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Molecular Biology

Evidence of Multiple Algal Replacement in the Hydra viridissima-Chlorella symbiosis

Alexya Aguilera ('12); Cassandra Owen ('14); Mentor: Daniel Martínez

Abstract: Green hydra form a long-recognized group in the genus Chlorohydra. They not only have a special color imparted by the intracellular algae, but they also have conspicuously small cells, nematocysts, and genome, the validity of the species being questioned. We collected green hydra from over 50 locations around the world and characterized them and their chlorella endosymbionts by sequencing nuclear and mitochondrial DNA. We find that several distinct clades of green hydra and the relation of these to taxonomic species remains to be determined. The algal endosymbionts of different strains of hydra are quite diverse and do not seem to belong to only one species of algae. We find that closely related hydra share the same type of algae. Analyses reveal that there is no obvious pattern of deep-rooted coevolution between hydra and their endosymbionts. The pattern observed in our trees suggests that there have been multiple replacements of the endosymbionts. We hypothesize that after the original symbiosis was established, there have been few, sporadic replacements of the algae.
Funding Provided by: Paul K. Richter and Evalyn E. Cook Richter Memorial Fund NIH

Identifying Novel Binding Partners with Drosophila Syndecan Cytoplasmic Domains

Julia Ayabe ('13); Mentor: Karl Johnson

Abstract: Syndecan, a transmembrane heparan sulfate proteoglycan with two highly conserved cytoplasmic domains, controls axon guidance at the midline and bouton growth at the synapse. While Syndecan’s effect on synapse growth has been observed, the downstream signaling pathways regulating Syndecan’s function are not well understood. Identification of novel binding partners with Syndecan’s cytoplasmic domain will help to elucidate the molecular mechanisms through which Syndecan functions. Several binding partners with Syndecan’s cytoplasmic domain have been identified in vertebrates through yeast two-hybrid screening. This project seeks to determine if these proteins are also binding partners with the cytoplasmic domains of Drosophila Syndecan using the GST pull-down biochemical binding assay. Thus far, we have induced protein expression of Synbindin, identified CAKI and Synectin, the Drosophila homologs of identified vertebrate binding partners and are currently constructing recombinant 6xHistidine tagged proteins of CAKI and Synectin for use in the GST pull-down.
Funding Provided by: National Science Foundation ARRA Grant #IOS-0841551 (KJ)

Characterization of a Novel Homing Endonuclease: The KlbA Intein

Jessica Barry ('12); Mentor: Len Seligman

Abstract: Inteins come from sequences of DNA that disrupt a gene and then splice out on the protein level. Some inteins contain homing endonucleases (HEs), which are enzymes that recognize and cleave specific sequences of DNA. They spread quickly throughout a species’ genome and are transmitted horizontally between species. Inteins are present in the KlbA gene of five archeal species, including Pyrococcus furiosus (Pfu), and Pyrococcus horikoshii (Pho). The target sites for the HEs in all five species are very similar on the amino acid level, making comparative studies possible. We have demonstrated that the inteins from Pfu and Pho both contain active HEs capable of cutting their own target site. Future studies will examine differences between the five HEs, including which target sites they can cut in addition to their own, and which base pairs must be present in the target site for the HE to recognize and cleave it.
Funding Provided by: Pomona College SURP

Studies into the Origins of the Adaptive Immune System

Stella Deng ('13); Sophie Wang ('14); Mentor: Jonathan Moore

Abstract: Jawless vertebrates, like the lamprey, have an adaptive immune system similar to that found in jawed vertebrates. Interestingly though, the immune response of the lamprey occurs without the presence of the typical players found in jawed vertebrate immune systems, such as immunoglobulin, RAG, and MHC genes, a spleen or a thymus. Instead, variable lymphocyte receptors (VLRs) have been identified as being responsible for producing the lamprey’s highly diverse immune response. One of these VLRs is expressed on the surface of lymphocyte-like cells and functions similarly to antibodies. We have now been investigating the evolutionary relationship between these two adaptive immune systems by looking at the regulation of this VLR gene by typical immunological transcription factors, in particular the Ets transcription factor family. We have been using reporter constructs to make transgenic lamprey embryos to determine important DNA elements for transcription of this gene. Our results are not yet conclusive, as we are still currently collecting data.
Funding Provided by: Pomona College SURP

Diversity and Phylogeny of Thermophilic Bacteria Found in the Domuyo Hot Springs of Argentina

Paloma Garcia ('12); Samira Nedungadi ('13); Glenn Freund; Mentor: Maria Christina Negritto, Daniel Martínez

