Search results for "Coding strand"
showing 5 items of 5 documents
Generation of reporter plasmids containing defined base modifications in the DNA strand of choice
2012
Physiological effects of DNA bases other than A, G, C, and T as well as ways of removal of such bases from genomes are studied intensely. Methods for targeted insertion of modified bases into DNA, therefore, are highly demanded in the fields of DNA repair and epigenetics. This article describes efficient procedures for incorporation of modified DNA bases into a plasmid-borne enhanced green fluorescent protein (EGFP) gene. The procedure exploits excision of a stretch of 18 nt from either the transcribed or nontranscribed DNA strand with the help of the sequence-specific nicking endonucleases Nb.Bpu10I and Nt.Bpu10I. The excised single-stranded oligonucleotide is then swapped for a synthetic …
Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence.
2013
8-Oxoguanine (8-oxoG) is a major product of oxidative DNA damage, which induces replication errors and interferes with transcription. By varying the position of single 8-oxoG in a functional gene and manipulating the nucleotide sequence surrounding the lesion, we found that the degree of transcriptional inhibition is independent of the distance from the transcription start or the localization within the transcribed or the non-transcribed DNA strand. However, it is strongly dependent on the sequence context and also proportional to cellular expression of 8-oxoguanine DNA glycosylase (OGG1)-demonstrating that transcriptional arrest does not take place at unrepaired 8-oxoG and proving a causal…
8-Oxo-7,8-dihydroguanine in DNA does not constitute a barrier to transcription, but is converted into transcription-blocking damage by OGG1.
2011
The common DNA base modification 8-oxo-7,8-dihydroguanine (8-oxo-G) affects the efficiency and fidelity of transcription. We constructed plasmid substrates carrying single 8-oxo-G residues, specifically positioned in the transcribed or the non-transcribed DNA strands, to investigate their effects on the expression of an EGFP reporter gene and to explore the role of base excision repair in the mechanism of transcription inhibition. We report that 8-oxo-G does not directly block transcription in cells, since a single 8-oxo-G in the transcribed DNA strand did not reduce the EGFP expression levels in repair-deficient (OGG1-null) mouse embryonic fibroblast cell lines. Rather, inhibition of trans…
Sequence, molecular organization and products of the Drosophila virilis homologs of the D. melanogaster nested genes lethal(2)tumorous imaginal discs…
1997
Abstract In this study, we describe the isolation of the Drosophila virilis (Dvir) 6201-bp genomic fragment homologous to a 7047-bp genomic region of D. melanogaster (Dmel) that harbors the nested genes lethal(2)tumorous imaginal discs (l(2)tid), lethal(2)neighbour of tid (l(2)not) and lethal(2)relative of tid (l(2)rot). The isolated fragment, which maps at the cytogenetic position 50A5 on chromosome 5, carries the Dvir homologs of the Dmel genes l(2)tid and l(2)not. In both cases, the interspecific comparison of the determined sequences reveals a high homology regarding the protein coding regions and a high degree of evolutionary divergence concerning the intronic parts of the genes. In th…
Mouse CSB protein is important for gene expression in the presence of a single-strand break in the non-transcribed DNA strand.
2010
CSB protein is required for strand-specific repair of bulky DNA lesions in transcribed genes and mediates transcription recovery after exposure to DNA-damaging agents. We enzymatically generated DNA single-strand breaks (SSBs) with 3'-OH and 5'-phosphate termini in defined positions of a plasmid-borne gene and measured their effect on transcription in cell lines with different statuses of the Csb gene. A single SSB in the transcribed region of the gene caused significant decrease of gene expression. In all tested cell lines of mouse and human origin, a SSB in the transcribed DNA strand was less harmful for gene expression than a SSB situated in the opposing DNA strand. CSB deficiency exhibi…