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At least 757 records · Page 42Linked to original sources
PubMed1995

The nucleotide sequence and proposed genome organization of oat chlorotic stunt virus, a new soil-borne virus of cereals.

The complete genomic sequence of a new virus, first found infecting oats in Wales, UK, has been determined. The genome is a positive-sense ssRNA molecule, 4114 nucleotides in length, examination of which indicates the presence of four ORFs. The first ORF initiating at the 5' terminus (ORF1) encodes a protein with a predicted M(r) of 23476 (p23). ORF2 extends through the amber termination codon of ORF1 to give a protein with a predicted M(r) of 84355 (p84). The readthrough domain of p84 contains amino acid sequence similarities with a number of putative RNA-dependent RNA polymerases. ORF3 is in a different reading frame from ORF1/2 and encodes a protein with an M(r) of 48231 (p48), identified as the coat protein by direct peptide sequencing. ORF4 nests within ORF3 but is in a different frame from it and codes for a protein with a predicted M(r) of 8220 (p8). Comparisons of peptide sequence, particularly within the putative polymerase region and within the S domain of the coat protein, highlight similarities with members of both the tombusvirus and carmovirus groups. The coat protein region shows most similarity with members of the tombusvirus group, whilst the size and predicted strategy of the genome seem to be intermediate between that of the carmovirus and tombusvirus groups. These features highlight possible evolutionary links with each group whilst being distinct from both. We propose the name of oat chlorotic stunt for this new virus.

Amino Acid Sequence↗
PubMed1996

Cloning and sequencing of the genome of chicken anaemia virus (CAV) TK-5803 strain and comparison with other CAV strains.

The complete genomic sequence of chicken anaemia virus (CAV) TK-5803 strain was determined. Comparisons of sequence data showed 37 nucleotide differences between TK-5803 and Cux-1, 38 nucleotide differences between TK-5803 and 26P4, and 48 nucleotide differences between TK-5803 and 82-2. There were 65 nucleotide differences in the largest open reading frame (ORF3) between TK-5803 and one Australian isolate. Base changes introduced amino acid changes at 6 positions in the C-terminal half of VP3, at 4 positions in the C-terminal quarter of VP2 and at 17 positions in VP1 among the strains. These indicate that the N-terminal half of VP3 and the N-terminal three quarter of VP2 are well conserved, and might sustain essential function of these proteins. The amino acid changes in VP1 which is thought to be the capsid protein may influence the antigenic character of different VP1s.

Amino Acid Sequence↗
PubMed2006

Gene identification and cloning by molecular marker mapping.

In the course of map-based cloning, mutant genes are identified through linkage to a sufficiently small region of the genetic map and subsequent DNA sequencing. This process has become fairly straightforward for Arabidopsis mutations, owing to the completed genome sequence and the discovery of many thousands of molecular markers. Initially, plants with the desired phenotype are identified in populations treated with ethylmethanesulfonate or other mutagens. Once the mutant phenotype has been discovered and confirmed, map-based identification of the mutated gene is a four-stage process: (1) mutant plants are crossed to another Arabidopsis ecotype and F2 seeds are generated; (2) approx 50 homozygous mutant F2 plants are genotyped to determine linkage to four or five molecular markers on each of the five chromosomes; (3) A larger F2 population (1000 to 2000 plants) is grown, genotyped, and phenotyped to determine fine-scale genetic linkage, ideally narrowing to a chromosomal region of about 40 kbp; and (4) sequencing of mutant and wild-type DNA is used to verify the identity of the mutated gene. Given a mutant phenotype that can be determined unambiguously in a single F2 plant, it is possible to complete an Arabidopsis map-based cloning project in about 1 yr.

Arabidopsis↗
PubMed2001

How do thermophilic proteins deal with heat?

