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Molecular Biology

Novel Glucose-6-Phosphate-Dehydrogenase-6-Phosphogluconolactonase Bifunctional Enzyme in Giardia Lamblia and Trichomomas Vaginalis

Thomas Dixon ('11); Andre Cavalcanti

The pentose phosphate pathway helps create reducing power and nucleotides both which are necessary during the infectious stages of many rapidly -duplicating pathogens. The first two enzymes of this pathway, Glucose-6-Phosphate-Dehydrogenase and 6-Phosphogluconolactonase, have been known to be fused in Plasmodium falpicarum since 2001. However, using BLAST searches we have found expressed sequence tags that show this fusion to also be present in Giardia lamblia and likely Trichomonas vaginalis. To determine the origin of this gene fusion we obtained 61 protein sequences for G6PD and 6PGL and aligned the sequences using Clustalx, cut non-homologous regions of the sequences using Jalview, and ran MrBayes v3.1 to resolve a phylogenetic tree. Since Giardia lamblia and Trichomonas vaginalis grouped with the prokaryotes rather than the eukaryotes in this analysis, we determined that both organisms obtained their G6PD genes from a lateral gene transfer event from an ancient prokaryote.
Funding provided by: Pomona College SURP

Synthetic Phenolic Compound Bha Causes a Significant Increase of Single-Strand Breaks in S. Cerevisiae 

Christian Gallardo ('10); Tina Negritto; Cynthia Selassie

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 (DSBs). Our preliminary PFGE results show that BHA, BHT, and 4-methoxyphenol (4-MP) do not induce DSBs in yeast chromosomes. To determine single-strand break (SSB) formation, an alkaline gel electrophoresis method was adopted, which showed a 12-fold increase in BHA-induced SSBs. Interestingly, BHA-treated mutant strains for the apn1/apn2 genes showed a marked reduction in SSBs compared to WT, suggesting an involvement of the base-excision repair pathway in the repair of BHA-induced DNA damage.
Funding provided by: Merck Institute for Science Education and AAAS

Reverse Aging?

Janice Joo ('11); Laura Hoopes

Saccharomyces cerevisiae can be induced into meiosis by nutrient starvation and then restored to mitotic growth when returned to rich media. Our lab has found that when aged 8 generation cells are induced into meiosis and then returned to mitotic growth, their life spans resemble those of young cells. This suggests that the aging clocks of the 8g cells are meiotically reset. Life span analyses indicate that the reset happens between 1 and 4 hours in meiosis. We hypothesize that the reset mechanism happens during DNA replication, but analysis using BrdU incorporation and attaching fluorescent antibodies has not yet yielded sufficient data to conclude that the time of DNA replication is between 1 and 4 hours.
Funding provided by: The Elgin Fund for Summer Student Research

Foci Appearance and Disappearance Following Induced UV Damage in Yeast Saccharomyces Cerevisiae

Afshin Khan ('11); Laura Hoopes

Nucleotide Excision Repair (NER) of DNA lesions requires the Rad1-Rad10 endonuclease. We measured intracellular concentrations of GFPRad1 in yeast with Rad1-GFP and Sik1-RFP. Delivery of 55 J of UV was followed by time-lapse examination in a) Untreated cells b) Cells treated with tetracycline to prevent production of new GFP-Rad1 c) Cells synchronized with Nocodazole. It was observed earlier that (4±1) foci per cell appear during the half hour after UV radiation. Time lapse microscopy of individual untreated cells attached to Concanavalin A treated slides showed foci of varying intensity in 90% at every interval examined. Cells treated with tetracycline showed foci at 4 min in 46% of cells, decreasing to 31% after 8 min. Synchronized cells arrested with nocodozole in G2/M and later released for 3 hours at the same phase in cell cycle show foci appearance in almost 100% of cells which decreases to 20% within 7 minutes.
Funding provided by: Pomona College SURP

A Fusion of the Subuntis Alpha and Beta of Succinyl-Coa Synthetase Can Be Used as a Phylogenetic Marker of Pezizomycotina Fungi

