The development of a diagnostic polymerase chain reaction (PCR) or quantitative PCR (qPCR) assay for universal detection of highly variable viral genomes is always a difficult task. The purpose of this chapter is to provide a guideline on how to align, process, and evaluate a huge set of homologous nucleotide sequences in order to reveal the evolutionarily most conserved positions suitable for universal qPCR primer and hybridization probe design. Attention is paid to the quantification and clear graphical visualization of the sequence variability at each position of the alignment. In addition, specific problems related to the processing of the extremely large sequence pool are highlighted. All of these steps are performed using an ordinary desktop computer without the need for extensive mathematical or computational skills.
V článku jsou prezentovány první výsledky charakterizace 29 invazivních kmenů Streptococcuspyogenes, izolovaných v České republice v první polovině roku 2003, metodou multilokusové sekvenačnítypizace (MLST). U žádného z 16 přítomných emm typů nebyla mezi kmeny našeho souboruzjištěna variabilita sekvenčních typů (ST). První výsledky MLST ukazují, že populace kmenůzpůsobujících závažná onemocnění v České republice je odlišná od kmenů izolovaných v zahraničí.U sedmi kmenů byly popsány nové sekvenční typy: ST134, ST308, ST336, 5T340, obsahující novékombinace známých alel a ST341 se třemi dosud nepopsanými alelami (gki 91, murI 65 a yqiL 60),zjištěný u třech kmenů. Nově popsané sekvenční typy byly registrovány v celosvětové databáziMLST S. pyogenes.
First results of multilocus sequence typing (MLST) for characterization of 29 invasive Streptococcuspyogenes strains isolated in the Czech Republic in the first half of 2003 are presented. None of 16emm types detected among the study strains showed sequence type (ST) variability. The MLSTresults are indicative of differences between the strains causing serious diseases in the CzechRepublic and those isolated in other countries. In seven strains, four new STs with known alleles innew combinations, ST134, ST308, ST336, ST340, and one new ST with three as yet undescribed alleles(gki 91, murI 65 and yqiL 60), ST341, were described. These newly described STs were submitted tothe web-based reference MLST database for S. pyogenes.
- MeSH
- Alleles MeSH
- Research Support as Topic MeSH
- Cloning, Molecular MeSH
- Humans MeSH
- Sequence Analysis MeSH
- Streptococcus pyogenes isolation & purification classification pathogenicity MeSH
- Check Tag
- Humans MeSH
- Publication type
- Database MeSH
- Review MeSH
- Comparative Study MeSH
- Geographicals
- Czech Republic MeSH
- MeSH
- Child MeSH
- Adult MeSH
- Gene Expression MeSH
- Research Support as Topic MeSH
- Bone Marrow MeSH
- Blood MeSH
- Leukemia diagnosis genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Cordocentesis MeSH
- Oligonucleotide Array Sequence Analysis methods MeSH
- Check Tag
- Child MeSH
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Review MeSH
- Comparative Study MeSH
Cystathionine beta-synthase [CBS; l-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains approximately 5 kb of the 5' flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5' UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3' UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (approximately 80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number of Alu repeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians. Copyright 1998 Academic Press.
- MeSH
- Alternative Splicing * genetics MeSH
- White People MeSH
- Cystathionine beta-Synthase * genetics MeSH
- Alu Elements genetics MeSH
- Exons genetics MeSH
- Cloning, Molecular MeSH
- Humans MeSH
- Minisatellite Repeats genetics MeSH
- Molecular Sequence Data MeSH
- Polymorphism, Genetic genetics MeSH
- Promoter Regions, Genetic genetics MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA MeSH
- Sequence Homology, Nucleic Acid MeSH
- Transcription Factors genetics MeSH
- Binding Sites genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
BACKGROUND: PsbO, the manganese-stabilising protein, is an indispensable extrinsic subunit of photosystem II. It plays a crucial role in the stabilisation of the water-splitting Mn4CaO5 cluster, which catalyses the oxidation of water to molecular oxygen by using light energy. PsbO was also demonstrated to have a weak GTPase activity that could be involved in regulation of D1 protein turnover. Our analysis of psbO sequences showed that many angiosperm species express two psbO paralogs, but the pairs of isoforms in one species were not orthologous to pairs of isoforms in distant species. RESULTS: Phylogenetic analysis of 91 psbO sequences from 49 land plant species revealed that psbO duplication occurred many times independently, generally at the roots of modern angiosperm families. In spite of this, the level of isoform divergence was similar in different species. Moreover, mapping of the differences on the protein tertiary structure showed that the isoforms in individual species differ from each other on similar positions, mostly on the luminally exposed end of the β-barrel structure. Comparison of these differences with the location of differences between PsbOs from diverse angiosperm families indicated various selection pressures in PsbO evolution and potential interaction surfaces on the PsbO structure. CONCLUSIONS: The analyses suggest that similar subfunctionalisation of PsbO isoforms occurred parallelly in various lineages. We speculate that the presence of two PsbO isoforms helps the plants to finely adjust the photosynthetic apparatus in response to variable conditions. This might be mediated by diverse GTPase activity, since the isoform differences predominate near the predicted GTP-binding site.
