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PubMed1998

Presence of Helicobacter species DNA in Swedish water.

Municipal water, treated waste-water and well-water from all 25 counties in Sweden were analysed for the presence of Helicobacter spp. DNA. Bacteria were concentration by immunomagnetic separation. Culture, Gram staining and urease tests were performed before lysis of bacteria. Two polymerase chain reaction (PCR) assay with high sensitivity (adhesin and 16S rRNA) were followed by Helicobacter spp. specific hybridization. Nine of 24 private wells, three of 25 municipal tapwater and three of 25 wastewater samples were positive for both PCR assays. Positive municipal and waste-water samples were positive for counties. Non-specificity of PCR methods due to the presence of unknown bacteria within the genus helicobacter cannot be totally ruled out. Thus, the clinical significance of findings Helicobater spp. DNA in drinking water needs to be further evaluated.

DNA, Bacterial↗
PubMed2003

A new method for mapping discontinuous antibody epitopes to reveal structural features of proteins.

Antibodies that bind to protein surfaces of interest can be used to report the three-dimensional structure of the protein as follows: Proteins are composed of linear polypeptide chains that fold together in complex spatial patterns to create the native protein structure. These folded structures form binding sites for antibodies. Antibody binding sites are typically "assembled" on the protein surface from segments that are far apart in the primary amino acid sequence of the target proteins. Short amino acid probe sequences that bind to the active region of each antibody can be used as witnesses to the antibody epitope surface and these probes can be efficiently selected from random sequence peptide libraries. This paper presents a new method to align these antibody epitopes to discontinuous regions of the one-dimensional amino acid sequence of a target protein. Such alignments of the epitopes indicate how segments of the protein sequence must be folded together in space and thus provide long-range constraints for solving the 3-D protein structure. This new antibody-based approach is applicable to the large fraction of proteins that are refractory to current approaches for structure determination and has the additional advantage of requiring very small amounts of the target protein. The binding site of an antibody is a surface, not just a continuous linear sequence, so the epitope mapping alignment problem is outside the scope of classical string alignment algorithms, such as Smith-Waterman. We formalize the alignment problem that is at the heart of this new approach, prove that the epitope mapping alignment problem is NP-complete, and give some initial results using a branch-and-bound algorithm to map two real-life cases. Initial results for two validation cases are presented for a graph-based protein surface neighbor mapping procedure that promises to provide additional spatial proximity information for the amino acid residues on the protein surface.

Actins↗
PubMed1999

Serotyping of adenoviruses on conjunctival scrapings by PCR and sequence analysis.

To detect and identify adenovirus (Ad), we investigated hypervariable regions (HVRs) of Ad by using a combination of PCR and direct sequencing (PCR-sequence) method. Primers for nested PCR to amplify the conserved region in the hexon protein containing HVRs were designed based on hexon gene sequences derived from GenBank. These two primer sets amplified a DNA fragment of 7 HVRs from 16 prototypes of Ad, which were divided into five subgenera, including seven serotypes that are the predominant causative agents of acute conjunctivitis in Japan, and from 31 recent conjunctival scraping specimens from patients with adenoviral conjunctivitis. HVR DNA sequences were determined by means of universal sequence primers. Analysis of the predicted amino acid homology of HVRs among Ad prototypes suggested three regions, HVR4, -5, and -7, to be candidates for the neutralization epitopes. The clinical serotype of specimens was determined by the PCR-sequence method with reference to these three HVRs. The serotype determined according to this method was identical to that obtained by culture isolation and the neutralization test (NT) in all scraping samples, whereas the results of this method did not match PCR and restriction fragment length polymorphism (PCR-RFLP) analysis in five samples. It took only three days to detect Ad and to identify the serotype, in contrast to culture isolation-NT, which took at least 2 weeks. These findings indicate that our newly developed PCR-sequence method is applicable for the detection and serotyping of human Ads.

Adenovirus Infections, Human↗
PubMed2006

Real-time polymerase chain reaction for quantitative detection of histamine-producing bacteria: use in cheese production.

