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PubMed2000

Phylogenetic position of the North American isolate of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, as inferred from 16S rDNA sequence analysis.

A 1341 bp sequence of the 16S rDNA of an undescribed species of Pasteuria that parasitizes the soybean cyst nematode, Heterodera glycines, was determined and then compared with a homologous sequence of Pasteuria ramosa, a parasite of cladoceran water fleas of the family Daphnidae. The two Pasteuria sequences, which diverged from each other by a dissimilarity index of 7%, also were compared with the 16S rDNA sequences of 30 other bacterial species to determine the phylogenetic position of the genus Pasteuria among the Gram-positive eubacteria. Phylogenetic analyses using maximum-likelihood, maximum-parsimony and neighbour-joining methods showed that the Heterodera glycines-infecting Pasteuria and its sister species, P. ramosa, form a distinct line of descent within the Alicyclobacillus group of the Bacillaceae. These results are consistent with the view that the genus Pasteuria is a deeply rooted member of the Clostridium-Bacillus-Streptococcus branch of the Gram-positive eubacteria, neither related to the actinomycetes nor closely related to true endospore-forming bacteria.

Animals↗

PubMed1996

Sequence analysis of the 2nd intron revealed common sequence motifs providing the means for a unique sequencing based typing protocol of the HLA-A locus.

We here present a sequencing strategy for the HLA-A locus which is generally applicable for all HLA class I genes. The typing strategy is based on a group-specific amplification according to the serologically defined antigens. The PCR products carry the typing-relevant polymorphic regions of the 2nd and 3rd exon including the 2nd intron. The sequencing primers were designed to match conserved sequence motifs in the 2nd intron allowing a nested sequencing approach in 3' and 5' direction. These conserved regions were identified after sequence compilation of the 2nd intron of 143 clinical samples and 48 cell lines mostly from the 9th and 10th IHWC representing all serologically defined groups of alleles. This strategy allowed the use of only one 5' and one 3' sequencing primer regardless of the amplified allele. Therefore, it was possible to use dye terminator as well as dye primer sequencing chemistry. The amplification strategy allowed the separation of the haplotypes in almost all cases. Thus, an assignment of heterozygous positions requiring high sequencing quality was not necessary, allowing the application of Sequenase as well as TaqPolymerase as sequencing enzyme. Concerning the resolution of heterozygosity it is obvious that this approach is superior to a typing system using a single pair of generic primers followed by direct sequencing, since the latter technique is not capable of defining the cis/trans linkage of polymorphic sequences and, hence, cannot exclude the presence of unknown alleles.

Alleles↗

PubMed1987

A high-performance liquid chromatography procedure for recovering subnanomole amounts of protein from SDS-gel electroeluates for gas-phase sequence analysis.

A high-performance liquid chromatographic procedure for recovering subnanomole amounts of protein from SDS/polyacrylamide gel electroeluates in a form suitable for gas-phase sequence analysis has been developed. By a judicious choice of reversed-phase column packing, proteins can be retained at high concentrations of n-propanol (90-100%) where sodium dodecylsulfate and acrylamide gel-related contaminants are washed through the column. Retained proteins can be recovered from the column in high yield (greater than 90%) by the simultaneous adding of an ion-pairing reagent into the mobile phase and elution with a gradient of decreasing n-propanol concentration (i.e. an 'inverse or negative gradient'). Furthermore, by using a steep gradient (e.g. 50%/min) at a low flow rate (20-200 microliters/min) the proteins can be recovered in less than 100 microliters and can be used for gas-phase sequence analysis without further manipulation. This procedure is independent of sodium dodecylsulfate concentration (up to 1.2% w/v) in sample loading volumes of up to 1.5 ml. Microbore columns (2.1 mm internal diameter) have been employed for recovering small amounts of protein (1-100 micrograms from electroeluates of protein-containing gel spots while conventional columns (4.6 mm internal diameter) were used for isolating larger amounts of protein (greater than 500 micrograms) from electroeluates of preparative gel bands. The general utility of this inverse-gradient high-performance liquid chromatography procedure has been demonstrated by its successful application in recovering a wide variety of proteins from sodium dodecylsulfate gel electroeluates in a form suitable for N-terminal sequence analysis in the 10-500 pmol range.

