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At least 577 records · Page 32Linked to original sources
PubMed1999

Molecular data from the chloroplast rpoC1 gene suggest a deep and distinct dichotomy of contemporary spermatophytes into two monophyla: gymnosperms (including Gnetales) and angiosperms.

Partial sequences of the rpoC1 gene from two species of angiosperms and three species of gymnosperms (8330 base pairs) were determined and compared. The data obtained support the hypothesis that angiosperms and gymnosperms are monophyletic and none of the recent groups of the latter is sister to angiosperms.

Amino Acid Sequence↗
PubMed1998

Identification of Trichosporon asahii by PCR based on sequences of the internal transcribed spacer regions.

Trichosporon asahii is a major causative agent of deep-seated trichosporonosis, which has a high mortality rate. To detect T. asahii, we have developed specific oligonucleotide primers based on the internal transcribed spacer regions of this organism's genome. Amplification products were selectively obtained from only T. asahii DNA; the DNAs of other Trichosporon species, as well as those of medically relevant yeasts such as Candida albicans, Cryptococcus neoformans, and Malassezia furfur, were not amplified. This detection system will be useful as a microbiological tool for the diagnosis of trichosporonosis.

DNA Primers↗
PubMed1995

Immunobiological activity of recombinant H antigen from Histoplasma capsulatum.

The H antigen from Histoplasma capsulatum is one of the known principal antigens of this fungus, but information regarding its identity and its participation in cellular immunity is largely unavailable. Therefore, we sought to determine both the nature of this antigen and the nature of its involvement in the cell-mediated immune response. The antigen was isolated from histoplasmin and digested with selected proteinases, and the cleavage products were subjected to reverse-phase high-performance liquid chromatography. Amino acid sequences of protein fragments were obtained by Edman degradation. A fragment of the gene encoding the H antigen was isolated by using degenerate primers in the PCR. This fragment was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence revealed homology to extracellular beta-glucosidases. A cDNA was generated by reverse transcription PCR and cloned into the expression vector pET 19b. Recombinant H antigen was isolated from inclusion bodies of Escherichia coli and tested for its ability to elicit and induce an in vitro cell-mediated immune response in BALB/c mice. Recombinant antigen stimulated splenocytes from mice immunized with viable yeast cells or with antigen suspended in adjuvant. Mice inoculated with H antigen were not protected against either a sublethal or a lethal inoculum of yeast cells. Thus, H antigen stimulates a cell-mediated immune response but does not induce a protective response to H. capsulatum.

Amino Acid Sequence↗
PubMed2003

Searching factors causing implausible non-monophyly: ssu rDNA phylogeny of Isopoda Asellota (Crustacea: Peracarida) and faster evolution in marine than in freshwater habitats.

This contribution addresses two questions: which alignment patterns are causing non-monophyly of the Asellota and what is the phylogenetic history of this group? The Asellota are small benthic crustaceans occurring in most aquatic habitats. In view of the complex morphological apomorphies known for this group, monophyly of the Asellota has never been questioned. Using ssu rDNA sequences of outgroups and of 16 asellote species from fresh water, littoral marine habitats and from deep-sea localities, the early divergence between the lineages in fresh water and in the ocean, and the monophyly of the deep-sea taxon Munnopsidae are confirmed. Relative substitution rates of freshwater species are much lower than in other isopod species, rates being highest in some littoral marine genera (Carpias and Jaera). Furthermore, more sequence sites are variable in marine than in freshwater species, the latter conserve outgroup character states. Monophyly is recovered with parsimony methods, but not with distance and maximum likelihood analyses, which tear apart the marine from the freshwater species. The information content of alignments was studied with spectra of supporting positions. The scarcity of signal (=apomorphic nucleotides) supporting monophyly of the Asellota is attributed to a short stem-line of this group or to erosion of signal in fast evolving marine species. Parametric boostrapping in combination with spectra indicates that a tree model cannot explain the data and that monophyly of the Asellota should not be rejected even though many topologies do not recover this taxon.

