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Molecular Biology

UV and H2O2 Sensitivity of Yeast Mutants RAD3-A596P and RAD3-R660C

Rocco Anthony Addante ('08), Tina Negritto

Mutations in human XPD, a subunit of TFIIH, can lead to the diseases XP, TTD, and CS, all of which exhibit increased susceptibility to cancer. We studied two novel mutations in Rad3, the yeast homolog of XPD, by exposing mutant strains to hydrogen peroxide and ultraviolet (UV) radiation. We found that rad3-R660C, a mutation in yeast that is homologous to a mutation found in TTD patients, increases UV sensitivity, but not hydrogen peroxide sensitivity. In contrast, rad3-A596P, which also shares a human homolog in TTD patients, does not lead to increased UV or hydrogen peroxide sensitivity. Further analysis of transcription levels and DNA stability by microarray and southern blot of yeast rad3 mutants may be instructive.
Funding provided by: HHMI

The ΔMSN2ΔMSN4 Mutant of Saccharomyces Cerevisiae Posseses a Shortened Lifespan

Eleanor Ruth Cameron ('08), Laura L.M. Hoopes

We hypothesized that mutant cells lacking the msn2 and msn4 genes would possess a shorter average lifespan than wild-type (WT) cells, as msn2 and msn4 show increased expression with age in wild type cells and have roles in vital processes such as stress response and metabolism. To test this, we performed lifespan analyses on both WT and mutant cells. We found the average lifespan of Δmsn2Δmsn4 mutant cells to be about 10.8 generations (g), while the WT cells possessed an average lifespan of about 17 g. This data agrees with our previous microarray data, analysis of which showed similarities between the 8 g Δmsn2Δmsn4 mutant and the 18-20 g wild type, and differences between the 8 g mutants and the 8 g wild type. We also saw in the mutants specific changes in gene regulation that are known to correspond with aging in S. cerevisiae. All of this implicates that Δmsn2Δmsn4 mutant cells age faster at the genetic level than wild type cells, and therefore also possess a shortened average lifespan.
Funding provided by: SURP (Richter)

Identification and Characterization of Novel Endonucleases

Diana Artour Koulechova ('08), Diana Chen ('07), Len Seligman, E.J. Crane

Homing endonucleases (HEs) induce double stranded DNA breaks upon recognition of a homing site, which can be anywhere from 12 - 40 base pairs in length. Previous work by our lab had been done with Cpa and I-CfrI, HEs coded for by introns within the chloroplast DNA of two Chlamydomonas species. Furthering this work, we clarified previously ambiguous observations about the activities of several mutant proteins and confirmed the optimal target site length for the wild-type protein. We made and assayed several Cpa double mutants, with inconclusive results. We also identified six archaeal HEs, consisting of three pairs of homologues between two related species, Pyroccocus abyssis and Pyroccocus furiosus, and are currently working to characterize these proteins.
Funding provided by: Merck/AAAS

Genes that control vesicular transport in Drosophila

Simiao Li ('08), C.M. Cheney

Abstract removed upon request.

Flavonoid Cytotoxicity: An Experiment in Troubleshooting

Erik Allen Lykken ('09), Tina Negritto, Cynthia Selassie

The overarching goal of this experiment is the determination of IC50 (50% growth inhibition) values for several different flavonoids. Growth assays are conducted in 96 well plates, and a spectrophotometer is used to determine optical density (O.D.), which is then correlated to growth. The data is then manipulated (backgrounds subtracted out, multiplied by the dilution factor, and normalized to data point [flavonoid]=0) and killing curves subsequently produced, plotting the normalized O.D. readings against the log([flavonoid]). IC values are then extracted from such curves. These values find further use as test concentrations in a DEL assay, which assesses the level of DNA damage through a recombinant phenotype (HIS3+). Actual experimentation proved challenging as flavonoids are not particularly soluble under yeast assay conditions. However, after devising new controls and reworking the protocol, some incomplete killing curves were produced for Quercetin, Morin, and (-) Epicatechin. ICs calculated from these curves are not wholly reliable as precipitation allows for unknown treatment conditions. However, a pilot DEL assay performed with Morin showed promise of significant (2-fold+) damage at IC50+. Little else can be said as much remains for future investigation.
Funding provided by: Merck/AAAS

Construction and Analysis of Mutant DNA Cognate Sites for the Homing Endonuclease I-CREI