Chromatography, High Pressure Liquid↗

PubMed1999

Genomic organization around the centromeric end of the HLA class I region: large-scale sequence analysis.

We previously sequenced two regions around the centromeric end of HLA class I and the boundary between class I and class III. In this paper we analyze the two regions of about 385 kb and confirm, giving a new line of evidence, that the following two pairs of the genomic segments were duplicated in evolution: (i) a 43-kb genomic segment including the HLA-B gene showing the highest polymorphism among the classical HLA class I loci (class Ia) and a 40-kb segment including the HLA-C locus showing the lowest polymorphism and (ii) a 52-kb segment including the MIC (MHC class I chain related gene) B and a 35-kb segment including MICA. We also found that repetitive elements such as SINEs, LINEs, and LTRs occupy as much as 47% of nucleotides in this 385-kb region. This unusually high content of repetitive elements indicates that repeat-mediated rearrangements have frequently occurred in the evolutionary history of the HLA class Ia region. Analysis of LINE compositions within the two pairs of duplicated segments revealed that (i) LINEs in these regions had been dispersed prior to both the duplication of the HLA-B and -C loci and the duplication of the MICB and MICA loci, and (ii) the divergence of the HLA-B and -C loci occurred prior to the duplication of the MICA and MICB loci. To find novel genes responsible for HLA class I-associated or other diseases, we performed computer analysis applying GenScan and GRAIL to GenBank's dbEST. As a result, at least five as yet uncharacterized genes were newly mapped on the HLA class I centromeric region studied. These novel genes should be analyzed further to determine their relationships to diseases associated with this region.

Base Sequence↗

PubMedDate not supplied

Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis.

Small subunit rRNA sequence data were generated for 27 strains of cyanobacteria and incorporated into a phylogenetic analysis of 1,377 aligned sequence positions from a diverse sampling of 53 cyanobacteria and 10 photosynthetic plastids. Tree inference was carried out using a maximum likelihood method with correction for site-to-site variation in evolutionary rate. Confidence in the inferred phylogenetic relationships was determined by construction of a majority-rule consensus tree based on alternative topologies not considered to be statistically significantly different from the optimal tree. The results are in agreement with earlier studies in the assignment of individual taxa to specific sequence groups. Several relationships not previously noted among sequence groups are indicated, whereas other relationships previously supported are contradicted. All plastids cluster as a strongly supported monophyletic group arising near the root of the cyanobacterial line of descent.

Cyanobacteria↗

PubMed1999

Identification of clinical isolates of Mycobacterium spp. by sequence analysis of the 16S ribosomal RNA gene. Experience from a clinical laboratory.

Twenty-one mycobacterial type strains and 334 clinical isolates of mycobacteria were identified by standardized sequence analysis using part of the gene encoding 16S rRNA. Apart from two clinical isolates, the resulting sequences corresponded to previously published sequences. The results of the molecular determinations of the type strains completely overlapped the identities obtained using conventional techniques (cultural characteristics, biochemical tests, commercial DNA probes, and gas chromatographic lipid profiles). Of 323 isolates conventionally identified as slow-growing mycobacteria, 318 (98.5%) were identified to the same species or group level by 16S rDNA sequence analysis, while 6 of the 11 strains of rapid growers obtained a corresponding identity with the two approaches. The sequencing protocol combined with a few cultural characteristics (i.e. growth rate, pigmentation and susceptibility testing) offers a rapid, reliable and usually definite identification of mycobacterial isolates.

Amino Acid Sequence↗

PubMed2003

RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis.

Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.

Apolipoproteins B↗

PubMed2000

Comparative phylogenetic analyses of members of the order Planctomycetales and the division Verrucomicrobia: 23S rRNA gene sequence analysis supports the 16S rRNA gene sequence-derived phylogeny.

