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Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis.

The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, Cystofilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.

Basidiomycota↗

Different types and rates of genome evolution detected by comparative sequence analysis of orthologous segments from four cereal genomes.

Orthologous regions in barley, rice, sorghum, and wheat were studied by bacterial artificial chromosome sequence analysis. General microcolinearity was observed for the four shared genes in this region. However, three genic rearrangements were observed. First, the rice region contains a cluster of 48 predicted small nucleolar RNA genes, but the comparable region from sorghum contains no homologous loci. Second, gene 2 was inverted in the barley lineage by an apparent unequal recombination after the ancestors of barley and wheat diverged, 11-15 million years ago (mya). Third, gene 4 underwent direct tandem duplication in a common ancestor of barley and wheat 29-41 mya. All four of the shared genes show the same synonymous substitution rate, but nonsynonymous substitution rates show significant variations between genes 4a and 4b, suggesting that gene 4b was largely released from the strong purifying selection that acts on gene 4a in both barley and wheat. Intergenic retrotransposon blocks, many of them organized as nested insertions, mostly account for the lower gene density of the barley and wheat regions. All but two of the retrotransposons were found in the regions between genes, while all but 2 of the 51 inverted repeat transposable elements were found as insertions in genic regions and outside the retrotransposon blocks.

Biological Evolution↗

Delineation of Borrelia burgdorferi sensu lato species by multilocus sequence analysis and confirmation of the delineation of Borrelia spielmanii sp. nov.

To evaluate multilocus sequence analysis (MLSA) for taxonomic purposes in the delineation of species within Borrelia burgdorferi sensu lato, seven relevant loci of various strains for which extensive DNA-DNA reassociation data were available were sequenced. MLSA delineation proved to be fully concordant with conventional methods. Our analysis confirmed the delineation of a novel species, Borrelia spielmanii sp. nov., previously known as 'Borrelia spielmani' Richter et al. 2004, with strain PC-Eq17N5T (=DSM 16813T = CIP 108855T) as the type strain.

Bacterial Typing Techniques↗

Inductive learning and biological sequence analysis. The PLAGE program.

Inductive learning, also called 'learning from examples', is a subfield of artificial intelligence. Inductive learning methods are able to deal with 'structural descriptions'. These portray objects as composite structures consisting of various components. The use of structural descriptions to represent biological objects is appealing. For instance, they have been used by Rawlings et al [1] for symbolically and comprehensively representing the folding of proteins. This paper shows how inductive learning techniques may be used for extracting information from biological objects. We briefly describe some general techniques for describing objects in a structural way and for learning from these descriptions. We present details of a program that we developed, PLAGE, and show the application of this program for a study on signal peptides, which was done in collaboration with A Danchin [2,3]. Finally, we survey some other approaches and applications of inductive learning to molecular biology.

Algorithms↗

Isolation and identification by sequence analysis of experimentally induced guinea pig amyloid fibrils.

Amyloidosis was produced experimentally in guinea pigs by multiple casein injections. Amyloid fibrils were isolated and fractionated and a protein obtained that had an amino acid composition comparable with A protein, a unique nonimmunoglobulin constituent of secondary amyloid deposits. N-terminal sequence analysis demonstrated a sequence homologous with that of A proteins from human and monkey preparations but preceded by a 5-residue peptide which had an N-terminal histidine. A definite species specificity in A protein from human and guinea pig was identified on immunologic analysis.

Amino Acid Sequence↗

Identification of a 35-kilodalton serovar-cross-reactive flagellar protein, FlaB, from Leptospira interrogans by N-terminal sequencing, gene cloning, and sequence analysis.

During the screening of antibodies to pathogenic leptospires, a murine monoclonal antibody (designated M138) was found to react with various serovars. An antigen of approximately 35 kDa from Leptospira interrogans serovar pomona, which reacted strongly with M138, was characterized by N-terminal amino acid sequencing and identified as a flagellin, a class B polypeptide subunit (FlaB) of the periplasmic flagella. The gene encoding the FlaB protein, flaB, was amplified from the genomic DNA of several pathogenic serovars by PCR with a single pair of oligonucleotide primers, suggesting that FlaB is highly conserved among these serovars. Cloning and sequence analysis of flaB from serovar pomona revealed that it contains an 849-bp open reading frame with a G + C content of 46.88% which encodes a 283-amino-acid protein with a calculated molecular mass of 31.297 kDa and a predicted pI of 9.065. A sequence comparison of flagellin proteins revealed that the amino acid sequence is most variable in the central portion of the serovar pomona FlaB, which is believed to contain specific sequence information and which may thus be useful in the design of DNA or synthetic peptide probes suitable for the detection of infection with pathogenic leptospires.

