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PubMed2002

Sequence analysis and genotypes of glutamate rich protein of Plasmodium falciparum isolates from different malaria endemic areas in China.

OBJECTIVE: To sequence the gene encoding glutamate rich protein (GLURP) and identify the genotypes of geographically different Plasmodium falciparum (P. f) isolates from China. METHODS: The gene of R2 repeat region of GLURP was amplified by nested polymerase chain reaction and cloned into T-vector. The nucleotide sequence of GLURP gene was determined by automatic sequencer (Dideoxy termination method) and analyzed by DNA Star software. RESULTS: At least 7 different GLURP genotypes ranging from 600 bp to 1,500 bp were found in Yunnan and Hainan provinces. R2 region of GLURP gene consisted of several repeat units. Each repeat unit was composed of 19-20 residues which were shown to be highly conserved. GLURP gene was also size polymorphic due to differences in the number of repeat units, whereas the repeat sequence was conserved. Sequence analysis showed that DNA sequences and deduced amino acid sequences were highly homologous among the geographically dispersed isolates or various isolates from the same geographical region. No obvious differences were found in the GLURP gene sequences among geographically different isolates. CONCLUSION: GLURP gene is highly structure conserved and size polymorphic, and so is useful in searching for malaria vaccine candidate antigen and developing a genotyping method for malaria research.

Amino Acid Sequence↗

PubMedDate not supplied

The applicability of recurrent neural networks for biological sequence analysis.

Selection of machine learning techniques requires a certain sensitivity to the requirements of the problem. In particular, the problem can be made more tractable by deliberately using algorithms that are biased toward solutions of the requisite kind. In this paper, we argue that recurrent neural networks have a natural bias toward a problem domain of which biological sequence analysis tasks are a subset. We use experiments with synthetic data to illustrate this bias. We then demonstrate that this bias can be exploitable using a data set of protein sequences containing several classes of subcellular localization targeting peptides. The results show that, compared with feed forward, recurrent neural networks will generally perform better on sequence analysis tasks. Furthermore, as the patterns within the sequence become more ambiguous, the choice of specific recurrent architecture becomes more critical.

Algorithms↗

PubMed2004

Algorithms for sequence analysis via mutagenesis.

MOTIVATION: Despite many successes of conventional DNA sequencing methods, some DNAs remain difficult or impossible to sequence. Unsequenceable regions occur in the genomes of many biologically important organisms, including the human genome. Such regions range in length from tens to millions of bases, and may contain valuable information such as the sequences of important genes. The authors have recently developed a technique that renders a wide range of problematic DNAs amenable to sequencing. The technique is known as sequence analysis via mutagenesis (SAM). This paper presents a number of algorithms for analysing and interpreting data generated by this technique. RESULTS: The essential idea of SAM is to infer the target sequence using the sequences of mutants derived from the target. We describe three algorithms used in this process. The first algorithm predicts the number of mutants that will be required to infer the target sequence with a desired level of accuracy. The second algorithm infers the target sequence itself, using the mutant sequences. The third algorithm assigns quality values to each inferred base. The algorithms are illustrated using mutant sequences generated in the laboratory.

Algorithms↗

PubMed1994

Sequence analysis of a 3.5 Kb EcoRI fragment from the left arm of Saccharomyces cerevisiae chromosome XI reveals the location of the MBR1 gene and a sequence related to a GTPase-activating protein.

We present the DNA sequence analysis of a region covering a 3.5 kb EcoRI fragment from the left arm of chromosome XI from Saccharomyces cerevisiae. This region contains five open reading frames (ORFs) which code for proteins of greater than 100 amino acids. ORF YKL425 codes for the previously sequenced Mbr1 (Valens et al., 1991; Daignan-Fornier et al., 1993) which participates in mitochondrial biogenesis. YKL424 has identity with a GTPase-activating protein of higher eukaryotes. The three remaining ORFs have no identity to known proteins within the databases screened and are not assigned ORF numbers as they are completely contained with ORFs YKL424 and YKL425.

Amino Acid Sequence↗

PubMed1990

Sequence analysis of acetylation and methylation in two histone H3 variants of alfalfa.

