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Unexpected genetic and structural relationships of a long-forgotten flavoenzyme to NAD(P)H:quinone reductase (DT-diaphorase)

A mammalian cytosolic FAD-dependent enzyme that catalyzes the reduction of quinones by N-ribosyl- and N-alkyldihydronicotinamides, but not by NADH, NADPH, or NMNH (reduced nicotinamide mononucleotide), was isolated from bovine kidney more than 30 years ago [S. Liao, J. T. Dulaney and H. G. Williams-Ashman (1962) J. Biol. Chem. 237, 2981-2987]. This enzyme is designated here as quinone reductase type 2 (QR2). Bovine QR2 is a homodimer that migrates on SDS/PAGE at approximately 22 kDa. Three tryptic peptides of bovine QR2 (representing 39 amino acids) showed 43% identity to human NAD(P)H:quinone reductase (DT-diaphorase; EC 1.6.99.2), here designated QR1 and 82% identity to a related human cDNA clone [called hNQO2 by A. K. Jaiswal, P. Burnett, M. Adesnik and O. W. McBride (1990) Biochemistry 29, 1899-1906], and designated here as hQR2. The protein encoded by the latter cDNA did not show QR activity when tested with conventional nicotinamide nucleotides. The unexpected high homology between the old flavoenzyme and hQR2 prompted us to clone and overexpress hQR2. The properties of hQR2 were identical to those of the flavoenzyme described by S. Liao and H. G. Williams-Ashman, thus establishing their genetic identity. Recombinant human QR2: (i) reacts with N-ribosyl- and N-alkyldihydronicotinamides, but not with NADH, NADPH, or NMNH; (ii) is very weakly inhibited by dicumarol or Cibacron blue; (iii) is very potently inhibited by benzo[a]pyrene. The x-ray crystal structure of rat QR1 shows that the 43 amino acid C-terminal tail of QR1 provides the binding site for the hydrophilic portions of NADH and NADPH. In the absence of this binding site in QR2, the enzyme retains the essential catalytic machinery, including affinity for FAD, but cannot bind phosphorylated hydride donors.

Amino Acid Sequence↗

Variation of female and male lineages in sub-Saharan populations: the importance of sociocultural factors.

In this paper, we present a study of genetic variation in sub-Saharan Africa, which is based on published and unpublished data on fast-evolving (hypervariable region 1 of mitochondrial DNA and six microsatellites of Y chromosome) and slow-evolving (haplogroup frequencies) polymorphisms of mtDNA and Y chromosome. Our study reveals a striking difference in the genetic structure of food-producer (Bantu and Sudanic speakers) and hunter-gatherer populations (Pygmies, Kung, and Hadza). In fact, the ratio of mtDNA to Y-chromosome Nupsilon is substantially higher in food producers than in hunter-gatherers as determined by fast-evolving polymorphisms (1.76 versus 0.11). This finding indicates that the two population groups differ substantially in female and male migration rate and/or effective size. The difference also persists when linguistically homogeneous populations are used and outlier populations are eliminated (1.78 vs 0.19) or when the jacknife procedure is applied to a paired population data set (1.32 to 7.84 versus 0.14 to 0.66). The higher ratio of mtDNA to Y-chromosome Nnu in food producers than in hunter-gatherers is further confirmed by the use of slow-evolving polymorphisms (1.59 to 7.91 versus 0.12 to 0.35). To explain these results, we propose a model that integrates demographic and genetic aspects and incorporates ethnographic knowledge. In such a model, the asymmetric gene flow, polyginy, and patrilocality play an important role in differentiating the genetic structure of sub-Saharan populations. The existence of an asymmetric gene flow is supported by the phylogeographic features of mtDNA and Y-chromosome haplogroups found in the two population groups. The role of polyginy and patrilocality is sustained by the evidence of a differential pressure of genetic drift and gene flow on maternal and paternal lineages of food producers and hunter-gatherers that is revealed through the analysis of mitochondrial and Y-chromosomal intrapopulational variation.

Africa South of the Sahara↗

A chemical, genetic, and structural analysis of the nuclear bile acid receptor FXR.

The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.

