A New Technical Approach To The Study Of Radiation-Induced "Bystander Effect" Using An X-Ray Microbeam. ANDREA DRAGER (City College of San Francisco, San Francisco, CA 94110) KATHLEEN BJORNSTAD (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

A.P. Drager 2, E.A. Blakely 1, R.I.Schwarz 1, K.A. Bjornstad 1, A.L. Thompson 1, P.Y. Chang 1, C.J. Rosen 1, R. Romano 1, B. Parvin 1, and D. Sudar 1 1 Lawrence Berkeley National Laboratory, Life Sciences Division 2 City College of San Francisco June 8 - August 13, 2004 Exposure to radiation can cause bystander effects" in neighboring cells that are outside of the radiation field (Azzam et al 2003). Significant levels of genetic changes, molecular damage and lethality have been observed in bystander cells of varying genetic background, lineage and organ origin. While evidence for such effects has been well established, a clear understanding of the basic biochemical and molecular processes by which they occur is only beginning to emerge. The goal of this project is to investigate the bystander effect in human mammary epithelial cells, using an X-ray Microbeam and serine -15 phosphorylation of TP53 as a marker of radiation-induced DNA damage (specifically, a double-strand break- see Saito et al. 2002) in irradiated and neighboring cells in a time course after irradiation. To do this we have quantitated the fluorescence signal associated with the TP53 ser15phosphorylation in a stripe of dose in a time course after irradiation. The ALS X-ray microbeam is a novel method to evaluate bystander effects that has potential for the evaluation of large cell numbers for statistical power. The effects of X-ray microbeam dose stripes broaden with time up to 3 hours to include bystander cells not in the radiation field. Intensity of fluorescence also increases with dose. Further statistical evaluation of the bystander effect is in progress using BioSig. Work is in progress to analyze this bystander effect at lower doses.

A Physical Map of the Heterochromatic Portion of the Drosophila melanogaster Genome. ASHLEY RYLES (Howard University, Washington, DC 20001) DR. ROGER HOSKINS (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

With the reports of Celera Genomics Inc., the Berkeley Drosophila Genome Project, and others, approximately 70% of the genome has been sequenced. This portion is referred to as the euchromatin. However, the remaining 30% of the genome has yet to be mapped and sequenced due to its high repetitive sequence content. It was originally thought that this part of the Drosophila genome was "junk DNA," though more recent studies have shown that the heterochromatin contains important molecular and genetic functions. Drosophila is an excellent model organism for understanding the structure and function of this part of the genome in other animals, including the human. A bacterial artificial chromosome (BAC)- based map was constructed of different chromosomes represented in Drosophila melanogaster. Sequence tagged site (STS) content, restriction fingerprinting, and in situ hybridization of radiolabelled DNA probes were all approaches used in order to produce these maps. The STS content map was used to divide the genome in segments and restriction fingerprints of BACs was used to assemble and edit the segment. Once the maps were compiled, a tiling path sequence was used to verify the results of the genome assembly. This project was designed to close gaps within the heterochromatic assembly. Mapping proceeded in an orderly manner across the 12Mb target. Data from approximately 192 probes was produced and analyzed. Of the probes used, 87% hybridized to BACs. The remaining 13% are not represented in the BAC library possibly due to high repeat content or restriction site distribution. In conclusion, a BAC-based physical map of 12Mb of Drosophila heterochromatin was successfully produced. The map will aid in positioning heterochromatin sequences on the chromosomes, and in finishing heterochromatin sequences to high accuracy. Most probes tested are represented in the BAC library, but some sequences are apparently absent from the library. Mapping these regions may requires a different approach. With this information, it should be possible to map and sequence a large fraction of the heterochromatin.

