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At least 271 records · Page 15Linked to original sources

PubMed2005

Transcripts from a human primordial follicle cDNA library.

BACKGROUND: Human primordial follicles (PFs) or the oocyte-pre-granulosa complex, constitute the earliest and most immature stage of human oogenesis. The factors, signalling networks and the precise role of the oocyte and the pre-granulosa cells in initiating growth and recruitment from this finite resting pool remain largely unknown at present. METHODS: To obtain a gene resource of this oogenesis stage and thereby determine a molecular blueprint of the human PF, a cDNA library was constructed from 50 isolated human PFs using the phagemid vector pTriplEx2. RESULTS: Sequence analysis showed that 46.67% of these clones corresponded to known genes while 29.48% were uncharacterized genes that included hypothetical proteins, human cDNA clones and novel genes. Bioinformatics analysis revealed a preponderance of mitochondrial genes and repeat elements followed by ribosomal proteins, transcription and translation genes. Transcripts for heat shock proteins, cell cycle, embryogenesis genes and apoptosis genes were identified. Members of the ubiquitin-proteasome pathway, MAPK, p38/JNK, GPCR, Wnt, NF-kappaB and notch signalling pathways were identified. A mitochondrial pathway and a transcription factor pathway in the human PF were generated. The gene networks in the transcription factor pathway provided a first glimpse of the balance between proliferation and cell death/apoptosis in this earliest stage of oogenesis. CONCLUSIONS: The abundance and diversity of retroviral elements and transcriptional repressor genes in the human PF suggest these could contribute to the maintainance of this oogenesis stage. The role of these genes in initial recruitment and in subsequent oogenesis stages will be greatly facilitated and elucidated by printing a human PF cDNA array of the sequenced clones and using it for gene profiling.

Cell Differentiation↗

PubMed2005

[Analysis of secreted proteases of Trichophyton rubrum].

Secreted proteases are thought as potential virulent factors of Tricophyton rubrum. Based on cDNA libraries of 6 physiological phases of Tricophyton rubrum, 9683 unique ESTs were obtained by DNA sequencing. By bioinformatic analysis, 18 ESTs of putative secreted proteases were obtained from the established ESTs library, including 4 secreted peptidases, 1 secreted metalloprotease, 2 extracellular serine proteases, 1 secreted aspartic proteinase, 9 secreted subtilisin-like proteases and 1 vacuolar serine protease. These secreted proteases are related to nutrient uptake, lesion extension and host immune responses elicitation in the process of T. rubrum infection. These results provide a clue to further research on the infection and pathogencity of T. rubrum.

Amino Acid Sequence↗

PubMed2004

Diagnostic protein discovery using proteolytic peptide targeting and identification.

Plasma protein profiling with mass spectrometry is currently being evaluated as a diagnostic tool for cancer and other diseases. These experiments consist of three steps: plasma protein fractionation, analysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and comparisons of the MALDI profiles to develop diagnostic fingerprints using bioinformatic techniques. While preliminary results appear promising in small sample groups, the method is limited by the sensitivity of MALDI-MS for intact proteins, the limited mass range of MALDI-MS, and difficulties associated with isolating individual proteins for identification to validate the diagnostic fingerprint. Here we present an alternative and improved method directed toward diagnostic protein discovery, which incorporates proteolytic peptide profiling, bioinformatic targeting of ion signals, and MALDI tandem mass spectrometry (MS/MS) peptide sequencing, rather than fingerprinting. Pancreatic cancer patients, pancreatitis patients, and controls are used as the model system. Profiling peptides after enzymatic digestion improves sensitivity and extends the accessible protein molecular weight range when compared to intact protein profiling. The first step is to extract and fractionate the proteins from plasma. Each fraction is digested with trypsin and subsequently analyzed by MALDI-MS. Rather than using bioinformatic analysis as a pattern-matching technique, peptides are targeted based on the disease to control peak intensity ratios measured in the averages of all mass spectra in each group and t-tests of the intensity of each individual peak. The targeted peptide ion signals are subsequently identified using MALDI-MS/MS in quadrupole-TOF and tandem-TOF instruments. This study found not only the proteins targeted and identified by a previous protein profiling experiment, but also detected additional proteins. These initial results are consistent with the known biology of pancreatic cancer or pancreatitis, but are not specific to those diseases.

