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Molecular Biology

Cheese as a model microbial ecosystem: Iron in rind community dynamics

Miriam Shiffman (2013); Additional Collaborator(s): Rachel Dutton*; Mentor(s): Julie Button*
*Harvard University, FAS Center for Systems Biology

Abstract: The rind of cheese is comprised of a dynamic community that may serve as an experimentally tractable model to study microbial ecosystems. Iron, a requirement for almost all life, is scarce in the rind environment. In order to assess the role of iron in the rind community, I screened 28 representative cheese isolates for production of siderophores. Additionally, I monitored the development of a five-species community on cheese curd agar under a variety of iron-poor and iron-rich conditions. I further investigated specific interspecies interactions within this community using spot-on-lawn and supernatant disc-on-lawn assays. Through these methods, I identified multiple microbes from diverse genera as siderophore producers, and demonstrated that iron availability impacted development of a model natural rind community. I also observed potential siderophore piracy, as well as toxin production, among members of this community. These results help illuminate the complex effects of nutrient limitation on the development of a microbial community over time.
Funding Provided by: FAS Center for Systems Biology; NIGMS grant P50GM068763

Structural and Functional Analysis of Related Enzymes G6PD and H6PD

Lauren Kershberg (2015); Student Collaborator(s): Aaron Altman (2013); Mentor(s): Andre Cavalcanti

Abstract: Glucose 6 Phosphate Dehydrogenase (G6PD) and Hexose 6 Phosphate Dehydrogenase (H6PD) are closely related enzymes that perform a similar function in vertebrate organisms. Both enzymes are involved in the pentose phosphate pathway and they produce NADPH. In a search for the difference both structurally and functionally between these two enzymes we have determined that G6PD and H6PD posses key similarities and differences within their amino acid sequence that suggest the presence of a structural glycollate ion in both enzymes and similar coenzyme functioning but differing substrate binding sites. Attempts at localizing the two enymes within the cell have been thus far unsuccessful, but in time we hope to support our hypothesis that H6PD is located within the ER and G6PD in the cytosol.
Funding Provided by: Pomona College Biology Department (LK); Kenneth T. and Eileen L. Norris Foundation (AA)

Analysis of Protein Length for Organisms with Differing Stop Codon Number

Samira Nedungadi (2013); Mentor(s): Andre Cavalcanti

Abstract: The standard genetic code contains 3 stop codons; some organisms reassign one or more of these stop codons to sense codons. This study examines the hypothesis that organisms utilizing codes with fewer stop codons should have, on average, longer proteins. Homologous proteins were identified among ciliates, which utilize 1 stop codon, and related protists that utilize 3 stop codons. For each organism, the mean length of these homologs were compared, and a nested ANOVA was used to determine whether stop codon number had a significant effect on mean protein length. It was found that while mean protein lengths varied significantly from organism to organism, stop codon number does not appear to contribute to this variation. For each pair of organisms, a test of proportions was also used to determine if one organism's proteins were consistently longer than the other's. This test also showed no correlation between protein length and stop codon number. Further studies will include expanding the examined dataset to include other organisms that reassign stop codons, such as Mycoplasma.
Funding Provided by: Sherman Fairchild Foundation

Genes that control vesicular transport in Drosophila - various research projects

Jessica Te-Ling Chiang (2013); Mentor(s): Clarissa Cheney

Vivian Chou (2013); Mentor(s): Clarissa Cheney

Will Evenson (2014); Mentor(s): Clarissa Cheney

Brian Fung (2013); Mentor(s): Clarissa Cheney

Howard Lee (2014); Mentor(s): Clarissa Cheney

Jin hoo Lee (2013); Mentor(s): Clarissa Cheney

Abstracts withheld upon request

Functional Characterization of Genomic Variants in CD4+ T Lymphocytes and CD14+CD16- Monocytes

Joseph Replogle (2013); Additional Collaborator(s): Barbara Stranger*; Towfique Raj*; Mentor(s): Johanna Hardin
*Harvard Medical School

Abstract: Genome-Wide Association Studies (GWAS) reveal a complex genetic basis underlying autoimmune diseases, immunologically-mediated diseases defined by an inflammatory immune response against self tissues, but fail to illuminate mechanisms by which genomic variants influence disease. To this end, we integrate GWAS and transcription data to provide novel insights into the function of genomic variants in primary immune cells, CD4+ lymphocytes and CD14+ monocytes. As ubiquitous genomic regulation of transcription has been previously characterized in expression quantitative trait loci (eQTL) studies, we assess the association between intragenic variants and alternative splicing to identify splicing quantitative trait loci (sQTL). We find 52 genes with significant SNP-exon correlations and replicate experimentally-verified sQTLs in OAS1 and CAST. Performing an overlap between our sQTLs and the GWAS Catalog, we find that rs1143674, a SNP associated with autism risk, correlates with alternative splicing of ITGA4. Future work hopes to replicate this finding in other cell-types and populations.
Funding Provided by: Paul K. Richter and Evalyn E. Cook Richter Memorial Funds; Harvard-MIT HST BIG

