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Biology

The Relationship Between Codon Bias and the Over-Representation of Tandem Stops Codons in S. Cerevisi

Marie Adachi ('09), Andre Cavalcanti

Tandem stop codons are in-frame stop codons found downstream of the primary stop codon. It has been proposed that they act as a safety net in the event of a translation read-through of the primary stop codon by causing translation termination immediately after the read-through occurs. As previously described in Liang et al (2005), we have found that genes with high codon bias have a significant over-representation of tandem stop codons in position 3. Additionally, we have also found that genes with low codon bias have a significant over-representation of tandem stop codons in position 1. We propose that the reason for this is that the nucleotide context around the stop codon is important for genes with high codon bias, so this blocks positions 1 and 2, preventing tandem stop codons from being in those positions. On the other hand, the nucleotide context does not matter as much for the genes with low codon bias, therefore allowing the tandem stop codons to be present in the first position. The same analysis was performed on the Tetrahymena and Paramecium tetraurelia genomes, in which the two the stop codons TAG and TAA have been reassigned to Glutamine. Reference: Liang H, Cavalcanti ARO, Landweber LF. (2005) Conservation of tandem stop codons in yeasts. Genome Biology, 6: R31.
Funding provided by: HHMI

Assessing Foraging Trip Durations of Little Auks: Pitfalls of Pit Tag Readers

Nell Wheeler Baldwin ('09), Derek Buchner (’09), Zachary Brown (’07), Nina Karnovsky

The purpose of this study was to assess how long it takes a breeding little auk (Alle alle) to find food for its chick. Foraging trip duration is a measure of food availability and oceanographic conditions. We hypothesized that when food densities are low, foraging trip duration would increase. We assessed foraging trip durations through two separate methods: Passive Integrated Transponder (PIT) readings and observations of marked birds. PIT tag readers register when the bird enters and exits the nest. Feed watch data shows when the birds enter and exit and how much time they spend outside of their nests before and after feeding the chicks in the nest. Here we compare the results from these two different methods. Understanding how these seabirds respond to environmental variability is important because they are considered an indicator species of climate change.
Funding provided by: Mellon Foundation (ZB); NSF (#ARC-06125504 NK)

Auks at Sea: Deep Diving Behavior of a Small Seabird

Derek Buchner ('09), Nell Baldwin (’09), Zachary Brown (’07), Nina Karnovsky

The purpose of this study was to assess the diving behavior of little auks (Alle alle). We attached Time Depth Recorders (TDRs) to foraging little auks. These instruments measure temperature and pressure every 5 seconds and when the auks are below 1.5 meters, they register these parameters every 0.2 seconds. Here we describe the diving behavior of a pair of breeding little auks who both wore these instruments for several days. Dives were frequent and to an average of 20 meters. We also compare the dive data to our observations of these birds at the colony to see if the times we saw them enter and exit their nests correspond to temperature readings of the TDRs that reflect entering the nests.
Funding provided by: Mellon Foundation (ZB); NSF (#ARC-06125504 NK)

Proteins Interacting with the Cytoplasmic Domain of Syndecan: A Yeast Two-Hybrid Screen

Julia Ann Chang ('08), Vivek Charu ('09), Karl Johnson

Syndecan (Sdc) is a transmembrane heparan sulfate proteoglycan essential for axon guidance at the Drosophila CNS midline and for growth of the neuromuscular junction. Sdc’s cytoplasmic domain consists of two unique regions that are evolutionarily conserved across phyla. To date, no research has examined protein-protein interactions between cytoplasmic Sdc and other proteins in the Drosophila embryo. Yet, elucidating these interactions is a critical part of understanding how Sdc regulates nervous system development. This summer, we have performed a yeast two-hybrid screen to identify proteins that interact with the cytoplasmic domain of Sdc. We have screened approximately 1 x 10^6 plasmid clones from a Drosophila embryonic cDNA library. The screen has resulted in the identification of 580 plasmids encoding putative interactors with Sdc. PCR amplification, sequencing, and BLAST analysis have allowed us to determine which positive hits from the experiment most likely reflect true protein interactions in the Drosophila embryo. To date, our yeast two-hybrid system has detected interactions between the cytoplasmic domain of Sdc and a number of actin-, sensory organ development-, and intracellular transport- related proteins that are of further interest.
Funding provided by: HHMI

