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PubMed1998

Identification of group VS116 strains among Borrelia burgdorferi sensu lato grown from the hard tick, Ixodes ricinus (Linnaeus, 1758) by off-coupled restriction fragment length polymorphism analysis.

A total of 67 spirochetal isolates grown from the hard tick, Ixodes ricinus were analysed by PCR amplification of the spacer region between two conserved structures, the 3' end of the 5S rRNA and the 5' end of the 23S rRNA genes of Borrelia burgdorferi sensu lato. A 246-255 bp amplicon was generated from 13 reference strains of B. burgdorferi sensu lato representing the three major genospecies, B. burgdorferi sensu stricto, B. garinii, and B. afzelii, and from all 67 spirochetal isolates from ticks but not from B. hermsii. As could be confirmed by DNA sequence analysis, restriction fragment length polymorphism (RFLP) analysis of the PCR product after cleavage with DraI and MseI distinguished between the three major genospecies: out of the 67 B. burgdorferi sensu lato isolates from ticks, 27 (40.3%) were typed as B. burgdorferi sensu stricto including five isolates with a unique DraI or MseI pattern. 26 isolates (38.8%) were typed as B. garinii and 6 (9.0%) as B. afzelii, respectively. A group of eight isolates (11.9%) displayed a unique MseI pattern identical to that described for a putative new European genospecies of Borrelia burgdorferi sensu lato designated VS116. DNA sequences of the PCR product of seven of these isolates tested were by less than 88.5% identical with the established European major genospecies but shared 98% to 100% homology with that of database-derived sequences of strain VS116 from Switzerland and strain UK from England which are both representatives of the European genomic group VS116.

Animals↗
PubMed1979

Sequence analysis of two yeast mitochondrial DNA fragments containing the genes for tRNA Ser UCR and tRNA Phe UUY.

Two restriction enzyme fragments containing yeast mitochondrial tRNA genes have been characterized by DNA sequence analysis. One of these fragments is 320 base pairs long and contains a tRNA Ser gene. The corresponding tRNA SER was isolated from yeast mitochondria and its nucleotide sequence also was determined. This mitochondrial tRNA is 90 nucleotides in length, has a G + C content of 38%, and has UGA as the anticodon. A portion of a 680-base-pair DNA fragment containing a tRNA Phe gene was also sequenced. The portion of this gene which codes for the mature tRNA is 75 base pairs in length, has a G + C content of 33%, and contains the anticodon GAA. Neither gene contains an intervening sequence or codes for the 3' CCA terminus. Both are surrounded by regions of more than 90% A + T. The significance of these sequences is discussed.

Base Sequence↗
PubMed2025

Taxogenomic analysis of Pichia senei sp. nov. and new insights into hybridization events in the Pichia cactophila species complex.

Three strains of a novel yeast species were isolated from necrotic cactus tissues of Cereus saddianus and Micranthocereus dolichospermaticus and from phytotelmata of Bromelia karatas. DNA sequence analysis of the Internal Transcribed Spacer (ITS) region and D1/D2 domains of the large subunit ribosomal RNA, along with whole genome phylogenomic analysis, showed that this yeast is most closely related to Pichia insulana, Pichia cactophila, and Pichia inconspicua. The new species differs by 10-13 nucleotide substitutions from these species in D1/D2 sequences and exhibits <90% genome-wide average nucleotide identity to them. The name Pichia senei sp. nov. is proposed for the novel species, which is homothallic and produces asci with one to four hat-shaped ascospores. The holotype is CBS 16311 (MycoBank MB 858723). Taxogenomic analyses of the P. cactophila species complex, including P. senei, provide new insights about the hybridizations events that shaped this group. Pichia insulana and P. inconspicua are identified as the parental lineages that originated P. cactophila, and P. senei also appears closely related to one of the progenitors of P. inconspicua. We assess phylogeny, heterozygosity, and ploidy to explore the processes shaping diversity, showing how genomic data support yeast species delimitation and reveal complex hybridization.

Phylogeny↗
PubMed1985

Analysis of DNA sequences using a single chemical cleavage procedure.

A novel approach to sequence analysis of end-labeled, defined DNA fragments, using a single chemical cleavage procedure and electrophoretic separation in a single lane, has been developed. Prolonged treatment with hot aqueous piperidine results in partial cleavage of the DNA at all positions; the relative propensity for this cleavage is different for the various bases in the DNA. The hydrolysate is resolved on a DNA sequencing gel, and the distribution of radioactivity in the electrophoretic lane is analyzed (a) in terms of differential peak heights of the radioactive bands and (b) in terms of the spacings between successive bands. Simultaneous application of these two base-characteristic criteria allows the deduction of the nucleotide sequence with an accuracy approaching that of the established four-lane methods of DNA sequencing.

