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PubMed1997

Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase.

The tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro, 460 amino acid residues) is a multifunctional protein involved in aphid-mediated transmission, genome amplification, polyprotein processing, and long-distance movement. To investigate the interrelationships between three of these functions, 25 alanine-scanning mutations affecting clusters of charged residues were introduced into the HC-Pro coding sequence. The resulting mutants were analyzed with respect to HC-Pro proteolytic activity in vitro, genome amplification in protoplasts, and long-distance movement in tobacco plants. Three classes of mutants were identified. Class I mutants (total of 17) were capable of genome amplification, long-distance movement, and HC-Pro proteolysis with efficiencies similar to parental virus. The class III mutant (total of 1) encoded a proteolytically debilitated HC-Pro and was replication-defective. Class II mutants (total of 7) encoded proteolytically active HC-Pro, but each exhibited a suppressed amplification phenotype that was characterized by a progressive shutoff during the course of infection in protoplasts. The class II mutants also exhibited defects in long-distance movement, accumulating to relative levels of 0 to 7.5% in noninoculated tissue. Wild-type HC-Pro supplied in trans was able to partially rescue the class II mutant amplification defects in protoplasts and long-distance movement defects in plants, although the extent of complementation of movement function varied for each mutant. Six of the seven class II mutations affected the central region of HC-Pro between residues 126 and 300, whereas only one affected the C-terminal proteolytic domain. These results indicate that the central region of HC-Pro is necessary for efficient genome amplification and long-distance movement, and that the one or more HC-Pro functions involved in these processes is at least partially trans-active. Additionally, the long-distance movement properties of a previously characterized HC-Pro-defective mutant (TEV-GUS/CCCE) were characterized further using grafted nontransgenic and HC-Pro-expressing transgenic plants. The results indicated that HC-Pro is required in both inoculated and noninoculated tissues to complement the TEV-GUS/CCCE movement defects.

Alanine↗

PubMedDate not supplied

[cDNA-microarrays in cartilage research - functional genomics of osteoarthritis].

Functional genomics represents a new challenging approach in order to analyze complex diseases such as osteoarthritis on a molecular level. The characterization of the molecular changes of the cartilage cells, the chondrocytes, enables a better understanding of the pathomechanisms of the disease. In particular, the identification and characterization of new target molecules for therapeutic intervention is of interest. Also, potential molecular markers for diagnosis and monitoring of osteoarthritis contribute to a more appropriate patient management. The DNA-microarray technology complements (but does not replace) biochemical and biological research in new disease-relevant genes. Large-scale functional genomics will identify molecular networks such as yet identified players in the anabolic-catabolic balance of articular cartilage as well as disease-relevant intracellular signaling cascades so far rather unknown in articular chondrocytes. However, at the moment it is also important to recognize the limitations of the microarray technology in order to avoid over-interpretation of the results. This might lead to misleading results and prevent to a significant extent a proper use of the potential of this technology in the field of osteoarthritis.

Cartilage↗

PubMed1993

Gene cluster involved in melanin biosynthesis of the filamentous fungus Alternaria alternata.

The filamentous fungus Alternaria alternata produces melanin, a black pigment, from acetate via 1,8-dihydroxynaphthalene. To isolate a fungal gene required for melanin biosynthesis, we transformed an A. alternata Brm1- (light brown) mutant with the DNA of a wild-type strain genomic library constructed by use of a cosmid carrying the hygromycin B phosphotransferase gene. When hygromycin B-resistant transformants were screened for melanin production, 1 of 1,363 transformants appeared to regain melanin production, as judged by black pigmentation of the cultured mycelia. The cosmid, named pMBR1, was recovered by packaging nuclear DNA of the melanin-producing transformant into lambda phage. The gene on pMBR1 that enables the Brm1- mutant to produce melanin was designated BRM1. In addition to the BRM1 gene, pMBR1 was found to carry two more genes involved in melanin biosynthesis. These two genes, designated ALM and BRM2, transformed A. alternata Alm- (albino) and Brm2- (brown) mutants, respectively, to the wild-type phenotype. The three genes are located within a ca. 30-kb genomic region in the order ALM-BRM1-BRM2. Analysis of the gene transcripts indicated approximate sizes of 7.2, 4.0, and 0.9 kb for ALM, BRM1, and BRM2, respectively. The BRM1 and BRM2 transcripts are generated from the same strand, but the ALM transcript is generated from the opposite strand. The three mRNA species accumulate in cultured mycelia of the wild-type strain synchronously with mycelial melanization. The essential roles of the three genes in melanin biosynthesis were confirmed by transformation-mediated gene disruption experiments.

