Tracking individual chromosomes with integrated arrays of Lacop sites and GFP-Laci repressor (Prot 15)
The visualisation of specific DNA sequences in living cells, achieved through the integration of lac operator arrays (lacop) and expression of a GFP-lac repressor fusion, has provided new tools to examine how the nucleus is organised and how basic events like sister chromatid separation occur (Straight et al. 1996; Belmont 2001). In contrast to other methods, such as fluorescence in situ hybridisation, the lacop/GFP-lac repressor (GFP-laci) technique is non-invasive, and therefore interferes minimally with nuclear structure and function. In addition, it facilitates analysis of the rapid dynamics of specific DNA loci (Gasser, 2002). Although this technique has been adapted to organisms from bacteria to man, the ease with which GFP fusions can be targeted to specific chromosomal sites depends on the organism's ability to carry out homologous recombination. This process is very efficient in budding yeast, allowing pairs of chromosomal loci to be analysed at the same time through the use of two bacterial repressors (laci and tetR) fused to different GFP variant. Given the relatively advanced state of the art in budding yeast, we present protocols optimised for this organism. These provide a starting point for adapting multi-locus tagging to other species. Moreover, the techniques described here for the quantitative analyses of locus dynamics are universally applicable.
The Friedrich Miescher Institute for Biomedical Research – Maulbeerstrasse 66 – CH-4058 Basel, Switzerland