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Human glutathione transferase zeta.

Zeta-class glutathione transferases (GSTZs) were recently discovered by a bioinformatics approach and the availability of human expressed sequence tag databases. Although GSTZ showed little activity with conventional GST substrates (1-chloro-2,4-dinitrobenzene; organic hydroperoxides), GSTZ was found to catalyze the oxygenation of dichloroacetic acid (DCA) to glyoxylic acid and the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate. Hence, GSTZ plays a critical role in the tyrosine degradation pathway and in alpha-haloacid metabolism. The GSTZ-catalyzed biotransformation of DCA is of particular interest, because DCA is used in the human clinical management of congenital lactic acidosis and because DCA is a common drinking water contaminant. Substrate selectivity studies showed that GSTZ catalyzes the glutathione-dependent biotransformation of a range of dihaloacetic acids along with fluoroacetic acid, 2-halopropanoic acids, and 2,2-dichloropropanoic acid. Human clinical studies showed that the elimination half-life of DCA increases with repeated doses of DCA; also, rats given DCA show low GSTZ activity with DCA as the substrate. DCA was found to be a mechanism-based inactivator of GSTZ, and proteomic studies showed that Cys-16 of human GSTZ1-1 is covalently modified by a reactive intermediate that contains glutathione and the carbon skeleton of DCA. Bioinformatics studies also showed the presence of at least four polymorphic variants of human GSTZ; these variants differ considerably in the rates of catalysis and in their susceptibility to inactivation by DCA. Finally, Gstz1(-/-) mouse strains have been developed; these mice fail to biotransform DCA or maleylacetone. Although the mice have no obvious phenotype, a high incidence of lethality is observed in young mice given phenylalanine in their drinking water. Gstz1(-/-) mice should prove useful in expanding the role of GSTZ in alpha-haloacid metabolism and in the tyrosine degradation pathway.

Acids↗

Faint lined (Fnl): a novel X-linked coat mutant in the mouse.

We report here a novel X-linked mutant, named faint lined (Fnl), which was discovered in the litter of an irradiated 3H1 male (Dr Bruce Cattanach, personal communication). The mutation is associated with fine dorsal striping in affected heterozygous females and prenatal lethality in males. Approximately 50% of Fnl/+ females die in utero and surviving animals have a reduced weight at birth and weaning. Histological studies failed to reveal the underlying basis of the phenotype or any gross structural abnormalities in internal organs (Fnl/+ x Mus spretus) F1 affected females were backcrossed to 3H1 males and haplotype analysis positioned Fnl in the proximal region of the mouse X chromosome distal to Ant2 and proximal to Hprt. Therefore, Fnl lies within a defined conserved segment and its human homologue can be predicted to lie in the ANT2-HPRT region in Xq25. Further genetic resolution of co-segregating markers flanking Fnl established that Fnl lies in a 7.6 +/- 2.6 cM interval between DXMit50 and DXMit82.

Animals↗

Association of the TSHR gene with Graves' disease: the first disease specific locus.

The development of autoimmune thyroid disease (AITD) is associated with autoantibodies directed against the thyroid stimulating hormone receptor (TSHR). Previous studies have failed to demonstrate a consistent association between the TSHR and AITD, or any of its sub-phenotypes. In the present study, we analysed the linkage disequilibrium (LD) structure encompassing the TSHR, to identify LD 'blocks' and SNPs, which capture the majority of intra-block haplotype diversity. The haplotype tagging SNPs, plus all common SNPs reported in previous studies were genotyped in 1,059 AITD Caucasian cases and 971 Caucasian controls. A haplotype, across two LD blocks, showed association (P<1 x 10(-6), OR 1.7) with Graves' disease (GD) but not autoimmune hypothyroidism (AIH). We replicated these findings by genotyping the most associated GD SNP, rs2268458, in a separate UK Caucasian cohort of 1,366 AITD cases and 1,061 controls (GD, P=2 x 10(-6), OR 1.3; AIH, P=NS). These results in two independent Caucasian data sets suggest that the TSHR is the first replicated GD-specific locus meriting further fine mapping and functional analysis to identify the aetiological variants.