Characterization of receptor interaction in bacterial chemotaxis. ISRAEL FIGUEROA (University of California, Berkeley, Berkeley, CA 94720) ADAM P. ARKIN (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Bacterial chemotaxis is the motion of bacteria toward chemical attractants or away from chemical repellents. It is one of the most studied biological systems, most notably in E.coli. Chemotaxis is a model system for signal transduction with its relative simplicity, wide dynamic range, high sensitivity and robustness. It remains unknown how E.coli is able to sense such a wide dynamic range of chemicals, while still maintaining such high sensitivities. We have studied the interactions between different chemical receptors types to find a possible answer. It is hypothesized that receptors synergistically interact to amplify signals and increase chemical sensitivity. E.coli strains were constructed that allow the number of Tar and Tsr receptors to be controlled via the PBAD promoter. Motility plate assays were then conducted with varying chemical conditions and receptor numbers. The expression of the Tar receptor was successfully controlled in the engineered strain, but the Tsr receptor was not - probably due to medium plasmid copy numbers and expression leakage inherent of the PBAD promoter. Preliminary results from motility plate assays show that the Tar and Tsr chemoreceptors do not interact.

Characterization of the Microbial Community in a Methane-Driven Denitrifying Bioreactor. ANA FERNANDEZ (University of Florida, Mayaguez, PR 681) WILLIAM STRINGFELLOW (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Denitrification is an important step of the wastewater bioremediation process. It involves the removal of nitrogenous compounds from wastewater, known to cause adverse effects to human health and the environment. Because denitrification is the last step in the wastewater bioremediation process, a supplemental source of organic carbon must be added at a significant cost to wastewater treatment plants. Methane may be an inexpensive alternative source of organic carbon for denitrification. Previous studies suggest that coupled anaerobic methane oxidation and denitrification may be an effective, low cost alternative to facilitate the denitrification process in wastewater treatment plants. Understanding the microbial community present in a coupled anaerobic methane oxidation denitrifying system may allow for optimization of denitrification in wastewater treatment facilities. In this study, a low oxygen, methane-oxidizing denitrifying bioreactor (MOR) is used to emulate a coupled anaerobic methane oxidation denitrifying system. Methanotrophic and denitrifying bacteria are expected to be the major constituents of the MOR. 16S rDNA sequencing via microarray hybridization is used to identify the microbial community of the MOR. Results show that five bacterial subgroups represent the major constituents of the microbial community of the MOR, including members of the Bacillus subdivision known to reduce nitrate to nitrite. Denitrifying bacteria are indeed the major constituents of the microbial community of the MOR. However, 16S rDNA microarray technology is unable to detect the presence of known methanotrophic bacteria in the MOR. The 16S rDNA microarray must be optimized to detect methanotrophic bacteria. Additionally, the microarray technology is used to identify the microbial communities of an ammonia-oxidizing bioreactor (AOR) and a hydrogen membrane bioreactor (HMBR). As expected, distinct microbial communities are found in each of the three bioreactors using the 16S rDNA microarray. Results show that the 16S rDNA microarray is effective for the identification of distinct microbial communities in different bioreactors.

Comparative Analysis of the ApoE gene for humans and five primates. MAYA HOVEY (City College of San Francisco, San Francisco, CA 94112) J.-F. CHENG (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Comparison of human sequences with the DNA of other primates is a means of identifying conserved functional elements in the human genome.The Comparative Genomics approach is the analysis and comparison of genomes from different species. The approach examines the counterparts of genes in different species by lining up multiple genomes and looking for conserved sequences. Conserved sequences represent regions of the genome that could serve common functions such as coding for proteins of similar function or regulatory sequences that control gene expression. The study of sequences in closely related species will determine conserved non-coding sequences in order to drive future research. This approach has been successfully used in comparing closely related species such as primates to identify functional domains of the DNA that are only present in the primate lineage. This approach will be used to detect potential regulatory sequences related to ApoE gene expression. The methods used in the finishing process were RCA, PCR and sequencing. The sequence for the Owl Monkey was completed. In multiple species comparison, known exons were shown to be conserved. Significant conservation in some non-coding regions suggests presence of regulatory elements. Humans and Old World monkeys share a duplication in the region of ApoC1, New World Monkeys do not.

