Belmont Lab
 Unversity of Illinois at Urbana-Champaign
Alumni Labs
 
Tagging chromosomes with Lac operator repeats

Our goal upon starting our laboratory many years ago was to develop the technology by which we could first visualize specific chromosome regions or even single chromosome loci in live cells and then after fixation at higher resolution by electron microscopy. Early experiments showed that while mock in situ hybridization procedures roughly preserved large-scale chromatin structure at light microscopy resolution the same conditions caused dramatic loss of ultrastructural preservation. Our strategy therefore was to use protein- DNA recognition as our tagging system, which we accomplished through creation of a 256mer tandem direct repeat of the lac operator sequence and either in vitro lac repressor binding or in vivo expression of the lac repressor (Figure). The fortuitious discovery of GFP soon after our successful demonstration of lacI staining of lac operator repeats greatly facilitated our extension of this technology to live cells. We accomplished this in mammalian cells using a GFP-lac repressor fusion protein; in parallel our collaborators Aaron Straight and Andrew Murray developed a GFP-lac repressor fusion protein for their extension of this lac op / repressor labeling technology to visualizing chromosome loci in yeast cells. Early work by us in mammalian cells suggested some additional chromosomal instability was caused by stable expression of the tetrameric wild type lac repressor. Similarly, work from the Murray laboratory showed that tetrameric lac repressor was capable of bridging sister chromatids during anaphase. Therefore we both switched to use of COOH deletions of the lac repressor which eliminated tetramer formation and instead assembled into dimers capable of binding only one lac operator.

In our initial publication of this system we demonstrated detection of single copies of the 256mer lac operator array in mammalian cells. In unpublished work from our laboratory we have been able to visualize as low as a single copy ~32 copy repeat in Drosophila imaginal disk diploid nuclei. The determining factor for detection sensitivity appears to be the background level of unbound EGFP-LacI. In collaboration with others this technology was soon demonstrated in prokaryotic cells, yeast, Drosophila, and C. elegans. Since then this technology has been widely adopted by a large number of laboratories and applied successfully to a diverse group of organisms.

Although an early motivation for our labeling system was to be able to identify specific chromosomal regions at the ultrastructural level, sensitivity of this tagging system using immunogold labeling for electron microscopy for many years lagged way behind the sensitivity demonstrated by light microscopy. A major problem was that fixation conditions which preserved large-scale chromatin structure prevented accessibility of gold labeled antibodies. More recently, however, we have shown the ability to visualize lac operator arrays using a novel in vivo immunogold labeling approach.

Currently we are applying this technology towards development of assays for the identification of cis and trans determinants of large-scale chromatin organization.

Selected Publications:

Kireev I, Lakonishok M, Liu W, Joshi VN, Powell R, Belmont AS. In vivo immunogold labeling confirms large-scale chromatin folding motifs. Nat Methods. 2008 Apr; 5 (4) :311-3. PubMed PMID:18345005.

Belmont AS. Visualizing chromosome dynamics with GFP. Trends Cell Biol. 2001 Jun; 11 (6) :250-7. PubMed PMID:11356361.

Belmont AS, Li G, Sudlow G, Robinett C. Visualization of large-scale chromatin structure and dynamics using the lac operator/lac repressor reporter system. Methods Cell Biol. 1999; 58:203-22. PubMed PMID:9891383.

Li G, Sudlow G, Belmont AS. Interphase cell cycle dynamics of a late-replicating, heterochromatic homogeneously staining region: precise choreography of condensation/decondensation and nuclear positioning. J Cell Biol. 1998 Mar 9; 140 (5) :975-89. PubMed PMID:9490713; PubMed Central PMCID: PMC2132695.

Belmont AS, Straight AF. In vivo visualization of chromosomes using lac operator-repressor binding. Trends Cell Biol. 1998 Mar; 8 (3) :121-4. PubMed PMID:9695822.

Marshall WF, Straight A, Marko JF, Swedlow J, Dernburg A, Belmont A, Murray AW, Agard DA, Sedat JW. Interphase chromosomes undergo constrained diffusional motion in living cells. Curr Biol. 1997 Dec 1; 7 (12) :930-9. PubMed PMID:9382846.

Webb CD, Teleman A, Gordon S, Straight A, Belmont A, Lin DC, Grossman AD, Wright A, Losick R. Bipolar localization of the replication origin regions of chromosomes in vegetative and sporulating cells of B subtilis. Cell. 1997 Mar 7; 88 (5) :667-74. PubMed PMID:9054506.

Straight AF, Belmont AS, Robinett CC, Murray AW. GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol. 1996 Dec 1; 6 (12) :1599-608. PubMed PMID:8994824.

Robinett CC, Straight A, Li G, Willhelm C, Sudlow G, Murray A, Belmont AS. In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol. 1996 Dec; 135 (6 Pt 2) :1685-700. PubMed PMID:8991083; PubMed Central PMCID: PMC2133976.

 

 

 

 

 

 

 

 

 

 

 

       
 
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