In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.
We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously 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 microns in length. Subsequent experiments demonstrated 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 detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.
Robinett
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CC
Department of Cell and Structural Biology, University of Illinois, Urbana-Champaign 61801, USA.
Straight
A
A
Li
G
G
Willhelm
C
C
Sudlow
G
G
Murray
A
A
Belmont
A S
AS
D000705:Anaphase / Q000502:physiology; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D002842:Chromatids / Q000502:physiology / Q000648:ultrastructure; D002843:Chromatin / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure*; D002875:Chromosomes / Q000502:physiology / Q000648:ultrastructure; D006224:Cricetinae; D004247:DNA / Q000032:analysis; D005784:Gene Amplification; D018628:Gene Dosage; D049452:Green Fluorescent Proteins; D017403:In Situ Hybridization; D007763:Lac Operon / Q000502:physiology*; D008164:Luminescent Proteins / Q000032:analysis; D008854:Microscopy, Electron; D008938:Mitosis / Q000502:physiology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis; D012091:Repetitive Sequences, Nucleic Acid; D013194:Staining and Labeling; D013997:Time Factors; D015003:Yeasts / Q000235:genetics
D002843:Chromatin; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004247:DNA