Crystallization of the Full-Length Nitrogenase Accessory Factor-Y (NafY) Protein. MEHNAZ MALEK (Diablo Valley College, Pleasant Hill, CA 94523) GERRY MCDERMOTT (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Nitrogenase Accessory Factor-Y (NafY) is found in the bacteria Azotobacter vinelandii and is involved in nitrogen fixation. Understanding the structure and function of NafY may lead to better nitrogen harvesting techniques as well as improvement of crops in the agricultural industry. It can bind either to the iron molybdenum cofactor (FeMo-co) or to apodinitrogenase and may be involved in the transfer of FeMo-co into apodinitrogenase. The crystal structure of the NafY core domain has been solved; however, the entire protein has not been crystallized until recently. Initial screening of protein with the Hampton Peg Ion Screen resulted in large, multiple crystals. Lowering of protein concentration and MnCl2 additive yielded semi-singular crystals in various trays; silver stain gel tests proved them to be full length NafY protein crystals. As they did not diffract very well when tested in an x-ray source, it was not possible to solve their structure. Attempts to optimize the quality of the crystals through techniques such as seeding, and under-oil growth are in progress.

Fatty Acid Methy Ester Analysis by Gas Chromatography. CRISTINA RAMIREZ (Austin Community College, Austin, TX 78730) TAMAS TOROK (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Cellular fatty acid compositions of 34 different filamentous fungi, including ascomycetes and deuteromycetes, were analyzed by the fatty acid methyl ester (FAME) method to determine if they can be differentiated from one another on this basis. The fungi were isolated from several geothermal sites in Kamchatka, Russia and the radiation contaminated soils in Chernobyl, Ukraine as part of an ongoing study of extremophiles and their potential for biotechnological applications. Many fungi were found to possess the same fatty acids but at different relative concentrations. Some fungi differed in both the fatty acids produced and their relative concentrations. Three cultures showed sufficient taxonomic differences to warrant further biochemical and genetic studies since taxonomic unrelatedness often signifies more interesting physiological attributes.

Feasibility of Using Fluorescence as an Accurate Measure of Algal Biomass. SUSAN REPON (Merritt College, Oakland, CA 94601) DR. WILLIAM STRINGFELLOW (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Algae were studied to determine the relationship between fluorescence and biomass. Scenedesmus sp. was isolated and cultured under laboratory conditions with a daily cycle of 14 hours light, 10 hours darkness. Fluorescence readings were taken every two minutes with an automated SCUFA unit, and manual cell counts were performed twice per day, during the light and dark conditions. The study found that fluorescence dropped significantly with increased light conditions, while cell counts remained statistically consistent.

Functional Survey of Conserved Noncoding Elements Near the PPARg Gene. NANCY HSU (University of California, Davis, Davis, CA 95616) JAN-FANG CHENG (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Cardiovascular patients commonly suffer from atherosclerosis, the narrowing of the coronary artery with low-density lipids. The human peroxisome proliferators-activated receptor gamma (PPAR), a fatty acid receptor, regulates the formation of fat cells and their ability to function normally. Studying the regulatory mechanisms of the PPAR gene may improve diagnosis and treatment of cardiovascular disease. One approach to understanding the functional content of the human DNA is comparative sequence analysis, also known as Comparative Genomics. By using various computational tools, such as VISTA (VISualization Tool for Alignment) and the University of California at Santa Cruz (UCSC) Genome Browser, conserved noncoding DNA sequences (CNS) surrounding the PPAR gene can be detected and studied for regulatory function. In this study, three noncoding sequences upstream to the PPAR, conserved among human, mouse, and chicken, were selected. They were subcloned from human genomic DNA into pWhsp68-lacZ reporter constructs, containing a minimal hsp68 promoter, and a lacZ reporter gene. Two insulators were also included to shield position effects. Accounting for both orientations of insertion, a total of six vectors were generated. In future work, the reporter constructs will be injected into mouse pronucleus zygotes for transient transgenic assay. If the conserved sequences can drive gene expression, then the lacZ gene, encoding -galactosidase, will be expressed in a tissue-specific fashion. At 12.5 days, the embryos recovered will undergo X-gal staining and will be examined for lacZ expression.