Hilary Sarah Parker ('08), Len Seligman, John Kloke

I-CreI is a homing endonuclease that is known to fully cleave its cognate DNA site of 22 base pairs. Protein crystallography of I-CreI with its DNA substrate shows that the protein does not make contact with the middle-four bases of the cognate site. I-CreI is also known to bind irreversibly to its DNA substrate. Previous work by Michael Brown (PO ’07) has shown that mutations in these middle four bases do affect cleavage. In this study, we further examined the effects of middle-four site mutations on the efficiency of I-CreI cleavage. First, we built a larger library of middle-four mutation sites that hinder I-CreI cleavage. Second, we used in vitro methods to study the cleavage efficiency of certain middle-four mutation sites, and analyzed them using logistic regression. We found that for all the middle-four mutation sites we studied, the parameters for the logistic regression differed significantly from those for the non-mutant strands. Finally, we studied the reversibility with which I-CreI binds to these mutant sites. We were able to find that I-CreI bound irreversibly to a middle-four mutant that it cleaved fully in vivo, and bound reversibly to a middle-four mutant that it cleaved only partially in vivo.
Funding provided by: HHMI

Extending the Reach of I-CREI: Furthering the Therapeutic Potential of a Homing Endonuclease

Laura Elizabeth Rosen ('08), Len Seligman

I-CreI is a homing endonuclease from the green algae Chlamydomonas reinhardtii that recognizes and cleaves a mostly-palindromic 22 base pair DNA sequence. It is a homodimer and has two DNA-binding domains. I propose that an I-CreI derivative engineered to contain four DNA-binding domains could interact with a longer target site and act with greater specificity than the wild-type enzyme. I-CreI derivatives already have a potential application in gene therapy because of the flexibility of the structure towards mutations, and this project could increase that potential by lengthening the recognizable target site. Towards this goal, plasmids are being created that contain two I-CreI open reading frames connected by linkers of varying length and content. These constructs are being assayed against DNA targets consisting of either two or four half-sites. So far there is evidence that the proteins engineered with four DNA-binding domains can fold correctly and have activity against target sites, but more research is needed to determine if the extra domains will be a help or a hindrance.
Funding provided by: Beckman Foundation

Recombination by Para-Substituted Phenols in Saccharomyces Cerevisiae

Jasmine Kaur Walia ('09), Christa Lovett (‘09), Cynthia Selassie, Tina Negritto

This project looks into how phenols induce DNA damage in Saccharomyces cerevisiae. Two different methods are used to measure the effect of the phenols tested. One measures the inhibition of cell growth caused by the phenol and the other the induction of homologous recombination due to DNA damage caused by the phenol. This project focuses on obtaining IC 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. This is done by using a DEL assay, created by Schiestl et al. In this assay the frequency of homologous recombination between two his3 repeat sequences flanking a LEU 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. For the compounds hydroquinone, p-phenolsulfonamide, and p-n pentyl phenol, the IC values were determined. However, a problem with the strain and solubility may cause for the project goals to shift slightly.
Funding provided by: Rose Hills Foundation

Investigation of FOXO and FOXJ Gene Expression in Hydra Magnipapillata

Darby Jean Walters ('08), Daniel Martinez

The Forkhead (FOX) family of genes consists of an important family of transcription factors that play key roles in development, metabolism, cancer, and aging. Specifically, the Class O Forkhead Box Transcription Factor (FOXO) is an essential protein in the insulin/IGF-1 signaling pathway that is largely conserved in metazoans. FOXO is implicated in DNA repair, cell cycle progression, defense against oxidative stress, apoptosis, and lifespan. In this study, we examine the phenotype that occurs with over expression of FOXO in Hydra magnipapillata. We are creating a plasmid that contains the FOXO gene with an actin promotor and a GFP molecule. In the future, we will inject this plasmid into Hydra eggs and observe the resulting overexpression. Additionally, we are studying the FOXJ gene. Little is known about its transcriptional properties, and only one transcription target has been identified. We are using the in situ procedure to look at the areas of FOXJ gene expression in Hydra in order to further elucidate its role in the aging process. We have currently found that the FOXJ gene is primarily expressed at the base of the tentacles and in the foot of Hydra under normal conditions.
Funding provided by: SURP

Gene Expression Changes and Methylation Genes in Replicative Aging of Sacchoromyces Cerevisiae

Gloria Yiu ('08), Laura L Mays Hoopes

Replicative aging of yeast is asymmetric; the mother ages 1 generation (g) per divsion, but the daughter is produced at 0 g. We have suggested that replication stalls contribute to replicative aging. We now report gene expression at 1,8,12, and 18/20 g. Significant findings were confirmed using RT PCR and data from the two assays agreed in direction of up or down regulation. Statistical analysis shows that just over 1,000 genes change expression during aging and many change progressively. Clustering and pathway analysis (GenMAPP2) show that changes in nucleolus/ribosome pathways, central metabolism, and stress response genes predominate. A novel finding is down regulation of methylation related genes. Via High Pressure Liquid Chromatography (HPLC) of nucleosides, we found that in wild type yeast, methylation of total RNA decreases from 1 g to 8 g and remains low at 12 g. In mutant strains containing one copy of the SAM genes, lifespan studies show no significant difference in aging from wild type strains with two copies of the SAM genes.
Funding provided by: Beckman Foundation (GY)

Research at Pomona