Case-Control Studies↗

Phylogeography of Kandelia candel in East Asiatic mangroves based on nucleotide variation of chloroplast and mitochondrial DNAs.

Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to long-range dispersal. To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia candel based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplast DNA (cpDNA) atpB-rbcL spacer, cpDNA trnL-trnF spacer, and mitochondrial DNA (mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals. Three data sets suggested consistent phylogenies recovering two differentiated lineages corresponding to geographical regions, i.e. northern South-China-Sea + East-China-Sea region and southern South-China-Sea region (Sarawak). Phylogenetically, the Sarawak population was closely related to the Ranong population of western Peninsula Malaysia instead of other South-China-Sea populations, indicating its possible origin from the Indian Ocean Rim. No geographical subdivision was detected within the northern geographical region. An analysis of molecular variance (AMOVA) revealed low levels of genetic differentiation between and within mainland and island populations (phiCT = 0.015, phiSC = 0.037), indicating conspicuous long-distance seedling dispersal across oceans. Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations as the major force causing phylogenetic discrepancy between mtDNA and the trnL-trnF spacer within the northern region. Instead, relative ages of alleles contributed to non-random chlorotype-mitotype associations and tree inconsistency. Widespread distribution and random associations (chi2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes indicated the panmixis of populations of the northern geographical region as a whole. In contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration.

Alleles↗

The mitochondrial genome of the olive fly Bactrocera oleae: two haplotypes from distant geographical locations.

The complete sequence of the olive fly (Bactrocera oleae) mitochondrial genome has been determined. Two independent haplotypes, from flies of distant geographical origin (Italy and Portugal) were completely sequenced. The molecule is 15815 bp long, and shows the gene content and organization typical of insects, namely thirteen protein coding genes (PCGs) encoding proteins involved in oxidative phosphorylation, two rRNAs, twenty-two tRNAs and a long (949 bp) noncoding region. The genomes of the two fly specimens share the same arrangement, differing by a mere thirty-one point mutations. The differences are mostly transitions (26) and synonymous substitutions in PCGs (21). The two new sequences are compared with others already present in the database.

Animals↗

Recognition specificity of self-incompatibility maintained after the divergence of Brassica oleracea and Brassica rapa.

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS46 (S46 in B. rapa) and BoS7 (S7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS12, and BrS8 and BoS32, having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS15 showed incompatibility with a BoS7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS7. These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.

Amino Acid Sequence↗

Susceptibility to severe ulcerative colitis is associated with polymorphism in the central MHC gene IKBL.

BACKGROUND & AIMS: IKBL gene lies telomeric of the tumor necrosis factor cluster in the central major histocompatibility complex and carries a structural polymorphism at position +738. In the Spanish white population, we found the IKBL+738(C) allele in haplotypes carrying either HLA-DRB1(*)1501 or -DRB1(*)0103. Because these HLA class II alleles may confer susceptibility to ulcerative colitis, we investigated an association between IKBL+738(C) and this disease. METHODS: DNA-based techniques were used to type individual alleles of HLA-DRB1 and IKBL+738. The frequencies of these alleles were compared among ethnically matched populations comprising 155 patients and 298 controls. RESULTS: IKBL+738(C) allele was exclusively increased in patients with extensive and/or intractable disease. HLA-DRB1(*)0103 was the only HLA-DRB1 allele to be significantly increased in frequency in patients with UC compared with controls. It was found in patients with extensive and distal disease. In the HLA-DRB1(*)0103-negative population, patients with extensive disease still had a significant association with IKBL+738(C). The difference between the 2 groups of patients was statistically significant (13.7% vs. 1.7% in patients with distal disease; odds ratio, 9.25; P = 0.01). CONCLUSIONS: HLA-DRB1(*)0103 is associated with susceptibility to ulcerative colitis, and IKBL+738(C) marks a propensity to extensive and more severe disease.

