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PubMed2005

Assembly of a gene sequence tag microarray by reversible biotin-streptavidin capture for transcript analysis of Arabidopsis thaliana.

BACKGROUND: Transcriptional profiling using microarrays has developed into a key molecular tool for the elucidation of gene function and gene regulation. Microarray platforms based on either oligonucleotides or purified amplification products have been utilised in parallel to produce large amounts of data. Irrespective of platform examined, the availability of genome sequence or a large number of representative expressed sequence tags (ESTs) is, however, a pre-requisite for the design and selection of specific and high-quality microarray probes. This is of great importance for organisms, such as Arabidopsis thaliana, with a high number of duplicated genes, as cross-hybridisation signals between evolutionary related genes cannot be distinguished from true signals unless the probes are carefully designed to be specific. RESULTS: We present an alternative solid-phase purification strategy suitable for efficient preparation of short, biotinylated and highly specific probes suitable for large-scale expression profiling. Twenty-one thousand Arabidopsis thaliana gene sequence tags were amplified and subsequently purified using the described technology. The use of the arrays is exemplified by analysis of gene expression changes caused by a four-hour indole-3-acetic (auxin) treatment. A total of 270 genes were identified as differentially expressed (120 up-regulated and 150 down-regulated), including several previously known auxin-affected genes, but also several previously uncharacterised genes. CONCLUSIONS: The described solid-phase procedure can be used to prepare gene sequence tag microarrays based on short and specific amplified probes, facilitating the analysis of more than 21,000 Arabidopsis transcripts.

Arabidopsis↗
PubMed2005

Dissecting the Rickettsia prowazekii genome: genetic and proteomic approaches.

The obligate nature of Rickettsia prowazekii intracellular growth places severe restrictions on the analysis of rickettsial gene function and gene expression. Fortunately, this situation is improving as methods for the genetic manipulation and proteomic analysis of this fascinating human pathogen become available. In this paper, we review the current status of rickettsial genetics and the isolation of rickettsial mutants using a genetic approach. In addition, the examination of rickettsial gene expression through characterization of the rickettsial proteome will be described. This will include a description of a high-throughput, accurate mass approach that has identified 596 rickettsial proteins in a complex rickettsial protein sample.

Bacteriological Techniques↗
PubMed2004

Delivery of herpes simplex virus-based vectors to stem cells.

In contrast to traditional drugs that generally act by altering existing gene product function, gene therapy aims to target the root cause of the disease by altering the genetic makeup of the cell to treat the disease. Researchers have adapted several classes of viruses as gene-transfer vectors, taking advantage of natural viral mechanisms designed to efficiently and effectively deliver DNA to the host-cell nucleus. Among these, the human herpesviruses are excellent candidate vectors for a variety of applications. Herpes simplex virus type 1 (HSV-1) is a particularly attractive gene-transfer vehicle because natural infection in humans includes a latent state in which the viral genome persists in a nonintegrated form without causing disease in an immune-competent host. HSV-1 is a large DNA virus with a broad host range that can be engineered to accommodate multiple or large therapeutic transgenes (4). HSV vectors may be generally useful for gene transfer to a variety of tissues in which short-term or extended transgene expression of therapeutic transgenes achieve a therapeutic effect. We have used therapeutic vectors to successfully treat human disease models in animals, including cancer, Parkinson's disease, and nerve damage (5-10).

Animals↗
PubMed2006

Gene disruption in the budding yeast Saccharomyces cerevisiae.

One essential step for the molecular dissection of gene function is gene inactivation. In the yeast Saccharomyces cerevisiae, elaborate tools for gene disruption are available. Gene disruption cassettes carrying completely heterologous marker genes flanked by short DNA segments homologous to the corresponding regions left and right of the gene to be deleted result in highly efficient one-step gene disruption events yielding usually more than 50% of the clones carrying the correctly disrupted gene. Presence of loxP sites flanking the disruption marker gene allows Cre recombinase-mediated marker rescue so that the marker can be used to disrupt another gene.

Base Sequence↗
PubMed2005

RNA interference remarkably suppresses bcl-2 gene expression in cancer cells in vitro and in vivo.

Bcl-2 is an anti-apoptotic protein. If the level of Bcl-2 protein can be reduced sufficiently in tumors using RNA interference (RNAi) to target the gene message, the apoptosis of tumor cells may be promoted. In this study, we synthesized 19 nucleotides (nts) small interference RNA (siRNA) constructs suppressing bcl-2 gene expression in human tumor cells (HeLaB2 and BGC-823 cell lines) in vitro. The bcl-2 gene expression levels were significantly reduced when these siRNA were transfected into experimental two tumor cells for 72 hours. The apoptosis process was also examined in the tumor cells. Here we synthesized siRNA from a DNA template under the control of the RNA polymerase III promoter in transfected tumor cells. Using this DNA vector-based approach, we found that the siRNA efficiently and specifically inhibited the synthesis of protein encoded by the bcl-2 gene in HeLaB2 and BGC-823 tumor cells. Tumor growth was inhibited by 66.5% with 2mg/kg pSilencer 3.1H1-bcl-2 in mouse liver tumor-bearing BALB/c mice. This approach may prove to be a valuable clinical technique for the analysis of specific gene functions and gene therapy of malignant tumors that utilize the bcl-2 gene via RNA interference.