Animals↗
PubMed2001

Molecular cloning, characterization and expression of the heat shock protein 60 gene from the human pathogenic fungus Paracoccidioides brasiliensis.

A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized. The hsp60 gene is composed of three exons divided by two introns. Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes. The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins. The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis. The HSP60 protein was isolated from Pb yeast cellular extracts. N-terminal amino acid sequencing of HSP60 confirmed that the cloned hsp60 gene correlated to the predicted protein in Pb. HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis. The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).

Amino Acid Sequence↗
PubMed2007

217 000-year-old DNA sequences of green sulfur bacteria in Mediterranean sapropels and their implications for the reconstruction of the paleoenvironment.

Deep-sea sediments of the eastern Mediterranean harbour a series of dark, organic carbon-rich layers, so-called sapropels. Within these layers, the carotenoid isorenieratene was detected. Since it is specific for the obligately anaerobic phototrophic green sulfur bacteria, the presence of isorenieratene may suggest that extended water column anoxia occurred in the ancient Mediterranean Sea during periods of sapropel formation. Only three carotenoids (isorenieratene, beta-isorenieratene and chlorobactene) are typical for green sulfur bacteria and thus do not permit to differentiate between the approximately 80 known phylotypes. In order to reconstruct the paleoecological conditions in more detail, we searched for fossil 16S rRNA gene sequences of green sulfur bacteria employing ancient DNA methodology. 540 bp-long fossil sequences could indeed be amplified from up to 217 000-year-old sapropels. In addition, such sequences were also recovered from carbon-lean intermediate sediment layers deposited during times of an entirely oxic water column. Unexpectedly, however, all the recovered 16S rRNA gene sequences grouped with freshwater or brackish, rather than truly marine, types of green sulfur bacteria. It is therefore feasible that the molecular remains of green sulfur bacteria originated from populations which thrived in adjacent freshwater or estuarine coastal environments rather than from an indigenous pelagic population.

Benzopyrans↗
PubMed1989

Base only binding of spermine in the deep groove of the A-DNA octamer d(GTGTACAC).

The crystal structure of a complex of spermine with the DNA octamer d(GTGTACAC) has been determined at 2.0-A resolution. The alternating sequence adopts an A-DNA conformation with a novel purine-purine extra-Watson-Crick hydrogen bond involving the central guanine G3 (G11) and adenine A13 (A5) in the deep groove. The oligocation spermine binds in the floor of the deep groove by interacting with the bases and assumes an S-shape. Its dyad is coincident with that of the DNA, reminiscent of repressor binding to B-DNA. The terminal and central ammonium groups of the top half of spermine form hydrogen-bonding interactions to the 5'-bases, GTG, of one strand; then the spermine winds across the groove to interact with the corresponding set of bases on the other strand. The methylene groups of spermine form a hydrophobic cluster with the methyl groups of the thymines and the O6 atoms of the guanines of the TGT sequences on either side of the dyad. The observed mode of binding of spermine to A-DNA can serve as a model for deep groove binding in RNA and DNA-RNA hybrids that show a propensity also for the A-conformation. It will be of interest to see if base binding of spermine to DNA is involved in the regulation of gene expression, since spermine and other oligocations are ubiquitous in cells and their concentration is coupled to stages in cell cycle.

DNA↗
PubMed2003

[Intraspecies diversity of Trichosporon asahii as the causative agent of opportunistic fungal infection and summer-type hypersensitivity pneumonitis].