Adaptor Proteins, Signal Transducing↗

Natural selection on mitochondrial DNA in Parus and its relevance for phylogeographic studies.

I examined mitochondrial ND2 sequences in species of tits (genus Parus) to determine whether substitutions were neutral or under selection. Haplotype trees in these species are shallow and geographically unstructured, which could be a signature of recent demographic expansion or purifying selection. McDonald-Kreitman tests revealed a pattern of excess replacement polymorphisms for closely related taxa. Replacement substitutions tended to be less common in the basal parts of haplotype trees, suggesting that they are selected against over evolutionary time. Thus, the pattern of selection is consistent with a model of mildly deleterious haplotypes. To investigate the significance of this selection, the ND2 gene was subdivided into surface and transmembrane portions, and synonymous-non-synonymous sites, and F(ST)-values (the amount of variance distributed among populations) computed for each. Among sampling localities in P. major, both partitions gave similar estimates of F(ST), which is consistent with demographic expansion as the cause of the shallow trees. In P. montanus, two localities contained high percentages of individuals that had a single replacement substitution in the transmembrane portion of the gene, which inflated F(ST)-values relative to the surface sites. Thus, the interpretation of the genetic differentiation of these two sites could be either geographical isolation or selection. Given that the sole substitution causing the high F(ST)-values was a replacement one, selection is implicated. However, this substitution occurred in only 2 of 12 localities, revealing that overall the phylogeographic pattern was not biased by selection, once the cause of the two high pairwise F(ST)-comparisons was recognized. Investigators should consider the source of significant genetic heterogeneity within species, to determine if the variation is likely caused by demographic isolation or selection. However, it seems unlikely that most phylogeographic analyses are invalidated by the effects of natural selection.

Animals↗

Identification of HLA-B*56 variant (B*560502) in the Korean population.

We report here a new HLA-B*56 allele, B*560502, identified by sequencing-based typing in the Korean population. HLA-B*560502 differs from B*560501 by a single nucleotide at position 141 in exon 2 (T(r)C). This single nucleotide substitution may not result in an amino acid difference in the alpha1 domain at residue 23. The putative haplotype involving B*560502 may be A*24-DRB1*1201-DQA1*0503-DQB1*0304-DPB1*0202.

Alleles↗

Examining genetic structure in a bogus yucca moth: a sequential approach to phylogeography.

Understanding the phylogeography of a species requires not only elucidating patterns of genetic structure among populations, but also identifying the possible evolutionary events creating that structure. The use of a single phylogeographic test or analysis, however, usually provides a picture of genetic structure without revealing the possible underlying evolutionary causes. We used current analytical techniques in a sequential approach to examine genetic structure and its underlying causes in the bogus yucca moth Prodoxus decipiens (Lepidoptera: Prodoxidae). Both historical biogeography and recent human transplantations of the moth's host plants provided a priori expectations of the pattern of genetic structure and its underlying causes. We evaluated these expectations by using a progression of phylogenetic, demographic, and population genetic analyses of mtDNA sequence data from 476 individuals distributed across 25 populations that encompassed the range of P. decipiens. The combination of these analyses revealed that much of the genetic structure has evolved more recently than suggested by historical biogeography, has been influenced by changes in demography, and can be best explained by long distance dispersal and isolation by distance. We suggest that performing a suite of analyses that focus on different temporal scales may be an effective approach to investigating the patterns and causes of genetic structure within species.

Animals↗

Incomplete cryptic speciation between intertidal and subtidal morphs of Acrocnida brachiata (Echinodermata: Ophiuroidea) in the Northeast Atlantic.