Characterization of the yeast mutants rad3-A596P and rad3-R660C

Gregory Elison (2013); Mentor(s): Tina Negritto

Abstract: The protein complex TFIIH, or transcription factor IIH, is known to be a critical part of the nucleotide excision repair (NER) pathway which repairs mainly UV damage to DNA. Several human disorders are caused by mutations in various subunits of TFIIH. Two mutations known to cause disease in humans had previously been created in S. cerevisiae, and we show that these mutations cause sensitivity to oxidative DNA damage in yeast using a survival assay. In addition to its role in NER, our lab has shown that TFIIH plays a direct role in double strand break (DSB) repair via recruitment to an induced DSB. Our goal is to determine if these mutations in rad3 change the recruitment pattern of TFIIH to a DSB by performing a chromatin immunoprecipitation (CHIP). A rad4 null strain has also been prepared and will be assayed for its potential role in recruitment of TFIIH to DSBs.
Funding Provided by: Jack Steller Summer Curriculum Enhancement Endowment

Designing a protocol to study the role of Rad3 in nucleotide excision repair

Ted Gkoo (2015); Mentor(s): Tina Negritto

Abstract: Mutations in the human XPD protein can cause numerous fatal diseases resulting from deficiencies in DNA repair. We worked to develop a protocol that allows us to to elucidate the role of Rad3, a yeast homolog of XPD, in Nucleotide Excision Repair (NER) of UV damage in DNA. Studying Rad3 mutants can help clarify how XPD is involved in NER. Through immunoblotting, we tested different Rad3 mutant strains for their ability to remove Thymine Dimers (TDs) caused by UV exposure. Yeast genomic DNA from cells exposed to UV was extracted, immobilized on a nylon membrane and detected with a primary antibody specific for TDs . The primary antibody was then detected with a secondary antibody fused to a horse radish peroxidase enzyme that cleaves a chemiluminescent substrate. This protocol will enable the quantification of TD removal over time in different mutant and WT strains.
Funding Provided by: Jack Steller Summer Curriculum Enhancement Endowment

The role of TFIIH, a nucleotide excision repair protein, in DNA double strand break repair: Analysis of the Rad3 and Kin28 subunits

Tori Holtestaul (2013); Mentor(s): Tina Negritto

Abstract: We are investigating the recruitment of TFIIH, a nucleotide excision repair protein, to the site of DNA double strand breaks in S. cerevisiae. Rad3, one subunit of TFIIH, has a human homologue, XPD, that is susceptible to mutations which can lead to xeroderma pigmentosum and tricothiodystrophy, diseases involved in skin cancer and mental retardation, respectively. The presence of Tfb1, a subunit of the TFIIH complex, has already been proven at the double strand break. We are seeking further confirmation of TFIIH’s recruitment through the presence of Kin28 and Rad3, two additional subunits, at the break site. In doing so, we will utilize chromatin immunoprecipitation, a technique involving the fixing of protein to DNA, precipitation of the protein using a specific antibody, and subsequent analysis of the DNA and protein present in the precipitated fraction. So far, we have optimized conditions for experimentation regarding Kin28, and shown that Rad3 may not be a viable protein to immunoprecipitate due to difficulties in tagging.
Funding Provided by: Jack Steller Summer Curriculum Enhancement Endowment

Optimizing the Detection of Short-Sequence Recombination by Insertion-Deletion Assay in Haploid S. cerevisiae Rad3 Targets

Johan Sebastian Martinez (2013); Mentor(s): Tina Negritto

Abstract: Yeast DNA helicase Rad3 from Saccaromyces cerevisiae has roles in transcription and DNA repair pathways and is homologous to human XPD, mutants of which are known to cause recessive genetic disorders such as Xeroderma Pigmentosum. Previous studies have shown that in an insertion deletion (ID) assay rad3-G595R mutants conduct higher levels of short-sequence recombination (SSR) than wild-type (WT) cells; however, the number of target sequences available for the repair process to occur in these assays seem to affect the sensitivity of the assay. We hypothesized that limiting the number of targets available for the repair process would yield a more sensitive ID assay and higher levels of SSR than previously recorded in rad3-G595R mutants. In our methodology, we constructed novel haploid rad3-G595R and WT yeast strains containing the ID targets sam2::his3::ura3::KANr::ura3::his3::sam2 and HIS3. The assay then consists of transforming a 1.2 kbp fragment of a URA3 gene flanked by a total of 127 bp of HIS3 homology. The fragment, once inside the haploid, will only have two potential targets with which it shares homology: a short one (HIS3 locus) and a long one (SAM2 locus). Relative rates of SSR can be determined by dividing the number recombinants at the HIS3 locus by the number of recombinants at the SAM2 locus. The ability to assay DNA repair proteins such as Rad3 44 emphasizes yeast as a model organism for elucidating the detailed mechanisms of human genetic disorders.
Funding Provided by: Pomona College SURP

