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At least 631 records · Page 35Linked to original sources
PubMed2001

Human sulfotransferase SULT1C1 pharmacogenetics: gene resequencing and functional genomic studies.

Sulfotransferase (SULT) enzymes catalyze an important phase II reaction in the biotransformation of many drugs and other xenobiotics. We previously cloned the human SULT1C1 cDNA and gene as steps toward pharmacogenetic studies. We have now 'resequenced' the exons, portions of introns flanking exons and approximately 315 bp of the 5' flanking region of SULT1C1 in 89 DNA samples from Caucasian subjects to identify common genetic polymorphisms. Nineteen separate polymorphisms were observed, including four nonsynonymous coding region single nucleotide polymorphisms (cSNPs) and five insertions/deletions. These data were also used to determine and/or infer common SULT1C1 haplotypes. Three of the four nonsynonymous cSNPs had allele frequencies greater than 1%, including one with a frequency of 6.7%. Expression constructs were created for all of the nonsynonymous cSNPs observed, and those constructs were used to transfect COS-1 cells. Three of the four SULT1C1 variant allozymes had significantly reduced enzyme activity when compared with the wild-type enzyme. Among the variant allozymes, apparent Km values for 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the sulfate donor for the reaction, varied 7-fold, and quantitative Western blot analysis showed variable levels of immunoreactive protein when compared to the wild-type enzyme. Therefore, mechanisms responsible for decreased activity involved both alterations in levels of enzyme protein and alterations in substrate kinetics. In summary, application of a 'genotype to phenotype' strategy has resulted in the identification of a series of functionally significant common genetic polymorphisms for SULT1C1. It will now be possible to evaluate the possible contribution of these polymorphisms to variation in the sulfate conjugation of drugs, other xenobiotics and/or disease pathophysiology.

Animals↗
PubMed1999

Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media.

DNA arrays of the entire set of Escherichia coli genes were used to measure the genomic expression patterns of cells growing in late logarithmic phase on minimal glucose medium and on Luria broth containing glucose. Ratios of the transcript levels for all 4,290 E. coli protein-encoding genes (cds) were obtained, and analysis of the expression ratio data indicated that the physiological state of the cells under the two growth conditions could be ascertained. The cells in the rich medium grew faster, and expression of the majority of the translation apparatus genes was significantly elevated under this growth condition, consistent with known patterns of growth rate-dependent regulation and increased rate of protein synthesis in rapidly growing cells. The cells grown on minimal medium showed significantly elevated expression of many genes involved in biosynthesis of building blocks, most notably the amino acid biosynthetic pathways. Nearly half of the known RpoS-dependent genes were expressed at significantly higher levels in minimal medium than in rich medium, and rpoS expression was similarly elevated. The role of RpoS regulation in these logarithmic phase cells was suggested by the functions of the RpoS dependent genes that were induced. The hallmark features of E. coli cells growing on glucose minimal medium appeared to be the formation and excretion of acetate, metabolism of the acetate, and protection of the cells from acid stress. A hypothesis invoking RpoS and UspA (universal stress protein, also significantly elevated in minimal glucose medium) as playing a role in coordinating these various aspects and consequences of glucose and acetate metabolism was generated. This experiment demonstrates that genomic expression assays can be applied in a meaningful way to the study of whole-bacterial-cell physiology for the generation of hypotheses and as a guide for more detailed studies of particular genes of interest.

Amino Acids↗
PubMed2001

Functional genomics of odor-guided behavior in Drosophila melanogaster.

The avoidance response to repellent odorants in Drosophila melanogaster, a response essential for survival, provides an advantageous model for studies on the genetic architecture of olfactory behavior. Transposon tagging in a highly inbred strain of flies in combination with a rapid and simple statistical behavioral assay enables the identification of not only large phenotypic effects, but also small aberrations from wild-type avoidance behavior. The recent completion of the sequence of the Drosophila genome facilitates the molecular characterization of transposon-tagged genes and correlation between gene expression and behavior in smell-impaired (smi) mutant lines. Quantitative genetic analyses of a collection of smi lines in a co-isogenic background revealed an extensive network of epistatic interactions among genes that shape the olfactory avoidance response. Candidate genes for several of these transposon-tagged smi loci implicate genes that mediate odorant recognition, including a novel odorant binding protein; signal propagation, including a voltage-gated sodium channel; and a protein containing multiple leucine rich repeats and PDZ domains likely to be involved in postsynaptic organization in the olfactory pathway. Several novel genes of unknown function have also been implicated, including a novel tyrosine-regulated protein kinase. The discovery and characterization of novel gene products that have major, hitherto unappreciated effects on olfactory behavior will provide new insights in the generation and regulation of odor-guided behavior. The identification and functional characterization of proteins encoded by smi genes that form part of the olfactory subgenome and correlation of polymorphisms in these genes with variation in odor-guided behavior in natural populations will advance our understanding of the genetic architecture of chemosensory behavior.

