06 Nov 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.
Medical Research: What implications for genetics and medicine in general do you envision from the use of the telomerator?
Dr. Mitchell: Construction of synthetic chromosomes and even synthetic genomes is quickly becoming mainstream due to the ever-decreasing cost of DNA synthesis and the availability of sequencing data. To put it another way, since we can both read and write DNA with relative ease we are now in the position to build new designer DNA molecules of large size. We think the telomerator can play an important tool to aid in the construction of synthetic chromosomes.
Medical Research: What future research are you planning as a result of this study?
Dr. Mitchell: Now that we have developed the telomerator and demonstrated its function in yeast, we plan to expand the concept for use in mammalian cells. Generally speaking, mammalian chromosomes are much larger and more complicated than yeast chromosomes. For instance unlike budding yeast that have small ‘point centromeres’ of ~125 base pairs, mammalian centromeres encodes 100 of kilobasepairs of highly repetitive DNA. Therefore, synthetic mammalian chromosome construction has its own unique set of challenges. However we believe that the telomerator mediated linearization of these molecules after construction can have an important impact on stability and gene expression.
Citation:
Last Updated on November 24, 2014 by Marie Benz MD FAAD