Analysis of acetylation in the two histone H3 variants of alfalfa by acid/urea/Triton-polyacrylamide gel electrophoresis has established that the minor variant H3.2 has a 2-fold higher level of acetylation than the major variant H3.1. Purification and sequence analysis of both variants showed sequence identity across the complete amino-terminal domain, which contains the 6 modified lysines 4, 8, 14, 18, 23, and 27. The two proteins have different distributions for acetylation: mono-, di-, and tri-methylation. The higher level of acetylation of H3.2 was confirmed in a wider pattern across all 6 lysines. Lysine modification levels varied for all sites in both proteins between 5 and 95%, with combinations of one to four types of modification co-existing at each residue. Additional sequence analysis of the H3.1 and H3.2 proteins and of tryptic core peptides established that the two histones differ only in residues 31, 41, 87, and 90. This indicates that major histone H3.1 is the product of the major alfalfa histone H3 gene and makes it likely that H3.2 is the product of the minor H3 gene, known from a partial cDNA clone. The variant-specific differences in lysine modifications in protein domains with identical primary structures suggest that the pattern and level of lysine modifications may be directed by the distinct chromatin environments of the two histone H3 variants.

Acetylation↗

PubMed1996

Elucidation of an HIV-1 transmission from mother to child in west Africa by sequence analysis.

A pregnant woman living in Germany went to Ghana for several months, where she received 4 blood transfusions. Her newborn child also received one blood transfusion in West Africa. After return to Germany, HIV-1 infection was detected in both of them. Serotyping with V3 peptides revealed that the sera reacted only poorly with the subtype B-specific antigens. To investigate whether the child had been infected by vertical or parenteral transmission, we amplified different proviral HIV-1 gene segments from samples obtained 1-3 years after infection. Sequence analysis of the hypervariable regions V1 and V2 of the proviral env gene was misleading, since the viral population of the mother was highly heterogeneous, whereas only one predominant viral variant was found in the child. In contrast, sequences of the gag p17 gene and the regulatory genes nef and vif were homogeneous and revealed a very high homology, suggesting that the child had been infected by the mother. This was confirmed by phylogenetic tree analysis showing that sequences of mother and child clustered together and that both were infected by HIV-1 subtype A which is common in West Africa. The results suggest that sequence analysis of the hypervariable regions V1 and V2 alone can lead to unclear results, especially if not single genomes are analysed but a mixture of quasi-species. It is recommended that investigations into HIV transmission should be based on sequence analysis of several HIV genes.

Africa, Western↗

PubMed2002

[Cloning and sequence analysis of aspartokinase genes from Corynebacterium crenatum].

Aspartokinase genes (ask) from wild-type Corynebacterium crenatum AS1.542 and an AEC-resistant mutant Corynebacterium crenatum CD945 were cloned and sequenced. Analysis of ask sequence shows a exchange in a single base pair at position 1199 from T to C, leading to an amino acid change in the beta subunit of Aspartokinase. Leu80 in the wild-type is converted to Pro80 in the feedback-resistant enzyme. The substitution is located in ACT domain, a region regulated by concentration of lysine. The ORF sequence of ask from C. crenatum AS 1.542 shows homologies of 97.23%, 97.55% and 97.62% to those from C. glutamicum, C. flavum and B. lactofermentum. And the amino acid sequence deduced from ORF displays homologies of 99.76%, 99.52% and 99.76%, respectively. But there is much variation in the upstream sequence of C. crenatum AS 1.542 ask promoter compared to those from other Corynebacteria.

Amino Acid Sequence↗

PubMed1995

Cloning and DNA sequence analysis of two abortive infection phage resistance determinants from the lactococcal plasmid pNP40.