Adult↗

PubMed2006

A novel exon duplication event leading to a truncating germ-line mutation of the APC gene in a familial adenomatous polyposis family.

Familial Adenomatous Polyposis (FAP) is an autosomal dominant condition predisposing to multiple adenomatous polyps of the colon. FAP patients frequently carry heterozygous mutations of the APC tumour suppressor gene. Affected individuals from a cohort of FAP families (n=22), where no germ-line APC mutation was detected by direct sequencing, were analysed by Multiplex Ligation-dependent Probe Amplification (MLPA). MLPA identified a previously unreported APC mutation involving duplication of exon 4. Subsequent analysis of cDNA from affected family members revealed expression of mutant mRNA species containing two copies of exon 4, resulting in a frameshift and premature stop codon. Bioinformatic analysis of the relevant APC genomic segment predicted a role for homologous recombination possibly involving Alu repeats in the generation of this genotype. Our results highlight the importance of MLPA as an adjunct to exon-by-exon sequencing in identifying infrequent mutational events in cancer predisposing genes.

Adenomatous Polyposis Coli↗

PubMed2005

A functional single nucleotide polymorphism site detected in nasopharyngeal carcinoma-associated transforming gene Tx.

Tx is a transforming gene cloned from a nasopharyngeal carcinoma (NPC) cell line CNE2. Sequence analysis revealed that Tx encoded an aberrant immunoglobulin kappa light chain, which is abnormally expressed in epithelial tumor cells and plays an important role in nasopharyngeal carcinogenesis. Bioinformatic analysis confirmed the presence of a single nucleotide polymorphism (SNP) site in the Tx gene as matched to the Cancer Genome Anatomy Project SNP clusters database, which predicted 8 candidate SNP sites. Distribution of the confirmed SNP site in the genomes of healthy individuals and NPC patients was analyzed by denaturing high performance liquid chromatography. Heteroduplex genotype (GC/CG) occurred in NPC patients with a frequency significantly higher (52.44%) than that detected in healthy individuals (33.75%). In contrast, homoduplex genotype (GG) was less frequent in NPC patients (31.70%) than in normal individuals (56.25%), suggesting that heteroduplex genotype of Tx gene might be a risk factor for NPC.

Adolescent↗

PubMed2004

Genome-wide analysis of gene expression in neuroblastomas detected by mass screening.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and the third most common pediatric cancer. Although numerous factors including patient age, disease stage and genetic abnormalities have been shown to be predictive of outcome, the mechanisms responsible for the highly variable clinical behavior of this tumor remain largely unknown. In order to gain new insights into the biology of this tumor, we performed microarray analysis and compared the gene expression patterns of NB detected by mass screening, characterized by highly probable spontaneous regression, versus stage 4 NB with poor prognosis. The bioinformatics analysis revealed a set of 19 discriminatory genes that may play a significant role in the natural progression of the disease. Validation of these results and further mechanistic studies would shed new light on the biology of tumor progression, and provide new tools to predict clinical outcome in children with NB.

Child, Preschool↗

PubMed2025

GSK3B inhibition partially reverses brain ethanol-induced transcriptomic changes in C57BL/6J mice: Expression network co-analysis with human genome-wide association studies.