Animals↗
PubMed2005

Comparative functional genomics revealed conservation and diversification of three enhancers of the isl1 gene for motor and sensory neuron-specific expression.

Islet-1 (Isl1) is a member of the Isl1 family of LIM-homeodomain transcription factors (LIM-HD) that is expressed in a defined subset of motor and sensory neurons during vertebrate embryogenesis. To investigate how this specific expression of isl1 is regulated, we searched for enhancers of the isl1 gene that are conserved in vertebrate evolution. Initially, two enhancer elements, CREST1 and CREST2, were identified downstream of the isl1 locus in the genomes of fugu, chick, mouse, and human by BLAST searching for highly similar elements to those originally identified as motor and sensory neuron-specific enhancers in the zebrafish genome. The combined action of these elements is sufficient for completely recapitulating the subtype-specific expression of the isl1 gene in motor neurons of the mouse spinal cord. Furthermore, by direct comparison of the upstream flanking regions of the zebrafish and human isl1 genes, we identified another highly conserved noncoding element, CREST3, and subsequently C3R, a similar element to CREST3 with two CDP CR1 recognition motifs, in the upstream regions of all other isl1 family members. In mouse and human, CRESTs are located as far as more than 300 kb away from the isl1 locus, while they are much closer to the isl1 locus in zebrafish. Although all of zebrafish CREST2, CREST3, and C3R activate gene expression in the sensory neurons of zebrafish, CREST2 of mouse and human does not have the sequence necessary for sensory neuron-specific expression. Our results revealed both a remarkable conservation of the regulatory elements regulating subtype-specific gene expression in motor and sensory neurons and the dynamic process of reorganization of these elements whereby each element increases the level of cell-type specificity by losing redundant functions with the other elements during vertebrate evolution.

Animals↗
PubMed2003

A functional genomics approach for the identification of putative tumor suppressor genes: Dickkopf-1 as suppressor of HeLa cell transformation.

We described previously the isolation and characterization of two non-tumorigenic revertants from the HeLa cervical carcinoma cell line, and demonstrated that loss of the transformed phenotype in these cells was the result of dominant somatic mutations. The goal of the present study was to use cDNA microarrays to identify candidate tumor suppressors among the set of genes whose increased expression correlated with loss of tumorigenicity in both revertants. Among the genes with significantly increased expression levels in both HA and HF revertants we identified Insulin Growth Factor Binding Protein-3 (IGFBP-3) and the Dickkopf-1 (DKK-1) genes. Both of these genes encode secreted proteins implicated in the modulation cell growth and differentiation, and IGFBP-3 was shown previously to have tumor suppressing activity. To test the hypothesis that increased expression of IGFBP-3 or the DKK-1 genes could have contributed to the suppression of tumorigenicity in the revertants, we expressed IGFBP-3 or DKK-1 in HeLa cells, and assessed their effects on anchorage dependent and independent growth, and tumor formation in athymic nude mice. Ectopic expression of IGFBP-3 or DKK-1 resulted in significantly decreased growth in soft agar. HeLa cells expressing ectopic IGFBP-3 or DKK-1 showed statistically significant differences in the kinetics of tumor formation. In any tumors that arose in animals injected with the IGFBP-3 expressing cells, there was a complete loss of IGFBP-3 activity, as measured by binding to IGF-1 and IGF-2 proteins. All tumors that arose after injection of cells expressing DKK-1, invariably showed almost a complete loss of ectopic DKK-1 expression. The observations that loss of DKK-1 expression or IGFBP-3 activity was required for tumorigenicity suggested that both proteins encode putative tumor suppressor genes. We also show that while DKK-1 expression does not affect cell growth in vitro, the protein does sensitize cells to apoptosis. We also demonstrated that effect of DKK-1 was not due to inhibition of beta-catenin/TCF4-regulated transcription. Taken together, our results indicate that somatic cell genetics combining with gene expression profiling may be a useful approach for the identification of functional suppressors of malignant cell growth.

Animals↗
PubMed2000

Fatty acid synthesis: from CO2 to functional genomics.