Animals↗
PubMed2000

Hodgkin and reed-sternberg cells represent an expansion of a single clone originating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription.

Single cell studies aimed at clarifying the nature and clonality of Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin's disease (HD) have so far produced conflicting results. Using an improved single cell procedure, the HRS cells of 25 patients with nodular sclerosing HD lacking B- and T-cell antigens, with and without Epstein-Barr virus infection, were analyzed for the presence of immunoglobulin (Ig) gene rearrangements. One patient with HD developed follicular lymphoma 2 years later. Both lymphomas originated from a common precursor identified as a germinal center B cell. The data show that all but one of the investigated cases harbored rearranged Ig genes, which were clonal in all instances and carried a high load of somatic mutations. The Ig coding capacity was preserved in 18 of the 24 cases (75%) with rearrangements. However, expression of Ig messenger RNA was not detectable in the HRS cells with the exception of Ig kappa light chain expression in some tumor cells of 1 case. The lack of Ig gene transcription in HRS cells was confirmed by analyzing the HD cell lines L428 and KM-H2 in transient transfection experiments. An Ig promoter/enhancer reporter construct showed virtually no activity in these cells compared to 5 control B-cell lines. We conclude that (1) classical HD is a B-cell lymphoma in most instances, (2) HRS cells are clonal without any exception, (3) they are derived from germinal center B-cells that (4) mostly lack crippling mutations but (5) have consistently lost their Ig gene transcription ability, due to functional defects in the Ig gene regulatory elements. (Blood. 2000;95:1443-1450)

Adolescent↗
PubMed2001

Selecting informative genes with parallel genetic algorithms in tissue classification.

Recent advances in biotechnology offer the ability to measure the levels of expression of thousands of genes in parallel. Analysis of such data can provide understanding and insight into gene function and regulatory mechanisms. Several machine learning approaches have been used to aid to understand the functions of genes. However, these tasks are made more difficult due to the noisy nature of array data and the overwhelming number of gene features. In this paper, we use the parallel genetic algorithm to filter out the informative genes relative to classification. By combing with the classification method proposed by Golub et al. and Slonim et al., we classify the data sets with tissues of different classes, and the preliminary results are presented in this paper.

Algorithms↗
PubMed2002

Microbial aspects of anaerobic BTEX degradation.

Combined with conventional methods, developments in both geochemical (delineation of redox processes) and molecular microbial methods (analysis of 16S rDNA genes and functional genes) have allowed us to study in details microorganisms and genes involved in the anaerobic degradation of benzene, toluene, ethylbenzene and xylene (BTEX) under specific redox conditions. This review summarizes recent research in this field. The potential for anaerobic BTEX degradation is widely spread. Specific groups of microorganisms appear to be involved in degradation under different redox conditions. Members of the Azoarcus/Thauera cluster perform BTEX degradation under denitrifying conditions, Geobacteraceae under Fe (III) reducing conditions and Desulfobacteriaceae under sulfate reducing conditions. The information so far obtained on biochemistry and molecular genetics of BTEX degradation indicates that each BTEX compound is funneled into the central benzyol-CoA pathway by a different peripheral pathway. The peripheral pathways of per BTEX compound show similarities among different physiological groups of microorganisms. We also describe how knowledge obtained on the microbial aspects of BTEX degradation can be used to enhance and monitor anaerobic BTEX degradation.

Bacteria, Anaerobic↗
PubMed2004

[Basic research on and application of RNA interference].

RNAi, a novel strategy of gene-silencing induced by dsRNA, has recently become an important topic of discussion. dsRNAs introduced into a living body are processed by an enzyme known as the Dicer and become fractions of 21-23 nt RNAs (siRNA), after which they form protein complexes known as RNA-induced silencing complexes (RISC). The resulting RISC recognizes cognate mRNAs and cleave them. There are 2 ways to introduce dsRNAs to a living body: direct introduction of synthetic dsRNAs, and introduction of DNA templates into cells, followed by production of dsRNAs by transcription. Now that we can apply RNAi technologies into a living body, RNAi has become a widely used tool due to its great simplicity and high efficiency. However, there are still problems to be solved for more efficient use of RNAi. There are a wide range of applications such as analysis of gene functions and gene therapy. It is suggested that small RNAs, called miRNAs, exist in a living body and bind mRNAs to repress their translations. miRNA plays a great role in gene regulation and control of the differentiation and development of cells.