Alcohol use disorder (AUD) is a chronic behavioral disease with greater than 50% of its risk due to complex genetic contributions. Existing pharmacological and behavioral treatments for AUD are minimally effective and underutilized. Animal model behavioral genetics and human genome-wide association studies have begun to identify individual genes contributing to the progressive compulsive consumption of ethanol that occurs with AUD, promising possible new therapeutic targets. Our laboratory has previously identified Gsk3b as a central member in a network of ethanol-responsive genes in mouse prefrontal cortex, which altered ethanol consumption with genetic manipulation and was also significantly associated with risk for alcohol dependence in human genome-wide association studies. Here we perform detailed brain RNA sequencing transcriptomic studies to characterize a highly specific and clinically available GSK3B pharmacological inhibitor, tideglusib, as a possible therapeutic for clinical trials on treatment of AUD. A model of chronic intermittent ethanol consumption was used to study gene expression changes in prefrontal cortex and nucleus accumbens in the presence or absence of tideglusib treatment. Multivariate analysis of differentially expressed genes showed that tideglusib largely reversed ethanol- induced expression changes for two prominent clusters of genes in both prefrontal cortex and nucleus accumbens. Bioinformatic analysis showed these genes to have prominent roles in neuronal functioning and synaptic activity. Additionally, mouse brain differential gene expression data was analyzed together with human protein-protein interaction and genome-wide association studies on AUD to derive networks responding to tideglusib and relevant to human genetic risk for alcohol dependence. These studies identified discrete networks significantly enriched with genes provisionally associated with AUD, and provide key information on central hubs of such networks. Together these studies document tideglusib as a major modulator of chronic ethanol consumption-evoked brain gene expression signatures, and identify possible new targets for therapeutic modulation of AUD.

Journal Article↗

PubMed2003

A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation.

A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

Adaptation, Physiological↗

PubMed2006

Analysis of expressed sequence tags from skeletal muscle-specific cDNA library of Chinese native Xiang pig.

A Longissimus Dorsi muscle cDNA library of Xiang Pig was constructed, and 131 randomly isolated clones were sequenced in this study. The results of bioinformatics analysis showed that 131 ESTs represented 109 unique clones sequences, of which 99 showed homology to previously identified genes in humans or other mammals, 3 matched other uncharacterized expressed sequence tags (ESTs), and 7 showed no significant matches to sequences already present in DNA databases. No protein matches were found for 10 ESTs. Functional analysis of the ESTs showed that a considerable proportion of them encoded proteins involved in gene/protein expression (45.46%). Other classes included genes involved in metabolism (10.10%), cell structure/motility (10.10%), cell/organism defense (5.05%), cell signaling/communication (2.02%), and cell division (0.0%). Unclassified genes constituted the remaining 27.27%. This study reported the results of the first gene expression profile analysis of Chinese native Xiang Pig skeletal muscle cells, thereby greatly facilitating the functional study of candidate genes involved in muscle growth as well as in the improvement of meat quality in domestic pigs.

Animals↗

PubMed2025

Bioinformatics for the Structural Genomics of Poxviruses.

Poxviruses are large, complex viruses, and their host species are widespread across the tree of life. As a result, the bioinformatics analysis of their genomes can be complex. Here we show how a few helpful tools and strategies can be used to inform the analysis, leading to a better understanding of the structural properties of poxvirus genomes and to a more accurate quality control of, or comparison between, assembled sequences.

Poxviridae↗

PubMed2005

Integration specificity of phage phiC31 integrase in the human genome.

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.

Animals↗

PubMed2002

Cloning and characterization of a novel human pantothenate kinase gene.

Pantothenate kinase (PanK) is a key regulatory enzyme in the CoA biosynthetic pathway in bacteria and mammalian cells. It catalyzes the first committed step in the universal biosynthetic pathway leading to CoA. Here we report the molecular cloning and characterization of a new human PanK gene. The gene encoded a protein of 314 amino acid residues, which share high homology to mouse pantothenate kinase (mPanK) 1 beta. Northern blot analysis revealed a transcript of the gene of 2.6 kb in human liver and kidney. The human PanK gene was located to human chromosome between 10q22.3 and 10q23.2 by bioinformatics analysis.

Amino Acid Sequence↗

PubMed2004

[Toxicoproteomics: first experiences in a BMBF-study].