Alternaria↗

PubMed2001

Using in silico transcriptomics to search for tumor-associated antigens for immunotherapy.

Immunotherapy approaches to fight cancer are based on the principle of mounting an immune response against a self-antigen expressed by the tumor cells. In order to reduce potential autoimmunity side-effects, the antigens used should be as tumor-specific as possible. A complementary approach to experimental tumor antigen discovery is to screen the human genome in silico, particularly the databases of "Expressed Sequence Tags" (ESTs), in search of tumor-specific and tumor-associated antigens. The public databases currently provide a massive amount of ESTs from several hundreds of cDNA tissue libraries, including tumoral tissues from various types. We describe a novel method of EST database screening that allows new potential tumor-associated genes to be efficiently selected. The resulting list of candidates is enriched in known genes, described as being expressed in tumor cells.

Antigens, Neoplasm↗

PubMed2002

A similarity-based method for genome-wide prediction of disease-relevant human genes.

MOTIVATION: A method for prediction of disease relevant human genes from the phenotypic appearance of a query disease is presented. Diseases of known genetic origin are clustered according to their phenotypic similarity. Each cluster entry consists of a disease and its underlying disease gene. Potential disease genes from the human genome are scored by their functional similarity to known disease genes in these clusters, which are phenotypically similar to the query disease. RESULTS: For assessment of the approach, a leave-one-out cross-validation of 878 diseases from the OMIM database, using 10672 candidate genes from the human genome, is performed. Depending on the applied parameters, in roughly one-third of cases the true solution is contained within the top scoring 3% of predictions and in two-third of cases the true solution is contained within the top scoring 15% of predictions. The prediction results can either be used to identify target genes, when searching for a mutation in monogenic diseases or for selection of loci in genotyping experiments in genetically complex diseases.

Algorithms↗

PubMed2003

Structural equation model-based genome scan for the metabolic syndrome.

BACKGROUND: The metabolic syndrome is characterized by the clustering of several traits, including obesity, hypertension, decreased levels of HDL cholesterol, and increased levels of glucose and triglycerides. Because these traits cluster, there are likely common genetic factors involved. RESULTS: We used a multivariate structural equation model (SEM) approach to scan the genome for loci involved in the metabolic syndrome. We found moderate evidence for linkage on chromosomes 2, 3, 11, 13, and 15, and these loci appear to have different relative effects on the component traits of the metabolic syndrome. CONCLUSION: Our results suggest that the metabolic syndrome components, diabetes, obesity, and hypertension, are under the pleiotropic control of several loci.

Chromosomes, Human, Pair 11↗

PubMed1996

Construction and screening of subgenomic library of chickpea (Cicer arietinum L.) for legumin genes and their analysis.

Legumin storage protein genes of chickpea were identified by hybridizing restricted chickpea genomic DNA transferred on Southern blot with heterologous pea legumin cDNA probes. Southern hybridization of restricted chickpea genomic DNA with pea legumin pDUB6 and pDUB8 cDNA probes indicated presence of pea legumin homologous sequences in chickpea. About 4-5 kb EcoRI fragment was hybridized with both the probes used. DNA corresponding to this size was isolated from the gel and used for construction of subgenomic library in lambda ZAPII vector. After amplification, library was screened for presence of legumin genes in chickpea. Out of clones screened (0.1 x 10(6)), three positive clones were obtained. Restriction analysis and hybridization studies showed presence of complete structural gene in each of the identified positive clones.