Amino Acid Sequence↗

Genetic consequences of human management in an introduced island population of red deer (Cervus elaphus).

We investigated phylogeography and spatial genetic structure in an introduced island population of red deer (Cervus elaphus) on the Isle of Rum, Scotland, experiencing spatial variation in management regime. Five different mitochondrial DNA (mtDNA) haplotypes were present among female red deer on Rum. These comprised two phylogenetically divergent groups, one of which clustered with red deer from Sardinia and North Africa, while the other four grouped with other Western European red deer. Recent and historical red deer management practices explain this result. The Rum population is descended from recent introductions from at least four different UK mainland populations, and translocation of red deer within the UK and across Europe is well documented. We found significant spatial genetic structure across Rum in both mtDNA haplotypes and microsatellite markers. Mitochondrial spatial structure was over an order of magnitude greater than structure in nuclear markers. This extreme difference is explained by the fact that the Rum population was introduced from different source populations, the highly male-biased dispersal patterns of red deer and the much smaller effective population size of mitochondrial compared to nuclear markers. Spatial structure in mtDNA conformed to a pattern of isolation by distance, while nuclear DNA did not. Apparent structure in the nuclear markers was driven by differences between the North Block and the rest of the island. We suggest that recent differences in the management regimes in different parts of the island have led to differences in effective male migration that would account for this observation.

Animal Migration↗

Structural and genetic studies on chicken 7S immunoglobulin allotypes. II. Distribution of allotypes on the 7S immunoglobulin of homozygous and heterozygous chickens.

We have previously reported that chicken 7S immunoglobulin (Ig) heavy (H) chain allotypes (CS-1 locus) segregate as phenogroups in F2 progeny. Specificity CS-1.1 formed a phenogroup with CS-1.4 in inbred chicken line UCD 2, and a second phenogroup with CS-1.3 in line UCD 3. To determine whether these phenogroups were formed by combinations of specificities on the same, or on separate subclasses of 7S Ig, their distribution on the 7S Ig molecules of birds homozygous for 7S Ig allotypes was analyzed by radioimmunoassay. Anti-CS-1.1 and anti-CS-1.3 alloantisera each bound more than 94% of line UCD 3 1252-7S Ig. Similar results were obtained with alloantisera to CS-1.1 and CS-1.4 WITH 125 I-7S Ig from line UCD 2. These results indicate that both phenogroups were formed by combinations of specificities present on the same H chain. Thus, each phenogroup described, probably is the product of a single structural gene which is responsible for more than 94% of the 7S Ig H chain constant regions. In F hybrids with the genotype CS-1.3, 1.3/CS-1.2, two populations of serum 7S Ig molecules were detected by direct and sequential binding analysis with specific alloantisera. One population of 7S Ig contained specificities CS-1.1 AND CS-1.3, but not CS-1.2; while the second population was exclusively the product of one parental allele. Consistent with a genetic regulatory mechanism involving allelic exclusion, no MS Ig containing allotypes produced by both alleles was detected.

Animals↗

Thysanophora penicillioides includes multiple genetically diverged groups that coexist respectively in Abies mariesii forests in Japan.

We investigated intraspecific diversity and genetic structures of a saprotrophic fungus--Thysanophora penicillioides--based on sequences of nuclear ribosomal internal transcribed spacer (ITS) in 15 discontinuous Abies mariesii forests of Japan. In such a well-defined morphological species, numerous unexpected ITS variations were revealed: 12 ITS sequence types detected in 254 isolates collected from 15 local populations were classified into five ITS sequence groups. Maximally, four ITS groups consisted of seven ITS types coexisting in one population. However, group 1 was dominant with approximately 65%; in particular, one haplotype, 1a, was most dominant with approximately 60% in respective populations. Therefore, few differences were recognized in genetic structure among local populations, implying that the gene flow of each lineage of the fungus occurs among local populations without geographic limitations. However, minor haplotypes in some ITS groups were found only in restricted areas, suggesting that they might expand steadily from their places of origin to neighboring A. mariesii forests. Aggregating sequence data of seven European strains and four North American strains from various substrates to those of Japanese strains, 18 ITS sequence types and 28 variable sites were recognized. They were clustered into nine lineages by phylogenetic analyses of the beta-tubulin and combined ITS and beta-tubulin datasets. According to phylogenetic species recognition by the concordance of genealogies, respective lineages correspond to phylogenetic species. Plural phylogenetic species coexist in a local population in an A. mariesii forest in Japan.

