Student Abstracts: Biology at ANL

Participation of src gene in Cadmium-induced bone changes in mice. ADETOWUN ALIMI (Malcolm X College, Chicago, IL 60612) MARYKA BHATTACHARYYA (Argonne National Laboratory, Argonne, IL 60439).
Cadmium is a heavy metal known to have an adverse effect on people exposed to it both in environmental and occupational settings. We hypothesize that cadmium acts by stimulating osteoclastic pathways to release calcium-45 from bone. Mice which are heterozygous for src gene deficiency were mated and produced a litter of four heterozygous wild type pups (+/0= +/+, +/-). Mother (dam) was administered calcium-45 drinking water for skeletal labeling. Calcium-45 was transferred to pups through breast milk during lactation. The pups were weaned into metabolism cages with powdered basal diet where fecal and urine collections were made. Two doses of cadmium solution were given by gavage, one on day 25 and the second on day 32. Fecal and urine collections were dissolved in hydrochloric acid and placed into a scintillation counter to determine amount of calcium-45 excreted in feces and urine. Upon analyzing the data, cadmium targeted the bone to cause a slight increase in fecal calcium-45 concentration of mice that received cadmium doses compared to the control mouse. These results show that cadmium on exposure acts on bone to cause the release in the calcium content of src normal mice (wild type). This experiment provided a mouse model to study the effect of cadmium on the release of calcium from bone in src or fos gene deficient mice.

Differential Detection of the Potyviruses: Potato Virus Y and Tobacco Etch Virus Through the Use of a Biochip Assay. LAVEDA CASTERLOW (North Carolina Agricultural and Technical State University, Greensboro, NC 27411) DR. DARRELL CHANDLER (Argonne National Laboratory, Argonne, IL 60439).
Through the use of a biochip, viral pathogens of the plant family Solanaceae including species of pepper, tobacco, tomato, and potato will be detected. Oligonucleotide sequences specific for Potato Virus Y (PVY) and Tobacco Etch Virus (TEVwere placed on the biochip. Using the National Center for Biotechnology Information website, primers for the seven viruses were selected and were used to generate probes. Capsid protein regions of each virus were blasted against those in the database. Those regions found in the database were placed into ClustalW software to align the nucleotide bases. Information was gathered using the Serpntn software to detect similarities amongst the viruses and provide the primers, which will serve as the template on the biochip. Only perfectly aligned oligonucleotides with fifty base pairs could be blasted into the NCBI software to ensure that the desired virus was the only plant virus obtained from that blast. Group and specific 20mers in the 5? to 3? direction were obtained by a blasting procedure that was specific for a particular isolate or group of isolates. All 20mers were then placed in the ClustalW window and aligned to make certain that each 20mer was specific for a particular group, strain, or virus. Each 20mer was then translated using Chargaff's rule and reversed to create 3? to 5? probes. The probes were placed into a table that consisted of accession number, type of protein (capsid or replicase), strain/isolate, location, melting temperature, and 20mer sequence.

Protein Production for Structural Genomics Project. POLY CHAN (Harry's Truman College, Chicago, IL 60440) SHIU MOY (Argonne National Laboratory, Argonne, IL 60439).
The structure and function of proteins is a continuing research that concerns scientists all over the world. The Humane Genome Project which mapped out all the genes of the human body has been accomplished. Currently scientists are interested with the structure and functions of those particular genes. Scientists understand that genes in a specific order develop into proteins and proteins are needed in order for our bodies to function correctly. For example, proteins are responsible for oxygen transport by hemoglobin from lungs to muscle cells, antigen which binds to an antibody that protects against disease agents, cells surface which transports food molecules into a cell, structural proteins which maintain the physical form of vertebrates includes the following extracellular varieties: elastin, and keratin; Collagen makes up about 25% of the proteins in humans which is a connective tissue found in ligaments, and muscle coverings called sarcomeres; Keratin is and intracellular protein forming the layer of dead skin cells; It is however found extracellularly as hair, birds, claws or most mammals. In other words, proteins are important in order for humans to exist. This research paper focuses on the steps of manual work on the process of transformation, expression, and solubility of proteins. The results obtain are protein samples that were successfully expressed and soluble.

