Student Abstracts: Biology at LBNL

Structural Classification of the Internal and External Loops Found in Ribosomal RNA. BRIANA COOK (Southern Utah University, Cedar City, UT 84720) STEPHEN HOLBROOK (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
As part of a project to classify RNA structure we have made a preliminary effort to group the internal and external loops of 5S, 16S and 23S RNA. We used a software package, AMIGOS, to calculate pseudo-conformational angles from the coordinates from the overall ribosomal RNA structure. These angles were plotted and common conformations were determined from clusters in the distribution of points. We then used computer graphics to visualize the three-dimensional structure of these conformations. We identified a common motif in which an S-shaped backbone results in base-base interaction on the same strand and forming a three base triple interaction with the opposite strand. We also identified a common structure in external loops consisting of five residues. In this structure the first and fourth residues were hydrogen bonding while the second residue in the loop was perpendicular to the first and the third protruded into solution. These conserved motifs will be utilized in our overall structure of classification of RNA.

ALCHEMY: the transmutation of matter. DOUGLAS DZIUBAN (Allan Hancock College, Santa Maria, CA 93454) TAMAS TOROK (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Abstract goals of the research: to identify the ability of microbes to reduce hexavalent chromium to it's less toxic trivalent state. approach: set up and conducted an experiment consisting of 32 trials. The variables for the trials were: organism, hexavalent chromium concentration, and addition of an iron source. Each trial was then sampled regularly for biomass growth and hexavalent chromium reduction and the resulting data was then compiled and analyzed. results: some key findings of the experiment were that some species of microbes have the ability to reduce hexavalent chromium when the concentrations were low (2ppm), and that some can tolerate hexavalent chromium even at high concentrations (200ppm) though they could not reduce it; leading to the conclusion that the ability to tolerate and the ability to reduce chromium are independent of one another. The experiment took a surprise turn when it became apparent that a contaminant in one of the control trials was adept at reducing chromium. Subsequent secondary experiments supported this microbe's ability, which surpassed the ability of the other microbes used in the experiment.

CYP1B1 Polymorphisms: Possible Risk Factors for Breast Cancer. KACEE FUJINAMI (Allan Hancock College, Santa Maria, CA 93454) REGINE GOTH-GOLDSTEIN (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Polycyclic aromatic hydrocarbons (PAHs) are a group of chemicals that contain many carcinogens. PAHs are prevalent in industrialized countries because they are formed during incomplete combustion of hydrocarbons in energy production. PAHs deposit in adipose tissues, such as those in the breast. PAH is altered in a two phase reaction in order to detoxify and excrete PAH from the body. Phase I of the reaction involves enzymes, particularly Cytochrome P450 1B1 (CYP1B1) in breast tissue, that convert PAH to a water-soluble, carcinogenic intermediate. Phase II involves enzymes that detoxify this intermediate for excretion. Two polymorphisms in exon 3 of the CYP1B1 gene are being investigated for their role in breast cancer risk. One variant, m1, is a single base change at codon 432 and causes Leucine to be substituted for Valine. The second variant, m2, is a single base change at codon 453 and causes Serine to be substituted for Aspargine. Both enzymes show higher oxidation levels of PAH than the wild-type enzyme. Genotypes are examined by isolating DNA from tissue, amplifying the CYP1B1 gene using polymerase chain reaction (PCR), digesting the PCR product with Eco57I (digests m1 variation) and Cac8I (digests m2 variation), and running the products in polyacrylamide gel electrophoresis (PAGE). A comparison of genotypes from tissue samples from reduction mammoplasty patients and mastectomy patients has not yet shown conclusive evidence for a specific genotype increasing breast cancer risk, although only a small sample size of 55 reduction mammoplasties and 97 mastectomies has been tested.

Constructng a Plasmid for the Expression of Arginine Rich Protein. ABRIL GARCIA (California State University, Fresno, Fresno, CA 93740-8026) JONI MOTT (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Abstract The extracellular matrix (ECM) is an organized network of extracellular material made of several types of glycoproteins such as collagen, proteoglycan, and fibronectins found beyond the plasma membrane. The ECM plays a key role in determining cell shape and activity. The ECM is degraded by matrix metalloproteinases (MMP), which are a family of zinc dependant endoproteinases. MMP are important in wound healing, implantation, organ involution, and growth and development. MMP are secreted by mammalian cells and are activated extracellularly by the cysteine switch where cysteine residue in the propeptide becomes uncoordinated with the catalytic zinc ion leaving the active site of MMP available for catalyses. Tissue inhibitor of metalloproteinase (TIMP's) are a family of protein inhibitors that regulate MMP activity. Until recently it was believed that TIMP's were the only inhibitors of MMP, research suggests other MMP inhibitors exist. A fragment of Arginine Rich Protein (ARP) was recently identified as a po tential non-TIMP MMP inhibitor. The purpose of this research was to create an expression vector for ARP for its expression in mammalian cells.

