3D Visualization of Inner Ear Hair Cell Machines by Electron Tomography. DANIELLE JORGENS (California State University Fresno Fresno, CA 93740) MANFRED AUER (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

The inner ear hair cells are responsible for the transduction of mechanical force into an electrical signal which is the starting point of hearing. Protruding from the apical surface of the hair cell is a hair bundle made up of multiple stereocilia and one kinocilium. The stereocilia are composed of actin filaments, unconventional myosins, ERM proteins and other actin or myosin binding proteins. The actin filaments are cross-linked by either espin or fimbrin, both of which are documented actin-bundling proteins. Fimbrin has been found to cross-link actin in vitro by either a slanted or straight pattern. The outer most actin filaments of the actin bundle are also suspected to be connected to the plasma membrane via linker proteins. To examine these two lines of thought we dissected the saccular maculae out of the bullfrog and subjected it to progressive lowering of temperature dehydration. This method of dehydration resulted in excellent preservation of the stereocilia and actin bundle. Using electron tomography, we three-dimensionally visualized, with a resolution of ~4-6 nm, the actin-actin structure and actin-membrane structure. We found surprisingly that the actin filaments were cross-linked in both slanted and straight patterns; however we were unable to conclusively identify the cross-linking protein. When we examined the connection between the actin bundle and the adjacent membrane, there were many regions whose size and shape were consistent with myosin. This first glimpse at the actin bundle of stereocilia in vivo is also evidence that stereocilia do not undergo shrinkage due to the resin embedding procedure. When actin filament models were docked to the tomogram we found that the regularly spaced actin filaments were 12 nm apart, supporting in vitro studies of actin filaments. For further study of these protein interactions and for certain indentification of such targeted proteins, double tilt-series tomography combined with labeling techniques should be employed.

An Observational Study of Guided Discovery Science & Math Activities that Promote Environmental Education. BRANDI MILLER (California State University Fresno Fresno, CA 93740) KATHY BARRETT (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

At the Lawrence Hall of science, where education is number one, I was able to be apart of a full summer of various observational studies of guided discovery science & math activities that promote environmental education. On a regional outreach program I assisted the Family First Health Department at LHS introduce healthy foods to kids and parents at a local elementary school in Hayward, CA. Additionally there were stations set up to teach them about what parts of the plants we eat (fruit, seed, flower, leaf, etc), what cereals are low in sugar, small activities to get your blood pumping, and taste testing! Another approach to guided discovery learning was the summer camps, both the local day camps and overnight regional camps. Both were activity based, with science (and or math) lessons and of course some fun time! They both exhibited environmental education and both showed to be exciting and fun for the participants (and for me as well). A new approach to teaching math was taught by the Growing Learning Community at a two week workshop. This included daily (sometimes multiple) "Math in the Garden" lessons, a approach to teaching K-6 math lessons in a Garden setting, rather than the normal classroom setting and using the garden as a learning tool. We, the workshop, designed a lesson using the Japanese style of Lesson Study and implemented it on a group of kids, courtesy of the UC Berkeley Botanical Garden summer education program. Then we reworked and revised the lesson based on the data we collected and implemented the lesson again on a different group of kids to see how our revisions worked out. In all the settings described above, environmental education adds a tangible aspect to learning and the kids respond well to the new environment as well as the new approach. As one kid told another after hearing her comment of, 'Hey this is fun, I like this!' "I thought you didn't like MATH!"…she didn't realize it was math and not just an activity.

Characterization of a TK6-Bcl-x_L gly-159-ala Human Lymphoblast Clone. LAWRENCE CHYALL (San Francisco City College San Francisco, CA 94720) AMY KRONENBERG (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

TK6 cells are a well-characterized human B-lympoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-x_L (TK6-Bcl-xL gly-159-ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6-thioguanine resistance (hypoxanthine phosphoribosyltransferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-x_L protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyltransferase (HPRT) locus were inconclusive. A mutant fraction for TK6-Bcl-x_L gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increases the mutant fraction of for TK6-Bcl-x_>L gly-159-ala clone #38 cells.