Ambushing the Ambush Hypothesis

Charlotte Hsien-Wei Chang ('10), Andre R.O. Cavalcanti

Translation is one of the most important and frequently performed processes in the cell. Errors waste energetic resources and also present the risk of cytotoxic products. One common error in protein translation is frameshifts caused by ribosomal slippage. In 2004, H. Seligmann and D. Pollock proposed the “Ambush Hypothesis” wherein a frameshift causes sense codons to form a nonsense codon. This project sought to examine the validity of the Ambush Hypothesis by randomizing genetic sequences and comparing the random and actual sequences. Our results challenge the Ambush Hypothesis.
Funding provided by: SURP

A Yeast-Two Hybrid Screen to Identify Novel Binding Partners for Cytoplasmic Syndecan

Vivek Charu ('09), Julia Chang ('08), Karl Johnson

Syndecan (Sdc) is a transmembrane heparan sulfate proteoglycan with two highly conserved cytoplasmic domains. Previous work on Sdc has revealed its importance in axon guidance at the midline, in the context of Slit/Robo signaling. Sdc has also been implicated in synapse formation by promoting pre-synaptic bouton growth via LAR, a conserved receptor tyrosine phosphatase. While these aspects of Sdc's affect on synaptic biology have been studied, much less is known about the precise molecular mechanisms through which Sdc functions. Specifically, the downstream signaling of the cytoplasmic domains of Sdc is very poorly understood. In this experiment, we utilized a GAL4 based yeast-two-hybrid screen to identify potential interactors with cytoplasmic Sdc from a Drosophila embryonic cDNA library containing three million unique plasmids. We have screened roughly 850,000 clones, with a resulting 580 potential interactors. We have isolated and sequenced sixty potential interactors from the library, and are currently characterizing others.
Funding provided by: HHMI

Genes that control vesicular transport in Drosophila

Kimberly Minh Chau ('10), Clarissa Cheney, Karl Johnson

Abstract removed upon request.

The Impact of Sea-Ice on the Recruitment of Antarctic Fish

Sonia Rosa Fang ('08), Nina Karnovsky, Delsa Anderl*, Dan Kimura*
*National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Alaska Fisheries Science Center, Seattle, WA.

The Antarctic silverfish (Pleuragramma antarcticum) and lanternfish Electrona antarctica are two major predators of krill and the main prey of seabirds that feed in the waters around the Antarctic Peninsula. Over the last few decades, dramatic declines in sea-ice around the Antarctic Peninsula have lead to an order of magnitude decrease in krill. To determine the influence of sea-ice on the recruitment of these species, the fish’s ages were determined from their otoliths, calcium carbonate earbones that form annual marks, retrieved from the stomach contents or fecal matter of seabirds. The otoliths of Electrona antarctica were surface read and Pleuragramma antarcticum otoliths were weighed and thin-sectioned to determine ages for subsamples (20%) from 2004-2006. Mean Electrona antarctica ages did not vary significantly between years (df=2, P=0.060), though there was a trend of increasing mean age over the years. The Electrona antarctica subsample produced a mean age of 5.6 years and a maximum age of 9 years. From otolith weight-based estimations of age, Pleuragramma antarcticum varied significantly (df=2, P=0.0083), with an older population in 2005. Weight-based age estimates and thin sections produced a mean age of approximately 13 years and a maximum age of 20 years.
Funding provided by: Rose Hills Foundation

What's in a Song?: Unraveling the Mutterings of House Wren Males

Julia Star Gleichman ('10), Jade McGill ('09), Rachel Levin, Andre Cavalcanti

Bird song is best understood when individual birds sing repertoires of discrete song types. In the house wren, Troglodytes aedon, however, males sing complex songs which either gradually or abruptly change in syntax and structure during a bout of singing. We began studying the structure and function of this type of song by recording the first hour of singing from several individual birds and displaying their songs using Raven software. Each song was then scored as a sequence of notes, and each note was given a unique ID number. We developed a clustering program to reveal associations of different note types within songs and to reveal how one type of song transitions to another within a bout of singing. In a second approach, we recorded the songs birds sang in response to our playback from within their territory of four different songs. We examined vocal response for evidence of playback-induced changes in song type and resemblance of respondent?s songs to the syntax and structure of those that we played back. These analyses revealed that house wren song repertoires consist of several “families” of song types and that song types might be selected to match territorial intruders.
Funding provided by: SURP