Base Sequence↗
PubMed2002

[Study on sequence difference and SNP pheomenon of rDNA ITS region in F type and H type population of Dendrobium officinale].

OBJECTIVE: To study rDNA ITS sequence differences between F type and that of H type of Dendrobium officinale in main habitat of China. METHOD: The population differences of the rDNA ITS region (including ITS1, ITS2, 5.8S) sequences of D. officinale were studied by the method of DNA sequences analysis. RESULT: There were two different sites between the rDNA ITS sequence of F type and that of H type. One was in ITS1 region, and the other was in 5.8S region. It was proved that there was some relativity between the character of rDNA ITS region and the life type of the populations. The phenomenon of single nucleotide polymorphism (SNP) existed in 5.8S region of rDNA ITS region between F type and H type. The sequences of rDNA ITS region of D. officinale were reported for the first time, and the sequences of ITS region ranged 634 bp (ITS1 231 bp, ITS2 240 bp, 5.8S 163 bp). CONCLUSION: The analysis of rDNA ITS of D. officinale deeply reveal the population differences of D. officinale of F type and H type.

Base Sequence↗
PubMed2005

Five new genome types of adenovirus type 37 caused epidemic keratoconjunctivitis in Sapporo, Japan, for more than 10 years.

Human adenovirus type 37 (HAdV-37) is a major cause of epidemic keratoconjunctivitis and has recently been the largest causative agent of keratoconjunctivitis in Japan. To investigate the genetic characteristics of HAdV-37 strains isolated in Sapporo, we analyzed the genome types and genetic relationships of 51 strains isolated there from 1990 through 2001. By using DNA restriction analysis, eight genome types (HAdV-37/D1, HAdV-37/D3, and HAdV-37/D6 to HAdV-37/D11) were identified, including five new ones. The restriction fragments of these genome types shared more than 95% identity with those of the prototype strain. By DNA sequence analysis, five and three single nucleotide substitutions, respectively, were found in partial sequences of the hexon and fiber genes. The combinations of mutations resulted in four hexon and fiber types (hx1 to hx4 and f1 to f4) and six hexon/fiber pairs (hx1/f1, hx2/f1, hx1/f2, hx1/f3, hx3/f4, and hx4/f4). The six pairs correlated well with certain genome types. In all three epidemics of keratoconjunctivitis to strike Sapporo in the past 12 years, specific genome types and fiber types were usually isolated: in the first epidemic, HAdV-37/D1 (f1) and HAdV-37/D3 (f1); in the second, HAdV-37/D6 (f2) and HAdV-37/D8 (f3); and in the third, HAdV-37/D10 (f4) and HAdV-37/D11 (f4). We conclude that mutations in the adenovirus genome occurred chronologically and that certain mutations were correlated with the epidemics of adenoviral keratoconjunctivitis.

Adenovirus Infections, Human↗
PubMed2000

Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages.

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.

Amino Acid Sequence↗
PubMed2000

The primary structure of the subunit in Bacillus thermoamyloliquefaciens KP1071 molecular weight 540,000 homohexameric alpha-glucosidase II belonging to the glycosyl hydrolase family 31.

The gene that coded for the subunit of an molecular weight (Mr) 540,000 homohexameric alpha-glucosidase II (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) produced by Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477) growing at 30 to 66 degrees C was expressed in Escherichia coli HB101. The resulting homohexameric enzyme had a half-life of 10 min at 80 degrees C. Its purification and characterization showed that the enzyme was identical with the native one except for the latter deleting 7 N-terminal residues found in the former. The primary sequence of the subunit with 787 residues and an Mr of 91,070 deduced from the gene was 24-34% identical to the corresponding sequences of 15 alpha-glucosidases in the glycosyl hydrolase family 31 from 14 eukaryotic origins and the archaeon Sulfolobus solfataricus 98/2. From the sequence analysis by the neural network method of Rost and Sander [Rost, B. and Sander, C., Proteins: Struct. Funct. Genet., 19, 55-72 (1994)], we inferred that alpha-glucosidase II might make each subunit of 3 secondary structural regions, i.e., one N-terminal beta region, one central alpha/beta region with two catalytic residues Asp407 and Asp484, and one C-terminal beta region.

Amino Acid Sequence↗
PubMed2000

Complete cDNA sequence and amino acid analysis of a bovine ribonuclease K6 gene.

The complete cDNA sequence of a ribonuclease k6 gene of Bos Taurus has been determined. It codes for a protein with 154 amino acids and contains the invariant cysteine, histidine and lysine residues as well as the characteristic motifs specific to ribonuclease active sites. The deduced protein sequence is 27 residues longer than other known ribonucleases k6 and shows amino acids exchanges which could reflect a strain specificity or polymorphism within the bovine genome. Based on sequence similarity we have termed the identified gene bovine ribonuclease k6 b (brk6b).

