A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase.

NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is a FAD-containing reductase that catalyzes a unique 2-electron reduction of quinones. It consists of 2 identical subunits. In this study, it was found that the carboxyl-terminal portion of the 2 subunits can be cleaved by various proteases, whereas the amino-terminal portion cannot. It was also found that proteolytic digestion of the enzyme can be blocked by the prosthetic group FAD, substrates NAD(P)H and menadione, and inhibitors dicoumarol and phenindione. Interestingly, chrysin and Cibacron blue, 2 additional inhibitors, cannot protect the enzyme from proteolytic digestion. The results obtained from this study indicate that the subunit of the quinone reductase has a 2-domain structure, i.e., an amino-terminal compact domain and a carboxyl-terminal flexible domain. A structural model of the quinone reductase is generated based on results obtained from amino-terminal and carboxyl-terminal protein sequence analyses and electrospray mass spectral analyses of hydrolytic products of the enzyme generated by trypsin, chymotrypsin, and Staphylococcus aureus protease. Furthermore, based on the data, it is suggested that the binding of substrates involves an interaction between 2 structural domains.     

Population structure in two sympatric species of the Lake Tanganyika cichlid tribe Eretmodini: evidence for introgression

Patterns of genetic differentiation were analysed and compared in two sympatric species of the endemic Lake Tanganyika cichlid tribe Eretmodini by means of mitochondrial DNA (mtDNA) sequences of the control region and six microsatellite DNA loci. The sample area covers a total of 138 km of mostly uninterrupted rocky shoreline in the Democratic Republic of Congo and includes the entire distribution range of Tanganicodus cf. irsacae that stretches over a distance of 35 km. Both markers detected significant genetic differentiation within and between the two species. T. cf. irsacae contained lower overall genetic variation than Eretmoduscyanostictus, possibly due to its more restricted range of distribution and its smaller effective population sizes. Complete fixation of Tanganicodus mtDNA haplotypes was observed in Eretmodus at two localities, while at two other localities some Tanganicodus individuals possessed Eretmodus mtDNA haplotypes. Taking into account the relatively large average sequence divergence of 6.2% between the two species, as well as the geographical distribution of mtDNA haplotypes in the lake, the observed pattern is more likely to be a consequence of asymmetric introgression than of shared ancestral polymorphism. As there is significant population differentiation between sympatric Tanganicodus and Eretmodus populations, the events of introgressions may have happened after secondary contact, but our data provide no evidence for ongoing gene flow and suggest that both species are reproductively isolated at present time.     

Discordant patterns of population structure for two co‐distributed snake species across a fragmented Ontario landscape

Aim The goal of our study was to investigate the effects of a fragmented landscape on the genetic population structure of two sympatric snake species that differ in habitat preference. The eastern garter snake (Thamnophis sirtalis sirtalis) is a common, habitat generalist, whereas the endangered eastern foxsnake (Mintonius [Elaphe] gloydi) is rarer, geographically restricted, and a marsh‐specialist. We were most interested in comparing the genetic population structure of both species and identifying any natural and human‐created features of the landscape that overlap with genetic disjunctions. Location Southwestern Ontario, Canada, surveying over half of the remaining range of the eastern foxsnake. Methods We utilized DNA microsatellite markers to examine genetic population structure of both species. The number of genetically distinct clusters for each species was determined using both Bayesian spatial assignment and spatial principal component analyses (sPCA). Genetic clusters were overlaid onto a habitat map to deduce possible physiognomic barriers to gene flow. Results Spatial assignment revealed three genetic clusters for garter snakes and five for foxsnakes. Each individual garter snake had a near equal probability of membership to two or more clusters with no cluster mapping onto a discrete geographic region, indicating that garter snakes comprise a single genetic population. The identified foxsnake clusters correspond to geographically circumscribed locations on the landscape, roughly coincident with isolated patches of suitable habitat. sPCAs revealed significant global allelic structure for foxsnakes, but not for garter snakes. No significant local structure was found for either species. Main Conclusions Our results imply that foxsnakes and garter snakes are differentially impacted by the same landscape or have dramatically different effective population sizes. Unsuitable intervening habitat such as agricultural tracts and roads between existing populations of foxsnakes appears to act as barriers to gene flow, while garter snake movement appears unrestricted by these features. Our findings have important implications for the management of eastern foxsnakes.     