Trichosporon asahii is the major causative agent of the opportunistic infection trichosporonosis, and also causes summer-type hypersensitivity pneumonitis (SHP). Random amplification of polymorphic DNA analysis was used to determine the intraspecies diversity of T. asahii isolates from clinical specimens,the houses of SHP patients, and environmental material. Clinical isolates formed a cluster, characterized by a 90% matching coefficient, but they did not cluster with strains isolated from SHP patients houses or environmental sources. The biochemical characteristics of T. asahii isolates from the three sources were compared, and a phenogram was constructed. One of the two clusters included most of the clinical isolates and strains isolated from the houses, and the other cluster included most of the environmental isolates. There was a remarkable difference in the abilities of the strains belonging to these clusters to utilize several compounds. These results suggest that the genetic diversity and biochemical characteristics of T. asahii are related to the source of the isolates. In addition, based on the IGS1 sequence, which is located between the 26S and 5S rRNA genes, we identified five genotypes of T. asahii, which is a major causative agent of deep-seated trichosporonosis. Of the five genotypes, three were isolates that originated in Japan, whereas two were American isolates. IGS sequence analysis shows great potential as a new epidemiological tool.

Alveolitis, Extrinsic Allergic↗
PubMed2004

Phylogenetic artifacts can be caused by leucine, serine, and arginine codon usage heterogeneity: dinoflagellate plastid origins as a case study.

Phylogenetic analyses of first and second codon positions (DNA1 + 2 analysis) and amino acid sequences (protein analysis) are often thought to provide similar estimates of deep-level phylogeny. However, here we report a novel artifact influencing DNA level phylogenetic inference of protein-coding genes introduced by codon usage heterogeneity that causes significant incongruities between DNA1 + 2 and protein analyses. DNA1 + 2 analyses of plastid-encoded psbA genes (encoding of photosystem II D1 proteins) strongly suggest a relationship between haptophyte plastids and typical (peridinin-containing) dinoflagellate plastids. The psbA genes from haptophytes and a subset of the peridinin-type plastids display similar codon usage patterns for Leu, Ser, and Arg, which are each encoded by two separated codon sets that differ at first or first plus second codon positions. Our detailed analyses clearly indicate that these unusual preferences shared by haptophyte and some peridinin-type plastid genes are largely responsible for their strong affinity in DNA analyses. In particular, almost all of the support from DNA level analyses for the monophyly of haptophyte and peridinin-type plastids is lost when the codons corresponding to constant Leu, Ser, and Arg amino acids are excluded, suggesting that this signal comes from rapidly evolving synonymous substitutions, rather than from substitutions that result in amino acid changes. Indeed, protein maximum-likelihood analyses of concatenated PsaA and PsbA amino acid sequences indicate that, although 19' hexanoyloxyfucoxanthin-type (19' HNOF-type) plastids in dinoflagellates group with haptophyte plastids, peridinin-type plastids group weakly with those of stramenopiles. Consequently our results cast doubt on the single origin of peridinin-type and 19' HNOF-type plastids in dinoflagellates previously suggested on the basis of psaA and psbA concatenated gene phylogenetic analyses. We suggest that codon usage heterogeneity could be a more general problem for DNA level analyses of protein-coding genes, even when third codon positions are excluded.

Animals↗
PubMed2000

Phylogenetic utility of the tyrosine kinase gene X-src for assessing relationships among representative cichlid fishes.

The nuclear gene X-src is a member of the tyrosine-kinase class of proto-oncogenes whose normal product is localized within the cytoplasm of the cell. The X-src gene has been used in only a few phylogenetic studies, each focusing on systematics of killifishes (Cyprinodontiformes). The present study is an attempt to examine the phylogenetic utility of X-src for uncovering relationships of representative cichlid fishes, especially the cichlids of Middle America. The family Cichlidae is a species-rich group of tropical freshwater fish made up of more than 1000 species which show a Gondwanan pattern of distribution. Cichlid fish have been the focus of numerous studies ranging from behavioral to biogeographical to systematic in nature. Particular emphasis has focused on the cichlids of the African Great Lakes and the explosive adaptive radiation of this group. However, Neotropical cichlids have received considerably less attention than their African counterparts. Our findings regarding the utility of X-src concur with those of previous phylogenetic analyses showing the exons of X-src to be highly conserved and useful mostly for revealing deep relationships among taxa. Like previous X-src studies, we also found the intron sequences of the gene to be variable in length and difficult to align across distantly related taxa but they provided useful information for resolving relationships among more closely related taxa. The X-src phylogeny supports the monophyly of Neotropical cichlids and cichlasomines groups A (=heroines) + B (=cichlasomines). A highly resolved tree is obtained within the heroines but little support is evident for most nodes based on the low number of unambiguous substitutions. The X-src gene is likely to be quite useful for resolving deep phylogenetic relationships such as those among major groups of actinopterygian fishes.