Amino Acid Sequence↗

Sequence analysis of light chain genes from human intestinal plasma cells demonstrates that lambda genes are almost all in-frame and highly mutated and most kappa genes are highly mutated when in-frame and minimally mutated when out-of-frame.

Around 80 % of immunoglobulin (Ig)-producing cells in man are located in the gut, with a preponderance of IgA- and IgM-producing cells that express heavily mutated IgVH genes. Here we describe the characteristics of Ig light chain genes isolated from human ileal and colonic lamina propria plasma cells. We focused on the properties of the two most commonly used light chain families, Vkappa1 and Vlambda2. Out-of-frame lambda rearrangements were very rare, suggesting that these lambda light chains may have undergone sequential rearrangements until successful conformation was achieved. This has not been observed in the human peripheral B cell population. The in-frame lambda gene rearrangements were highly mutated, with a frequency of mutation that was indistinguishable from that observed in many groups of heavy chain variable regions used by intestinal plasma cells. The in-frame kappac chain rearrangements were also highly mutated, but contained a subgroup of genes (27.3 %) that showed over 98 % homology with the germ-line gene. The majority of unused kappa chain genes were unmutated. A strong tendency for preferential mutation of G over C nucleotides was observed. Detailed analysis of the sequences in which the biases were observed suggested that this was likely to be due to selection, rather than a characteristic of the mechanism introducing the mutations.

Aged↗

Comparison of the oral bacterial flora in saliva from a healthy subject and two periodontitis patients by sequence analysis of 16S rDNA libraries.

The oral bacterial flora in the saliva from two patients with periodontitis and from a periodontally healthy subject were compared using a sequence analysis of 16S rDNA libraries without cultivation. 16S rDNAs were amplified from salivary DNA by PCR and cloned. Randomly selected clones were partially sequenced. On the basis of sequence similarities, the clones were classified into several clusters corresponding to the major phylum of the domain Bacteria. The major phylum in the libraries was the low G+C Gram-positive bacteria. There was no clonal sequence affiliated with periodontopathic bacteria in the salivary sample from the healthy subject, while a number of periodontal pathogens such as Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis and Treponema socranskii were detected in the salivary samples from the patients with periodontitis. In addition, a number of previously uncharacterized and uncultured microorganisms were recognized. These organisms may have some role in periodontal disease. This study reveals some potential for a molecular-biological technique to analyze the oral microflora associated with periodontal disease, including previously uncharacterized and uncultured microorganisms, without cultivation.

Adult↗

Sequence analysis of genomic regions containing trinucleotide repeats isolated by a novel cloning method.

A method was developed for effective isolation of trinucleotide repeats from genomic DNA. This method is based on the DNA polymerase reaction, which is restricted with only two or three nucleotide substrates and primed by biotinylated oligonucleotide probes. Sequences are then isolated by a streptavidin biotin-trapping method. More than 80% of the clones from each library contained more than eight trinucleotide repeats. Sequence analysis showed that the characteristic dinucleotide flanking sequences usually confronting various trinucleotide repeats are not found in the vicinity of CAG repeats, suggesting that CAG repeats may have been generated through a mechanism different from that of other trinucleotide repeats.

Blood↗

Isolation and sequence analysis of a Caenorhabditis elegans cDNA which encodes a 14-3-3 homologue.

We report the cloning of the Caenorhabditis elegans homologue of a 14-3-3 protein-encoding gene which is located within the unc-22 gene cluster on chromosome IV. Sequence analysis reveals that the cDNA-encoded product is 78% identical to both the Drosophila melanogaster and bovine 14-3-3 proteins. Our cDNA hybridizes to at least three major transcripts of 1.5, 1.35 and 0.9 kb, which are all more abundant in fertile hermaphrodites than in those lacking germ cells.

