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Results for “Complete genome characterization”

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PubMed2006

Toward a global picture of development: lessons from genome-scale analysis in Caenorhabditis elegans embryonic development.

Development is the result of complex events, including cascades of transcriptional programs and numerous molecular interactions. Traditionally, research focus has been given to the characterization of individual mutants, regulators, or interactions. With the availability of complete genome sequences and high-throughput (HT) experimental techniques, probing development on a system level has become feasible. Pioneering work initiated in invertebrate model systems such as Caenorhabditis elegans has provided first drafts of catalogs of essential components, transcriptional regulatory diagrams and molecular interaction networks underlying developmental processes. Integrating these drafts approximates a system-level picture of development and provides local models for protein/gene functions. Here we summarize the progress toward elucidating developmental processes on a system level, including the applications of genomic technologies and computational analyses. We discuss C. elegans embryonic development in case studies to illustrate how various HT approaches can be integrated and how biological insights can be gained from these approaches.

Animals↗

PubMed2003

Avermectin: biochemical and molecular basis of its biosynthesis and regulation.

Avermectin and its analogues, produced by Streptomyces avermitilis, are major commercial antiparasitic agents in the field of animal health, agriculture, and human infections. They are 16-membered pentacyclic lactone compounds derived from polyketide and linked to a disaccharide of the methylated deoxysugar l-oleandrose. Labeling studies, analyses of the biosynthetically blocked mutants, and the identification of the avermectin gene cluster allows characterization of most of the biosynthetic pathway. Recent completion of S. avermitilis genome sequencing is also expected to help in revealing the precise biosynthetic sequence and the complicated regulatory mechanism for avermectin biosynthesis, which has been long-awaited to be elucidated. The well characterized avermectin biosynthetic pathway and availability of S. avermitilis genome information in combination with the recent development of combinatorial biosynthesis should allow us to redesign more potent avermectin analogues and to engineer S. avermitilis as a more efficient host for the production of important commercial analogues.

Antiparasitic Agents↗

PubMedDate not supplied

Molecular characterization of a cloned dolphin mitochondrial genome.

DNA clones have been isolated that span the complete mitochondrial (mt) genome of the dolphin, Cephalorhynchus commersonii. Hybridization experiments with purified primate mtDNA probes have established that there is close resemblance in the general organization of the dolphin mt genome and the terrestrial mammalian mt genomes. Sequences covering 2381 bp of the dolphin mt genome from the major noncoding region, three tRNA genes, and parts of the genes encoding cytochrome b, NADH dehydrogenase subunit 3 (ND3), and 16S rRNA have been compared with corresponding regions from other mammalian genomes. There is a general tendency throughout the sequenced regions for greater similarity between dolphin and bovine mt genomes than between dolphin and rodent or human mt genomes.

Animals↗

PubMed1989

Characterization of the repetitive DNA elements in the genome of fish lymphocystis disease viruses.

The complete DNA nucleotide sequence of the repetitive DNA elements in the genome of fish lymphocystis disease virus (FLDV) isolated from two different species (flounder and dab) was determined. The size of these repetitive DNA elements was found to be 1413 bp which corresponds to the DNA sequences of the 5' terminus of the EcoRI DNA fragment B (0.034 to 0.052 m.u.) and to the EcoRI DNA fragment M (0.718 to 0.736 m.u.) of the FLDV genome causing lymphocystis disease in flounder and plaice. The degree of DNA nucleotide homology between both regions was found to be 99%. The repetitive DNA element in the genome of FLDV isolated from other fish species (dab) was identified and is located within the EcoRI DNA fragment B and J of the viral genome. The DNA nucleotide sequence of one duplicate of this repetition (EcoRI DNA fragment J) was determined (1410 bp) and compared to the DNA nucleotide sequences of the repetitive DNA elements of the genome of FLDV isolated from flounder. It was found that the repetitive DNA elements of the genome of FLDV derived from two different fish species are highly conserved and possess a degree of DNA sequence homology of 94%. The DNA sequences of each strand of the individual repetitive element possess one open reading frame.

Amino Acid Sequence↗

PubMed2000

Biological function made crystal clear - annotation of hypothetical proteins via structural genomics.