Animals↗
PubMed2001

An increased expression of Ca(2+) channel alpha(1A) subunit immunoreactivity in deep cerebellar neurons of rolling mouse Nagoya.

Rolling mouse Nagoya (RMN) is an ataxic mutant and carries a mutation in the gene coding for the alpha(1A) subunit of the P/Q-type Ca(2+) channel. We examined the immunohistochemical expression of the alpha(1A) subunit in deep cerebellar nuclei of RMN. The antibody used recognized residues 865-883 of the mouse alpha(1A) subunit not overlapping the altered sequences in RMN. In RMN, many neurons exhibited definite alpha(1A) subunit-staining in the medial nucleus, interposed nucleus, and lateral nucleus of deep cerebellar nuclei. The number of positive neurons in these nuclei was significantly higher in RMN than in controls. Increased expression of the alpha(1A) subunit in deep cerebellar neurons might compensate for the altered function of the P/Q-type Ca(2+) channel of RMN.

Animals↗
PubMed1996

Mitochondrial DNA sequence variation in the eastern house mouse, Mus musculus: comparison with other house mice and report of a 75-bp tandem repeat.

The control region and flanking tRNAs were sequenced from 139 Mus musculus mitochondrial DNAs (mtDNAs) from mice collected at 44 localities extending from Germany to Japan. Among the 36 types of M. musculus mtDNA resolved, five have an added 75-bp direct repeat; the two copies within an individual differ by two to four base substitutions. Among 90 M. domesticus mtDNAs sequenced, 12 new types were found; 96 M. domesticus types have now been identified by sequencing this segment. Representative mtDNAs from M. castaneus, M. macedonicus, M. spicilegus and M. spretus were also sequenced. A parsimony tree for the M. musculus mtDNAs is about half as deep as the tree for the M. domesticus mtDNAs, which is consistent with the idea that M. musculus is genetically less diverse and younger than M. domesticus. The patterns of variation as a function of position are similar but not identical in M. musculus and M. domesticus mtDNAs. M. castaneus and M. musculus mtDNAs are allied, at a tree depth about three times as great as the start of intra-M. musculus divergence. The coalescence of the M. musculus and M. castaneus mtDNAs is about half as deep as their coalescence with the M. domesticus mtDNA lineages. The mtDNAs of the aboriginal M. macedonicus and M. spicilegus are each other's closest relatives, at a tree depth greater than the deepest intracommensal node. The mtDNA results support the view that the aboriginal M. spretus is the sister group of the other five species.

Animals↗
PubMed2006

Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi).

