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Molecular Biology

Research Presentation Video

Watch Molly Kupfer '14 discuss her research project.

Ciliate Dynamics: Selection and Senescence

David Morgens (2014); Mentor(s): Andre Cavalcanti

Abstract: Ciliates are single cellular eukaryotes whose unusual life cycle and nuclear dimorphism can give rise to novel dynamics on both an evolutionary and population level. We use simulations and modeling to address the effects on chromosomal imbalances as well as population genetics. This gives us insight into a variety of processes such as protein evolution, effective population size, senescence, regulation, and evolutionary patterns.
Funding Provided by: Howard Hughes Medical Institute

Studies on Vesicle Transport in Drosophila

Alan Chen (2014); Student Collaborator(s): Karen Leung (2014 CMC); Michelle Ozaki (2016 SCR); Additional Collaborator(s): Zhaohua Tang (W.M. Keck Science Center of the Claremont Colleges); Ruye Wang (HMC); Mentor(s): Clarissa Cheney

Abstract withheld upon request.
Funding Provided by: Howard Hughes Medical Institute

Studies on Vesicle Transport in Drosophila

Mary (Molly) Horgan (2014); Mentor(s): Clarissa Cheney

Abstract withheld upon request.
Funding Provided by: Pomona College SURP

Studies on Vesicle Transport in Drosophila

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

Abstract withheld upon request.
Funding Provided by: Corwin Hansch and Bruce Telzer Fund

An Epigenetic Analysis of Apis mellifera

Jingyuan Liao (2015); Student Collaborator(s): Tobin Ivy (2013 HMC); Sherry Zhang (2015 HMC); Mentor(s): Robert Drewell

Abstract: Apis mellifera, the European honey bee, has a caste system consisting of one fertile female queen, fertile males (drones) and infertile female (workers). Differentiation of the three is suspected to be through epigenetic modification of DNA methylation performed by the queen when she lays eggs. Previous work has shown differential DNA methylation between the egg and sperm of A.mellifera. Further DNA methylation analysis of A.mellifera genomic DNA is performed in this project to identify the differential methylated genes (DMGs) involved in worker sterility. However, future work is needed to ensure the proper amplification of our targeted DMGs.
Funding Provided by: Howard Hughes Medical Institute

The Role of PDGF Signaling in Epicardial Cell Activation/Migration and Blood Vessel Formation in Regenerating Zebrafish Hearts after Cryoinjury

Luis Francisco Díaz (2015); Additional Collaborator(s): Jieun Kim (Saban Research Institute, Children’s Hospital Los Angeles); Gayatri Nair (Saban Research Institute, Children’s Hospital Los Angeles); Mentor(s): Ellen Ching-Ling; M. Cristina Negritto

Abstract: Approximately 600,000 people die of heart disease in the United States alone every year according to the Centers for Disease Control and Prevention (CDC). Myocardial infarction (MI) in humans causes severe heart damage leading to scarring of the heart, which leads to impaired cardiac function and eventual heart failure. Unlike mammals, zebrafish have to ability to regenerate their hearts after severe injury, however, the mechanism for this is not well understood. During zebrafish heart regeneration epicardial cells proliferate in a platelet-derived growth factor (PDGF) manner and can undergo an epithelial-mesenchymal transition (EMT) contributing to the formation of blood vessels. In this study we investigated the role PDGF signaling plays in epicardial migration/activation and blood vessel formation during zebrafish heart regeneration after cryoinjury. We used the cryoinjury model to injure the zebrafish hearts (n=19) as doing so causes their hearts to scar making it more relevant to the manner in which mammalian hearts respond to MI. In our study, we blocked PDGF signaling using the platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor V (0.25µM). To monitor epicardial migration/activation in regenerating zebrafish hearts we used transgenic fish expressing fluorescent reporters under the control of two epicardial markers, tcf21 and wt1. To monitor blood vessel formation we used the endothelial cell marker fli1a. We used confocal microscopy to observe blood vessel formation and epicardial migration/activation using these transgenic fish.
Funding Provided by: Office of Minority Health Research Coordination (OMHRC) in the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH)

Studying the role of the mtlA 5’-UTR in the regulation of the mannitol transporter by the small RNA MtlS in Vibrio cholera