Many of the gene products of completely sequenced organisms are 'hypothetical' - they cannot be related to any previously characterized proteins - and so are of completely unknown function. Structural studies provide one means of obtaining functional information in these cases. A 'structural genomics' project has been initiated aimed at determining the structures of 50 hypothetical proteins from Haemophilus influenzae to gain an understanding of their function. Each stage of the project - target selection, protein production, crystallization, structure determination, and structure analysis - makes use of recent advances to streamline procedures. Early results from this and similar projects are encouraging in that some level of functional understanding can be deduced from experimentally solved structures.

Bacterial Proteins↗

PubMed2004

Evolutionary genomics of nuclear receptors: from twenty-five ancestral genes to derived endocrine systems.

Bilaterian animals are notably characterized by complex endocrine systems. The receptors for many steroids, retinoids, and other hormones belong to the superfamily of nuclear receptors, which are transcription factors regulating many aspects of development and homeostasis. Despite a diversity of regulatory mechanisms and physiological roles, nuclear receptors share a common protein organization. To obtain the broad picture of bilaterian nuclear hormone receptor evolution, we have characterized the complete set of nuclear receptor genes from nine animal genome sequences and analyzed it in a phylogenetic framework. In addition, expressed sequence tags from key lineages with no available genome sequence were also searched. This allows us to date the evolutionary events that led from an ancestral nuclear receptor gene, in an early metazoan, to present day diversity. We show that there were approximately 25 nuclear receptor genes in Urbilateria, the ancestor of bilaterians, at which point the fundamental diversity of the subfamily was already established. Surprisingly, differential gene loss played an important role in the evolution of different nuclear receptor sets in bilaterian lineages. The nuclear receptor distribution was also shaped by periods of gene duplication, essentially in vertebrates, as well as a lineage-specific duplication burst in nematodes. Our results imply that the genes for major receptors such as steroid receptors or thyroid hormone receptors were present in Urbilateria.

Animals↗

PubMed1995

Genomic organization of the neurofibromatosis 1 gene (NF1).

Neurofibromatosis 1 maps to chromosome band 17q11.2, and the NF1 locus has been partially characterized. Even though the full-length NF1 cDNA has been sequenced, the complete genomic structure of the NF1 gene has not been elucidated. The 5' end of NF1 is embedded in a CpG island containing a NotI restriction site, and the remainder of the gene lies in the adjacent 350-kb NotI fragment. In our efforts to develop a comprehensive screen for NF1 mutations, we have isolated genomic DNA clones that together harbor the entire NF1 cDNA sequence. We have identified all intron-exon boundaries of the coding region and established that it is composed of 59 exons. Furthermore, we have defined the 3'-untranslated region (3'-UTR) of the NF1 gene; it spans approximately 3.5 kb of genomic DNA sequence and is continuous with the stop codon. Oligonucleotide primer pairs synthesized from exon-flanking DNA sequences were used in the polymerase chain reaction with cloned, chromosome 17-specific genomic DNA as template to amplify NF1 exons 1 through 27b and the exon containing the 3'-UTR separately. This information should be useful for implementing a comprehensive NF1 mutation screen using genomic DNA as template.

Amino Acid Sequence↗

PubMed2002

Genetic characterization of wild-type genotype VII hepatitis A virus.

The complete genome sequence of the only identified genotype VII hepatitis A virus (HAV), strain SLF88, was obtained from PCR amplicons generated by a modified long PCR approach. There was 90% nucleotide identity in the 5' untranslated region compared to other known HAV sequences. In the remainder of the genome containing the long open reading frame, there was about 85% nucleotide identity to human HAV genotypes IA and IB and 80% identity to simian HAV genotype V. Compared to HAV strain HM-175, the capsid amino acids were highly conserved, with only four homologous amino acid changes, while an increasing number of amino acid differences was seen in the P2 and P3 genome regions. While nucleotide variability within the three functional coding regions did not differ, the P3D region was found to have the largest number of amino acid changes compared to HM-175.

Base Sequence↗

PubMed2005

L1 integration in a transgenic mouse model.