Almost complete 23S rRNA gene sequences were obtained from 13 planctomycete strains, the fimbriated, prosthecate bacterium Verrucomicrobium spinosum and two strains of the genus Prosthecobacter. The 23S rRNA genes were amplified by the PCR, using modified primers. The majority of the planctomycete strains investigated were shown to have 23S rRNA genes that were not linked to the 16S rRNA genes. Amplification of the 5'-termini of these genes was achieved using a novel primer-design strategy. Comparative phylogenetic analyses were performed using the 23S rRNA gene sequences determined in this study and previously determined 16S rRNA gene sequences. The phylogenetic dendrograms constructed from both datasets showed that the planctomycetes form a coherent group and distinct lineage within the domain Bacteria. Analysis of 23S rRNA gene sequences of Verrucomicrobium spinosum, Prosthecobacter fusiformis and Prosthecobacter sp. strain FC-2 showed that these organisms cluster together, as was also shown here and previously by analysis of 16S rRNA gene sequences. The distinct phylogenetic position of the division Verrucomicrobia was also supported by analysis of the 23S rRNA gene sequences, and no statistically significant phylogenetic relationship between the division Verrucomicrobia and the planctomycetes was found. The analyses presented in this study also provide further evidence that the chlamydiae are no more related to members of the order Planctomycetales and the division Verrucomicrobia than they are to members of other bacterial lineages.

Bacteria↗

PubMed2000

Expression of four genes of bacteriophage MB78 from contiguous open reading frames: the genomic organization as deduced by sequence analysis.

Four proteins of bacteriophage MB78 having apparent molecular weights as 35, 14, 21 and 16 kDa are expressed from 3.9 kb SalI-HindIII fragment located almost in the middle of the phage genome. Analysis of the sequence supported by some experimental evidences suggest that these four proteins are expressed from polycistronic message without any intercistronic gap. Stop and start codons of consecutive ORFs overlap and rare initiation codons are used. Computer analysis of the sequence suggests the presence of two more open reading frames within the ORFs of 35 and 16 kDa proteins but in the opposite orientation, i.e. in the complementary strand.

Amino Acid Sequence↗

PubMed2006

Sequence analysis of VP7 gene of Indian bluetongue virus serotype-23 shows its close phylogenetic relationship to Australian and Chinese serotypes.

Bluetongue, an arthropod borne viral disease of wild and domestic ruminants, causes heavy economic losses throughout the world. In the present study, full-length VP7 gene of Indian bluetongue virus (BTV) serotype 23 was sequenced and compared with prototype strains of BTV reported from different countries. Nucleotide sequence analysis of VP7 gene revealed Indian BTV serotype 23 to have 1154 nucleotides with the deletion of two nucleotides at 3' non-coding region and a unique amino acid change 211S-N. The Indian virus also demonstrated a maximum similarity of 94.2% with Australian serotype 1 and a minimum similarity of 67.4% with Australian serotype 15. However, at deduced amino acid level, it had maximum similarity of 99.7% and a minimum of 82.5% with Chinese serotypes 1, 2 and 4 and Australian serotype 15, respectively. Deduced amino acid sequence analysis of putative receptor binding domain (121-249) revealed all the nine hydrophilic domains to be conserved across the serotypes. Functional motifs present in VP7 protein were also conserved in almost all the BTV serotypes including Indian serotype 23. Phylogenetic analysis based on VP7 gene sequence revealed Indian BTV serotype 23 segregating into a monophyletic group along with Australian serotype 1 and Chinese serotypes 1, 2 and 4, indicating its close evolutionary relationship with these Australian and Chinese serotypes.

Amino Acid Sequence↗

PubMed2006

The MIGenAS integrated bioinformatics toolkit for web-based sequence analysis.

We describe a versatile and extensible integrated bioinformatics toolkit for the analysis of biological sequences over the Internet. The web portal offers convenient interactive access to a growing pool of chainable bioinformatics software tools and databases that are centrally installed and maintained by the RZG. Currently, supported tasks comprise sequence similarity searches in public or user-supplied databases, computation and validation of multiple sequence alignments, phylogenetic analysis and protein-structure prediction. Individual tools can be seamlessly chained into pipelines allowing the user to conveniently process complex workflows without the necessity to take care of any format conversions or tedious parsing of intermediate results. The toolkit is part of the Max-Planck Integrated Gene Analysis System (MIGenAS) of the Max Planck Society available at www.migenas.org (click 'Start Toolkit').

Animals↗

PubMed2006

Immunoglobulin heavy chain sequence analysis in Waldenstrom's macroglobulinemia and immunoglobulin M monoclonal gammopathy of undetermined significance.