The brittle-star Acrocnida brachiata (Montagu) lives in sandy-bottom habitat of both intertidal and subtidal zones along the coasts of the northwestern Europe. An allozyme frequency-based survey (five enzyme loci) was combined with a mitochondrial (mt) COI haplotype analysis (598-bp sequences) on 17 populations to trace back past colonization pathways from the actual population structure of the species. Both genetic markers display a sharp genetic break between intertidal (clade I) and subtidal populations (clade S). This break corresponds to an allele frequency inversion at three enzyme loci (Hk, Pgm and Pgi) and a deep divergence of about 20% in mtCOI sequences between most of the intertidal populations and other samples. The geographic distribution of clade I seems to be more restricted than clade S as it is absent from the intertidal of the eastern English Channel and North Sea and may be replaced by clade S in south Brittany. Applying previously published rates of mutation, divergence between the two clades is estimated to pre-date 5 million years ago and may be due to allopatric speciation processes at the Mio-Pliocene transition. The occurrence of putative hybrids in a few localities, however, suggests incomplete cryptic speciation with secondary contact zones. The relative importance of colonization history vs. habitat specialization are discussed in the light of neutral evolution as tested from mtCOI gene sequences. While differential selection seems to have contributed little to the separation of the lineages, it may have played a role in the emergence of adaptive polymorphisms in the hybrid zone. Furthermore, congruent spatial patterns of differentiation were observed in both clades suggesting a recent increase in population size. These findings are in agreement with a recent expansion of the populations during or after the formation of the English Channel, from a southern refuge for the subtidal clade whereas the intertidal clade may have persisted further north. As previously suspected for a species with a very short pelagic larval phase, contemporary gene flow between distant or adjacent populations appears to be extremely reduced or even absent.

Animals↗

Unique epitopes of glutamic acid decarboxylase autoantibodies in slowly progressive type 1 diabetes.

Disease-specific epitope profiles of glutamic acid decarboxylase (GAD)65 autoantibodies (GAD65Ab) were studied in slowly progressive type 1 (insulin-dependent) diabetes mellitus (SPIDDM) and acute onset type 1 (insulin-dependent) diabetes mellitus (AIDDM) using seven kinds of GAD65/67 chimeric molecules. Sera obtained from Japanese SPIDDM (n = 17) and AIDDM (n = 46) patients followed prospectively were analyzed by immunoprecipitation, ELISA, and Western blotting. GAD65Ab in all SPIDDM samples reacted specifically with an N-terminal linear epitope located on the membrane anchoring domain between amino acids 17-51 and C-terminal conformational epitope between amino acids 443-585 of GAD65. The binding of GAD65Ab with N-terminal 83 residues in SPIDDM inversely correlated with the period in which insulin was not required. GAD65Ab in AIDDM did not react with N-terminal epitope located between amino acids 1-83, irrespective of the titer of GAD65Ab. A novel epitope of GAD65Ab in AIDDM residing between amino acids 244-360 was identified in 17% (8 of 46) of patients whose age of onset was younger than other AIDDM patients. In conclusion, GADAb in SPIDDM has unique N-terminal linear epitopes that are located on the anchoring domain of GAD65 molecules. Association is suggested between GAD65Ab targeted to this region and slowly progressive beta-cell failure in SPIDDM.

Autoantibodies↗

A universal T cell epitope-containing peptide from hepatitis B surface antigen can enhance antibody specific for HIV gp120.

