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At least 721 records · Page 40Linked to original sources
PubMed2005

Genome analysis of Cryphonectria hypovirus 4, the most common hypovirus species in North America.

Many different viruses that reduce virulence and alter the phenotype to varying extents have been identified in the chestnut blight fungus Cryphonectria parasitica. Most viruses identified in this fungus fall within the Hypoviridae family of positive-sense RNA viruses, which contains one genus and four species. Different species predominate in different geographic locations in chestnut-growing areas around the world. In this paper, we describe the genome organization and some variants of Cryphonectria hypovirus 4 (CHV-4), the species most commonly found in eastern North America. CHV-4 is distinguished from other hypoviruses by having little effect on fungal virulence and colony morphology. The 9.1-kb genome of strain CHV-4/SR2 is the smallest of any member of the family characterized to date. Like the recently characterized species CHV-3, a single ORF was predicted from deduced translations of CHV-4/SR2. Sequence analysis revealed the presence of a putative glucosyltransferase domain in both CHV-4 and in CHV-3, but no such homolog was detected in the more thoroughly examined CHV-1 or in CHV-2. Alignments with 8 other CHV-4 isolates from different regions of eastern North America revealed sequence diversity within the species and the likelihood that RNA recombination has led to this diversity.

Ascomycota↗
PubMed2001

Genome partitioning and whole-genome analysis.

Standard DNA marker-based approaches to mapping genes that influence complex traits typically consider a limited number of hypotheses. Most of these hypotheses concentrate on the effect of a single individual locus (or relatively few loci) on the trait of interest. Although of tremendous importance scientifically, such hypotheses do not accommodate the full range of genetic phenomena that may contribute to phenotypic expression. We present novel approaches to complex trait analysis that make as complete use of marker information as is possible. The proposed methodologies can be used to entertain a wide variety of hypotheses, including those that engage, for example, the contribution of a particular chromosome, genome-wide heterozygosity, and multiple genomic regions, to phenotypic expression. We consider a number of possible extensions of the proposed methods as well as their limitations. Although we discuss many methodological details in the context of quantitative trait locus mapping involving sampling units such as human pedigrees and hybrids resulting from crosses between inbred strains of model organisms, our procedures can be easily adapted to standard sibpair and other sampling unit-based designs. Ultimately, the proposed approaches not only have the potential to increase power to identify individual loci that harbor trait-influencing genes, but also present a framework for testing a number of hypotheses about the nature of the genetic determinants of phenotypes in general.

Alleles↗
PubMed2005

Full genome analysis of hepatitis B virus genotype E strains from South-Western Africa and Madagascar reveals low genetic variability.

The eight genotypes of hepatitis B virus (HBV), A-H, have a characteristic geographical distribution. The high prevalence of genotype E within a wide expanse of Africa makes it important to characterize this genotype. Although the preS/S region of a large number of genotype E isolates has been sequenced, to date only eight complete genotype E genomes have been published. The aim of this study, therefore, was to sequence and characterize the complete genome of genotype E isolates obtained from geographically distinct regions in Africa, including Namibia, Angola, and Madagascar. The nine newly sequenced genotype E isolates were closely related with an intra-group nucleotide divergence of 1.2%, reflecting their close geographical origin. All genotype E strains have the same characteristics, an in-frame deletion of three nucleotides (one amino acid) in the 5' pre-S1, a signature pattern of amino acids in the pre-S1 region and the serological subtype ayw4. In addition, we have identified another unique attribute of genotype E strains, the introduction of another start codon Met(83) in the pre-S1 region that may result in the translation of an elongated middle hepatitis B surface protein (MHBs).

Adult↗
PubMed2005

Genetic and genomic analysis of the AT-rich centromere DNA element II of Saccharomyces cerevisiae.

Centromere DNA element II (CDEII) of budding yeast centromeres is an AT-rich sequence essential for centromere (CEN) function. Sequence analysis of Saccharomyces cerevisiae CDEIIs revealed that A(5-7)/T(5-7) tracts are statistically overrepresented at the expense of AA/TT and alternating AT. To test the hypothesis that this nonrandom sequence organization is functionally important, a CEN library in which the CDEII sequences were randomized was generated. The library was screened for functional and nonfunctional members following centromere replacement in vivo. Functional CENs contained CDEIIs with the highly biased A(n)/T(n) run distribution of native centromeres, while nonfunctional CDEIIs resembled those picked from the library at random. Run content, defined as the fraction of residues present in runs of four or more nucleotides, of the functional and nonfunctional CDEII populations differed significantly (P < 0.001). Computer searches of the genome for regions with an A + T content comparable to CDEIIs revealed that such loci are not unique to centromeres, but for 14 of the 16 chromosomes the AT-rich locus with the highest A(n > or =4) + T(n > or =4) run content was the centromere. Thus, the distinctive and nonrandom sequence organization of CDEII is important for centromere function and possesses informational content that could contribute to the determination of centromere identity.