The lactococcal plasmid pNP40, from Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, confers complete resistance to the prolate-headed phage phi c2 and the small isometric-headed phage phi 712 in L. lactis subsp. lactis MG1614. A 6.0-kb NcoI fragment of pNP40 cloned in the lactococcal Escherichia coli shuttle vector pAM401 was found to confer partial resistance to phi 712. Subcloning and deletion analysis of the recombinant plasmid pPG01 defined a 2.5-kb ScaIHpaI fragment as conferring phage insensitivity. Sequence analysis of this region confirmed the presence of two overlapping open reading frames (ORFs). Further subcloning of pNP40 to characterize the resistance determinant active against phi c2 identified a 5.6-kb EcoRV fragment of pNP40 which, when cloned in pAM401, conferred partial resistance to both phi c2 and phi 712. Subcloning and deletion analysis of the recombinant plasmid pCG1 defined a 3.7-kb EcoRV-XbaI fragment as encoding phage insensitivity. DNA sequence analysis of this region revealed the presence of a single complete ORF. The introduction of a frameshift mutation at the unique BglII site within this ORF disrupted the phage resistance phenotype, confirming that this ORF is responsible for the observed phage insensitivity. The mechanisms encoded by pPG01 and pCG1 in L. lactis subsp. lactis MG1614 conformed to the criteria defining abortive infection and were designated AbiE and AbiF, respectively. Analysis of the phage DNA content of phi 712-infected hosts containing AbiF demonstrated that it inhibited the rate of phage DNA replication, while AbiE had little effect on phage DNA replication, suggesting a later target of inhibition. The predicted protein product of abiF shows significant homology to the products of two other lactococcal abortive infection genes, abiD and abiD1.

Amino Acid Sequence↗

PubMed2005

Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile.

A multilocus sequence analysis of ten virulence-associated genes was performed to study the genetic relationships between 29 Clostridium difficile isolates of various origins, hosts and clinical presentations, and selected from the main lineages previously defined by multilocus sequence typing (MLST) of housekeeping genes. Colonization-factor-encoding genes (cwp66, cwp84, fbp68, fliC, fliD, groEL and slpA), toxin A and B genes (tcdA and tcdB), and the toxin A and B positive regulator gene (tcdD) were investigated. Binary toxin genes (cdtA and cdtB) were also detected, and internal fragments were sequenced for positive isolates. Virulence-associated genes exhibited a moderate polymorphism, comparable to the polymorphism of housekeeping genes, whereas cwp66 and slpA genes appeared highly polymorphic. Isolates recovered from human pseudomembranous colitis cases did not define a specific lineage. The presence of binary toxin genes, detected in five of the 29 isolates (17 %), was also not linked to clinical presentation. Conversely, toxigenic A-B+ isolates defined a very homogeneous lineage, which is distantly related to other isolates. By clustering analysis, animal isolates were intermixed with human isolates. Multilocus sequence analysis of virulence-associated genes is consistent with a clonal population structure for C. difficile and with the lack of host specificity. The data suggest a co-evolution of several of the virulence-associated genes studied (including toxins A and B and the binary toxin genes) with housekeeping genes, reflecting the genetic background of C. difficile, whereas flagellin, cwp66 and slpA genes may undergo recombination events and/or environmental selective pressure.

Alleles↗

PubMed1996

Nucleotide sequence analysis of pOg32, a cryptic plasmid from Leuconostoc oenos.

Analysis of the nucleotide sequence of Leuconostoc oenos plasmid pOg32 (2544 bp) revealed three open reading frames (ORFs) in one of the DNA strands preceded by putative promoters and ribosome binding sites. ORF1 corresponded to a polypeptide similar to the replication proteins (Rep) of pT181 plasmid family. A nucleotide sequence like the double strand origin (DSO) of the same plasmid family was found inside the presumptive rep gene. ORF2 is capable of encoding a protein resembling the recombination enzyme (Pre) of two lactobacilli plasmids. Immediately upstream of the presumed pre gene, a sequence similar to the consensus RSA regions of those plasmids was encountered. Between ORF3 and ORF1, a sequence comparable to the RSB consensus region of the single strand origin ssoA was detected. The putative RSB region was located at the 3'-end of an imperfect palindrome which may be a single stranded origin. The described features, common to other small plasmids of Gram-positive bacteria, suggest that pOg32 replicates by the rolling circle mechanism. The amino acid sequence encoded by ORF3 was not similar to any known protein.

Amino Acid Sequence↗

PubMed1993

Rabbit masseter expresses the cardiac alpha myosin heavy chain gene. Evidence from mRNA sequence analysis.