Beta-1 Integrin Protein Expression in Differentiating Human Lens Epithelial Cells Following X Irradiation. MICHAEL GARCIA (Allan Hancock College, Santa Maria, CA 93455) DR. ELEANOR BLAKELY (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
B1-Integrin is a cell adhesion molecule which has an essential role in anchorage of cells to the extracellular matrix (ECM). We have preliminary immunofluorescence evidence indicating that ionizing radiation modulates expression of B1-Integrin in differentiating Human Lens Epithelial cells (HLE). In the present study, we compared b1-Integrin levels in protein extracts from x-irradiated and non-irradiated, control HLE. HLE were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and 5 ng/ml FGF-2. HLE at four different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5, 10 and 15 days post-confluence. Cultures were irradiated with a 4 Gy, single dose of x-ray (150 kVp). Total protein was harvested from samples at various times (30 minutes to 12 hours) after irradiation, and analyzed by Western analysis using SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis). B1-Integrin was detected using a mouse monoclonal antibody. Western analysis revealed that B1-Integrin from HLE produced two distinct protein bands. There was no obvious difference in expression levels of B1-Integrin in exponential HLE after 4 Gy compared to controls. The relative intensities of the two b1-Integrin bands changed during the differentiation of HLE. We will compare the expression of B1-Integrin in HLE observed by Western analysis of extracted protein samples, with results obtained previously by immunofluorescence analysis of fixed cells.

High Resolution Imaging with the Soft X-ray Microscope, XM-1. ADEN HABTEAB (San Jose State University, San Jose, CA 95192) GREG DENBEAUX (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Microscopy has taken great strides in its evolution since Aton van Leeuwenhoek assembled simple microscopes and used them to study microorganisms. The Center for X-ray Optics (CXRO) is a major contributor to the advancement of microscopy. It built a new high- resolution, soft X-ray microscope, the XM-1, at the Advanced Light Source (ALS) facility. The XM-1 uses bending magnet radiation from a synchrotron as a light source. The optical set-up for the XM-1 allows for high spatial resolution. The resolution is primarily determined by the outer- most zone width of the fresnel zone plate lens used for imaging. Other components of the XM-1, such as the external visible light microscope (VLM) and a Zeiss Axioplan visible light microscope (ALM), contribute to the precision and the user friendliness of the XM-1. XM-1 has applications in biology, environmental science, material science, and magnetic materials. In addition to these applications, optics testing is continuously conducted on the XM-1 to ensure its efficiency and also to expand its capabilities. Testing of the condenser zone plate's illumination revealed an error in its focusing ability. We replaced the zone plate with a new one. The level of damage to the CCD camera due to high-energy photons was revealed through dark current imaging. Currently, testing on stray light, which reduces the contrast of images, is being conducted. There is now a new interactive slide show for visitors of XM-1 at the beam line. Optics testing is necessary for ensuring the optimal performance of XM-1.

Construction of BAC Resources for Mapping and Sequencing Mammalian Genes. MICHAEL LAM (City College of San Francisco, San Francisco, Ca 94112) JAN-FANG CHENG (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Part of the process for mapping and sequencing genes in the complex mammalian genome involves the construction of bacterial artificial chromosome (BAC) resources. An organism's genomic DNA is partially digested with EcoRI and size-fractionated by pulsed-field gel electrophoresis. The DNA fragments (100-200kb) are ligated to the EcoRI cloning site of vector pBACe3.6, which will then be transformed into competent E. coli cells for constructing a genomic library. Once the genomic library is completed, high-density filters are generated for hybridization. Screening each filter by hybridizing probes labeled with radioactive 32P isotopes locates the clones containing segments of the initial genomic DNA encoding the gene of interest. BACs of the positive clones are extracted and digested by restriction enzyme Bst171 to generate smaller size fragments for restriction mapping. Restriction digest can also be used to determine the size distribution of the library using the endonuclease NotI. Advance machineries integrated in the process such as the Genetix QPix for colony picking and the BioGrid for generating filters greatly increase the efficiency in constructing the BAC resources. Ultimately, the information derived from mapping and sequencing other mammalian genes enables scientists and researchers to generate cross-species comparative analysis on human gene sequences acquired from the Human Genome Project. The studies of highly conserved non-coding sequences found in both species increase the understanding of important regulatory elements and roles for gene expression, which ultimately leads to significant medical applications.