Genetic Polymorphisms in Glutathione S-Transferase Genes and Implications in Breast Cancer. SARAH WILSON (City College of San Francisco, San Francisco, CA 94102) REGINE GOTH-GOLDSTEIN (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

The role of the glutathione S-transferase (GST) enzyme family is to detoxify environmental toxins and carcinogens and to protect organisms from their adverse effects, including cancer. The genes GSTM1 and GSTT1 code for two GSTs involved in the detoxification of carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and benzene. Epidemiological studies indicate that GST polymorphisms increase the level of carcinogen-induced DNA damage and several studies have found a correlation of polymorphisms in one of the GST genes and an increased risk for certain cancers. We examined the role of polymorphisms in genes coding for these two GST enzymes in breast cancer. A breast tissue collection consisting of specimens of breast cancer patients and non-cancer controls was analyzed by polymerase chain reaction (PCR) for the presence or absence of the GSTM1 and GSTT1 genes. We found that GSTT1 deletions occurred more frequently in cases than in controls, but found no relation between GSTM1 deletions in our raw data. The superior detoxifier (putative low-risk) genotype (defined as presence of both GSTM1 and GSTT1 genes) was less frequent in cases than controls (28% vs. 37% respectively). However, the sample size of this study was too small to provide conclusive result

High Throughput Analysis of Stress Response in Metal and Radionuclide Reducing Bacteria. NATALIE KATZ KATZ (Seattle Central Community College, Seattle, WA 98122) TERRY C. HAZEN (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

The United States Department of Energy (DOE) has three million cubic meters of buried radioactive and hazardous waste, 75 million cubic meters of contaminated soil, 475 billion gallons of contaminated groundwater, and 120 nuclear weapons production facilities contaminated with radioactive materials, hazardous chemicals, asbestos, and lead. Bioremediation has proven to be a valuable and cost-effective way of eliminating and containing these hazardous substances through the use of microorganisms to reduce, eliminate, contain, or transform the contaminants to nonhazardous or less hazardous forms. The Center for Environmental Biotechnology at Lawrence Berkeley National Laboratory is conducting research as a part of the Virtual Institute for Microbial Stress and Survival (VIMSS). The VIMSS group aims to identify stress response pathways induced by various environmental factors, such as temperature, pH, oxygen, nitrate and nitrite concentration, and metal and radionuclide concentration. Desulfovibrio vulgaris is the model organism of choice because it is a known sulfate-reducing bacterium capable of reducing the metals of interest at these contaminated DOE sites, such as radioactive uranium (IV) and the heavy metal chromium(VI). D. vulgaris cultures were grown in LS4D defined media, and then used to inoculate media solutions containing different concentrations of nitrate, nitrite, or NaCl. These solutions were then used to inoculate a 96-well plate. The plates were placed in the Omnilog plate reader and allowed to incubate for 150 hours. VIMSS was looking for the minimum inhibitory concentration (MIC), defined as the amount of stressor needed to double the organism’s generation time. The MIC determined for nitrate was 3500 ppm. The MIC determined for nitrite was 75 ppm. The MIC determined for NaCl was 250 mM. Hexavalent chromium and radioactive uranium (IV) will be added to the media solutions to see how the MIC’s are affected in the presence of a heavy metal or radionuclide. These studies will be used to design time series experiments for the VIMSS project for rapid deduction of stress response pathways in metal and radionuclide reducing bacteria by simultaneous analyses using proteomics, metabolomics, lipidomics, transcriptomics, and phenomics.

Isolation and Growth of Hyperthermophilic Microorganisms. AARON RAMIREZ (Austin Community College, Austin, TX 78613) TAMAS TOROK (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Recent improvements in the techniques to culture microorganisms in the laboratory have made the study of microorganisms from extreme environments possible. Extremophiles, thermophilic microorganisms among them, may possess novel and highly valuable genetic traits that agriculture, industry, and the biomedical field can benefit from. In this feasibility study fourteen environmental samples collected from Kamchatka, Russia, and four samples collected from Lake Baikal, Russia, were used to investigate the presence of microorganisms capable of growing at a temperature of 80ºC. These hyperthermophiles are of interest because they may contain an enzyme, a-galactosidase, capable of digesting raffinose series oligosaccharides (RSO). This activity has a potential application in plant biotechnology. Dietary supplementation with a-galactosidase is theorized to reduce viscosity and improve nutrient digestion by swine that are fed soybean meal (SBM) as their main food source. This project investigates the potential of using a high temperature a-galactosidase of microbial origin in transgenic soybeans activated by elevated temperature during a post-harvest processing. Preliminary results obtained during this internship provided evidence for the presence of hyperthermophiles in stored environmental samples. After approximately fourteen days growth was observed in six of the samples. Genomic DNA extraction was successful. PCR amplification was still pending at the time when the internship concluded. In follow up experiments the presence of thermostable a-galactosidase in these hyperthermophilic microorganisms will be elucidated. Enzyme activity will be tested using a p-nitrophenyl-a-D-galactopyranoside (pNP-a-gal) assay. Active enzymes will be tested for thermostability and substrate specificity with regard to soybean meal oligosaccharides. Then, with cloned PCR products plant transformation will be carried out to test the use of the heterologous microbial enzyme in post-harvest processing.

