|
Experimental Biophysics
Humboldt-Universität zu Berlin
|
 |
| Nuclear gene targeting in Chlamydomonas |
|
Homologous DNA recombination (HR) allows the deletion (knock out), repair (rescuing) and modification of a selected gene thereby rendering a functional analysis of the gene product possible. However, targeting of nuclear genes has been an extremely inefficient process in most eukaryotes including algae, plants and animals due to the dominance of integration of the applied DNA into non-homologous regions of the genome. We have shown for the green alga Chlamydomonas reinhardtii by repairing a previously introduced truncated aminoglycoside 3’-phosphotransferase gene aphVIII that single-stranded DNA can recombine with a homologous endogenous DNA-region of interest. Non-homologous DNA-integration appeared to be more than 300 fold reduced compared with the use of double-stranded DNA, thus allowing isolation of the homologous recombinants. We propose that this method will be applicable to direct targeting of nuclear C. reinhardtii genes. In the future we will hopefully improve the recombination efficiency in Chlamydomonas by expression of several protein that are involved in recombination as it occurs during the natural mating process. Moreover, we will test the promotion of DNA recombination by selected proteins in vitro (in cooperation with Vladislav Lanzov, St. Petersburg). 
Exchange of one strand of a double stranded DNA (red) by a homologous single stranded DNA (green). Proteins like RecA are assisting this process. The figure is taken from Voet & Voet “Biochemistry” more links: http://www.uni-jena.de/content_lang_en_page_5920.html http://www.biology.duke.edu/chlamy/ |
 |
 |
|
![Log-In Area [protected]](./etc/img/index/key.gif)
|
||
 |
 |