Biology Abstracts:

A Comparison of DNA Damage Probes in Human Mammary Epithelial Cells with 150 kVp X-Rays. CHRISTY WISNEWSKI (University of California, Davis, Davis, CA, 95616) ELEANOR BLAKELY AND KATHLEEN BJORNSTAD (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

In this study we investigated 53BP1 and H2AX, DNA damage markers, to look at genetic mechanisms underlying responses to radiation insult. Two human mammary epithelial cell (HMEC) lines, one subtype of HMEC 184 with a finite lifespan and S1 with an infinite lifespan were investigated to research the role of immortalization in DNA marker expression. Cells were irradiated with 50 cGy, fixed after 1 hour with 4% paraformaldehyde, and processed through immunofluorescence. Cells were imaged using an immunofluorescent microscope and digitally captured using Image Pro Plus software. 8-bit images were analyzed using Image J and counted. The 184 cells showed more positive response within the irradiated samples than the S1 samples. It was observed that the S1 had a previous peak time of 30 minutes with an alternative DNA damage probe; this could explain the decrease in signal for S1 for both probes used in this research. We also noted that the H2AX response was more punctate in the 184 cells, whereas the 53BP1 response was punctate in both cell lines. We hope to expand the dose and time course studied in the hope that this will broaden the knowledge obtained from the preliminary data of this research. It is important to understand whether the process of transformation to immortalization compromises the DNA damage sensor and repair process.

A New Method for Protein Sequence Characterization Using Hidden Markov Models. HARSH SHAH (University of Illinois at Chicago, Chicago, IL, 60607) GYORGY BABNIGG (Argonne National Laboratory, Argonne, IL, 60439)

Predicted protein sequences of newly sequenced species are normally analyzed for similarity to existing protein sequences using BLAST. The sequences are also characterized by searching with Hidden Markov Models (HMMs) of existing protein families in order to assist function assignment. While the BLAST searches are performed quickly, the more accurate searches using HMMs are computation intensive and might take a long time. Once new sequences are identified that belong to a certain protein family, these sequences should be incorporated into family profile (HMM) in order to represent these new members. The new HMM in return should be used to search the protein space again for inclusion of potential new members not found previously or simply rebuilding statistical data for the existing family members. While HMMs are very powerful for the detection of protein family members, the dynamic construction of them is computationally prohibitive. We have explored the possibility of constraining the protein space and therefore speeding up searches with HMMs using two methods: 1) using a regular expression (REGEX) obtained from the HMM or 2) using PSI-BLAST with a position-specific scoring matrix generated from the HMM to select candidate sequences from the protein space (about 3.5 million sequences). The smaller proteins sequence database was used for the more accurate search by the HMM. We have compared execution times and the accuracy using a direct HMM search approach with the two-step techniques. We have determined that while the current implementation of the REGEX-based approach was resulting in fast execution times, its accuracy was greatly affected by the type of HMM and was only applicable for a small subset of cases. The second, PSI-BLAST-based approach resulted in fast execution times and high accuracy when compared to the HMMSearch standard. Using the combination of PSI-BLAST and HMMSearch programs (PSI-HMMER) improved accuracy even further with small impact on execution times. The software developed during this project (PSI-HMMER) enables the searches of protein sequences databases about 200 times faster than traditional tools with negligible impact on search accuracy. This tool will be incorporated into the currently used bioinformatics pipelines.

Acetyltransferase activity of the 11-amino acid peptide cofactor of the adenovirus proteinase. YUE LIANG (University of California, Berkeley, Berkeley, CA, 94704) WALTER F. MANGEL (Brookhaven National Laboratory, Upton, NY, 11973)

The 11-amino acid peptide pVIc (GVQSLKRRRCF) from the C-terminus of the precursor to adenovirus protein VI has a transacetylase activity. Acetylation is an important physiological process in eukaryotic cells. For example, it is involved in DNA replication, transcription and repair. pVIc was incubated with acetyl coenzyme-A (Ac-CoA) at pH 7.0 for 15 minutes and the reaction was fractionated by high performance liquid chromatography (HPLC). A new peak appeared whose mass corresponded to mono-acetylated pVIc as determined by Matrix-Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry. The kinetics of acetylation were linear for at least 21 minutes. The equilibrium dissociation constant (Kd) for the interaction of Ac-CoA with pVIc was 1.1 mM. When purified mono-acetylated pVIc obtained at pH 7.7 was incubated at pH 10.3 for 15 minutes, multiple peaks were observed upon HPLC. MALDI analysis of each peak indicated one peak contained pVIc, another the original mono-acetylated pVIc, two other peaks contained mono-acetylated pVIc, three peaks contained di-acetylated pVIc and one peak contained tri-acetylated pVIc. The reaction at pH 10.3 was repeated but in the presence of the C-to-A mutant of pVIc. The mutant pVIc did not become acetylated. Thus, the new acetylations at pH 10.3 occurred in cis, except for the trans-acetylation between cysteine residues on different pVIc. A long-term goal is to find out the effects of acetylation on pVIc and, therefore, on viron assembly, and further discover anti-viral agents that can interfere with viral assembly.

Amplification and Tagging of Sulfolobus solfataricus Genes for Recombinant Expression. STEPHANIE PETERSON (Del Mar College, Corpus Christi, TX, 78404) STEVEN M. YANNONE (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

In organisms that thrive at moderate temperatures, many biological processes such as DNA repair occur through transient protein interactions. Understanding these interactions and the temporary protein complexes that they form is vital to understanding how cells function especially in how they repair damaged DNA. Protein interactions within hyperthermophiles like Sulfolobus solfataricus may be stabilized at moderate temperatures. The work presented in this study provides the initial steps towards thermally trapping otherwise short-lived protein complexes. Genes associated with DNA repair were selected from the S. solfataricus P2 genome and were modified with a directional tag on the N-terminus and a six-histidine tag (6x-hist) on the C-terminus using gene specific primers. Genes were amplified, cloned into entry vectors, and transformed into E. coli cells. Colonies were then selected and grown in liquid culture. Plasmid DNA was isolated using alkaline-lysis extraction method and constructs were confirmed with restriction digestion. Sixteen out of twenty-nine constructs were successfully confirmed by restriction digestion and fragment pattern on 1% agarose gels. These constructs will be further studied through two different expression systems: E. coli expression and S. solfataricus expression. E. coli expression should provide insight into independent protein structure and function. Native expression will not only provide information about the structure and function but will also identify obligate protein partners in their native organism. In addition, this approach will identify the root causes of difficulties that arise from recombinant expression.

Analysis of Cell Wall Mutations in Maize using Pyrolysis Molecular Beam Mass Spectrometry. BRIANNA HARP (Metropolitan State College of Denver, Denver, CO, 0) MARK DAVIS (National Renewable Energy Laboratory, Golden, CO, 89401)

This study applies Pyrolysis Molecular Beam Mass Spectrometry (PyMBMS) as a high throughput screen of cell wall substrates to characterize cell wall mutations. We had 3 goals in our study: improve PyMBMS technique, confirm cell wall mutations, and characterize differences between mutant lines and controls. We tested two methods of extraction: the Accelerated Solvent Extraction (ASE) and a simplified, rapid ethanol/acetone extraction using PyMBMS and multivariate statistical analysis to determine which method is most effective for removing extraneous cell wall material. This investigation found the ASE extraction to be most effective. A second study was performed using a combination of ASE extracted and untreated whole mutant 33_’00FL-041-39 and 34_02S-1030-22 samples and controls to identify mutant lines and characterize differences among the samples, while analyzing benefits of the ASE extraction technique. The results of this study showed no significant differences in cell wall chemistry or advantage in using the ASE extraction technique. Our third investigation sampled six different mutants using only whole samples. We found that the mutant line 27_02s-1137-40 had a significant increase in C5 and C6 sugars. The results of our analysis of mutant line 39_’00FL-042-20 (387, 388, 393, 395, 396, 399) is inconclusive.

Analyzing the Structure and Function of Novel Cytochromes from a Natural Microbial Community. ANNA SIEBERS (University of California, San Diego, La Jolla, CA, 92093) MICHAEL P. THELEN (Lawrence Livermore National Laboratory, Livermore, CA, 94550)

The Richmond mine in Iron Mountain, California, provides an unusual ecosystem suitable for the growth of microbial biofilms which produce many unique proteins. Through iron oxidation, these proteins facilitate acid mine drainage (AMD). Because this habitat is extremely acidic, survival is an extraordinary feat and the process of environmental selection is rare. In order to understand the mechanisms by which these organisms oxidize iron and gain electrons for energy, biochemical studies were applied. More specifically, column chromatography, spectrophotometry, and gel electrophoresis were used to determine the proteins present in different biofilms. Two specific locations of the mine researched were the AB drift and Ultraback C (UBC), which were both found to contain at least five different types of protein and a large amount of heme-bound cytochromes. Another application of these methods was to investigate proteins playing a major role within the community; one protein selected was cytochrome 579 (Cyt₅₇₉) due to its abundance in the biofilm, iron oxidizing potential, and signature absorbance of 579nm. The structure and function of Cyt₅₇₉ could be characterized by the isolation of its heme, which was completed using column chromatography; however, one of the challenges has been liberating the heme from the column. Further research, including acid-base and temperature profiling of Cyt₅₇₉ should help elucidate its structural changes within alternate environments and metabolism within the community.