Urate Deposits as Possible Nitrogen Stores in Armadillidium Vulgare

Caitlin Grace Howe ('09), Jonathan C. Wright

The purpose of this study was to determine the function of urate deposits found on the epidermis of Armadillidium vulgare, a terrestrial isopod. It was hypothesized that these deposits serve as nitrogen stores for developmental processes requiring high levels of nitrogen for protein synthesis, such as molting or oogenesis. An alternative hypothesis was that due to the importance of water conservation in A. vulgare, these animals may revert to storage excretion by converting nitrogen waste to urate during particularly dry periods, rather than excreting nitrogen waste as ammonia gas. To test this, animals were kept in high humidity chambers and their mean nitrogen contents were monitored over time, using HPLC. The effects of dietary nitrogen on animal urate contents were simultaneously tested by supplementing low nitrogen diets of oak leaf litter with high-protein white beans. Animals undergoing oogenesis did not exhibit varying urate contents, but molting animals showed significant decreases in whole-animal urate from intermolt to pre- molt, consistent with mobilization of urate nitrogen for protein synthesis. Most likely urate deposits form due to a combination of storage excretion and nitrogen storage during molting.
Funding provided by: SURP

A Molecular Phylogeny of the Genus Hydra

Abril Rivas Iniguez ('08), Daniel Martinez, Kassia Percell, Julia Willner, James Signorovitch, Richard Campbell

Although individual species of the freshwater polyp Hydra have been difficult to define, morphological features have allowed for the categorization of hydra into four groups (Campbell 1987). Our goal is to elucidate the phylogenetic relationships between species within the genus Hydra. We have previously sequenced ITS1, 5.8S, and ITS2 regions of rDNA and a fragment of the 16S rDNA gene for approximately 250 strains of hydra from around the world. More recently, we have sequenced a fragment of the COX1 gene. Together, the three regions were used to generate a Maximum Likelihood tree. The main findings of our study are: Campbell’s four morphologically distinct groups appear as separate clades. There exist several distinct clades of H.viridissima. H.hymane appears basal to H.circumcincta and H.utahensis. H.circumcincta, which consists of two distinct clades, is also present in North America. H.oligactis is widely distributed in both Eurasia and North America. European H.oxycnida and North American H.canadensis appear as sister species. There exist at least four distinct H.vulgaris clades that correspond to geographical regions. European and Asian hydra of the Vulgaris group appear in a single clade with little genetic variation, thus, the current distinction between European H.vulgaris and Japanese H.magnipapillata may be unwarranted.
Funding provided by: NSF

Preliminary Survey of Estrogenic Compounds in Surface Water in Los Angeles, San Bernardino, Riverside and Orange Counties Measured by Radioimmunoassay

Meredith Anne Lee ('08), Gene Fowler

This study was a preliminary survey of Los Angles region waterways to determine if there were measurable levels of estrogenic compounds in them. There have been numerous studies on the effects of estrogenic compounds estradiol, ethynlestradiol, estrone and other forms of estrogen and synthetic chemicals that act like estrogen on aquatic species. It has also been observed that during the dry season and particularly during drought years, that rivers can have relatively large and biologically active levels of estrogenic compounds, especially downstream from sewage waste treatment facilitates. In this study, water samples were collected from several points on waterways in Los Angeles, San Bernardino, and Orange counties. Estrogenic compounds were extracted from these samples using dichloromethane and measured using a radioimmunoassay to determine the presence and amount of these compounds. The antibody used binds specifically to estradiol (E2), but will also bind estrone (E1) and possibly a number of synthetic chemicals that biologically act similarly to estradiol. Low levels of estrogenic compounds were found in most surface water tested, but there were a number of waterways that had biologically active amounts of estrogenic compounds.
Funding provided by: Mellon Foundation

Correlations Between Seabirds and Oceanographic Patterns in the North Water Polynya

Jessica Kang Lee ('09), Nina Karnovsky

The distribution of the Arctic seabirds Alle alle, Uria lomvia, Cepphus grylle, Rissa tridactyla, Fulmaris glacialis, Larus hyperboreus, and Pagophila eburnea in the North Water Polynya were examined in order to observe correlations between their distributions and other patterns such as prey distribution and ice cover. This was done through the program ArcGIS, which allows one to examine spatial patterns. As of now, the population distributions of the seabirds have been mapped on ArcGIS, which reveals how the distributions vary seasonally. However, the project is still underway as there are significantly more data left to be worked with.
Funding provided by: Rose Hills Foundation

Are House Wren Males Deadbeat Dads?