Abies↗

A comparison of neurological, metabolic, structural, and genetic evaluations in persons at risk for Huntington's disease.

We compared four diagnostic data sets for the assessment of individuals at risk for Huntington's disease. Fifty-four chorea-free persons were evaluated by neurological examination, positron emission tomography measurement of glucose metabolism, radiographic computerized tomographic measurement of caudate size, and genetic testing at the polymorphic DNA loci D4S10, D4S43, and D4S125. Twelve (22%) persons had abnormal caudate metabolism, 6 (11%) had subtle abnormalities of motor control, and 7 (13%) had computed tomographic evidence of caudate atrophy, compared with an expected gene frequency of 34% for this population. In 20 persons with unambiguous genetic test results or the subsequent phenotypic expression of Huntington's disease (chorea), there was a greater sensitivity of the positron emission tomographic measurement of caudate metabolism (75%) relative to computed tomography (33%) or the clinical examination (17%) for the determination of a subpopulation of probable Huntington's disease gene carriers. Hypometabolism of the putamen and globus pallidus, and hypermetabolism of the precentral gyrus were also associated with a high probability of carrying the Huntington's disease gene. The findings support the hypothesis that abnormalities of cerebral metabolism precede clinical or structural (computed tomographic) abnormalities in gene-positive individuals at risk for Huntington's disease.

Atrophy↗

Gene flow between natural and domestic populations of Lutzomyia longipalpis (Diptera: Psychodidae) in a restricted focus of American visceral leishmaniasis in Venezuela.

The epidemiology of the visceral leishmaniasis in the Americas is associated with both a natural and a domestic cycle. The existence of reproductively isolated populations of Lutzomyia longipalpis (Lutz & Neiva), and the scarcity of records of this species from natural habitats in areas where it has been associated with domestic habitats indicated that natural populations could be genetically distinct from domestic ones. Therefore, we compared the genetic structure and estimated the gene flow between L. longipalpis from domestic and peridomestic habitat and from an adjacent undisturbed natural environment along a 1.2-km transect. The analyses were performed on electrophoretic data from eight isozyme loci. The absence of fixed differences in the diagnostic loci Ak and Hk indicated that all specimens belonged to one of the two cryptic species identified in Venezuela. The average number of alleles per locus ranged from 2.0 to 2.9 and the average heterozygosity ranged from 7.8 to 13.4%. No differences were detected in the genetic structure of this species from domestic or peridomestic habitats and those trapped as far as 1.2 km from human dwellings. Nm, estimated from Wright's Fst, indicated that at least 208 individuals per generation migrated between the peridomestic habitat and a 1.2-km distant point to maintain the observed similarities in allelic frequencies. This high rate of gene flow indicated that this species has high migration rates between domestic and natural environments, and has the potential to transport for Leishmania from natural to domestic environments.

Adenylate Kinase↗

A human pancreatic islet inwardly rectifying potassium channel: cDNA cloning, determination of the genomic structure and genetic variations in Japanese NIDDM patients.

Ligand gated potassium channels, such as the ATP-regulated potassium channel, play crucial roles in coupling of stimuli to insulin secretion in pancreatic beta cells. Mutations in the genes might lead to the insulin secretory defects observed in patients with non-insulin-dependent diabetes mellitus (NIDDM). We isolated a cDNA encoding a putative subunit of a ligand gated potassium channel from a human islet cDNA library. The channel, which we designated hiGIRK2, appeared to be an alternative spliced variant and a human homologue of recently reported mbGIRK2, KATP-2/BIR1. Transcripts were detected in human brain and pancreas, but not in other tissues including cardiac muscle. The sizes of transcripts in the pancreas differed from those in the brain, suggesting tissue-specific alternative splicing and possible isoforms. We then isolated human genomic clones, determined the complete genomic structure and localized the gene to chromosome 21 (21q22). The gene was comprised of four exons and the protein was encoded by three exons. The entire coding region of the hiGIRK2 gene was scanned by polymerase chain reaction-single strand conformation polymorphism analysis in 80 Japanese NIDDM patients. We found five nucleotide substitutions; three were silent mutations of the third base of codons, one in the first intron, 9 bases upstream of exon 2, and one in the 3'-untranslated region. We conclude that mutations in the gene encoding hiGIRK2, a (subunit of) ligand gated potassium channel, is not a major determinant of the susceptibility to NIDDM in Japanese.