Abstract: This study examined the population of thermophiles in various microbial mats of the Domuyo hot springs, with an intent to investigate their phylogenetic relationship to those in other hot springs of the world. Bacteria present in the mat samples were identified and grouped into taxa based on 16S rRNA analysis. All samples were determined to be thermophilic bacteria, with the majority of sequences belonging either to the cyanobacterial genus Fischerella or the green nonsulfur genus Chloroflexus. Phylogenetic trees were constructed for the taxa corresponding to Fischerella and Chloroflexus; these trees were in accord with current literature. Bootstrap analysis supported the Chloroflexus tree significantly more than the Fischerella tree. We will continue to construct phylogenetic trees with strong bootstrap support for all five taxa.
Funding Provided by: Pomona College SURP (PG) Paul K. Richter and Evalyn E. Cook Richter Memorial Fund (SN)

Characterization of Two Intein-encoded Homing Endonucleases

Andrew Quinn ('12); Miriam Shiffman ('13); Michael Koenig ('12); Jess Barry ('12); Mentor: Len Seligman

Abstract: Homing endonucleases genes are selfish genetic elements that code for enzymes with high specificities called homing endonucleases (HEs). I isolated and characterized HEs (2 total) found in: 1. The DNA topoisomerase I (topA) intein in Thermococcus kodakaraensis (Tko) 2. The replication factor C (RFC) intein in Methanococcus janaschii (Mja). The Mja RFC-2 HE cuts its native target site and the Tko TopA HE cuts both its native site as well as that of the Mja gyr gene. This HE also exhibited some promiscuity. Continued characterization of these HEs in the context of related archaeal species will be a topic of future research.
Funding Provided by: Pomona College SURP (AQ, JB) Rose Hills Foundation (MK) Walter Stutzman Summer Undergraduate Research (MS)

Characterizing Homing Endonucleases in Extremophilic Archaea

Miriam Shiffman ('13); Mentor: Len Seligman

Abstract: Homing endonucleases (HEs) are restriction enzymes that target DNA sequences of sufficient length to occur once within a complex genome. They are encoded by selfish genetic elements with a unique method of spreading themselves among members of a species, and even to other species. HE evolution is governed by two opposing forces: HEs must be promiscuous enough to cleave homologous sites in other species, yet specific enough to avoid cleavage of ectopic sites within the host genome. To explore this theory, I expressed and purified two pairs of homologous HEs from different species of extremophilic archaea. Each HE cleaved all four nonnative sites tested in addition to its own, demonstrating promiscuity. In fact, competition assays revealed that each HE had a higher affinity for some sites beside its own. Surprisingly, having an intein in vivo was not predictive of relative affinity for that site, and affinity for a particular site did not always correspond to more efficient cleavage.
Funding Provided by: Walter Stutzman Summer Undergraduate Research

Transcription Factor IIH’s (TFIIH) Roles in Basal Transcription and NucleotideExcision Repair (NER) of Damaged Bases

Brian Ssembajjwe ('12); Mentor: Maria Christina Negritto

Abstract: The Transcription Factor IIH’s (TFIIH) plays critical roles in basal transcription and nucleotide excision repair (NER) of damaged bases, especially those caused by UV damage. In humans, mutations in the TFIIH’s helicase subunit, XPD, results in UV light sensitive diseases Xeroderma Pigmentosum (XP), Cockayne syndrome and Trichothiodystrophy. The Saccharomyces cerevisiae homologue to XPD is Rad3. Because the sequences between Rad3 and XPD are highly conserved, Rad3 can be mutated to mimic the mutations that cause these human diseases. The homologous mutations tested are rad3-A596P, rad3-R66OC which generate TTD when mutated in XPD, and rad3-G595R, which is not associated with any mutations cause disease (Figure 1). In addition to its role in the NER pathway, Rad3 has been shown to affect Short Sequence Recombination (SSR). The mutatant rad3-G595R shows heightened levels of SSR as well as decreased single stranded DNA degradation at double strand breaks (Bailis et al. 1995). This study will investigate the levels of SSR in the three mutants through an insertion deletion assay as well as the extent of single stranded DNA degradation in the pulsed field gel electrophoresis assay when compared to Wild-Type Rad3.
Funding Provided by: Pomona College SURP

Senescence, Stress, and Sexual Reproduction in Hydra Oligactis

Adrienne Stormo ('12); Glenn Freund; Diane Bridge*; Mentor: Daniel Martínez, Gang Chen
*Elizabethtown College, Biology Dept, Elizabethtown, PA

