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PubMed2005

Modification of endogenous natural genes by gene targeting in rice and other higher plants.

The capability to modify a genomic sequence into a designed sequence is a powerful tool for biologists and breeders to elucidate the function of an individual gene and its cis-acting elements of multigene families in the genome. Gene targeting refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome. In higher plants, however, the overwhelming occurrence of the random integration of transgenes by non-homologous end-joining is the main obstacle to develop efficient gene targeting. Two approaches have been undertaken to modify a genomic sequence in higher plants- chimeric RNA/DNA oligonucleotide-directed gene targeting to generate a site-specific base conversion, and homologous recombination-dependent gene targeting to produce either a base change or a gene replacement in a sequence-specific manner. The successful and reproducible targeting of an endogenous gene by homologous recombination, independently of gene-specific selection by employing a strong positive-negative selection, has been demonstrated for the first time in rice, an important staple food and a model plant for other cereal species. This review addresses the current status of targeting of an endogenous natural gene in rice and other higher plants and discusses possible models for Agrobacterium- mediated gene targeting by homologous recombination using a strong positive-negative selection.

Arabidopsis↗
PubMed1997

Ectopic expression of target genes may represent an inherent limitation of RT-PCR assays used for micrometastasis detection: studies on the epithelial glycoprotein gene EGP-2.

Our objective was to develop and study the feasibility of a quantitative, nested reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of micrometastatic, epithelial tumor cells using the epithelial glycoprotein EGP-2 gene as a target. Several carcinoma cell lines and peripheral blood samples of 10 healthy volunteers were screened for levels of EGP-2 mRNA. The assay included EGP-2 competitor molecules, carrying an internal deletion, that had been titrated by limiting dilution. Seven carcinoma cell lines showed a wide spectrum of EGP-2 mRNA expression levels, with the highest values (20-100 molecules/cell) seen in 3 breast-cancer cell lines. Unexpectedly, a consistent low level of EGP-2 mRNA expression (0.0004 molecules/cell) was observed in peripheral blood mononuclear cells, probably representing ectopic non-functional expression. Because of this background level, spiking experiments with T47D breast-carcinoma cells added to blood mononuclear cells exhibited a detection limit that was not better than approximately one tumor cell in 2 x 10(4) normal cells. Together with the considerable variation of EGP-2 transcript levels that is observed in different carcinoma cell lines, the extent of expression in normal blood cells would prevent a reliable estimation of low numbers of carcinoma cells in clinical samples. A similar situation might well apply for other target genes. This emphasizes the need for a critical evaluation of the different steps involved in the methods used for RT-PCR detection of micrometastatic tumor cells.

Adolescent↗
PubMed2006

Zinc finger transcription factor Slug is a novel target gene of aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. We previously showed that AhR localizes predominantly in the cytoplasm under high cell densities of a keratinocytes cell line, HaCaT, but accumulates in the nucleus at low cell densities. In the current report, we show that the Slug, which is a member of the snail/slug family of zinc finger transcriptional repressors critical for induction of epithelial-mesenchymal transitions (EMT), is activated transcriptionally in accordance with nuclear accumulation of AhR. By reporter assay of the promoter of the Slug gene, gel shift and chromatin immunoprecipitation analyses showed AhR directly binds to xenobiotic responsive element 5 at -0.7 kb of the gene. AhR-targeted gene silencing by small interfering RNA duplexes led to the abolishment of not only CYP1A1 but also Slug induction by 3-methycholanthrene. The Slug was co-localized to the AhR at the wound margins of HaCaT cells, where apparent nuclear distribution of AhR and Slug was observed. The induced Slug was associated with reduction of an epithelial marker of cytokeratin-18 and with an increase in the mesenchymal marker, fibronectin. Taken together, these findings suggest that AhR participated in Slug induction, which, in turn, regulates cellular physiology including cell adhesion and migration.

Animals↗
PubMed2002

CASK and its target gene Reelin were co-upregulated in human esophageal carcinoma.

