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PubMed2000

Multiple data sets, congruence, and hypothesis testing for the phylogeny of basal groups of the lizard genus Sceloporus (Squamata, Phrynosomatidae).

Several data partitions, including nuclear and mitochondrial gene sequences, chromosomes, isoenzymes, and morphological characters, were used to propose a new phylogeny and to test previously published hypotheses about the phylogenetic positions of basal clades of the lizard genus Sceloporus and the relationship of Sceloporus to the former genus "Sator". In accord with earlier studies, our results grouped "Sator" as internal to Sceloporus, and both support a hypothesis of transgulfian vicariance for the origin of the former genus "Sator" on islands in the Sea of Cortez. Robustness of support for internal nodes in our best tree was established though widely used indices (bootstrap proportions, decay values) but also through congruence among independent data partitions. Several deep nodes in the tree recovered by several methods, including equally weighted and differentially weighted parsimony and maximum likelihood models, are only weakly supported by the traditional indices. This methodological concordance is taken as evidence for insensitivity of the deep structure of the topology to alternative assumptions.

Animals↗
PubMed1999

Phylogeographical structure of the Dover sole Microstomus pacificus: the larval retention hypothesis and genetic divergence along the deep continental slope of the northeastern Pacific Ocean.

Population genetic variability and phylogeographical structure of the Dover sole, Microstomus pacificus (Teleostei: Pleuronectidae), were examined along the continental slope of the north-eastern Pacific Ocean using sequences of the left domain of the mitochondrial DNA (mtDNA) control region. Six sites were sampled in three primary biogeographical provinces (based on surface water temperatures), ranging from the subarctic to the warm temperate zones. Many haplotypes were discerned, totalling 90 for 110 individuals. Neighbour joining and parsimony analyses of the entire data set showed that some groups of haplotypes of adult Dover sole were widely distributed, suggesting either panmixia or homoplasy and reversals. However, a substantial number of groups of related haplotypes were geographically circumscribed, and there were significant differences among sites in the distribution of restricted haplotypes (based on a reduced number of characters), supporting geographical structure. Geographical differentiation of populations appeared to be consistent with the member-vagrant hypothesis for retention of the pelagic larvae in currents and recruitment to adult habitats, reducing long-distance dispersal. Results of this study indicate phylogeographical structure among some populations, despite extended pelagic larval periods, and apparent environmental homogeneity in the adult habitats along the continental slope.

Animals↗
PubMed2002

Shewanella denitrificans sp. nov., a vigorously denitrifying bacterium isolated from the oxic-anoxic interface of the Gotland Deep in the central Baltic Sea.

Three strains of denitrifying estuarine bacteria, OS217(T), 05220 and OS226, were characterized for their physiological and biochemical features, fatty acid profiles and their phylogenetic position based on 16S rDNA sequences. The strains were isolated from the oxic-anoxic interface of an anoxic basin of the central Baltic Sea. Phylogenetic analyses of the 16S rDNA sequences revealed a clear affiliation with members of the genus Shewanella of the gamma-Proteobacteria. The closest sequence similarity was seen with Shewanella baltica, Shewanella putrefaciens and Shewanella frigidimarina (95-96%). The dominant fatty acids were 16:1omega7c, 15:0 iso, 16:0 and 13:0 iso. The G+C content of the DNA ranged from 46.8 to 48.1 mol%. The strains were unpigmented, polarly flagellated, mesophilic, facultatively anaerobic and able to use nitrate, nitrite and sulphite as electron acceptors. Growth was observed at salinities from 0 to 6%, with an optimum between 1 and 3%. According to their morphology, physiology, fatty acid composition and 16S rRNA sequences, the described bacteria fitted well into the genus Shewanella, but could be easily distinguished from the Shewanella species described to date. Because of their capacity for vigorous denitrification, the name Shewanella denitrificans sp. nov. is suggested for the Baltic isolates, for which the type strain is OS217(T) (= DSM 15013(T) = LMG 21692(T)).

Baltic States↗
PubMed2006

Bacterial and archaeal phylotypes associated with distinct mineralogical layers of a white smoker spire from a deep-sea hydrothermal vent site (9 degrees N, East Pacific Rise).

A diffusely venting chimney spire from the East Pacific Rise (9 degrees N) was analysed by petrographic thin sectioning and 16S rRNA gene cloning and sequencing in parallel, to correlate microbial community composition with mineralogy and inferred in situ conditions within the chimney mineral matrix. Both approaches indicated a zonation of the chimney spire into distinct microhabitats for different bacteria and archaea. The thermal gradient inferred from the mineral composition and porosity of the chimney was consistent with the distribution of bacterial and archaeal phylotypes in the chimney matrix. A novel phylogenetic lineage of euryarchaeota was found that co-occurred with clones related to cultured hyperthermophilic archaea. A few phylotypes related to mesophilic bacteria were found in the hot core of the chimney, indicating that seawater influx during retrieval and cooling of these highly porous structures can entrain microorganisms into chimney layers that are not their native habitat.