Protein Crystallography. BRIANNE LAWRENCE (Fresno City College, Fresno, CA 93741) THOMAS EARNEST (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
The Wnt signaling pathway merits further research because it plays a critical role in determining the final fate of a cell during embryogenesis and the proliferation of cells in adult tissues. (Cell proliferation is the multiplying of cells.) Many proteins are involved in the Wnt pathway. An important protein in the pathway, called beta-catenin, interacts with other proteins in the pathway to continue cell proliferation. A destruction complex, comprised of several proteins, breaks down beta-catenin when proliferation needs to be stopped or slowed. Inappropriate activation of the pathway has been found to play a role in various human cancers. During embryogenesis, inappropriate activation of the pathway causes mutations such as multiple organs and other body parts, or the complete relocation of the growth of a body part. In adult humans, overproduction of proteins can lead to the development of colon cancer and breast cancer. Before developing methods and medications to control the pathway when it is found to be faltering, the function of the proteins in the pathway must be identified. In the study of the Wnt pathway proteins, it is necessary to purify each of the proteins and crystallize them. Once crystallized, the protein's structure can be determined using x-ray diffraction. A computer generates a three-dimensional figure of the protein. Further examination of the protein's structure will lead to a better understanding of its function.

Classification of Protein Function from a Global Parametrization of Amino Acid Sequence Using Support Vector Machines. RICHARD MERAZ (California State University Long Beach, Long Beach, CA 90840) STEPHEN R. HOLBROOK (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
The exponential growth of sequence data in protein sequence repositories makes necessary the development of rapid and accurate tools for annotating protein function from amino acid sequence alone. The prevalent techniques for annotating function involve the use of various hybrids of sequence homology comparison algorithms to deduce similarities between newly sequenced proteins and previously annotated entries in the databases. These techniques are unable to detect proteins that may have common functionality but lack sufficient sequence similarity. We investigate the use of machine learning methods for the empirical classification of protein function from appropriately parameterized representations of amino acid sequence. Specifically, we trained support vector machines on sequence databases assembled according to the molecular functions of the Genome Ontology (GO). The current results are for the classification of nucleic acid binding proteins in the radioresistant bacterium Deinococcus radiodurans.

Efficiency of Fatty Acid Extractions. TESSIE NG (Bowdoin College, Brunswick, ME 04011) TAMAS TOROK (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Identification of microorganisms and characterization of microbial communities are often based on fatty acid analysis. Although published extraction protocols have not been systematically compared, it is well understood that these methods extract fatty acids from different cellular domains. It is uncertain whether they are being extracted sufficiently and this consideration becomes critical, especially since different fatty acid composition profiles will lead to inaccurate identification of isolates and imprecise characterization of communities. Here we assessed the extraction efficiency of fatty acid methyl ester (FAME) and phospholipid fatty acid (PLFA) extraction techniques, with and without the use of a pressurized accelerated hot solvent extractor (DIONEX ASE 200), on isolates. Four bacterial and two fungal isolates were systematically analyzed. Analysis of the profiles was carried out using Sherlock, Microbial Identification System'. The TSBA40 and FUNGI methods of the analysis software were tested for fidelity of positive peak and species identifications of known microbial species (four bacteria and two fungi). The profiles indicate that there is no one technique that is able to extract all the fatty acids present in all three methods. The PLFA/DIONEX technique, however, was faster and able to increase the yield of a representative 16:0 fatty acid when compared to the traditional method of PLFA extractions for all but one of the bacteria.

The Effects of Trifluoroacetic Acid on Mixed Waste Biodegradation. ANGELA PROCTOR (Southern Utah University, Cedar City, UT 84720) WILLIAM T. STRINGFELLOW (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Mixed wastes generated by the biomedical community contain both hazardous organic compounds and radioactive isotopes. These wastes have high water content (80%) and a high organic content (less than or equal to 20%). Some of the major hazardous organic compounds in these mixed wastes are acetonitrile and trifluoroacetic acid. Due to conflicting disposal regulations, incineration is the only option for disposal of these mixed wastes, creating a problem because incineration releases the radioactive isotopes directly into the environment. We have proposed that biodegradation could be an alternative treatment for the mixed waste stream that would not result in the release of radioactivity during treatment. The purpose of this study was to determine if mixed wastes containing acetonitrile and trifluoroacetic acid could be treated biologically. Previous study has shown that the acetonitrile component of the mixed waste is biodegradable. In this study, we tested the effect of trifluoroacetic acid on acetonitrile degradation. Trifluoroacetic acid is present in the mixed wastes in low concentrations (0.1%) but the chemical was suspected of having a toxic effect on acetonitrile bacteria degradation. An oxygen uptake measurement as a function of trifluoroacetic acid concentration was used to determine the effects of trifluoroacetic acid on the acetonitrile degrading culture. The results of this study demonstrate that trifluoroacetic acid is not toxic to the acetonitrile degradation process and that mixed wastes containing trifluoroacetic acid will be amenable to biological treatment.