To study integration of the human LINE-1 retrotransposon (L1) in vivo, we developed a transgenic mouse model of L1 retrotransposition that displays de novo somatic L1 insertions at a high frequency, occasionally several insertions per mouse. We mapped 3' integration sites of 51 insertions by Thermal Asymmetric Interlaced PCR (TAIL-PCR). Analysis of integration locations revealed a broad genomic distribution with a modest preference for intergenic regions. We characterized the complete structures of 33 de novo retrotransposition events. Our results highlight the large number of highly truncated L1s, as over 52% (27/51) of total integrants were <1/3 the length of a full-length element. New integrants carry all structural characteristics typical of genomic L1s, including a number with inversions, deletions, and 5'-end microhomologies to the target DNA sequence. Notably, at least 13% (7/51) of all insertions contain a short stretch of extra nucleotides at their 5' end, which we postulate result from template-jumping by the L1-encoded reverse transcriptase. We propose a unified model of L1 integration that explains all of the characteristic features of L1 retrotransposition, such as 5' truncations, inversions, extra nucleotide additions, and 5' boundary and inversion point microhomologies.

Animals↗

PubMed2000

Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA cloned as a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant.

The complete genome of Marek's disease virus serotype 1 (MDV-1) strain 584Ap80C was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the U(S)2 locus of the MDV-1 genome by homologous recombination. Viral DNA containing the BAC vector was used to transform Escherichia coli strain DH10B, and several colonies harboring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identified. DNA from various MDV-1 BACs was transfected into chicken embryo fibroblasts, and from 3 days after transfection, infectious MDV-1 was obtained. Growth of MDV-1 recovered from BACs was indistinguishable from that of the parental virus, as assessed by plaque formation and determination of growth curves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB) were deleted by one-step mutagenesis using a linear DNA fragment amplified by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbored a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MDV-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-negative virus reported here represents the first MDV-1 mutant with a deletion of an essential gene and demonstrates the power and usefulness of BACs to analyze genes and gene products in slowly growing and strictly cell-associated herpesviruses.

Animals↗

PubMed1997

Genomic cloning of hGSTP1*C, an allelic human Pi class glutathione S-transferase gene variant and functional characterization of its retinoic acid response elements.

The complete hGSTP1*C, consisting of 7 exons and 6 introns contained in 3116 base pairs, was isolated from a cosmid genomic library of a glioblastoma multiforme cell line. Although the promoter of hGSTP1*C was identical to that of the previously reported GST-Pi gene, several of its structural features had not been previously described. These include several nucleotide transitions and transversions. Transitions of A --> G at +1404 and C --> T at +2294 in exons 5 and 6, respectively, changed codons Ile104 to Val104 and Ala113 to Val113. The gene also contained a guanine insertion at +51 in the insulin response element in intron 1 and six tandem repeats and one palindromic retinoic acid response element (RARE) consensus half-sites, A(G)GG(T)TC(G)A in intron 5. Retinoic acid (RA) treatment increased GST-Pi gene expression significantly in MGR3 cells. GST-Pi gene constructs with and without RARE deletion were used to show the RARE requirement for GST-Pi gene induction by RA. The isolation of the hGSTP1*C gene and the evidence that it contains functional RAREs should contribute to a better understanding of the molecular regulation of the GST-Pi gene in human cells.

Alleles↗

PubMed2003

Proteome analysis of the plant pathogen Xylella fastidiosa reveals major cellular and extracellular proteins and a peculiar codon bias distribution.

The bacteria Xylella fastidiosa is the causative agent of a number of economically important crop diseases, including citrus variegated chlorosis. Although its complete genome is already sequenced, X. fastidiosa is very poorly characterized by biochemical approaches at the protein level. In an initial effort to characterize protein expression in X. fastidiosa we used one- and two-dimensional gel electrophoresis and mass spectrometry to identify the products of 142 genes present in a whole cell extract and in an extracellular fraction of the citrus isolated strain 9a5c. Of particular interest for the study of pathogenesis are adhesion and secreted proteins. Homologs to proteins from three different adhesion systems (type IV fimbriae, mrk pili and hsf surface fibrils) were found to be coexpressed, the last two being detected only as multimeric complexes in the high molecular weight region of one-dimensional electrophoresis gels. Using a procedure to extract secreted proteins as well as proteins weakly attached to the cell surface we identified 30 different proteins including toxins, adhesion related proteins, antioxidant enzymes, different types of proteases and 16 hypothetical proteins. These data suggest that the intercellular space of X. fastidiosa colonies is a multifunctional microenvironment containing proteins related to in vivo bacterial survival and pathogenesis. A codon usage analysis of the most expressed proteins from the whole cell extract revealed a low biased distribution, which we propose is related to the slow growing nature of X. fastidiosa. A database of the X. fastidiosa proteome was developed and can be accessed via the internet (URL: www.proteome.ibi.unicamp.br).