Animals↗
PubMed2005

Targeting epigenetic changes in acute myeloid leukemia.

Recent advances in the understanding of acute myeloid leukemia (AML) and myeloid leukemogenesis have begun to narrow the focus of therapy to specific molecular aberrations that characterize subsets of this disease. Rather than embracing "one-fits-all" cytarabine-based chemotherapy as a standard approach for all patients, ongoing clinical research in AML focuses on drugs that target unique genomic aberrations, protein structure/functional alterations, or dysregulated cellular pathways that are specific for molecular subgroups of patients. Epigenetic modifications causing gene transcriptional repression have been associated with malignant transformation and are intriguing new targets in the treatment of AML. In contrast to genetic deletions causing irreversible loss of gene function, epigenetic gene silencing mediated by DNA methylation and histone deacetylation can be reversed via pharmacologic inhibition of DNA methyltransferases and histone deacetylases, respectively. When this occurs, normal patterns of gene expression, hematopoietic differentiation, and apoptosis may be restored and disease response obtained. In this review, we focus on the clinical applicability of epigenetic targeting in the treatment of patients with AML.

Acetylation↗
PubMed1987

[Molecular genetic study of the cut locus of Drosophila melanogaster. V. The suffix sequence found in the locus is involved in the 3'-end maturation of different Drosophila mRNAs].

The nucleotide sequences of 8 genomic and 2 mRNA copies of the suffix were studied. It was found that this short repeat (265 bp) forms the last exon (73 bp) in different developmentally regulated Drosophila genes. The functioning genes contain short insertions carrying polyadenylation signals and polyadenylation sites at the same position of the suffix. It was shown that the suffix sequence is directly involved in the formation of the last splicing site and 3'-end maturation of mRNA.

Animals↗
PubMed1996

Evaluation of promoter strength for hepatic gene expression in vivo following adenovirus-mediated gene transfer.

Transgene expression in studies of both gene function and gene therapy may be assisted considerably through the use of transcriptional regulatory elements which permit high-level, and/or tissue-specific gene expression. We have therefore evaluated the transcriptional activities of a series of viral and cellular enhancer/promoter elements, both in vitro and in vivo. The five enhancer/promoter elements showing either high-level or hepatocyte-specific expression following transient transfection into hepatoma cells were incorporated into recombinant adenoviruses expressing human alpha 1-antitrypsin (hAAT) for in vivo studies in the liver of immunodeficient and immunocompetent mice. The human elongation factor 1 alpha gene promoter produced 2 mg/ml serum level of hAAT, which is physiologic in humans and will be therapeutic for patients with AAT deficiency. This and all other enhancer/promoters except that of the CMV-IE gene yielded persistent hAAT expression in SCID mice. These findings demonstrate that adenovirus vectors provide an effective system for studies designed to evaluate enhancer/promoter activities in vivo. Several of the enhancer/promoters examined in this study will have significant utility in adenovirus-mediated gene therapy for alpha 1-antitrypsin deficiency and other genetic disorders.

Adenoviruses, Human↗
PubMed2001

Cloning the human and mouse MMS19 genes and functional complementation of a yeast mms19 deletion mutant.

The MMS19 gene of the yeast Saccharomyces cerevisiae encodes a polypeptide of unknown function which is required for both nucleotide excision repair (NER) and RNA polymerase II (RNAP II) transcription. Here we report the molecular cloning of human and mouse orthologs of the yeast MMS19 gene. Both human and Drosophila MMS19 cDNAs correct thermosensitive growth and sensitivity to killing by UV radiation in a yeast mutant deleted for the MMS19 gene, indicating functional conservation between the yeast and mammalian gene products. Alignment of the translated sequences of MMS19 from multiple eukaryotes, including mouse and human, revealed the presence of several conserved regions, including a HEAT repeat domain near the C-terminus. The presence of HEAT repeats, coupled with functional complementation of yeast mutant phenotypes by the orthologous protein from higher eukaryotes, suggests a role of Mms19 protein in the assembly of a multiprotein complex(es) required for NER and RNAP II transcription. Both the mouse and human genes are ubiquitously expressed as multiple transcripts, some of which appear to derive from alternative splicing. The ratio of different transcripts varies in several different tissue types.

Alternative Splicing↗
PubMed1993

Sex-lethal, master and slave: a hierarchy of germ-line sex determination in Drosophila.