Archaea↗
PubMed2003

Reconstitution and characterization of NtrC protein in a deep-sea piezophilic bacterium, Shewanella violacea strain DSS12.

NtrC protein of piezophilic Shewanella violacea was overexpressed and purified, to confirm the protein-DNA interaction. An electrophoretic mobility shift assay demonstrated that the NtrC recognizes the sequence for NtrC binding within the region upstream of the glnA operon. Western blot analysis also showed that the NtrC is expressed at a higher level under high-pressure conditions than under atmospheric pressure conditions.

Bacterial Proteins↗
PubMed2000

Using antibody catalysis to study the outcome of multiple evolutionary trials of a chemical task.

Catalytic aldolase antibodies generated by immunization with two different, but structurally related, beta-diketone haptens were cloned and sequenced to study similarities and differences between independently evolved catalysts. Kinetic and sequence analysis coupled with mutagenesis, structural, and modeling studies reveal that the defining event in the evolution of these catalysts was a somatic mutation that placed a lysine residue in a deep, yet otherwise unrefined, hydrophobic pocket. We suggest that covalent chemistries may be as readily selected from the immune repertoire as the traditional noncovalent interactions that have formed the basis of immunochemistry until this time. Further, we believe that these experiments recapitulate the defining events in the evolution of nature's enzymes, particularly as they relate to chemical mechanism, catalytic promiscuity, and gene duplication.

Amino Acid Sequence↗
PubMed1996

Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis.

The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers IAf and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the alpha-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed marked variation from the remainder in specific regions of the sequence. From a comparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe was used to screen primary populations of PCR clones, and all of those that were probe negative were checked for the presence of inserts, which were then sequenced. By using this strategy, further novel methanogen 16S rRNA variants were identified and analyzed. The sequences recovered from the peat formed two clusters on the end of long branches within the methanogen radiation that are distinct from each other. These cannot be placed directly with sequences from any cultured taxa for which sequence information is available.

Base Sequence↗
PubMed2001

Epsilon-proteobacterial diversity from a deep-sea hydrothermal vent on the Mid-Atlantic Ridge.

The prokaryotic phylogenetic diversity was determined for a sample associated with an in situ growth chamber deployed for 5 days on a Mid-Atlantic Ridge hydrothermal vent (23 degrees 22'N, 44 degrees 57'W). The DNA was extracted from the sample and the 16S rDNA amplified by PCR. No Archaea were detected in the sample. Eighty-seven clones containing bacterial 16S rDNA inserts were selected. Based on restriction fragment length polymorphism analysis, 47 clones were unique, however, based on comparative sequence analysis some of these were very similar, and thus only 22 clones were selected for full sequence and phylogenetic analysis. The phylotypes were dominated by epsilon-Proteobacteria (66%). The remainder formed a novel lineage within the Proteobacteria (33%). One clone formed a distinct deeply branching lineage, and was a distant relative of the Aquificales. This report further expands the growing evidence that epsilon-Proteobacteria are important members in biogeochemical cycling at deep-sea hydrothermal ecosystems, participating as epibionts and free living bacteria.

Ecosystem↗
PubMed1991

[Ethologic selection studies with early weaned piglets kept in pens with different use of straw. 3. Results of studies of the position sequence of area size and stall air temperature].

In continuation of our earlier investigations on the range of environmental factors in housing systems with perforated floors and the experiments with or without free choice on litter or deep litter the effects of the size of the area and the environmental temperatures on the behaviour of early weaned piglets (8 kg to 30 kg) kept on litter and deep litter have been investigated. Both factors had a crucial but diametrical influence. While for the total activity of the pigs the size of the compartment proved more important than room temperature, for the total inactivity it was the other way around. Therefore some essential requirements have to be obeyed even for housing systems with litter or deep litter, which can be considered more suitable for the housing of pigs from the start on. Because of their different importance for the activity (20% of the total behaviour) and the inactivity (80%) the factors can't replace each other. For housing systems with litter size allocation shouldn't amount less than 0.45 m2/animal and with deep litter not less than 0.70 m2/animal with room temperatures starting at about 25 degrees C and 20 degrees C, respectively.