Antioxidants↗

PubMed1995

Characterization of a YAC contig spanning the pseudoautosomal region.

Due to its unique biology of partial sex linkage and high recombination rates, the pseudoautosomal region (PAR1) on both X and Y chromosomes has attracted considerable interest. In addition, an extremely high level of YAC instability has been observed in this region. We have derived 82 YAC clones from six different YAC libraries mapping to this 2.6-Mb region. Of these a subset of 22 YACs was analyzed in detail. YAC contigs were assembled using 67 pseudoautosomal probes, of which 64 were unambiguously ordered. All markers are well distributed over the entire region, including the middle part of the region, which has previously been found difficult to contig. Two gaps of less than 50 kb within the genomic locus of CSF2RA and around XE7 remain, which could not be covered with YACs, cosmids, or phages. This YAC contig anchored on the physical map of PAR1 represents one of the best characterized large regions of the human genome with a map completion greater than 90% at 100-kb resolution and has permitted the accurate localization of all known genes within this region.

Chimera↗

PubMed1991

Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain Hällnäs B1 coding for the viral RNA polymerase.

The L RNA segment of the nephropathia epidemica virus (NEV) strain Hällnäs B1 was characterized by molecular cloning of the corresponding cDNA and subsequent determination of the DNA nucleotide sequence. The L RNA segment is 6550 nucleotides long with complementarity of 20 bases at the 3' and 5' termini. The viral messenger sense RNA contains one major open reading frame (ORF) with a coding capacity of 2156 amino acid residues encoding a protein with a calculated molecular weight of 246 kDa and an IEP of pH 7.4. Comparison of the deduced amino acid sequences from NEV hantavirus and Bunyamwera virus (BWV) L segment messenger sense RNAs, revealed a high degree of diversity (overall amino acid identity, 17%). However, three clusters of 30-40% amino acid identity were detected. One of these domains, containing an Asp-Asp motif found in many RNA polymerases, also shares amino acid sequence homology with the PB1 polymerase component of influenza type A. These results indicate that the L RNA segment of the NEV codes for the viral RNA-dependent RNA polymerase. The data presented here complete our previous studies on the characterization of the NEV genome by cDNA sequencing of the viral M and S RNA segments.

Amino Acid Sequence↗

PubMed2003

Nucleotide sequence and phylogenetic classification of candidate human papilloma virus type 92.

From a basal cell carcinoma (BCC) the complete genome of candidate human papillomavirus (HPV) type 92 was characterized. Phylogenetically, the candidate HPV 92 was relatively distantly related to other cutaneous HPV types within the B1 group. By quantitative real time PCR, 94 viral copies were present per cell in the BCC and another BCC contained 1 viral copy per cell. Lower copy numbers were found in two solar keratoses (1 copy per 33 cells and 1 copy per 60 cells) and two squamous cell carcinomas (1 copy per 436 cells and 1 copy per 1143 cells). The high viral load of HPV 92 in two BCCs differs from the low amount of HPV DNA reported from nonmelanoma skin cancers.

Basal Cell Carcinoma↗

PubMed2003

Evolution of Cryptosporidium parvum lactate dehydrogenase from malate dehydrogenase by a very recent event of gene duplication.

