Experiments we perform

We use a variety of modern molecular and genetic approaches to understand how yeast cells recognise and respond to chromosome damage.  We often analyse the effects of a temperature sensitive mutation affecting an essential telomere capping protein, Cdc13. 
In this genetic background telomere uncapping is efficiently induced by raising the temperature from room temperature (23oC) to higher temperatures (27oC and above)  Telomere uncapping induces checkpoint control (cell cycle arrest) and degradation of telomeres generating single stranded DNA. 

In the image above on the right a culture of cdc13-1 mutants has been incubated at 36oC for 2 hours, the DNA stained with a blue dye and the cells photographed using a microscope.  Most cells show two buds and a single bright blue DNA mass because in these cells the telomeres are uncapped and segregation of DNA in mitosis is stopped by DNA damage checkpoint pathways.

Spot tests are a simple way to measure genetic interactions between telomere capping mutations and DNA damage responses. 
Serial dilutions of yeast cultures are spotted onto agar plates.  The spots dry and the plates are incubated under different conditions and colonies form.    Checkpoint genes (RAD9, RAD24) and nuclease genes (EXO1), which respond to uncapped telomeres clearly inhibit the growth of cdc13-1 mutants at high temperatures. 

Use of automated camera system and image analysis allows us to convert spot tests (as above) into growth curve.  We can perform thousands of such measurements in parallel, in Quantitative Fitness Analysis.


We use many other methods too.  Several years ago we developed QAOS (Quantitative Amplification of Single Stranded DNA), a real time PCR based method to measure single stranded DNA arising in yeast cells.   QAOS and many of the other experimental approaches we use are described in our publications.