 ^j . Based on serological analyses of the inbred and B10.W lines, the polymorphism of theIA gene (or genes) is estimated to be at a minimum of 15 alleles and theIE gene (or genes) at a minimum of 4 alleles. These results indicate that theIA subregion is more polymorphic than theIE subregion. By combining the I-region typing data with theH-2K andH-2D region typing data reported previously, a total of 11 new natural recombinants of inbredH-2 alleles were detected among the B10.W lines.     

Evolution of thedec-1 eggshell locus inDrosophila. I. Restriction site mapping and limited sequence comparison in themelanogaster species subgroup

Summary We have analyzed 18 kb of DNA in and upstream of thedefective chorion-1 (dec-1) locus of the eight known species of themelanogaster species subgroup ofDrosophila. The restriction maps ofD. simulans, D. mauritiana, D. sechellia, D. erecta, andD. orena are shown to have basically the restriction map ofD. melanogaster, whereas the maps ofD. teissieri andD. yakuba were more difficult to align. However, the basic amount of DNA and sequence arrangement appear to have been conserved in these species. A small deletion of varying length (65–200 bp) is found in a repeated sequence of the central transcribed region ofD. melanogaster, D. simulans, andD. erecta. Restriction site mapping indicated that thedec-1 gene is highly conserved in themelanogaster species subgroup. However, sequence comparison revealed that the amount of nucleotide and amino acid substitution in the repeated region is much larger than in the 5 translated region. The 5 flanking region showed noticeable restriction site polymorphisms between species. Based on calculations from the restriction maps a dendrogram was derived that supports earlier published phylogenetic relationships within themelanogaster species subgroup except that theerecta-orena pair is placed closer to themelanogaster complex than toD. teissieri andD. yakuba.     

Structural and evolutionary comparisons of four alleles of the mouse immunoglobulin kappa chain gene,Igk-VSer

The mouseIgK-VSer gene encodes an immunoglobulin light chain variable region which gives rise to two phenotypic polymorphisms of mouse chains. The nucleotide sequences of coding and flanking regions of theIgk-VSer ^c andIgk-VSer ^d alleles found in recently inbred strains of wild mice are compared with those of theIgk-VSer ^a andIgk-VSer ^b alleles described previously. Results suggest that the gene is evolving randomly and that framework 2 and complentarity determining region 2 are preserved, presumably for overall light chain structure. Results indicate that all four allels have an octamer motif upstream of the gene which should be functional and allow prediction of whether or not the product of the germ line gene will be detectable as either the I_B-peptide or Ef1^a phenotypic polymorphism. Southern hybridization of genomic DNA using as probe a 1-kbXba I-Xba I fragment located approximately 4 kb upstream of the BALB/cIgk-VSer ^b coding region demonstrated the presence of homologous DNA in mice bearing theIgk-VSer ^a allele and absence from mice bearing theIgk-VSer ^c andIgk-VSer ^d alleles. Nucleotide sequence comparison of BALB/c and SK/CamRk (Igk-VSer ^d ) DNA in this region demonstrated that BALB/c contained an insertion 2.4 kb in length which was absent from SK/CamRk. Both strains contain DNA homologous to the reverse complement of the mouse Bam5 repetitive element at the point of the insertion, with BALB/c containing approximately 70 nucleotides more of the element than SK/CamRk. Surprisingly, the strains containing DNA related to theXba I-Xba I probe are not those determined to be the most similar by nucleotide sequence comparisons and by the Phylogenetic Analysis Using Parsimony program. The evolutionary relationship of the alleles and a possible basis for the inconsistency presented by theXba I-Xba I fragment-related DNA are discussed.     