Howard Chang (2014); Mentor(s): Jane Liu

Abstract: Small, non-coding RNAs (sRNAs) may contribute to the ability of V. cholerae, the causative agent of cholera, to adapt quickly to different environments. The MtlS sRNA, expressed in non-mannitol carbon sources, is hypothesized to repress translation of the mannitol transporter MtlA by binding to the 5’-untranslated region (UTR) of the mtlA mRNA. We investigated whether the mtlA 5’-UTR is sufficient for knockdown of MtlA expression by MtlS in Escherichia coli and V. cholerae, or if the full-length mtlA sequence is required. The following plasmids were used: pXGmtlA5’UTR-gfp (reporter, with a fusion between the mtlA 5’-UTR and gfp), pXGmtlAfull-gfp (reporter, with a fusion between the entire mtlA gene and gfp), pMtlS (allows for inducible expression of MtlS), and pVector (control). We observed by fluorescence assays that E. coli cells with pXGmtlA5’UTR-gfp and pMtlS had a ≥3-fold reduction in fluorescence compared to the control. The same experiment in V. cholerae resulted in a modest reduction in fluorescence. When only the reporter plasmid (pXGmtlAfull-gfp) was used, V. cholerae cells in glucose had reduced fluorescence compared to cells grown in mannitol; prior research showed no reduction in fluorescence under the same conditions using cells with pXGmtlA5’UTR-gfp. Our results suggest that the mtlA 5’-UTR is sufficient for repression of MtlA synthesis in V. cholerae when MtlS is expressed in cis to the mtlA 5’-UTR or when MtlS is over-expressed in trans to the mtlA 5’-UTR.
Funding Provided by: Rose Hills Foundation

Post-translational Regulation of the Mannitol Transporter by the small RNA MtlS

Maxwell Coyle (2014); Additional Collaborator(s): Daniel O'Leary; Sonja Hess (Caltech); Mentor(s): Jane Liu

Abstract: Vibrio cholerae regulates the influx of mannitol through the small RNA MtlS – if mannitol is removed from the Vibrio environment, MtlS is transcribed and decreased concentrations of the mannitol-specific transporter MtlA are observed. The same reduction in MtlA levels is seen in V. cholerae grown in mannitol media where MtlS is overexpressed. Since MtlA is a stable protein (half-life > 100 minutes), I hypothesized that reduction of MtlA levels proceeds through active proteolysis of MtlA involving increased translation of a protease or adaptor protein and directed by the expression of MtlS. To test this hypothesis, I treated V. cholerae cells grown in mannitol with chloramphenicol (Cm), a translational inhibitor, and subsequently induced MtlS transcription. In many trials, Cm-treatment rescued degradation of MtlA, although the induced transcription of MtlS in Cm¬treated samples was impaired compared to control samples. These results suggest that Cm-treatment paired with induced transcription of MtlS is an ineffective way to study the MtlA/MtlS system. I now plan to use mass spectrometry-based quantitative proteomics to identify changes in protein expression following MtlS induction. I have demonstrated that “heavy” Lys and Arg can be incorporated into V. cholerae proteins, indicating that I will be able to use stable incorporation of labeled amino acids in cell culture (SILAC) to study changes to the V. cholerae proteome in relation to changing levels of MtlS.
Funding Provided by: Dale N. Robertson Fund

MtlS, an sRNA in Vibrio cholerae, Regulates Translation of mtlA in vitro

John Replogle (2014); Mentor(s): Jane Liu

Abstract: As the causative agent of cholera, Vibrio cholerae moves between human small intestines and aquatic environments throughout its life cycle. In order to thrive, this bacterium must use a variety of carbon sources for energy. We previously showed that a small RNA (sRNA) expressed by V. cholerae, MtlS, affects the pathogen’s gene expression and metabolism. MtlA is the mannitol transporter protein necessary for V. cholerae to metabolize mannitol and is produced exclusively in the presence of mannitol. On the other hand, MtlS sRNA is only produced in the absence of mannitol and appears to post-transcriptionally downregulate expression of mtlA. We hypothesized that since it is complementary to the 5’ untranslated region (UTR) of mtlA mRNA, MtlS may affect expression of MtlA by forming an mtlA-MtlS complex that blocks translation of this mRNA. We used in vitro methods in order to examine the role MtlS plays in mtlA regulation. Using a gel shift assay, we showed that the sRNA is able to bind specifically to the mtlA mRNA. Next, we used an in vitro translation assay with varying amounts of MtlS to demonstrate that MtlA production decreases with increasing levels of MtlS. Thus, these results are consistent with our hypothesis that MtlS is sufficient to block ribosomes from translating mtlA mRNA. These findings will allow us to further understand how mannitol metabolism is regulated in V. cholerae and how these bacteria quickly adapt to new environments.
Funding Provided by: Sherman Fairchild Foundation