Measuring the radiocarbon of three soil density fractions. ERIN HANLON (St. John's College, Santa Fe, NM 87505) MARGARET S. TORN (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Measuring carbon-14 isotopes (14C) in different soil components is a useful technique for tracing the pathways of carbon in soil. This study investigates the incorporation of 14C into different soil C pools at the 0-15cm depth. Soil samples were taken from Walker Branch Watershed in Oak Ridge, Tennessee. Soil samples were processed using physical fractionation techniques and separated using 1.7 g/mL sodium polytungstate (NaPT) into three density fractions; free light, occluded light and heavy. Each density fraction was assayed for '´14C ? content using an accelerator mass spectrometer and for C and N with an elemental analyzer. The carbon in the occluded light fraction was found to be older than the carbon in the free light fraction. These results support the idea that organic matter that is occluded within aggregates is protected from decomposition, leading to longer turnover times and increased stabilization of C. Understanding the role soil aggregates play in stabilizing soil organic carbon can increase our knowledge of the methodology of carbon sequestration in a particular environment.

Modeling DNA repair: approaching in vivo techniques in the hyperthermophile Sulfolobus solfataricus. JESSICA BLANTON (Amherst College, Amherst, MA 1002) JILL FUSS (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Archaea are found in some of the most extreme environments on earth and represent a third domain of life distinct from Eukarya and Eubacteria. The hyperthermophilic archaeon Sulfolobus solfataricus was isolated from acidic hot springs (80oC, pH 3) in Yellowstone National Park. Archaeal DNA repair mechanisms are more similar to those of eukaryotes than prokaryotes. This similarity, together with its relatively simple genome, make S. solfataricus an ideal model system for characterizing these eukaryal processes. DNA repair is essential to all domains of life and requires coordinated protein-protein interactions that are frequently transient. Protein complexes that are transient at extreme temperatures where archaea thrive may be more stable at room temperature, allowing for the characterization of otherwise short-lived complexes. However, characterization of these systems in archaea has been limited by the absence of a stable in vivo transformation and expression system. The work presented here is a pilot study in gene cloning and recombinant protein expression in S. solfataricus. Three genes associated with DNA repair were selected for expression: MRE11, a putative CSB homologue, and PCNA1. Though preparation of these recombinant genes followed standard methods, preparation of a suitable vector proved more challenging. The shuttle vector pSSV64, derived from the SSV1 virus and the E. coli vector pBSSK+, was most successfully isolated from the DH5a E. coli strain. Currently, alternative vectors are being designed for more efficient genetic manipulations in S. solfataricus.

Role of XPG in Global Repair of Oxidative Damaged DNA. SAM ZENHARI (Contra Costa College, San Pablo, CA 94806) BRETT HALTIWANGER (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Oxidative DNA damage is removed by the base excision repair pathway(BER). In this experiment we look at the specific repair of DNA containing Thymine glycol damage, in human cells. We are investigating the role of XPG which is a protein that has both essential role in nucleotide excision repair and a role in BER to see if there is a significant difference in repair between the cells that have XPG compared to ones that lack XPG. In previous experiments it was shown that XPG stimulates the DNA repair activity of some specific BER enzymes in vitro, notably hNTH1. We are hoped to see in support of this idea by introducing a plasmid containing Thymine glycol damage into cells. This experiment was done by treating plasmid DNA with OsO4 and tranfecting the damaged plasmid into VA13 a cell line that contains XPG and 94RD27 a cell line that is XPG deficient.