Calcium/calmodulin-dependent serine protein kinase (CASK) showed overexpression in human esophageal carcinoma by suppression subtractive hybridization. The upregulation of CASK gene and its target gene Reelin in human esophageal carcinoma tissues versus corresponding normal tissues was revealed by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry or Western blot. Moreover, RT-PCR results indicated that the expression patterns of CASK and Reelin in human gastric carcinoma and colon carcinoma were different with those in esophageal carcinoma. Therefore, it suggested that CASK and Reelin were associated with tumorigenesis of esophagus and they were co-upregulated in human esophageal carcinoma.

Blotting, Western↗
PubMed2003

Target genes for OBP3, a Dof transcription factor, include novel basic helix-loop-helix domain proteins inducible by salicylic acid.

Overexpression of a salicylic-acid (SA)-inducible Arabidopsis DNA binding with one finger (Dof) transcription factor, called OBF-binding protein 3 (OBP3; AtDof3.6), has previously been shown to result in growth defects. In this study, suppressive subtraction hybridization (SSH) was used to isolate genes induced in an OBP3-overexpression line and several putative clones, called OBP3-responsive genes (ORGs), were isolated. The link with the induced expression levels of these genes and OBP3 overexpression was confirmed by analysing additional OBP3-overexpression lines. ORG1 through ORG4 are novel genes, while ORG5 is an extensin gene, AtExt1. While ORG4 has no similarity with other proteins in the database, ORG1 has weak similarity in different regions of the predicted protein with CDC2 and fibrillin. ORG2 and ORG3 share 80% overall identity in their deduced amino acid sequences and contain a basic helix-loop-helix DNA-binding domain, suggesting that ORG2 and ORG3 may be transcription factors. The expression of the ORG1, ORG2 and ORG3 genes was co-regulated under all conditions examined including upregulation by SA and downregulation by jasmonic acid (JA). Fifteen OBP3-silenced lines were generated to further explore the function of OBP3. Although there were no visible phenotypic changes in any of these lines, the expression of ORG1, ORG2 and ORG3 was reduced. Among the ORG genes, ORG1, ORG2 and ORG3 contained the highest number of potential Dof-binding sites in the promoter region, and their expression was significantly increased within 3 h after induction of OBP3 expression using an inducible promoter system, and closely reflected the expression levels of the exogenous OBP3 protein. The results from the gain-of-function and loss-of-function experiments suggest that the ORG1, ORG2 and ORG3 genes are direct target genes of OBP3.

Amino Acid Sequence↗
PubMed2000

Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli.

Mutations in loci other than genes for the target topoisomerases of fluoroquinolones, gyrA and parC, may play a role in the development of fluoroquinolone resistance in Escherichia coli. A series of mutants with increasing resistance to ofloxacin was obtained from an E. coli K-12 strain and five clinical isolates. First-step mutants acquired a gyrA mutation. Second-step mutants reproducibly acquired a phenotype of multiple antibiotic resistance (Mar) and organic solvent tolerance and showed enhanced fluoroquinolone efflux. None of the second-step mutants showed additional topoisomerase mutations. All second-step mutants showed constitutive expression of marA and/or overexpressed soxS. In some third-step mutants, fluoroquinolone efflux was further enhanced compared to that for second-step mutants, even when the mutant had acquired additional topoisomerase mutations. Attempts to circumvent the second-step Mar mutation by induction of the mar locus with sodium salicylate and thus to select for pure topoisomerase mutants at the second step were not successful. At least in vitro, non-target gene mutations accumulate in second- and third-step mutants upon exposure to a fluoroquinolone and typically include, but do not appear to be limited to, mutations in the mar or sox regulons with consequent increased drug efflux.

Anti-Infective Agents↗
PubMed2005

Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption.

Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24 degrees C resulted in high frequencies of transformation (> 100 transformants/10(7) conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Deltaalb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.

Agrobacterium tumefaciens↗
PubMed2006

Global analysis of microRNA target gene expression reveals that miRNA targets are lower expressed in mature mouse and Drosophila tissues than in the embryos.