Amino Acid Sequence↗
PubMed1999

Protein S gene analysis reveals the presence of a cosegregating mutation in most pedigrees with type I but not type III PS deficiency.

DNA sequence analysis of the protein S gene (PROS1) in 22 Spanish probands with type I or III PS deficiency, has allowed the identification of 10 different mutations and 2 new sequence variants in 15 probands. Nine of the mutations, 8 of which are novel, cosegregate with type I or quantitative PS deficiency in 12 of the 13 pedigrees analyzed. One of these mutations (Q238X) also cosegregates with both type I and III PS-deficient phenotypes coexisting in a type I/III pedigree. Another mutation identified in a pedigree with these two PS phenotypes is the missense mutation R520G, present in the homozygous form in the type I propositus and in the heterozygous form in his type III relatives. By contrast, no cosegregating PROS1 mutation has been found in any of the six families with only type III phenotypes. Three of these families, as well as the two families with type I and I/III phenotypes where no other PROS1 mutation has been identified, segregate the P allele of the S460P variant, although this allele does not always cosegregate with the deficient phenotype. From these results we conclude that while mutations in PROS1 are the main cause of type I PS deficiency, the molecular basis of the type III phenotype is probably more complex, with many cases not being explained by a PROS1 mutation.

Adolescent↗
PubMed2006

Isolation and cloning of an endo-beta-1,4-mannanase from Pacific abalone Haliotis discus hannai.

An endo-beta-1,4-mannanase was isolated from digestive fluid of Pacific abalone, Haliotis discus hannai, by successive chromatographies on TOYPEARL CM-650M, hydroxyapatite, and TOYOPEARL HW50F. The abalone mannanase, named HdMan in the present paper, showed a molecular mass of approximately 39,000 Da on SDS-PAGE, and exhibited high hydrolyic activity on both galactomannan from locust bean gum and glucomannan from konjac at an optimal pH and temperature of 7.5 and 45 degrees C, respectively. HdMan could degrade either beta-1,4-mannan or beta-1,4-mannooligosaccharides to mannotriose and mannobiose similarly to beta-1,4-mannanases from Pomacea, Littorina, and Mytilus. In addition, HdMan could disperse the fronds of a red alga Porphyra yezoensis into cell masses consisting of 10-20 cells that are available for cell engineering of this alga. cDNAs encoding HdMan were amplified by polymerase chain reaction from an abalone-hepatopancreas cDNA library. From the nucleotide sequences of the cDNAs, the sequence of 1232 bp in total was determined and the amino-acid sequence of 377 residues was deduced from the translational region of 1134 bp locating at nucleotide positions 15-1148. The N-terminal region of 17 residues except for the initiation Met, was regarded as the signal peptide of HdMan because it was absent in the HdMan protein and showed high similarity to the consensus sequence for signal peptides of eukaryote secretory proteins. Accordingly, mature HdMan was considered to consist of 359 residues with the calculated molecular mass of 39,627.2 Da. HdMan is classified into glycoside hydrolase family 5 (GHF5) on the basis of sequence homology to GHF5 enzymes.

Amino Acid Sequence↗
PubMed1994

Atypical regions in large genomic DNA sequences.

Large genomic DNA sequences contain regions with distinctive patterns of sequence organization. We describe a method using logarithms of probabilities based on seventh-order Markov chains to rapidly identify genomic sequences that do not resemble models of genome organization built from compilations of octanucleotide usage. Data bases have been constructed from Escherichia coli and Saccharomyces cerevisiae DNA sequences of > 1000 nt and human sequences of > 10,000 nt. Atypical genes and clusters of genes have been located in bacteriophage, yeast, and primate DNA sequences. We consider criteria for statistical significance of the results, offer possible explanations for the observed variation in genome organization, and give additional applications of these methods in DNA sequence analysis.

Bacteriophage lambda↗
PubMed2001

Exact distribution for the local score of one i.i.d. random sequence.

Let X(1)...X(n) be a sequence of i.i.d. positive or negative integer-valued random variables and H(n) = max(0 < or = i < or = j < or = n)(X(i) +...+ X(j)) be the local score of the sequence. The exact distribution of H(n) is obtained using a simple Markov chain. This result is applied to the scoring of DNA and protein sequences in molecular biology.

Computational Biology↗
PubMed2004

Novel archaeal alanine:glyoxylate aminotransferase from Thermococcus litoralis.