For over 25 years there has been uncertainty over the pathway from CO(2) to acetyl-CoA in chloroplasts. On the one hand, free acetate is the most effective substrate for fatty acid synthesis by isolated chloroplasts, and free acetate concentrations reported in leaf tissue (0.1-1 mM) appear adequate to saturate fatty acid synthase. On the other hand, a clear mechanism to generate sufficient free acetate for fatty acid synthesis is not established and direct production of acetyl-CoA from pyruvate by a plastid pyruvate dehydrogenase seems a more simple and direct path. We have re-examined this question and attempted to distinguish between the alternatives. The kinetics of (13)CO(2) and (14)CO(2) movement into fatty acids and the absolute rate of fatty acid synthesis in leaves was determined in light and dark. Because administered (14)C appears in fatty acids within < 2-3 min our results are inconsistent with a large pool of free acetate as an intermediate in leaf fatty acid synthesis. In addition, these studies provide an estimate of the turnover rate of fatty acid in leaves. Studies similar to the above are more complex in seeds, and some questions about the regulation of plant lipid metabolism seem difficult to solve using conventional biochemical or molecular approaches. For example, we have little understanding of why or how some seeds produce >50% oil whereas other seeds store largely carbohydrate or protein. Major control over complex plant biochemical pathways may only become possible by understanding regulatory networks which provide 'global' control over these pathways. To begin to discover such networks and provide a broad analysis of gene expression in developing oilseeds, we have produced microarrays that display approx. 5000 seed-expressed Arabidopsis genes. Sensitivity of the arrays was 1-2 copies of mRNA/cell. The arrays have been hybridized with probes derived from seeds, leaves and roots, and analysis of expression ratios between the different tissues has allowed the tissue-specific expression patterns of many hundreds of genes to be described for the first time. Approx. 10% of the genes were expressed at ratios > or = 10-fold higher in seeds than in leaves or roots. Included in this list are a large number of proteins of unknown function, and potential regulatory factors such as protein kinases, phosphatases and transcription factors. The arrays were also found to be useful for analysis of Brassica seeds.

Acetyl Coenzyme A↗
PubMed1998

GeneCards: a novel functional genomics compendium with automated data mining and query reformulation support.

MOTIVATION: Modern biology is shifting from the 'one gene one postdoc' approach to genomic analyses that include the simultaneous monitoring of thousands of genes. The importance of efficient access to concise and integrated biomedical information to support data analysis and decision making is therefore increasing rapidly, in both academic and industrial research. However, knowledge discovery in the widely scattered resources relevant for biomedical research is often a cumbersome and non-trivial task, one that requires a significant amount of training and effort. RESULTS: To develop a model for a new type of topic-specific overview resource that provides efficient access to distributed information, we designed a database called 'GeneCards'. It is a freely accessible Web resource that offers one hypertext 'card' for each of the more than 7000 human genes that currently have an approved gene symbol published by the HUGO/GDB nomenclature committee. The presented information aims at giving immediate insight into current knowledge about the respective gene, including a focus on its functions in health and disease. It is compiled by Perl scripts that automatically extract relevant information from several databases, including SWISS-PROT, OMIM, Genatlas and GDB. Analyses of the interactions of users with the Web interface of GeneCards triggered development of easy-to-scan displays optimized for human browsing. Also, we developed algorithms that offer 'ready-to-click' query reformulation support, to facilitate information retrieval and exploration. Many of the long-term users turn to GeneCards to quickly access information about the function of very large sets of genes, for example in the realm of large-scale expression studies using 'DNA chip' technology or two-dimensional protein electrophoresis. AVAILABILITY: Freely available at http://bioinformatics.weizmann.ac.il/cards/ CONTACT: cards@bioinformatics.weizmann.ac.il

Algorithms↗
PubMed2003

A functional genomic study to identify differential gene expression in the preterm and term human myometrium.

The mechanisms that lead to the onset of human parturition are still unknown, although selected critical factors have been identified. To investigate the changes in myometrial gene expression associated with parturition, we used two macroarrays each containing 1176 different complementary human cDNA clones. Methods involving hierarchical clustering and conventional statistical analysis allowed us to generate a profile of genes expression at three stages of late pregnancy: preterm (29 wk amenorrhea); full term, not in labor (38 wk amenorrhea); and full term in labor (39 wk amenorrhea). Only 4% of the genes investigated were differentially expressed between the preterm and term groups (P < 0.05). These genes could be clustered as groups of either down-regulated or up-regulated transcripts. The changes in transcript abundance were particularly marked between the preterm and term stages of gestation, whereas the differences between term not in labor and term in labor were less pronounced. The parturition was characterized by a massive down-regulation of a large panel of developmental, cell adhesion molecule and proliferation-related genes, along with the up-regulation of inflammatory, contraction and apoptosis associated genes. We propose that the mechanisms of parturition consist primarily in the arrest of the processes of myometrial development, a step that might be essential to allow the uterus to recover appropriate contractile function before delivery.

Adult↗
PubMed2002

Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis.