Alternative Splicing↗

Genetic population structure of the prosobranch snail Potamopyrgus antipodarum (Gray) in Denmark using PCR-RAPD fingerprints.

Parthenogenetic species are often more widely distributed geographically than their sexual relatives. This success in colonizing can be explained either by dispersal of one or a few clones of wide physiological tolerance or by the distribution of many locally adapted clones. Here we test the hypothesis that successfully invading clones of Potamopyrgus antipodarum (Gray) are composed of a few broadly adapted genotypes by using polymerase chain reaction random amplified polymorphic DNA (PCR-RAPD) fingerprinting on six different populations of P. antipodarum from Denmark and three morphotypes of P. antipodarum from Britain. We detected two genotypes of P. antipodarum in six populations examined across Denmark using four decamer primers. The two genotypes were found to be morphologically and genetically indistinguishable from British P. antipodarum. In five of the six Danish populations only one genotype was found; at the remaining site, the two genotypes occurred sympatrically. The present study suggests that P. antipodarum successfully invaded Europe by the proliferation of very few clones.

Animals↗

Identification of bacterial groups preferentially associated with mycorrhizal roots of Medicago truncatula.

The genetic structures of bacterial communities associated with Medicago truncatula Gaertn. cv. Jemalong line J5 (Myc+ Nod+) and its symbiosis-defective mutants TRV48 (Myc+ Nod-) and TRV25 (Myc- Nod-) were compared. Plants were cultivated in a fertile soil (Châteaurenard, France) and in soil from the Mediterranean basin showing a low fertility (Mas d'Imbert, France). Plant growth, root architecture, and the efficiency of root symbiosis of the three plant genotypes were characterized in the two soils. Structures of the bacterial communities were assessed by automated-ribosomal intergenic spacer analysis (A-RISA) fingerprinting from DNA extracted from the rhizosphere soil and root tissues. As expected, the TRV25 mutant did not develop endomycorrhizal symbiosis in any of the soils, whereas mycorrhization of line J5 and the TRV48 mutant occurred in both soils but at a higher intensity in the Mas d'Imbert (low fertility) than in the Châteaurenard soil. However, modifications of plant growth and root architecture, between mycorrhizal (J5 and TRV48) and nonmycorrhizal (TRV25) plants, were recorded only when cultivated in the Mas d'Imbert soil. Similarly, the genetic structures of bacterial communities associated with mycorrhizal and nonmycorrhizal plants differed significantly in the Mas d'Imbert soil but not in the Châteaurenard soil. Multivariate analysis of the patterns allowed the identification of molecular markers, explaining these differences, and markers were further sequenced. Molecular marker analysis allowed the delineation of 211 operational taxonomic units. Some of those belonging to the Comamonadaceae and Oxalobacteraceae (beta-Proteobacteria) families were found to be significantly more represented within bacterial communities associated with the J5 line and the TRV48 mutant than within those associated with the TRV25 mutant, indicating that these bacterial genera were preferentially associated with mycorrhizal roots in the Mas d'Imbert soil.

Betaproteobacteria↗

Population structure and genetic diversity of Actinobacillus actinomycetemcomitans strains isolated from localized juvenile periodontitis patients.

