POSTER #25:
Molecular Species Identification of Pelagic Fish Egg Samples
Lani Gleason
Dr. Ron Burton
Eggs of two commercially important fish species, chub mackerel (Scomber japonicus) and hake (Merluccius productus), are morphologically identical. Here we demonstrate the use of molecular techniques for egg identification. Two mitochondrial genes, 16S rRNA and cytochrome oxidase subunit I (COI) genes of fish egg samples were amplified, sequenced, and compared to sequences from GenBank and the Fish-BOL (Barcoding of Life) Database. Positive identification of individual eggs collected during CalCOFI surveys and stored for years in ethanol was achieved.
POSTER #26:
Expression and characterization of recombinant Plasmepsin X, an aspartic protease of P. falciparum
Kenneth Petterson
Dr. Joseph Vinetz
Malaria is considered to be among the most successful parasites plaguing humans. Plasmodium, the parasite responsible for malaria, has a life cycle that consists of both vertebrate and invertebrate hosts. It invades and subsequently occupies multiple tissue types in both organisms. Numerous proteins secreted by Plasmodium are vital for its survival and successful propagation throughout its complex life cycle. Plasmepsin X is an aspartic protease believed to play an essential role in the invasiveness of Plasmodium falciparum, which causes the most lethal form of malaria. The objective of this project is to express active, recombinant protease in order to characterize its function. So far, the catalytic domain has been cloned. Soluble protein has been successfully expressed and is being assayed for activity. Cloning of the full protein is underway. Success in expressing active, recombinant Plasmepsin X could lead to a novel anti-malarial drug, which is much needed due to widespread resistance of the most common anti-malarial drugs.
POSTER #27:
Exploring DNA Methyltransferase 1 in Human Colorectal Cancer Cell Line
Audrey Kim
Dr. Bing Ren
Gene expression is negatively regulated by DNA methylation, a type of epigenetic modification. Hypermethylating normal tumor suppressor genes can inactivate the genes, therefore causing tumorigenesis. DNA methyltransferase (DNMT1) is one of the enzymes involved in maintaining DNA methylation in normal cells. However, DNMT1’s involvement in it is unclear how DNMT1 contributes to the altered epigenetic state in cancer cells. We determined the localization of DNMT1 in HCT116 cells. Additionally, we try to identify DNMT1 interacting partners by Co-IP from HCT116 nuclear extract. The location and binding partners of DNMT1 could help us understand DNMT1 mechanism.
POSTER #28:
Development of a Transient Assay to Monitor Circadian Rhythms in Plants
Jasmine King
Dr. Steve A. Kay
The circadian clock plays a critical role in regulating essential aspects of growth, metabolism, and behavior in many species. The goal of this project is to develop a method to rapidly screen transcription factors for effects on the circadian clock in plants. Through the use of agrobacterium-mediated infiltration, we were able to transiently transfect Arabidopsis core-clock promoter driven luciferase reporters into tobacco leaves. These bioluminescent reporters were shown to display robust, ~24 hour oscillations consistent with previous data from stable Arabidopsis transgenic lines. The oscillatory behavior of the bioluminescent reporter was observed for several days after the leaves were excised and imaged using an automated CCD-camera system. By co-injecting known effectors of the clock and the reporters, we were able to observe changes for both period and phase consistent with prior knowledge from stably transfected Arabidopsis lines. This method is currently being used to test both novel promoters and effectors of the clock, leading to a significant increase in the rate at which new clock components can be identified and studied.
POSTER #29:
Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis by Arachidonic Acid Metabolizing Proteins Alox15 & Cyp4B1
Damian Ng
Dr. Lorraine Pillus
Cystic fibrosis is a hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane chloride channel. These mutations lead to CFTR misfolding and premature degradation. Patients with cystic fibrosis are characterized with altered fatty acid metabolism, particularly showing increased arachidonic acid levels. We investigate the effects of arachidonic acid-metabolizing proteins 12/15-lipoxygenase (12/15-LO) and cytochrome P450 Cyp4B1 on CFTR biogenesis. Preliminary overexpression studies suggest that 12/15-LO has no effect on CFTR, while Cyp4B1 reduced CFTR maturation. Western blot data in COS-7 cells overexpressing CFTR and Cyp4B1 show reduced mature CFTR (c band), and an accumulation of immature CFTR (b band). Additionally, western blot data in COS-7 cells overexpressing CFTR and Cyp4B1 treated with proteasome inhibitor MG-132 did not reveal any differences in polyubiquitinated CFTR; this suggests that Cyp4B1 is not implicated in CFTR ubiquitination and degradation. Further experiments are in progress to determine the specific mechanism for Cyp4B1 inhibition of CFTR biogenesis and maturation.