Cosmic Ray Detector. GUADALUPE AMEZQUITA (Reedley College Reedley, CA 93654) TOM KNIGHT (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Cosmic rays are energetic particles that enter the earth's atmosphere from space. They are produced by the interactions of high-energy particles from space the sun and supernovae. Scientists can not determine the specific source and direction of the charged particle because the magnetic fields in space distort their direction of origin. The cosmic ray detector is made to measure the muon flux as a function of elevation, in these flux muons and anti-muons are produced equally. This is the process in which muons are made: A charged particle in space collides with atoms in the earth's atmosphere, from these interactions pions are given off. Pions decay rapidly but they may interact first and make even more pions, when the pions decay they create high-energy muons and two (unseen) neutrinos. Muons decay slower than the pions. Muons decay into electrons and neutrinos, but the electrons and neutrinos do not have enough energy to be detected by the scintillators in the detector. Muons are the only particles with enough energy to be detected above the noise of the environment. Muons are able to penetrate large amounts of materials without interacting. An example of these materials would be mountains and concrete which were part of our experiment. We took the detector to the Caldecott Tunnel to see if concrete and the mountains on top of the tunnel decreased the amount of muons that would be detected. The result was that the counts did decrease which meant that the concrete and the mountain slowed down the muons. Also, we took the detector to Mt. Diablo to test if the altitude increase the counts of the muons being detected. In result the higher we got on Mt. Diablo the higher the counts were.

Determining the 3-Dimensional Structure of Proteins From Mycobacterium tuberculosis. DINICIO DELGADO (Fresno State Fresno, CA 93722) DR. EVAN BURSEY (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Dr. Hung leads the X-ray Crystallography Center for the Mycobacterium tuberculosis Structural Genomics Consortium. The role of the center is to conduct experiments to determine the 3-dimensional structure of various proteins from the bacterium Mycobacterium tuberculosis. To identify the 3-dimensional structure of the Mycobacterium tuberculosis protein we use a technique called x-ray crystallography. This technique requires a source of x-rays and crystallized protein. The Advance Light Source, here at Lawrence Berkeley National Laboratory, supplies the x-rays. Dr. Hung's collaborators at Lawrence Livermore National Laboratory provide the crystallized proteins. The crystallized protein is irradiated with x-rays. The x-rays bounce off of the electrons in the protein. The experiment consists of collecting the x-rays that scatter off of the crystallized proteins. If we can describe the x-rays going into the protein crystal, and if we can describe the x-rays going out of the crystal, then we can mathematically determine what has to be inside the crystal. In other words, we can determine the position of all the atoms in the crystal.This Research is important so that Scientist can develop antibiotics for tuberculosis disease. I spent time working on our protein expression system, and also participating in x-ray diffraction data collection when we have time at the Advanced Light Source (approximately twice a month). I gained experience with Polymerase Chain Reaction techniques, protein expression techniques (in vivo and in vitro), and basic protein analysis methods such as dot blots & western blots. I learned to use the automated liquid-handling robot. I participated in the wet lab to learn to make buffer reagents, use the PCR thermo cycle, performed gel electrophoresis, and toke inventory when needed.. I became a crucial asset in a team environment, along side Dr. Hung, Dr. Bursey, Dr Yu and Dr. Radhakannan who provided me with a memorable experience in research.

Domain analysis of the DNA repair proteins TFB5, XPD, and CSB. JESSICA BLANTON (Amherst College Amherst, MA 01002) JILL FUSS (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Damage to an organism's DNA can cause severe defects in gene function, and to counter such damage, all organisms have developed mechanisms of DNA repair. The nucleotide excision repair pathway (NER) is responsible for the removal of helix-distorting lesions in the DNA, both for global genome repair (GG-NER) that maintains the genome and transcription coupled repair (TCR-NER) that preferentially removes damage that blocks transcription. In humans, mutations in either the GG-NER or the TC-NER pathway lead to three severe but rare autosomal recessive disorders: Xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and Trichothiodystrophy (TTD). Three proteins implicated in these diseases are investigated in this study: the recently identified protein TFB5, associated with TTD; the DNA helicase XPD, associated with XP, CS and TTD; and CSB, from the SWI/SNF family of DNA-dependent ATPases, which is associated with CS. Standard molecular and biochemical techniques were used to express in E. coli and purify these proteins for biochemical analysis. TFB5, at 8kDa, has proven to be relatively simple to clone and express. Expression of the larger XPD (86 kDa) has been moderately successful, with three of four truncations proving to have straightforward amplification and cloning, and two of those three yielding soluble protein expression in E. coli cells. The truncations of the 170kDa CSB protein thus far appear to express as soluble protein, but further testing must be done to verify presence of the full-length segments. Continued progress and large-scale expression of these proteins will hopefully provide material for structural and functional studies, in turn enlightening further the mechanisms of NER and human DNA repair associated disorders.