The phylogeny of 20 Actinobacillus actinomycetemcomitans strains isolated from patients with localized juvenile periodontitis (LJP) was investigated by using partial sequence analysis of 16S rRNA genes, arbitrarily primed PCR (AP-PCR), and four additional PCR assays that amplified polymorphic regions in the leukotoxin (lkt), cytolethal distending toxin (cdt), major fimbrial subunit (flp-1), and serotype-specific O polysaccharide gene clusters. Our analysis also included four strains isolated from healthy subjects and nine reference strains. We found that A. actinomycetemcomitans strains comprised three major phylogenetic lineages. One lineage consisted of serotype b strains, a second lineage consisted of serotype c strains, and a third lineage consisted of serotype a, d, e, and f strains. 16S rRNA sequences within each lineage were highly conserved (<1% base substitutions), whereas sequences between lineages were exceptionally divergent (1.9 to 5.0% substitutions). Two strains exhibited 16S rRNA sequences that were even more distantly related to those of the three major lineages (2.7 to 6.7% substitutions), indicating that additional minor lineages or variants exist. The distribution of 16S rRNA sequences and lkt, cdt, flp-1, and AP-PCR genotypes was consistent with a clonal population structure, with little evidence of assortative recombination between strains of different serotypes. Strains from all three major lineages were recovered from LJP patients, suggesting that phylogenetically diverse strains of A. actinomycetemcomitans carry pathogenic potential.

Actinobacillus Infections↗

Mitochondrial DNA polymorphisms among Hindus: a comparison with the Tharus of Nepal.

The polymorphisms of mitochondrial DNA for the restriction enzymes HpaI, BamHI, HaeII, MspI, AvaII and HinecII were studied in a sample of 79 Hindus, 45 from New Delhi (India) and 34 from Terai (Nepal), both to characterize another Caucasian population and to investigate some possible Hindu component in the genetic structure of the Tharus, a Nepalese population, the anthropological position of which is still disputed. 1. A new BamHI polymorphism was detected: about 5% of the Hindu mtDNAs have lost the site at 14258 bp and lack any BamHI site. Once again a BamHI polymorphism was found in a Caucasian population. 2. New site mutations were found to yield morphs previously described (MspI-7, AvaII-18). 3. Variant morphs for two different enzymes were found due to a shared mutation (morphs BamHI-0/AvaII-30 and morphs MspI-7Hindu/AvaII-18Hindu). 4. Comparison between Hindu and Tharu data does not show any evidence of a specific Indian component in the Tharu genetic structure and allows us to conclude that Tharus are clearly differentiated from modern Hindus.

Adult↗

Further genetic studies on the Katsunuma population of Drosophila melanogaster.

Changes in the genetic structure of the Katsunuma natural population of Drosophila melanogaster have been examined during the past 35 years. The frequency of recessive lethal genes on the second chromosome once increased from 15% to 30% in the early 1970s, then decreased to about 24% in the late 1970s, and thereafter showed no significant changes. Sterility genes, the frequency of which is always less than the lethals, showed a similar tendency. The SD (segregation distorter) mutant gene disappeared but some others such as rbl (reduced bristle) and bw (brown) persisted in the population. The frequency of inversion-carrying chromosomes gradually decreased in the period, such that the standard chromosome frequency in the second and third chromosomes increased from about 40% to more than 80%. Coincident with these frequency changes is the invasion of a transposable element P into the Katsunuma population. The P element should have invaded into Katsunuma in the late 1960s. It spread over the population apparently inducing deleterious mutations, causing the decrease in the allelism rate, and hence increasing the effective population size. Soon, however, most flies became resistant to the P element-mediated transposition as they began to harbor defective P elements. During the course of spreading, the P element must also have induced deleterious mutations on the polymorphic inversions, breaking up the heterotic gene complexes along the chromosomes, which probably caused the reduction in the frequency of inversion chromosomes. Temporal invasion of D. simulans, a sibling species of D. melanogaster, into Katsunuma occurred several times after 1978, and the species seems to have been settled since 1990. This, however, did not have any effect on the genetic structure of D. melanogaster population.

Animals↗

Structural and genetic analysis of laminin-nidogen interaction.