Biogenic amines are toxic substances that appear in foods and beverages as a result of AA decarboxylation. The enzyme histidine decarboxylase catalyzes the decarboxylation of histidine to histamine, the biogenic amine most frequently involved in food poisoning. The aim of the present work was to develop a real-time quantitative PCR assay for the direct detection and quantification of histamine-producing strains in milk and cheese. A set of primers was designed, based on the histidine decarboxylase gene sequence of different gram-positive bacteria. The results show the proposed procedure to be a rapid (total processing time < 2 h), specific and highly sensitive technique for detecting potential histamine-producing strains. Chromatographic methods (HPLC) verified the capacity of real-time quantitative PCR to correctly quantify histamine accumulation.

Animals↗
PubMed1998

Characterization of the CSN1AG allele of the bovine alpha s1-casein locus by the insertion of a relict of a long interspersed element.

The bovine CSN1AG allele is associated with a lower proportion of alpha s1-casein in milk and is characterized by an insertion in the last noncoding exon (19th). According to DNA sequence analysis, the location, length, and origin of the insertion characterizing the bovine CSN1AG allele have been identified. This insertion interrupts the 19th exon between nucleotides 58 and 59, is 371 bp long, and has a high level of homology with relicts of long interspersed elements of retropositional origin. Northern blot analysis shows that the inserted element is transcribed and that the number of transcripts of the CSN1AG allele is less than one-half of the number of transcripts of the CSN1AB allele. The lower amount of alpha s1-casein in milk obtained from cows carrying the CSN1AG allele can be explained by a reduced mRNA stability. Furthermore, a method has been developed using polymerase chain reaction for identification of the carriers of the CSN1AG allele.

Alleles↗
PubMed2003

Analysis of low-density lipoprotein receptor gene mutations in a Chinese patient with clinically homozygous familial hypercholesterolemia.

OBJECTIVE: To screen the point mutation of the low-density lipoprotein receptor (LDL-R) gene in Chinese familial hypercholesterolemia (FH) patients, characterize the relationship between the genotype and the phenotype and discuss the molecular pathological mechanism of FH. METHODS: A patient with clinical phenotype of homozygous FH and her parents were investigated for mutations in the promoter and all eighteen exons of the LDL-R gene. Screening was carried out using Touch-down PCR and direct DNA sequencing; multiple alignment analysis by DNASIS 2.5 was used to find base alteration, and the LDL-R gene mutation database was searched to identify the alteration. In addition, the apolipoprotein B gene (apo B) was screened for known mutations (R3500Q) that cause familial defective apo B100 (FDB) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Two new heterozygous mutations in exons 4 and 9 of the LDL-R gene were identified in the proband (C122Y and T383I) as well as her parents. Both of the mutations have not been published in the LDL-R gene mutation database. No mutation of apo B100 (R3500Q) was observed. CONCLUSION: Two new mutations (C112Y and T383I) were found in the LDL-R gene, which may result in FH and may be particularly pathogenetic genotypes in Chinese people.

Adult↗
PubMed1996

Comparative analysis of the secondary structural motifs of P450BM-3 and the regions located upstream of the calmodulin-binding domain in the nitric oxide synthases.

The proposed method of multiple alignment of secondary structures makes it possible to estimate the multidomain enzymes' structural similarity in those cases where, following alignment, the identity appears to be inadequate or too insignificant to estimate protein relationship in spite of their functional analogy. Multiple alignment of sequences, representing the secondary structural elements of the nitric oxide synthase (NOS) N-terminal "tails", localized upstream of the calmodulin (CAM)-binding domain, and the P450 superfamily representative P450BM-3 has revealed the existence of a common secondary structural motif, standing of 60% identity between the polypeptide chain packing of NOS and the full sequence of P450BM3. This fact may point to the existence of their common ancestor. Presence of the linker olygopeptides within NOS permitted us to determine the boundary of the cytochrome-like part of NOS.

Amino Acid Sequence↗
PubMed1999

Molecular phylogenetic studies on Theileria parasites based on small subunit ribosomal RNA gene sequences.