POSTER #30:
3D Models of DHT Analogs in the Wild-Type Androgen Receptor and the T877A Mutant Androgen Receptor: Development of Drugs that can Treat Prostate Cancer
Paola Ortiz
Dr. Michael Baker
One of the most common mutations found in prostate cancer patients is the replacement of Threonine 877 with Alanine. The mutant AR is activated by binding to Cortisol and anti-androgens such as flutamide, which promotes prostate cancer cell growth. In this study, the interaction of human AR with different analogs of DHT, such as RB2, RB161, RB166, RB238, and RB243 (Fig.4a-4e), were studied in order understand the differences in their activation of wt-AR and the T877A mutant. These DHT analogs were synthesized and characterized in the laboratory of Dr. Brigitte Frey. Studies of their relative binding affinities (RBA ligand concentration 10-6M, RBA of DHT=100) with the mutant form T877A. RB2 has a RBA of 51 with the wild type form and a RBA of 81 with the mutant form, RB 161, RB238, RB166, and RB 243, show no RBA with the wild-type AR, but have RBAs of 20, 51, 19, and 32 respectively, with the mutant T877A. Certain patterns were observed while studying RB 2, RB 161, RB 166, RB 238, and RB 243. First, it was observed that while RB 161, RB 166, and RB 238 always show an interaction with Ser 778 when bound to the WT_AR and none when bound to the M_AR, RB 243 shows the opposite (an interaction with Ser 778 when it is bound to the mutant and no interaction when bound to the WT_AR). Second, all RB’s that were inserted in the WT_AR never interacted with amino acid Phe 891, but interacted with all the mutant forms of all RB ligands. Third, all RB ligands seem to show the weaker interactions with T877A when bound to the mutant (3.8 Å), while when these are bound to the WT_AR, they all show H-bonding (2.8-3.0 Å). Fourth, when RB 166 is bound to the M_AR it is the only one that has an interaction with Met 780.
POSTER #31:
Changes in Mitochondrial DNA Copy Number and Damage that Occur with Aging
Kacie Paik
Dr. Robert Naviaux
When cells age, mitochondrial DNA (mtDNA) damage can occurs. This results in mtDNA copy number abnormalities, deletions, point mutations, oxidative adducts, and many intermediates of mtDNA damage repair such as formamidopyrimidine (FAPY) residues. These abnormalities of mtDNA have been implicated in number of age related diseases: Alzheimer, and Parkinson disease, cataracts, muscle weaknesses, kidney failure, heart failure, hearing loss, and many others. There is a close relationship between changes of mtDNA copy number and occurrence of mutations in mtDNA genome during aging. We have found that mitochondrial DNA copy number increases during the developmental stage, plateaus until the mid-age, and decreases from mid to the end of life. Also the mtDNA copy number change occurs in tissue specific manner. Therefore we have concluded that changes in mtDNA copy number and mtDNA damage are sensitive biomarkers of tissue-specific aging and the rate of mtDNA copy number changes and damage accumulation with age is also tissue-specific.
POSTER #32:
ISG15 plays important role in Interferon-stimulated transduction pathway by conjugating to STAT1
Nhat Pham
Dr. Michael David
Interferon Stimulated Gene 15 (ISG15) is a 15 kDa protein induced in the presence of type I interferons. It is an important member of the ubiquitin-like proteins family. Due to its homology to ubiquitin, ISG15 is recognized by ubiquitin conjugating system (UBE1, UBE2, UBE3), and with the help of these enzymes, ISG15 has been found to be conjugated to important cellular proteins involved in several processes such as glycolysis, cell motility, stress and immune responses. ISG15 is also an important participant in interferon-induced immunity. ISG15 knock-out mice showed increased susceptibility to influenza, herpes and Sindbis virus infections, and the condition is rescued when wild-type ISG15 gene is reintroduced into the mouse genome. In the presence of interferon (IFN), ISG15 is upregulated and conjugated to other proteins. One of the important target proteins in the interferon-induced transduction pathway is STAT1, a member of the Signal Transducer and Activator of Transcription family. The primary purpose of this project is to identify the mechanism, localization and function of ISG15 conjugation to STAT1. Given that ubiquitin-like proteins are typically conjugated to a lysine residue on the target protein, we generated lysine to arginine mutants of the 60 potential conjugation sites for ISG15 on STAT1. These mutants are currently being screened for their ability to undergo ISG15 conjugation upon stimulation with IFNβ;. The mutant identified to lack ISG15 conjugation will be introduced into STAT1 knockout cells to investigate the roles of STAT1-ISG15 conjugation in interferon biology.