AT Rich Sequence↗
PubMed1987

Genome analysis and reverse transcriptase activity of human teratocarcinoma-derived retroviruses.

Five of five human teratocarcinomas cultured in vitro could be induced to synthesize retrovirus-like particles, albeit in extremely low amounts. The accumulation and purification of the human teratocarcinoma-derived retrovirus (HTDV) from one of these cell lines allowed characterization of its genomic material as high mol. wt. (60S) RNA. Partial purification of HTDV-associated RNA-dependent DNA polymerase (reverse transcriptase) has also been achieved. HTDVs are easily distinguishable from the exogenous human T-lymphotropic viruses and human immunodeficiency viruses by morphological, biological and immunological criteria.

Humans↗
PubMed2004

Functional characterisation and genomic analysis of an epithelial calcium channel (ECaC) from pufferfish, Fugu rubripes.

An orthologue to the mammalian epithelial calcium channels, ECaC1 (TRPV5) and ECaC2 (TRPV6), was cloned from gill of pufferfish (Fugu rubripes) and characterised, demonstrating that this gene predates the evolution of land-living vertebrates. The F. rubripes ECaC (FrECaC) protein displays all structural features typical for mammalian ECaCs including three ankyrin repeats, six transmembrane domains, and a putative pore region between TM V and TM VI. Functional expression of FrECaC in Madin-Darby canine kidney (MDCK) cells confirmed that the channel mediates Ca(2+) influx. FrECaC was also permeable to Zn(2+) and, to a small extent, to the Fe(2+) ion. Thus, in addition to a role in Ca(2+) uptake FrECaC might serve as a pathway for zinc and iron acquisition. FrECaC mRNA was highly abundant in the gill, but sparsely present in the intestine. Calcium absorption via FrECaC in pufferfish may be subject to the regulation of 1.25(OH)(2)D(3), estrogen and progesterone as consensus cis regulatory elements for the respective steroid hormone receptors were found in the upstream regulatory region of the FrECaC gene. FrECaC gene organisation is very conserved when compared with mammalian ECaCs. Only one ECaC gene seems to exist in the F. rubripes genome, and the corresponding protein clusters together with ECaC2 from mammals upon phylogenetic analysis. Thus, the two mammalian ECaC genes may originate from a single ancestral ECaC2 gene in vertebrates appearing early in evolution.

Amino Acid Sequence↗
PubMed2002

Genomic analysis of Hox clusters in the sea lamprey Petromyzon marinus.

The sea lamprey Petromyzon marinus is among the most primitive of extant vertebrates. We are interested in the organization of its Hox gene clusters, because, as a close relative of the gnathostomes, this information would help to infer Hox cluster organization at the base of the gnathostome radiation. We have partially mapped the P. marinus Hox clusters using phage, cosmid, and P1 artificial chromosome libraries. Complete homeobox sequences were obtained for the 22 Hox genes recovered in the genomic library screens and analyzed for cognate group identity. We estimate that the clusters are somewhat larger than those of mammals (roughly 140 kbp vs. 105 kbp) but much smaller than the single Hox cluster of the cephalochordate amphioxus (at more than 260 kb). We never obtained more than three genes from any single cognate group from the genomic library screens, although it is unlikely that our screen was exhaustive, and therefore conclude that P. marinus has a total of either three or four Hox clusters. We also identify four highly conserved non-coding sequence motifs shared with higher vertebrates in a genomic comparison of Hox 10 genes.

Amino Acid Sequence↗
PubMed2002

Genome analysis: RNA recognition motif (RRM) and K homology (KH) domain RNA-binding proteins from the flowering plant Arabidopsis thaliana.

