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At least 613 records · Page 34Linked to original sources
PubMed2007

Mutagenesis and repair in Bacillus anthracis: the effect of mutators.

We have generated mutator strains of Bacillus anthracis Sterne by using directed gene knockouts to investigate the effect of deleting genes involved in mismatch repair, oxidative repair, and maintaining triphosphate pools. The single-knockout strains are deleted for mutS, mutY, mutM, or ndk. We also made double-knockout strains that are mutS ndk or mutY mutM. We have measured the levels of mutations in the rpoB gene that lead to the Rif(r) phenotype and have examined the mutational specificity. In addition, we examined the mutational specificity of two mutagens, 5-azacytidine and N-methyl-N'-nitro-N-nitroso-guanidine. The mutY and mutM single knockouts are weak mutators by themselves, but the combination of mutY mutM results in very high mutation rates, all due to G:C --> T:A transversions. The situation parallels that seen in Escherichia coli. Also, mutS knockouts are strong mutators and even stronger in the presence of a deletion of ndk. The number of sites in rpoB that can result in the Rif(r) phenotype by single-base substitution is more limited than in certain other bacteria, such as E. coli and Deinococcus radiodurans, although the average mutation rate per mutational site is roughly comparable. Hotspots at sites with virtually identical surrounding sequences are organism specific.

Amino Acid Substitution↗
PubMed2006

rmlB and rmlC genes are essential for growth of mycobacteria.

The rhamnose-GlcNAc disaccharide linker is fundamental to the structural integrity of mycobacterial cell wall. The donor dTDP-rhamnose is synthesized by four enzymes (RmlA, B, C, and D) beginning with dTTP and glucose-1-phosphate. We generated M. smegmatis rmlB gene knockout mutant (transcription of downstream rmlC gene was blocked because of a polar effect) by homologous recombination. When the Mycobacterium tuberculosis (Tb) rmlB rescue plasmid carrying a temperature-sensitive replication origin and Tb rmlC bearing plasmid with a normal replication origin were present in the mc(2)155 rmlB knockout mutant, the mutant was unable to grow at non-permissive temperature (42 degrees C) where the Tb rmlB rescue plasmid is lost. While the Tb rmlC rescue plasmid carrying a temperature-sensitive replication origin and Tb rmlB bearing plasmid with a normal replication origin were present in the mc(2)155 rmlB knock out mutant, this mutant was also unable to grow at the non-permissive temperature where the Tb rmlC rescue plasmid is lost. These results demonstrate that rmlB and rmlC genes are essential for mycobacterial growth, therefore, RmlB and RmlC are essential targets to develop new anti-tuberculosis drugs.

Cell Proliferation↗
PubMed2005

[Genetically modified animals as models for psychiatric diseases].

Animal models of mental disorders as well as physical disorders have been useful tools to elucidate pathophysiology of the complex disorders and develop new therapeutics. Transgenic mice including gene knockout animals have been produced by gene targeting technology. Monoamine system which is target of antipsychotics and antidepressants has very important roles in pathology of functional psychosis. In this review, we would like to introduce monoamine transporter knockout mice that we had generated as animal models of mental disorders including schizophrenia and drug abuse.

Animals↗
PubMed1997

Immunopathologic responses to Aspergillus antigen in interleukin-4 knockout mice.

Two strains of interleukin-4 (IL-4) gene knockout mice were studied and compared with wild strains to determine the role of IL-4 in the immunopathogenesis of murine allergic aspergillosis. Animals immunized intraperitoneally were subsequently challenged with Aspergillus antigen intranasally. The animals were evaluated for total serum immunoglobulin E (IgE) levels, Aspergillus-specific IgG antibody isotypes, peripheral blood eosinophils, cytokine and chemokine mRNA transcripts in spleen cells, and pulmonary histology. No serum IgE was detected in animals deficient in the IL-4 gene. Aspergillus-specific IgG1 was detected in all animals, while enhanced levels of IgG2a were detected in IL-4 knockout animals challenged with A. fumigatus antigen. There were no differences in the peripheral blood or lung eosinophils in the two groups of mice exposed to A. fumigatus. These results indicate that lung injury in Aspergillus-antigen challenged animals may be the result of the eosinophil mediators and that IgE-mediated injury may not be significant in this model, which may be a significant variation between the model and human allergic aspergillosis.

Animals↗
PubMed2005

Hormonal regulation of male reproductive tract development.