Structural analysis of Nudix hydrolases from Deinococcus radiodurans. ALLEN RASSA (North Idaho College, Coeur D' Alene, ID 83815) STEPHEN R. HOLBROOK (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Nudix hydrolases, a family of proteins ubiquitous to all kingdoms of life are found in larege numbers in the bacterium Deinococcus radiodurans and are suspected to be a part of this organisms remarkable resistance to ionizing radiation. These proteins have been shown to specifically bind to toxic and mutagenic substrates and contribute extensively to DNA repair and genome maintenance. Structural studies of one of these proteins (DR1025) shows that it forms an intertwined homodimer, with each monomer containing the characteristic Nudix fold. Crystal structures of seven forms of the DR1025 protein were solved at high resolution (1.4-1.6 Angstroms) in the absences or presence of magnesium or samarium and either a non-hydrolyzable GTP analog or Ap4A, two putative substrates. Structural comparisons were conducted on the seven proteins and are presented here.

Three-Dimensional Histopathology of the Mammary Gland. ABBEY HARTLAND (Shasta College, Redding, CA 96049) CARLOS ORTIZ DE SOLORZANO (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Breast cancer researchers today are faced with the extensive task of discovering and describing the mechanisms that drive the initiation and progression of mammary cancers. To correctly define these mechanisms, they must be studied within the context of the tissue where they appear and develop. Several techniques exist to detect or quantify proteins and genes involved in this neoplastic process, however none have been able to both identify and effectively preserve these chemicals within their histological context. In response to these difficulties, our laboratory has developed a computer-assisted microscopy system to transform serial sections of tissue into a three-dimensional (3D) image, thus allowing us freedom to both identify breast cancer markers and to visualize the context where these markers are found ex vivo. This system was utilized to reconstruct the mammary ductal trees of two peripubertal female mice: one transgenic c-neu overexpressing and one wild-type control. Combining this reconstruction with current immunohistochemical techniques, we mapped three major receptor proteins known to have a crucial role in the normal development of the gland: estrogen receptor (ER)-á, progesterone receptor (PR), and the growth factor receptor neu. Numerical analyses indicate that the transgenic animal over-expresses HER2, resulting in an under-expression of ER and subsequent inhibition of ductal elongation. PR is expressed differentially in terminal end buds (TEBs), the lowest levels found in proliferating end buds of the wild-type animal, while the highest levels are found in the prematurely developed lobuloalveolar structures of the c-neu mouse, suggesting that PR expression in TEBs increases proportionately to the relative level of maturity. The data obtained from current and future analyses will enable us to determine changes in the morphology and the pattern of receptor expression associated with cancer initiation and development.

Use of 28S rRNA gene D1/D2 Domain Sequences for the Identification and Phylogenetic Analysis of Filamentous Fungi Isolated from Extreme Environments. ERICA BOUDREAU (University of New Hampshire, Durham, NH 3824) SUZANNE NITTA (California State University Fresno, Fresno, CA 93740) TAMAS TOROK (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Microorganisms have been found to thrive in some of the most extreme environments on earth, exposed to high radiation, chemical contamination, high salinity, acidic or alkaline pH conditions, or exceptionally low or high temperatures. Due to the vast diversity of extremophillic microorganisms, they have become a valuable bioprospecting target. An important aspect of bioprospecting is the taxonomic identification of these microorganisms, for this provides a greater understanding of their diversity, unique metabolism, and ecological function. An ongoing study in this laboratory focuses on the taxonomic characterization by molecular comparison of thousands of filamentous fungi isolated from sediments of Lake Baikal and geothermal sites in Kamchatka, Russia, and from the 30-km "exclusion zone" surrounding the failed nuclear power plant in Chernobyl, Ukraine. Pure cultures of the filamentous fungi were grown under standardized conditions. The genomic DNA was extracted using the BIO 101 FastDNA kit and protocol (Q-BIOgene, CA). The D1/D2 domain of the 28S rRNA gene was amplified by polymerase chain reaction (PCR). Amplicons were separated by gel electrophoresis in a 1.2 % agarose in a 1x TBE buffer. Prior to sequencing, PCR products were run through MicroSpin™ silica gel columns (Amersham Pharmacia, NJ) and ethanol precipitated. The University of California at Berkeley DNA Sequencing Facility sequenced the PCR products. Raw sequences were edited with EditView Ver. 1.0.1.1 (ABI, CA), aligned using the online site MultAlin (http://prodes.toulouse.inra.fr/multalin/multalin.html), and blasted against the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/BLAST) to determine taxonomical relatedness of the filamentous fungi.