Amanda Koire ('13); Andre R.O. Cavalcanti

Gene fusions, yielding formation of multidomain proteins, are rare evolutionary events that can be utilized as phylogenetic markers. We described a fusion gene comprising the alpha and beta subunits of succinyl coA synthetase in Pezizomycotina fungi. Succinyl-CoA synthetase is an enzyme of the citrate cycle and catalyzes the reversible transformation of succinyl CoA, orthophosphate, and GDP to succinate, GTP, and coenzyme A . The fusion is present in all Pezizomycotines with complete genome sequences and is absent from all other organisms. A phylogenetic tree for each subunit was created on MrBayes v3.1.2 and through Maximum Likelihood (PhyML) analysis on TOPALi v.2.5. Both forms of analysis supported the monophyly of the fusion copies separate from nonfused copies, suggesting that a duplication of each subunit in a Pezizomycotina ancestor preceded the fusion event. These results support that the fusion can be used as a molecular marker for membership in the Pezizomycotina group.
Funding provided by: Howard Hughes Medical Institute

Biochemical Characterization of Novel Homing Endonucleases

Katie Soe ('10); Lenny Seligman

Encoded within self-splicing introns or protein inteins, homing endonucleases (HEs) target and form double-strand DNA breaks at specific 14 to 40 base pair homing sites. We have studied six putative HEs from homologous hyperthermophilic archaea Pyrococcus abyssi (Pab) and Pyrococcus furiosus (Pfu). Two of these enzymes, PfuVMA and PabVMA, demonstrated activity in vitro. Their specificities appear flexible, as each cut not only its own, but also the other’s (homologous) recognition site. These two sites differ by 10 base pairs along their 30 base pair sequences. PabVMA demonstrated increased endonuclease activity with increasing protein concentration and temperature, while PfuVMA appeared unaffected by changes in temperature.
Funding provided by: Pomona College SURP

Para-Substituted Phenols Induce DNA Damage in Saccharomyces Cerevisiae

Clarissa Valdez ('10); Jasmine Walia (’09); Cynthia Selassie; Maria Christina Negritto

Phenols are used in a wide variety of household products, but their potential to form radicals has led to controversy over their possible toxicity and carcinogenic properties. This project examines how phenols induce DNA damage in S. cerevisiae through two different methods: the inhibition of cellular growth (IC) and increase in homologous recombination. Decrease of cellular growth due to phenols was measured by obtaining IC 20, 50, and 80 values, indicating 20%, 50%, and 80% cell death. Yeast cells are then treated at these values and the level of DNA damage determined indirectly in a DEL Assay. The Del Assay measures the frequency of deletion events due to homologous recombination between direct repeat sequences. The frequency obtained indirectly measures the amount of DNA damage induced by the phenols. Results indicate that Methoxyphenol, Hydroquinone, Phenol and BHA cause DNA damage in Saccharomyces cerevisiae.
Funding provided by: Merck Institute for Science Education and AAAS

Selection of Yeast Aging Genes

Jocelyn Young ('11); Laura Mays Hoopes

Identification of genes involved in the aging of Saccharomyces cerevisiae may be accomplished through the use of bar code strains, a collection of deletion mutants in which each gene is replaced by a unique bar code of DNA flanked by two PCR handles that are utilized to amplify the bar code. By comparing the DNA barcodes from pre- and post-aging strains on a microarray, deletion strains that are being depleted and those being enriched over the aging process may be determined. To gain more insight, lifespan dissections were completed for ACE2, MAC1, ELM1, and SRB2, genes found to be considerably affected by aging. The ELM1 deletion strain appears to have experienced a decreased lifespan while the MAC1 deletion strain seems to have experienced an increased lifespan based on the predicted lifespan of the wild-type strain for the mutants. Thus, ELM1 and MAC1 may play significant roles in yeast aging.
Funding provided by: Wig Grant (LH)

Research at Pomona