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At least 1,099 records · Page 61Linked to original sources
PubMed2004

Profiling DNA methylation by bisulfite genomic sequencing: problems and solutions.

The surge of interest in DNA methylation during the last two decades has triggered an urgent need for an effective method to detect the methylation status of the cytosines in the genome. Bisulfite genomic sequencing is the most attractive choice so far for many laboratories. Various protocols have been established, but difficulties are often encountered, particularly by individuals who have limited experience in this field. This analysis presents a simple protocol that has consistently worked well in our laboratory. Discussions of potential technical problems and corresponding solutions are also included to facilitate the reproducibility of this protocol.

Chemical Precipitation↗
PubMed2004

Nontemplated nucleotide addition prior to polyadenylation: a comparison of Arabidopsis cDNA and genomic sequences.

A comparison of Arabidopsis DNA sequences revealed that the final nucleotides at the 3' end of approximately half of the Arabidopsis mRNAs, immediately upstream of the poly(A) tail, differ from the corresponding genomic sequences. This suggests that extra nucleotides were added to these mRNAs at their 3' termini prior to polyadenylation. Among the mRNAs containing additional nucleotides, approximately 65% had a single additional nucleotide, with the nucleotide C added most often. This nontemplated addition before the addition of the poly(A) sequence could be a major contributing factor to the often observed heterogeneity in transcription products. These findings should be helpful in the elucidation of the mechanisms of mRNA 3'-end processing.

Arabidopsis↗
PubMed2004

Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors.

The bacterial family Enterobacteriaceae is notable for its well studied human pathogens, including Salmonella, Yersinia, Shigella, and Escherichia spp. However, it also contains several plant pathogens. We report the genome sequence of a plant pathogenic enterobacterium, Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043, the causative agent of soft rot and blackleg potato diseases. Approximately 33% of Eca genes are not shared with sequenced enterobacterial human pathogens, including some predicted to facilitate unexpected metabolic traits, such as nitrogen fixation and opine catabolism. This proportion of genes also contains an overrepresentation of pathogenicity determinants, including possible horizontally acquired gene clusters for putative type IV secretion and polyketide phytotoxin synthesis. To investigate whether these gene clusters play a role in the disease process, an arrayed set of insertional mutants was generated, and mutations were identified. Plant bioassays showed that these mutants were significantly reduced in virulence, demonstrating both the presence of novel pathogenicity determinants in Eca, and the impact of functional genomics in expanding our understanding of phytopathogenicity in the Enterobacteriaceae.

Base Sequence↗
PubMed2001

Computational analysis of full-length mouse cDNAs compared with human genome sequences.

Although the sequencing of the human genome is complete, identification of encoded genes and determination of their structures remain a major challenge. In this report, we introduce a method that effectively uses full-length mouse cDNAs to complement efforts in carrying out these difficult tasks. A total of 61,227 RIKEN mouse cDNAs (21,076 full-length and 40,151 EST sequences containing certain redundancies) were aligned with the draft human sequences. We found 35,141 non-redundant genomic regions that showed a significant alignment with the mouse cDNAs. We analyzed the structures and compositional properties of the regions detected by the full-length cDNAs, including cross-species comparisons, and noted a systematic bias of GENSCAN against exons of small size and/or low GC-content. Of the cDNAs locating the 35,141 genomic regions, 3,217 did not match any sequences of the known human genes or ESTs. Among those 3,217 cDNAs, 1,141 did not show any significant similarity to any protein sequence in the GenBank non-redundant protein database and thus are candidates for novel genes.

Algorithms↗
PubMed1996

Evidence of presence of poliovirus genomic sequences in cerebrospinal fluid from patients with postpolio syndrome.