Cytotoxicity of Butylated Hydroxyanisole, Butylated Hydroxytoluene, and Bisphenol A in S. Cerevisiae

Joshua Rothman (2013); Mentor(s): Tina Negritto

Abstract: Phenolic compounds, which exist in nature as phytochemicals, are commonly used in the industrial synthesis of consumer products. Previous studies have made it difficult to determine the health effects of exposure to phenols because some evidence suggests that they have protective properties while other evidence suggests that the phenols themselves cause DNA damage. In this study, we evaluate the cytotoxic effects of bisphenol A (BPA), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), three phenols to which humans are commonly exposed. We found that yeast cells treated with BPA, BHA, and BHT all had an increase in the number of Rad52 foci compared to control. This result suggests that DNA breaks were induced more frequently when yeast cells are exposed to phenols. To understand the cellular targets and the pathways involved in the response to phenol toxicity we screened for growth defects in DNA repair mutant strains. Our results show that phenols are targeting DNA and causing damage. We also confirmed that some mutant strains are more affected by phenol exposure than others. DNA repair mechanisms are similar in yeast and humans and thus the findings of this study will help identify the repair mechanisms involved in repairing phenol damage and understand the potential detrimental effects phenols can have on health.
Funding Provided by: The Rose Hills Foundation

Characterization of the Iron Transport Proteins NFeoB and FeoA in Shewanella Oneidensis

Kevin Wang (2013); Mentor(s): Matthew Sazinsky

Abstract withheld upon request

Selectively Mutating Residues in Toluene Monooxygenase to Enhance Terminal Hydroxylation of Alkanes

Jieyi Zhuang (2014); Mentor(s): Matthew Sazinsky

Abstract withheld upon request

Investigation into the relationship between structure and activity of flavonoids and BHT derivatives

Stella Deng (2013); Mentor(s): Cynthia Selassie

Abstract: This study uses DPPH and MTT assays in a structure-activity approach to determine the features that enhance the antioxidant and/or toxicity behaviors of two sets of compounds, including a series of four flavonoids with different numbers of hydroxyl groups and a series of 22 phenolic compounds that are derivatives of butylated hydroxytoluene (BHT). With the four flavonoids, antioxidant activity increased with greater numbers of hydroxyl groups. For the BHT derivatives, a quantitative structure-activity relationship model was calculated: log "1" ⁄"EC50" = -1.301σ+ + 1.921. This shows that electronics, represented by by σ+, appears to be the largest determining factor in antioxidant activity as compounds with electron-donating groups displayed much greater antioxidant activity than those with electron-withdrawing groups. Future experiments involve continuing cytotoxicity assays to observe the effects of flavonoids on human leukemia cells. Preliminary results have shown that high concentrations of quercetin increase cell growth while small concentrations appear to be inhibitory.
Funding Provided by: Kenneth T. and Eileen L. Norris Foundation

Functional Characterization of the Novel Pyrococcus horikoshii Lon Homing Endonuclease

Frances Hundley (2013); Mentor(s): Lenny Seligman; Matthew Sazinsky

Abstract withheld upon request

Characterization of Two Novel Homing Endonucleases, Can They Make the Cut?

Nicholas Murphy (2013); Mentor(s): Lenny Seligman

Abstract: Homing endonucleases (HEs) are selfish genetic elements found in inteins of conserved genes. Inteins are peptides that post-translationally excise themselves from an extein. HEs target 14-40 bp DNA recognition sites for cleavage; this target site is in the middle of a gene conserved across species. Once cut, cells repair cleaved DNA by using a homologous template, which results in copying the allele with the intein from which the HE came. HEs generally act as harmless parasites, inserting themselves into genomes without causing trauma to the cell. They are able to recognize and cleave target sites outside of the species from which they come, however, they must not be too promiscuous as this might result in interrupting vital proteins and incapacitating a cell. Thus, there is a minimal recognition site that the HE will recognize so that it can continue to invade new species without nonspecifically cutting important genes. Tko, Mja, and Maeo helicase-a HE’s share a common insertion site in the helicase-a gene. We have isolated and expressed these HEs to characterize their targeting. Tko and Mja have both shown cleavage activity on their native sites while Maeo has not exhibited cutting activity. Mja is less promiscuous in its cutting than Tko which cuts the three native sites as well as the hypothetical insertion sites in Pfu, Pab and Pho. We are currently determining the minimal recognition sites for Mja and Tko. 
Funding Provided by: Sherman Fairchild Foundation; Pomona College Molecular Biology Program