Characteristic colonization of poplar trees with gfp expressing bacterial endophytes. ERIKA FREIMUTH (Cornell University, Ithaca, NY, 14850) DANIEL VAN DER LELIE (Brookhaven National Laboratory, Upton, NY, 11973)

Endophytic bacteria that colonize the internal systems and spaces of plants contribute valuable functions and services to the plant both individually and as a community. The enhancement of plant growth and metabolic processes by endophytic bacteria offers the opportunity to mediate both increased production of plant material and the sequestration of toxins within its tissues. Cuttings of poplar trees were inoculated with selected strains of endophytic bacteria (Pseudomonas putida W619 and Enterobacter sp. 638) genetically modified to stably express a green fluorescent protein and resistance to kanamycin. The colonization behavior and effects of these bacteria on the plant were monitored biweekly using fluorescent microscopy as well as by grinding and plating different sections of plant tissue (including sections of the rhizosphere, roots, shoots, and leaves). By thus tracking select endophytic bacterial strains marked with gfp through the plant’s development, the colonization efficiency and patterns of these endophytes in poplar can be utilized in various applications. Poplar biomass production and chemical sequestration efficiency can be maximized via endophytic bacteria for use in biofuels, carbon management and recycling, and the phytoremediation of environmental pollutants.

Characterizing the Role of the Nell1 Gene in Cardiovascular Development. LEAH LIU (Pennsylvania State University, University Park, PA, 16802) CYMBELINE CULIAT (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Nell1^6R is a chemically-induced point mutation in a novel cell-signaling gene, Nell1, which results in truncation of the protein and degradation of the Nell1^6R transcript.  Earlier studies revealed that loss of Nell1 function  reduces expression of numerous extracellular matrix (ECM) proteins required for differentiation of bone and cartilage precursor cells, thereby causing severe skull and spinal defects.  Since skeletal and cardiovascular development are closely linked biological processes, this research focused on: a) examining Nell1 mutant mice for cardiovascular defects, b) determining Nell1 expression in fetal and adult hearts, and c) establishing how ECM genes affected by Nell1 influence heart development.   Structural heart defects in Nell1^6R mutant fetuses  were analyzed by heart length and width measurements on formalin-fixed specimens and standard histological methods (haematoxylin and eosin staining).  Nell1 expression was assayed in fetal and adult hearts using reverse transcription polymerase chain reaction (RT-PCR).  A comprehensive bioinformatics analysis using public databases (UCSC Genome Browser, Mouse Genome Informatics, Integrated Cartilage Gene Database, PUBMED) was undertaken to investigate the relationship between cardiovascular development and each of the 28 ECM genes affected by Nell1.  Nell1-deficient mice have significantly enlarged hearts (particularly the heart width), dramatically reduced blood flow out of the heart and unexpanded lungs. Isolation of total RNAs from hearts of adult (control and heterozygote) and fetal (control and homozygous mutant) mice have been completed and RT-PCR assays are in progress.  The bioinformatics analysis showed that the majority of ECM genes with reduced expression in Nell1-deficient mice are normally expressed in the heart (80%; 22/28), blood vessels (71%;  20/28) and bone marrow (61%; 17/28).  Moreover, mouse mutations in seven of these genes (Col15a1, Osf-2, Bmpr1a, Pkd1, Mfge8, Ptger4, Notch3) manifest abnormalities in cardiovascular development.  These data demonstrate for the first time that Nell1 has a role in early mammalian cardiovascular development, mediated by its regulation of ECM proteins necessary for normal cardiovascular growth and differentiation.  In addition, the identification of Nell1 and its associated ECM genes can provide future targets for treatment of heart and blood vessel defects.

Chytrid Fungus and Ranavirus on Long Island. KATIE HEISER (University of Maryland, College Park, MD, 20740) TIM GREEN (Brookhaven National Laboratory, Upton, NY, 11973)

Amphibian populations worldwide have been devastated by Chytridiomycosis, a disease caused by Chytrid fungus (Bactrachytrium dendrobatidis).  Having likely originated in Africa, it has been found on nearly every continent and can be spread by a number of hosts. Many amphibian species do not have any tolerance to the fungus and populations can  quickly disappear following its introduction to the area. However, some can survive the disease and will act as carriers to spread it. Iridovirus is also a threat to local populations and was found at the  Brookhaven National Lab last year. As such, this project was conducted to investigate the potential link that these diseases may have with a recent island wide decline and possible extirpation of the southern leopard frog (Rana sphenocephala).  In their absence, several common and related potential vector frog species were sampled from various Long Island wetlands. Frogs were swabbed and the swabs were tested for Chytrid and Iridovirus. PCR and gel electrophoresis will be used to confirm the presence or absence of the diseases. The presence of chytrid or iridovirus in local populations would serve as a possible lead in the decline of Southern Leopard Frogs on Long Island. This was a data collection study and will not include the final results.

Cloning of DNA Repair Genes from a Hydrothermal Vent Worm. ANABEY CORNEJO (Contra Costa College, San Pablo, CA, 94806) JILL O. FUSS (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

The primary structure of DNA is highly reactive with molecular by-products of metabolism as well as UV radiation from the sun, and these reactions alter and damage the human genome. Alterations to the DNA structure and chemistry result not only from natural physical agents but also from man made chemicals, although to a lesser extent. If these alterations are not detected and either corrected or removed, a mutation can be fixed in the genome potentially leading to cancer and aging. Through evolutionary adaptation, cells have developed a series of mechanisms which allow them to remove the damage and restore the normal nucleotide sequence and DNA structure. One such mechanism is Nucleotide Excision Repair (NER) which removes oligonucleotides, which are short nucleotide segments that contain damaged bases. NER is further categorized based on the location of the repair. GG-NER or global genome repair refers to NER taking place in DNA not undergoing transcription. TC-NER or transcription-coupled repair refers to NER occurring in the transcribed strand of active genes. If the NER pathway is compromised, a number of human genetic diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome (CS) and Trichothiodystrophy (TTD) may result. These diseases are characterized by causing premature aging or a predisposition for cancer. To investigate the human DNA mechanisms of repair, we are studying an organism highly genetically homologous to humans, in this case Alvinella pompejana or Pompeii worm. The effectiveness behind using the Pompeii worm as a model for studying the process of DNA repair is due to the fact that most of its protein activity occurs at temperatures as high as 80C. The Pompeii worm inhabits geysers found along underwater volcanic mountain ranges known as hydrothermal vents. These underwater formations release jets of water reaching temperatures as high as 300C. Not only does it serve as a great organism for comparative studies due to its genetic similarity to the human genome but also because of its extreme heat tolerance. This would imply that its proteins will be fairly stable at room temperature which will allow for extensive in vitro study. More specifically to investigate DNA repair genes involved in NER, through the cloning of these genes from Alvinella pompejana as well as constructing plasmid vectors for recombinant protein expression, protein purification, protein to protein interaction studies, transfection of cultured human cells with expression vectors followed by assays for reporter gene expression, and analyzing particular proteins as a function of the cell cycle in mammalian cells.

Comparison of Fragmentation-Directing Properties of Amino Acids. DANNY TAASEVIGEN (Montana State University, Bozeman, MT, 59715) WILLIAM R CANNON (Pacific Northwest National Laboratory, Richland, WA, 99352)

Proteomics is the study of proteins and their function by the use of high, throughput methods such as mass spectrometry. With the use of molecular theory and computation, our group helps to develop more accurate peptide identification methods. Taking into account the potential energy of the system and the forces that affect peptide formation, simulations can be run to determine the probability of obtaining a configuration conducive to proton transfer to the respective carbonyl oxygen along the side chain. These simulations are run in the gas phase with a total time of 5 nanoseconds. Once complete, radial distribution functions provide us with the probability of proton donation along the chain. This data will be compared to predicted spectra in hopes of identification. The peptides are composed of various proportions of 20 naturally occurring amino acids and were derived from polyalanine, which proves to be the basis of analysis. However, these peptides have to have specific charges and the simulations have certain specifications that must be set to properly match the spectra. If detection of peptides is successful in this manner, it is likely to be scaled up to the genome-wide scale.