Female sex determination in the germ line of Drosophila melanogaster is regulated by genes functioning in the soma as well as genes that function within the germ line. Genes known or suspected to be involved in germ-line sex determination in Drosophila melanogaster have been examined to determine if they are required upstream or downstream of Sex-lethal+, a known germ-line sex determination gene. Seven genes required for female-specific splicing of germ-line Sex-lethal+ pre-mRNA are identified. These results together with information about the tissues in which these genes function and whether they control sex determination and viability or just sex determination in the germ line have been used to deduce the genetic hierarchy regulating female germ-line sex determination. This hierarchy includes the somatic sex determination genes transformer+, transformer-2+ and doublesex+ (and by inference Sex-lethal+), which control a somatic signal required for female germ-line sex determination, and the germ-line ovarian tumor genes fused+, ovarian tumor+, ovo+, sans fille+, and Sex-lethal+, which are involved in either the reception or interpretation of this somatic sex determination signal. The fused+, ovarian tumor+, ovo+ and sans fille+ genes function upstream of Sex-lethal+ in the germ line.

Animals↗
PubMed2002

Origin of plant glycerol transporters by horizontal gene transfer and functional recruitment.

Gene-family evolution mostly relies on gene duplication coupled with functional diversification of gene products. However, other evolutionary mechanisms may also be important in generating protein diversity. The ubiquitous membrane intrinsic protein (MIP) gene family is an excellent model system to search for such alternative evolutionary mechanisms. MIPs are proteins that transport water, glycerol, and small solutes across cell membranes in all living organisms. We reconstructed the molecular phylogeny of MIPs based on amino acid sequence data by using neighbor-joining, maximum-likelihood, and Bayesian methods of phylogenetic inference. The recovered trees show an early and distinct separation of water and glycerol transporters, i.e., aquaporins (AQPs), and aquaglyceroporins. The latter are absent from plants. As expected, gene duplication and functional diversification account for most of the diversity of animal and plant members of the family. However, in contrast to this model, we find that the sister group of plant glycerol transporters are bacterial AQPs. This relationship suggests first that plant glycerol transporters may resulted from a single event of horizontal gene transfer from bacteria, which we have estimated to have occurred approximately 1,200 million years ago, at the origin of plants, and second that bacterial AQPs were likely recruited to transport glycerol in plants because of their absence of aquaglyceroporins. This striking example of adaptive evolution at the molecular level was demonstrated further by finding convergent or parallel replacements at particular amino acid positions related to water- and glycerol-transporting specificity.

Amino Acid Sequence↗
PubMed1990

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine.

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.

Aldose-Ketose Isomerases↗
PubMed2001

Systematic approaches to mouse mutagenesis.

A major challenge in post-genomics is the systematic determination of mammalian gene function. A variety of mouse mutagenesis technologies, both gene- and phenotype-driven, are being used to underpin systematic and comprehensive approaches to mammalian gene function studies. Recently, a number of centres have completed large-scale ENU mutagenesis programmes that employ a phenotype-driven approach to the generation of mouse mutants. The use of ENU mutagenesis represents a powerful and efficient approach to mammalian gene-function studies, but many parallel developments are needed in downstream technologies to properly harness the new enlarged mouse-mutant resources that are being created.

Animals↗
PubMed2004

Microsatellites within genes: structure, function, and evolution.

Recently, increasingly more microsatellites, or simple sequence repeats (SSRs) have been found and characterized within protein-coding genes and their untranslated regions (UTRs). These data provide useful information to study possible SSR functions. Here, we review SSR distributions within expressed sequence tags (ESTs) and genes including protein-coding, 3'-UTRs and 5'-UTRs, and introns; and discuss the consequences of SSR repeat-number changes in those regions of both prokaryotes and eukaryotes. Strong evidence shows that SSRs are nonrandomly distributed across protein-coding regions, UTRs, and introns. Substantial data indicates that SSR expansions and/or contractions in protein-coding regions can lead to a gain or loss of gene function via frameshift mutation or expanded toxic mRNA. SSR variations in 5'-UTRs could regulate gene expression by affecting transcription and translation. The SSR expansions in the 3'-UTRs cause transcription slippage and produce expanded mRNA, which can be accumulated as nuclear foci, and which can disrupt splicing and, possibly, disrupt other cellular function. Intronic SSRs can affect gene transcription, mRNA splicing, or export to cytoplasm. Triplet SSRs located in the UTRs or intron can also induce heterochromatin-mediated-like gene silencing. All these effects caused by SSR expansions or contractions within genes can eventually lead to phenotypic changes. SSRs within genes evolve through mutational processes similar to those for SSRs located in other genomic regions including replication slippage, point mutation, and recombination. These mutational processes generate DNA changes that should be connected by DNA mismatch repair (MMR) system. Mutation that has escaped from the MMR system correction would become new alleles at the SSR loci, and then regulate and/or change gene products, and eventually lead to phenotype changes. Therefore, SSRs within genes should be subjected to stronger selective pressure than other genomic regions because of their functional importance. These SSRs may provide a molecular basis for fast adaptation to environmental changes in both prokaryotes and eukaryotes.

Codon↗