The presence of myosin alpha heavy chain in the rabbit masseter has been previously suggested at the protein level [(1991) Basic App. Myol. 1, 23-34; (1991) Histochem. J. 23, 160-170]. To confirm this finding, we cloned most of the mRNA corresponding to the myosin heavy chain S2 subfragment. PCR analysis and subsequent nucleotide sequence determination of the amplified cDNA demonstrates the presence of a myosin alpha heavy chain mRNA in rabbit masticatory muscles.

Amino Acid Sequence↗

PubMed2006

Full-length sequence analysis of SIVmus in wild populations of mustached monkeys (Cercopithecus cephus) from Cameroon provides evidence for two co-circulating SIVmus lineages.

Mustached monkeys (Cercopithecus cephus), which form a significant component of primate bushmeat in west central Africa, are infected with simian immunodeficiency virus (SIVmus). We identified and genetically characterized five new SIVmus strains infecting wild living mustached monkeys from Cameroon. Phylogenetic analysis of partial pol sequences revealed that SIVmus strains form two distinct groups within the clade comprised of lentiviruses isolated from Cercopithecus nictitans (SIVgsn), Cercopithecus mona (SIVmon) and C. cephus (SIVmus). Characterisation of three full-length SIVmus genomes confirmed the presence of two distinct lineages infecting mustached monkeys. These two variants of SIVmus, here designated SIVmus-1 and SIVmus-2, were isolated from animals sharing habitats within the same geographic region. Phylogenetic analyses showed that the diversification of SIVmus, SIVgsn and SIVmon involved inter-lineage recombination, and suggested that one of the SIVmus lineages likely resulted from cross-species transmission and recombination involving SIVmus and an as yet uncharacterized SIV. These results indicate that cross-species transmission and recombination play a major role in the evolution of primate lentiviruses among sympatric primate species.

Animals↗

PubMed1994

Genomic cloning and complete sequence analysis of a highly divergent African human immunodeficiency virus isolate.

Analysis of the complete sequence of a human immunodeficiency virus (HIV) isolate (Ant70) obtained from a Cameroonian patient indicates that this virus is the most divergent strain within the HIV-1 family hitherto described. Comparison of the Pol protein, usually highly conserved within the HIV-1 family, shows only about 73% similarity with the HIVmm isolate, whereas for the more variable proteins such as envelope, similarities of 50% or lower are found. The principal neutralizing determinant (V3 loop) and the immunodominant region within gp41 also contain some unusual substitutions, which may have implications for protein function as well as for serological assays based on these regions. Phylogenetic analyses show that this isolate occupies a unique position relative to the human HIV-1 isolates and the recently described SIVcpz virus, indicating that this Cameroonian isolate may provide us with new insights into the origins of the HIV-1 family.

Amino Acid Sequence↗

PubMed1997

Phylogenetic relationship of the twenty-one DNA groups of the genus Acinetobacter as revealed by 16S ribosomal DNA sequence analysis.

The inter- and intrageneric relationships of members of the genus Acinetobacter were investigated by performing a comparative sequence analysis of PCR-amplified 16S ribosomal DNAs (rDNAs) from 21 strains representing all of the DNA groups that have been described. Phylogenetic treeing confirmed that Acinetobacter spp. form a coherent cluster within the gamma subdivision of the class Proteobacteria that includes strains with overall levels of 16S rDNA sequence similarity of more than 94%. The analysis of intrageneric relationships suggested that the majority of the strains cluster in five clearly distinguishable clusters, and this conclusion was supported by the results obtained with the different methods used for phylogenetic analysis (i.e., the maximum-likelihood, parsimony, and distance matrix methods). The first cluster contains the representatives of DNA groups 2 (Acinetobacter baumannii) and TU13, whereas the second cluster comprises representatives of DNA groups 3, "Close To TU13," and "between 1 and 3." The representatives of closely related Acinetobacter DNA groups 8 (Acinetobacter twoffii) and 9 belong to the third cluster, which includes the representative of DNA group 6 as well. The fourth cluster is formed by DNA groups BJ15, BJ16, and BJ17, and the fifth cluster comprises DNA groups 1 (Acinetobacter calcoaceticus), BJ14, 10, and 11. Within the fifth cluster the 16S rDNA sequences of DNA group 10 and 11 strains are nearly identical. The representatives of DNA groups 4 (Acinetobacter haemolyticus), 5 (Acinetobacter junii), 7 (Acinetobacter johnsonii), 12 (Acinetobacter radioresistens), TU14, and TU15 form individual branches that are not significantly affiliated with any of the five clusters identified. Apart from the clustering of the most closely related DNA groups, the general topology of the distance dendrogram revealed some discrepancy with previous DNA-DNA hybridization data, which may point to the inadequacy of comparative 16S rDNA sequence analysis for reflecting true evolutionary relationships of closely related bacterial taxa. Important, however, was the presence of unique sequence motifs in each of the 21 different DNA groups studied, which may be useful for rapid differentiation of DNA groups of the genus Acinetobacter.