Gabriela Jade Novas McGill ('09), Julia Gleichman ('10), Musafare Ruswa ('10), Caroline King ('07), Jacqueline King ('07), Elicia Whittlesey ('07), Allison Bailey ('07), Rachel N. Levin

One prevailing hypothesis suggests that differences in predation rates at different locations drive differences in parental behavior. We have previously shown that in temperate zone, but not tropical, populations of house wrens, males feed less as chicks mature and that this effect may be enhanced when males have access to supplemental food. This suggests that temperate zone males may actually abandon their chicks when conditions are good enough for females to provide all parental care. To test this interpretation, we observed parental response to placement of a rubber snake on nest boxes early (day 6) and late (day 12+) in nestling development. We predicted that male response to nest predators would decline over time, but that female response would not. We compared responses within and between breeding seasons and found that male response dropped significantly over time, due, primarily, to a decrease in song and flights, rather than attacks. Female response did not change over time, within or between years. These results suggest that male investment in chicks may indeed drop over time in temperate populations.
Funding provided by: SURP (Richter)

Density and Floral Herbivory in Eriastrum Sapphirinum

Seana Alexandra McNamara ('10), Sarah Cusser ('06), Robert Mendenhall (’08), Frances Hanzawa

Floral herbivory, or the consumption of flower tissue, may influence the number, or the viability of seeds produced by plants, possibly affecting future plant traits. As both pollinators and floral herbivores forage in flowers, floral herbivores may locate plants in a similar way to pollinators. Some species of pollinators may approach denser patches of flowering plants, or plants with more flowers, more frequently than less dense patches of plants, or plants with fewer flowers. This study examined whether floral herbivores of Eriastrum sapphirinum, a native annual, show patterns of selective foraging similar to these pollinators. We hypothesized that high levels of floral herbivory are linked to high local densities of E. sapphirinum flowers. Each day from May 20th to July 9th, we counted the number of plants in flower at each of our six study sites, located at the Bernard Field Station, and scored new flowers as either undamaged or damaged. While the proportion of flowers damaged differed significantly between study sites, rates of per plant herbivory were unrelated to population size, local density, the number of plants in flower that day, or the number of flowers produced by each plant. Therefore, the floral herbivores of E. sapphirinum do not forage selectively.
Funding provided by: SURP, The Schenck Fund for Botanical Research.

Assessing Riparian Birds in the Short Grass Prairie of Northeastern Montana

Sabrina McNew ('09), Nina Karnovsky, John Carlson*
* Bureau of Land Management, Glasgow, MT

This summer I participated in the founding of a new Monitoring Avian Productivity and Survivorship (MAPS) station in northeastern Montana, one of the last locations of intact prairie, and the home of several species of threatened grassland birds. In addition to establishing and operating the 10 mist nets that make up a MAPS station, we analyzed the productivity of the nets based on their location in four distinct habitats so that we may both increase yield of mist nets next season and determine where birds are living within our study site. We caught birds in nets in three different cover areas: interior locations, within dense ash groves, medium-cover areas, within a riparian cottonwood corridor, and edge positions between the grasslands and the woodlands. We expected higher individual yields in the interior nets, but because of the intersection of different habitats, we predicted we would catch greater species diversity in the edge nets. Although our conclusions are limited by a limited data set, this year the interior nets caught the most species and individuals, but this is likely because mist nets do not function as well in edge habitats, and may not be because of a superior habitat in the interior.
Funding provided by: JSD Mellon Grant

Patterns of Codon Usage in Two Ciliates that Reassign the Genetic Code: Tetrahymena Thermophila and Paramecium Tetraurelia

Hannah Mariah Warner Salim ('09), Karen Ring ('07), Andre Cavalcanti

We used the recently sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia to analyze the codon usage patterns in both organisms. We used Perl scripts to analyze codon usage bias, Gln codon usage, GC content and the nucleotide contexts of initiation and termination codons in Tetrahymena and Paramecium. We also studied how these trends change along gene segments and in subsets of highly and lowly expressed genes. Our results corroborate some of the trends previously described in Tetrahymena, but also negate several specific observations. In both genomes we found a strong bias toward codons with low GC content, that codon bias increases along gene segments and in highly expressed genes, and that the contexts surrounding initiation and termination codon contexts are always AT rich. Our results also indicate that the reassignment of stop codons to Gln is highly efficient.
Funding provided by: HHMI