Classification of Theileria parasites of south-east Asian countries is still ambiguous due to the lack of basic studies, especially their molecular genetic information. In this study, we included 6 known species and 14 unclassified Theileria parasite isolates: Theileria annulata, Theileria parva, Theileria taurotragi, Theileria sergenti, Theileria buffeli, Theileria types Sable, Theileria types A, B, B1, B2, C, D, E, F, G, G1, Theileria type Medan (Indonesia), Theileria type Ipoh (Malaysia) and Theileria type Thong Song (Thailand). Small subunit ribosomal RNA (srRNA) nucleotide sequence data were collected by PCR, cloning and dideoxy sequencing. The srRNA nucleotide sequences were aligned and analyzed by distance methods, maximum parsimony algorithms and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the strength of the different phylogenetic reconstructions. The data indicated that all of the tree-building methods gave very similar results. This study identified two groups of Theileria, the pathogenic and benign groups, which are strongly supported by bootstrap analysis. The analysis also indicated that three subgroups (A, B and C) were generated within the benign Theileria group whereas the classification of Theileria type D and Thong Song is questionable. However, more basic information such as life cycle differences, vectors, modes of transmission, virulent and genetic/sexual compatability is essential for clearer taxonomic definition of the benign Theileria parasites.

Animals↗
PubMed1993

Prediction of protein secondary structure at better than 70% accuracy.

We have trained a two-layered feed-forward neural network on a non-redundant data base of 130 protein chains to predict the secondary structure of water-soluble proteins. A new key aspect is the use of evolutionary information in the form of multiple sequence alignments that are used as input in place of single sequences. The inclusion of protein family information in this form increases the prediction accuracy by six to eight percentage points. A combination of three levels of networks results in an overall three-state accuracy of 70.8% for globular proteins (sustained performance). If four membrane protein chains are included in the evaluation, the overall accuracy drops to 70.2%. The prediction is well balanced between alpha-helix, beta-strand and loop: 65% of the observed strand residues are predicted correctly. The accuracy in predicting the content of three secondary structure types is comparable to that of circular dichroism spectroscopy. The performance accuracy is verified by a sevenfold cross-validation test, and an additional test on 26 recently solved proteins. Of particular practical importance is the definition of a position-specific reliability index. For half of the residues predicted with a high level of reliability the overall accuracy increases to better than 82%. A further strength of the method is the more realistic prediction of segment length. The protein family prediction method is available for testing by academic researchers via an electronic mail server.

Mathematical Computing↗
PubMed2003

Significance of interspecies matches when evolutionary rate varies.

We develop techniques to estimate the statistical significance of gap-free alignments between two genomic DNA sequences, using human-mouse alignments as an example. The sequences are assumed to be sufficiently similar that some but not all of the neutrally evolving regions (i.e., those under no evolutionary constraint) can be reliably aligned. Our goal is to model the situation in which the neutral rate of evolution, and hence the extent of the aligning intervals, varies across the genome. In some cases, this permits the weaker of two matches to be judged as less likely to have arisen by chance, provided it lies in a genomic interval with a high level of background divergence. We employ a hidden Markov model to capture variations in divergence rates and assign probability values to gap-free alignments using techniques of Dembo and Karlin, which are related to those used for the same purpose by BLAST. Our methods are illustrated in detail using a 1.49 Mb genomic region. Results obtained from the analysis of human chromosome 22 using these techniques are also provided.

Animals↗
PubMedDate not supplied

[Symptomatic Black Queen Cell Virus infection of drone brood in Hessian apiaries].

The Black Queen Cell Virus (BQCV) can affect brood of the honey bee (Apis mellifera). In general queen cells are endangered showing dark coloured cell walls as typical symptoms. Worker- and dronebrood can be infected by BQCV but normally without clinical symptoms. This paper describes for the first time a symptomatic BQCV-infection of diseased drone brood found on two bee yards in Hessen/Germany in 2001. The drone larvae were seriously damaged and some of them were dead. Samples of the affected brood were tested for BQCV by the PCR detection method. A BQCV specific nucleic acid fragment was found. The PCR product were sequenced and aligned with the relevant GenBank entry. At the nucleic acid level as well as at the deduced protein level the isolate showed a high similarity with the south african isolate noted in GenBank.