Animals↗
PubMed1999

Multiplicity of glutamic acid decarboxylases (GAD) in vertebrates: molecular phylogeny and evidence for a new GAD paralog.

The evolution of chordate glutamic acid decarboxylase (GAD; EC 4.1.1.15), a key enzyme in the central nervous system synthesizing the neurotransmitter gamma-amino-butyric acid (GABA) from glutamate, was studied. Prior to this study, molecular data of GAD had been restricted to mammals, which express two distinct forms, GAD65 and GAD67. These are the products of separate genes and probably are derived from a common ancestral GAD following gene duplication at some point during vertebrate evolution. To enable a comprehensive phylogenetic analysis, molecular information of GAD forms in other vertebrate classes was essential. By reverse transcriptase-polymerase chain reaction (RT-PCR), partial nucleotide sequences of GAD were cloned from brains of zebra finch (Taeniopygia guttata), turtle (Trachemys scripta), goldfish (Carassius auratus), zebrafish (Danio rerio), and armoured grenadier (Coryphaenoides (Nematonurus) armatus, a deep-sea fish), and from the cerebral ganglion plus neural gland of Ciona intestinalis, a protochordate. Whereas GAD65 and GAD67 homologs were expressed in birds, reptiles, and fish, only a single GAD cDNA with equal similarities to both vertebrate GAD forms was found in the protochordate. This indicates that the duplication of the vertebrate GAD gene occurred between 400 and 560 million years ago. For both GAD65 and GAD67, the generated phylogenetic tree followed the general tree topology for the major vertebrate classes. In turtle, an alternative spliced form of GAD65, putatively encoding a truncated, nonactive GAD, was found. Furthermore, a third GAD form, which is equally divergent from both GAD65 and GAD67, is expressed in C. (N.) armatus. This third form might have originated from an ancient genome duplication specific to modern ray-finned fishes.

Alternative Splicing↗
PubMed2005

Culturing and environmental DNA sequencing uncover hidden kinetoplastid biodiversity and a major marine clade within ancestrally freshwater Neobodo designis.

Bodonid flagellates (class Kinetoplastea) are abundant, free-living protozoa in freshwater, soil and marine habitats, with undersampled global biodiversity. To investigate overall bodonid diversity, kinetoplastid-specific PCR primers were used to amplify and sequence 18S rRNA genes from DNA extracted from 16 diverse environmental samples; of 39 different kinetoplastid sequences, 35 belong to the subclass Metakinetoplastina, where most group with the genus Neobodo or the species Bodo saltans, whilst four group with the subclass Prokinetoplastina (Ichthyobodo). To study divergence between freshwater and marine members of the genus Neobodo, 26 new Neobodo designis strains were cultured and their 18S rRNA genes were sequenced. It is shown that the morphospecies N. designis is a remarkably ancient species complex with a major marine clade nested among older freshwater clades, suggesting that these lineages were constrained physiologically from moving between these environments for most of their long history. Other major bodonid clades show less-deep separation between marine and freshwater strains, but have extensive genetic diversity within all lineages and an apparently biogeographically distinct distribution of B. saltans subclades. Clade-specific 18S rRNA gene primers were used for two N. designis subclades to test their global distribution and genetic diversity. The non-overlap between environmental DNA sequences and those from cultures suggests that there are hundreds, possibly thousands, of different rRNA gene sequences of free-living bodonids globally.

Animals↗
PubMed2005

Laterally transferred elements and high pressure adaptation in Photobacterium profundum strains.

BACKGROUND: Oceans cover approximately 70% of the Earth's surface with an average depth of 3800 m and a pressure of 38 MPa, thus a large part of the biosphere is occupied by high pressure environments. Piezophilic (pressure-loving) organisms are adapted to deep-sea life and grow optimally at pressures higher than 0.1 MPa. To better understand high pressure adaptation from a genomic point of view three different Photobacterium profundum strains were compared. Using the sequenced piezophile P. profundum strain SS9 as a reference, microarray technology was used to identify the genomic regions missing in two other strains: a pressure adapted strain (named DSJ4) and a pressure-sensitive strain (named 3TCK). Finally, the transcriptome of SS9 grown under different pressure (28 MPa; 45 MPa) and temperature (4 degrees C; 16 degrees C) conditions was analyzed taking into consideration the differentially expressed genes belonging to the flexible gene pool. RESULTS: These studies indicated the presence of a large flexible gene pool in SS9 characterized by various horizontally acquired elements. This was verified by extensive analysis of GC content, codon usage and genomic signature of the SS9 genome. 171 open reading frames (ORFs) were found to be specifically absent or highly divergent in the piezosensitive strain, but present in the two piezophilic strains. Among these genes, six were found to also be up-regulated by high pressure. CONCLUSION: These data provide information on horizontal gene flow in the deep sea, provide additional details of P. profundum genome expression patterns and suggest genes which could perform critical functions for abyssal survival, including perhaps high pressure growth.