The rapid development of molecular toxicology is providing innovative approaches to an improved investigation and recognition of toxic substances. Proteome analysis offers, with 2DE/MS (two-dimensional gel electrophoresis and mass spectrometry) and SELDI (surface enhanced laser desorption/ionisation), a promising discipline to classify molecular changes caused by toxic exposure. The Rat Liver Foci Bioassay (RLFB) is a detailed, well-described model for the investigation of liver carcinogenesis induced by chemical substances. Based on this model, we examined whether proteomic methods of molecular toxicology can be used for the early recognition of toxic and/or carcinogenic characteristics of toxic substances. In addition, identification and subsequent prevalidation of new hepatocellular biomarkers was performed, enabling better prediction of toxic and/or carcinogenic effects. This could lead to a more meaningful RLFB and thus to an improved risk assessment of chemicals. 2DE analysis in this study showed that deregulated proteins are assigned to mainly anabolic and catabolic metabolism pathways in the cell. Beyond this, individual proteins were identified which play a key role in the carcinogenic process. A comparison of the differentially expressed proteins in tissue from tumour-bearing animals and tissue derived from the start of the study revealed that protein expression changes (biomarkers) were already detectable shortly after exposure. In addition, analysis by SELDI clearly showed several differentially expressed proteins and/or derived masses. The spectra represented specific differences in tissues, which could be assigned to the same histopathological endpoints. With bioinformatics analysis it was possible to identify individual discriminating mass peaks, which were indicative of tumour formation. Group specific changes can be illustrated and/or represented in more detail with further cluster analysis methods. These results give hope for an improved prediction of hepatotoxicity and carcinogenicity by means of protein markers, which could in the future lead to a shortening of carcinogenicity studies and to a reduction in the use of experimental animals.

Animals↗

PubMed2004

[Molecular cloning for an alternatively splicing cDNA of human PKNOX1 gene and it's expression analysis].

Down's syndrome (DS) is the most common chromosomal abnormality in live born infants. The cloning and characterization of genes on HC21 are necessary steps for the understanding of the molecular basis of Down's syndrome. To search for new Down's syndrome related genes, we have used bioinformatics analysis and rapid amplification of cDNA ends to identify an alternatively splicing cDNA of human PKNOX1 in Down's syndrome critical region on the long arm of human chromosome 21. The alternatively splicing full length cDNA of human PKNOX1, named PKNOX1B, is 2 793 bp in length and encodes a 405 amino acid protein. Bioinformatical studies show PKNOX1B gene spans over 58 kb on chromosome 21q22.3 and contains 11 exons and 10 introns. The predicted molecule weight of PKNOX1B is 44.628 kDa, and the deduced iso-electric point is 6.28. Compared with PKNOX1 gene, PKNOX1B is 30 aa shorter at the C terminus than PKNOX1 owing to alternative splice between (10th exon and 11th exon) last two exons. PKNOX1B posses a same homeobox-domain as PKNOX1, so the isoform of PKNOX1 may be involved in the genetic control of development like other members of homeobox-containing gene family. RT-PCR results showed that PKNOX1B is expressed in all examined tissues except in marrow tissue. By Northern blot, PKNOX1 has three transcripts in adult testis, one about 5 kb, second about 2.9 kb, and third about 2 kb. The two larger transcript is expressed in all examined tissues, but the smallest transcript is only expressed actively in adult testis, so that it may play potential roles in spermatogenesis.

Alternative Splicing↗

PubMed2005

Electron cryomicroscopy of biological machines at subnanometer resolution.

Advances in electron cryomicroscopy (cryo-EM) have made possible the structural determination of large biological machines in the resolution range of 6-9 angstroms. Rice dwarf virus and the acrosomal bundle represent two distinct types of machines amenable to cryo-EM investigations at subnanometer resolutions. However, calculating the density map is only the first step, and much analysis remains to extract structural insights and the mechanism of action in these machines. This paper will review the computational and visualization methodologies necessary for analysis (structure mining) of the computed cryo-EM maps of these machines. These steps include component segmentation, averaging based on local symmetry among components, density connectivity trace, incorporation of bioinformatics analysis, and fitting of high-resolution component data, if available. The consequences of these analyses can not only identify accurately some of the secondary structure elements of the molecular components in machines but also suggest structural mechanisms related to their biological functions.

Capsid Proteins↗

PubMed2002

Genetic determinants of coxsackievirus B3 pathogenesis.