Slit Localization to CNS Axons: An Empirical Examination of Slit Diffusion

Joseph Gardner Wilson ('10), Karl Johnson

In Drosophila, the repellant Slit accumulates on central nervous system (CNS) axons with the aid of the Robo co-receptor Syndecan, but the mechanism of Slit localization is unknown. Two models are possible: a diffusion model and a contact-dependent model that would require direct axon-midline contact. Comm mutants have absolutely no axon-midline contact and were tested to see whether or not Slit localized to CNS axons. Results clearly showed that indeed it did, demonstrating that Slit localization to axons does not require axon-midline contact and instead occurs as a result of diffusion. Additionally, Comm mutants exhibited an unexpected lack of Slit staining on midline glial cells, suggesting that existing models for Slit’s role in midline repulsion must be modified.
Funding provided by: SURP (Seaver)

Metabolism in the Oniscidea: Allometry in Relation to Pleopodal Lungs

Grace Wu ('08), Jonathan Wright

Kleiber’s law states that metabolic rate (MR) scales to the 0.75 power of mass. Resting metabolic rate (RMR) is in large part determined by efficiency of oxygen delivery. We examined the MR of six species of Oniscidea (terrestrial isopods) that vary widely in development of pleopodal lungs to test the hypothesis that inefficient gas exchange limits metabolism, and thus more elaborate lung design would result in greater RMR, greater metabolic scope, and metabolic scaling independent of surface area. Results fail to support this hypothesis and instead demonstrate that isopods possessing the most elaborate lungs, T. punctatus and A. vulgare, had lower metabolism. The interspecific allometric exponent was similar to that of insects, but RMR was only half that of insects. Gravid L. occidentalis, the only species studied that provisions marsupium fluid exogenously rather than endogenously, had greater RMR than non-gravid animals, suggesting that metabolic cost of brood carrying is contingent on how marsupium fluid is provisioned.
Funding provided by: Rose Hills Foundation

The Correlation of DNA Damage in Older Generations of Saccharomyces Cerevisiae to the SIR2 Gene

Yimin Amelia Yu ('09), Laura Hoopes, Cynthia Selassie

Aging in Saccharomyces cerevisiae is an asymmetric process in which daughter cells bud off from the mother cells, effectively increasing the mother’s age by one generation and starting the daughter’s age at generation zero. Recent research has shown that having an extra copy of the SIR2 gene lengthens yeast lifespan while having no copy shortens lifespan. Aging has also been positively correlated with DNA breaks and the formation of extrachromosomal rDNA circles (ERCs). This project sought to determine a correlation between the amount of DNA damage with age and the SIR2 gene. SIR2EX were expected to have less damage with respect to wild type and sir2∆ more damage as compared to wild type. Older generations of yeast were obtained through a magnetic age sort and confirmed with average bud scar counts. DNA damage was assessed through the TUNEL reaction and under a fluorescent microscope. Results confirmed SIR2EX strains to show significantly less nuclear fluorescence, and thus less damage, than either wild type or sir2∆ strains and that sir2∆ strains contained more damage than either SIR2EX or wild type. Current research looking at induced DNA damage by phenols and flavonoids in young, wild type yeast is underway.
Funding provided by: Merck/AAAS

Meiotic Resetting of the Replicative Aging Clock in Yeast

Ann Feng Zhao ('09), Rachel Allen (’10), Laura Hoopes

Yeast exhibit asymmetric aging during mitosis, with the mother aging one generation, and the daughter having its replicative aging clock reset to zero. The Hoopes lab has shown that during meiosis in wild type yeast, the aging clock is reset to zero. We have suggested that DMC1, which repairs double-strand breaks during recombination, may influence this resetting. Both sporulating and non-sporulating, young and old, ndt80/ndt80 cells, the control strain, were dissected; life spans show that the sporulating cells have reset by the time this mutant arrests, which is late in prophase I. PCR-based gene targeting is being used to create a deletion of DMC1; further dissections will be done with this mutant strain. If the dmc1 strain does not reset, as predicted, it would suggest that some part of DNA is directly involved in the aging process. To further confirm that this resetting occurs during (or prior to) the pachytene stage of prophase I, a wild type return to growth experiment was done, which shows that the resetting has been accomplished by 4 hours after the cells are placed on sporulation medium, very soon after the cells have entered into meiosis.
Funding provided by: HHMI

Research at Pomona