Creating Information System of Oncological Methylated DNA data. REGINALD GABRIEL (Cheyney Unversity, Cheyney, PA, 19319) DR. SEAN R. MC CORKLE (Brookhaven National Laboratory, Upton, NY, 11973)

DNA methylation is the addition of a methyl group to the cytosine ring of the CpG dinucleotide, forming methyl-cytosine and is catalysed by DNA methyltransferases. An information system is needed for biologists to retrieve empirical data and relate it to DNA methylation sequences experimentally identified in human cancer cells. This project is intended to create such information system available for biologists. Information was collected from biomedical article reviews and existing Internet database, such as RefSeq (NCBI), the UCSC Genome database. PostgreSQL database server running on MAC OsX (DARWIN – BSD) system was established. The tool NetBeans Integrated Development Environment (IDE) was use to create the Graphical User Interface (GUI), the client application with the Java programming language. The Java client can run virtually on any computer because of the benefits of Java; “write once and run anywhere”. Communication between the Java client and PostgreSQL database server was established through the Java Data-Base Connectivity (JDBC) package. Object Oriented Techniques (OOT) is ideal for connecting various sub-components of the system: network connections and GUI application. A class PostJDBC was implemented to connect to the database, issue queries, and retrieve resultant data. UserGui, the user interface, relies heavily on inheritance and polymorphism techniques to achieve dynamic data manipulation and graphical display of the output. The client application presents the user with a list of gene symbols which are contained in the database, and allows the user to browse for more detailed information by mouse selection. This project can be easily expanded by including information about different types of features in DNA sequences and their relation to human cancer cells.

Crystal Structure of Mn2+ Bound Escherichia coli L-arabinose Isomerase Complex: Its Implications in Protein Catalytic Mechanism and Thermo-Stability. WEISHA ZHU (Cornell University, Ithaca, NY, 14853) BABU MANJASETTY (Brookhaven National Laboratory, Upton, NY, 11973)

Escherichia coli L-arabinose isomerase (ECAI) is responsible for the initial stage of L-arabinose catabolism, converting arabinose into ribulose in vivo. This enzyme also plays a crucial role in catalyzing the conversion of galactose into tagatose (low calorie natural sugar) in vitro. The crystal structure of its native form has been recently solved. This project aims to determine the crystal structure of enzyme in complex with co-factor metal ions and candidate substrates/inhibitors to further study structure-function relationships important for the enzyme’s biological mechanism. High-throughput techniques were adopted for protein structure analysis. Crystals of different complexes were grown using hanging drop vapor diffusion method. Diffraction datasets were collected at X4C, X6A and X29 beamlines, National Synchrotron Light Source, Brookhaven National Laboratory. The method of molecular replacement was utilized in structure solving using the native structure as the initial model. A series of crystallographic software packages were used to build and refine the structural model. The analysis of the structure of Mn²⁺ bound ECAI protein complex completed to 2.8 Å resolution with the R-factor of 23% and the structure was deposited to Protein Data Bank (PDB ID 2HXG). The overall fold and biological arrangement of the complex are conserved. Active site of ECAI is located at the subunit interface and forms deep cleft. Mn²⁺ ion is identified at the active site surrounding with an octahedral coordination. Comparison between apo and holo structures reveals variations in the metal coordination sphere, conformational changes associated with the active site residues and increased number of interactions between subunits. The available biochemical and structural data provides the structural basis for efficient protein catalytic mechanism and increase in thermo-stability of the enzyme induced by the presence of Mn²⁺. This study will benefit optimization of low-calorie sugar production. The target of this project is one of the many selected under investigation by New York Structural Genomics Research Consortium (NYSGXRC ID T2031).

Deletion of a Chitin Synthase Gene in a Citric Acid Producing Strain of Aspergillus niger. TORRI RINKER (Oregon State University, Corvallis, OR, 97331) SCOTT BAKER (Pacific Northwest National Laboratory, Richland, WA, 99352)

Fungi have the potential to convert biomass into high-value chemical products. Filamentous fungi produce organic acids, such as citric, lactic, and fumaric acid. Citric acid production by the filamentous fungus Aspergillus niger is carried out in a process that causes the organism to drastically alter its morphology. This altered morphology includes hyphal swelling and highly limited polar growth resulting in clumps of swollen cells that eventually aggregate into pellets of approximately 100 microns in diameter. In pelleted form, A. niger has increased citric acid production rates when compared to those in filamentous form. Production of citric acid by A. niger serves as a model in which a filamentous fungus can take on a particular morphology and increase product output. Chitin, a polysaccharide in fungal cell walls, plays a central role in the morphology of fungi. A gene coding for chitin synthase with a myosin-motor domain (csmA) was deleted from the genome of A. niger using a PCR-based gene deletion construct. The mutant was tested on minimal media with and without osmotic stabilization. In the absence of osmotic stabilization, the germlings of the deletion strain (csmA) were abnormally swollen and highly vacuolated. This suggests that chitin synthase is important for germination of spores, which in turn impacts morphology and may affect citric acid production rates. Genetic manipulation, such as gene deletion, can be used in the future in other strains of filamentous fungi to obtain desired morphologies and optimized product output.

Detecting and Quantifying Heavy Metal Contamination in Water. MATTHEW LARSEN (Brigham Young University - Idaho, Rexburg, ID, 83460) ANGELICA STORMBERG (Idaho National Laboratory, Idaho Falls, ID, 83415)

The DOE has a goal of remediating environmental contaminants. We must first know where the contaminants are, and in what concentration. For detection of heavy metals in aqueous media a transgenic nematode has been developed. C. elegens releases a Green Flourescent Protein (GFP) upon contact with heavy metals. Our work was to calibrate the amount of flouresence with the concentration of heavy metal contamination. Water samples can be analyzed in 24 hours, and cost less than 10 cents per sample. We were able to analyze five metals: Zinc, Lead, Mercury, Cadmium, and Nickel. Each was analyzed at 4 different concentrations. The calibration curves developed may be used to test a water sample and determine the concentration of the heavy metal contaminants.

Detection of botulinum toxin with an automated fluidics system using quantum dots as the fluorophore. ABBY TYLER (Utah State University, Logan, UT, 84321) MARVIN WARNER (Pacific Northwest National Laboratory, Richland, WA, 99352)

New technologies able to detect biotoxins are in demand as the threat of bioterrorism grows. Effective detection systems have a high sensitivity, allow rapid detection and be automated and accurate. Automated fluidics systems are being developed at PNNL to fulfill all these requirements. The fluidics system performs a sandwich immunoassay that ultilizes high affinity antibodies to detect botulinum toxin. A column of beads, which are conjugated to an antibody specific for a single epitope on the botulinum neurotoxin, are packed above a rotating rod. Next, a liquid sample containing the toxin analyte is perfused over the column. As the sample passes over the column toxin molecules bind to the antibodies on the beads. Then, a dye-labeled antibody that is specific for a second epitope on the toxin is perfused over the column to facilitate binding to the toxin immobilized on the column. Unbound dye is flushed off the column with buffer. Finally, the column is exposed to light to excite the dye and signal the absence or presence of the target toxin. A photo multiplier tube is used to measure the fluorescence of the dye particles left in the column. Many fluorescent dyes are available, and in a series of parallel experiments we have been investigating the use of semicondutor quantum dots as the fluorophore in this detection system. Quantum dots, or semiconductor nanocrystals, are a fluorphore that is becoming widely used in bioimaging though their use in biodetection is relatively new. Some of the advantages that quantum dots have over molecular dyes are that they have a broad excitation spectrum and a narrow emission spectra that is highly red-shifted compared to the excitation wavelength. Further, the wavelength at which they emit can be controlled by the size of the quantum dot. Detection of botulinum toxin using quantum dots in an automated fluidics system was the goal of this research, and is currently under development. Future work will involve improving the overall performance of the system by investigating characteristics such as the non-specific binding of the quantum dots to the column as well as the stability of the antibody coupled materials.

Determination of a Role for Cellular XPG in Repair of Oxidative Damage to DNA. EMILY FOX (City College of San Francisco, San Francisco, CA, 94112) HELEN BUDWORTH (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Mutation of XPG can cause the debilitating diseases Xeroderma Pigmentosum (XP) and Cockayne’s Syndrome (CS), which result from a deficiency in DNA repair. XPG cuts 3’ to DNA lesions during nucleotide excision repair (NER), as well performing the non-catalytic roles of recognizing stalled RNA polymerase II and binding transcription-sized bubbles in transcription coupled repair. Through in vitro tests with purified proteins, XPG has been found to stimulate hNth1, which removes oxidized pyrimidines in the base excision repair (BER) pathway. In this study, whole cell extracts from XPG-deficient cells obtained from patients with XP-G/CS were found to be defective in incision of 5,6-dihydrouracil (DHU). This defect was corrected by the addition of purified XPG, suggesting that the mutated XPG in XP-G/CS cells is unable to stimulate hNth1. In addition, cells from XP-G/CS patients were found to be slightly sensitive to X-rays and hydrogen peroxide, as determined by colony formation survival assays. shRNA against XPG was used to knockdown XPG in normal cells in order to provide another model for XPG deficiency in which the only variation from control cells is reduced levels of XPG.