We have employed gene knockout technology in mice to probe gene function in various stages of mouse male sexual differentiation. Insulin-like factor (Insl3) is prominently expressed in Leydig cells. Mutation of this gene leads to fully penetrant cryptorchidism. Single mutation in each of the three known insulin family receptor tyrosine kinases alone has limited effects on sexual differentiation; however, compound mutations result in formation of ovotestes, and triple mutations cause male-to-female sexual reversal. The implications of our mouse models are discussed.

Animals↗
PubMed2006

Psychosis pathways converge via D2high dopamine receptors.

The objective of this review is to identify a target or biomarker of altered neurochemical sensitivity that is common to the many animal models of human psychoses associated with street drugs, brain injury, steroid use, birth injury, and gene alterations. Psychosis in humans can be caused by amphetamine, phencyclidine, steroids, ethanol, and brain lesions such as hippocampal, cortical, and entorhinal lesions. Strikingly, all of these drugs and lesions in rats lead to dopamine supersensitivity and increase the high-affinity states of dopamine D2 receptors, or D2High, by 200-400% in striata. Similar supersensitivity and D2High elevations occur in rats born by Caesarian section and in rats treated with corticosterone or antipsychotics such as reserpine, risperidone, haloperidol, olanzapine, quetiapine, and clozapine, with the latter two inducing elevated D2High states less than that caused by haloperidol or olanzapine. Mice born with gene knockouts of some possible schizophrenia susceptibility genes are dopamine supersensitive, and their striata reveal markedly elevated D2High states; suchgenes include dopamine-beta-hydroxylase, dopamine D4 receptors, G protein receptor kinase 6, tyrosine hydroxylase, catechol-O-methyltransferase, the trace amine-1 receptor, regulator of G protein signaling RGS9, and the RIIbeta form of cAMP-dependent protein kinase (PKA). Striata from mice that are not dopamine supersensitive did not reveal elevated D2High states; these include mice with knockouts of adenosine A2A receptors, glycogen synthase kinase GSK3beta, metabotropic glutamate receptor 5, dopamine D1 or D3 receptors, histamine H1, H2, or H3 receptors, and rats treated with ketanserin or aD1 antagonist. The evidence suggests that there are multiple pathways that convergetoelevate the D2High state in brain regions and that this elevation may elicit psychosis. This proposition is supported by the dopamine supersensitivity that is a common feature of schizophrenia and that also occurs in many types of genetically altered, drug-altered, and lesion-altered animals. Dopamine supersensitivity, in turn, correlates with D2High states. The finding that all antipsychotics, traditional and recent ones, act on D2High dopamine receptors further supports the proposition.

Animals↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed2003

Lack of association between serotonin 5-HT1B receptor gene polymorphism and suicidal behavior.

A genetic susceptibility to suicide attempts has been repeatedly suggested by family-, twin-, and adoption-studies. Because elevated impulsive aggression is one of the most prominent characteristics of suicide attempters and aggressive behavior has been reported in 5-HT1B receptor gene knockout mice, the serotonin receptor 1B gene (5-HT1B) is an attractive candidate. The distribution of a polymorphism (G861C) in the 5-HT1B gene was examined in 148 consecutively hospitalized German suicide attempters, and 327 German healthy volunteers randomly recruited from the general population. The controls and their first degree relatives had no history of mental disorders or suicidal behavior. We found no significant difference in allele or genotype frequency between patients and controls. The results did not differ when the patients were divided into several subgroups (gender, suicide attempters with a violent method or suicide attempters with unipolar-, bipolar-, borderline personality-, and schizophrenia spectrum disorders). These findings suggest that the 5-HT1B polymorphism is unlikely to play a major role in the genetic susceptibility to suicide attempts.

Adolescent↗
PubMed2002

Development of the neuroendocrine hypothalamus.

The development of the neuroendocrine hypothalamus has been studied using a variety of neuroanatomical and molecular techniques. Here, the major findings that mold our understanding of hypothalamic development are reviewed. The rat hypothalamus is generated predominantly from the third ventricular neuroepithelium in a "lateral early to medial late" pattern dictated perhaps by the medially receding third ventricle. Neuroendocrine neurons seem to exhibit a delayed migrational strategy, showing relatively early birthdates, although they are located in the latest-generated, periventricular nuclei. Several homeobox genes seem to play a role in hypothalamic development, and gene knockout experiments implicate a number of genes of importance in the generation of the neuroendocrine cell type.

Animals↗
PubMed2006

Genes for the peptidoglycan synthesis pathway are essential for chloroplast division in moss.