The development of high throughput genomic and bioinformatic analysis tools, coupled with established molecular techniques, has allowed new insights into the pathogenesis of infectious diseases. In humans, coxasackievirus B3 (CVB3) is the primary etiological agent of viral myocarditis, an inflammatory disease process involving the heart muscle. Early host cellular survival and apoptotic mechanisms during viral infections, as well as immune events, affect myocarditis progression and outcome. Therefore, our laboratory has been keenly interested in infectomics, defined here as the transcriptional events of both virus and host. We first elucidated up- or downregulated transcriptional activities in CVB3-infected hearts by mRNA differential display. Further characterization of these regulated genes including Nip21, IP10, and IGTPase, and study of their role in CVB3-infection are underway. In further dissection of the stages of myocarditis-peak viremia, inflammatory infiltration and tissue repair-we used cDNA microarrays to probe differential gene expression in the myocardium following virus infection. Following virus infection, there are global decreases in metabolic and mitochondrial genes, increases in signaling genes and distinctive patterns in other functional groups. To establish early gene expression profiles in infected cells by themselves, we also used oligonucleotide arrays in an in vitro model of CVB3 infection. Notably, we have found increased expression of transcription factors c-fos and c-jun down-stream of extracellular signal-related kinase, a pathway which is crucial for virus replication and pathogenesis. Our investigations based on gene profiling following CVB3 infection have thus far been fruitful in providing new experimental leads. High throughput genetic analysis has allowed us to simultaneously try on greater than 12,000 potential genetic "glass slippers." Our in vitro experimental plan has enabled us to chart prominent patterns of gene expression, analyzed by novel bioinformatic approaches, and to separate varied and potentially significant gene expression events.

Computational Biology↗

PubMed2001

Functional Analysis of six novel ORFs on the left arm of Chromosome XII of Saccharomyces cerevisiae reveals three of them responding to S-starvation.

Six novel Open Reading Frames (ORFs) located on the left arm of the chromosome XII (YLL061w, YLL060c, YLL059c, YLL058w, YLL057c and YLL056c) have been analysed using either short-flanking homology (SFH) or long-flanking homology (LFH) gene replacement. Sporulation and tetrad analysis showed none of these ORFs to be essential for vegetative growth. The standard EUROFAN growth tests failed to reveal any obvious phenotypes resulting from deletion of each of the ORFs. Bioinformatic analysis revealed that YLL061w is probably an amino acid permease for S-methylmethionine and that YLL060c encodes a glutathione transferase which is involved in cellular detoxification, while YLL058w may play a role in sulphur-containing amino-acid metabolism, YLL057c in sulphonate catabolism and YLL056c in stress response. The transcription of three ORFs (YLL061w, YLL057c and YLL056c) has been shown to increase more than 10-fold under sulphate starvation. Replacement cassettes, comprising the kanMX marker flanked by each ORF's promoter and terminator regions, were cloned into pUG7. All the cognate clones, were generated using direct PCR products amplified from genomic DNA or using gap-repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).

Amino Acid Transport Systems↗

PubMed2002

Genomic and proteomic analysis of the myeloid differentiation program: global analysis of gene expression during induced differentiation in the MPRO cell line.

We have used an approach using 2-dimensional gel electrophoresis with mass spectrometry analysis combined with oligonucleotide chip hybridization for a comprehensive and quantitative study of the temporal patterns of protein and mRNA expression during myeloid development in the MPRO murine cell line. This global analysis detected 123 known proteins and 29 "new" proteins out of 220 protein spots identified by tandem mass spectroscopy, including proteins in 12 functional categories such as transcription factors and cytokines. Bioinformatic analysis of these proteins revealed clusters with functional importance to myeloid differentiation. Previous analyses have found that for a substantial number of genes the absolute amount of protein in the cell is not strongly correlated to the amount of mRNA. These conclusions were based on simultaneous measurement of mRNA and protein at just a single time point. Here, however, we are able to investigate the relationship between mRNA and protein in terms of simultaneous changes in their levels over multiple time points. This is the first time such a relationship has been studied, and we find that it gives a much stronger correlation, consistent with the hypothesis that a substantial proportion of protein change is a consequence of changed mRNA levels, rather than posttranscriptional effects. Cycloheximide inhibition also showed that most of the proteins detected by gel electrophoresis were relatively stable. Specific investigation of transcription factor mRNA representation showed considerable similarity to those of mature human neutrophils and highlighted several transcription factors and other functional nuclear proteins whose mRNA levels change prominently during MPRO differentiation but which have not been investigated previously in the context of myeloid development. Data are available online at http://bioinfo.mbb.yale.edu/expression/myelopoiesis.

Animals↗