Abstract: Unlike Hydra vulgaris, which show negligible senescence, Hydra oligactis, show inducible senescence when transferred from 18°C to 10°C, which also initiates a switch from sexual to asexual reproduction. To examine the role of gametogenesis in senescence, polyps were returned to 18°C after varying periods of time at 10°C. Preliminary results suggest that after 5 weeks at 10°C, some animals will senesce once at 18°C. Before this point, full reversion and no mortality is observed, suggesting that gametogenesis is not sufficient for senescence. Also of interest is FoxO, a transcription factor correlated with lifespan. Current findings may indirectly implicate FoxO in heat shock-related mortality: Hydra vulgaris overexpressing HIlP2, a FoxO inhibitor, die upon heat shock treatment like oligactis, but unlike wild type vulgaris, which typically survive this treatment. Work is underway to produce a FoxO probe for oligactis, which will further elucidate the role of FoxO in Hydra senescence.
Funding Provided by: Paul K. Richter and Evalyn E. Cook Richter Memorial Fund National Institute of Health Grant # (DM)

Exploring the Toxicity and Cellular Damage of Phenols in Saccharomyces cerevisiae

Julia Ticus ('13); Mentor: Maria Christina Negritto

Abstract: Because phenols are both common in everyday goods and have been shown to have toxic and carcinogenic properties, this study explores the toxicity and cellular targets of three phenols (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and bisphenol A (BPA). Particularly of interest is the identification of DNA repair genes in Saccharomyces cerevisiae responsible for the response to phenol treatment. Through pinning assays and microscopy studies, growth inhibition or stimulation as well as damaged DNA can be detected and quantified. Results have shown that many S. cerevisiae DNA repair mutants are either significantly sensitive or resistant to treatment with BHA, BHT, or BPA. It has also been observed that these phenolic compounds induce damage to DNA.
Funding Provided by: Bruce Telzer Summer Undergraduate Research Fellowship (Underwritten by Dr. Emil Kakkis, '82)

Microbial Community of Terrestrial Mud Volcanoes Near the Salton Sea, California

Joseph Tseng ('12); Mentor: E. J. Crane

Abstract: At the edge of the Salton Sea, near the town of Niland, California, there is a field of active geothermic mud volcanoes that continuously discharge anaerobic mud containing CO2-rich gases, hydrocarbons, and sulfur compounds at about 65 degrees Celsius. Mud volcanoes host populations of unique microorganisms that may be involved in the sulfur cycle and hydrocarbon metabolism, but the microbial communities of mud volcanoes have not been well characterized. In this study, DNA was extracted from mud collected from the Salton Sea mud volcanoes, followed by amplification and cloning of bacterial and archael 16S ribosomal RNA gene sequences. Overall, the microbes reveal a strong link with sulfur respiration, many of which may represent novel species. Currently, degenerate primers for a sulfur-reducing protein are being designed and utilized in order to offer greater understanding about not only the activity of the protein, but also it's genetic makeup and variance.
Funding Provided by: Rose Hills Foundation

Recruitment of Transcription Factor IIH to DNA double-strand breaks in S. Cerevisiae

Paige Wolstencroft ('12); Mentor: Maria Christina Negritto

Abstract: Double-strand breaks (DSBs) in DNA can cause extensive problems for an organism if not corrected. We propose that transcription factor IIH (TFIIH), with known functions in the nucleotide excision repair pathway (NER) and transcription initiation, also has the ability to repair DSBs in eukaryotic cells. We have studied the recruitment of TFIIH to the site of DSBs in both wildtype and rad3-G595R mutant strains of S. cerevisiae. Our results indicate that in the wildtype strains the level of TFIIH at the site of the break increases after a DSB has been induced and then gradually declines once the break stops being induced. However in the rad3-G595R mutants there is no significant increase in TFIIH localization following DSB induction. By verifying the presence of TFIIH at DSBs a role for this complex in dealing with the repair of DSBs has been confirmed, and the exact role it plays is being investigated.
Funding Provided by: Pomona College SURP

In-Frame Deletion Mutagenesis of the NADH-Dependent Persulfide Reductase in Shewanella loihica PV-4

Zhen Zhen (Jane) Xu (’12); Mentor: E.J. Crane

Abstract: The facultative anaerobe Shewanella loihica PV-4 is a gram-negative bacteria capable of using a wide variety of compounds, such as oxygen and sulfur, as electron acceptors. NADH-dependent persulfide reductase (Npsr) has been shown to be involved in sulfur respiration of PV-4 by catalyzing the reduction of persulfides to hydrogen sulfide. However, its role remains somewhat controversial. This study sought to perform a knock-out deletion of the Npsr gene to confirm its importance in sulfur respiration by examining the effects of this deletion on the organism’s ability to reduce sulfur. Successful knock-out of the Npsr gene is near completion and awaits confirmation by sequencing. Further investigation is required to determine if sulfur respiration in S. loihica has been affected in any way.
Funding Provided by: Rose Hills Foundation

Research at Pomona