High-affinity binding of nidogen to laminins involves a single binding site on the laminin gamma 1 chain and is thus a property shared by almost all laminin isoforms. This binding mediates the connection of laminins to the collagen IV network, perlecan and other proteins and is considered to be an essential step in the stabilization of basement membranes. Nidogen binding has been located to a single LE module (gamma 1III4) by recombinant analysis. Site-directed mutagenesis and X-ray crystallography demonstrated that three amino acids (Asp, Asn, Val) in loop a of gamma 1III4 are crucial for binding and are supported by some other residues. A restricted complementary binding region seems to exist on nidogen domain G3. A mutant laminin gamma 1 chain gene that lacks the region encoding gamma 1III4 was prepared in mouse embryonic stem (ES) cells by homologous recombination. ES cells homozygous for this defect were shown to assemble laminin-1 into a cruciform structure and to secrete it properly. Yet the mutant laminin failed to associate with nidogen. The mutant ES cells were still able to form embryoid bodies with a similar differentiated histology as the wild type. Immunofluorescence, however, indicated an impaired deposition of nidogen into basement membrane-like structures.

Amino Acid Sequence↗

A genetic fine structure analysis of the suppressor 3 locus in Saccharomyces.

A meiotic fine structure map of a yeast tyrosine-inserting ochre suppressor, SUP3-omicron, was constructed. This was accomplished by examining ten intragenic suppressor-inactive revertants for their relationship to each other and to the original SUP3-omicron mutation. The second-site revertants map on both sides of the SUP3-omicron mutation. The meiotic map length based on the summation of short intervals is 45+/10(5) asci.

Chromosome Mapping↗

Structural and genetic heterogeneity of the receptor mediating translocation of immunoglobulin A dimer antibodies across epithelia in the rabbit.

Secretory component (SC), synthesized as a transmembrane protein, acts as the receptor that binds IgA dimers and mediates their transepithelial transport. Cleavage of the receptor (membrane SC) apparently occurs during transport and a fragment, the secreted form, is generated, which remains tightly bound to the IgA dimer. In the rabbit, variation in the size of membrane SC is observed with both a high and a low molecular weight family, each composed either of two or of four distinct polypeptides depending on the individual rabbit. The same degree of size heterogeneity is observed for secreted SC. Part of this size heterogeneity is related to genetic polymorphism. The milk of individual rabbits typed with anti-SC-allotype sera reveals three different banding patterns. The simplest pattern, found in t61/t61 and t62/t62 homozygotes, consists of an upper and a lower doublet. Since each band of these two doublets in the t62 allotype migrates slightly faster than its counterpart in the t61 allotype, a composite pattern is observed in the heterozygotes (t61/t62). Within a given allotypic group, all SC polypeptide chains expressed the identical allotypic specificity. The 2000 to 4000 difference in molecular weight between the two forms of a doublet probably reflects differences in the number of glycosylated asparagine residues, since individual bands of a doublet show identical peptide maps. High and low molecular weight families are also structurally related to each other as shown by one-dimensional peptide maps and identical NH2- and COOH-terminal amino acid sequences. These results indicate that the 25-kDa size difference between SC from the high and low molecular weight families reflects an intramolecular deletion.

Amino Acid Sequence↗

Impact of crop management on intraspecific diversity of Pseudomonas corrugata in bulk soil.

The genetic structure of Pseudomonas corrugata populations was investigated in bulk soil to evaluate the impact of crop management on the intraspecific diversity of this bacterium stimulated in plant rhizosphere. As these bacteria are rare in bulk soil, an immunotrapping assay was developed to isolate them from soils located in Grignon (France), where adjacent plots with similar soil features were cultivated under maize/wheat crop rotation or continuous wheat cropping. Genomic fingerprinting of 291 isolates was performed using repetitive sequence-based polymerase chain reaction (ERIC-PCR). Most of these isolates (236 out of 291) were assigned to P. corrugata on the basis of an amplified ribosomal DNA restriction analysis (ARDRA) with 12 restriction enzymes. Data were analyzed by Pearson's chi-squared tests and genetic diversity was evaluated with specific indices for richness and evenness. Comparison of richness or evenness diversity indices (global diversity) showed no significant difference, whereas ERIC-PCR fingerprinting data (intraspecific diversity) showed that the genetic structure of P. corrugata was significantly affected by crop management.

Journal Article↗