Recent years have witnessed an explosion of sequence and structural information for proteins from hyperthermophilic and thermophilic organisms. Complete genome sequences are available for many hyperthermophilic archaeons. Here, we review some recent studies on protein thermostability along with work from our laboratory. A large number of sequence and structural factors are thought to contribute toward higher intrinsic thermal stability of proteins from these organisms. The most consistent are surface loop deletion, increased occurrence of hydrophobic residues with branched side chains and an increased proportion of charged residues at the expense of uncharged polar residues. The energetic contribution of electrostatic interactions such as salt bridges and their networks toward protein stability can be stabilizing or destabilizing. For hyperthermophilic proteins, the contribution is mostly stabilizing. Macroscopically, improvement in electrostatic interactions and strengthening of hydrophobic cores by branched apolar residues increase the enthalpy change between the folded and unfolded states of a thermophilic protein. At the same time, surface loop deletion contributes to decreased conformational entropy and decreased heat capacity change between the folded and unfolded states of the protein.

Archaea↗
PubMed1999

Isolation of salt-induced periplasmic proteins from Synechocystis sp. strain PCC 6803.

Periplasmic proteins were obtained from control cells and salt-adapted cells of the cyanobacterium Synechocystis sp. PCC 6803 using the method of cold osmotic shock. Two of these proteins (PP 1, apparent mol. mass 27.6 kDa, and PP 3, apparent mol. mass 39.9 kDa) were accumulated in high amounts in the periplasm of salt-adapted cells, while the major periplasmic protein (PP 2, apparent mol. mass 36.0 kDa) was accumulated independently from salt. After isolation from gels and partial sequencing, the proteins could be assigned to proteins deduced from the complete genome sequence of Synechocystis. Neither salt-induced periplasmic proteins (PP 1, Slr0924 and PP 3, Slr1485) exhibited sequence similarity to proteins of known function from databases. The major protein (PP 2-Slr0513) showed significant sequence similarities to iron-binding proteins. All proteins included typical leader sequences at their N-terminus.

Adaptation, Physiological↗
PubMed1998

Mycoplasma pneumoniae and Mycoplasma genitalium: a comparison of two closely related bacterial species.

The rapid progress in sequencing large quantities of DNA will provide an increasing number of complete genome sequences of closely related bacterial species as well as of pairs of isolates from the same species with different features, such as a pathogenic and an apathogenic representative. This opens the way to apply subtractive comparative analysis as a tool to select from the large pool of all bacterial genes a relatively small set of genes that can be correlated with the expression of a certain phenotype. These selected genes can then be the target for further functional analyses.

Bacterial Proteins↗
PubMed2003

Expression defect of an HLA-A*24 allele associated with DNA methylation in a normal individual.

Non-expressed HLA alleles become a potential problem in the transition of the HLA typing methodology from serologic typing to more accurate DNA typing. In this study, a novel nonexpressed A*24 allele identified from two members of a Korean family was characterized. At the DNA sequence level, the nonexpressed allele (A*24023) is apparently normal; the complete genomic sequence was identical to HLA-A*2402101, from the 5'-upstream region to the 3'-downstream region, except for a single silent substitution at codon 211 (GCG-->GCA) in exon 4. A DNA methylation analysis using methylation-sensitive restriction enzymes, however, showed that the nonexpressed A*24023 allele from an apparently normal individual was highly methylated in regions covering exons and introns as well as the 5'-upstream region. This result suggests that hypermethylation of the HLA-A gene may induce gene inactivation in the normal individuals.

DNA Methylation↗
PubMed1999

The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase.

Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a beta-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.

Adenosine Monophosphate↗
PubMed1993

Cloning and characterization of the C. elegans histidyl-tRNA synthetase gene.

In this paper, we report the cloning and sequencing of the C. elegans histidyl-tRNA synthetase gene. The complete genomic sequence, and most of the cDNA sequence, of this gene is now determined. The gene size including flanking and coding regions is 2230 nucleotides long. Three small introns (45-50 bp long) are found to interrupt the open reading frame. The open reading frame translates to 523 amino acids. This putative protein sequence shows extensive homology with the human and yeast histidyl-tRNA the histidyl-tRNA synthetase gene is a single copy gene. Hence, it is very likely that it encodes both the cytoplasmic and the mitochondrial histidyl-tRNA synthetases. It is likely to be trans-spliced since it contains a trans-splice site in its 5' untranslated region.