TheD17Tu5 locus in thet complex: implications for the origin oft haplotypes and inbred strains

The mouse × Chinese hamster cell line R4 4-1 contains only one mouse chromosome, the bulk of which corresponds toMus musculus chromosomes 17 and 18 (MMU17 and MMU18, respectively). A genomic library was prepared from the R4 4-1 DNA, and a mouse clone was isolated from the library, which—with the help of somatic cell hybrids-could be mapped to the MMU17. A locus defined by a 2.7-kb longBam HI probe from this clone was designatedD17Tu5 (Tu for Tübingen). The locus proved to be polymorphic among inbred strains and wild mice. By testing of recombinant inbred strains and partialt haplotypes, theD17Tu5 locus could be mapped to a position between theD17Leh66E andD17Rp17 loci within thet complex. Two alleles were found at this locus,D17Tu5 ^a andD17Tu5 ^b , defined byTaq I restriction fragment length polymorphism. Both alleles are present among inbred strains and wild mice of the speciesM. domesticus. All completet haplotypes tested carry theD17Tu5 ^a allele and all tested wild mice of the speciesM. musculus, with the exception of those bearingt haplotypes, carry theD17Tu5 ^b allele. Additional alleles are found in some populations of wild mice and in other species of the genusMus. The distribution of the two alleles among the inbred strains correlates well with their known or postulated genealogy. Their distribution between the two species ofMus and among the mice withT haplotypes suggests a relatively recent origin of thet haplotypes.     

Molecular analysis of the MHC class II region inDR4, DR7, andDR9 haplotypes

Within the class 11 region of the major histocompatibility complex (MHC) the amount of DNA in theDR-DQ interval has been shown to be haplotype dependent, with those carrying the DR4, DR7, and DR9 specificities having been reported to contain 110–160 kilobases (kb) more DNA than haplotypes carrying the DR3 specificity. Certain subtypes of haplotypes carrying particular DR specificities are more closely associated with autoimmune diseases than others. With the prospect of the DNA perhaps containing a disease susceptibility locus, we have mapped eight DR4 and two DR7 homozygous cell lines and a DR7/9 heterozygous cell line together with a control DR3 cell line using pulsed field gel electrophoresis (PFGE) with the enzymesBss H II,Pvu I, andNot I/Nru I. Our results, however, show that the presence and amount of the extra DNA is constant irrespective of the subtype. We have also tried to narrow down the position of insertion of the extra DNA using eight further rare-cutting enzymes but, due to the polymorphic nature of sites and/or differences in methylation in this region, it was not possible to refine it further than between DRA and DQA1/B1. This polymorphic nature of theDR-DQ region is unusual, considering the uniformity of rare cutter sites that has been observed within the rest of the class II, and class III, regions. The presence of this, and other, haplotype dependent variations in the DNA content of theDR subregion may be important with respect to recombination and will be particularly interesting if the additional DNA is found to contain novel genes.     

Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: Selection of mutants defective in photosynthesis

Summary Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Km^r) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Km^r gene into an AvaII restriction site of the gene psbD-1. All other harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.     

Heterogeneity inHLA-DR2-relatedDR,DQ haplotypes in eight populations of Asia-Oceania

The relative distributions of 480DR2-relatedDR, DQ haplotypes have been determined in Australian Aborigines, Papua New Guinean Highlanders, coastal Melanesians, Micronesians, Polynesians, Javanese, and Southern and Northern Chinese. Using sequence-specific oligonucleotides (SSOs) for hybridization of polymerase chain reaction (PCR) products from DRBI,DRBS,DQA1, andDQBI genes, 15 differentDR2-related haplotypes were identified. The predominantDR2 haplotype in Oceania involved a novel combination ofDRBI ^* 1502,DRB5 ^* 0101 alleles; this haplotype occurred sporadically in Java, but not in China. In Southern China, the most frequent DR2 haplotype involved the unusual arrangementDRB1 ^* 1602,DRB5 ^* 0101; alternatively,DRB1 ^* 1602 was associated with a newDRB5 SSO pattern. This study has important implications for molecular HLA-typing protocols that assume particularDRB1 DRB5 orDR,DQ linkage relationships. Further, the novelDRBI,DRB5 haplotype in Oceania suggests that the mixed lymphocyte culture (MLC) determinants Dw2 and Dw12 are discriminated by codon 86 at theDRBI locus.