Subcloning of the H+-ATPase pmh1 in
Chlamydomonas reinhardtii
Lindsay Cohen, Davidson College, Davidson NC 28036
Summary
Chlamydomonas reinhardtii, or Chlamy, is a haploid, unicellular, biflagellated green alga. Chlamy differentiates into two mating types that attach flagella, fuse, and form a zygote only in the absence of nitrogen. It is an ideal organism used to study cell-cell interactions because of this unique mating system. In 1992, Dr. Ginger Armbrust isolated a mutant strain of Chlamy that cannot attach flagella. In subsequent experiments, Dr. Armbrust identified the wild-type version of the affected gene. From the sequence data that have been obtained, the genomic DNA appears to code for an H+-ATPase. Earlier this year, the wild-type gene was divided into two halves and was cloned into two different plasmids. Using the two halves, I constructed several intermediate plasmids. I used restriction enzymes to cleave the DNA at specific sites and then either religated the remaining plasmid or inserted the cleaved section into another plasmid. I also used PCR to add about 500 bp. to the gene's 3' end to include the polyadenylation signal. Although I did not combine the two halves to form the final constructs, I successfully shortened the two halves and cloned other constructs that were sequenced. The ultimate goal of the project is to transform the final wild-type constructs into the mutant Chlamy cells; this will tell us 1) if the wild-type genomic DNA can complement the mutation to allow the cells to mate and 2) how long the genomic DNA needs to be to do this.
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