Fabaceae↗

PubMed2000

Mutation screening of human 5-HT(2B)receptor gene in early-onset obsessive-compulsive disorder.

The serotonin receptor 2B gene (HTR2B; MIM 601122) is a pharmacological and positional candidate gene in early-onset obsessive-compulsive disorder. Sequences of a putative promoter region and splice regions were first elucidated, then sequenced along with HTR2B coding regions. Probands from seven families included in a previous genome scan in which one of the strongest linkage findings was to a region including HTR2B, along with two genomic DNA pools of 10 unrelated control subjects and 10 unrelated autism probands were screened. One single nucleotide polymorphism was found in intron 1, that may be useful as a marker in genetic linkage and association studies. It does not appear likely to affect splicing. No evidence for functional mutation was found in the sequenced regions of HTR2B.

5' Untranslated Regions↗

PubMed2025

Operationalizing the Wilson-Jungner principles for the genomics era: Consensus recommendations from the International Consortium on Newborn Sequencing.

PURPOSE: For decades, the selection of disorders included in newborn screening (NBS) programs has been guided by principles published by Wilson and Jungner in 1968. As research explores the expansion of conditions included in NBS through genomic sequencing, there is a critical need for updated recommendations to address the opportunities and complexities of genomic data. METHODS: The International Consortium on Newborn Sequencing includes leaders from over 16 research projects investigating genomic NBS across the United Kingdom, Europe, United States, and Oceania. Consortium members were invited to participate in a modified Delphi study, aggregating opinion on the selection of conditions for genomic NBS through 3 rounds of online questionnaires, with feedback provided to participants between rounds. RESULTS: In round 1, 94 participants completed the questionnaire, and 10 of 43 statements reached consensus. In round 2, 81 participants completed the questionnaire, and 14 of 27 statements reached consensus. In round 3, 68 participants completed the questionnaire, and all 10 statements reached 72% or more consensus. CONCLUSION: The 10 consensus recommendations developed in this study can guide future research and public health programs performing genomic NBS. This process also identified key areas of participant discordance, highlighting important topics for future research.

Humans↗

PubMed1997

A short linear sequence in the pre-S domain of the large hepatitis B virus envelope protein required for virion formation.

Envelopment of the hepatitis B virus (HBV) nucleocapsid depends on the large envelope protein L, which is expressed as a transmembrane polypeptide at the endoplasmic reticulum membrane. Previous studies demonstrated that the cytosolic exposure of the N-terminal pre-S domain (174 amino acids) of L was required for virion formation. N-terminal truncations of L up to Arg 103 were tolerated. To map sites in the remaining C-terminal part of pre-S important for virion morphogenesis, a series of 11 L mutants with linker substitutions between Asn 98 and Pro 171 was generated. The mutants formed stable proteins and were secreted in transfected cell cultures, probably as components of subviral hepatitis B surface antigen particles. All four constructs with mutations between Asn 98 and Thr 125 were unable to complement in trans the block in virion formation of an L-negative HBV genome in cotransfected HuH7 cells. These mutants had a transdominant negative effect on virus yield in cotransfections with the wild-type HBV genome. In contrast, all seven mutants with substitutions downstream of Ser 124 were able to envelop the nucleocapsid and to secrete HBV. The sequence between Arg 103 and Ser 124 is highly conserved among different HBV isolates and also between HBV and the woodchuck hepatitis virus. Point mutations in this region introducing alanine residues at conserved positions blocked virion formation, in contrast to mutations at nonconserved residues. These results demonstrate that the pre-S sequence between Arg 103 and Ser 124 has an important function in HBV morphogenesis.

Alanine↗

PubMed2004

Theoretical aspects of genomic variation screening using DNA microarrays.