Regulation of gene expression at the post-transcriptional level is mainly achieved by proteins containing well-defined sequence motifs involved in RNA binding. The most widely spread motifs are the RNA recognition motif (RRM) and the K homology (KH) domain. In this article, we survey the complete Arabidopsis thaliana genome for proteins containing RRM and KH RNA-binding domains. The Arabidopsis genome encodes 196 RRM-containing proteins, a more complex set than found in Caenorhabditis elegans and Drosophila melanogaster. In addition, the Arabidopsis genome contains 26 KH domain proteins. Most of the Arabidopsis RRM-containing proteins can be classified into structural and/or functional groups, based on similarity with either known metazoan or Arabidopsis proteins. Approximately 50% of Arabidopsis RRM-containing proteins do not have obvious homologues in metazoa, and for most of those that are predicted to be orthologues of metazoan proteins, no experimental data exist to confirm this. Additionally, the function of most Arabidopsis RRM proteins and of all KH proteins is unknown. Based on the data presented here, it is evident that among all eukaryotes, only those RNA-binding proteins that are involved in the most essential processes of post-transcriptional gene regulation are preserved in structure and, most probably, in function. However, the higher complexity of RNA-binding proteins in Arabidopsis, as evident in groups of SR splicing factors and poly(A)-binding proteins, may account for the observed differences in mRNA maturation between plants and metazoa. This survey provides a first systematic analysis of plant RNA-binding proteins, which may serve as a basis for functional characterisation of this important protein group in plants.

Amino Acid Motifs↗
PubMed2004

A genomic analysis of Drosophila somatic sexual differentiation and its regulation.

In virtually all animals, males and females are morphologically, physiologically and behaviorally distinct. Using cDNA microarrays representing one-third of Drosophila genes to identify genes expressed sex-differentially in somatic tissues, we performed an expression analysis on adult males and females that: (1) were wild type; (2) lacked a germline; or (3) were mutant for sex-determination regulatory genes. Statistical analysis identified 63 genes sex-differentially expressed in the soma, 20 of which have been confirmed by RNA blots thus far. In situ hybridization experiments with 11 of these genes showed they were sex-differentially expressed only in internal genital organs. The nature of the products these genes encode provides insight into the molecular physiology of these reproductive tissues. Analysis of the regulation of these genes revealed that their adult expression patterns are specified by the sex hierarchy during development, and that doublesex probably functions in diverse ways to set their activities.

Animals↗
PubMed2005

Comparative genomic analysis of the pPT23A plasmid family of Pseudomonas syringae.

Members of the pPT23A plasmid family of Pseudomonas syringae play an important role in the interaction of this bacterial pathogen with host plants. Complete sequence analysis of several pPT23A family plasmids (PFPs) has provided a glimpse of the gene content and virulence function of these plasmids. We constructed a macroarray containing 161 genes to estimate and compare the gene contents of 23 newly analyzed and eight known PFPs from 12 pathovars of P. syringae, which belong to four genomospecies. Hybridization results revealed that PFPs could be distinguished by the type IV secretion system (T4SS) encoded and separated into four groups. Twelve PFPs along with pPSR1 from P. syringae pv. syringae, pPh1448B from P. syringae pv. phaseolicola, and pPMA4326A from P. syringae pv. maculicola encoded a type IVA T4SS (VirB-VirD4 conjugative system), whereas 10 PFPs along with pDC3000A and pDC3000B from P. syringae pv. tomato encoded a type IVB T4SS (tra system). Two plasmids encoded both T4SSs, whereas six other plasmids carried none or only a few genes of either the type IVA or type IVB secretion system. Most PFPs hybridized to more than one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors. The overall gene contents of individual PFPs were more similar among plasmids within each of the four groups based on T4SS genes; however, a number of genes, encoding plasmid-specific functions or hypothetical proteins, were shared among plasmids from different T4SS groups. The only gene shared by all PFPs in this study was the repA gene, which encoded sequences with 87 to 99% amino acid identityamong 25 sequences examined. We proposed a model to illustrate the evolution and gene acquisition of the pPT23A plasmid family. To our knowledge, this is the first such attempt to conduct a global genetic analysis of this important plasmid family.

DNA Helicases↗
PubMed1999

Optical PCR: genomic analysis by long-range PCR and optical mapping.

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors.

DNA Restriction Enzymes↗
PubMed2005

Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells.

Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNA's side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.

Antiviral Agents↗
PubMed2006

A comprehensive proteomics and genomics analysis reveals novel transmembrane proteins in human platelets and mouse megakaryocytes including G6b-B, a novel immunoreceptor tyrosine-based inhibitory motif protein.