Peptide-based vaccines that directly target T cell or B cell epitopes may have significant advantages over conventional vaccines. Further, synthetic chimeric peptides that combine strong T cell epitopes with poorly immunogenic, but immunodominant, B cell epitopes or strain-conserved B cell epitopes may be useful in eliciting antibody to such important regions. Here we characterize a human T cell epitope analyzed in 54 individuals immunized with a hepatitis B virus surface Ag vaccine. Primary cultures from a total of 59 immunized donors were assessed for their ability to respond to hepatitis B virus surface Ag and peptides, and five were non-responders (8.5%). T cell lines were established from the remaining 54 responders. Of the responders, it was found that the peptide representing amino acids 19 through 33 (19-33) elicited significant proliferation in lines derived from 50 donors. This "universal" T cell epitope, which was recognized in donors of many different HLA-DR and -DQ haplotypes, was then used to construct a chimeric peptide containing 19-33 and the third V region loop structure (V3 loop) of HIV-1 envelope gp 120, in an attempt to augment the immune response to the V3 loop peptide. The V3 loop is the region to which significant neutralizing antibody is directed. Thus, a strong immune response to a synthetic peptide that contains the strain-conserved V3 loop region could have significant therapeutic implications. The V3 loop/19-33 peptide was then used to prime mice, to determine whether V3 loop-specific antibody could be induced. The peptide elicited potent 19-33-specific proliferation in T cells isolated from draining lymph nodes, and in six of six mice anti-V3 loop antibody was elicited. Further, V3 loop/19-33-primed animals made significant levels of antibody that bound rgp120. These data suggest that, when a major T cell epitope is synthesized in tandem with the V3 loop, a significant immune response against the loop can be elicited. Thus, given the finding that neutralizing antibody may play a role in the control and/or prevention of HIV infection, an HIV vaccine composed of a T cell epitope-containing peptide may prove effective. In addition, this type of approach can be generalized to the design of peptide-based vaccines.

Amino Acid Sequence↗

[The effect of marriage migration on the genetic structure of the Taimyr Nganasan population: genealogical analysis inferred from MtDNA markers].

The marriage structure of Nganasans during the time period from 1796 to 1991 and genealogy of carriers of mitochondrial DNA haplotypes was studied in a sample of 280 individuals. It was shown that, from the beginning of its formation to the late 1970s, the population exhibited high endogamy (1976, 83.8%; 1926, 88.4%; 1976, 74.3%). The main source of traditional marriage migration (preferentially female) was populations of Entsy and, indirectly, Nentsy. Intense assimilation of Nganasans by the immigrant population, and to a lesser extent, by Dolgans, in the second half of the 20th century resulted in a reduction of endogamy index in Avam Nganasans to 42.5% by 1991. Assimilation by the immigrants was predominantly paternal, promoting preservation of the historically formed genetic diversity of the Nganasan mitochondrial gene pool. Genealogical analysis of mtDNA haplotypes showed that a relatively high total frequency of Western Eurasian mtDNA haplogroups (20.4%) in the Mongoloid (according to anthropological type) Nganasan population is explained not only by the common ethnic origin with Entsy and Nentsy, but also by direct marriage migration from the Entsy population and indirect marriage migration, from the Nentsy population. This migration led to accumulation of Entsy-Nentsy maternal lineages in the genealogy of Avam Nganasans (38.9% of the total number). Of all mtDNA haplotypes, 28.6% were introduced to Avam Nganasans by female Entsy and Nentsy, whereas the total frequency of these haplotypes was 0.204. Genetic diversity of mitochondrial DNA haplotypes was 0.935.

DNA, Mitochondrial↗

Phylogenetic conservation of a class III major histocompatibility complex antigen, factor B. Isolation and nucleotide sequencing of mouse factor B cDNA clones.

The complement protein factor B is a novel serine protease which is encoded within the major histocompatibility complex in man, guinea pig, and mouse. To determine the structure of mouse factor B, cDNA clones were isolated from mouse strains of two different major histocompatibility complex haplotypes, H-2k and H-2d, and clones of 0.9 and 1.5 kilobases, respectively, were sequenced. The H-2d clone includes the H-2k clone sequence and spans 94% of the Bb-coding sequence. No differences in sequence or in restriction enzyme sites were observed between the H-2k and H-2d clones. The H-2d clone displays 83% nucleotide homology and 83% (derived) amino acid homology with that of human factor B; there are no insertions or deletions. Comparison of the mouse and human factor B sequence reveals extensive regional homology at the catalytic residues and in the NH2-terminal portion of the Bb fragment.

Amino Acid Sequence↗

[Genetic studies of panic disorder].