The postpolio syndrome (PPS) is characterized by new neuromuscular symptoms occurring 30 to 40 years after the acute episode of poliomyelitis paralysis. The presence of the poliovirus RNA genome in the cerebrospinal fluid from 10 patients with PPS and from 23 control patients was sought by using reverse transcription and a PCR specific for polioviruses and/or other enteroviruses. Poliovirus-specific genomic sequences in the 5' untranslated region and in the capsid region (VP1) were detected by reverse transcription PCR in 5 of 10 patients with PPS but in none of the control patients. Sequencing confirmed the presence of mutated poliovirus sequences. This finding suggests persistent viral infection in the central nervous system related to the presence of poliovirus genomes.

Adult↗
PubMed2004

The complete mitochondrial genome sequence of the haptophyte Emiliania huxleyi and its relation to heterokonts.

The complete nucleotide sequence of the mitochondrial genome of Emiliania huxleyi (Haptophyta) was determined. E. huxleyi is the most abundant coccolithophorid, key in many marine ecosystems, and plays a vital role in the global carbon cycle. The mitochondrial genome contains genes encoding three subunits of cytochrome c oxidase, apocytochrome b, seven subunits of the NADH dehydrogenase complex, two ATPase subunits, two ribosomal RNAs, 25 tRNAs and five ribosomal proteins. One potentially functional open reading frame was identified, with no counterpart in any other organism so far studied. The cox1 gene transcript is apparently spliced from two distant segments in the genome. One of the most interesting features in this mtDNA is the presence of the dam gene, which codes for a DNA adenine methyltransferase. This enzyme is common in bacterial genomes, but is not present in any studied mitochondrial genome. Despite the great age of this group (ca. 300 Ma), little is known about the evolution of haptophytes or their relationship to other eukaryotes. This is the first published haptophyte organellar genome, and will improve the understanding of their biology and evolution and allow us to test the monophyly of the chromoalveolate clade.

Base Sequence↗
PubMed1997

Complete genomic sequence of the human PK-L/R-gene includes four intragenic polymorphisms defining different haplotype backgrounds of normal and mutant PK-genes.

The human pyruvate kinase L/R-gene has been completely sequenced in unrelated normal individuals and in pyruvate kinase-deficient patients by a PCR-based direct genomic sequencing approach and analyzed for polymorphisms. The total length of the gene is 8409 nucleotides. Four polymorphic sites have been detected: C/A1705 and C/T1992 in exon 12, a T-stretch in intron 1 occurring in the two polymorphic forms (T)10 and (T)19 and an (ATT)n microsatellite in intron J which has been found in the variation (ATT)11-17. Haplotype analysis using these four markers has been applied to trace the genetic background in PK-deficiencies. The results support the idea of a single origin of most of the individual PK-mutations.

Anemia, Hemolytic↗
PubMed2007

Genome sequence and analysis of the soil cellulolytic actinomycete Thermobifida fusca YX.

Thermobifida fusca is a moderately thermophilic soil bacterium that belongs to Actinobacteria. It is a major degrader of plant cell walls and has been used as a model organism for the study of secreted, thermostable cellulases. The complete genome sequence showed that T. fusca has a single circular chromosome of 3,642,249 bp predicted to encode 3,117 proteins and 65 RNA species with a coding density of 85%. Genome analysis revealed the existence of 29 putative glycoside hydrolases in addition to the previously identified cellulases and xylanases. The glycosyl hydrolases include enzymes predicted to exhibit mainly dextran/starch- and xylan-degrading functions. T. fusca possesses two protein secretion systems: the sec general secretion system and the twin-arginine translocation system. Several of the secreted cellulases have sequence signatures indicating their secretion may be mediated by the twin-arginine translocation system. T. fusca has extensive transport systems for import of carbohydrates coupled to transcriptional regulators controlling the expression of the transporters and glycosylhydrolases. In addition to providing an overview of the physiology of a soil actinomycete, this study presents insights on the transcriptional regulation and secretion of cellulases which may facilitate the industrial exploitation of these systems.

Actinomycetales↗
PubMed2003

A cattle-human comparative map built with cattle BAC-ends and human genome sequence.

