IN-VIVO LOCALIZATION OF DNA-SEQUENCES AND VISUALIZATION OF LARGE-SCALE CHROMATIN ORGANIZATION USING LAC OPERATOR REPRESSOR RECOGNITION/

Citation
Cc. Robinett et al., IN-VIVO LOCALIZATION OF DNA-SEQUENCES AND VISUALIZATION OF LARGE-SCALE CHROMATIN ORGANIZATION USING LAC OPERATOR REPRESSOR RECOGNITION/, The Journal of cell biology, 135(6), 1996, pp. 1685-1700
Citations number
37
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cell Biology
Journal title
ISSN journal
0021-9525
Volume
135
Issue
6
Year of publication
1996
Part
2
Pages
1685 - 1700
Database
ISI
SICI code
0021-9525(1996)135:6<1685:ILODAV>2.0.ZU;2-4
Abstract
We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructur e and direct, in vivo observations, 256 direct repeats of the lac oper ator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characte rized by visualization of chromosome homogeneously staining regions (H SRs) produced by gene amplification using a dihydrofolate reductase (D HFR) expression vector with methotrexate selection. Using electron mic roscopy, most HSRs showed similar to 100-nm fibers, as described previ ously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 mu m in length. Subsequent experiments demo nstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detecti on of a single 256 lac operator repeat and its stability during mitosi s was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the g reen fluorescent protein-lac repressor protein allowed extended, in vi vo observations of the operator-tagged chromosomal DNA. Future applica tions of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.