POSTER #33:
Toll- Like Receptor- 4 Protection Against Leptospirosis, an Infection of the Urban Slums
Kenneth Wong
Dr. Joseph Vinetz
From urban Baltimore to the shanty-towns of Peru, barefooted people are often infected with leptospirosis when a skin break is exposed to rat or dog urine. Yet, nearly 150 years after the discovery of Leptospira bacteria, we still do not understand why leptospirosis is more threatening to some people than to others. Some people just experience mild ‘flu-like’ illness. Sadly, the less fortunate die from bleeding lungs. My research is currently determining the role of a class of toll-like receptor (or “antennae”) on the body’s macrophages (or “defense cells”). Previous studies have established that the variation of symptoms to leptospirosis has a large genetic component. With the guidance of my mentors, I am investigating how toll-like receptor- 4 has an immunomodulatory function. It prevents an excessive inflammatory response, which harms the host. We are determining TLR-4’s mechanism by examining the gene expression changes occurring during infection with Leptospira. By June, we will have obtained the gene expression profiles of infected wild-type (“normal”) and TLR-4 deficient mice with Toll-Like Receptor Signaling Pathway PCR arrays. Quantitative polymerase chain reaction (qPCR) will also have been used to confirm gene expression levels of select genes in order to validate the PCR array data. Much down the road, related research will be able to correlate polymorphisms in human toll-like receptors to leptospirosis outcome.
POSTER #34:
Phytoplankton Community Dynamics
Asako Yamamoto & Ana Freed
Dr. Ron Burton
*I will also be presenting this poster with another BISP 199 student in the Burton Lab, Ana Freed. She is also pursuing a BS. We are studying Phytoplankton Community Dynamics by utilizing molecular techniques. I use hybridization methods using microscopic beads that are coupled to probes which hybridize to species specific DNA amplified by PCR. The Luminex machine detects the flourescence emitted from the labeled DNA which has hybridized to the probe, and in theory the more DNA (more organisms of a specific species) is present in a water sample, the higher the flourescence would appear. We have been concentrating on the species Lingulodinium polyedrum which can form harmful algal blooms. Ana has been also trying to quantitate cell density of specific species of phytoplankton in a given water sample but uses the Quantitative Real-Time PCR method (qPCR). We both run our tests on the same water sample that we collect off of the Scripps Institute of Oceanography Pier. We would like to present our data as a means of comparing our different methods.
POSTER #35:
Analysis of the ET08 Genome
Jason Ho & Theresa Wong
Dr. Kit Pogliano & Dr. Joe Pogliano
This poster explores the major differences between two of the major gene-calling programs, GeneMark and GLIMMER. In the fall of 2008, our Phage Genomics Research Initiative class isolated a novel phage infecting the bacterium Mycobacterium smegmatis, which we called ET08. After the genome was sequenced, our class worked as a team to annotate the potential genes with the ET08’s genome. A key part of annotating a genome involves correctly identifying all of the open reading frames. We used two programs, Glimmer and GeneMark, to automatically predict the location of each open reading frame. We then manually examined each prediction. To determine which, if any, of the predictions were likely to be correct, we searched for potential ribosome binding sites, also called Shine-Delgarno sequences, upstream of the putative start codons. We also used BLAST searches to identify related genes within the public gene databanks online. During the course of our annotations, we noticed that GeneMark and GLIMMER occasionally disagreed in their gene predictions. Here we describe some of differences that we observed between the two programs. Ultimately, we found that GeneMark predicted more genes than GLIMMER because GLIMMER scored potential genes with more accuracy and discretion.