Atmospheric Pressure↗
PubMed1997

Distribution of motoneurons supplying feline neck muscles taking origin from the shoulder girdle.

A combination of fluorescent retrograde tracers and horseradish peroxidase (HRP) was used to compare the spinal distributions of motoneurons supplying shoulder muscles with attachments to the skull and cervical spinal cord that suggest a significant role in head movement. Two muscles, the rhomboideus and the levator scapulae, were innervated by multiple segmental nerve bundles that entered the muscles at different rostrocaudal locations. Motoneurons that were labelled retrogradely from rhomboideus nerve bundles formed a single, long column in the ventral horn from C4 to C6, lateral to previously studied motor nuclei supplying deep neck muscles. When different tracers were used to differentiate motoneurons supplying specific nerve bundles, discrete subnuclei could be identified that were organized in a rostrocaudal sequence corresponding to the rostrocaudal order of the nerve bundles. Levator scapulae motoneurons formed a second elongate column immediately lateral to the rhomboidues motor nucleus. Three other muscles, that trapezius, sternomastoideus, and cleidomastoideus, were supplied by cranial nerve XI. Labelled motoneurons from these muscles formed a single column from the spinomedullary junction to middle C6. Within this column, the three motor nuclei supplying the sternomastoideus, cleidomastoideus, and trapezius were laminated mediolaterally. Sternomastoideus and cleidomastoideus motoneurons were confined to upper cervical segments, whereas trapezius motoneurons were found from C1 to C6. In C1 and C6, the motoneuron column was located centrally in the gray matter, but, between C2 and C5, the column lay on the lateral wall of the ventral horn in a position dorsolateral to motor nuclei supplying the rhomboideus and the deeper neck muscles. The findings in this study suggest that descending and propriospinal systems responsible for coordinating head movement may have to descend as far caudally as C6 if they are to project onto muscles controlling the mobility of the lower neck.

Accessory Nerve↗
PubMed2005

New ratios for the detection and classification of CJD in multisequence MRI of the brain.

We present a method for the analysis of deep grey brain nuclei for accurate detection of human spongiform encephalopathy in multisequence MRI of the brain. We employ T1, T2 and FLAIR-T2 MR sequences for the detection of intensity deviations in the internal nuclei. The MR data are registered to a probabilistic atlas and normalised in intensity prior to the segmentation of hyperintensities using a foveal model. Anatomical data from a segmented atlas are employed to refine the registration and remove false positives. The results are robust over the patient data and in accordance to the clinical ground truth. Our method further allows the quantification of intensity distributions in basal ganglia. sCJD patient FLAIR images are classified with a more significant hypersignal in caudate nuclei (10/10) and putamen (6/10) than in thalami. Defining normalised MRI measures of the intensity relations between the internal grey nuclei of patients, we robustly differentiate sCJD and variant CJD (vCJD) patients, as an attempt towards the automatic detection and classification of human spongiform encephalopathies.

Algorithms↗
PubMed2004

Isolation and disruption of the melanin pathway polyketide synthase gene of the softwood deep stain fungus Ceratocystis resinifera.

Ceratocystis resinifera hyphae produce a black melanin pigment causing a deep stain in softwood logs. We exploited the homology of polyketide synthases to clone PKS1, a gene responsible for dihydroxynaphthalene-melanin biosynthesis in C. resinifera. Sequence analysis indicated that PKS1 has two introns near its 5(') end and encodes a 2188-amino acid polypeptide with five functional domains: beta-ketoacyl synthase, acyl transferase, two acyl carrier proteins and a thioesterase/Claisen cyclase. A gene disruption construct designed to replace a portion of PKS1 with a hygromycin resistance cassette was transformed into C. resinifera through Agrobacterium tumefaciens-mediated transformation. PKS1 null mutants had an albino phenotype, and pigmentation was restored by the addition of scytalone, a melanin pathway intermediate. The disruption of PKS1 and restoration of pigmentation with scytalone confirmed the presence of a dihydroxynaphthalene-melanin pathway in C. resinifera. The transformation method described in this paper is the first reported for a Ceratocystis species.

Amino Acid Sequence↗
PubMed2004

Crystal structure of MshB from Mycobacterium tuberculosis, a deacetylase involved in mycothiol biosynthesis.