Determining Cellular Localization of Candidate Cilia Proteins in Transgenic Caenorhabditis elegans. SANDRA MCGILL (Clayton State University, Morrow, GA, 30281) EDWARD J. MICHAUD (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Primary cilia are small organelles that protrude from cell surfaces and are conserved in most eukaryotes, including nematodes, mice, and humans. In humans primary cilia have vital sensory functions; flaws in these organelles lead to many diseases. Based on comparative genomics and proteomics studies it has been estimated that the cilia proteome consists of 300-500 proteins, but the functions of most are unknown. Disruption of the homologous genes in mice is an effective approach for determining function. However, prior to performing lengthy and costly experiments in mice, it is desirable to verify that these candidate genes do indeed localize to primary cilia. An efficient method for determining the in vivo cellular localization of candidate cilia proteins is to perform translational GFP assays in transgenic nematodes. In a previous project, translational fusion vectors were constructed in which two nematode candidate cilia genes, E02H1.5 and R148.1, were cloned in-frame with the Green Fluorescence Protein (gfp) reporter gene in a plasmid vector, pPD95.81. In this project, the two vectors are being microinjected into the gonads of nematodes, where transcription and translation of the fusion vector results in the nematode protein being tagged with the GFP protein. Visualizing the GFP marker under a fluorescent microscope reveals the locations of the fusion proteins in nematodes. Three independent lines of transgenic nematodes were established for E02H1.5, but examination of the transgenic nematodes did not reveal any GFP expression. Based on this result, and on further examination of the genomic locus encompassing the E02H1.5 gene, it appears that E02H1.5 may be one gene in a four-gene operon. Approximately 10% of all worm genes exist in operons, where one promoter region directs the expression of all genes in the operon. The entire E02H1.5 operon is now being cloned into the pPD95.81 vector and will be used to generate new lines of transgenic nematodes. Microinjection of the R148.1 vector into nematodes is under way. Additionally, translational fusion vectors are being made for two other candidate cilia genes, K07G5.3 and C02H7.1, which will also be used to generate transgenic nematodes to determine protein localization. If these four proteins are shown to localize to cilia in nematodes, the stage will be set for analysis of the functions of the homologous proteins in mice and humans.

Developing EPIMODEL2: A Computer Program for Teaching Population Growth Modeling. DANIEL HEIDFELD (Michigan Technological University, Houghton, MI, 49931) FORREST W. NUTTER JR. (Ames Laboratory, Ames, IA, 50011)

EPIMODEL is a computer teaching program that is currently being used in more than 40 universities worldwide to teach students biological concepts concerning population growth modeling. EPIMODEL has become outdated because it was originally created in Quick BASIC for MS-DOS. Therefore, the goal of this project was to rewrite EPIMODEL using a computer language that would support Windows™. The programming language Java was chosen to develop EPIMODEL2 because Java is capable of operating on many platforms, including Windows™. EPIMODEL2 was programmed with the aid of various online manuals to develop algorithms that implement features within the Java environment. EPIMODEL2 was designed to mimic all of the features of the old version of EPIMODEL, but the new program implements a Graphical User Interface (GUI). The new program, EPIMODEL2, is a valuable and more versatile replacement for the outdated version, and the development of a new version in Java successfully accomplished the goal of this internship.

Developing software within Gatan’s Digital Micrograph in order to create automated STEM tomography. MICHAEL MYLENSKI (State University of New York at Albany, Albany, NY, 12222) HUILIN LI (Brookhaven National Laboratory, Upton, NY, 11973)

Automated Scanning Transmission Electron Microscope (STEM) tomography follows the ideals of Jianglin Feng, a researcher in the Department of Molecular Physiology and Biological Physics at the University of Virginia. Feng’s automated version of STEM tomography revolves around the ideas of auto-tracking and auto-focusing. An automated STEM tomography program will be the first of its kind at Brookhaven National Laboratory (BNL). The development of the automated STEM tomography program was based on scripts created using Digital Micrograph (DM), which is the program of choice for communication between the researchers at BNL and the JEOL 2010F microscope. When creating these scripts, JEOL’s FasTEM Communication Kit (FTCOMM) and Digital Micrograph’s Software Developers Kit (DMSDK) were essential in creating new functions to implement auto-tracking and auto-focusing. Feng’s paper provided the basic algorithm for automated STEM tomography and also a gradient formula in order to implement auto-focusing. Overall, there were five scripts and a Dynamic Link Library (DLL) that were developed in order to implement automated STEM tomography. The DLL file was the means of communication between the microscope and DM using FTCOMM and DMSDK, and it was developed using Microsoft’s Visual Studio .NET 2003. The Graphical User Interface (GUI) was created using functions within DM’s scripting language in order to benefit the user. The arithmetic for the auto-tracking and auto-focusing is primarily based in the DM scripts. When they communicated with the microscope, there were functions created in the DLL file that used FTCOMM functions in order to link the microscope and the DM scripts. The program that was completed includes the basics of an automated STEM tomography system with simple implementations of auto-tracking and auto-focusing. This project must be continued in the future in order for this version of automated STEM tomography to fully benefit researchers at Brookhaven Nation Laboratory in their study of structural biology.

Development of a Biosensor for Measuring Antibody-Antigen Interactions. CASSANDRA ARMSTEAD-WILLIAMS (Washington University School of Medicine, St. Louis, MO, 63110) KARA KRUSE (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

In recent years the Nanoscale Science and Devices group at Oak Ridge National Laboratory has developed and continues to study how microcantilever technology can be used to create high-throughput, label-free tests for biological macromolecules. The Vascular Research Laboratory at the University Of Tennessee Medical Center of Knoxville is studying the effects of certain drugs on the concentrations of Matrix Metalloproteases (MMP’s) in the blood. The Vascular Research Laboratory and the Nanoscale Devices and Sciences group are working together to create a dependable, high-throughput, label-free system for quantitatively measuring concentrations of MMP’s. Microcantilevers bend due to a change in the entropy and energy on one side of the cantilevers and not the other. This bending can be monitored optically by reflecting a laser beam off of the cantilevers and onto a position sensitive detector (PSD). The PSD translates the optical signal into an electronic signal for real-time monitoring of microcantilever bending. For this experiment, gold-coated silicon microcantilevers were immobilized with 3,3-Dithiobis(sulfosuccinimidylpropionate) (DTSSP)-a homobifunctional, amine reactive cross linking agent. Immobilizing the cantilevers with a DTSSP monolayer allows selective attachment of biochemical molecules onto the gold and silicon/silicon nitride microcantilever substrate. MMP specific probe antibodies were then attached to the microcantilevers via a captavidin-biotin linkage system. Surface amine groups of captavidin were reacted with the succinimide terminals of DTSSP. The biotin-conjugated, MMP specific probe antibodies selectively adsorbs onto the captividin protein layer. After functionalization (immobilization), solutions containing unknown concentrations of MMP’s are introduced to the microcantilevers while bending is being monitored. Using a model antibody system, Current tests indicate that DTSSP and antibodies can reliably be immobilizing on to the cantilevers surfaces. However, we have not been able to determine the antibodies’ range of sensitivity for protein concentration detection. The reliability of the DTSSP and antibody immobilization shows that this detection system can work. We are currently modifying our detection system and probe immobilization procedures to find the optimal working range for this technology.