As a step toward the goal of adding the cattle genome to those available for multispecies comparative genome analysis, 40,224 cattle BAC clones were end-sequenced, yielding 60,547 sequences (BAC end sequences, BESs) after trimming with an average read length of 515 bp. Cattle BACs were anchored to the human and mouse genome sequences by BLASTN search, revealing 29.4% and 10.1% significant hits (E < e-5), respectively. More than 60% of all cattle BES hits in both the human and mouse genomes are located within known genes. In order to confirm in silico predictions of orthologyand their relative position on cattle chromosomes, 84 cattle BESs with similarity to sequences on HSA11 were mapped using a cattle-hamster radiation hybrid (RH) panel. Resulting RH maps of BTA15 and BTA29 cover approximately 85% of HSA11 sequence, revealing a complex patchwork shuffling of segments not explained by a simple translocation followed by internal rearrangements. Overlay of the mouse conserved syntenies onto HSA11 revealed that segmental boundaries appear to be conserved in all three species. The BAC clone-based comparative map provides a foundation for the evolutionary analysis of mammalian karyotypes and for sequencing of the cattle genome.

Animals↗
PubMed2004

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species.

BACKGROUND: Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature. RESULTS: We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs. CONCLUSIONS: Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.

Anti-Bacterial Agents↗
PubMed2025

Orthopoxvirus Genome Sequencing, Assembly, and Analysis.

Poxviruses have exceptionally large genomes compared to most other viruses, which represent unique challenges to sequencing and assembly due to complex features such as repeat elements and low complexity sequences. The 2022 global mpox outbreak led to an unprecedented level of poxvirus sequencing as public health and research institutions faced with large sample numbers and demand for fast turnaround, merged NGS protocols designed for small RNA viruses with poxvirus expertise. Traditional manual assembly, checking, and editing of genomes was not feasible. Here, we present a protocol for metagenomic sequencing and orthopoxvirus genome assembly directly from DNA extracted from a patient lesion swab with no viral enrichment or host depletion. This sequencing approach is cost effective when using high throughput sequencing instruments and allows for detection of genomic insertions, deletions, and large rearrangement with confidence. We describe usage of two publicly available bioinformatic pipelines for genome assembly, quality control, annotation, and submission to sequence repositories.

Orthopoxvirus↗
PubMed2005

The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences.

BACKGROUND: The genus Corynebacterium includes Gram-positive microorganisms of great biotechnologically importance, such as Corynebacterium glutamicum and Corynebacterium efficiens, as well as serious human pathogens, such as Corynebacterium diphtheriae and Corynebacterium jeikeium. Although genome sequences of the respective species have been determined recently, the knowledge about the repertoire of transcriptional regulators and the architecture of global regulatory networks is scarce. Here, we apply a combination of bioinformatic tools and a comparative genomic approach to identify and characterize a set of conserved DNA-binding transcriptional regulators in the four corynebacterial genomes. RESULTS: A collection of 127 DNA-binding transcriptional regulators was identified in the C. glutamicum ATCC 13032 genome, whereas 103 regulators were detected in C. efficiens YS-314, 63 in C. diphtheriae NCTC 13129 and 55 in C. jeikeium K411. According to amino acid sequence similarities and protein structure predictions, the DNA-binding transcriptional regulators were grouped into 25 regulatory protein families. The common set of DNA-binding transcriptional regulators present in the four corynebacterial genomes consists of 28 proteins that are apparently involved in the regulation of cell division and septation, SOS and stress response, carbohydrate metabolism and macroelement and metal homeostasis. CONCLUSION: This work describes characteristic features of a set of conserved DNA-binding transcriptional regulators present within the corynebacterial core genome. The knowledge on the physiological function of these proteins should not only contribute to our understanding of the regulation of gene expression but will also provide the basis for comprehensive modeling of transcriptional regulatory networks of these species.

Carbohydrates↗
PubMed2006

Genomic sequence and spatiotemporal expression comparison of zebrafish mbx1 and its paralog, mbx2.