MicroRNAs (miRNAs) are non-coding small RNAs of approximately 22 nt that regulate the gene expression by base pairing with target mRNAs, leading to mRNA cleavage or translational repression. It is currently estimated that miRNAs account for approximately 1% of predicted genes in higher eukaryotic genomes and that up to 30% of genes might be regulated by miRNAs. However, only very few miRNAs have been functionally characterized and the general functions of miRNAs are not globally studied. In this study, we systematically analyzed the expression patterns of miRNA targets using several public microarray profiles. We found that the expression levels of miRNA targets are lower in all mouse and Drosophila tissues than in the embryos. We also found miRNAs more preferentially target ubiquitously expressed genes than tissue-specifically expressed genes. These results support the current suggestion that miRNAs are likely to be largely involved in embryo development and maintaining of tissue identity.

Animals↗
PubMed2004

In vivo mitochondrial p53 translocation triggers a rapid first wave of cell death in response to DNA damage that can precede p53 target gene activation.

p53 promotes apoptosis in response to death stimuli by transactivation of target genes and by transcription-independent mechanisms. We recently showed that wild-type p53 rapidly translocates to mitochondria in response to multiple death stimuli in cultured cells. Mitochondrial p53 physically interacts with antiapoptotic Bcl proteins, induces Bak oligomerization, permeabilizes mitochondrial membranes, and rapidly induces cytochrome c release. Here we characterize the mitochondrial p53 response in vivo. Mice were subjected to gamma irradiation or intravenous etoposide administration, followed by cell fractionation and immunofluorescence studies of various organs. Mitochondrial p53 accumulation occurred in radiosensitive organs like thymus, spleen, testis, and brain but not in liver and kidney. Of note, mitochondrial p53 translocation was rapid (detectable at 30 min in thymus and spleen) and triggered an early wave of marked caspase 3 activation and apoptosis. This caspase 3-mediated apoptosis was entirely p53 dependent, as shown by p53 null mice, and preceded p53 target gene activation. The transcriptional p53 program had a longer lag phase than the rapid mitochondrial p53 program. In thymus, the earliest apoptotic target gene products PUMA, Noxa, and Bax appeared at 2, 4, and 8 h, respectively, while Bid, Killer/DR5, and p53DinP1 remained uninduced even after 20 h. Target gene induction then led to further increase in active caspase 3. Similar biphasic kinetics was seen in cultured human cells. Our results suggest that in sensitive organs mitochondrial p53 accumulation in vivo occurs soon after a death stimulus, triggering a rapid first wave of apoptosis that is transcription independent and may precede a second slower wave that is transcription dependent.

Animals↗
PubMed2005

Cell type-specific regulation of RE-1 silencing transcription factor (REST) target genes.

RE-1 silencing transcription factor (REST) is a transcriptional repressor that represses neuronal gene transcription in non-neuronal cells. REST target genes are expressed in neurons and in neuroendocrine cells. Here, we show that treatment with the histone deacetylase inhibitor trichostatin A (TSA) or expression of a mutant of REST (DP-REST:ER) that contains a transcriptional activation domain enhanced expression of the REST target genes encoding synaptophysin and secretogranin II in neuronal as well as in neuroendocrine cells. These data indicate that the synaptophysin and secretogranin II genes are similarly regulated in neuronal and neuroendocrine cells. In contrast, expression of the connexin36 gene was inducible by TSA or DP-REST:ER only in pancreatic alpha and beta cells, but not in neuronal and pituitary cells, indicating that transcriptional repression by REST functions in a cell type-specific manner. Expression of the BDNF and GluR2 genes, both described as targets of REST, was not induced by either TSA or expression of DP-REST:ER in neuronal or neuroendocrine cells. Chromatin immunoprecipitation experiments using antibodies directed against methylated histone H3Lys4 or H3Lys9 showed a perfect correlation between expression of REST target genes in different cell types and nucleosomal modifications that distinguish active from inactive genes. We conclude that the cell type-specific microenvironment, in particular the cell type-specific structure of the chromatin, is crucial for the ability of REST to control gene transcription.

Adrenocorticotropic Hormone↗
PubMed2000

Mechanisms regulating target gene selection by the homeodomain-containing protein Fushi tarazu.