Amino Acid Sequence↗
PubMed2004

Characterization of the cis-acting regulatory element controlling HrpB-mediated activation of the type III secretion system and effector genes in Ralstonia solanacearum.

The ability of Ralstonia solanacearum to cause disease on plants depends on its type III secretion system (TTSS) encoded by hrp genes. The expression of hrp genes and known TTSS substrates is coordinately regulated by HrpB, a member of the AraC family of transcriptional regulators. Two HrpB-regulated promoters (hrpY and popABC) were characterized by deletion analysis, and the HrpB-dependent activation of these promoters was found to be conferred by a 25-nucleotide DNA element, the hrp(II) box (TTCGn16TTCG), which is present in other hrp promoters. The hrp(II) box element is an imperfect plant inducible promoter box, an element which was originally found in hrp promoters of Xanthomonas campestris (S. Fenselau and U. Bonas, Mol. Plant-Microbe Interact. 8:845-854, 1995) but which was not characterized at the molecular level. Site-directed mutagenesis showed that the hrp(II) box is essential for hrpY promoter activation in vivo. Functional analysis of the hrp(II) box element identified critical parameters that are required for HrpB-dependent activity. Further mapping analyses of several other hrpB-dependent promoters also indicated that the position of the hrp(II) box is conserved, at -70 to -47 bp from the transcriptional start. As a first step toward identifying novel TTSS effectors, we used the hrp(II) box consensus sequence to search for potential HrpB-regulated promoters in the complete genome sequence of R. solanacearum strain GMI1000. Among the 114 genes identified, a subset of promoters was found to have a structural relationship with hrp promoters, thus providing a pool of candidate genes encoding TTSS effectors.

Bacterial Proteins↗
PubMed1987

The genome of herpes simplex virus: structure, replication and evolution.

The objectives of this paper are to discuss the structure and genetic content of the genome of herpes simplex virus type 1 (HSV-1), the nature of virus DNA replicative processes, and aspects of the evolution of the virus DNA, in particular those bearing on DNA replication. We are in the late stages of determining the complete sequence of the DNA of HSV-1, which contains about 155,000 base pairs, and thus the treatment is primarily from a viewpoint of DNA sequence and organization. The genome possesses around 75 genes, generally densely arranged and without long range ordering. Introns are present in only a few genes. Protein coding sequences have been predicted, and the functions of the proteins are being pursued by various means, including use of existing genetic and biochemical data, computer based analyses, expression of isolated genes and use of oligopeptide antisera. Many proteins are known to be virion structural components, or to have regulatory roles, or to function in synthesis of virus DNA. Many, however, still lack an assigned function. Two classes of genetic entities necessary for virus DNA replication have been characterized: cis-acting sequences, which include origins of replication and packaging signals, and genes encoding proteins involved in replication. Aside from enzymes of nucleotide metabolism, the latter include DNA polymerase, DNA binding proteins, and five species detected by genetic assays, but of presently unknown functions. Complete genome sequences are now known for the related alphaherpesvirus varicella-zoster virus and for the very distinct gammaherpesvirus Epstein-Barr virus. Comparisons between the three sequences show various homologies, and also several types of divergence and rearrangement, and so allow models to be proposed for possible events in the evolution of present day herpesvirus genomes. Another aspect of genome evolution is seen in the wide range of overall base compositions found in present day herpesvirus DNAs. Finally, certain herpesvirus genes are homologous to non-herpesvirus genes, giving a glimpse of more remote relationships.

Base Sequence↗
PubMed2026

Development and evaluation of a one-pot RPA-Cas12a assay based on a primer-driven reverse screening strategy for preliminary screening of megalocytivirus-related viruses.

