Cloning of "Animal Cryptochrome" cDNA from the Model Organism <i>CHLAMYDOMONAS REINHARDTII</i> for Functional Analysis of Its Protein Product

<i>reinhardtii</i>, a unicellular green alga, is a model organism to study the circadian clock. Cryptochromes are the blue light photoreceptors that entrain the clock in some organisms. The CPH1 protein of <i>C. reinhardtii</i> resembles the cryptochromes of the plant model <i>Arabidopsis</i>, but whether CPH1 entrains the circadian clock in <i>C. reinhardtii</i> is not yet known. Recent reports have suggested the existence of one more cryptochrome in <i>C. reinhardtii</i>, which resembles the cryptochromes of animals. However, the amino acid sequence of this protein shows even higher sequence similarity with the 6-4 DNA photolyase of <i>Arabidopsis</i>. DNA photolyases are involved in the repair of UV light-induced DNA damage using the energy of blue light. In order to determine, if the “animal cryptochrome” gene of <i>C. reinhardtii</i> actually encodes a 6-4 DNA photolyase rather than a photoreceptor, an experimental design was developed to test whether the protein product is able to rescue an <i>E. coli</i> mutant defective in its DNA photolyase gene. The design is as follows: In a first step, the coding region of the “animal cryptochrome” cDNA is cloned. In a second step, the cDNA is inserted in-frame into an <i>E. coli</i> expression vector. In a third step, the construct is transformed into an <i>E. coli</i> photolyase mutant, its expression induced, and the strain tested for better survival after UV light exposure. To accomplish the first step, the cloning of “animal cryptochrome” cDNA, total RNA was successfully extracted from <i>C. reinhardtii</i> 4 hrs into the light phase of a 12 h light/12 h dark cycle and reverse transcribed into cDNA using oligo(dT) primers. After initially unsuccessful attempts at amplifying animal cryptochrome from cDNA or genomic template with a variety of primers and conditions, a short fragment with the expected size of 186 bp was amplifiable with both templates. However, even this fragment was not reliably obtained in every PCR assay. Because of this difficulty, real-time PCR was finally performed in the presence of DMSO (Dimethylsulfoxide) and Betaine. These two adjuvants were reported to improve amplifications particularly for GC-rich templates. <i>C. reinhardtii</i> DNA is especially GC-rich with an average of 64% Gs and Cs. The improved conditions allowed the reliable amplification of the 186 bp fragment from genomic template. It also enabled the amplification of a larger fragment of 528 bp from the same template. The results suggest that a combination of 5% DMSO and 1M Betaine is optimal for the amplification of <i>C. reinhardtii</i> DNA and thus can serve as the basis for successful amplification of the entire 1788 bp coding region of the animal cryptochrome cDNA.

Name thesis:degree_name

Master of Science

Discipline thesis:degree_discipline

Department of Biology

Year

2009

Author dc:creator

• Silparasetty, Shobha Lavanya

Contributors dc:contributor

• Dr. Sigrid Jacobshagen (Director), Dr. Claire A. Rinehart, Dr. Cheryl Davis

• genomics
• circadian rhythm
• biological chemistry
• Biochemistry
• Biochemistry, Biophysics, and Structural Biology
• Cell Biology
• Genetics and Genomics

Repository record dc:identifier

https://digitalcommons.wku.edu/theses/117

OAI identifier oai:identifier

oai:digitalcommons.wku.edu:theses-1117