Homeodomain proteins are DNA-binding transcription factors that control major developmental patterning events. Although DNA binding is mediated by the homeodomain, interactions with other transcription factors play an unusually important role in the selection and regulation of target genes. A major question in the field is whether these cofactor interactions select target genes by modulating DNA binding site specificity (selective binding model), transcriptional activity (activity regulation model) or both. A related issue is whether the number of target genes bound and regulated is a small or large percentage of genes in the genome. In this study, we have addressed these issues using a chimeric protein that contains the strong activation domain of the viral VP16 protein fused to the Drosophila homeodomain-containing protein Fushi tarazu (Ftz). We find that genes previously thought not to be direct targets of Ftz remain unaffected by FtzVP16. Addition of the VP16 activation domain to Ftz does, however, allow it to regulate previously identified target genes at times and in regions that Ftz alone cannot. It also changes Ftz into an activator of two genes that it normally represses. Taken together, the results suggest that Ftz binds and regulates a relatively limited number of target genes, and that cofactors affect target gene specificity primarily by controlling binding site selection. Activity regulation then fine-tunes the temporal and spatial domains of promoter responses, the magnitude of these responses, and whether they are positive or negative.

Animals↗
PubMed1998

Different frequency of gene targeting events by the RNA-DNA oligonucleotide among epithelial cells.

A unique hybrid oligonucleotide composed of both RNA and DNA has been shown to correct a point mutation in a site-specific and inheritable manner in extrachromosomal and chromosomal targets. In order to develop new gene therapeutics for skin, we tested two oligonucleotides that were shown to create a point mutation in alkaline phosphatase and beta-globin genes in several epithelial cell types. Highly transformed epithelial cells (HeLa) exhibited a conversion frequency of 5% by both RNA-DNA oligonucleotides. In comparison, other immortalized epithelial cells (HaCaT) or human primary keratinocytes did not show any detectable level of gene conversion by the restriction fragment length polymorphism analysis, indicating less than 1% conversion frequency. The concentration of the oligonucleotide in the nuclei of HeLa cells was similar to that of HaCaT or human primary keratinocytes measured by a radiolabeled or a fluorescein-conjugated oligonucleotide. Moreover, the RNA-DNA oligonucleotide exhibited a prolonged stability in the nucleus. Thus, neither uptake nor nuclear stability of the oligonucleotide appears to be a limiting factor in gene targeting events under our experimental conditions. These results indicate that the frequency of gene targeting varies among different cells, suggesting that cellular recombination and DNA repair activities may be important.

Alkaline Phosphatase↗
PubMed2001

The chromatin remodelling factor Brg-1 interacts with beta-catenin to promote target gene activation.

Wnt-induced formation of nuclear Tcf-beta-catenin complexes promotes transcriptional activation of target genes involved in cell fate decisions. Inappropriate expression of Tcf target genes resulting from mutational activation of this pathway is also implicated in tumorigenesis. The C-terminus of beta-catenin is indispensable for the transactivation function, which probably reflects the presence of binding sites for essential transcriptional coactivators such as p300/CBP. However, the precise mechanism of transactivation remains unclear. Here we demonstrate an interaction between beta-catenin and Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodelling complexes. A functional consequence of reintroduction of Brg-1 into Brg-1-deficient cells is enhanced activity of a Tcf-responsive reporter gene. Consistent with this, stable expression of inactive forms of Brg-1 in colon carcinoma cell lines specifically inhibits expression of endogenous Tcf target genes. In addition, we observe genetic interactions between the Brg-1 and beta-catenin homologues in flies. We conclude that beta-catenin recruits Brg-1 to Tcf target gene promoters, facilitating chromatin remodelling as a prerequisite for transcriptional activation.

Amino Acid Substitution↗
PubMed1998

Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques.

Gene targeting in mouse embryonic stem (ES) cells generally includes the analysis of numerous colonies to identify a few with mutations resulting from homologous recombination with a targeting vector. Thus, simple and efficient screening methods are needed to identify targeted clones. Optimal screening approaches require probes from outside of the region included in the targeting vector to avoid detection of the more common random insertions. However, the use of large genomic fragments in targeting vectors can limit the availability of cloned DNA, thus necessitating a strategy to obtain unique flanking sequences. We describe a rapid method to identify sequences adjacent to cloned DNA using long-range polymerase chain reaction (PCR) amplification from a genomic DNA library, followed by direct nucleotide sequencing of the amplified fragment. We have used this technique in two independent gene targeting experiments to obtain genomic DNA sequences flanking the mouse cholecystokinin (CCK) and gastrin genes. The sequences were then used to design primers to characterize ES cell lines with CCK or gastrin targeted gene mutations, employing a second long-range PCR approach. Our results show that these two long-range PCR methods are generally useful to rapidly and accurately characterize allele structures in ES cells.