POSTER #36:
Genes likely involved in DNA synthesis, Protein Synthesis and Gene Expression during replication of phage ET08
Yunjeong Yang, Sahrish Ekram, Kevin He, Bridget Guiza, Diana Li, & Danielle Wang
Dr. Kit Pogliano & Dr. Joe Pogliano
Lyticbacteriophage subvert the hosts biosynthetic apparatus to maximize phage production. We investigated the ET08 genome to identify gene products involved in transcription, DNA metabolism and protein synthesis. We identified two DNA binding proteins that are found in the ET genome and in all related mycobacteriophage. The first was a Helix-turn-Helix protein that aids in recognition of and binding to DNA. The second was a ParBprotein which is produced from ParCBAoperon and acts as a nuclease. It is possible that these proteins regulate gene expression during ET replication. We identified 29 tRNAs in the ET genome. Many large bacteriophage encode tRNAs that read codons that are highly used by phage genes, but are rare in host genes. ET08 also encodes a tmRNA that releases stalled nascent chains from the ribosome and a tRNApeptidylhydrolase that cleaves the tRNA from nascent peptides that are released from stalled ribosomes. These features are important to the fitness of phage survival because the phage takes advantage of all the resources available in the host cell, by maximizing the potential for protein synthesis to occur. We investigated the ET08 genome for genes encoding DNA polymerases and other enzymes involved in DNA replication. This analysis allowed us to identify helicase, DNA primase, and DNA pol III alpha, which includes most of the enzymes required for replication of bacteriophage T4. However we were unable to identify other genes involved in DNA metabolism such as a gene for thioredoxin, nor were we able to identify the genes responsible for nucleotide salvage in ET08. Our analysis allowed us to identify several ET proteins that are likely involved in protein and DNA synthesis and which are conserved in all of the cluster C mycobacteriophage
POSTER #37:
Genes involved in morphogenesis of Mycobacteriophage ET08
Tina Lu, Leila Haghighat, Emilie Nguyen, Kayla Busby, Phillip Wu, & Crystal Estrada
Dr. Kit Pogliano & Dr. Joe Pogliano
We analyzed the genome of the newly isolated Mycobacteriophage ET08 to identify proteins involved in morphogensis, by comparison to previously studied and structurally similar phage. To analyze tail assembly and functionality, we compared ET08 with coliphage T4, the current model for bacteriophage capsid and contractile tail assembly. We observed that, while some tail genes are clustered together in both genomes, BLAST searches do not identify many predicted morphogenesis genes; indeed, we found few homologs to ET08 genes in other bacteriophage outside of the mycobacteriophage C1 cluster. We also searched the genome for proteins involved in host cell lysis and found cell wall degradation proteins, some of which contain the LysM domain. These proteins are likely to promote lysis by rupturing the lipid cell membrane and peptidoglycan cell wall. In addition, we identified a unique secretion pathway in ET08 that is conserved in other mycobacteriophages. Twin-arginine protein translocase is a ubiquitous secretion pathway that targets proteins with a twin-arginine motif in the signal peptide. We identified a possible TatC protein in ET08 that is found in all of the Cluster C1 bacteriophages, as well as two potential TAT substrates. We conclude that further genetic studies are needed to assign functions to conserved genes related to morphogenesis in the Myobacteriophage, which are genetically distinct from the well studied E. coli phage T4.
POSTER #38:
Phinding Phage: Isolation and Characterization of Novel Bacteriophages
Hanna Wang and Katrina Nguyen
Dr. Kit Pogliano & Dr. Joe Pogliano
In the fall of 2008, the students of BIMM171A isolated, purified, and characterized novel phage infecting host Mycobacterium smegmatis. Mycobacteriophage were abundant in compost and could be isolated from other environments after enrichment. Nineteen bacteriophage were isolated and characterized using plaque assays, DNA restriction analysis and electron microscopy. Plaque assays suggested that we had isolated both lytic and lysogenic phage. Electron Microscopy demonstrated that all of the phage were double stranded DNA phage of the families Siphoviridae or Myoviridae, which correspond to the families of the well studied E. coli phage lambda and T4, respectively. One bacteriophage, ET08, was selected for genome sequencing and annotation due to its predicted large genome size and because it was the only Myoviridae identified. ET08 was shown to belong to the Cluster C1 contractile tailed mycobacteriophage family.
POSTER #39:
Comparative analysis of ET08 with related Mycobacteriophage of the C1 cluster.
Tin Tang, Jon Tsay, Brad Segal, & Andrew Ang
Dr. Kit Pogliano & Dr. Joe Pogliano
ET08 is a newly isolated phage belonging to the Mycobacteriophage C1 cluster. Since ET08 shares many genes with other C1 phage, much information can be drawn by comparing it genome to other C1 phage. We used an online program called mauve to align ET with other C1 phage. This provided a qualitative inter-genome comparison. A second program, Phamerator, was used to grade the percent likeness between the proteins in phamily 85, as 91.7% in the cluster C1 phage. A phylogenetic analysis was conducted to compare the related of ET to other cluster C1 phage, including Bxz1, Rizal, Spud, ScottMcG, Catera, Cali and LRRHood. Genome comparisons revealed the presence of several inteins in ET and related phage. Inteins, short for “internal proteins”, are mobile elements found in genomes of all kingdoms of life and also in viral proteins. Similar to introns, inteins are coded in the middle of a genetic sequence, and are most likely found in expressed genes. However, unlike introns, which are removed before translation, inteins are translated and the product is spliced to give two separate proteins, the free intein and the target protein. Inteins are often considered selfish or “parasitic” genetic elements because they do not provide any known function to the host protein and are not known to confer any selectable advantage. However, due to their genetic mobility from homing endonuclease domains, any co-infection by phage that contains genes with and without inteins will produce progeny that contain the intein domains.