We present a theoretical model for typical microarray-based single nucleotide polymorphism (SNP) assay of small genomic DNA amount. We derived the adsorption isotherm expressing the on-array hybridization efficiency in terms of genomic target sequence and concentration, oligonucleotide probe sequence and surface density, hybridization buffer, and temperature. This isotherm correctly describes the surface probe density effects, the sensitivity peak, and the melting temperature depression, and is in accord with published experiments. We discuss optimization of parallel SNP genotyping. Our estimates show that SNP detection at a single temperature in aqueous hybridization buffer is restricted by DNA regions that differ by less than 20% in GC content. We predict that the variety of genotyped SNPs could be substantially extended using an assay design with high probe density and a large fraction of probes hybridized.

Genetic Testing↗

PubMed2005

Genome-wide screening using array-CGH does not reveal microdeletions/microduplications in children with Kabuki syndrome.

Kabuki syndrome (KS) is a rare multiple congenital anomaly/mental retardation syndrome. It is characterized by a distinct facial appearance, mental retardation, postnatal growth retardation, skeletal anomalies, unusual dermatoglyphics and fetal fingertip pads. It has previously been speculated that KS is caused by a microdeletion or duplication. In a recent report, an interstitial microduplication of 8p22-23.1 was presented in several cases with this disorder. We investigated 10 Caucasian patients diagnosed with KS by fluorescence in situ hybridization and microsatellite markers located on 8p22-23.1. Using the same clones that were previously reported to be duplicated on chromosome 8p, we could exclude the duplication in all our patients. In addition, we performed a genome-wide screening on this group of patients using array-based comparative genomic hybridization containing BAC clones spaced at approximately 1 Mb intervals across the genome and could not find any evidence for gene dose alterations. The characteristics of KS are variable, a fact that complicates the diagnosis of this disorder. It is possible that we will find genetic heterogeneity among Kabuki patients, and therefore it is unlikely that all patients have an interstitial 8p duplication. We conclude that the etiology of KS remains to be solved and further genetic studies are necessary to delineate its genetic cause.

Adolescent↗

PubMed1998

Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs.

We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number and size of the porcine chromosome fragments retained in each hybrid clone were checked by fluorescence in situ hybridization with a SINE probe or by primed in situ labeling (PRINS) with SINE-specific primers. A strategy based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) was developed for selected clones to determine if the large fragments painted by the SINE probe corresponded to one pig chromosome or to different fragments of several chromosomes. This strategy was buttressed by a double PRINS approach using primers specific for alpha-satellite sequences of two different groups of swine chromosomes. Genome retention frequency was estimated for each clone by PCR with 32 markers localized on different porcine chromosomes. Of the 152 hybrids produced, 126 were selected on the basis of cytogenetic content and chromosome retention frequency to construct a radiation hybrid map of swine chromosome 8. Our initial results for this chromosome indicate that the resolution of the radiation hybrid map is 18 times higher than that obtained by linkage analysis.

Animals↗

PubMed2006

Identification of ciliary and ciliopathy genes in Caenorhabditis elegans through comparative genomics.

BACKGROUND: The recent availability of genome sequences of multiple related Caenorhabditis species has made it possible to identify, using comparative genomics, similarly transcribed genes in Caenorhabditis elegans and its sister species. Taking this approach, we have identified numerous novel ciliary genes in C. elegans, some of which may be orthologs of unidentified human ciliopathy genes. RESULTS: By screening for genes possessing canonical X-box sequences in promoters of three Caenorhabditis species, namely C. elegans, C. briggsae and C. remanei, we identified 93 genes (including known X-box regulated genes) that encode putative components of ciliated neurons in C. elegans and are subject to the same regulatory control. For many of these genes, restricted anatomical expression in ciliated cells was confirmed, and control of transcription by the ciliogenic DAF-19 RFX transcription factor was demonstrated by comparative transcriptional profiling of different tissue types and of daf-19(+) and daf-19(-) animals. Finally, we demonstrate that the dye-filling defect of dyf-5(mn400) animals, which is indicative of compromised exposure of cilia to the environment, is caused by a nonsense mutation in the serine/threonine protein kinase gene M04C9.5. CONCLUSION: Our comparative genomics-based predictions may be useful for identifying genes involved in human ciliopathies, including Bardet-Biedl Syndrome (BBS), since the C. elegans orthologs of known human BBS genes contain X-box motifs and are required for normal dye filling in C. elegans ciliated neurons.