A novel alanine:glyoxylate aminotransferase was found in a hyperthermophilic archaeon, Thermococcus litoralis. The amino acid sequence of the enzyme did not show a similarity to any alanine:glyoxylate aminotransferases reported so far. Homologues of the enzyme appear to be present in almost all hyperthermophilic archaea whose whole genomes have been sequenced.

Amino Acid Sequence↗
PubMed2003

The four TCR genes of teleost fish: the cDNA and genomic DNA analysis of Japanese flounder (Paralichthys olivaceus) TCR alpha-, beta-, gamma-, and delta-chains.

We have isolated and identified all four TCR alpha, beta, gamma, and delta cDNAs and genomic clones from a Japanese flounder leukocyte cDNA library and bacterial artificial chromosomal genomic library. Numerous TCR transcripts were sequenced to examine the variability against antigenic peptide, and were shown hypervariability on their complementarity-determining region 3 (CDR3) loops. Among CDR3s, CDR3 delta showed a long and broad length distribution, indicating greater similarity to that of Ig. From cDNA sequences and genomic gene analysis of each chain, we found that flounder TCR beta, gamma, and delta have two different C gene segments, while the TCR alpha C region exists as a single segment. The flounder C gammas and C deltas showed different lengths in the connecting peptide (CP) region between the different types of polypeptides. The C delta 1 gene consists of two exons, one that encodes an extracellular Ig-like domain (exon 1) and the other that encodes either a very short or possibly a lacking CP region, a transmembrane region, and a cytoplasmic tail (exon 2); these are located within TCR alpha gene locus. Southern blot analysis, using the bacterial artificial chromosomal genomic DNA clones, revealed that the C delta 2 gene segment, which has a long CP region and different genomic organization to the C delta 1 gene, exists on same gene locus as the TCR gamma-chain. This suggests that the flounder possesses very unique genomic DNA organization and gene loci for TCR, C alpha/C delta 1, and C gamma/C delta 2.

Amino Acid Sequence↗
PubMed2002

A novel asparaginase-like protein is a sperm autoantigen in rats.

A novel asparaginase-like protein (ALP) of spermatozoa was cloned from rat and human testis cDNA libraries on the basis of reactivity with antibodies produced after vasectomy. Although obstruction of the male reproductive tract is known to cause an immunologic response, few of the sperm antigens responsible for the generation of autoantibodies have been characterized. We are identifying proteins of interest by coring autoantigenic protein spots from two-dimensional (2-D) gels of rat sperm extracts and microsequencing them by mass spectrometry. The peptide sequences from ALP, a 28 kDa, pI 5.7 protein, matched to a single partial length rat EST. These peptide sequences were used to clone a cDNA encoding a novel 333 amino acid open reading frame. The new protein had a similarity to portions of L-asparaginases of plants (43%) and to glycosylasparaginases in animal cells (32%). Human ALP cDNA was subsequently cloned. It showed 77% identity to the rat ALP sequence and the gene, ASRGL1 (asparaginase-like 1), mapped to chromosome locus 11q12.3. Purified recombinant rat ALP (rALP), expressed in E. coli, was used to raise polyclonal antiserum in guinea pigs. Two observations verified that the correct protein had been cloned: 1) the anti-rALP antibody reacted with both rALP and rat sperm; and 2) post-vasectomy sera bound rALP. Anti-rALP antibody stained the midpiece of rat and human sperm coincident with staining by MitoTracker Green FM, suggesting that ALP is associated with the mitochondria. Northern analysis revealed that rat ALP message was abundantly expressed in the testis but was also present in heart, brain, liver, skeletal muscle, and kidney.

Amino Acid Sequence↗
PubMed2001

Application of denaturing high-performance liquid chromatography for mapping of single nucleotide polymorphisms in barley (Hordeum vulgare L.).

Recent advances in DNA sequence analysis and the establishment of high-throughput assays have provided the framework for large-scale discovery and analysis of DNA sequence variation. In this context, single nucleotide polymorphisms (SNPs) are of particular interest. To initiate a systematic approach to develop an SNP map of barley (Hordeum vulgare L.), we have employed denaturing high-performance liquid chromatography (DHPLC) to analyse segregating SNP patterns in a doubled-haploid (DH) mapping population. To this end, SNPs between the parental genotypes were identified using a direct sequencing approach. Once a SNP was established between the parents, the optimal melting temperature of the PCR fragment containing the SNP was predicted for its analysis by DHPLC. Following the detection of the optimal temperature, the DH lines were analysed for the presence of either of the alleles. To test the utility of the analysis, data from previously mapped RFLP markers from which these SNPs were derived were compared. Results from these experiments indicate that DHPLC can be efficiently employed in analysing SNPs on a high-throughput scale.

Chromatography, High Pressure Liquid↗