The expression of midbrain homeobox-1 (mbx1) defines a discrete region in the vertebrate neural plate that will give rise to the mesencephalon, as well as subregions of the diencephalon and retinal field. Here, we report on the identification and cloning of a second Mbx gene in zebrafish, termed mbx2. Genomic sequence comparison suggests that mbx1 and mbx2 are derived from the duplication of a single putative ancestral gene that is conserved in other vertebrates as a single copy gene. Furthermore, phylogenetic analyses indicate that the mbx genes belong to a novel subgroup of paired-like homeobox genes. Finally, quantitative reverse transcriptase-PCR and whole mount in situ hybridization experiments revealed a pattern of partial spatiotemporal expression divergence between the mbx paralogs that correlates with sequence divergence in noncoding regulatory domains. Our data support a subfunctionalization model that may explain the retention of duplicate mbx genes in teleosts.

Amino Acid Sequence↗
PubMed1997

Completion of the impatiens necrotic spot virus genome sequence and genetic comparison of the L proteins within the family Bunyaviridae.

The nucleotide sequence of the large (L) genome segment of impatiens necrotic spot virus (INSV) has been determined, and herewith the complete nucleotide sequence of the whole, tripartite genome of this important tospovirus has been elucidated. The L RNA is 8776 nucleotides long and of negative polarity, containing one large ORF on the viral complementary strand. Comparison of the deduced amino acid sequence of the INSV L RNA primary translation product (330.3 kDa) with those of the L RNAs of other members of the family Bunyaviridae reveals that this protein represents the putative viral RNA-dependent RNA polymerase. A cluster dendrogram of the (putative) RNA polymerases indicates that the genera Tospovirus and Tenuivirus, though both encompassing ambisense plant-infecting viruses, have different affinities to the animal-infecting Bunyaviridae, tospoviruses being most closely related to the genus Bunyavirus, and tenuiviruses to the genus Phlebovirus.

Amino Acid Sequence↗
PubMed2006

PCR-based detection and partial genome sequencing indicate high genetic diversity in Bangladeshi begomoviruses and their whitefly vector, Bemisia tabaci.

The population diversity of Bangladeshi begomoviruses and their vector, Bemisia tabaci was analysed by PCR-based detection and partial genome sequencing. B. tabaci adults and plants expressing symptoms of virus infection were collected from locations representing diverse agro-ecological regions of the country. Universal and species-specific primers were used to detect begomoviruses in seven crops (chilli, okra, papaya, pumpkin, sponge gourd, tomato and yardlong bean) and two common weeds (Ageratum conyzoides and Croton bonplandianum). At least five distinct species of tomato leaf curl viruses infected tomato and other host-plants. Phylogenetic analyses of their nucleotide sequences ( approximately 530 bases) from the intergenic region and capsid protein of DNA-A indicated the existence of five distinct clusters of begomoviruses. Begomoviruses infecting tomato, chilli and dolichos have been reported previously, and those infecting Ageratum, Croton, okra, papaya, pumpkin and yardlong bean are described for the first time. Phylogenetic analyses based on mitochondrial cytochrome oxidase I gene sequences of 21 B. tabaci from Bangladesh and other reference sequences grouped them into at least two independent clusters. Some sequences from different countries, e.g., Bangladesh, China, India, Nepal, Pakistan and Thailand were almost identical while others collected from plants within the same field diverged by as much as 15%, indicating high diversity even at the local level. None of the B. tabaci from Bangladesh grouped with the reference B- and Q-biotype sequences, thus these two aggressive biotypes were apparently absent from Bangladesh.

Animals↗
PubMed2004

Distinct genomic sequence of the CNR/Pcdhalpha genes in chicken.