We have expressed the L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (malDH) genes from the apicomplexan Cryptosporidium parvum (CpLDH1 and CpMalDH1) as maltose-binding protein (MBP) fusion proteins in Escherichia coli. The substrate specificities, enzymatic kinetics, and oligomeric states of these two parasite enzymes have been characterized. By taking advantage of recently completed and ongoing apicomplexan genome sequencing projects, we identified additional MalDH genes from Plasmodium spp., Toxoplasma gondii, and Eimeria tenella that were previously unavailable. All apicomplexan MalDHs appeared to be cytosolic and no organellar homologs were identified from the completely sequenced P. falciparum genome and other ongoing apicomplexan genome-sequencing projects. Using these expanded apicomplexan LDH and MalDH sequence databases, we reexamined their phylogenetic relationships and reconfirmed their relationship to alpha-proteobacterial MalDHs. All LDH and MalDH enzymes from apicomplexans were monophyletic within the LDH-like MalDH group (i.e., MalDH resembling LDH) as a sister to alpha-proteobacterial MalDHs. All apicomplexan LDHs, with the exception of CpLDH1, formed a separate clade from their MalDH counterparts, indicating that these LDHs were evolved from an ancestral apicomplexan MalDH by a gene duplication coupled with functional conversion before the expansion of apicomplexans. Finally, CpLDH1 was consistently placed together with CpMalDH1 within the apicomplexan MalDH cluster, confirming an early working hypothesis that CpLDH1 was probably evolved from the same ancestor of CpMalDH1 by a very recent gene duplication that occurred after C. parvum diverged from other apicomplexans.

Amino Acid Sequence↗

PubMed1995

Characterization and genomic mapping of the ZNF80 locus: expression of this zinc-finger gene is driven by a solitary LTR of ERV9 endogenous retroviral family.

ERV9 is a low repeated family of human endogenous retroviral elements, which has close to 50 members, in addition to at least 4000 solitary LTRs. Previous work has shown that randomly selected LTRs can promote transcription of reporter genes, raising the possibility that these sequences may affect the expression of adjacent cellular genes. We describe here the structure of the ZNF80 cDNA clone putatively coding for a zinc-finger protein, whose 5' terminus starts from within an ERV9-LTR. Characterization of the single copy genomic locus indicates that a complete ERV9-LTR element is present upstream of the ZNF80 coding region and that this element acts as a functional promoter in both in vivo and in vitro experiments. A 2.6 kb long transcript is selectively expressed only in some hematopoietic cell lineages. Interestingly we mapped the ZNF80 locus to the 3q13.3 band, a region involved in karyotype rearrangements associated with myelocytic disorders. We have also analyzed the ZNF80 genomic organization in African green monkey and we show that this lower primate does not harbour an ERV9 element at this locus. Our findings strongly suggest that the expression of a zinc finger gene, which is highly conserved during evolution of primates, is regulated in humans by an LTR element of the ERV9 family.

Animals↗

PubMed2026

Genotype II Live-Attenuated ASFV Vaccine Bearing 24 Genes Deletion in 3 Independent Regions Is Able to Provide Complete Protection Against Homologous Lethal Challenge.

African swine fever (ASF) is an acute, febrile, and highly contagious infectious disease of swine with the etiological agent of African swine fever virus (ASFV). The mortality rate of virulent strains is as high as 100%. Strengthening biosafety is so far the most effective way to prevent and control ASF. Therefore, it is urgent to develop a safe and effective vaccine. In this study, a Genotype II live-attenuated ASF vaccine bearing 24 genes deletion in 3 independent regions was constructed based on the highly virulent Eurasian strain ASFV CN/GS 2018 backbone. The resulting mutant ASFV-Δ24 is characterized by complete deletion of 24 genes distributed in 3 genomic positions of 852 to 11 468, 19 732 to 22 929, and 179 519 to 180 617, among which MGF100 and whole MGF300 families are pioneeringly removed. The ASFV-Δ24 displayed a delayed and reduced replication kinetics as well as aberrant icosahedral empty particles devoid of a nucleoid when compared to the parental virus. Animal experiments showed that ASFV-Δ24 was completely attenuated in animals as evidenced by stable body temperature and no ASF-compatible clinical signs in vaccinated pigs. The ASFV-Δ24 could provide complete homologous protection against lethal challenge, as vaccinated pigs demonstrated boosted antibody response, transient but low levels of viremia in blood and virus titers in organs as well as almost undetectable viral shedding. Gene deletions in multiple regions are helpful for prevention of virulence reversion. These results indicate that ASFV-Δ24 can be used as an effective and promising candidate vaccine to control the spread of ASFV.

Animals↗