Morphological and molecular evidence for two new species of Tetragonopterus (Characiformes: Characidae) from central Brazil

The combination of morphological and molecular data of Tetragonopterus species collected in the Rio Araguaia basin allows the recognition of two undescribed species that are presented in this article. These species are distinguished from their congeners (Tetragonopterus anostomus, Tetragonopterus argenteus, Tetragonopterus carvalhoi, Tetragonopterus chalceus and Tetragonopterus rarus) by characters related to the number and morphology of the teeth, the numbers of gill rakers on the upper and lower limbs of the first gill arch, the number of predorsal scales and the overall colour pattern. In addition, the analysis of mitochondrial DNA sequences identified an accentuated genetic distance between these two new species and their congeners. A discussion of the phylogenetic relationships within Tetragonopterus is provided.     

Defining the bacteriophage T4 DNA packaging machine: evidence for a C-terminal DNA cleavage domain in the large terminase/packaging protein gp17

Double-stranded DNA packaging in bacteriophage T4 and other viruses occurs by translocation of DNA into an empty prohead by a packaging machine assembled at the portal vertex. Coordinated with this complex process is the cutting of concatemeric DNA to initiate and terminate DNA packaging and encapsidate one genome-length viral DNA. The catalytic site responsible for cutting, and the mechanisms by which cutting is precisely coordinated with DNA translocation remained as interesting open questions. Phage T4, unlike the phages with defined ends (e.g. lambda, T3, T7), packages DNA in a strictly headful manner, and exhibits no strict sequence specificity to initiate or terminate DNA packaging. Previous evidence suggests that the large terminase protein gp17, a key component of the T4 packaging machine, possesses a non-specific DNA cutting activity. A histidine-rich metal-binding motif, H382-X(2)-H385-X(16)-C402-X(8)-H411-X(2)-H414-X(15)-H430-X(5)-H436, in the C-terminal half of gp17 is thought to be involved in the terminase cleavage. Here, exhaustive site-directed mutagenesis revealed that none of the cysteine and histidine residues other than the H436 residue is critical for function. On the other hand, a cluster of conserved residues within this region, D401, E404, G405, and D409, are found to be critical for function. Biochemical analyses showed that the D401 mutants exhibited a novel phenotype, showing a loss of in vivo DNA cutting activity but not the DNA packaging activity. The functional nature of the critical residues and their disposition in the conserved loop region between two predicted beta-strands suggest that these residues are part of a metal-coordinated catalytic site that cleaves the phosphodiester bond of DNA substrate. The data suggest that the T4 terminase consists of at least two functional domains, an N-terminal DNA-translocating ATPase domain and a C-terminal DNA-cutting domain. Although the DNA recognition mechanisms may be distinct, it appears that T4 and other phage terminases employ a common catalytic paradigm for phosphodiester bond cleavage that is used by numerous nucleases.     

Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site

The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.     

When east meets west: population structure of a high-latitude resident species,the boreal chickadee (Poecile hudsonicus)

The population genetic structure of northern boreal species has been strongly influenced both by the Quaternary glaciations and the presence of contemporary barriers, such as mountain ranges and rivers. We used a combination of mitochondrial DNA (mtDNA), nuclear microsatellites and spatial distribution modelling to study the population genetic structure of the boreal chickadee (Poecile hudsonicus), a resident passerine, and to investigate whether historical or contemporary barriers have influenced this northern species. MtDNA data showed evidence of eastern and western groups, with secondary admixture occurring in central Canada. This suggests that the boreal chickadee probably persisted in multiple glacial refugia, one in Beringia and at least one in the east. Palaeo-distribution modelling identified suitable habitat in Beringia (Alaska), Atlantic Canada and the southern United States, and correspond to divergence dates of 60–96 kya. Pairwise F_ST values for both mtDNA and microsatellites were significant for all comparisons involving Newfoundland, though mtDNA data suggest a more recent separation. Furthermore, unlike mtDNA data, nuclear data support population connectivity among the continental populations, possibly due to male-biased dispersal. Although both are significant, the isolation-by-distance signal is much stronger for mtDNA (r²=0.51) than for microsatellites (r²=0.05), supporting the hypothesis of male-biased dispersal. The population structure of the boreal chickadee was influenced by isolation in multiple refugia and contemporary barriers. In addition to geographical distance, physical barriers such as the Strait of Belle Isle and northern mountains in Alaska are restricting gene flow, whereas the Rocky Mountains in the west are a porous barrier.     