CNR/Pcdhalpha family proteins have been first identified as a receptor family that corporate with Fyn, a family of the Src family of tyrosine kinase, and known as synaptic cadherins. Here we report the complete genomic sequence and organization of the chicken (Gallus gallus) CNR/Pcdhalpha The total length of chicken CNR/Pcdhalpha is 177kb. The chicken CNR/Pcdhalpha cluster encodes 12 variable and 3 constant exons. The genomic organizations of the chicken, rat, mouse, and human CNR/Pcdhalpha are basically orthologous. The constant-region exons (CP1, CP2, and CP3) are highly conserved between chicken and mammals, with percent identities of 90.9%, 90.7%, and 91.8% at the amino-acid level for chicken versus rat, mouse, and human, respectively. In contrast, the percent identities of the variable-region exons between chicken and mammals were lower: 51.8%, 51.3%, and 52.7%, on average, for chicken versus rat, mouse, and human, respectively, at the amino-acid level. Moreover, the chicken variable-region exons (from v1 to v12) are highly conserved paralogously (91.4%: nucleic acid, 92.4%: amino acid) in comparison with those of mammals. The CG content of each variable exon in the chicken (v1 to v12) is 74% on average and the CpG dinucleotide frequency in each variable-region exon is twice that of mammals. Due to the high CG content, chicken variable exons (from v1 to v12) encode 3 to 4 frame-shifted open reading frames, which span 1.5-3.0kb, in both the sense and anti-sense orientations.

Animals↗
PubMed1999

Contributions of genome sequencing to understanding the biology of Helicobacter pylori.

About half of the world's population carries Helicobacter pylori, a gram-negative, spiral bacterium that colonizes the human stomach. The link between H. pylori and, ulceration as well as its association with the development of both gastric cancer and mucosa-associated lymphoid tissue lymphoma in humans is a serious public health concern. The publication of the genome sequences of two stains of H. pylori gives rise to direct evidence on the genetic diversity reported previously with respect to gene organization and nucleotide variability from strain to strain. The genome size of H. pylori strain 26695 is 1,6697,867 bp and is 1,643,831 bp for strain J99. Approximately 89% of the predicted open reading frames are common to both of the strains, confirming H. pylori as a single species. A region containing approximately 45% of H. pylori strain-specific open reading frames, termed the plasticity zone, is present on the chromosomes, verifying that some strain variability exists. Frequent alteration of nucleotides in the third position of the triplet codons and various copies of insertion elements on the individual chromosomes appear to contribute to distinct polymorphic fingerprints among strains analyzed by restriction fragment length polymorphisms, random amplified polymorphic DNA method, and repetitive element-polymerase chain reaction. Disordered chromosomal locations of some genes seen by pulsed-field gel electrophoresis are likely caused by rearrangement or inversion of certain segments in the genomes. Cloning and functional characterization of the genes involved in acidic survival, vacuolating toxin, cag-pathogenicity island, motility, attachment to epithelial cells, natural transformation, and the biosynthesis of lipopolysaccharides have considerably increased our understanding of the molecular genetic basis for the pathogenesis of H. pylori. The homopolymeric nucleotide tracts and dinucleotide repeats, which potentially regulate the on- and off-status of the target genes by the strand-slipped mispairing mechanism, are often found in the genes encoding the outer-membrane proteins, in enzymes for lipopolysaccharide synthesis, and within DNA modification/restriction systems. Therefore, these genes may be involved in the H. pylori-host interaction.

Amino Acid Sequence↗
PubMed1993

Presence of respiratory syncytial virus genomic sequences in middle ear fluid and its relationship to expression of cytokines and cell adhesion molecules.

The presence of respiratory syncytial virus (RSV) and several cytokines and cell adhesion molecules in middle ear effusions and mucosal tissues was evaluated using polymerase chain reaction. RSV genomic sequences were detected in 23 (52.7%) of 44 middle ear effusions tested. The sequences were detectable at an even higher rate (82.4%) in effusions of children in whom infectious virus was detected in the nasopharynx. All samples with the RSV genome contained the mRNA for interleukin-1 beta and -6 and tumor necrosis factor-alpha. The messages for these cytokines, together with intercellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1, were detected in human middle ear mucosal organ cultures infected in vitro with RSV. Our results suggest that the enhanced synthesis of proinflammatory cytokines and cell adhesion molecules in the middle ear infected with RSV may contribute to the inflammatory processes in otitis media.

Base Sequence↗