Author Interviews, Genetic Research, Nature, NYU / 06.11.2014
Telomerator Tool Enhances Construction of Designer DNA
MedicalResearch.com Interview with:
Leslie Mitchell, PhD
New York University Langone Medical Center
Boeke Lab, Institute for Systems Genetics
NY NY, 10016
Medical Research: What is the background for this study? What are the main findings?
Dr. Mitchell: One of our major interests is building synthetic chromosomes. Typically we construct synthetic chromosomes using a bottom-up approach, first designing the sequence in silico and then synthesizing and piecing together the DNA to build the designer molecule. While eukaryotic chromosomes are usually linear in structure, oftentimes we build our designer synthetic chromosomes as circular molecules to take advantage of cloning technologies available in E. coli, an organism that tolerates only circular chromosomes. We developed the telomerator as a means by which to convert circular synthetic chromosomes into linear molecules, which more closely resemble the native chromosomes found in eukaryotic cells.
We discovered that the telomerator is an extremely effective tool for generating linear derivatives of circular synthetic chromosomes. There are two main reasons for this.
- First, the action of the telomerator can be assessed using a simple phenotypic assay so it is easy to differentiate cells that encode linear synthetic chromosomes from those with the circular format.
- Second, the telomerator encodes ‘telomere seed sequences’ that are exposed and recognized by the cell upon linearization, thus the ends of a newly linearized chromosome are protected, which ensures its stability over generations. We put the telomerator to the test by integrating it in 54 different positions on a circular synthetic yeast chromosome called synIXR (Dymond et al. 2011). In 51 of the 54 positions we could successfully linearize the synIXR chromosome and recover viable cells, however many of the different linear derivatives conferred growth defects. We determined the mechanism underlying both the growth defects and lethality associated with linearization to be telomere position effect. In other words, when essential genes were re-positioned near telomeres their reduction in expression due to subtelomeric silencing was detrimental to the cell.