We review the literature on genetic factors in anxiety disorders and present data from a study in which we used the HLA system to evaluate distribution of different antigens in the members of a family with high morbidity of Panic Disorder. Our aim was to look for HLA haplotype sharing among the affected subjects. All members of the family were interviewed with the SCID interview to detect any psychiatric disorder. Third generation members under 18 were evaluated with a structured interview especially designed to identify separation anxiety disorder. In all cases we assessed 11 HLA-A, 16 HLA-B and 5 HLA-Cw antigens. The results suggest a genetic component for Panic Disorder, based on the presence of the same haplotype (A3B18) in the six members of the family suffering from Panic Disorder and Agoraphobia, compared with its absence in the others who were free of such disorders.

Adult↗

Two divergent routes of evolution gave rise to the DRw13 haplotypes.

The HLA class II genes and haplotypes have evolved over a long period of evolutionary time by mechanisms such as gene conversion, reciprocal recombination and point mutation. The extent of the diversity generated is most clearly evident in an analysis of the HLA class II alleles present within DRw13 haplotypes. This study uses cDNA sequencing to examine the first domains of DRB1, DRB3, DQA1, and DQB1 alleles from several American black individuals expressing seven different DRw13 haplotypes, five with undefined HLA-D specificities (i.e., not Dw18 or Dw19). Two new DRw13 alleles described in this study are the first examples of convergent evolution of DR alleles in which gene conversion has apparently combined segments of DRB1 alleles encoding DRw11 and DRw8 to generate two new DRB1 alleles, DRB1*1303 and DRB1*1304, that encode molecules bearing serologic determinants of a third allele, DRw13. These new DRw13 alleles are found embedded in haplotypes of DRw11 origin distinct from haplotypes encoding previously identified DRw13 alleles, DRB1*1301 and DRB1*1302. These data suggest that two evolutionary pathways may have given rise to two subgroups of alleles encoding molecules that share DRw13 serologic determinants yet which possess different structural and, likely, functional motifs. Reciprocal gene recombination events resulting in different DR, DRw52 and DQ allele combinations also appear to have played a crucial role in augmenting the level of diversity found in DRw13 haplotypes. Recombination has resulted in the association of one of the new DRw13 alleles with a DQw2 allele normally found associated with DR7 and the association of the DRw52c-associated DRw13 allele (DRB1*1302) with three different DQw1 alleles. The seven DRw13 haplotypes that have resulted from the effect of recombination on haplotypes formed by the two pathways of DRw13 allelic diversification have resulted in different repertoires of class II molecules and, most likely, different immune response profiles in individuals with these haplotypes.

Alleles↗

Genetic structure and relationships in the snail species complex Littorina arcana hannaford ellis, L. compressa Jeffreys and L. saxatilis (Olivi) in the British isles using SSCPs of cytochrome-b fragments.

Snails of the Littorina saxatilis species complex are ubiquitous and important members of hard shore intertidal communities in the North Atlantic. The complex includes L. arcana, L. compressa and L. saxatilis. We investigated species and population structure among these species from six locations in Ireland and Britain using a nonradioactive single strand conformational polymorphism analysis of a 375 base pair fragment of the cytochrome-b gene. Variability was high with 38 haplotypes found in 591 individuals. The most common haplotype in L. arcana and L. compressa was absent from L. saxatilis and the most common haplotype in L. saxatilis was found in low numbers in L. arcana and L. compressa. Haplotypes restricted to L. arcana and L. compressa formed a cluster separate from L. saxatilis haplotypes in maximum parsimony and multidimensional scaling analyses. In analyses examining populations, L. arcana and L. compressa formed a group separate from L. saxatilis. Both the populations in the L. arcana/L. compressa group and in L. saxatilis show similar geographical structuring in that the North England populations were separated from a cluster of Cornwall (South-west England) and Irish populations. We suggested that Pleistocene sea level changes accounted for these congruent genetic patterns. We concluded that L. arcana and L. compressa are more closely related to each other than either is to L. saxatilis and offer this as a resolution to a previous trichotomy.

Animals↗