Dialysis or Column Exchange: Developing an Efficient and Quantifiable Protocol for Detergent Exchange Prior to Crystallization of Membrane Proteins. NICK IMPELLITTERI (UW Stevens Point, Stevens Point, WI, 54481) PILIP D. LAIBLE (Argonne National Laboratory, Argonne, IL, 60439)

The goal of this study was to develop quantitative methods to replace detergents used for the solublization and purification of a membrane protein with a diverse range of detergents that could potentially increase the success rate of the proteins characterization and crystallization. Previously it has been anecdotally perceived that detergent exchange could be accomplished either by dialysis or by extensively washing and eluting column bound membrane protein with an alternate buffer containing a different, yet desired, detergent. Rhodobacter sphaeroides reaction centers (RCs), as well as foreign affinity-tagged Escherichia coli membrane protein APC00809 (809) were solublized, concentrated, and purified using the detergents N,N-Dimethyldodecylamine-N-Oxide (LDAO) and deriphat-160, respectively. These two membrane protein’s detergents were exchange into the following detergents using both on column exchange and dialysis: LDAO, Triton X-100, n-Octyl-B-D-Glucopyranoside (OG), Tetraethylene Monooctyl Ether (C8E4), Deriphat-160, and CHAPS. Column-bound membrane proteins were eluted and analyzed after being washed with 1, 5, 10, and 20 column volumes of buffer containing the desired detergent, while membrane proteins were left in dialysis tubing with large reservoirs for 1,2,5, or 7 days. After concentrating all samples to ~10 mg\mL and analyzing them using thin layer chromatography (TLC), iodine staining and Image J software, the results of this experiment explicitly show the superiority of column exchange over dialysis in terms of accomplishing complete exchange of detergents with every combination attempted. For every trial, detergent exchange for RCs is more complete after 20 column volumes of detergent buffer than after 7 days of dialysis. Column exchange yielded a complete detergent exchange for all detergents except deriphat, which did not exchange well with LDAO in RCs in either column exchange or dialysis. Though all dialysis and most column exchange samples for 809 were lost in concentration, the column exchange samples that were concentrated and analyzed clearly showed that column exchange was fast and effective. In this experiment it clearly shows that column exchange is the most suitable means by which to exchange one detergent with many others of varying CMC. Dialysis, as this experiment shows, can only yield up to a 90% exchange after 7 days when exchanging with detergents of extremely low CMC. These results or this experiment will lead to more defined and reproducible protein-detergent complexes for input into structural and functional studies.

Effectiveness of Driving Surveys to Locate Burrowing Owls (Athene cunicularia). SARITA INCE (Lane Community College, Eugene, OR, 97401) MICHAEL SACKSCHEWSKY (Pacific Northwest National Laboratory, Richland, WA, 99352)

The burrowing owl (Athene cunicularia) is classified as a Species of Concern in the Columbia Basin by the U.S. Fish and Wildlife Service, a candidate species by the Washington Department of Fish and Wildlife, and as Endangered in Canada. A standardized, reliable, and time-effective population survey method has yet to be developed for burrowing owls. Without a reliable survey method, long term population trends cannot be accurately estimated. Driving surveys were evaluated as a method to locate burrowing owls in shrub steppe habitat and estimate relative population density. Burrowing owls are tolerant of human activities and are often found near roadways, making them an ideal candidate for driving surveys. Surveys were conducted on pre-determined routes at a prescribed speed of 20 mph. Seven routes within the Department of Energy’s Hanford Site near Richland, Washington, were chosen based on habitat type and previous burrowing owl sightings. The surveyed routes consist of shrub steppe habitat with short grass and sagebrush cover ranging from dense to none. Surveys were undertaken for two to three hours during the owls’ most active times of day, after sunrise and before sunset. Driving surveys require two people, a driver and an observer who carefully watched one side of the road for burrowing owls. At the end of the route, the driver turns around and the observer watches the other side of the road. When a burrowing owl was observed, the location from the road was recorded using a GPS (Global Positioning System). Burrowing owls were sighted on all but one of the seven routes. Burrows and owls were found very close to the road and over 100 meters off road. Proximity to human activities seemed to have little effect on burrowing owl sightings. Driving surveys are effective in locating burrowing owls on the Hanford Site and should be repeated in future years as part of an on-going population monitoring effort. Relatively few of the owl groups were seen during both surveys of each route, indicating that more then two surveys per route is required to assure that all of the owls along each route are found. Driving surveys also may prove useful for gathering population density data on other animals within the Hanford Site.

Effects of Low-Level Cadmium on Cyr61 Expression in pre-Osteoclastic Cells. KATHRYN TORMOS (Benedictine University, Lisle, IL, 60532) MARYKA H. BHATTACHARYYA (Argonne National Laboratory, Argonne, IL, 60439)

Cadmium is a heavy-metal element that is a major component of orange or red pigments and is naturally occurring in trace amounts in air, water, and soil. It is well known that high, unnatural exposures to cadmium can result in toxic effects in human beings, in particular in the kidneys, liver, and lungs. However, the discovery that cadmium is linked to the severe bone-breaking Itai-Itai disease has shed light on the potential impact of cadmium on bone systems as well. Previous microarray experimentation of cadmium’s effects on genes expressed in bone cells in vivo in mice has shown the most up regulated gene early after cadmium treatment to be cyr61. The Cyr61 protein has been linked to such molecular processes as angiogenesis, ossification, embryogenesis, and cell migration. Preliminary immunohistochemistry staining and RNA isolation in bone cell cultures have shown potential Cyr61 protein expression in osteoclasts (bone resorbing cells); this expression appeared to be greater than in cultured osteoblasts (bone matrix excreting cells). This summer, we continue to test the hypothesis that cadmium triggers osteoclastic precursors cells to secrete Cyr61, increasing their migration, aggregation, and fusion into mature osteoclasts. Using the pre-osteoclastic RAW264.7 cell line, Cyr61 protein expression is being analyzed using Western blotting and immunoprecipitation, and cyr61 gene expression is being analyzed using RT-PCR technology. We aim to conclusively demonstrate whether the Cyr61 protein is significantly up regulated in cadmium’s presence in RAW 264.7 cells. Showing Cyr61’s role in osteoclastic precursors in the presence of cadmium will pinpoint a new, previously unresearched, role for the protein, and will also help our laboratory continue to construct a hypothetical pathway proving cadmium’s central role in bone resorption.

Environmental Health Studies on Peconic River Headwaters: Water and Sediment Chemistry. HA WANNA ST. CYR (Southern University at New Orelans, New Orleans, La, 70141) DR. MURTY KAMBHAMPATI (Brookhaven National Laboratory, Upton, NY, 11973)

The purpose of this research was to collect scientific environmental health data on water and sediments from remediated and natural sites of the Peconic River (PR) headwaters at the Brookhaven National Laboratory (BNL) and to compare results with available earlier findings. The specific objectives were to: (a) analyze samples for physico-chemical factors; (b) compile and analyze data statistically; and (c) to identify the interrelationships between abiotic factors. We hypothesized that waters of the PR would be acidic with excessive turbidity, nutrient poor, low dissolved oxygen (DO) levels, and free of contaminants. We have collected 54 surface samples (<15cm deep at 150m intervals) randomly from 7 experimental sites (LH1-7). Experimental sites were plotted using eXplorist 200 Global Positioning System (GPS) and ArcInfo Geographic Information Systems (GIS). Field data were obtained on DO, temperature, pH, turbidity, and conductivity using Yellow Spring Instruments, Inc. (YSI) probe. Water samples were analyzed using Hach DR890 colorimeter. Filtered and acidified water samples (pH<2) were used to estimate metal content using Inductively Coupled Plasma Spectrometer (ICP). Sediment samples were air dried, sieved, and saved in Ziploc bags. Macro and micronutrients were measured using LaMotte Soil Test Kits. Sediments were acidic (6.00±0.00 to 6.25±0.94 at LH3 and LH5, respectively) and nutrient poor. Water was acidic (4.61±0.10 to 5.87±0.04 at LH2 and LH5, respectively) and low in DO (1.49±0.17 to 5.67±0.70mg/L at LH3 and LH1, respectively). Samples had traces to zero chlorides, nitrates, and sulfates. Alkalinity ranged from 10.5±5.65 to 83.13±3.26mg/L. Sediment ANOVA results showed significant mean differences (P<0.05 and P<0.01) between elements, aluminum (Al), iron (Fe), lead (Pb), and chromium (Cr). In conclusion, experimental results were in partial agreement with our hypothesis (nutrient poor sediments and water; and low DO and high turbidity in water). However, we reject the null hypothesis, since the mean differences between groups were significant. Also, water and sediments of the PR natural sites have greater metal content (Al, Fe, Pb) than in the remediated sites. In some instances, however, current elemental contents of Al, Cd, Fe, Pb, Mg, and K in sediments of remediated sites exceeded the earlier findings.

Established an Information System Comprised of CpG Methylated DNA Data in Cancers. WENYI BI (Cheyney University of Pennsylvania, Cheyney, PA, 19319) SEAN MCCORKLE (Brookhaven National Laboratory, Upton, NY, 11973)

Determining the global pattern of DNA methylation, or the methylome and its variation in cells has become an area of considerable interest primarily because of its potential use as an early diagnostic biomarker for cancers. For time being, the research papers, results and literatures on this subject have grown rapidly and scattered in different systems. However, because there is lack of a comprehensive information system available to gain all correlated information into one picture, it forces the researchers to manually search through scores of biomedical journals and related websites. To meet the requirements to acquire them automatically, we developed an information system consisting of database system (back-end), and web application (front-end) by querying from a gene level to the related to methylated genes, cancers and literatures to gain all information by one-short deal. Our research went through data source collection, database system establishment, and web application coding. In addition, we developed a novel algorithm to identify genes near the diTag found in the experiment data.