Environmental Chemical Exposure and Genetic Polymorphisms of the CYP1B1 Gene in Breast Cancer Susceptibility. JOHN DEACON (Contra Costa College San Pablo, CA 94806) REGINE GOTH-GOLDSTEIN (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Polycyclic Aromatic Hydrocarbons (PAHs) are common lipophilic organic chemicals metabolized in the body through the Cytochrome P-450 1B1 (CYP1B1) enzyme pathway, where CYP1B1 metabolizes PAHs into electrophilic intermediates that can form adducts with DNA. CYP1B1 is strongly expressed in mammary tissue where PAH-DNA adducts can cause mutation and cancer. Single Nucleotide Polymorphisms (SNPs) are versions of genes differing by one base-pair and can differ from each other in function. Difference in catalytic function between SNPs of CYP1B1 may extend the electrophilic presence, thus effecting cancer susceptibility. An ongoing epidemiologic study in Brazil compares CYP1B1 genotype, CYP1B1 expression levels, and PAH-DNA adduct levels in normal breast tissue of breast cancer patients and controls, and estimates participant PAH exposure by questionnaire. The genotyping of participants for two CYP1B1 SNPs has produced a partial data set and is herein discussed. DNA was isolated from mammary tissues of participants and genotyped for two SNPs by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR /RFLP). PCR was used to amplify a segment of the gene and RFLP involved use of enzymes to digest SNPs for visualization by electrophoresis. 42 genotyped samples show 38 samples with SNP M1 (90.5%) and 11 samples with SNP M2 (26.2%). The M1 and M2 genotypic and allelic frequencies are consistent with other studies of European populations. These data represent only 1/3 of the study's total participants. Once all data is collected, a case v. control comparison may show correlation between these parameters and breast cancer incidence.

Expression of Parkin. NICHOLAS LEIBY (Massachusetts Institute of Technology Cambridge, MA 02139) KENNETH DOWNING (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Parkinson's Disease is the second most common neurodegenerative disorder, affecting 1-2% of the population over 60 years of age. The pathogenesis of the disease is still unknown, but it's believed that a complex interaction of genetic and environmental influences are involved. Mutations in the park2 gene, encoding the parkin protein, account for up to 50% of familial recessive cases of the disease. Previous studies have shown that parkin binds strongly with microtubules, but the specifics of the bond between are unknown. A better understanding of this interaction may help to determine the pathogenesis of Parkinson's Disease. We tried 2 different methods to isolate parkin protein: E. coli and cell-free expression. E. coli: We transformed an IPTG inducible, GST-tagged pGEX-6P1 vector containing park2 into BL21 E. coli, grew a 2L culture of cells, then induced expression by adding IPTG to a concentration of 1 mM. We spun down the cells, resuspended them in 20mL of PBS, and lysed by sonication. We then centrifuged the lysate at 120 G for 45 minutes and mixed the supernatant with GST bind resin. The beads were washed with PBS and then the bound substrates were eluted with 60mM reduced glutathione in PBS. The elution was concentrated using a centrifugal concentrator. Cell-free: We used the Invitrogen Expressway Plus cell-free kit. Park2 was inserted into a pEXP1-DEST vector, and the reaction was run using different plasmid concentrations. A pEXP-lacZ vector was used as a positive control and a reaction without DNA was used to determine background. The reaction product from the cell-free system and the concentrated elution from the E. coli expression were denatured in SDS at 90 degrees C for 15 minutes and run out on gels. Some gels were stained with coomassie blue, and others were used to run western blots using anti-parkin antibody. Our results were inconclusive in both cases. There was too much background protein in the cell-free system coomassie stain to be certain there was parkin, and the E. coli expression bead wash was not stringent enough to remove all the background protein. The western blots were also inconclusive. The anti-parkin antibody was polyclonal and not selective enough for our purposes- multiple bands were visible in both cases. More work will have to be done to isolate parkin. The Westerns will have to be redone using Xpress and GST antibodies, and the GST column will have to be run with a stronger wash.