Alleles↗
PubMed1992

Vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo.

Infection by vesicular stomatitis virus (VSV) results in a rapid inhibition of host cell transcription and translation. To determine whether the viral matrix (M) protein was involved in this inhibition of host cell gene expression, an M protein expression vector was cotransfected with a target gene vector, encoding the target gene, encoding chloramphenicol acetyltransferase (CAT). Expression of M protein caused a decrease in CAT activity in a gene dosage-dependent manner, and inhibition was apparent by 12 h posttransfection. The inhibitory effect of M protein was quite potent. The level of M protein required for a 10-fold inhibition of CAT activity was less than 1% of the level of M protein produced during the sixth hour of VSV infection. Northern (RNA) analysis of cotransfected cells showed that expression of M protein caused a reduction in the steady-state level of the vector-encoded mRNAs. Expression of both CAT and M mRNAs was reduced in cells cotransfected with a plasmid encoding M protein, indicating that expression of small amounts of M protein from plasmid DNA inhibits further expression of both M and CAT mRNAs. Nuclear runoff transcription analysis demonstrated that expression of M protein inhibited transcription of the target genes. This is the first report of a viral gene product which is capable of inhibiting transcription in vivo in the absence of any other viral component.

Animals↗
PubMed1988

Homologous integration in mammalian cells without target gene selection.

Homologous integrations into a nonselectable target locus have been highly enriched for following DNA transfections into mammalian cells. The target gene, the SV40 early region in COS1 cells, provides transcription signals to activate a defective selectable marker, the gpt gene. We find that nearly half of the selected clones have integrated the gpt gene at the homologous sequence in the COS1 genome. This is an estimated 100-fold enrichment for homologous events compared with transfections in which the gpt gene is transcriptionally active. As shown for yeast integration events, a double-strand break at a position of homology between the transfected DNA and the genomic target is necessary to achieve a high frequency of homologous integrations. Furthermore, the arrangement of sequences at the integration site includes a repair of the double-strand gap, which was present on the transfected DNA, suggesting that similarities exist between yeast and mammalian integrations. The experimental design, in which a defective marker is activated following a homologous integration, may have general applications for gene targeting in mammalian cells.

Animals↗
PubMed2005

A versatile and efficient gene-targeting system for Aspergillus nidulans.

Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuA delta) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA delta and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.

Antigens, Nuclear↗
PubMed2006

[Selection and quantitative detection of target genes in oral squamous cell carcinoma].

OBJECTIVE: To select and identify the target genes related to oral squamous cell carcinoma (OSCC) and provide target genes for designing oligo-nucleotide functional microarray of OSCC. METHODS: Genes possibly related to oral squamous cell carcinoma were selected from the 5 years' published data of differently expressed profiles with microarray testing in OSCC. Then mRNA expression of selected genes were evaluated by real time quantitative polymerase chain reaction (RT-PCR) in 22 cases of OSCC, including tumor tissues and paried normal mucosas and quantified according to an internal control GAPDH. RESULTS: Eight genes were tested. The overexpression of SPARC, PDGF-A, SERPINE1, TGF-beta(1) and VEGF-C genes were measured in 16, 18, 16, 20, 18 cases of tumor specimens, respectively. The expression of CK15 gene was lower than that of its normal tissue. There were overexpression of CCND1, BIRC3 in tumor tissues, but there was no significant difference of CCND1 and BIRC3 expression between tumor tissue and normal tissue (P > 0.05). CONCLUSIONS: SPARC, PDGF-A, SERPINE1, TGF-beta(1), VEGF-C and CK15 genes were closely related to tumor progress of OSCC. They can be used as the target genes for designing oligo-nucleotide functional microarray of OSCC.

Adult↗