Animals↗
PubMed1996

How similar must a template protein be for homology modeling by side-chain packing methods?

Given the correct backbone coordinates of a globular protein, side-chain packing methods can be generally expected to predict the side-chain coordinates of the buried core residues accurately. In the context of a study in modeling a family of bacteriophage DNA-binding proteins, we observed that when the coordinates of the actual perfect backbone are not available, the side-chain packing methods are still of predictive value using homologous but imperfect backbones. This is the situation in practical homology modeling where a target protein sequence is modeled from template structures of known protein homologs. In order to assess the quality and degree of accuracy of such predictions and their dependence on the extent of homology, we have now extended these studies to a well characterized family of globin structures that span a much wider range of sequence-structure similarity. The collective results show a clear relationship that is independent of protein family between side-chain prediction accuracy and the level of similarity between the template and target proteins. We judge this similarity in terms of sequence identity and the backbone r.m.s. deviation of the template structure used for modeling and the actual target structure in cases where the target structures are available. In summary, as sequence identity drops from 100% to about 50%, or when the backbone r.m.s. deviation between template and target structures increases from 0 A to about 1 A, the overall average r.m.s. error for the buried-core residues rises from 1.2 A to 1.5 A while the chi 1 prediction accuracy drops from 85% to 70-75% and the chi 2 prediction accuracy drops from 80% to 60-65%. When the sequence identity drops below 50% or the backbone r.m.s. deviation rises above 1 A, all 3 measures of prediction accuracy decrease rapidly. When the sequence identity edges to the so-called twilight zone of sequence similarity at around 22%, or when the backbone r.m.s. deviation exceeds 2 A, the prediction accuracy approaches the values to be expected for random predictions, namely, 3.1 A for average r.m.s. error, 22% and 29% for accuracy of chi 1 and chi 2 prediction. These observations provide a practical evaluation of the side-chain packing methods and are of value to the homology-modeler. The extent and degree to which the backbone topology of a protein fold can constrain internal side-chain orientation gives insight into the plasticity of the sequence-structure relationship found in the architecture of proteins.

Amino Acid Sequence↗
PubMed2002

TLFAM--a new set of protein family databases.

PFAM is a popular and effective database of Hidden Markov Models (HMMs), which represent a wide range of protein families. Here, we introduce TLFAM as a more specific set of HMM databases. Analyses of bacterial genomes using TLFAM-Pro show better scores, E-values, and alignment lengths than those using the more generalized PFAM. Since PFAM will still find hits that TLFAM-Pro will not, we recommend that they be used jointly, rather than exclusively. This method provides the best features of both databases. This method has been extended to a number of other organism types, such as archaea, and the databases are freely available to interested researchers.

Databases, Protein↗
PubMed1994

Drosophila melanogaster genes encoding three troponin-C isoforms and a calmodulin-related protein.

Using low-stringency hybridization and polymerase chain reaction (PCR)-based DNA amplification, we have isolated three Drosophila melanogaster genes that encode troponin-C isoforms and one specifying a protein that is closely related to calmodulin. Two of the troponin-C genes, located within the 47D and 73F subdivisions of chromosomes 2 and 3, respectively, encode very closely related isoforms. That specified by the 47D gene accumulates almost exclusively in larval muscles, while that encoded by the 73F gene is present in both larvae and adults. The third gene, located within the 41C subdivision of chromosome 2, encodes a more distantly related troponin-C isoform that accumulates only within adults. The gene that encodes the calmodulin-related protein is located within the 97A subdivision of chromosome three. The protein encoded by this gene has a different primary sequence from that of conventional calmodulin, which is specified by a gene located within the 49A subdivision of chromosome 2. Our report is the first to describe insect troponin-C isoforms and further avails genetic methods for investigating the in vivo functions of the troponin-C/myosin light-chain/calmodulin protein superfamily.