Folate Receptor Binding nanoparticles Targeted Against Ovarian Cancer. SONIA REVECO (Contra Costa College San Pablo, CA 94806) FANQING CHEN (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

The Folate Receptor Protein (FR) is found in over 90% of ovarian carcinomas. Ovarian cancer is one of the deadliest types of cancer due to few symptoms until advanced stages. Since the FR has a high affinity for folic acid, nanoparticles can be conjugated with folic acid and targeted specifically to only ovarian cancer cells. The goal of this research was to attach aminated DNA conjugated to folic acid to a dendrimer, and then attach thiolated DNA conjugated to Doxorubicin (a cancer treatment pharmaceutical) to the same dendrimer. Then the dendrimer was to be tested on live cells. After each conjugation, agarose gels were run and then photographed under ultraviolet light. The gel from the conjugation of folic acid to aminated DNA showed a distinctly brighter band. Unfortunately the gel with the dendrimer and DNA showed no DNA bands under ultraviolet light. The gel with Doxorubicin and thiolated DNA gave inconclusive results. Although the folic acid/DNA conjugation was successful, further testing using radioactive labeling is needed to determine the success of the dendrimer. This work is a portion of a larger project that would use the completed dendrimer to kill only ovarian cancer cells in vitro and finally in vivo.

High Throughput Analysis of Stress Response in Shewanella oneidensis MR-1. NATALIE KATZ (Laney College Oakland, CA 94607) DR. TERRY C. HAZEN (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Shewanella oneidensis MR-1 has shown extraordinary metabolic diversity through its use of a variety electron acceptors. It can grow both aerobically and anaerobically, and can use nitrate, fumarate, sulfur compounds, and oxidized metals as electron acceptors. It has the ability to grow in almost any environment, and its entire genome has been sequenced and most of the genes annotated, which makes S. oneidensis an excellent candidate for environmental remediation of sites contaminated with toxic metals and radionuclides. The Natural and Accelerated Bioremediation Research (NABIR) Field Research Center (FRC) in Oak Ridge, Tennessee, like most other US Department of Energy uranium contaminated sites, has very high concentrations of nitrate and various salts from the metal processing wastes that contained nitric acid. To rapidly screen the response of S. oneidensis to stressors of interest, e.g. nitrate, nitrite, and osmotic stress- the Biolog Omnilog Phenotype Microarray system was utilized. The Omnilog uses digital imagery sensing technology to track the changes in turbidity of cultures growing in individual wells of a 96-well microarray plate over a given time period. The Omnilog was calibrated with two other methods in order to determine the final cell yield of S. oneidensis by correlating the digital readings produced by Omnilog with OD600 readings and direct cell counts. After the calibration, S. oneidensis was subjected to varying concentrations of each stressor of interest, coupled with either sodium or potassium as the carrier salt. Inhibition of growth was determined by calculating the maximum cell yield achieved by the control and stressed cultures to see which concentration of stressor decreased the final yield by at least fifty percent. In general, final cell yields decreased with increasing concentrations of stressor. A fifty percent inhibition of the final cell yield for nitrate stress was seen at 250 and 6000 ppm nitrate for NaNO3 and KNO3, respectively. Fifty percent inhibition of the final cell yield for nitrite stress was seen at 125 ppm nitrite for both NaNO2 and KNO2. Fifty percent inhibition of the final cell yield for salt stress was seen at 100 and 250 mM salt for NaCl and KCl, respectively.