The general consensus is that a cyanobacterium phagocytosed by a host cell evolved into the plastids of red and green algae, land plants, and glaucophytes. In contrast to the plastids of glaucophytes, which retain a cyanobacterial-type peptidoglycan layer, no wall-like structures have been detected in plastids from other sources. Although the genome of Arabidopsis thaliana contains five genes that are essential for peptidoglycan synthesis, MurE, MurG, two genes for D-Ala-D-Ala ligase (Ddl), and the gene for translocase I (MraY), their functions have not been determined. We report that the moss Physcomitrella patens has nine homologous genes related to peptidoglycan biosynthesis: MurA, B, C, D, E, and F, Ddl, genes for the penicillin-binding protein Pbp, and dd-carboxypeptidase (Dac). Corroborating a computer prediction, analysis of the GFP fusion proteins with the N terminus of PpMurE or of PpPbp suggests that these proteins are located in the chloroplasts. Gene disruption of the PpMurE gene in P. patens resulted in the appearance of macrochloroplasts both in protonema and in leaf cells. Moreover, gene knockout of the P. patens Pbp gene showed inhibition of chloroplast division in this moss; however, no Pbp gene was found in A. thaliana.

Arabidopsis↗
PubMed1997

Specific and redundant functions of retinoid X Receptor/Retinoic acid receptor heterodimers in differentiation, proliferation, and apoptosis of F9 embryonal carcinoma cells.

We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)alpha and retinoic acid receptor (RAR)alpha genes or the RXRalpha and RARgamma genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRalpha-/-, RARalpha-/-, and RARgamma-/- cells. RXRalpha-/-/ RARalpha-/- cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRalpha-/- cells. In contrast, RXRalpha-/-/RARgamma-/- cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRalpha and RARgamma represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype- and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.

Animals↗
PubMed2003

Site-specific selfish genes as tools for the control and genetic engineering of natural populations.

Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.

Animals↗
PubMed2000

Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway.

The ability to stimulate recombination in a site-specific manner in mammalian cells may provide a useful tool for gene knockout and a valuable strategy for gene therapy. We previously demonstrated that psoralen adducts targeted by triple-helix-forming oligonucleotides (TFOs) could induce recombination between tandem repeats of a supF reporter gene in a simian virus 40 vector in monkey COS cells. Based on work showing that triple helices, even in the absence of associated psoralen adducts, are able to provoke DNA repair and cause mutations, we asked whether intermolecular triplexes could stimulate recombination. Here, we report that triple-helix formation itself is capable of promoting recombination and that this effect is dependent on a functional nucleotide excision repair (NER) pathway. Transfection of COS cells carrying the dual supF vector with a purine-rich TFO, AG30, designed to bind as a third strand to a region between the two mutant supF genes yielded recombinants at a frequency of 0.37%, fivefold above background, whereas a scrambled sequence control oligomer was ineffective. In human cells deficient in the NER factor XPA, the ability of AG30 to induce recombination was eliminated, but it was restored in a corrected subline expressing the XPA cDNA. In comparison, the ability of triplex-directed psoralen cross-links to induce recombination was only partially reduced in XPA-deficient cells, suggesting that NER is not the only pathway that can metabolize targeted psoralen photoadducts into recombinagenic intermediates. Interestingly, the triplex-induced recombination was unaffected in cells deficient in DNA mismatch repair, challenging our previous model of a heteroduplex intermediate and supporting a model based on end joining. This work demonstrates that oligonucleotide-mediated triplex formation can be recombinagenic, providing the basis for a potential strategy to direct genome modification by using high-affinity DNA binding ligands.

Animals↗
PubMed1998

Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice.

A transgenic mouse approach was used to examine the mechanism of principal cell-specific expression of aquaporin-2 (AQP2) within the renal collecting duct. RT-PCR and immunocytochemistry revealed that murine AQP2 was expressed in principal cells in the renal collecting duct, epithelial cells of the vas deferens, and seminiferous tubules within testis. The vas deferens expression was confirmed in rats. RT-PCR and immunocytochemistry showed that 14 kb of the human 5'-flanking region confers specific expression of a nucleus-targeted and epitope-tagged Cre recombinase in the principal cells within the renal collecting duct, in the epithelial cells of the vas deferens, and within the testis of transgenic mice. These results suggest that cell-specific expression of AQP2 is mediated at the transcriptional level and that 14 kb of the human AQP2 5'-flanking region contain cis elements that are sufficient for cell-specific expression of AQP2. Finally, renal principal cell expression of Cre recombinase is the first step in achieving cell-specific gene knockouts, thereby allowing focused examination of gene function in this cell type.