Amino Acid Sequence↗
PubMed1998

Amplification of genomic DNA fragments of Sarcocystis muris (Apicomplexa) cyst merozoites encoding a thiol (cysteine) proteinase.

Parasites of the phylum Apicomplexa (Sporozoa) cause diseases such as malaria, toxoplasmosis, or intestinal coccidiosis. Invasive stages possess typical apical organelles such as dense granules that harbor a broad range of polypeptides that are believed to take part in the parasite-host cell interaction. In previous studies a 26-kDa polypeptide of dense granules from Sarcocystis muris cyst merozoites (bradyzoites) was characterized as a thiol (cysteine) proteinase. In this paper a method is demonstrated to amplify DNA fragments from genomic DNA of S. muris cyst merozoites by polymerase chain reaction, which probably code for the 26-kDa antigen.

Amino Acid Sequence↗
PubMed2005

Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California.

Monilinia laxa is a pathogen of brown rot of stone fruit and almond in California, causing blossom blights and fruit rots. In this study, low-level resistance to the benzimidazole fungicides benomyl and thiophanate-methyl was detected in field isolates of M laxa collected from stone fruits and almonds in California. Low-resistant (LR) isolates grew in potato dextrose agar (PDA) plates amended with benomyl and thiophanate-methyl at 1 and 5 microg ml(-1), respectively, but not in plates amended with benomyl at 5 microg ml(-1) or thiophanate-methyl at 50 microg ml(-1). The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the beta-tubulin gene. The LR isolates showed high-temperature sensitivity, being sensitive to 1 microg ml(-1) of benomyl at 28 degrees C but resistant at 8-24 degrees C. Analysis of the DNA sequence of the beta-tubulin gene showed that the LR isolates had a point mutation at the amino-acid position 240, causing substitution of leucine by phenylalanine. Based on the point mutation, a pair of allele-specific PCR primers was developed for rapid detection of LR isolates of M laxa. In addition, a pair of PCR primers specific to M laxa was developed on the basis of the differences in the DNA sequence of the intron 6 of beta-tubulin gene from M laxa, M fructicola and other fungal species. The primer pair amplified the expected 376-bp DNA fragment from all M laxa isolates tested, but not from 14 other fungal species isolated from stone fruit and almond crops. The restriction endonuclease BsmA I recognized the sequence GTCTCC in the PCR products from sensitive (S) isolates only, but not the GTTTCC sequence in the PCR products from LR isolates. The endonuclease digested the 376-bp PCR products from S isolates to produce two bands (111 and 265 bp) on agarose gels. Thus, both allele-specific PCR and the PCR-restriction fragment length polymorphism (PCR-RFLP) methods could be useful for rapidly detecting benzimidazole-resistant isolates of M laxa from stone fruit and almond crops in California.

Ascomycota↗
PubMed2001

Trypanosoma vivax: characterization of the spliced-leader gene of a Brazilian stock and species-specific detection by PCR amplification of an intergenic spacer sequence.

The sequence of the spliced-leader gene repeat of a Brazilian Trypanosoma vivax stock from cattle showed high similarity to sequences of West African T. vivax in both intron and intergenic sequences. This is the first evidence based on DNA sequences of close-relatedness between Brazilian and West African T. vivax stocks. A T. vivax-specific diagnostic PCR assay based on spliced-leader gene intergenic sequences was able to amplify DNA from T. vivax stocks from South America (Brazil, Bolivia, and Colombia) and West Africa. Species-specificity of this method was confirmed by results obtained by testing 15 other trypanosomes, including other species and subspecies that can also infect cattle. The PCR assay developed presented high sensitivity, detecting the DNA content of only one parasite and also revealing T. vivax infection in asymptomatic animals without detectable parasitemia by microhematocrit or in Giemsa-stained blood smears. Use of crude preparations from field-blood samples collected on both filter paper and glass slides as DNA template suggested that this method could be useful for the diagnosis of T. vivax in large epidemiological studies.

Animals↗