High Throughput Analysis of Stress Response in Shewanella oneidensis MR-1. NATALIE KATZ (Laney College Oakland, CA 94607) DR. TERRY C. HAZEN (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Shewanella oneidensis MR-1 has shown extraordinary metabolic diversity through its use of a variety electron acceptors. It can grow both aerobically and anaerobically, and can use nitrate, fumarate, sulfur compounds, and oxidized metals as electron acceptors. It has the ability to grow in almost any environment, and its entire genome has been sequenced and most of the genes annotated, which makes S. oneidensis an excellent candidate for environmental remediation of sites contaminated with toxic metals and radionuclides. The Natural and Accelerated Bioremediation Research (NABIR) Field Research Center (FRC) in Oak Ridge, Tennessee, like most other US Department of Energy uranium contaminated sites, has very high concentrations of nitrate and various salts from the metal processing wastes that contained nitric acid. To rapidly screen the response of S. oneidensis to stressors of interest, e.g. nitrate, nitrite, and osmotic stress- the Biolog Omnilog Phenotype Microarray system was utilized. The Omnilog uses digital imagery sensing technology to track the changes in turbidity of cultures growing in individual wells of a 96-well microarray plate over a given time period. The Omnilog was calibrated with two other methods in order to determine the final cell yield of S. oneidensis by correlating the digital readings produced by Omnilog with OD₆₀₀ readings and direct cell counts. After the calibration, S. oneidensis was subjected to varying concentrations of each stressor of interest, coupled with either sodium or potassium as the carrier salt. Inhibition of growth was determined by calculating the maximum cell yield achieved by the control and stressed cultures to see which concentration of stressor decreased the final yield by at least fifty percent. In general, final cell yields decreased with increasing concentrations of stressor. A fifty percent inhibition of the final cell yield for nitrate stress was seen at 250 and 6000 ppm nitrate for NaNO₃ and KNO₃, respectively. Fifty percent inhibition of the final cell yield for nitrite stress was seen at 125 ppm nitrite for both NaNO₂ and KNO₂. Fifty percent inhibition of the final cell yield for salt stress was seen at 100 and 250 mM salt for NaCl and KCl, respectively.

Multimodality Nanopartcle for Targeted in Vivo Imaging with Xe NMR and Fluorescence. LESLEY;LESLEY LARA;LARA (Contra Costa College San Pablo, CA 94806) DR. FANQING FRANK CHEN (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

One of the major challenges for antibody-based therapeutics is the lack of sensitive and convenient methods for in vivo imaging that track the distribution, metabolism, movement of the drug delivery system, and provide an effective means to monitor the treatment efficacy of the drugs. The lack of sensitivity also made early detection of cancerous tumors unrealistic. Currently, radiolabels are the most sensitive labeling technology; however, radioactivity labels are undesirable for large-scale use due to the harmful effects of ionizing radiation to both the technicians and the patients. Current generation MRI contrasting reagents work in a very high concentration range of several millimolars, and there is a high false positive rate. To solve this problem, we have constructed a novel class of imaging reagent that uses near-infrared CdSe nanocrystals. The nanocrystals are clustered with Gd-based MRI contrasting reagents for regular MRI imaging, or with a novel zero- to low-field MRI agent. This dual modality nanoparticle composite would be detectable with both deep tissue near infrared in vivo imaging and MRI/zero-field MRI. To target this to breast cancer, the nanoparticle also uses single-chain antibody against ErbB2, which is a protein in the EGFR family over expressed in 15% to >50% of breast cancers, depending on the stage of the disease. The nanoparticle is highly fluorescent with a high quantum yield and the clustering of the Gd chelating compound or zero-field MRI agent is demonstrated to be at least 500 per nanoparticle. This new class of nanoparticle based imaging solution can be applied to diagnostic and monitory imaging of other cancers, or even other diseases.

Nanoplasmonic molecular ruler for DNA-protein interaction. BIPASHA MUKHERJEE (University of California, Berkeley Berkeley, CA 94720) FANQING CHEN (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

One of the major challenges of quantitative biochemistry and molecular biology is to monitor enzymatic activity within a femtoliter volume in real time. We have constructed a novel nanoscale plasmonic probe-based molecular ruler, which can perform label-free, real-time, and sensitive monitoring of DNA length during nuclease enzymatic reactions. The bionanoplasmonic molecular ruler was fabricated by tethering specificly-designed double-stranded DNA to single Au nanoparticles. Nuclease enzymatic activity was tracked via the evolution of the plasmon signal of a single Au-DNA nanoconjugate, which reflects DNA size changes introduced through site-specific DNA digestion by endonuclease. The scattering spectra of individual Au-DNA nanoconjugates are measured continuously in real time during nuclease incubation. The scattering spectra of Au-DNA nanoconjugates show a blue-shift of the plasmon resonance wavelength, as well as decrease in intensity and a time-resolved dependence on the reaction dynamics. With a series of enzymes that generate DNA incisions at different sites, the shifts of the plasmon resonance wavelength are observed to correlate closely with the positioning of the nuclease-targeted sites on the DNA, demonstrating DNA axial resolution in nanometer precision (5 nm of wavelength shift per nm of DNA length change, or 1.4 nm wavelength shift per base pair difference). DNA length differences of as little as 2 nm (6 base pairs) after nuclease digestion are differentiated by the corresponding plasmon resonance shifts of the Au-DNA nanoconjugate. Based on the mapping relationship between the DNA length and the plasmon resonance wavelength of the nanoconjugate, we further develop the nanoparticles into a new DNase footprinting platform. This DNase footprint mapping is demonstrated through the binding of DNA repair enzyme XPG to DNA bubbles. This work promises a novel molecular ruler that can monitor nuclease enzymatic reactions with single-particle sensitivity in real-time. It suggests the possibility of developing ultra-high density nanoarrays for parallel enzyme activity measurement in functional proteomic studies or biofunctional nanoprobe for intracellular enzymatic studies.

