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Molecular Biology

Genes that control vesicular transport in Drosophilia

Ayabe, Reed ('09);  Cheney, Clarissa; M.Rab GT

Abstract removed upon quest.

The Hydra Viridissima-Chlorella Symbiosis: Evidence for Multiple Algal Replacements

Chen, Diana ('09);  Martinez, Daniel; Iniguez, Abril ('08)

Freshwater cnidarians of the genus Hydra can be divided into two main groups, green and brown hydra. Green hydra (Hydra viridissima) contain endosymbiotic algae, presumably of the genus Chlorella. Brown hydra lack algal endosymbionts. The origin of the hydra-algal symbiosis is presumably ancient but its true evolutionary history is unknown. Potential evolutionary hypotheses involve one or several endosymbiotic origins and potential algal replacements after an initial symbiotic establishment. To test these hypotheses we are building molecular phylogenies for the green hydra and their endosymbiotic algae using the genomic sequences of the Internally Transcribed Spacers 1 and 2 of nuclear ribosomal RNA and the small subunit ribosomal RNA, respectively. Our preliminary results suggest that: 1. Most of the green hydra endosymbionts belong to the genus Chorella sensu stricto;  2. Algal replacements have occurred in a few cases;  and 3. Even though replacements do occur, symbiosis tends to be relatively stable.
Funding provided by: Rose Hills Foundation;  The Schenk Fund

Engineering I-CREI to Recognize Trinucleotide Repeat: Exploring the Potential for Gene Targetting

Cohen, Andrea ('09);  Seligman, Lenny

The homing endonuclease I-CreI binds and cleaves a specific 22 base pair DNA sequence. Previously, work has been done in Seligman lab to attempt to engineer a derivative of I-CreI that cleaves CAG repeat sequences, the expansion of which leads to some trinucleotide repeat diseases including Huntington's disease. We aim to expand our investigation of I-CreI by engineering derivatives that target GAA repeats, which underlie Friedrich's ataxia. Mutant I-CreI proteins that are predicted to target DNA sites containing GAA repeat sequences have been produced. To verify that the engineered proteins function as predicted, cognate DNA sites were also made. None of the predicted target sites tested to date appear to be efficiently recognized by corresponding mutant proteins.
Funding provided by: Howard Hughes Medical Institute

Synthetic Phenolic Compounds BHA and BHT are Not a Significant Cause of Double-Strand Breaks in S. Cerevisiae

Gallardo Garcia Freir, Christian ('10);  Negritto, Tina; Selassie, Cynthia

Students working in the Negritto/Selassie collaborative project have shown that treating S.cerevisiae with synthetic phenolic compounds results in higher recombination frequencies indicative of DNA lesions. In addition, Goldman (‘08) has shown that BHA and BHT generate strand breaks in yeast, possibly as a result of apoptosis. We used Pulsed-field gel electrophoresis (PFGE) to assess if phenol-induced DNA lesions manifested as double-strand breaks. PFGE is a technique that separates individual chromosomes into distinct bands via alternating electrical field. Our preliminary PFGE results fail to identify BHA or BHT as compounds that induce double-strand breaks in yeast chromosomes. Southern Blots of our PFGE gels will be used to assert the validity of these results. We hypothesize that the higher frequencies of recombination observed in yeast cells treated with these compounds could be due to the generation of single-strand breaks, which cannot be visualized via regular PFGE. A modified PFGE technique will be used in which DNA is denatured in order to determine the existence of single-strand breaks.
Funding provided by: Pomona College SURP

Investigating the Role of RAD3 in DNA Double Strand Break Repair in Saccharomyces Cerevisiae

Liu, Tiffany ('09);  Negritto, Tina

In S. cerevisiae, DNA double strand breaks (DSB) are usually repaired by homologous recombination. However since there exist many repeated sequences throughout a genome, spontaneous recombination of short repeated sequences (SSR) can also occur. SSR can lead to chromosomal abnormalities such as translocations, inversions, and deletions that can lead to cell death. Since these abnormalities do not seem to be frequently caused by spontaneous recombinations within the cell, there must be a cellular response that can activate HR and suppress SSR events at double strand break sites. It has been shown that Rad3 helicase, a subunit of the TFIIH transcription factor protein complex, suppresses short sequence recombination. In this study, we propose to find the molecular mechanism by which Rad3 plays this role. To study the direct effects, we used chromatin immunoprecipitation (ChIP) assays to see if Rad3 physically localized to DSB ends.
Funding provided by: Howard Hughes Medical Institute

Understanding the Interactions Between the Three R's: Resveratrol, the Retrograde Response, & ROS

Lykken, Erik ('09);  Hoopes, Laura

Two overarching goals exist for this study. (1) Determine if any explicit link exists between resveratrol’s lifespan extension capability and the retrograde response. Lifespan determinations for strains with deletions in RTG2 and RAS2 will provide the evidence for such a link. (2) ROS profiling to assess the impact of resveratrol, aging, the retrograde response, and all combinations of the three. Flow cytometry measurements of DCF fluorescence provide low sensitivity profiles but many more observations. ESR spectra will provide high sensitivity, quantitative ROS profiles. Actual experimentation has proved challenging. However, after reworking the flow cytometry protocol reproducible data were obtained and suggest that high concentrations (100μM) of resveratrol increase the mean fluorescence (1.2-1.5 fold) and the number of cells fluorescing in the second decade (2-2.5 fold). Little else can be said as much remains for future investigation.
Funding provided by: The Paul K. Richter and Evalyn E. Cook Richter Award

Discovery and Characterization of Novel Homing Endonucleases from Extremophilic Archaea

Soe, Katherine ('10);  Seligman, Len

Homing endonucleases (HEs) are enzymes that target long, specific 14 to 40 base pair homing sites where they create double-stranded DNA breaks. Six putative HEs from three pairs of homologous genes in the hyperthermophilic archaea Pyrococcus abyssi (Pab) and Pyrococcus furiosus (Pfu) are studied to determine whether they are functional in Escherichia coli. The results of this study may help us better understand the role of these HE genes in early genome evolution, as well as the biological dissemination of protein inteins. In vivo assays illustrate protein activity against the cognate target site for four of the six HEs studied, one of which shows temperature-dependent activity and another of which also appears toxic. A fifth HE demonstrates activity, manifested as toxicity, elsewhere in the E. coli genome.
Funding provided by: Merck Institute for Science Education and AAAS

Effects of Para-Substituted Phenols in Saccharamyces Cerevisiae

Walia, Jasmine ('09);  Valdez, Clarissa ('10); Negritto, Tina;  Selassie, Cynthia

This project looks into how phenols induce DNA damage in Saccharomyces cerevisiae. Two different methods are used to measure the effect of the phenols. One measures the inhibition of cell growth and the second, the induction of homologous recombination due to DNA damage caused by the phenol. This project focuses on obtaining the IC (inhibition of cell growth) 20, 50, and 80 values, which indicates there is an 80%, 50%, and 20% cell death (respectively). Using the IC values the level of DNA damage caused by each phenol is assayed using a DEL assay. In this assay the frequency of homologous recombination between two his3 repeat sequences flanking a URA marker to generate a functional HIS3 gene is determined. The frequency obtained is then used as an indirect measure of the amount of DNA breaks induced in the yeast genomic DNA of the cells treated with the different phenols.
Funding provided by: Merck Institute for Science Education and AAAS (JW);  Howard Hughes Medical Institute (CV)

Research at Pomona