Determining the Role of Tfb4 in DSB Repair via TAP-tag Immunopreciptation in Yeast

Colin Belanger (2014); Mentor(s): M. Cristina Negritto

Abstract: The repair of DNA damage is an extraordinarily complex process vital to overall cell function and survival, actively studied in the model organism Saccharomyces cerevisiae. One of the primary mediators suggested for this process include the TFIIH complex, a previously characterized transcription factor that may act through its Rad3 subunit to promote DNA end degradation at double-strand breaks. To further determine the extent of TFIIH involvement at DNA breaks, three subunits of the larger complex (Tfb4, SSL1, and Rad3) were labeled with the previously-characterized TAP tag and assayed for recruitment to a known DSB using a combination of both chromatin immunoprecipitation and quantitative real-time PCR. Preliminary results indicate a significant fold-increase in recruitment of the Tfb4::TAP subunit to a known DSB relative to intact control regions of DNA, as expected. Reproducing this trend for TAP-tagged SSL1 and Rad3 subunits will further confirm the notion that TFIIH is recruited to and actively involved with the DSB repair in vivo.
Funding Provided by: Pomona College Department of Molecular Biology; Jack Steller Curricular Development Fund

Toxicity Analysis of Novel Fluorescent Nucleic Acid Probe DANPY-1 and Traditional Intercalator Dyes

Emma Carroll (2014); Mentor(s): M. Cristina Negritto; Lewis Johnson

Abstract: Novel fluorescent nucleic acid probe DANPY-1 has a high affinity for nucleic acids, is soluble in biologically relevant solvents, and exhibits superior photostability. The DEL assay is a well-established and robust technique for assessing intrachromosomal recombination in S. cerevisiae caused by DNA damage in the presence of potential mutagens. Here, we use the DEL assay to assess mutagenicity of DANPY-1 and comparable nucleic acid probes ethidium bromide, SYBR® Green I, and SYBR® Safe and an additional control dye, DAST. Due to its relatively small size, DANPY-1 was hypothesized to exhibit lower mutagenicity and toxicity than traditional intercalator dyes. We find in our preliminary results that rates of intrachromosomal recombination caused by DNA damage in the presence of DANPY-1 are reduced when compared to recombination rates of classic DNA intercalators ethidium bromide and SYBR® Green, indicating that DANPY-1 may be a potentially less toxic alternative to traditional fluorescent nucleic acid probes. Furthermore, SYBR® Green I, marketed as a desirable alternative to ethidium bromide, exhibits the highest rates of intrachromosomal recombination and DNA damage in the study. These results highlight the need for additional toxicity testing of DANPY-1 and traditional fluorescent probes using assays for different types of DNA damage, such as frameshifts and gross chromosomal rearrangement, and reveal the potential to use QSAR techniques based on dye structure/toxicity relationships to design less toxic fluorescent nucleic acid probes.
Funding Provided by: Howard Hughes Medical Institute

Recruitment of TFIIH to DSBs in Saccharomyces cerevisiae by Rad4

Tyler Oe (2014); Mentor(s): M. Cristina Negritto

Abstract: Our lab focuses on TFIIH and its recruitment to double strand breaks (DSB) in Saccharomyces cerevisiae. We have shown that a subunit of TFIIH is recruited to DSBs and propose that the complex in its entirety is recruited as well. We also propose that TFIIH is responsible for the resection of DNA ends at DSB sites. In this study, we focus on what may be recruiting TFIIH to the DSB itself. RAD4 is known to recruit TFIIH during NER and we propose that it is also associated with TFIIH’s recruitment at DSB sites. We analyzed the recruitment of TFIIH by means of ChIP and qPCR in a rad4 null mutant. If RAD4 is imperative for TFIIH recruitment, we expect to see decreased TFIIH recruitment to a DSB in the null mutant compared to the wildtype.
Funding Provided by: Sherman Fairchild Foundation; Pomona College Department of Biology

Patched family member PTR-2 implicates cortical granules in formation of the C. elegans eggshell permeability barrier

Alexander Bell (2014); Mentor(s): Sara Olson

Abstract withheld upon request
Funding Provided by: Jack Steller Summer Curriculum Enhancement Fellowship

Investigating the Role of Sugar Modification Enzymes in Formation of the C. elegans Eggshell Permeability Barrier