Preparation, Purification, and Crystallization of ORFan Proteins from Pathogenic Bacteria. ELIZABETH OSTRANDER (University of Missouri Columbia, MO 65211) STEPHEN R. HOLBROOK (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Open Reading Frame orphan proteins (ORFans), proteins unique to a single organism or close family members, are potential targets for new drug therapies such as inhibitors designed to interfere with the roles of enzymes essential to the life cycle of infectious bacteria. Lacking sequence homology with other proteins, ORFans of unknown function can be crystallized and subjected to X-ray diffraction to determine their structure and thereby infer their function. We targeted two ORFans: MPN675 a member of the PFAM protein family denoted as "Domain of Unknown Function 16" found only in Mycoplasma pnuemoniae; and BA2578, a Nucleotide Diphosphate linked to X (Nudix) hydrolase of unknown function found only in Bacillus anthracis and close relatives. A selenomethionine derivative of MPN675 was successfully purified through a two-step process of metal affinity chromatography and ion exchange chromatography. Initial crystals of the purified derivative were obtained and diffracted to a resolution of 4.2Å. Data from this crystal as well as native and samarium derivative crystals are being analyzed in order to obtain phases and thereby solve the protein structure. Growth experiments using different E. coli background strains determined the induction conditions most suitable to obtaining expression of BA2578. The strain BL21-AI induced at 37 C with arabinose and isopropyl ß-D-1-thiogalactopyranoside (IPTG) gave the least amount of background expression. BA2578 was localized and enriched in the insoluble fraction. Solubilization and purification experiments are in progress.

Radiation Effects on Connexin 50 Expression in Human Lens Epithelial Cells. LYDIA MCCLURE (Carleton College Northfield, MN 55057) ELEANOR BLAKELY AND KATHY BJORNSTAD (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Astronauts encounter risks of chronic exposure to ionizing radiation, which is potentially damaging to sensitive cells of the body such as the lens of the eye, causing a heightened risk for cancer and cataracts. An estimated medical risk for astronauts in space is uncertain without an understanding of the effects of ionizing radiation. The lens of the eye includes two distinct cell types. Nucleated epithelial cells differentiate as they approach the equator of the lens, losing their nucleus and other organelles while becoming fully differentiated fiber cells. An internal circulation of fluid and ions flows away from the center of the lens at the equator, through connexons, and then inward at the poles, creating a current that washes the cells in essential solutes and metabolites. Connexons are molecular aggregates of intercellular channels between cells composed of a pair of hexameric assemblies of integral membrane proteins belonging to the connexin (Cx) family. Three connexins are expressed in the mammalian lens: Cx43 (a1) is predominant in epithelial cells, Cx46 (a3) and Cx50 (a8) are more prevalent in fiber cells. To examine the specific role of Cx50 during normal cell growth and differentiation after radiation exposure, human lens epithelial (HLE) cell cultures were irradiated with 4 and 0.5 Gy 600 MeV/amu Fe ions at Brookhaven National Laboratory and processed for immunofluorescence, RNA and protein. The cells were fixed for immunofluorescence at varying times, post-irradiation, using 4% paraformaldehyde. Cx50 was identified using secondary antibodies conjugated to red Cy3. A Zeiss Axiovert 200M immunofluorescent optical microscope was used to digitally capture fluorescent images. Additionally, protein lysates were separated using SDS-PAGE and analyzed with a Western blot. The irradiated HLE cells demonstrated increased expression of Cx50 protein within 5-7 hours after exposure as evidenced by immunofluorescence and Western analysis. However, this effect is not significant after a similar 0.5 Gy exposure time course. Since Cx50 is more prevalent in lens fiber cells, the radiation-induced increase in Cx50 in HLE cells may be linked to an aberrant induction of differentiation. Increased expression of Cx50 may play a role in the effects of radiation exposure experienced by astronauts in space.