Expression and Purification of a Yeast Hypothetical Protein. TIFFANY JORDAN (Elizabeth City State University, Elizabeth City, NC, 27909) S. SWAMINATHAN (Brookhaven National Laboratory, Upton, NY, 11973)

Solving protein crystal structures by X-ray diffraction or NMR is essential for doing protein chemistry and novel drug design. This project involves several aspects of macromolecular crystallography; for example expression and purification of proteins, optimization of crystallization conditions by use of robot, crystallization by sitting drop vapor diffusion method, observation and analysis of the crystals under the microscope and lastly x-ray diffraction of the crystals. During our ten weeks stay at Brookhaven National Laboratory, we were successful in expressing and purifying a 29 kDa hypothetical Yeast protein by His-tag purification using nickel chelated columns and by the size exclusion method of protein purification by using a FPLC machine. We ran 1D SDS-protein gels for analyzing and confirming our results.

Expression of Cyr61 in pre-Osteoclastic cells as a result of Cadmium exposure? MARYN VALDEZ (University of Maryland, College Park, MD, 20742) MARYKA BHATTACHARYYA (Argonne National Laboratory, Argonne, IL, 60439)

Cadmium (Cd) is a natural metal commonly present in paint, plastics, batteries, the protective coating of steel, fertilizers, and in cigarettes. Cd has been shown to start having adverse effects on bone organ and cell culture systems at levels ranging from 10nM-100nM. However, the mechanism by which Cd acts on the bone system to cause bone loss has yet to be deciphered and thus needs to be further studied. This is the ultimate goal of our research. Past experiments and the literature have led us to think that Cry61 plays a pivotal role in the pathway. Cyr61 is a ligand to the integrins aVß3. We hypothesize that Cd provokes expression of Cry61 in Osteoblast(OB) or Osteoclast(OC) cells, which binds to aVß3 located on the surface of OC precursors. The binding eventually leads to responses that lead to bone resorption. Specifically in this paper we are trying to determine whether Cd triggers OC precursor cells to secrete elevated levels of Cry61. Total Cell lysate(TL), extracellular matrix(ECM), and concentrated media(CM) samples collected after exposure to 100nM Cd for 2-48hrs, were analyzed for Cry61 expression using western blots. Most of our time was spent working out the proper protocol and we feel it has been developed to the best of our ability. We concluded that fetal calf serum (FCS ) does indeed cause Cry61 expression, as reported by others. We tentatively concluded, that at 24 hours and possibly at 48 hours, Cd is causing an increase in Cry61 expression in the media, but we can not say Cd is inducing Cry61 expression in TL or ECM. Perhaps a different cell line needs to be used-- One in which Cry61 is overexpressed to begin with, or perhaps we need to look at the pre-OB cell lines. More experiments that include more than 0.1% serum and more negative controls also need to be done. Immunhistochemical staining experiments may also be needed. Thus we made progress, but much more work needs to be done.

Expression of Type I Cohesin and Type I Dockerin Domains from Clostridia thermocellum for Nano Patterning. CATHEIRNE COFFMAN (University of Tennessee Chattanooga, Chattanooga, TN, 37403) JENNIFER MORRELL-FALVEY (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Cellulosomes are multienzymatic complexes from bacteria that have been studied for their ability to break down the cellulose in plant walls yielding ethanol as a byproduct. The scaffoldin protein is a significant component of the cellulosome structure because it binds to both the cellulose and cellulose degrading enzymes in the most advantageous manner for hydrolysis. In the assemblage of scaffoldin, nine Cohesin domains, in the presence of Calcium, mediate binding to enzymes that contain a Dockerin domain. This investigation examines the specific binding of the Type I Cohesin (CohI) and Type I Dockerin (DocI) molecules, which are components of the scaffoldin in the cellulosome. The objective is to artificially generate and assemble CohI and DocI using their unique binding specificity as an affinity pair. A 6x Histadine (His) tag will be added to the CohI so that it can bind to a nickel surface. Green florescent protein (GFP) and a strep tag are added to DocI. The interaction between the CohI and DocI would be visible through the GFP, and the strep tag allows for purification of the DocI fusion protein. Thus far, the CohI and DocI domains have been identified in the sequences of CipA and CelS respectively in Clostridia thermocellum. Using genomic material from C. thermocellum, the CohI and DocI domains were PCR amplified. Expression vectors were constructed with the CohI and DocI inserts. A GFP with no stop codon was also ligated into the vector containing DocI so that, when expressed, the GFP forms a fusion protein with DocI. The ligated plasmids were transformed into E. coli strain BL21(DE3) where the CohI and DocI-GFP can be expressed. After the cell produces the proteins, they can be purified using the attached His and strep tags. This affinity pair can potentially be useful in nano patterning as CohI can be positioned using nickel beads and binding events can be mediated through the Cohesin-Dockerin specificity. Binding can also be controlled since the CohI and DocI do not bind unless calcium is present in the system.

Extracting Methylated DNA using His-Strep Tagged Clones of McrB, MBD2b and MBD3LI Proteins. DANNY KOHUT (New York University, New York, NY, 10003) JESSICA POLICASTRO (University of Scranton, Scranton, PA, 18510) DR. JOHN J. DUNN (Brookhaven National Laboratory, Upton, NY, 11973)

Epigenetic modification of DNA by the addition of a methyl group to the 5 position of the cytosine ring, the methylome, is the only common covalent modification of mammalian DNA. This modification has been shown to control the expression of various genes. Changes in methylation patterns have been observed in both brain cells exposed to cocaine and in developing tumor cells. McrB, a GTP-powered protein, binds to sites containing methylated cytosines preceded by either guanine or adenine residues. This protein was cloned from E.coli into the pET28 vector between NdeI and BamHI sites, expressed and purified for use. In order to be effectively used in methylome pull-downs, McrB was cloned onto a His-Strep tag previously ligated onto the pET28 vector between the NcoI and NdeI restriction enzyme sites. The tag will aid in binding the strepavidin-coated beads used in pull-downs. MBD2b binds methylated cytosines followed by guanines, which are found in CpG islands, and does so even more tightly when in the presence of MBD3L1. These proteins were also bound to the His-Strep tag as part of this novel approach in extracting methylomes. The efficiency of the synthesized McrB protein will be compared with that of MBD2b and also with the MBD2b-MBD3L1 protein complex. Each protein may pull down varying or very similar sections of a genome, whether a mixture of two or more must be used for effective pull-downs will be decided. The cloning of McrB into pET28 was successful; sequencing of the clone revealed no mutations or transformations created during PCR. Ligating the Strep tag onto the digested pET28 vector has also been successful. With the construction of functioning methylome pull-down proteins, drug addiction studies and the early detection of cancer may be improved.

EZ-Viz version 2.0, Further Simplification and Increased Functionality of Pymol Made EZ. BRETT HANSON (Rochester Institute of Technology, Rochester, NY, 14623) CHARLIE WESTIN (Rochester Institute of Technology, Rochester, NY, 14623) PAUL CRAIG (Brookhaven National Laboratory, Upton, NY, 11973) LEN SLATEST (Brookhaven National Laboratory, Upton, NY, 11973)

PyMol, a powerful open source molecular modeling tool, has a notoriously difficult user interface. In order to decrease the steep learning curve associated with PyMol, Laura Grell and Chris Parkin developed PyMol made EZ, (EZ-Viz), a plug-in user interface, to simplify the use of PyMol. Despite the simplicity of the new plug-in, it lacked many of PyMol’s powerful features, severely limiting its versatility. To alleviate these shortcomings, numerous functions were added, including: Enhanced movie making ability, multiple view modes, electron density map importing and controls, Ramachandran plot fetcher, ray trace options, amino acid reference guide, mouse modes, multiple perspective options, polar contacts, and roving functions. With the new user in mind, buttons, sliders, and various other widgets were implemented to provide greater ease of use and efficiency. Instead of entering multiple character commands, a mere push of a button now renders the same outcome with much less frustration and time. Beyond these implementations, over 20 known catalytic active site motifs have been defined in EZ-Viz and are shown differentiated from the rest of the enzyme. Once the motif has been selected and returned for a PDB file, the user can easily show surface contacts, polar contacts, residue labels, and bound substrates. The prediction algorithms are based on measurements between specific amino acid atoms in relation to the other catalytic residues. The predefined measurement range can be modified by the user to increase or decrease precision of motif return. It is also possible for a user to define their own motif and exert a great deal of selectivity in searching for their domain of interest. Further pursuits regarding EZ-Viz will entail the implementation of complex algorithms for the prediction of ligand and protein docking, easier sequence alignment, and inclusion of a functional hydrogen bonding scheme. Utilization of EZViz’s abilities will allow researchers to easily investigate sites of interest within proteins, enabling quicker and more efficient research. It will also provide instructors with the tools to present more detailed and visually appealing overviews of protein structure and function.