A phylogeny of the fungal phylum Basidiomycota is presented based on a survey of 160 taxa and five nuclear genes. Two genes, rpb2, and tef1, are presented in detail. The rpb2 gene is more variable than tef1 and recovers well-supported clades at shallow and deep taxonomic levels. The tef1 gene recovers some deep and ordinal-level relationships but with greater branch support from nucleotides compared to amino acids. Intron placement is dynamic in tef1, often lineage-specific, and diagnostic for many clades. Introns are fewer in rpb2 and tend to be highly conserved by position. When both protein-coding loci are combined with sequences of nuclear ribosomal RNA genes, 18 inclusive clades of Basidiomycota are strongly supported by Bayesian posterior probabilities and 16 by parsimony bootstrapping. These numbers are greater than produced by single genes and combined ribosomal RNA gene regions. Combination of nrDNA with amino acid sequences, or exons with third codon positions removed, produces strong measures of support, particularly for deep internodes of Basidiomycota, which have been difficult to resolve with confidence using nrDNA data alone. This study produces strong boostrap support and significant posterior probabilities for the first time for the following monophyletic groups: (1) Ustilaginomycetes plus Hymenomycetes, (2) an inclusive cluster of hymenochaetoid, corticioid, polyporoid, Thelephorales, russuloid, athelioid, Boletales, and euagarics clades, (3) Thelephorales plus the polyporoid clade, (4) the polyporoid clade, and (5) the cantharelloid clade. Strong support is also recovered for the basal position of the Dacrymycetales in the Hymenomycetidae and paraphyly of the Exobasidiomycetidae.

Alleles↗
PubMed1998

Complete DNA sequence and detailed analysis of the Yersinia pestis KIM5 plasmid encoding murine toxin and capsular antigen.

Yersinia pestis, the causative agent of plague, harbors at least three plasmids necessary for full virulence of the organism, two of which are species specific. One of the Y. pestis-specific plasmids, pMT1, is thought to promote deep tissue invasion, resulting in more acute onset of symptoms and death. We determined the entire nucleotide sequence of Y. pestis KIM5 pMT1 and identified potential open reading frames (ORFs) encoded by the 100,990-bp molecule. Based on codon usage for known yersinial genes, homology with known proteins in the databases, and potential ribosome binding sites, we determined that 115 of the potential ORFs which we considered could encode polypeptides in Y. pestis. Five of these ORFs were genes previously identified as being necessary for production of the classic virulence factors, murine toxin (MT), and the fraction 1 (F1) capsule antigen. The regions of pMT1 encoding MT and F1 were surrounded by remnants of multiple transposition events and bacteriophage, respectively, suggesting horizontal gene transfer of these virulence factors. We identified seven new potential virulence factors that might interact with the mammalian host or flea vector. Forty-three of the remaining 115 putative ORFs did not display any significant homology with proteins in the current databases. Furthermore, DNA sequence analysis allowed the determination of the putative replication and partitioning regions of pMT1. We identified a single 2,450-bp region within pMT1 that could function as the origin of replication, including a RepA-like protein similar to RepFIB, RepHI1B, and P1 and P7 replicons. Plasmid partitioning function was located ca. 36 kb from the putative origin of replication and was most similar to the parABS bacteriophage P1 and P7 system. Y. pestis pMT1 encoded potential genes with a high degree of similarity to a wide variety of organisms, plasmids, and bacteriophage. Accordingly, our analysis of the pMT1 DNA sequence emphasized the mosaic nature of this large bacterial virulence plasmid and provided implications as to its evolution.

Antigens, Bacterial↗
PubMed2003

Archaeal diversity associated with in situ samplers deployed on hydrothermal vents on the East Pacific Rise (13 degrees N).

To evaluate possible compositional changes in archaeal communities at a deep-sea hydrothermal vent field scale, we examined five different samples obtained after deploying in situ collectors for different times on three spatially separated venting sulphide structures on the East Pacific Rise (13 degrees N). Direct cell counts and whole-cell hybridizations with fluorescently labelled 16S rRNA-based oligonucleotide probes revealed that the relative abundance of archaeal populations represented from 14 to 33% of the prokaryotic community. 16S rDNA sequence analysis of the archaeal clone libraries indicated that a large percentage of clones were closely related to known archaeal isolates recovered from similar habitats. Among the 24 different phylotypes identified, Thermococcales-related sequences were dominant in all the libraries that also included representative genera of orders Methanopyrales, Methanococcales, Archaeoglobales and Desulfurococcales. The presence of most of these phylogenetic groups was confirmed in enrichment cultures performed at temperatures from 60 to 90 degrees C. Additional sequences with no known cultivated relatives grouped with the Marine group I Crenarchaeota, Korarchaeota and Deep-sea Hydrothermal Vent Euryarchaeota (DHVE) within which a novel lineage was identified. Furthermore, the archaeal community composition was distinct from vent to vent within the same vent field and varied within short time scales. This study provides new insights into microbial diversity and distribution at deep-sea hydrothermal vents.