Animals↗

PubMed1999

Using recursive partitioning for exploration and follow-up of linkage and association analyses.

We first conducted a genome-wide screen for association with discordant sibships using the multi allelic and diallelic SDT. Markers at D4S1628, D8S1109, D9S66 and D7S1797 showed multi-allelic association. Deleterious diallelic association was found for markers at D1S1613, D1S534, D3S2459, D7S1817, and D9S131. Protective association was found at markers D8S1109, D8S1136, and D9S66. We then incorporated these findings with previous linkage findings in the exploration of oligogenes and epistasis using recursive partitioning. We conclude that recursive partitioning can be a useful adjunct to traditional linkage and association analyses in the exploration of these effects.

Alcoholism↗

PubMed2003

Production of Bacillus anthracis protective antigen is dependent on the extracellular chaperone, PrsA.

Protective antigen (PA) is a component of the Bacillus anthracis lethal and edema toxins and the basis of the current anthrax vaccine. In its heptameric form, PA targets host cells and internalizes the enzymatically active components of the toxins, namely lethal and edema factors. PA and other toxin components are secreted from B. anthracis using the Sec-dependent secretion pathway. This requires them to be translocated across the cytoplasmic membrane in an unfolded state and then to be folded into their native configurations on the trans side of the membrane, prior to their release from the environment of the cell wall. In this study we show that recombinant PA (rPA) requires the extracellular chaperone PrsA for efficient folding when produced in the heterologous host, B. subtilis; increasing the concentration of PrsA leads to an increase in rPA production. To determine the likelihood of PrsA being required for PA production in its native host, we have analyzed the B. anthracis genome sequence for the presence of genes encoding homologues of B. subtilis PrsA. We identified three putative B. anthracis PrsA proteins (PrsAA, PrsAB, and PrsAC) that are able to complement the activity of B. subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be PrsA-dependent.

Amino Acid Sequence↗

PubMed2001

Mutational analysis of the proteinase function of Potato leafroll virus.

cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513-515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.

Amino Acid Motifs↗

PubMed2005

A snapshot of viral evolution from genome analysis of the tectiviridae family.

The origin, evolution and relationships of viruses are all fascinating topics. Current thinking in these areas is strongly influenced by the tailed double-stranded (ds) DNA bacteriophages. These viruses have mosaic genomes produced by genetic exchange and so new natural isolates are quite dissimilar to each other, and to laboratory strains. Consequently, they are not amenable to study by current tools for phylogenetic analysis. Less attention has been paid to the Tectiviridae family, which embraces icosahedral dsDNA bacterial viruses with an internal lipid membrane. It includes viruses, such as PRD1, that infect Gram-negative bacteria, as well as viruses like Bam35 with Gram-positive hosts. Although PRD1 and Bam35 have closely related virion morphology and genome organization, they have no detectable sequence similarity. There is strong evidence that the Bam35 coat protein has the "double-barrel trimer" arrangement of PRD1 that was first observed in adenovirus and is predicted to occur in other viruses with large facets. It is very likely that a single ancestral virus gave rise to this very large group of viruses. The unprecedented degree of conservation recently observed for two Bam35-like tectiviruses made it important to investigate those infecting Gram-negative bacteria. The DNA sequences for six PRD1-like isolates (PRD1, PR3, PR4, PR5, L17, PR772) have now been determined. Remarkably, these bacteriophages, isolated at distinctly different dates and global locations, have almost identical genomes. The discovery of almost invariant genomes for the two main Tectiviridae groups contrasts sharply with the situation in the tailed dsDNA bacteriophages. Notably, it permits a sequence analysis of the isolates revealing that the tectiviral proteins can be dissected into a slowly evolving group descended from the ancestor, the viral self, and a more rapidly changing group reflecting interactions with the host.

Amino Acid Sequence↗