Genetic linkage and phenotypic analysis of Chlamydomonas reinhardtii SEC24B insertional mutants

COPII is a highly conserved protein group which is responsible for small membrane vesicles formation in the membrane of the endoplasmic reticulum. SEC24 gene is one of the components of COPII protein group and involved in the formation of COPII vesicles. SEC24 gene ise considered as the primary subcomponent responsible for binding to the cargo protein which is located in ER membrane. There are two SEC24 gene models in the Chlamydomonas reinhardtii genome that are possible to play a role in the formation of COPII vesicles. These gene models are: SEC24A and SEC24B. In this thesis we aimed to determine the function of SEC24B gene in C. reinhardtii with genetic linkage and phenotypic characterization which is not studied in literature before. sec24b mutant strain of C. reinhardtii was used in this study. To determine the region of the insertion site, genomic DNA from the sec24b mutant strain clones was isolated and the site where the insertion was located was amplified by PCR. To determine the genetic linkage, the mutant strain was crossed with the wild type and the progeny were tested for paromomycin resistance phenotype. Presence of SEC24B mRNA is determined by RT-PCR. Arylsulfatase and alkaline phosphatase activities tested colorimetrically determine whether there is a secretion function in sec24b mutant strain clones. As a result of these studies, a single insertion was detected in the genome of the sec24b mutant strain and 50% of the progeny was resistant to paromomycin. The presence of insertion in the gene has been determined in the progeny which are resistant to paromomycin. We hypothesize that in the mutant progeny, due to a problem in the transfer of proteins from ER to Golgi cells grow slower than normal cells. The data obtained in this thesis is aimed to provide information about the function of C. reinhardtii SEC24B gene to the literature.