A primer-driven reverse-screening strategy was used to identify an RPA-Cas12a target suitable for the rapid preliminary screening of megalocytivirus-related viruses. The ISKNV reference genome NC_003494.1 was used as the initial template, and candidate amplification units were designed according to RPA primer-design requirements, primer physicochemical properties, and the availability of Cas12a protospacer-adjacent motif (PAM) sites and crRNA target sequences. Following preliminary amplification assessment, the retained candidate primers were aligned individually against 75 complete genome sequences of megalocytivirus-related viruses. Of these, 67 sequences met the predefined criteria for target-region integrity, primer-binding-site compatibility, and Cas12a recognition. Retrospective mapping to the reference genome located the candidate amplification region within ORF057L. Based on the resulting candidate detection unit, a one-pot RPA-Cas12a assay incorporating a commercially available lyophilized RPA amplification module was developed. Optimization showed that 400 nM reporter and 80 nM crRNA-1 provided relatively stable fluorescence output. A cut-off value of 1281.6 relative fluorescence units (RFU) was established as the mean plus three standard deviations of the endpoint fluorescence values obtained from 20 qPCR-negative samples. In analytical sensitivity testing, the assay generated fluorescence signals above the negative control at low plasmid copy numbers. However, because only a limited number of replicates were tested at these low template concentrations, these findings were not used to define a formal limit of detection. ISKNV, RSIV, and TRBIV samples tested positive, whereas the MRV sample produced an endpoint fluorescence value below the cut-off. Repeatability analysis of the same sample in six independent reactions yielded a coefficient of variation of 8.03%. Among the 39 samples examined, no discordant qualitative results were observed between the RPA-Cas12a assay and qPCR. These findings support the use of the ORF057L-targeted one-pot RPA-Cas12a assay as a rapid preliminary screening tool for megalocytivirus-related viruses. Nevertheless, its formal limit of detection, inter-batch stability, cross-reactivity with additional non-target pathogens, and clinical diagnostic performance require further evaluation.

Lyophilized RPA↗
PubMed2005

Reduced representation sequencing: a success in maize and a promise for other plant genomes.

Plant, and particularly cereal genomes, are challenging to sequence due to their large size and high repetitive DNA content. Gene-enrichment strategies are alternative or complementary approaches to complete genome sequencing that yield, rapidly and inexpensively, useful sequence data from large and complex genomes. The maize genome is large (2.7 Gbp) and contains large amounts of conserved repetitive elements. Furthermore, the high allelic diversity found between maize inbred lines may necessitate sequencing several inbred lines in order to recover the maize "gene pool". Two gene-enrichment approaches, methylation filtration (MF) and high C(o)t (HC) sequencing have been tested in maize and their ability to sample the gene space has been examined. Combined with other genomic sequencing strategies, gene-enriched genomic sequencing is a practical way to examine the maize gene pool, to order and orient the genic sequences on the genome, and to enable investigation of gene content of other complex plant genomes.

Alleles↗
PubMed1999

Complete nucleotide sequences of garlic viruses A and C, members of the newly ratified genus Allexivirus.

Complete genomic sequences of garlic viruses A (Gar V-A) and C (Gar V-C), members of an unassigned virus group recently identified in garlic plants, were determined. Their respective genomes consist of 8 660 and 8 405 nucleotides. The genomic structure and organization of these viruses are similar to shallot virus X (ShVX) which is the type species of the newly ratified genus Allexivirus. Phylogenetic analysis based on the amino acid sequences of the putative proteins including RNA-dependent RNA polymerase (RdRP), DNA helicase, or viral coat protein showed that the GarV-type viruses should be included in the genus Allexivirus. Furthermore, the amino acid sequence in the RdRP hypervariable region is highly divergent among the viruses in the genus Allexivirus, suggesting that they evolved independently from a hypothetical ancestor virus(es).

3' Untranslated Regions↗
PubMed2000

DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae.