Using Balancer Chromosomes and Phenotypic Markers to Map the Locations of Drosophila melanogaster Genes. JOSHUA PURVES (California State University Fresno, Fresno, CA 93740) DR. SUSAN CELNIKER (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Drosophila melanogaster has a relatively small genome that is carried by three autosomal chromosomes (designated 2, 3, and 4) and two sex chromosomes (X and Y). The genome of D. melanogaster has two key features that simplify the creation of genetic crosses and facilitate the mapping of genes within it: (1) balancer chromosomes exist for each chromosome except the 4th, and (2) a plethora of phenotypic markers. Balancer chromosomes are lethal when homozygous and contain multiple inversions that prevent viable crossover products from forming between wild-type chromosomes and their balancers. Chromosome 4 does not have a balancer since its small size and high heterochromatin content virtually prohibit homologous recombination from occurring. Phenotypic markers associated with visible fruit fly characteristics such as the eye, body, wing, and bristles, are also plentiful within the Drosophila genome. Balancer chromosomes are constructed to contain some of these visible markers and, as a result, they allow geneticists to follow the segregation of the balancer or its normal homolog by looking for the presence or absence of the marker itself. A number of genetic crosses were created to map the locations of transposons within the Drosophila genome. The transposons, called P-elements, were engineered to contain regulatory DNA sequences as well as a mini w+ gene. The mini w+ gene puts pigment in the eye of otherwise white-eyed mutants (which carry the mutation w1118). In the first mapping cross, males containing the transposon (and therefore having wild-type eye color) were mated to virgin, white-eyed (w1118) females. These crosses resulted in progeny where both male and female flies exhibited the wild-type (w+) or the white-eyed phenotype (w1118), indicating that the transposon in question could not be located on the X chromosome. If this transposon were on the X chromosome of the parental male fly, only the female progeny could have had the wild-type (w+) phenotype. Further crosses to map the location of this transposon are not yet complete. A second set of crosses has been established between w+ males and w1118 females that are also carrying a balancer (CyO) for the 2nd chromosome. It can be concluded that the eye color gene in question is on the 2nd chromosome if all of the CyO flies from this second cross are white-eyed (w1118) and all of the non-CyO flies are w+. If this is not the case, further crosses must be performed to complete the mapping process.

Using image analysis in 3D to quantify the relative gene expression in Drosophila melanogaster blastoderms. BENJAMIN GARMAN (Shasta College, Redding, CA 96080) DAVID W. KNOWLES (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

While many studies of the genome and of specific genes of the organism Drosophila melanogaster have been conducted, there is a lack of available information to explain the connection between gene expression and the transcription network. As part of the Berkeley Drosophila Transcription Network Project (BDTN), we are aiding in the understanding of gene transcription in D. melanogaster by providing three-dimensional spatial data sets of gene expression for stage 5.8 blastulae, at cellular resolution. Based on biological and developmental factors, embryos are being staged and stained to provide flourescent samples that are within a tight 2-3 minutes developmental period. Staining is done through an intensive in-situ hybridization process using tyramide signal amplification protocols to attach the fluorophores coumarin, cy3 or cy5 to multiple gene products within the same embryo. Nuclear stain, sytox green, is also attached to reveal all nuclei. The resulting fluorescent embryo is then imaged using multi-channel 2-photon confocal microscopy to produce three-dimensional images, acquired at 1024x1024x100 pixels. The dimensions of each data point resolves to approximately one micron of real spatial distance. These images are then stored in a database and analyzed. Analysis techniques include mapping of each cell and its nuclei, creation of a mesh model of the embryo surface for projection and comparison analysis, figuring percentage expression of specific gene patterns in any individual cell, and future developments. Currently, the gene expression patterns snail, fushi tarazu, even skipped, sloppy paired 1, rhomboid, and knirps are being acquired and studied, but the list will slowly be expanded to include all active gene expression patterns within, and then beyond, this stage of the organisms development. The overall goal is to map the early transcription network of Drosophila melanogaster.