In this study, we used IGH sequence analysis to assess the maturational status of Waldenstrom's (WM) macroglobulinemia and its putative precursor immunoglobulin (Ig)-M monoclonal gammopathy of undetermined significance (MGUS). IGH sequence analysis was performed using standard methods in 23 cases (20 WM and 3 IgM MGUS as defined by consensus panel criteria). Waldenstrom's macroglobulinemia cases were characterized by heavily mutated IGH genes (median, 6.3%; range, 3.8%-13.9%) but without intraclonal variation (ICV). IgM MGUS was similarly characterized by somatic hypermutation (median, 7.5%; range, 7%-7.7%), but ICV was evident in 1 of the 3 cases. We would therefore conclude that WM is characterized by somatic hypermutation without ICV, which supports a derivation from postgerminal center/memory B cells. IgM MGUS is also characterized by somatic hypermutation but, in a manner similar to IgA/IgG MGUS, can be associated with ICV, although the significance of this remains unclear.

Aged↗

PubMed1992

Nucleotide sequence analysis of an HTLV-I isolate from a Chilean patient with HAM/TSP.

Isolates of HTLV-I have been characterized from a number of different regions of the world; however, there has not been a nucleotide sequence analysis of an HTLV-I isolate from a South American country. Reported here is an individual from Chile identified with the HTLV-I-associated neurological disease HAM/TSP. The sera and the nucleic acid sequence of the HTLV-I present in peripheral blood lymphocytes from this Chilean HAM/TSP patient over a two year period are characterized. During this time, the patient's condition grew progressively worse. While the serological profile of this patient was unremarkable in comparison with other HAM/TSP patients previously described, nucleic acid sequence analysis identified two nucleotide positions which contained nucleotides unique to this Chilean isolate. The nucleotide sequence analysis also indicates that the Chilean HTLV-I isolate is more closely related to Caribbean and Japanese isolates of HTLV-I than to the African and U.S. isolates described so far.

Base Sequence↗

PubMed1997

Genetic characterization of ruminant pestiviruses: sequence analysis of viral genotypes isolated from sheep.

Historically, the genus pestivirus was believed to contain three species of viruses; bovine viral diarrhea virus (BVDV), border disease virus (BDV) and classical swine fever virus (CSFV). However, based on limited sequence analysis of a small number of pestiviral isolates from domestic livestock, evidence has recently emerged indicating that at least four distinct genotypes exist. In an attempt to gain a better understanding of the degree of viral variation among ruminant pestiviruses, the entire structural gene coding region of an ovine pestivirus. BD31, genome encompassing 3358 nucleotides was cloned and sequenced. Sequence analysis revealed that BD31 shares less than 71% nucleotide similarity with other pestiviruses, suggesting, that BD31 is distinct from BVDV, CSFV as well as other ovine and bovine pestiviruses currently referred to as BVDV type II. Based on this data, BD31 is the first North American pestivirus isolate that falls under the category true BDV. Results from the analysis of the nucleotide sequence of the E0-E1 coding region of six additional ruminant pestiviruses identified the existence of three distinct virus genotypes in North America. Thus, among ruminent pestiviruses, bovine isolates can be grouped into two genotypes, namely types 1 and 4, whereas ovine isolates fall into genotypes 1, 3 and 4.

Amino Acid Sequence↗

PubMed1991

Sequence analysis and expression of the human parainfluenza type 1 virus nucleoprotein gene.

The nucleotide sequence of the human parainfluenza type 1 (Pl1) virus nucleoprotein (NP) gene was determined from cDNA clones of the mRNA. A cDNA clone, 4-31, containing a 1.7-kb insert, was identified as a Pl1 NP-specific clone by partial nucleotide sequence analysis and sequence comparison with Sendai virus (SV) NP. Using a vaccinia virus transient expression system, a polypeptide with electrophoretic mobility similar to that of Pl1 NP was synthesized from this clone, which reacted specifically with both polyclonal and monoclonal antibodies against Pl1 virus and Pl1 NP, respectively. The complete nucleotide sequence of this clone was determined and was found to contain a single open reading frame that can encode a protein of 524 amino acids with a predicted molecular weight of 57,547. Comparison of the amino acid sequence of Pl1 NP with that of other paramyxoviruses showed that two conserved amino acid sequences found within other paramyxoviruses are also present in Pl1 NP. Although Pl1 and SV showed a high sequence homology, approximately 100 amino acids at the C-terminal region were highly divergent as found also among other paramyxoviruses.