14-3-3 Proteins↗

Sequence analysis and molecular detection of mouse hepatitis virus using the polymerase chain reaction.

Sequence analysis of the nucleocapsid protein genes of five strains of mouse hepatitis virus (MHV) disclosed that the 3' region of the nucleocapsid protein gene contains highly conserved sequences unique to MHV. We designed a pair of primers to amplify cDNA from such sequences of MHV by using the polymerase chain reaction (PCR). Six isolates of wild-type MHV, as well as prototype viruses, were amplified successfully and detected in ethidium bromide-stained agarose gels. The sequence identity of PCR products was readily verified by confirming target size and a MflI site within the target. The sensitivity of our PCR assay was estimated to be sufficient to detect a single cell infected with MHV. This new approach may permit more sensitive and rapid detection of MHV in biologic materials than current methods such as virus isolation, the infant mouse bioassay, and the mouse antibody production test.

Base Sequence↗

Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F.

Previous studies have shown that the capsular polysaccharide synthesis (cps) locus of the type 19F Streptococcus pneumoniae strain SSZ was closely linked to a copy of the insertion sequence IS1202 (J.K. Morona, A. Guidolin, R. Morona, D. Hansman, and J.C. Paton, J. Bacteriol. 176:4437-4443, 1994). In the present study, we used plasmid insertion and rescue and inverse PCR to clone 6,322 bp of flanking DNA upstream of IS1202. Sequence analysis indicated that this region contains six complete open reading frames (ORFs) and one partial ORF that are arranged as a single transcriptional unit. Chromosomal disruption of any of these ORFs in a smooth-type 19F strain leads to a rough (unencapsulated) phenotype, indicating that this operon is essential for capsule production. The ORFs have therefore been designated cps19fA to cps19fG, where cps19fA is the first gene of the type 19F cps locus. Furthermore, many of the gene products from this incomplete operon exhibit strong similarities to proteins known to be involved in the production of capsular polysaccharide, exopolysaccharide, teichoic acid, enterobacterial common antigen, and lipopolysaccharide from numerous other bacterial species. This has allowed us to propose functions for many of the type 19F cps gene products. Southern hybridization studies reveal that cps19fA and cps19fB are conserved among all 12 pneumococcal serotypes tested, whereas genes downstream of cps19fB are conserved among some, but not all, of the serotypes tested.

Amino Acid Sequence↗

Identification and sequence analysis of the replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503.

The replication region of the phage resistance plasmid pCI528 from Lactococcus lactis subsp. cremoris UC503 was localised to within a 10-kb HindIII restriction fragment. A 6.3-kb BglII-HindIII subclone of this fragment, cloned into a replication probe vector, allowed replication in Lactococcus but not in Bacillus or Lactobacillus. Sequence analysis revealed an ORF of 1152 bp preceded by a putative ori region containing a 22-bp sequence tandemly repeated three and three-quarter times, a second smaller direct repeat and two inverted repeats. Extensive homology was observed with the well characterised replication region of the small cryptic plasmid pCI305 (Hayes, F., Vos, P., Fitzgerald, G.F., deVos, W. and Daly, C. Plasmid 25, 16-26).

Amino Acid Sequence↗

Characterization of the genome of molluscum contagiosum virus type 1 between the genome coordinates 0.045 and 0.075 by DNA nucleotide sequence analysis of a 5.6-kb HindIII/MluI DNA fragment.