Karen Hou (2014); Mentor(s): Sara Olson

Abstract withheld upon request
Funding Provided by: Kenneth T. and Eileen L. Norris Foundation

Recreating the Corneal Extracellular Matrix: The Effects of Material and Alignment on Corneal Fibroblasts

Molly Kupfer (2014); Student Collaborator(s): Jennifer Zheng (2015 HMC); Mentor(s): Elizabeth Orwin; Laura Hoopes

Abstract: Corneal blindness affects more than 10 million people worldwide. An artificially grown corneal tissue construct would help satisfy the worldwide need for corneal transplants and serve as a model for further corneal research. Corneal transparency is a function of the arrangement of type I collagen fibers in the extracellular matrix and the presence of the corneal crystalline proteins transketolase (TKT) and aldehyde dehydrogenase class 1A1 (ALDH1A1) in the fibroblasts of the corneal stroma. Fibroblasts in a healthy cornea exhibit the quiescent keratocyte phenotype and express these corneal crystallins. When the cornea is wounded, cells can differentiate into myofibroblasts that express high levels of alpha-smooth muscle actin (α-SMA) stress fibers, decreasing corneal transparency. In this study we used electrospinning to create aligned and unaligned fibrous scaffolds on which to seed rabbit corneal fibroblasts. Using three materials—collagen, gelatin, and laminin—we sought to compare the relative importance of scaffold material and fiber alignment in controlling cell phenotype, with the goal of reverting cells from the wound-healing phenotype to the quiescent phenotype. SEM imaging and analysis revealed that fiber density and diameter, both of which can affect cell phenotype, were not uniform among the electrospun samples. In some cases, cells cultured for a week on aligned scaffolds could be seen aligning in the direction of the fibers. Future work will focus on obtaining more uniform samples and comparing expression of TKT, ALDH1A1, and α-SMA among the six conditions.
Funding Provided by: Kenneth T. and Eileen L. Norris Foundation; Engman Foundation

Understanding the Cytotoxicity of Permalloy Disks

Aleksandra Karapetrova (2015); Student Collaborator(s): Sam Ciocys (2015 Drexel University); Brian Flores (2013 California State University, Long Beach); Philip Gach (Postdoctoral Student, Argonne National Laboratory); Additional Collaborator(s): Dr. Elena Rozhkova (Argonne National Laboratory-Center for Nanoscale Materials); Dr. Valentin Novosad (Argonne National Laboratory-Materials Science Division); Mentor(s): Matthew Sazinsky

Abstract: Nanomagnetic materials offer exciting opportunities to remotely control biological processes. For example, ferromagnetic microdisks induce apoptosis in cancer cells via external magnetomechanical stimulus. Iron-nickel permalloy disks, which have these capabilities, are fabricated using optical lithography and metal deposition. They are removed from an array using organic solvents and are transferred to aqueous mediums for further applications. The permalloy disks can be made with a layer of gold on the top and bottom sides for the purpose of surface functionalization. The disks can bond with fluorescent dyes and biological compounds to be used as probes or drug deliverers in biological systems. Since the magnetic cores of the disks consist of a transition metal alloy, there is a possibility of reactive oxidative species (ROS) forming in aqueous solution. This occurs through a Fenton reaction and ROS are considered harmful. The chemical stability of permalloy disks not coated with a gold layer were studied. ROS formation was detected using fluorescent probe hydroxyphenyl fluorescein, X-ray fluorescence microscopy, and Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). No significant levels of hydroxyl radicals were detected at neutral pH. However, X-Ray fluorescence did detect leaching of nickel and iron from the disks on a model cancer tissue.
Funding Provided by: Argonne National Laboratory

Engineering and Purifying ToMO Mutants to Enhance Terminal Hydroxylation of Alkanes

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

Abstract: Toluene/o-xylene monooxygenase (ToMO) belongs to a family of proteins called Bacterial Multicomponent Monooxygenase (BMM). Proteins of this family are capable of hydroxylating many different organic compounds despite having similar and sometimes even identical active sites. However, ToMO demonstrates poor terminal alkane hydroxylation capability as it hydroxylates toluene to form mainly cresol (99%), and a small amount of benzyl alcohol (1%) (Pikus, et. al., 1997). Mutations can be made on ToMO, however, to improve its ability in terminal hydroxylation. In order to accomplish these mutations, site directed mutagenesis is used to create a combination of single, double, and triple ToMO mutants Q141C, E214G, T201S. Additional single mutants Q141S, Q141A, Q141G, H96A, and H111D were also made. These mutant strains were then inserted into cell line Top 10, expressed, and screened for terminal hydroxylation activity using Whole Cell Assay and a Purpald Screen. Mutants that showed promising hydroxylation capabilities went through a series of protein purification steps. Hanging drop crystallization was subsequently performed on the isolated protein in an attempt to crystallize the structure of the respective ToMO mutant.
Funding Provided by: Kenneth T. and Eileen L. Norris Foundation

