Search results for "Reporter"
showing 10 items of 166 documents
Cockayne syndrome: varied requirement of transcription-coupled nucleotide excision repair for the removal of three structurally different adducts fro…
2014
Hereditary defects in the transcription-coupled nucleotide excision repair (TC-NER) pathway of damaged DNA cause severe neurodegenerative disease Cockayne syndrome (CS), however the origin and chemical nature of the underlying DNA damage had remained unknown. To find out, to which degree the structural properties of DNA lesions determine the extent of transcription arrest in human CS cells, we performed quantitative host cell reactivation analyses of expression vectors containing various synthetic adducts. We found that a single 3-(deoxyguanosin-N 2-yl)-2-acetylaminofluorene adduct (dG(N 2)-AAF) constitutes an unsurmountable obstacle to transcription in both CS-A and CS-B cells and is remov…
Excision of Uracil from Transcribed DNA Negatively Affects Gene Expression
2014
Uracil is an unavoidable aberrant base in DNA, the repair of which takes place by a highly efficient base excision repair mechanism. The removal of uracil from the genome requires a succession of intermediate products, including an abasic site and a single strand break, before the original DNA structure can be reconstituted. These repair intermediates are harmful for DNA replication and also interfere with transcription under cell-free conditions. However, their relevance for cellular transcription has not been proved. Here we investigated the influence of uracil incorporated into a reporter vector on gene expression in human cells. The expression constructs contained a single uracil opposi…
Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay).
2003
Abstract The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described. Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots. Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity. The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria. MucAB was cloned into the test plasmid to enhance error-prone DNA-repair. The conventional reversion principle ha…
LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli
2002
The function of the LysR-type regulator LrhA of Escherichia coli was defined by comparing whole-genome mRNA profiles from wild-type E. coli and an isogenic lrhA mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80. When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant. In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent K-D approximate to 20 nM), whereas the promoters of…
Neuronal precursor-specific activity of a human doublecortin regulatory sequence.
2005
The doublecortin (DCX) gene encodes a 40-kDa microtubule-associated protein specifically expressed in neuronal precursors of the developing and adult CNS. Due to its specific expression pattern, attention was drawn to DCX as a marker for neuronal precursors and neurogenesis, thereby underscoring the importance of its promoter identification and promoter analysis. Here, we analysed the human DCX regulatory sequence and confined it to a 3.5-kb fragment upstream of the ATG start codon. We demonstrate by transient transfection experiments that this fragment is sufficient and specific to drive expression of reporter genes in embryonic and adult neuronal precursors. The activity of this regulator…
IL-6 stimulates annexin 1 expression and translocation and suggests a new biological role as class II acute phase protein.
1998
Annexin 1 (Ax 1), a protein whose synthesis and secretion are induced during the inflammatory response, has been proposed as a mediator of the anti-inflammatory action of glucocorticoids. To gain insight into a broader role of Ax 1 during the inflammatory response, the authors have investigated how pro-inflammatory cytokines [interleukin 1 (IL-1), IL-6 and tumour necrosis factor alpha (TNF-alpha)] affect Ax 1 expression and regulation at transcriptional and translational levels. The authors show that induction of the Ax 1 protein and its translocation to the cell membrane are stimulated by interleukin 6. However neither IL-1 nor TNF-alpha display these effects. Analysis of 5'-deletion mutan…
Sea urchin HSF activity in vitro and in transgenic embryos.
1997
Evidence is provided for the presence at the physiological temperature of 20 degrees C of a heat shock transcriptor factor, HSF, in the nuclei of P.lividus embryos. This HSF is able to specifically bind in vitro the heat shock element, HSE, of the promoter of the hsp70 gene i.v., as suggested by DNA-protein binding reactions and DNAse I protection assays. Upon heat-shock, at the temperature of 31 degrees C, its ability to bind the HSE units becomes much higher. The HSF activated by heat-shock drives in vivo the transcription of the beta-galactosidase reporter gene in transgenic sea urchin gastrulae. An ATF-like transcription factor, widely described in other organisms but not at all in sea …
In silico characterization of the neural alpha tubulin gene promoter of the sea urchin embryo Paracentrotus lividus by phylogenetic footprinting
2011
During Paracentrotus lividus sea urchin embryo development one alpha and one beta tubulin genes are expressed specifically in the neural cells and they are early end output of the gene regulatory network that specifies the neural commitment. In this paper we have used a comparative genomics approach to identify con- served regulatory elements in the P. lividus neural alpha tubulin gene. To this purpose, we have first isolated a genomic clone containing the entire gene plus 4.5 Kb of 5 0 upstream sequences. Then, we have shown by gene transfer experiments that its non-coding region drives the spatio- temporal gene expression corresponding substantially to that of the endogenous gene. In addi…
Further development of the β-lactamase MutaGen assay and evaluation by comparison with Ames fluctuation tests and theumu test
2005
A rapid, high-throughput bacterial mutagenicity test system has been developed (MutaGen test) that detects reversions of inactivating base-pair substitutions and frameshifts in a TEM-1 class A β-lactamase (ampicillinase) gene. To quickly and sensitively detect mutagens, the system utilises a series of plasmids that contain the mutated ampicillinase gene and the mucAB operon. Inactivating mutations in the ampicillinase gene include frameshifts integrated into repetitive GC-sequences and G-runs known to be mutagenic hot-spots, and base-pair substitutions inserted in or around the β-lactamase active site. Frameshift mutations completely inactivated the enzyme only when located downstream of th…
Ras-pathway has a dual role in yeast galactose metabolism
2007
AbstractIn the yeast Saccharomyces cerevisiae the genes involved in galactose metabolism (GAL1,7,10) are transcriptionally activated more than a 1000-fold in the presence of galactose as the sole carbon source in the culture media. In the present work, we monitored the activity of the GAL10 gene promoter in different Ras-cAMP genetic backgrounds. We demonstrate that overexpression of C-terminus of the nucleotide exchange factor Cdc25p stimulates GAL10 transcription in yeast strains carrying the contemporary deletion of both RAS genes. Moreover, the deletion of the chromosomal CDC25 gene provokes impaired growth on galactose based media in yeast strain lacking both RAS genes and adenylate cy…