scholarly journals In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.

1996 ◽  
Vol 135 (6) ◽  
pp. 1685-1700 ◽  
Author(s):  
C C Robinett ◽  
A Straight ◽  
G Li ◽  
C Willhelm ◽  
G Sudlow ◽  
...  
Keyword(s):  
Gene Amplification ◽  
Dna Sequences ◽  
Cho Cells ◽  
Large Scale ◽  
Lac Repressor ◽  
Chromatin Organization ◽  
Yeast Cells ◽  
Chromosomal Dna ◽  
Lac Operator

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.

scholarly journals In Vivo HP1 Targeting Causes Large-Scale Chromatin Condensation and Enhanced Histone Lysine Methylation

2005 ◽  
Vol 25 (11) ◽  
pp. 4552-4564 ◽  
Author(s):  
Pernette J. Verschure ◽  
Ineke van der Kraan ◽  
Wim de Leeuw ◽  
Johan van der Vlag ◽  
Anne E. Carpenter ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
Large Scale ◽  
Chromosome Region ◽  
Chromatin Condensation ◽  
Regulation Of Gene Expression ◽  
Lac Repressor ◽  
Homologous Proteins ◽  
Lac Operator

ABSTRACT Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) α (HP1α) and HP1β on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1α and HP1β in vivo targeting. HP1α targeting causes the recruitment of endogenous HP1β to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1α and HP1β targeting is sufficient to induce heterochromatin formation.

scholarly journals Interphase Cell Cycle Dynamics of a Late-Replicating, Heterochromatic Homogeneously Staining Region: Precise Choreography of Condensation/Decondensation and Nuclear Positioning

1998 ◽  
Vol 140 (5) ◽  
pp. 975-989 ◽  
Author(s):  
Gang Li ◽  
Gail Sudlow ◽  
Andrew S. Belmont
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Sequences ◽  
Large Scale ◽  
S Phase ◽  
Interphase Cell ◽  
Compact Mass ◽  
Lac Operator ◽  
Cell Cycle Dynamics

Recently we described a new method for in situ localization of specific DNA sequences, based on lac operator/repressor recognition (Robinett, C.C., A. Straight, G. Li, C. Willhelm, G. Sudlow, A. Murray, and A.S. Belmont. 1996. J. Cell Biol. 135:1685–1700). We have applied this methodology to visualize the cell cycle dynamics of an ∼90 Mbp, late-replicating, heterochromatic homogeneously staining region (HSR) in CHO cells, combining immunostaining with direct in vivo observations. Between anaphase and early G1, the HSR extends approximately twofold to a linear, ∼0.3-μm-diam chromatid, and then recondenses to a compact mass adjacent to the nuclear envelope. No further changes in HSR conformation or position are seen through mid-S phase. However, HSR DNA replication is preceded by a decondensation and movement of the HSR into the nuclear interior 4–6 h into S phase. During DNA replication the HSR resolves into linear chromatids and then recondenses into a compact mass; this is followed by a third extension of the HSR during G2/ prophase. Surprisingly, compaction of the HSR is extremely high at all stages of interphase. Preliminary ultrastructural analysis of the HSR suggests at least three levels of large-scale chromatin organization above the 30-nm fiber.

scholarly journals Triple-Helical DNA in Drosophila Heterochromatin

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 227 ◽  
Author(s):  
Eduardo Gorab
Keyword(s):  
Nucleic Acids ◽  
Dna Sequences ◽  
Detection Methods ◽  
Triplex Dna ◽  
Thiazole Orange ◽  
Chromosomal Dna ◽  
Mitotic Chromosomes ◽  
Satellite Repeats

Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the Drosophila melanogaster chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the brown dominant allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by Drosophila correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.

In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator

1991 ◽  
Vol 219 (4) ◽  
pp. 623-634 ◽  
Author(s):  
Anastasia M. Khoury ◽  
Harry S. Nick ◽  
Ponzy Lu
Keyword(s):  
Escherichia Coli ◽  
Lac Repressor ◽  
Lac Operator

scholarly journals Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.