Purification and crystallization of intein-encoded homing endonucleases in P. horikoshii and M. jannaschii

William Reilly (2014); Mentor(s): Len Seligman; Matthew Sazinsky

Abstract: This study aims to employ X-ray crystallography to discern the structure of archaeal homing endonucleases (HEs) encoded by inteins within the Lon protease of P. horikoshii and replication factor C of M. jannaschii. To purify the proteins, we engineered a construct to add an N-terminal Strep-tag and a C-terminal 6xHis tag to both proteins. Following successful tagging of the P. horikoshii HE, large-scale expression and sequential affinity chromatography steps using Nickel-NTA and Strep-Tactin columns were completed. The eluate from the final column was estimated via protein gel to be ~95% pure (i.e. sufficiently pure for crystallization), so following size-exclusion concentration to 5 mg/mL, crystallization was attempted with 384 ‘core’ conditions outlined by the Joint Center for Structural Genomics. 16 conditions were found to promote protein microcrystal growth, and variations of these conditions are currently being tested to grow single crystals suitable for X-ray crystallography. Furthermore, in an attempt to co¬crystallize our HE with its DNA binding site, a 22 b.p. DNA strand derived from the protein binding site was prepared and added to the purified protein. Crystallization using this sample was attempted, and promising early results suggest that microcrystals have grown under a number of conditions.
Funding Provided by: Sherman Fairchild Foundation

Novel Surgical Aid: Fluorescent Nerve Peptide Labeling of Degenerate Neural Tissue

Melina Mastrodimos (2016); Additional Collaborator(s): Roger Tsien (UCSD School of Medicine -Department of Chemistry & Pharmacology); Mentor(s): Quyen Nguyen (UCSD School of Medicine)

Abstract: One of the most significant obstacles surgeons face in modern times is visual ambiguity within the human body. Various pathologies can obscure normal anatomy, making identification and preservation of normal structures challenging for the surgeon. With the use of a series of newly engineered, fluorescent peptides designed to bind to and illuminate specific anatomic structures, however, easy identification of obscured or damaged tissues is possible. In this study a comprehensive look was taken at the interaction between these fluorescent peptides and chronically denervated peripheral nerves. Using a series of these probes, nerves were highlighted at different locations, and stages of function. Degenerate distal branches were imaged 1-9 months post proximal surgical transection. At just 72 hours post transection, nerve electrical function shuts down and nerve stumps cannot be stimulated with electromyography (EMG) monitoring. Without the aid of stimulation feedback, most surgeons abandon any attempt at surgical repair, leaving patients with little to no options for recovery from paralysis. In our study a fluorescence microscope was utilized to obtain images of the degenerate nerves otherwise invisible to the eye with white light alone. With this specific targeting and fluorescence illumination capability, this study indicates the treatment window for paralysis could be extended up to 9 months post injury. To test this claim, he next phase of this study will explore fluorescence guided repair via surgical neurorrhaphy.
Funding Provided by: Howard Hughes Medical Institute; National Institute of Health (UCSD) SUR2695 -Award Title: Testing Fluorescently Labeled Probes for Nerve Imaging During Surgery

Eukaryotic evolutionary relationships in the methionine salvage pathway

Bianca Villavicencio (2016); Mentor(s): Andre Cavalcanti

Abstract: The methionine salvage pathway (MSP) is a nuclear encoded pathway present in all eukaryotic supergroups, responsible for recycling methionine from 5’¬methylthioadenosine. Some eukaryotic species have gene fusions resulting in multi-domain proteins, such as mtnKA and mtnBD in Tetrahymena thermophila, and mtnBC in Arabidopsis thaliana. In this project we used similarity searches to characterize the MSP proteins in 270 eukaryotes with fully sequenced genomes. MSP and its many fusion genes might give insight on eukaryotic evolution and phylogenetic relationships.
Funding Provided by: Pomona College SURP

Research at Pomona