Acinetobacter↗

PubMed2001

Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database: an expanded data model integrating natural language text and sequence analysis programs.

The HIV Reverse Transcriptase and Protease Sequence Database is an on-line relational database that catalogs evolutionary and drug-related sequence variation in the human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease enzymes, the molecular targets of anti-HIV therapy (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences, including submissions from International Collaboration databases and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the antiretroviral drug treatment history of the individual from whom the isolate was obtained. During the past year 3500 sequences have been added and the data model has been expanded to include drug susceptibility data on sequenced isolates. Database content has also been integrated with didactic text and the output of two sequence analysis programs.

Amino Acid Sequence↗

PubMed1992

A new method for sequence analysis of glycosaminoglycans from heavily substituted proteoglycans reveals non-random positioning of 4- and 6-O-sulphated N-acetylgalactosamine in aggrecan-derived chondroitin sulphate.

We have developed a new procedure for the sequence analysis of glycosaminoglycans, which is particularly suitable for the analysis of chains from heavily substituted proteoglycans. The procedure has been applied to various aggrecan-derived chondroitin sulphates. The glycans are released from the core protein by alkaline scission of the xylose-serine bond, subjected to reductive amination using p-aminobenzoic acid and finally radioiodinated at an acidic pH. Sequence analysis is performed by using various enzymic degradations, partial or complete, followed by high-resolution polyacrylamide gel electrophoresis, blotting and autoradiography to identify segments extending from the labelled reducing end to the point of cleavage. By using chondroitin C lyase to identify the location of 6-O-sulphated hexosamines, we find that chondroitin sulphate from tracheal cartilage has its 6-O-sulphated repeats concentrated to the extreme non-reducing terminal portion of the chain. In chondroitin sulphates derived from intervertebral discs (nucleus pulposus), the 6-O-sulphated repeats have a biphasic distribution; they occur mostly near the linkage region (i.e. the reducing end), but also in the non-reducing portion of the chain. Chondroitin sulphate from nasal cartilage, which is mostly 4-O-sulphated, displays considerable heterogeneity in the linkage region. Three or possibly more charge variants are observed.

4-Aminobenzoic Acid↗

PubMed1993

Toward multistrategy parallel and distributed learning in sequence analysis.

Machine learning techniques have been shown to be effective in sequence analysis tasks. However, current learning algorithms, which are typically serial main-memory-based, are not capable of handling the vast amounts of information being generated by the Human Genome Project. The multistrategy parallel learning approach presented in this paper is an attempt to scale existing learning algorithms. Learning speed is improved through running multiple learning processes in parallel and prediction accuracy is improved through multiple learners. Our approaches are independent of the learning algorithms used. This paper focuses on one of the MSPL approaches and preliminary empirical results that we present are encouraging.

Algorithms↗

PubMed1994

Gnome--an Internet-based sequence analysis tool.

Gnome (GenomeNet Open Mail-service Environment) is a sequence analysis tool that enables an end-user to make use of several Internet- (mainly e-mail) based services with an easy-to-use graphical user interface. Users can conduct homology and motif searches, and database-entry retrieval against the latest databases by emitting search requests to and receiving their results form a search-server by e-mail. The search results are viewed and managed efficiently with this system. The Macintosh and X (Motif) versions of the Gnome client and the UNIX version of the Gnome server are available to academic users free of charge.

Computer Communication Networks↗