1992 ◽  
Vol 12 (10) ◽  
pp. 4271-4278 ◽  
Author(s):  
D E Syroid ◽  
R I Tapping ◽  
J P Capone
Keyword(s):  
Mammalian Cells ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Lac Repressor ◽  
Trna Genes ◽  
Coding Region ◽  
Lac Operator ◽  
Cell Extracts

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

scholarly journals Dicentric Chromosome Stretching during Anaphase Reveals Roles of Sir2/Ku in Chromatin Compaction in Budding Yeast

2001 ◽  
Vol 12 (9) ◽  
pp. 2800-2812 ◽  
Author(s):  
Douglas A. Thrower ◽  
Kerry Bloom
Keyword(s):  
Budding Yeast ◽  
Lac Repressor ◽  
Yeast Cells ◽  
Dicentric Chromosome ◽  
Direct Role ◽  
Chromatin Compaction ◽  
Sir Proteins ◽  
Lac Operator ◽  
Mother And Daughter ◽  
Green Fluorescent

We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor–green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles ∼50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70,yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 μm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.

scholarly journals Requirement for End-Joining and Checkpoint Functions, but NotRAD52-Mediated Recombination, after EcoRI Endonuclease Cleavage of Saccharomyces cerevisiaeDNA

1998 ◽  
Vol 18 (4) ◽  
pp. 1891-1902 ◽  
Author(s):  
L. Kevin Lewis ◽  
Jakob M. Kirchner ◽  
Michael A. Resnick
Keyword(s):  
Nuclear Dna ◽  
Double Strand Breaks ◽  
Yeast Cells ◽  
Chromosomal Dna ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Damaging Agents ◽  
Physical And Chemical ◽  
Type Strains

ABSTRACT RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired inrad52 mutants and in G1-phase Rad+cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly,rad52 mutants were not more sensitive toEcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly whenEcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.

scholarly journals Aberrant Chromatin Remodeling by Retinoic Acid Receptor α Fusion Proteins Assessed at the Single-Cell Level

2007 ◽  
Vol 18 (10) ◽  
pp. 3941-3951 ◽  
Author(s):  
Jihui Qiu ◽  
Ying Huang ◽  
Guoqiang Chen ◽  
Zhu Chen ◽  
David J. Tweardy ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Nuclear Receptor ◽  
Fusion Proteins ◽  
Large Scale ◽  
Retinoic Acid Receptor ◽  
Chromatin Condensation ◽  
Promyelocytic Leukemia ◽  
Lac Repressor ◽  
Acid Receptor ◽  
Lac Operator

Acute promyelocytic leukemia (APL) is characterized by specific chromosomal translocations, which generate fusion proteins such as promyelocytic leukemia (PML)-retinoic acid receptor (RAR)α and promyelocytic leukemia zinc finger (PLZF)-RARα (X-RARα). In this study, we have applied lac operator array systems to study the effects of X-RARα versus wild-type RARα on large-scale chromatin structure. The targeting of these enhanced cyan fluorescent protein-lac repressor-tagged RARα-containing proteins to the gene-amplification chromosomal region by lac operator repeats led to local chromatin condensation, recruitment of nuclear receptor corepressor, and histone deacetylase complex. The addition of retinoic acid (RA) induced large-scale chromatin decondensation in cells expressing RARα; however, cells expressing X-RARα, especially PML-RARα, demonstrated insensitive response to this effect of all-trans retinoic acid (ATRA). Although we did not reveal differences in RA-dependent colocalization of either silencing mediator for retinoid and thyroid or steroid receptor coactivator (SRC)-1 with RARα versus X-RARα, the hormone-independent association between SRC-1 and X-RARα on the array has been identified. Rather, compared with cells expressing RARα, fluorescence recovery after photobleaching of live transfected cells, demonstrated decreased mobility of SRC-1 on the X-RARα–bound chromatin. Thus, the impaired ability of APL fusion proteins to activate gene transcription in response to ATRA corresponds to their reduced ability to remodel chromatin, which may link to their ability to impair the mobility of key nuclear receptor coregulators.

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