Genetic structure of the beaked whale genus Berardius in the North Pacific,with genetic evidence for a new species

There are two recognized species in the genus Berardius, Baird's and Arnoux's beaked whales. In Japan, whalers have traditionally recognized two forms of Baird's beaked whales, the common “slate‐gray” form and a smaller, rare “black” form. Previous comparison of mtDNA control region sequences from three black specimens to gray specimens around Japan indicated that the two forms comprise different stocks and potentially different species. We have expanded sampling to include control region haplotypes of 178 Baird's beaked whales from across their range in the North Pacific. We identified five additional specimens of the black form from the Aleutian Islands and Bering Sea, for a total of eight “black” specimens. The divergence between mtDNA haplotypes of the black and gray forms of Baird's beaked whale was greater than their divergence from the congeneric Arnoux's beaked whale found in the Southern Ocean, and similar to that observed among other congeneric beaked whale species. Taken together, genetic evidence from specimens in Japan and across the North Pacific, combined with evidence of smaller adult body size, indicate presence of an unnamed species of Berardius in the North Pacific.     

The crystal structure of a (-) gamma-lactamase from an Aureobacterium species reveals a tetrahedral intermediate in the active site

The structure of the recombinant (-) gamma-lactamase from an Aureobacterium species has been solved at 1.73A resolution in the cubic space group F23 with unit cell parameters a=b=c=240.6A. The trimeric enzyme has an alpha/beta hydrolase fold and closely resembles the cofactor free haloperoxidases. The structure has been solved in complex with a covalently bound ligand originating from the host cell and also in the unligated form. The associated density in the former structure has been interpreted as the two-ring ligand (3aR,7aS)-3a,4,7,7a-tetrahydro-benzo [1,3] dioxol-2-one which forms a tetrahedral complex with OG of the catalytic Ser98. Soaks of these crystals with the industrial substrate gamma-lactam or its structural analogue, norcamphor, result in the displacement of the ligand from the enzyme active site, thereby allowing determination of the unligated structure. The presence of the ligand in the active site protects the enzyme from serine hydrolase inhibitors. Cyclic ethylene carbonate, the first ring of the ligand, was shown to be a substrate of the enzyme.     

Primary structure of a chitinase-encoding gene (chi1) from the filamentous fungus Aphanocladium album: similarity to bacterial chitinases.

Chitinase 1 (Chil) is the major extracellular chitinase from the hyperparasitic fungus, Aphanocladium album. We determined the complete sequence of the chromosomal and cDNA copies of the structural gene (chi1) coding for Chil. The coding region is interrupted by three short introns (55, 53 and 49 bp long). Chil is 423 aa long and begins with a stretch of 34 aa not found in the mature protein. The Chil sequence presents overall similarities with bacterial chitinases from Serratia marcescens and Bacillus circulans. Compared with other chitinases, A. album Chi1 has only two short similarity regions (12 and 8 aa long), which are also found in bacterial, yeast and some plant chitinases.     

Phylogeography of two European Reticulitermes (Isoptera) species: the Iberian refugium

Chemical, i.e. cuticular hydrocarbons, and molecular data were used to probe the phylogeography of Reticulitermes termites collected from various parts of France, Spain and Portugal. Phylogenetic relationships were inferred from sequences of the internal transcribed spacer (ITS2) of nuclear ribosomal RNA genes as well as from two partial mitochondrial DNA segments, the cytochrome oxidase II gene and a sequence combining the tRNA-Leu gene and fragments of the NADH dehydrogenase I and ribosomal 16S genes. Two species, namely, R. grassei and R. banyulensis, were identified based on an analysis of cuticular hydrocarbons and the identification was confirmed by ITS2 haplotyping. However, phylogeny based on the analysis of mitochondrial DNA was not completely in agreement with the conclusions drawn from the chemical and nuclear data. An analysis of 56 R. grassei colonies revealed intraspecific differentiation into two major lineages with distinct geographical ranges. Whereas analysis of cuticular hydrocarbons showed that R. banyulensis was chemically distinct from R. grassei, analysis of mitochondrial DNA showed its close kinship with the R. grassei lineage occurring in southern Spain. This kinship could be explained by their evolution from a common polymorphic ancestor species in this ice age refugium.     