Archaea↗
PubMed1993

The phylogenetic utility of cytochrome b: lessons from bufonid frogs.

The mitochondrial cytochrome b gene is widely used in systematic studies to resolve divergences of many different ages. To investigate phylogenetic relationships among frogs of the large family Bufonidae, and to explore the utility of cytochrome b for this purpose, approximately one-third of the gene was sequenced from representatives of this group. Samples were chosen to represent a range of divergence levels within Bufonidae: (1) deep (= old), among species from around the world; (2) middle, among North American species; and (3) shallow (= young), within a single species group (the Bufo boreas group). The inferred amino acid sequences of cytochrome b are highly similar in these frogs although most pairwise comparisons of the nucleotide sequences are 15-20% different. Consequently insufficient information is available to generate robust phylogenetic hypotheses for the older divergences; silent differences are saturated and yet almost no informative replacement differences exist. Among the younger divergences, silent differences are not saturated and some resolution is possible. These results show that (1) the amino acid sequence of cytochrome b evolves differently in Bufonidae than expected based on other vertebrates (2) it consequently provides surprisingly little information about old divergences in Bufonidae, and (3) phylogenetic studies applying particular genes to new groups should begin with preliminary surveys of exemplar taxa representing the range of divergence times within the group to estimate the likely phylogenetic utility of that gene in that group.

Amino Acid Sequence↗
PubMedDate not supplied

Evaluation of competing diagnostic tests: sequences for the diagnosis of pulmonary embolism, Part II.

Tests for pulmonary embolism (PE) and its most frequent source, deep vein thrombosis, include angiography, ventilation-perfusion nuclear medicine scans, and contrast and radionuclide venography. Although selective angiography is the definitive procedure for diagnosis of PE, its risk of death, although small, as well as the morbidity associated with contrast injections, is high enough that alternative, less accurate but safer, diagnostic procedures are preferred. Part I of this report described the methodology for selection of the single test having the lowest effective cost based on the test cost, its risks, and diagnostic performance. Other than the pulmonary angiogram, however, no single test has a sufficiently high diagnostic performance to be clinically realistic: incorrect diagnoses can result in sudden death. Sequences of tests or algorithms can be used to lower the overall risk of the tests to patients while increasing correct decision making to reach a clinically acceptable level. These points are illustrated by comparing three commonly recommended algorithms for the diagnosis of PE. Additionally, the prevalence of PE affects the choice of the algorithm, and we found that no single algorithm is best for all values of prevalence. In summary, we found that the most cost-effective strategy was to select the particular algorithm having the lowest effective cost for the relevant prevalence value. Use of algorithms also decreased the overall risk of diagnostic test complications and the number of incorrect diagnoses.

Algorithms↗
PubMed1993

Study of a protein S gene polymorphism at DNA and mRNA level in a family with symptomatic protein S deficiency.

A protein S gene polymorphism, detectable by restriction analysis of amplified exonic sequences, was investigated in a family with members affected by protein S deficiency, deep vein thrombosis and ictus. The clinical laboratory findings as well as RFLP analysis were consistent with the presence of a type WP III protein S deficiency clearly marked by a polymorphic allele, thus enabling us to determine the carrier status in several subjects. The RFLP analysis, extended to platelet mRNA after reverse transcription and amplification, demonstrated that the mRNA produced by the putative defective gene was present in a subject affected by thrombosis.

Adult↗