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Multiple Alignment (multiple + alignment)

Distribution by Scientific Domains

Life Sciences	57%

Selected Abstracts

Involvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilization

FEBS JOURNAL, Issue 13 2000
Vincent Monchois
Multiple alignment of deduced amino-acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well-conserved catalytic domain. A portion of this domain displays homology to members of the ,-amylase family (glycoside hydrolase family 13), which all have a (,/,)8 barrel structure. In the glucansucrases, however, the ,-helix and ,-strand elements are circularly permuted with respect to the order in family 13. Previous work has shown that amino-acid residues contributing to the active site of glucansucrases are situated in structural elements that align with those of family 13. In ,-amylase and cyclodextrin glucanotransferase, a histidine residue has been identified that acts to stabilize the transition state, and a histidine is conserved at the corresponding position in all other members of family 13. In all the glucansucrases, however, the aligned position is occupied by glutamine. Mutants of glucosyltransferase I were constructed in which this glutamine, Gln937, was changed to histidine, glutamic acid, aspartic acid, asparagine or alanine. The effects on specific activity, ability to form glucan and ability to transfer glucose to a maltose acceptor were examined. Only histidine could substitute for glutamine and maintain Michaelis,Menten kinetics, albeit at a greatly reduced kcat, showing that Gln937 plays a functionally equivalent role to the histidine in family 13. This provides additional evidence in support of the proposed alignment of the (,/,)8 barrel structures. Mutation at position 937 altered the acceptor reaction with maltose, and resulted in the synthesis of novel gluco-oligosaccharides in which ,1,3-linked glucosyl units are joined sequentially to maltose. [source]

Evolution and phylogenetic relationships of chitin synthases from yeasts and fungi

FEMS YEAST RESEARCH, Issue 4 2002
José Ruiz-Herrera
Abstract Chitin, the structural component that provides rigidity to the cell wall of fungi is the product of chitin synthases (Chs). These enzymes are not restricted to fungi, but are amply distributed in four of the five eukaryotic ,crown kingdoms'. Dendrograms obtained by multiple alignment of Chs revealed that fungal enzymes can be classified into two divisions that branch into at least five classes, independent of fungal divergence. In contrast, oomycetes and animals each possess a single family of Chs. These results suggest that Chs originated as a branch of ,-glycosyl-transferases, once the kingdom Plantae split from the evolutionary line of eukaryotes. The existence of a single class of Chs in animals and Stramenopiles, against the multiple families in fungi, reveals that Chs diversification occurred after fungi departed from these kingdoms, but before separation of fungal groups. Accordingly, each fungal taxon contains members with enzymes belonging to different divisions and classes. Multiple alignment revealed the conservation of specific motifs characteristic of class, division and kingdom, but the strict conservation of only three motifs QXXEY, EDRXL and QXRRW, and seven isolated amino acids in the core region of all Chs. Determination of different structural features in this region of Chs brought to light a noticeable conservation of secondary structure in the proteins. [source]

Identification and characterization of the transcription factors involved in T-cell development, t-bet, stat6 and foxp3, within the zebrafish, Danio rerio

FEBS JOURNAL, Issue 1 2010
Suman Mitra
The discovery of cytokines expressed by T-helper 1 (Th1), Th2, Th17 and T-regulatory (Treg) cells has prompted speculation that these types of responses may exist in fish, arising early in vertebrate evolution. In this investigation, we cloned three zebrafish transcription factors, T-box expressed in T cells (t-bet), signal transducer and activator of transcription 6 (stat6) and fork-head box p3 (foxp3), in which two transcripts are present, that are important in the development of a number of these cell types. They were found within the zebrafish genome, using a synteny approach in the case of t-bet and foxp3. Multiple alignments of zebrafish t-bet, stat6 and foxp3 amino acids with known vertebrate homologues revealed regions of high conservation, subsequently identified to be protein domains important in the functioning of these transcription factors. The gene organizations of zebrafish t-bet and foxp3 were identical to those of the human genes, with the second foxp3 transcript lacking exons 5, 6, 7 and 8. Zebrafish stat6 (21 exons and 20 introns) was slightly different from the human gene, which contained 22 exons and 21 introns. Immunostimulation of zebrafish head kidney and spleen cells with phytohaemagglutinin, lipopolysaccharide or Poly I:C, showed a correlation between the expression of t-bet, stat6 and foxp3 with other genes involved in Th and Treg responses using quantitative PCR. These transcription factors, together with many of the cytokines that are expressed by different T-cell subtypes, will aid future investigations into the Th and Treg cell types that exist in teleosts. [source]

Evolution and phylogenetic relationships of APSES proteins from Hemiascomycetes

FEMS YEAST RESEARCH, Issue 4 2008
Bernardo Ramírez-Zavala
Abstract Available complete genomic sequences of hemiascomycetous yeast species were analysed in order to identify the APSES protein family, which belongs to transcriptional factors of the basic helix,loop,helix (bHLH) class. Phylogenetic analyses of the amino acid sequences revealed that a similar set of proteins were present in all yeast species studied. The genome duplication event of Saccharomycetales allows the acquisition of complementary functions between the APSES proteins. Putative ancestors, such as Ashbya gossypii, the Kluyveromyces group and filamentous fungi, only have one APSES protein. Conserved gene order relationships allow the possibility of tracing the evolution of this family and the detection of duplication events. Multiple alignments revealed strict conservation of the APSES motif, although other regions of the APSES proteins were diversified. This review focuses on the evolution of the gene family of APSES proteins in related Hemiascomycetes species; the comparisons could shed light on the functional overlap of these proteins with regard to the regulation of morphogenetic processes and their involvement in the virulence of pathogenic microorganisms. [source]

Evolution and phylogenetic relationships of chitin synthases from yeasts and fungi

Characterization of grouper nervous necrosis virus (GNNV)

JOURNAL OF FISH DISEASES, Issue 1 2001
S C Chi
Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper larvae, Epinephelus sp., using a fish cell line GF-1. The present study describes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) after 12 h post-infection (p.i.), although no cytopathic effect (CPE) appeared at that time. Clear CPE developed on the third day, and complete disintegration of the monolayer occurred over the subsequent two days. The infectivity of GNNV can be blocked following treatment at 60 °C for 1 h. GNNV was sensitive to pH 3 and pH 10,12 with a 4 log10 drop in infectivity. Purified GNNV was analysed by SDS,PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions and analysed. The molecular weights of genomic RNAs were 1.02 × 106 and 0.50 × 106 Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 sequence of two GNNV isolates with four genotypes of fish nodaviruses revealed that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution of GNNV in naturally infected grouper larvae was investigated by in situ hybridization using a dig-labelled probe, which showed that GNNV was not only detected in the brain and retina, but also in the gill, skeletal muscle, liver, pyloric gland, intestine and blood cells in the heart. [source]

Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction

JOURNAL OF MEDICAL VIROLOGY, Issue 10 2006
Tomoichiro Oka
Abstract Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI,GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5,×,107 to 2.5,×,101 copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses. J. Med. Virol. 78:1347,1353, 2006. © 2006 Wiley-Liss, Inc. [source]