Crystal structure of a metal-dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis: Computational prediction and experimental validation of phosphoesterase activity

The crystal structures of an unliganded and adenosine 5′‐monophosphate (AMP) bound, metal‐dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis are reported at 2.4 and 1.94 Å, respectively. Functional characterization of this enzyme was guided by computational analysis and then confirmed by experiment. The structure consists of a polymerase and histidinol phosphatase (PHP, Pfam: PF02811) domain with a second domain (residues 105‐178) inserted in the middle of the PHP sequence. The insert domain functions in binding AMP, but the precise function and substrate specificity of this domain are unknown. Initial bioinformatics analyses yielded multiple potential functional leads, with most of them suggesting DNA polymerase or DNA replication activity. Phylogenetic analysis indicated a potential DNA polymerase function that was somewhat supported by global structural comparisons identifying the closest structural match to the alpha subunit of DNA polymerase III. However, several other functional predictions, including phosphoesterase, could not be excluded. Theoretical microscopic anomalous titration curve shapes, a computational method for the prediction of active sites from protein 3D structures, identified potential reactive residues in YP_910028.1. Further analysis of the predicted active site and local comparison with its closest structure matches strongly suggested phosphoesterase activity, which was confirmed experimentally. Primer extension assays on both normal and mismatched DNA show neither extension nor degradation and provide evidence that YP_910028.1 has neither DNA polymerase activity nor DNA‐proofreading activity. These results suggest that many of the sequence neighbors previously annotated as having DNA polymerase activity may actually be misannotated. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Microsatellite markers for two species of dampwood termites in the genus Zootermopsis (Isoptera: Termopsidae)

Dampwood termites in the genus Zootermopsis inhabit forested areas in western North America. To better understand the colony composition and breeding structure of Zootermopsis, we identified polymorphic microsatellite loci to use in population analysis. Microsatellite loci were isolated from Zootermopsis nevadensis nevadensis (Hagen); however, all primers amplified homologous loci in Zootermopsis angusticollis (Hagen) and Zootermopsis nevadensis nuttingi (Hagen). Twelve loci were polymorphic in one or more of the above subspecies and species. The number of alleles per locus ranged from one to six, with some allelic differences among subspecies and species. We are currently utilizing the microsatellite markers to investigate the population genetics of Zootermopsis.     

Parentage of endemic Sorbus L. (Rosaceae) species in the British Isles: evidence from plastid DNA

The parentage of polyploid Sorbus species in the British Isles was investigated using plastid DNA microsatellites. Four hundred and fifty-three samples from 30 taxa were screened using six microsatellite fragments, which gave 28 haplotypes. The haplotypes formed groups clearly related to the ancestral diploids Sorbus aria , Sorbus aucuparia , and Sorbus torminalis . Species in the Sorbus aria group all had Aria haplotypes (with the exception of one English S. aria ), species in the Sorbus anglica group had an Aucuparia haplotype, and species in the Sorbus latifolia group had a Torminalis haplotype. Sorbus intermedia had an Aucuparia haplotype. This indicated that the hybridization events that led to the formation of species in the S. anglica and S. latifolia groups usually did so with S. aria s.l . as the pollen-donating (paternal) parent. The polyploids S. anglica , Sorbus bristoliensis , Sorbus croceocarpa , Sorbus decipiens , Sorbus devoniensis , Sorbus hibernica , Sorbus lancastriensis , Sorbus leptophylla , Sorbus leyana , Sorbus minima , Sorbus rupicola , Sorbus subcuneata , Sorbus vexans , Sorbus whiteana , Sorbus wilmottiana , and three unnamed taxa may each be derived from a single maternal lineage. The polyploids Sorbus eminens , Sorbus porrigentiformis , and S. latifolia have multiple maternal lineages. The two primary diploid hybrids S . ×  thuringiaca and S . ×  vagensis have arisen many times independently.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 291–304.     