The platelet surface is poorly characterized due to the low abundance of many membrane proteins and the lack of specialist tools for their investigation. In this study we identified novel human platelet and mouse megakaryocyte membrane proteins using specialist proteomics and genomics approaches. Three separate methods were used to enrich platelet surface proteins prior to identification by liquid chromatography and tandem mass spectrometry: lectin affinity chromatography, biotin/NeutrAvidin affinity chromatography, and free flow electrophoresis. Many known, abundant platelet surface transmembrane proteins and several novel proteins were identified using each receptor enrichment strategy. In total, two or more unique peptides were identified for 46, 68, and 22 surface membrane, intracellular membrane, and membrane proteins of unknown subcellular localization, respectively. The majority of these were single transmembrane proteins. To complement the proteomics studies, we analyzed the transcriptome of a highly purified preparation of mature primary mouse megakaryocytes using serial analysis of gene expression in view of the increasing importance of mutant mouse models in establishing protein function in platelets. This approach identified all of the major classes of platelet transmembrane receptors, including multitransmembrane proteins. Strikingly 17 of the 25 most megakaryocyte-specific genes (relative to 30 other serial analysis of gene expression libraries) were transmembrane proteins, illustrating the unique nature of the megakaryocyte/platelet surface. The list of novel plasma membrane proteins identified using proteomics includes the immunoglobulin superfamily member G6b, which undergoes extensive alternate splicing. Specific antibodies were used to demonstrate expression of the G6b-B isoform, which contains an immunoreceptor tyrosine-based inhibition motif. G6b-B undergoes tyrosine phosphorylation and association with the SH2 domain-containing phosphatase, SHP-1, in stimulated platelets suggesting that it may play a novel role in limiting platelet activation.

Animals↗
PubMed1996

Whole genome analysis of hepatitis B virus from four cases of fulminant hepatitis: genetic variability and its potential role in disease pathogenicity.

The precore stop-codon variant of hepatitis B virus (HBV) has been associated with fulminant hepatitis but is also found in patients with persistent infection and chronic hepatitis. We have examined the possibility that the severe outcome of infection in patients with fulminant disease may be a result of additional genomic variation. We sequenced the entire HBV genome from three patients of Greek and one patient of Chinese origin with fulminant hepatitis, and from two patients with hepatitis B e antigen (HBeAg) positive chronic infection from the same regions, using direct sequencing of amplified viral DNA. Three of the fulminant cases were infected with the precore stop-codon variant HBeAg negative) and the fourth with the wild-type (HBeAg) positive virus. We compared sequences from our four fulminant isolates, and an additional fulminant isolate reported by others, with HBeAg positive carriers from the same regions and 12 published HBV genomes. There was a higher number of nucleotide and amino-acid substitutions throughout the HBV genome in the precore variant fulminant sequences than in the wild type. A cluster of mutations previously identified in the X region (126-132) in sequences reported in Japanese patients and encompassing the Enhancer II-Core Promoter region (1751-1768), were not found in our patients. We conclude that although there are no changes common to all sequences of HBV isolates from fulminant cases, some of these changes are in recognized cis-acting regulatory elements, whilst others are in the immediate vicinity of such elements. The effect of these mutations on viral genome transcription must now be determined.

Amino Acid Sequence↗
PubMed2001

Complete genome analysis of enterovirus 71 isolated from an outbreak in Taiwan and rapid identification of enterovirus 71 and coxsackievirus A16 by RT-PCR.

Hand, foot, and mouth disease (HFMD) and encephalomyelitis were two major clinical manifestations during the 1998 enterovirus 71 (EV71) epidemic in Taiwan. To investigate whether different clinical patterns were caused by alterations in EV71 genomes, the complete nucleotide sequences of four EV71 strains associated with HFMD or encephalomyelitis were compared. Among these viral strains, two or three nucleotide differences were found within the 5'-noncoding region, and two or four amino acid differences were found within the regions encoding viral polyproteins; however, none of these differences were correlated with either clinical manifestation. Because coxsackievirus A16 was another major causative agent of HFMD, a reverse transcription-polymerase chain reaction assay, with high sensitivity and specificity for identification of EV71 (both 100%) and coxsackievirus A16 (100 and 98.8%), was developed for the rapid differential identification of these two viruses in HFMD patients.

5' Untranslated Regions↗