The complete DNA nucleotide sequence of a HindIII/MluI genomic DNA fragment (0.045-0.075 viral map units) from molluscum contagiosum virus type 1 (MCV-1) was determined. The HindIII/MluI DNA fragment comprises 5,646 bp with a base composition of 64.4% G + C and 35.6% A + T. The DNA sequence contains many perfect direct repeats. A cluster of three repetitive DNA elements R1, R2 and R3, with a complex structural arrangement was detected between nucleotide positions 1802 and 2107. The unit length (box) of the repetitive DNA sequences was found to be 6 bp (15 boxes) and 9 bp (24 boxes) for R1 and R2, respectively. The repetitive DNA element R3 is organized in fifteen boxes (15 bp) in which a unit length of R1 is combined with a unit length of R2. The arrangement of the repetition R3 within the DNA sequences of this particular region of the MCV-1 genome was found to be (5 x R3) + (2 x R2) + (1 x R3) + (6 x R2) + (1 x R3) + (1 x R2) + (8 x R3). Twenty-three open reading frames (ORFs) of 60-1,175 amino acid (AA) residues were detected. The largest ORF (number 17) comprises 1,175 AA with a predicted molecular weight of 126 kD. This ORF harbors a promoter signal which is located 21 nucleotides upstream from the start codon and is very similar to the early promoter signals known for vaccinia virus. This putative protein contains glutamine-enriched regions between AA residues 427 and 682 which show homologies to the corresponding glutamine-enriched regions of a variety of cellular genes like human transcriptional initiation factor (TFIID: TATA box factor).

Amino Acid Sequence↗

Molecular cloning and sequence analysis of a chondroitin sulfate proteoglycan cDNA.

We report the identification and DNA sequence of a chondroitin sulfate proteoglycan core protein cDNA. A cDNA clone, pPG1, was selected from a rat yolk sac tumor poly(A)+RNA-derived cDNA library by using synthetic oligonucleotides predicted from the NH2-terminal peptide sequence of the mature chondroitin sulfate proteoglycan. The resulting sequence analysis demonstrated that the 874-base-pair pPG1 clone contained the complete coding region of the mature proteoglycan core protein as well as 5' and 3' flanking sequences. The 104 amino acid proteoglycan core protein sequence reveals that the core protein is composed of three regions, the most striking of which is the central 49 amino acid region composed of alternating serine and glycine residues. This region clearly functions as the acceptor site for the attachment of chondroitin sulfate side chains. The serine-glycine repeat region is flanked by a 14 amino acid NH2-terminal region identical to the NH2-terminal sequence of the proteoglycan obtained by amino acid sequencing and a 41 amino acid COOH-terminal region. RNA transfer blot hybridizations of poly(A)+ mRNA from rat yolk sac tumor cells with nick-translated pPG1 reveal a single mRNA of approximately equal to 1300 nucleotides. The possibility of detecting mRNAs and genomic sequences for other proteoglycans with a serine-glycine repeat by using this cDNA clone is discussed.

Amino Acid Sequence↗

Cloning and sequence analysis of human butyrophilin reveals a potential receptor function.

Human butyrophilin was cloned and sequenced from a human breast cDNA library. The derived amino acid sequence shows 84% sequence identity and identical domain arrangements with the previously reported bovine sequence. Sequence analysis reveals an immunoglobulin constant (IgC) domain that was not previously identified in the bovine sequence. The extracellular domain composition of butyrophilin suggests a cell surface receptor function.

Amino Acid Sequence↗

Catalase gene of the yeast Candida tropicalis. Sequence analysis and comparison with peroxisomal and cytosolic catalases from other sources.

A clone harbouring the genomic DNA sequence for the peroxisomal catalase of an n-alkane-utilizable yeast, Candida tropicalis, has been isolated by the hybrid-selection method and confirmed with a probe of catalase partial cDNA. Nucleotide sequence analysis of the cloned DNA disclosed that the gene fragment coding for catalase had a length of 1455 base pairs (corresponding to 485 amino acids; m = 54937 Da), and that the size of this enzyme was the smallest among all catalases reported hitherto. No intervening sequence was found in this coding region and some portions coincided with the amino acid sequences obtained from the analysis of the purified catalase. The comparison with three peroxisomal catalases from rat liver, bovine liver and human kidney, and one cytosolic catalase from Saccharomyces cerevisiae has revealed that catalase from C. tropicalis was more homologous to the peroxisomal enzymes than to the cytosolic one. C. tropicalis used the codons of the high-expression type. Amino acid residues were all conserved at the active and heme-binding sites. In the N and C-terminal regions there was no characteristic signal sequence or consensus sequence. However, a noticeable region, which can be discriminated between peroxisomal and cytosolic catalases, was proposed.

Amino Acid Sequence↗