Finding Multiple Alignments and Common Motifs for Orthologous Rhodopseudomonas palustris genes. ARIELLA BARHEN (Miami University, Oxford, OH, 45056) LOREN HAUSER (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

There is an ongoing problem of being able to find consistent regulatory motifs in unaligned DNA sequences, including MEME, PhyloCon, and ClustalW. In one approach, orthologous input sequences are used to identify well conserved regions by phylogenetic footprinting. When numerous genomes are available, orthologous sequences can be aligned and conserved regions can be identified. These results are then submitted to various motif finders. To obtain a multiple alignment, ClustalW was used. ClustalW is a multiple sequence alignment program for DNA or proteins, and it calculates the best match for specific sequences and lines them up so that the sequences can be compared and contrasted. Phylogenetic trees were also constructed from the multiple alignments. ClustalW was used to provide multiple alignment sequences for specific genes from four different Rhodopseudomonas palustris strains. Based on the results, conserved sequences can be identified and motifs can be found. The complete sequences genome of Rhodopseudomonas palustris was used to compare R. palustris BisA53, R. palustris BisB18, R. palustris BisB5, and R. palustris HaA2. FASTA files were collected from Artemis (they contained an average of about 200 base pairs upstream from the start codon of the specific genes) and compiled using the editor “Kate.” ClustalW lines up the specific sequences and similarities and differences can be seen between the orthologous genes. Common motifs were able to be identified as well as possible promoter sequences. These identifications can be of high importance when trying to verify evolutionary relationships. Further work could be done to compare the actual transcription factors for the orthologous genes.

Fluorescence Imaging of Living Cells. CARMEN RODRIGUEZ (Washimgton State University Tri-cities, Tri-cities, WA, 99354) GALYA ORR (Pacific Northwest National Laboratory, Richland, WA, 99352)

The investigation of molecular and cellular processes has been strongly relying on the ability to observe these processes in real time and space using fluorescence imaging of the living cell. Fluorescence microscopy of living cells requires sterile techniques for growing and manipulating the cells, and the use of fluorescent dyes that are targeted at specific cellular compartments and organelles. During my internship, I have learned these techniques while participating in an ongoing study of particle-cell interactions. The influence of submicron particles on human health and the environment has been increasing as new chemicals are introduce to our environment and as a result of particles produced by combustion and industrialization processes. The mechanism of penetration of these particles into the human body is not entirely known. The research that I have participated in has focused on the uptake mechanism of particles by the living cell using fluorescence microscopy. We exposed the cells to 500 nm fluorescent particles and watched their internalization by the cells while alternating between fluorescence and DIC imaging over time. Using Matlab routines, we tracked the particles from one image to another to better understand their internalization process.

Gene Cloning and Expression in the Hyperthermophile Sulfolobus solfataricus. MEGAN HOCH (Del Mar College, Corpus Christi, TX, 78404) STEVEN M. YANNONE (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Sulfolobus solfataricus is a hyperthermophile Archaeon that lives in a very extreme environment, and for this it is considered an extremophile. This organism lives in acidic hot springs of Yellowstone National Park. S. solfataricus has become a model system for studying human DNA repair. Protein interactions that are needed to study DNA repair are sometimes transient. Protein interactions that are transient in high temperatures where Archaea live might be more stable at room temperature. This would allow a better look at and understanding of the protein complexes involved. Also, it is accepted that Archaea are more closely related to humans than bacteria, and the DNA replication and translation of the two is very similar. A group of genes was selected for cloning, and primers were designed for each gene. The genes were amplified using the polymerase chain reaction (PCR) method. The PCR products for the gene of interest, were cloned into a directional topoisomerase I (TOPO^®) cloning vector. These vectors were transformed into E. coli cells from Invitrogen. The cells were then plated on Luria-Bertani (LB) agar plates using sterile techniques. Clones were picked from the plates and a culture was grown overnight. The plasmid DNA was separated from the cells using alkaline lysis. Restriction enzyme digests were set up to confirm that the correct gene was inserted into the vector. The digest was visualized on a 1% agarose gel. This study successfully cloned sixteen out of the original twenty-nine genes selected. Some of the clones that grew on the LB plates did not contain any gene at all; it was simply the vector alone. After several restriction enzyme digests, there were about eight genes whose digests were not clear enough to confirm the prescense of the correct insert. These constructs will need to be further digested with different restriction enzymes to confirm the gene. Currently methods are being developed for expressing these genes in E. coli and S. solfataricus. In future studies, proteins expressed from S. solfataricus will be studied and characterized to understand the protein-to-protein interactions that are occurring.

Generation of Translational Fusion Vectors for Candidate Cilia Genes. SANDRA MCGILL (Clayton State University, Morrow, GA, 30260) EDWARD J. MICHAUD, III (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Cilia are small organelles that protrude from the surfaces of cells and are conserved in most eukaryotes, including nematode worms, laboratory mice, and humans. Cilia fall into two broad classes: motile cilia, which occur in clusters on mammalian cells and move fluids over cell surfaces; and primary cilia, which are found singly on virtually all cell types in mammals. It was recently discovered that primary cilia have vital sensory functions in many human tissues, and that flaws in these organelles lead to numerous human diseases. Comparative genomics and proteomics studies have determined that the cilia proteome consists of some 300-500 proteins. However, the functions of most of these candidate cilia proteins are unknown. The goal of this project was to select two of these candidate proteins identified by computational methods and to construct plasmid vectors in which the worm genes are cloned in-frame with the green fluorescence protein (gfp) reporter gene. Two candidate cilia genes in C. elegans nematodes, E02H1.5 and R148.1, were selected for cloning into the GFP reporter vector, pPD95.81. The DNA sequence of each gene was obtained from the University of California at Santa Cruz Genome Browser, and polymerase chain reaction (PCR) primers were chosen using the MacVector 6.5.3 sequence analysis program. These primers were used to amplify the promoter and coding regions of each gene from C. elegans DNA. Each gene was amplified by PCR and cloned in-frame with gfp in the pPD95.81 vector, resulting in two translational vectors, E02H1.5::GFP and R148.1::GFP. The E02H1.5::GFP and R148.1::GFP translational vectors were subjected to restriction enzyme digestions and DNA sequence analysis, which confirmed that the two C. elegans genes were cloned into the pPD95.81 vector in the correct orientation and in-frame with gfp. Future work will involve microinjection of these generated transgene vectors into the gonads of nematodes to produce transgenic worms, which will be used to determine experimentally if these proteins localize to primary cilia. These worms will be examined under a fluorescence microscope to determine if the E02H1.5::GFP and R148.1::GFP fusion proteins localize only to ciliated cells. If they do localize to ciliated cells exclusively, E02H1.5 and R148.1 will be confirmed as cilia proteins. This will set the stage for analysis of the functions of the homologous proteins in mice and humans.

Global Analysis of Iron Response and Fur Regulon in Shewanella oneidensis MR-1. DANIEL HARRIS (Albion College, Albion, MI, 49224) JIZHONG ZHOU (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Iron is an essential micronutrient for both prokaryotic and eukaryotic organisms. In addition to structural roles in proteins, the iron redox potential makes it a useful cofactor for proteins functioning in respiration, electron transport, photosynthesis, nitrogen fixation, DNA biosynthesis and other important processes. However, accumulation of free Fe(II) will catalyze Fenton reactions, which produce highly reactive oxygen species that damage cellular components. Levels of intracellular iron must therefore be precisely regulated to meet metabolic needs while minimizing risk of iron toxicity. Most bacteria moderate iron though the use of the Ferric Uptake Regulator (Fur), an iron-responsive global transcriptional factor that represses transcription when iron is present. In this study, the iron response network was analyzed in Shewanella oneidensis MR-1, and in a fur deletion mutant derived from MR-1. Cells were harvested at various intervals after addition of iron chelator (2,2’-dipyridyl), and then after iron repletion with iron sulfate (FeSO4). Total RNA from extracted from each strain at 13 time intervals, reverse transcribed, fluorescently labeled, and hybridized to DNA microarray slides. Four biological replicates were collected for each mutant for a total of 102 microarray hybridizations. Our results indicate that transcription of proteins involved in iron uptake and storage are mediated by Fur, and that these proteins are highly sensitive to iron availability. Proteins involved in energy metabolism are also regulated by iron availability and Fur, though to a lesser extent. Interestingly, Fur not only acts globally as a repressor, but also activates certain genes in response to fluctuations in extracellular iron concentration. Most of these genes have unknown function. In addition, we also identified regulatory genes controlled by Fur. Further investigations into the interaction between regulatory genes and Fur would be interesting and significantly increase our understanding of transcriptional regulation in S. oneidensis, an important metal-reducing bacterium with potential in the bioremediation of contaminated sites.