Screening Bacillus spp. for Crystalline δ-Endotoxin Production. HAIG KASSABIAN (Diablo Valley College Pleasant Hill, CA 94523) TAMAS TOROK (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Members of the bacterial genus Bacillus are microorganisms that, when deprived of nutrients, have the capability to develop endospores and concurrently produce δ-endotoxins. The crystalline δ-endotoxins are toxic to insects but not to humans because insects have an enzyme, which breaks down the crystalline structure and releases the toxic protein. The high insect specificity of the toxins makes the producing bacilli good candidates for their use as natural bio-insecticides. In this study, extremophilic Bacillus spp. were screened for the production of crystalline δ-endotoxins. The strains were grown in 96-well plates containing TB medium for one day and stressed in C2 medium for six days. We used high-throughput screening to detect crystalline toxins. Crystal-positive strains were grown in larger volume for further screening of the novel compounds. So far, 30.5% of the 583 strains screened showed positive crystalline toxin production. The two most prevalent toxin producing species were B. subtilis and B. megaterium. The area where the highest numbers of positive crystal producing bacilli were collected was Solovetskie Island, Russia, with 30.3% of the 178 strains screened. Continuing work will concentrate on screening more toxin producing strains, sequencing the novel proteins, and detecting their new insect specificities that will benefit crop protection.

Selective Breeding of Drosophila melanogaster to Achieve Gene Excision. BRESHELLE FARB (California State University Fresno Fresno, CA 93710) BILL FISHER (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Modeling is an important part of Cancer research. It allows researchers to better understand the function of cancer genes by using animal systems which can be altered via powerful genetic tools. Using D. melanogaster as our model we strove to discover the function of two genes. Both genes have been found to be over expressed in human breast cancer cells. We selected Drosophila cognates for each gene. In an effort to understand the function of the genes Trap 220 and Ppla-96A special mutations were planned. The goal of these mutations was to make the genes non-functional. By using a transposable element near each gene we hoped to create a small deficiency which will knock out gene function. The transposable elements are known as P-elements. P-elements need the enzyme transposase to mobilize. A four-generation cross scheme was developed to introduce transposase into flies carrying the P-elements. We scored and collected the progeny that were positive for the phenotypic marker telling us a P-element jump had occurred. Out of 100 parental crosses 18 excisions were found for P-element EY01286. Whereas, 58 excisions were found for P-element EY012810. The F1 generational crosses also had low success rates. Low success rates coupled with faltering supplies of the TM3/TM6 virgins prevented the completion of the final cross. It was not previously known, however, it has now been confirmed that P-element EY01286 does not jump as well as other p-elements. Since P-element EY01286 has a lower success rate this cross scheme will need to be repeated with a substantial increase in the number of parental crosses to obtain the desired amount of progeny with the intended genotype. An increase initial parental crosses coupled with better care of weak TM3/TM6 strains is likely to produce a more desirable result.

Sequence-Based Identification of Extremophilic Filamentous Fungi. SHAMEKA BERRY (Jackson State University Jackson, MS 39217) TAMAS TOROK (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Fungi have been found to survive and grow at environmental extremes of low pH, high temperatures, and high radiation. These extremophilic fungi have become of great interest to scientists because of their ability to survive such great extremes. To understand fungi and their ecological role, they first have to be identified taxonomically. Currently, research is being conducted at Berkeley Lab to identify fungi based on biomarker gene sequencing. Fungal isolates from the exploded Unit-4 and the 10-km "exclusion zone" surrounding the failed nuclear power plant in Chernobyl, Ukraine, are being characterized using this method. The strains were grown up and genomic DNA was extracted from these pure cultures. Polymerase chain reaction (PCR) was used to amplify the D1/D2 domains of the 28S rRNA- coding gene, and agarose gel electrophoresis was applied to analyze the amplicons. DNA sequencing was done at the University of California at Berkeley DNA Sequencing Facility. Raw sequences were edited online using FinchTV Version 1.3.1. The edited sequences were blasted against the National Center for Biotechnology Information (NCBI) database for preliminary identification. Of the 48 fungal strains, the identification of 75% of the strains is pending due to a lack of available DNA sequence data for filamentous fungi in the database. For the remaining 25%, about one-third matched with their classical identification. Our final results show the limitation of available public databases for fungal identification. Many of the strains that were included in this study do not have submitted sequences in databases. In the future, as more species of fungi are entered into online databases, the efficiency of fungal identification will improve. In addition, a wider selection of fungal species may prove in the future that the biomarker D1/D2 domains alone are not sufficient for identification. If additional biomarker genes were used, the identification of these fungi would become less of a problem.