- MeSH
- Amino Acids metabolism MeSH
- Species Specificity MeSH
- Photosystem II Protein Complex chemistry metabolism MeSH
- Phylogeny * MeSH
- Magnoliopsida genetics metabolism MeSH
- Models, Molecular MeSH
- Open Reading Frames genetics MeSH
- Protein Isoforms chemistry metabolism MeSH
- Genes, Plant MeSH
- Protein Structure, Secondary MeSH
- Amino Acid Sequence MeSH
- Amino Acid Substitution MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The genus Geosmithia Pitt (Ascomycota: Hypocreales) comprises cosmopolite fungi living in the galleries built by phloeophagous insects. Following the characterization in Geosmithia species 5 of the class II hydrophobin GEO1 and of the corresponding gene, the presence of the geo1 gene was investigated in 26 strains derived from different host plants and geographic locations and representing the whole phylogenetic diversity of the genus. The geo1 gene was detected in all the species tested where it maintained the general organization shown in Geosmithia species 5, comprising three exons and two introns. Size variations were found in both introns and in the first exon, the latter being due to the presence of an intragenic tandem repeat sequence corresponding to a stretch of glycine residues in the deduced proteins. At the amino acid level the deduced proteins had 44.6 % identity and no major differences in the biochemical parameters (pI, GRAVY index, hydropathy plots) were found. GEO1 release in the fungal culture medium was also assessed by turbidimetric assay and SDS-PAGE, and showed high variability between species. The phylogeny based on the geo1 sequences did not correspond to that generated from a neutral marker (ITS rDNA), suggesting that sequence similarities could be influenced by other factors than phylogenetic relatedness, such as the intimacy of the symbiosis with insect vectors. The hypothesis of a strong selection pressure on the geo1 gene was sustained by the low values (<1) of non synonymous to synonymous nucleotide substitutions ratios (Ka/Ks), which suggest that purifying selection might act on this gene. These results are compatible with either a birth-and-death evolution scenario or horizontal transfer of the gene between Geosmithia species.
- MeSH
- Ascomycota genetics isolation & purification MeSH
- DNA, Fungal chemistry genetics MeSH
- Exons MeSH
- Fungal Proteins genetics MeSH
- Phylogeny MeSH
- Genetic Variation * MeSH
- Introns MeSH
- Membrane Proteins genetics MeSH
- DNA, Ribosomal Spacer chemistry genetics MeSH
- Molecular Sequence Data MeSH
- Amino Acid Sequence MeSH
- Sequence Analysis, DNA MeSH
- Sequence Homology, Amino Acid MeSH
- Cluster Analysis MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Bacterial genotyping is a crucial process in outbreak investigation and epidemiological studies. Several typing methods such as pulsed-field gel electrophoresis, multilocus sequence typing (MLST) and whole genome sequencing are currently used in routine clinical practice. However, these methods are costly, time-consuming and have high computational demands. An alternative to these methods is mini-MLST, a quick, cost-effective and robust method based on high-resolution melting analysis. Nevertheless, no standardized approach to identify markers suitable for mini-MLST exists. Here, we present a pipeline for variable fragment detection in unmapped reads based on a modified hybrid assembly approach using data from one sequencing platform. RESULTS: In routine assembly against the reference sequence, high variable reads are not aligned and remain unmapped. If de novo assembly of them is performed, variable genomic regions can be located in created scaffolds. Based on the variability rates calculation, it is possible to find a highly variable region with the same discriminatory power as seven housekeeping gene fragments used in MLST. In the work presented here, we show the capability of identifying one variable fragment in de novo assembled scaffolds of 21 Escherichia coli genomes and three variable regions in scaffolds of 31 Klebsiella pneumoniae genomes. For each identified fragment, the melting temperatures are calculated based on the nearest neighbor method to verify the mini-MLST's discriminatory power. CONCLUSIONS: A pipeline for a modified hybrid assembly approach consisting of reference-based mapping and de novo assembly of unmapped reads is presented. This approach can be employed for the identification of highly variable genomic fragments in unmapped reads. The identified variable regions can then be used in efficient laboratory methods for bacterial typing such as mini-MLST with high discriminatory power, fully replacing expensive methods such as MLST. The results can and will be delivered in a shorter time, which allows immediate and fast infection monitoring in clinical practice.