The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response to DL-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

Bacillus subtilis↗
PubMed2002

Endogenous sodium-potassium ATPase inhibition related biochemical cascade in trisomy 21 and Huntington's disease: neural regulation of genomic function.

The isoprenoid pathway related cascade was assessed in trisomy 21 and Huntington's disease. Membrane Na+-K+ ATPase activity, serum magnesium and ubiquinone were decreased while HMG CoA reductase activity, serum digoxin and dolichol levels were increased in both the disorders. There were increased levels of tryptophan catabolites (nicotine, strychnine, quinolinic acid and serotonin) and decreased levels of tyrosine catabolites (dopamine, noradrenaline and morphine) in both trisomy 21 and Huntington's disease. There was an increase in dolichol levels, carbohydrate residues of glycoproteins, glycolipids, total/individual GAG fractions and lysosomal enzymes in both disorders. Reduced levels of ubiquinone, reduced glutathione and free radical scavenging enzymes as well as increased lipid peroxidation products and nitric oxide were noticed in both the disorders. The role of hypothalamic digoxin and a disordered isoprenoid pathway in the pathogenesis of trisomy 21 and Huntington's disease is discussed.

Down Syndrome↗
PubMed2006

Chromosome organization and chromatin modification: influence on genome function and evolution.

Histone modifications of nucleosomes distinguish euchromatic from heterochromatic chromatin states, distinguish gene regulation in eukaryotes from that of prokaryotes, and appear to allow eukaryotes to focus recombination events on regions of highest gene concentrations. Four additional epigenetic mechanisms that regulate commitment of cell lineages to their differentiated states are involved in the inheritance of differentiated states, e.g., DNA methylation, RNA interference, gene repositioning between interphase compartments, and gene replication time. The number of additional mechanisms used increases with the taxon's somatic complexity. The ability of siRNA transcribed from one locus to target, in trans, RNAi-associated nucleation of heterochromatin in distal, but complementary, loci seems central to orchestration of chromatin states along chromosomes. Most genes are inactive when heterochromatic. However, genes within beta-heterochromatin actually require the heterochromatic state for their activity, a property that uniquely positions such genes as sources of siRNA to target heterochromatinization of both the source locus and distal loci. Vertebrate chromosomes are organized into permanent structures that, during S-phase, regulate simultaneous firing of replicon clusters. The late replicating clusters, seen as G-bands during metaphase and as meiotic chromomeres during meiosis, epitomize an ontological utilization of all five self-reinforcing epigenetic mechanisms to regulate the reversible chromatin state called facultative (conditional) heterochromatin. Alternating euchromatin/heterochromatin domains separated by band boundaries, and interphase repositioning of G-band genes during ontological commitment can impose constraints on both meiotic interactions and mammalian karyotype evolution.

Animals↗
PubMed1999

Functional genomics: clinical effect and the evolving role of the surgeon.

The genetic and molecular revolution that has occurred over the last 2 decades has dramatically increased our understanding of basic disease processes and will undoubtedly lead to improved detection methods and treatment. This will occur at an even more rapid rate after the completion of the Human Genome Project in the next 2 to 3 years. While these remarkable technological advances offer great hopes for novel therapeutic modalities, complicated medical, ethical, and legal issues will need to be addressed. This article briefly describes the advances that have occurred and their future ramifications for the field of surgery. Most assuredly, we will all be affected by these changes. Surgeons have the opportunity to be active participants and real leaders in the research and complex decisions regarding the optimal treatment of patients. However, formal training in these techniques and their potential applications will be required. Surgeons, as well as all physicians, must rise to the occasion or, otherwise, we will be relegated to a bystander status with clinical and moral decisions being made by nonclinicians.

Ethics, Medical↗
PubMed2000

Ultradian clocks in eukaryotic microbes: from behavioural observation to functional genomics.

Period homeostasis is the defining characteristic of a biological clock. Strict period homeostasis is found for the ultradian clocks of eukaryotic microbes. In addition to being temperature-compensated, the period of these rhythms is unaffected by differences in nutrient composition or changes in other environmental variables. The best-studied examples of ultradian clocks are those of the ciliates Paramecium tetraurelia and Tetrahymena sp. and of the fission yeast, Schizosaccharomyces pombe. In these single cell eukaryotes, up to seven different parameters display ultradian rhythmicity with the same, species- and strain-specific period. In fission yeast, the molecular genetic analysis of ultradian clock mechanisms has begun with the systematic analysis of mutants in identified candidate genes. More than 40 "clock mutants" have already been identified, most of them affected in components of major regulatory and signalling pathways. These results indicate a high degree of complexity for a eukaryotic clock mechanism. BioEssays 22:16-22, 2000.

Activity Cycles↗