First evidence of cryptic species diversity and significant population structure in a widespread freshwater nematode morphospecies (Tobrilus gracilis)

Free‐living nematodes are ubiquitous and highly abundant in terrestrial and aquatic environments, where they sustain ecosystem functioning by mineralization processes and nutrient cycling. Nevertheless, very little is known about their true diversity and intraspecific population structure. Recent molecular studies on marine nematodes indicated cryptic diversity and strong genetic differentiation of distinct populations, but for freshwater nematode species, analogous studies are lacking. Here, we present the first extensive molecular study exploring cryptic species diversity and genetic population structure of a widespread freshwater nematode morphospecies, Tobrilus gracilis, from nine postglacially formed European lakes. Taxonomic species status of individuals, analysed for fragments of the mitochondrial COI gene and for the large (LSU) and small (SSU) ribosomal subunits, were determined by morphological characteristics. Mitochondrial and nuclear markers strongly supported the existence of three distinct genetic lineages (Tg I–III) within Tobrilus gracilis, suggesting that this morphospecies indeed represents a complex of highly differentiated biological species. High genetic diversity was also observed at the population level. Across the nine lakes, 19 mitochondrial, and seven (LSU) and four (SSU) nuclear haplotypes were determined. A phylogeographical analysis revealed remarkable genetic differentiation even among neighbouring lake populations for one cryptic lineage. Priority and persistent founder effects are possible explanations for the observed population structure in the postglacially colonized lakes, but ask for future studies providing direct estimates of freshwater nematode dispersal rates. Our study suggests therefore that overall diversity of limnetic nematodes has been so far drastically underestimated and challenges the assumed ubiquitous distribution of other, single freshwater nematode morphospecies.     

中国松毛虫属八个种和亚种亲缘关系的DNA指纹证据

利用DNA指纹谱方法探讨了中国松毛虫属8个种和亚种〔马尾松毛虫Dendrolimus punctatus punctatus (Walker),德昌松毛虫D. punctatus tehchangensis Tsai et Liu,文山松毛虫D. punctatus wenshanensis Tsai et Liu,思茅松毛虫D. kikuchii Matsumura,赤松毛虫D. spectabilis Butler,油松毛虫D. tabulaeformis Tsai et Liu,落叶松毛虫D. superans (Butler),云南松毛虫D. houi Lajonquiere之间的亲缘关系。13个随机引物在8种松毛虫中共检测到168个多态分子标记。分析表明,这8个种间的遗传距离的变化范围为0.3780～0.7360;马尾松毛虫与其亚种德昌松毛虫的遗传距离最近,为0.3780,与其另一亚种文山松毛虫以及油松毛虫的遗传距离次之,皆为0.5233;赤松毛虫、落叶松毛虫、云南松毛虫与马尾松毛虫的遗传距离则再次之,分别为0.6362,0.6770和0.6944;与马尾松毛虫遗传距离最远的是思茅松毛虫,为0.7360。8种松毛虫间具体的亲缘关系为: (D. superans (D. tabulaeformis (D. p. wenshanensis (D. p. tehchangensis, D. p. punctatus)(D. Kikuchii (D. spectabilis, D. houi))。     

Primary structure of a chitinase-encoding gene (chi1) from the filamentous fungus Aphanocladium album: similarity to bacterial chitinases

Chitinase 1 (Chil) is the major extracellular chitinase from the hyperparasitic fungus, Aphanocladium album. We determined the complete sequence of the chromosomal and cDNA copies of the structural gene (chi1) coding for Chil. The coding region is interrupted by three short introns (55, 53 and 49 bp long). Chil is 423 aa long and begins with a stretch of 34 aa not found in the mature protein. The Chil sequence presents overall similarities with bacterial chitinases from Serratia marcescens and Bacillus circulans. Compared with other chitinases, A. album Chi1 has only two short similarity regions (12 and 8 aa long), which are also found in bacterial, yeast and some plant chitinases.