All living species require protection against the damaging effects of the reactive oxygen species that are a natural by-product of aerobic life. In most organisms, glutathione is a critical component of these defences, maintaining a reducing environment inside cells. Some bacteria, however, including pathogenic mycobacteria, use an alternative low molecular mass thiol compound called mycothiol (MSH) for this purpose. Enzymes that synthesize MSH are attractive candidates for the design of novel anti-TB drugs because of the importance of MSH for mycobacterial life and the absence of such enzymes in humans. We have determined the three-dimensional structure of MshB (Rv1170), a metal-dependent deacetylase from Mycobacterium tuberculosis that catalyses the second step in MSH biosynthesis. The structure, determined at 1.9A resolution by X-ray crystallography (R=19.0%, R(free)=21.4%), reveals an alpha/beta fold in which helices pack against a seven-stranded mostly parallel beta-sheet. Large loops emanating from the C termini of the beta-strands enclose a deep cavity, which is the location of the putative active site. At the bottom of this cavity is a metal-binding site associated with a sequence motif AHPDDE that is invariant in all homologues. An adventitiously bound beta-octylglucoside molecule, used in crystallization, enables us to model the binding of the true substrate and propose a metal-dependent mechanistic model for deacetylation. Sequence comparisons indicate that MshB is representative of a wider family of enzymes that act on substituted N-acetylglucosamine residues, including a deacetylase involved in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors in eukaryotes.

Amidohydrolases↗
PubMed2005

New insights into the phylogeny of fig pollinators using Bayesian analyses.

The interaction between figs and fig pollinators is one of the most species-specific mutualisms. Recently, phylogenies of both partners based on molecular data provided insights into a wide spectrum of co-evolutionary questions. However, for the phylogeny of fig pollinators, there are some discrepancies between different studies and left some relationships unresolved, especially for deep nodes. The phylogenetic uncertainties of pollinators prohibit our further understanding of the history of the mutualism. Here, we present phylogenetic analyses of a larger COI sequence dataset that includes previously published datasets and our sequences from 20 species using Bayesian method and maximum parsimony. The analyses using different methods share similar topologies. Bayesian analyses provide high level of confidence for most internal nodes in terms of posterior probability. This study also clarifies some discrepancies between previous studies. After rooting with Tetrapus, other pollinators split into two clades. Wiebesia and Blastophaga are at basal positions in respective clade. Ceratosolen is not monophyletic because Kradibia and Liporrhopalum fall inside this group. Three subgenera of Ceratosolen: subgen. Ceratosolen, subgen. Rothropus, and subgen. Strepitus are not supported. Therefore, Ceratosolen is suggested to be re-divided into three groups. Urostigma pollinators (including Dolichoris and Blastophaga psenes) are clustered together. The monophylies of Wiebesia, Blastophaga, Dolichoris are not supported in this analysis. This study also provides a new framework for re-evaluating character evolution and re-inspecting the definition of some genera.

Animals↗
PubMed2025

Discovery of a MET -driven monogenic cause of steatotic liver disease.

BACKGROUND AND AIMS: Metabolic dysfunction-associated steatotic liver disease affects about a third of adults worldwide and is projected soon to be the leading cause of liver cirrhosis. It occurs when fat accumulates in hepatocytes and can progress to metabolic dysfunction-associated steatohepatitis, liver cirrhosis, and HCC. Metabolic dysfunction-associated steatotic liver disease pathogenesis is believed to involve a combination of genetic and environmental risk factors. Single nucleotide polymorphisms have been implicated, but non-syndromic monogenic causes are lacking. APPROACH AND RESULTS: We identified a novel genetic variant in a familial case of metabolic dysfunction-associated steatohepatitis and performed deep variant functional analysis, including protein modeling, dynamics, and cell-based assays to assess molecular mechanisms of dysfunction and altered cellular signaling. We analyzed exome sequencing data of 3904 individuals with steatotic liver disease (SLD) to identify additional cases and establish the link between specific gene variants and SLD diagnosis. We discovered and functionally validated the NM_000245.4:c.3505A>T; p.(Ile1169Phe) variant in the MET (mesenchymal-epithelial transition) kinase domain as a monogenic cause of SLD. Subsequently, we detected additional ultra-rare, previously uninterpreted, and likely deleterious variants in MET from screening sequencing data. Among individuals with confirmed SLD based on electronic record review, 1.1% (45/3904) had rare predicted deleterious MET variants. Eight of 45 (17.7%) individuals had predicted deleterious variants in the MET kinase domain confirmed to be functionally like the familial case variant. CONCLUSIONS: We report the first germline nonmalignant rare MET -driven disease, a monogenic form of SLD.

Adult↗