Sequence-Based Identification of Extremophilic Filamentous Fungi. LAMEKA BERRY (Jackson State University Jackson, MS 39217) TAMAS TOROK (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Fungi have been found to survive and grow at environmental extremes of low pH, high temperatures, and high radiation. These extremophilic fungi have become of great interest to scientists because of their ability to survive such great extremes. To understand fungi and their ecological role, they first have to be identified taxonomically. Currently, research is being conducted at Berkeley Lab to identify fungi based on biomarker gene sequencing. Fungal isolates from the exploded Unit-4 and the 10-km "exclusion zone" surrounding the failed nuclear power plant in Chernobyl, Ukraine, are being characterized using this method. The strains were grown up and genomic DNA was extracted from these pure cultures. Polymerase chain reaction (PCR) was used to amplify the D1/D2 domains of the 28S rRNA- coding gene, and agarose gel electrophoresis was applied to analyze the amplicons. DNA sequencing was done at the University of California at Berkeley DNA Sequencing Facility. Raw sequences were edited online using FinchTV Version 1.3.1. The edited sequences were blasted against the National Center for Biotechnology Information (NCBI) database for preliminary identification. Of the 48 fungal strains, the identification of 75% of the strains is pending due to a lack of available DNA sequence data for filamentous fungi in the database. For the remaining 25%, about one-third matched with their classical identification. Our final results show the limitation of available public databases for fungal identification. Many of the strains that were included in this study do not have submitted sequences in databases. In the future, as more species of fungi are entered into online databases, the efficiency of fungal identification will improve. In addition, a wider selection of fungal species may prove in the future that the biomarker D1/D2 domains alone are not sufficient for identification. If additional biomarker genes were used, the identification of these fungi would become less of a problem.

X-Ray Microbeam Bystander Effects: Cell-Type Dependent?. CHRISTY WISNEWSKI (Las Positas Community College Livermore, CA 94551) ELEANOR BLAKELY (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

When a tumor is irradiated, the normal tissue surrounding it exhibits a response to radiation called bystander effect. Bystander effect occurs when normal cells adjacent to irradiated cells exhibit radiation damage through DNA double-strand breaks. One way to identify the crosstalk within bystander effect is to tag protein markers within the ATM/TP53 signaling pathway (a pathway that is initiated through DNA damage). To do this, human mammary epithelial cells and fibroblasts were irradiated with 100 cGy using a Pantak 150 kVp X-ray machine and up to 50 cGy in a 100 µm wide stripe using a 12.5 keV X-ray microbeam at beamline 10.3.1 at the Advanced Light Source. The cells were fixed at varying times, post-irradiation, using -20^oC/100% methanol. Two protein markers within the signaling pathway, TP53 and ATM, were identified using immunofluorescence with secondary antibodies that were tagged with green FITC fluorescent dye. Alexa-fluor 488 anti-rabbit was used for detecting TP53 and Alexa-fluor 488 anti-mouse was used for detecting ATM. A Zeiss Axiovert 200M immunofluorescent optical microscope was used to digitally capture fluorescent images. The 100 µm dose stripes were visible as low as 10 cGy in the epithelial cells tagged with anti-TP53 and in the fibroblasts tagged with anti-ATM. Additionally, there was no stripe that was visually detected in the epithelial cells tagged with anti-ATM or in the fibroblasts tagged with anti-TP53 at less than 400 cGy. While quantitation of the images is still in progress, there is the suggestion of a greater bystander effect in the ATM-positive fibroblasts, than there is in the TP53-positive epithelial cells.