Exposure to Environmental Tobacco Smoke. JANE QI (Contra Costa College, San Pablo, CA 94508) BRETT SINGER (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Environmental tobacco smoke, or "secondhand smoke", is a complex mixture formed from sidestream smoke and the smoke exhaled by the smoker. Sidestream smoke is the escaping smoke of a tobacco product. The health risks of ETS include heart disease, lung cancer, asthma and impaired respiratory function. It is uncertain which components of ETS fine particles, specific vapors, or some combination affect the health outcomes. Quantifying exposures to organic vapors from ETS is challenging. It involves dynamics of organic vapor concentration. Therefore, improving quantitative data on ETS toxic exposure is an initial step to further understand the health impacts on exposed nonsmokers. In our research, we focus on the study of indoors toxic air contamination from sidestream smoke, which is the major contributor to ETS. The target of this project is to improve methods estimating the organic exposure vapors and particle in ETS and to measure emissions of a range of individual organic vapors. We place special emphasis on nicotine exposure, which is used as a tracer of ETS. It may pose a health risk to nonsmokers. We will compare the emission mass of daily smoking with the non-repeated smoking in 24-hour. The result will tell us the effect of sorption and re-emission on the daily smoking in indoor ETS exposure .The calculated AEFs might estimate a more accurate organic vapor exposure in ETS, when daily smoking is applied in a realistic indoor model.

Design, Construction and Analysis of Single-lesion Containing Shuttle Vectors for Use in Studies of Transcription-Coupled DNA Repair. JESSICA ZELLHOEFER (Cornell University, Ithaca, NY 14853) DR. PRISCILLA COOPER (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Our goal is to design a shuttle vector that contains a unique, site-specific lesion in order to study transcription-coupled repair of human DNA. In our system, the lesion is introduced by insertion of a synthesized 8-oxoguanine-containing oligomer into a pS189-derived plasmid at either of two locations: within the t-antigen (Tag) intron 400 bases beyond the ATG translation start codon, or at the end of the Tag, after the polyadenylation signal. The pS189 shuttle vector was modified to increase the transcription frequency of the Tag, prevent plasmid replication, and distinguish between Tag derived from SV40-transformed cells and that from the shuttle vector. Initial studies were undertaken to optimize the transfection conditions and also to verify the various plasmid alterations. Preliminary RT-PCR of mRNA harvested 24 hours after plasmid transfection has demonstrated that use of primers tuned to the Tag modifications do successfully distinguish plasmid from cellular RNA. Replication assays using methylation-sensitive endonucleases have verified the competence of engineered mutations in the SV40 ori in achieving preclusion of plasmid replication. RT-PCR has also shown low amplification near the Bgl II site, suggesting its removal during the processing of mature mRNA. It will therefore be necessary to construct a new site for lesion insertion before the poly-adenylation signal. In conclusion, with the competency of the pS189-derived plasmids confirmed by RT-PCR, both the shuttle vector and the transfection protocol have been optimized for TCR studies, and we are ready to insert the 8-oxoG-containing oligomer.

PAHs & Estrogens Effect On Gene CYP1B1 Expression. YILI ZHEN (University of California at Berkeley, Berkeley, CA 94704) REGINE GOTH-GOLDSTEIN (Ernest Orlando Lawrence Berkley National Laboratory, Berkley, CA 94720) .
Polycyclic aromatic hydrocarbons (PAHs) are known carcinogens ubiquitous in the environment. Inhaled or ingested PAHs are metabolically activated to exert their oncogenic effects. Cytochrome P4501 B1, encoded by the gene CYP1B1, is a major activating enzyme involved in PAH metabolism. In previous studies, CYP1B1 was shown to have a high level of expression in breast tissue. The amount of the CYP1B1 enzyme is controlled at the level of transcription by the Ah Receptor. The Ah receptor presents in the cytoplasm, is activated by the PAHs (or also dioxin) binding to it. The activated Ah complex binds to regulatory regions of various genes involved in PAH metabolism including the CYP1B1 gene and results in increased transcription of these genes. We compare the efficiency of various PAHs in inducing the CYP1B1 expression through the the Ah receptor pathway in cells in culture treated with the PAH by measuring expression by RT-PCR. The compounds to be compared are benzo[a]pyrene, benzo[c]fluorene and coal tar, a mixture of PAHs representative of PAHs occurring in the environment. It is well establised that PAHs and dioxin have an antiestrogenic effect and there is a "crosstalk" between Ah receptor and estrogen receptor. So, estrogens, which are also the substrates for CYP1B1 and they may increase the expression of gene CYP1B1. CYP1B1 expression with PAHs treatments was increased but there were no obvious changes in CYP1B1 expression of the estrogen treatment.