Here we determine the complete genomic sequence of the gram negative, gamma-Proteobacterium Vibrio cholerae El Tor N16961 to be 4,033,460 base pairs (bp). The genome consists of two circular chromosomes of 2,961,146 bp and 1,072,314 bp that together encode 3,885 open reading frames. The vast majority of recognizable genes for essential cell functions (such as DNA replication, transcription, translation and cell-wall biosynthesis) and pathogenicity (for example, toxins, surface antigens and adhesins) are located on the large chromosome. In contrast, the small chromosome contains a larger fraction (59%) of hypothetical genes compared with the large chromosome (42%), and also contains many more genes that appear to have origins other than the gamma-Proteobacteria. The small chromosome also carries a gene capture system (the integron island) and host 'addiction' genes that are typically found on plasmids; thus, the small chromosome may have originally been a megaplasmid that was captured by an ancestral Vibrio species. The V. cholerae genomic sequence provides a starting point for understanding how a free-living, environmental organism emerged to become a significant human bacterial pathogen.

Base Sequence↗
PubMed2003

MuGeN: simultaneous exploration of multiple genomes and computer analysis results.

MOTIVATION: The availability of increasing amounts of sequence data about completely sequenced genomes spurs the development of new methods in the fields of automated annotation, and of comparative genomics. Tools allowing the visualization of results produced by analysis methods, superimposed on possibly annotated sequence data, and enabling synchronized navigation in multiple genomes, provide new means for interactive genome exploration. This kind of visual inspection can be used as a basis to assess the quality of new analysis algorithms, or to discover genome portions to be subjected to in-depth studies. RESULTS: We propose a software package, MuGeN, built for navigating through multiple annotated genomes. It is capable of retrieving annotated sequences in several formats, stored in local files, or available in databases over the network. From these, it then generates an interactive display, or an image file, in most common formats suitable for printing, further editing or integrating in Web pages. Genome maps may be mixed with computer analysis results loaded from XML files, whose format is generic enough to be adapted to a majority of sequence oriented analysis methods. AVAILABILITY: MuGeN is available at http://www-mig.jouy.inra.fr/bdsi/MuGeN.

Computer Graphics↗
PubMed2000

WIT: integrated system for high-throughput genome sequence analysis and metabolic reconstruction.

The WIT (What Is There) (http://wit.mcs.anl.gov/WIT2/) system has been designed to support comparative analysis of sequenced genomes and to generate metabolic reconstructions based on chromosomal sequences and metabolic modules from the EMP/MPW family of databases. This system contains data derived from about 40 completed or nearly completed genomes. Sequence homologies, various ORF-clustering algorithms, relative gene positions on the chromosome and placement of gene products in metabolic pathways (metabolic reconstruction) can be used for the assignment of gene functions and for development of overviews of genomes within WIT. The integration of a large number of phylogenetically diverse genomes in WIT facilitates the understanding of the physiology of different organisms.

Databases, Factual↗
PubMed1995

Genomic organization of the human PAX3 gene: DNA sequence analysis of the region disrupted in alveolar rhabdomyosarcoma.

Mutations in the human PAX3 gene have previously been associated with two distinct diseases, Waardenburg syndrome and alveolar rhabdomyosarcoma. In this report we establish that the normal human PAX3 gene is encoded by 8 exons. Intron-exon boundary sequences were obtained for PAX3 exons 5, 6, 7, and 8 and together with previous work provide the complete genomic sequence organization for PAX3. Difficulties in obtaining overlapping genomic clone coverage of PAX3 were circumvented in part by RARE cleavage mapping, which showed that the entire PAX3 gene spans 100 kb of chromosome 2. Sequence analysis of the last intron of PAX3, which contains the previously mapped t(2;13)(q35;q14) translocation breakpoints of alveolar rhabdomyosarcoma, revealed the presence of a pair of inverted Alu repeats and a pair of inverted (GT)n-rich microsatellite repeats within a 5-kb region. This work establishes the complete structure of PAX3 and will permit high-resolution analyses of this locus for mutations associated with Waardenburg syndrome, alveolar rhabdomyosarcoma, and other phenotypes for which PAX3 may be a candidate locus.

Alleles↗