Water stress impacts on the net leaf 18O isoflux: Experimental results and model predictions. ERIN HANLON (St. John's College, Santa Fe, NM 87505) MARGARET S. TORN (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Water-stressed leaves have lower stomatal conductance and a lower photosynthetic rate than leaves not subjected to water stress. The Isotope Land Surface Model (ISOLSM) was developed from the Land Surface Model (LSM) to predict the isotopic fluxes of CO2 and H2O (isoflux) from vegetation and soil. Previous publications revealed ISOLSM's effectiveness at predicting the isoflux from plants under normal conditions. Until now the model had not been used to predict the consequences of water stress on the isoflux from photosynthesizing plants. We examine both causes and effects of water stress in the leaf. The model was set up to predict the effects of drought stress and we looked at the results from that prediction.

Whole Gland Hormone Receptor Analysis of Mammary Development. ABBEY HARTLAND (Shasta College, Redding, CA 94049) CARLOS ORTIZ DE SOLÓRZANO (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Steroid hormones, signaling through their cognate cellular receptors, are known to play an important role in the development of the breast and its tendency to become carcinogenic. Both the number of cells that express hormone receptors and their distribution throughout the gland contribute to the homeostatic equilibrium that allows normal development, maintenance and function of the gland. Many recent developments in diagnostic tools have provided for quick assessment of the receptors in clinical specimens, but do not account for the rich cellular heterogeneity within the tissue sample. In response to these difficulties, our laboratory has developed software capable of reconstructing the mammary ductal tree from serially sectioned glands and mapping proteins on that reconstruction. In this study, we have combined this software with immunohistological techniques to analyze estrogen receptor (ER)-a and progesterone receptor (PR) expression in wild-type murine mammary glands from 6-48 weeks of age. We have quantified the expression of the receptors in each of the mammary glands and shown the differences in number of receptor expressing cells between different morphological structures and developmental stages. These results stress the importance of examining cells within their three-dimensional histological context.

XPG Protein Levels and Protein Interactions After Exposure to Various DNA Damaging Agents. ANI TEJIRIAN (Diablo Valley College, Pleasant Hill, CA 94523) BRETT MAHER HALTIWANGER (Lawrence Berkeley National Laboratory, Berkley, CA 94720)

Exposure to ionizing radiation (IR) and hydrogen peroxide (H2O2) cause several types of DNA damage: DSBs, SSBs, and base damage, some of which can lead to stalled replications forks. Different proteins are known to be involved in each of the various repair pathways which occur. Preliminary evidence suggests that XPG interacts with other proteins know to be involved in the repair of replication forks, and may have role in base excision repair (BER), a process important in repairing base damage due to IR H2O2. There is evidence that BER is inducible and may be important to the same process as the adaptive response. The adaptive response is induced in cells which are treated with a priming dose of low IR or H2O2 and allowed to repair before treated with a challenge dose of higher IR or H2O2. These cells exhibit less damage then cells treated with a high dose of IR or H2O2 alone. The study was done to determine (A) the degree of colocalization of H2AX and XPG, and (B) whether XPG levels increase with increasing doses of H2O2 in cell lines known to induce an adaptive response. Colocalization between XPG and H2AX was determined by immunofluorescence. U2OS XPG/V5 tagged osteosarcoma cells were treated with 0, 40, 100 M of H2O2 or 0, 1, 4 Gy of IR. Lymphoblastoid cell lines +/- for an adaptive response were treated with 0, 40, 100 M of H2O2 and analyzed by both western and IF. Our results revealed the number, brightness, and size of H2AX and XPG foci seemed to increase for both IR and H2O2 doses. In the U2OS cells exposed to IR the degree of colocalization seemed to increase with increasing dose. In lymphocyte, the XPG staining seemed concentrated in the cytoplasm of the cells. The summary of western results revealed XPG levels to increase in one adaptive cell line (10), and remained the same in the other two lines. The increase of colocalization and XPG supports some interaction occurring at the site of damage. However, further research must be done to solidify these conclusions.