Amino Acid Sequence↗

PubMed1993

Genomic structure and sequence analysis of Drosophila melanogaster HSC70 genes.

We report the identification of two new members of the Drosophila melanogaster HSP70 gene family, HSC3 and HSC5. DNA sequence analysis predicts that HSC3 encodes a 72-kDa protein with a hydrophobic leader sequence and a C-terminal retrieval tetrapeptide, KDEL, characteristics associated with luminal endoplasmic reticulum (ER) proteins. Sequence analysis predicts that HSC5 encodes a 74-kDa protein with a characteristic mitochondrial leader sequence. We report the deduced amino acid (aa) sequence for the previously identified gene, HSC1. HSC1 encodes a 70-kDa protein lacking a leader sequence and is presumed to have a cytoplasmic localization. A comparison of the deduced aa sequences of these and hsc70 proteins from different species indicates that hsc70 proteins residing in the same intracellular compartment in different organisms are more similar to each other than are hsc70s from the same organism, but different organelles.

Amino Acid Sequence↗

PubMed1987

Sequence analysis on microcomputers.

Overall, each of the program packages performed their tasks satisfactorily. For analyses where there was a well-defined answer, such as a search for a restriction site, there were few significant differences between the program sets. However, for tasks in which a degree of flexibility is desirable, such as homology or similarity determinations and database searches, DNASTAR consistently afforded the user more options in conducting the required analysis than did the other two packages. However, for laboratories where sequence analysis is not a major effort and the expense of a full sequence analysis workstation cannot be justified, MicroGenie and IBI-Pustell offer a satisfactory alternative. MicroGenie is a polished program system. Many may find that its user interface is more "user friendly" than the standard menu-driven interfaces. Its system of filing sequences under individual passwords facilitates use by more than one person. MicroGenie uses a hardware device for software protection that occupies a card slot in the computer on which it is used. Although I am sympathetic to the problem of software piracy, I feel that a less drastic solution is in order for a program likely to be sharing limited computer space with other software packages. The IBI-Pustell package performs the required analysis functions as accurately and quickly as MicroGenie but it lacks the clearness and ease of use. The menu system seems disjointed, and new or infrequent users often find themselves at apparent "dead-end menus" where the only clear alternative is to restart the entire program package. It is suggested from published accounts that the user interface is going to be upgraded and perhaps when that version is available, use of the system will be improved. The documentation accompanying each package was relatively clear as to how to run the programs, but all three packages assumed that the user was familiar with the computational techniques employed. MicroGenie and IBI-Pustell further complicated their documentation by mixing instructions for the version based on floppy disk operation with that for the hard disk version.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed2000

Sequence analysis and clinical significance of the iceA gene from Helicobacter pylori strains in Japan.

The Helicobacter pylori iceA gene was recently identified as a genetic marker for the development of peptic ulcer in a Western population. To assess the significance of iceA subtypes of H. pylori in relation to peptic ulcer, 140 Japanese clinical isolates (88 from Fukui and 52 from Okinawa) were characterized. Sequence analysis of the iceA1 gene from 25 representative Japanese strains was also carried out to identify the differences in iceA between the ulcer group and the gastritis group. The iceA1 genotype was not correlated with the presence of peptic ulceration in either area. In addition, sequence analysis led to identification of five deletions and five point mutations (a nonsense mutation or a 1-bp insertion) within the iceA1 open reading frame corresponding to previously published sequences. These mutations were identified in both clinical groups (ulcer and gastritis groups) in each area. Local DNA sequence analysis revealed that the endpoints of all five deletions coincided with direct repeats. We also found four strains that carried longer iceA1 open reading frames compared with that for strain 60190. In conclusion, carriage of an iceA1 strain does not seem to be a risk factor for peptic ulcer in Japanese subjects. The critical mutations in the iceA1 gene in some isolates from patients with peptic ulcers suggested that IceA does not participate in the pathogenesis of peptic ulcer in Japan. We also found deletion hot spots that were associated with direct repeats in iceA1 and that favored a small-deletion model of slipped mispairing events during replication. We showed that iceA1 sequence variations may be useful tools for analysis of the population genetics of H. pylori.

Bacterial Proteins↗