High-Throughput X-ray Protein Crystallography. BINH NGUYEN (Contra Costa College, San Pablo, CA, 94806) DR. MINMIN YU (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Proteins play an integral function in cells, hence understanding them is critical, especially in medical research. Structural analysis of proteins is particularly important to the field of Structural Genomics, which aims to study protein molecules in nature to provide a fundamental understanding in biology. Knowing the three dimensional structures of proteins will enable the grouping of fold patterns and family of proteins that can lead to clues of how the proteins work. High-throughput protein crystallography allows structural determination of the protein via x-ray diffraction. Proteins are crystallized and the resulting crystals are analyzed with X-ray to create diffraction patterns that can be determined into three dimensional structures. The first important step in this process is we subject our proteins to series of crystallization matrices to find the right crystallization condition. We utilized various crystal screens from Hampton Research and Emerald Biosystems. The initial crystal hits-crystal formation-leads to further optimization of the crystallization solution so as to obtain crystals that have reasonable diffraction quality. Our crystals are analyzed mostly with the synchrotron at the Advance Light Source (ALS) within the Lawrence Berkeley National Lab (LBNL). Through protein 3-D structures, the folding topologies and local conformations of the proteins can be analyzed. The Li-Wei Hung Lab is currently analyzing various proteins for the Integrated Center for Structure and Function Innovation (ISFI) and TB Structural Genomics Consortium (TBSGC). These proteins are derived from interesting targeted DNA sequences of various sources, mostly species causing human diseases. Solved protein structures are deposited on our ISFI/TBSGC database (www.tbgenomics.org, http://techcenter.mbi.ucla.edu) and the Protein Data Bank (http://www.pdb.org).

Human adenovirus precursor protein VI and mature protein VI : Cloning, expression and characterization. MICHELLE LOUIE (The George Washington University, Washington, DC, 20037) WALTER F. MANGEL (Brookhaven National Laboratory, Upton, NY, 11973)

More than 50 different serotypes of adenovirus are known to infect humans, causing upper respiratory tract, gastrointestinal, and eye infections. The adenovirus proteinase (AVP) becomes activated inside nascent virions and subsequently cleaves virion precursor proteins thereby rendering a virus particle infectious. AVP requires two viral cofactors for maximal activity - adenovirus DNA and an 11-amino-acid peptide known as pVIc derived from the C-terminus of the precursor to protein VI (pVI). The interaction of AVP and DNA with pVI and VI has not been characterized. In order to characterize these interactions, the genes for pVI and its mature form VI must be cloned, expressed in bacterial hosts, and the resultant recombinant proteins purified. Cloning of pVI and VI from genomic DNA and insertion of restriction sites into the genes were accomplished by polymerase chain reaction (PCR) using gene-specific primers. Purified pVI and VI PCR products and the pET13a expression vector were then digested with Nde-1 and BamHI enzymes. Ligation of the pVI and VI DNA into the expression vector is completed before the plasmid is transformed into bacterial hosts and expression of the protein is attempted. pVI and VI DNA were successfully cloned. pVI was ligated into the pET28a-HS vector and transformed into the bacterial host. VI has been successfully inserted into the pET13a plasmid and transformed into a bacterial host. Expression of the pVI and VI proteins was observed in BL21-codon plus(DE3)-RIPL cells. Currently, optimal conditions for recombinant protein purification of protein pVI and VI are being investigated. Experiments using purified recombinant pVI and VI will determine how AVP liberates the pVIc cofactor from pVI. This study will determine if the C-terminus of pVI binds to AVP to produce an activated complex that subsequently removes pVIc from pVI or if AVP binds to DNA forming an active complex that then liberates pVIc from pVI. In addition, proteins derived from this investigation will help to reveal the crystal structures of pVI and VI and determine the equilibrium dissociation constants and stoichiometries of binding of pVI and VI to DNA. Finally, the products of this investigation will also be used to elucidate the sites of interaction of pVI and VI with AVP and with DNA to ultimately determine potential drug targets for anti-viral agents.

Isolation of Independent Spontaneous Thymidine Kinase-Deficient Mutants and an Estimation of the Mutation Rate at the Thymidine Kinase Locus in a Human B-Lymphoblast Clone. LAWRENCE CHYALL (University of California, Berkeley, Berkeley, CA, 94720) AMY KRONENBERG (Lawrence Berkeley National Laboratory, Berkley, CA, 94720)

Programmed cell death, or apoptosis, is tightly regulated by signals originating from both within the cell and its surroundings. The BCL-2 family of proteins helps modulate the balance between the life and death of cells. BCL-X_L (a BCL-2-like protein) assists in limiting apoptosis by titrating the concentration of pro-apoptotic proteins through the formation of a heterodimer. TK6-Bcl-x_L gly-159-ala #38 is a TK6 human B-lymphoblast cell line that was engineered to express BCL-X_L gly-159-ala, a mutated form of the BCL-X_L protein that does not have anti-apoptotic activity. A fluctuation experiment was used to estimate the mutation rate of TK6-Bcl-x_L gly-159-ala #38 cells. The mutation rate was found to be closer to the historical results for TK6-neo #1 cells than cells expressing the wild-type BCL-X_L protein, TK6-Bcl-x_L #4. The plating efficiency of TK6-Bcl-x_L gly-159-ala #38 cells was found to be the same as historical results for TK6-neo #1 cells and TK6-Bcl-x_L #4 cells. Thirty-four early-arising and sixty-three late-arising spontaneous TK1-deficient mutants of the TK6-Bcl-x_L gly-159-ala #38 cell line were isolated. DNA from each of these mutants was extracted for future analysis.

Membranes, surfactants, and membrane proteins: Successful partnering to facilitate structure and function studies. AARON BOWLING (University of Illinois, Champaign, IL, 61820) PHIL LAIBLE (Argonne National Laboratory, Argonne, IL, 60439)

Despite recent revolutionary advances in science and medicine, the structures and intricate molecular workings of membrane proteins remain one of the least understood aspects of any cell. These specialized cellular machines, which comprise an estimated 70% of all pharmaceutical targets, are so dependent on the membrane which accommodates them that their extraction into aqueous buffers for study, typically causes degradation. Previous investigations have yielded several methods leading to extraction of these proteins into detergent micelles, yet there is no rational method to partner a given protein to a surfactant that will work successfully. Herein, a method of analysis is outlined that suggests to researchers the most appropriate surfactant for use with membrane proteins under experimental consideration. This method classifies detergents based on their 'strength’ using several complexes of varied susceptibility found in photosynthetic bacteria. This protocol was coupled with an ink-based spectroscopy approach designed to accurately determine the critical micelle concentration of a surfactant, information vital to the strength analysis. These techniques were applied to several libraries of commercially available surfactants never before considered for use in structural biology. It is hoped that with this system of surfactant classification, the ability to obtain the structural knowledge of membrane proteins will be greatly enhanced; which in turn, will support the foundation for substantial advances in pharmaceuticals.

Methods and Protocols for Production of ScFvs from mRNA. LAURA MOON (University of Cincinnati, Cincinnati, OH, 45221) CHERYL BAIRD (Pacific Northwest National Laboratory, Richland, WA, 99352)

Since its discovery in 1997, Yeast Surface Display (YSD) has been indispensable in engineering antibodies to bind certain antigens at the desired affinities. Because yeast is highly compatible with FACS (Fluorescence Activated Cell Sorter), smaller fragments of antibodies displayed on the surface of yeast can be quickly analyzed in large quantities. In this way, we no longer need full-length purified IgG in our studies. The antibody format of scFv is highly favored because it is the smallest form that retains the antigen specificity of the original IgG molecule from which it was derived. By combining techniques for scFv production with Yeast Surface Display (YSD), we have developed an efficient method for scFv production and characterization. This manual describes in close detail the methods that we have developed for the production of scFvs from RNA extracted from cells. These protocols are intended to help standardize scFv production to increase efficiency and provide a foundation to be improved on.

Multimodality Nanoparticle for Targeted in Vivo Imaging with Xe NMR and Fluorescence. LESLEY LARA (Contra Costa College, Richmond, CA, 94805) 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 re