Animals↗
PubMed2006

Arabidopsis thaliana floral dip transformation method.

Arabidopsis floral dip transformation is notable for a number of reasons. First, it is strikingly simple to perform. Agrobacterium is applied to flowering Arabidopsis plants that subsequently set seed, and transgenic plants are then selected among the progeny seedlings. Because no plant tissue culture is required, somaclonal variation is avoided, and the procedure can be performed easily by nonspecialists. Success rates are high: it is common that 1% of the progeny seedlings are transgenic. The biology behind the method is interesting: Arabidopsis and some related Brassicaceae are apparently unique in allowing exogenously applied Agrobacterium to colonize the interior of developing ovaries, where female gametophyte cell lineages are transformed. The availability of the method has had a transformative effect on the overall practice of plant molecular biology, as the generation and analysis of large numbers of transgenic plants is now routine in hundreds of laboratories. The method has been exploited in a genomics context to make stable gene knockout plant lines for most Arabidopsis genes.

Agrobacterium tumefaciens↗
PubMed2006

Genetic dissection of ethanol tolerance in the budding yeast Saccharomyces cerevisiae.

Uncovering genetic control of variation in ethanol tolerance in natural populations of yeast Saccharomyces cerevisiae is essential for understanding the evolution of fermentation, the dominant lifestyle of the species, and for improving efficiency of selection for strains with high ethanol tolerance, a character of great economic value for the brewing and biofuel industries. To date, as many as 251 genes have been predicted to be involved in influencing this character. Candidacy of these genes was determined from a tested phenotypic effect following gene knockout, from an induced change in gene function under an ethanol stress condition, or by mutagenesis. This article represents the first genomics approach for dissecting genetic variation in ethanol tolerance between two yeast strains with a highly divergent trait phenotype. We developed a simple but reliable experimental protocol for scoring the phenotype and a set of STR/SNP markers evenly covering the whole genome. We created a mapping population comprising 319 segregants from crossing the parental strains. On the basis of the data sets, we find that the tolerance trait has a high heritability and that additive genetic variance dominates genetic variation of the trait. Segregation at five QTL detected has explained approximately 50% of phenotypic variation; in particular, the major QTL mapped on yeast chromosome 9 has accounted for a quarter of the phenotypic variation. We integrated the QTL analysis with the predicted candidacy of ethanol resistance genes and found that only a few of these candidates fall in the QTL regions.

Chromosome Mapping↗
PubMed1998

Transgenic approaches to the increase of disease resistance in farm animals.

Molecular genetics and reproductive biology techniques enable the transfer of foreign DNA into mammals. Novel approaches to modify disease resistance or susceptibility in livestock are justified by economic and animal welfare concerns. Current research on the improvement of disease resistance by gene transfer focuses on three main strategies, as follows: a) somatic gene transfer, i.e., nucleic acid vaccines b) deletive germ-line gene transfer, i.e., gene knockout c) additive germ-line gene transfer. These strategies aim at either the transient or stable expression of components known to influence non-specific or specific host defence mechanisms, or the disruption of genes known to cause susceptibility to disease. Referring to the source of the effective agent and the site and mode of action, the strategies are termed 'genetic', 'congenital', 'intracellular' and 'extracellular' immunisation. Each gene transfer experiment has to be evaluated carefully with respect to the potential to create novel cases of pathogen resistance or to lose species barriers or cell-type restrictions.

Animals↗
PubMed2001

D-RNAi (messenger RNA-antisense DNA interference) as a novel defense system against cancer and viral infections.

D-RNAi (Messenger RNA-antisense DNA interference), a novel posttranscriptional phenomenon of silencing gene expression by transfection of mRNA-aDNA hybrids, was originally observed in the effects of bcl-2 on phorbol ester-induced apoptosis in human prostate cancer LNCaP cells. This phenomenon was also demonstrated in chicken embryos and a human CD4(+) T cell line, H9. The in vivo transduction of beta-catenin D-RNAi was shown to knock out more than 99% endogenous beta-catenin gene expression, while the in cell transfection of HIV-1 D-RNAi homolog rejected viral gene replication completely. D-RNAi was found to have long-term gene knockout effects resulting from a posttranscriptional gene silencing mechanism that may involve the homologous recombination between intracellular mRNA and the mRNA components of a D-RNAi construct. These findings provide a potential intracellular defense system against cancer and viral infections.

DNA, Antisense↗