Structural Analysis of Native Membrane Proteins and Expression of Foreign Membrane Proteins in Rhodobacter sphaeroides. ADAM CRAWFORD (Ripon College, Ripon, WI 54971) PHIL LAIBLE (Argonne National Laboratory, Argonne, IL 60439).
Membrane proteins, while integral components of cellular processes, are limited in terms of knowledge about their structures because of difficulties that arise when isolating and purifying these proteins in preparation for structural and functional analysis. The structure of the membrane-bound photosynthetic reaction center (RC) of Rhodobacter (R.) sphaeroides has been determined by molecular replacement, a technique in which phases are determined by comparison of similar X-ray diffraction patterns of previously solved proteins. This particular structure has yet to be determined by isomorphous replacement, in which introduction of a heavy atom in a protein crystal determines the phases by causing diffraction pattern changes relative to a crystal without the heavy atom substitution. In my research, RCs with the heavy atom derivative selenomethionine incorporated in its amino acid chain were crystallized and used by staff crystallographers to determine unique experimental phases and unmatched electron density maps of the R. sphaeroides photosynthetic reaction centers. Success of Argonne's Rhodobacter expression system with respect to native proteins has suggested that the system can be used for expression and purification of foreign proteins as well. In another aspect of my research, efforts were made at expression in Rhodobacter of eight membrane proteins from the syphilis causing spirochete, Treponema (T.) pallidum.

The Development of a Biochip for the Detection of Potato Virus X and Tobacco Mosaic Virus. JASMIN FEIMSTER (North Carolina Agricultural and Technical State University, Greensboro, NC 27411) DARRELL CHANDLER (Argonne National Laboratory, Argonne, IL 60439).
Through the use of a biochip, viral pathogens of the plant family Solanaceae including species of pepper, tobacco, tomato, and potato will be detected. Oligonucleotide sequences specific for Potato Virus X (PVX) and Tobacco Mosaic Virus (TMV) were placed on the biochip. Using the National Center for Biotechnology Information website, primers for the viruses were selected and were used to generate probes. Capsid protein regions of each virus were blasted against those in the database. Those regions found in the database were placed into ClustalW software to align the nucleotide bases. Information was gathered using the Serpntn software to detect similarities amongst the viruses and provide the primers, which will serve as the template on the biochip. Only perfectly aligned oligonucleotides with fifty base pairs could be blasted into the NCBI software to ensure that the desired virus was the only plant virus obtained from that blast. Group and specific 20mers in the 5? to 3? direction were obtained by a blasting procedure that was specific for a particular isolate or group of isolates. All 20mers were then placed in the ClustalW window and aligned to make certain that each 20mer was specific for a particular group, strain, or virus. Each 20mer was then translated using Chargaff's rule and reversed to create 3? to 5? probes. The probes were placed into a table that consisted of accession number, type of protein (capsid), strain/isolate, location, melting temperature, and 20mer sequence.

The Effect of Cadmium on the Calcium Content of src deficient mice. SARITA HOEKZEMA (Hope College-graduate, Holland, MI 49422) MARKYA BHATTACHARYYA (Argonne National Laboratory, Argonne, IL 60439).
Cadmium has been found to increase the break down of bone by increasing the number of osteoclasts found within the bone in cell culture. Src deficient mice have osteoclastic cells but are found to be inactive. Using this information an experiment was designed to see whether cadmium could increase the breakdown of bone in the src deficient mouse. Mice known to be heterozygous for the src gene were bred. When pups were produced, a solution of calcium-45 water was given to the dam to label the pup's skeletons. The pups were weaned into metabolism cages where fecal and urine collections were made. A cadmium solution was administered by gavage on day 25 and 32. The fecal and urine collections were dissolved and placed into scintillation vials and counted within a scintillation counter to determine calcium concentration. After analyzing the results, it was found that a response to cadmium can be seen as a slight increase in fecal calcium concentration for animals receiving the cadmium gavage compared to a downward trend in the control. This difference follows our hypothesis for this experiment thus showing that cadmium caused an increase in the amount of calcium excreted from the body. Although the first round of breeding produced only wild-type mice, this experiment developed a protocol that is going to be used for follow-on experiments testing the same hypothesis with src-deficient mice.

Examining Processes to Fabricate a Compound Refractive X-ray Lens from Lithium . JONATHON LEUNG (Richard J. Daley College, Chicago, IL 60652) ? (Argonne National Laboratory, Argonne, IL 60439).
There has been ongoing research to design refractive optics that can focus x-rays which have the potential to significantly enhance micro-imaging techniques used today. The focusing is done by placing a number of lenses in series (a compound refractive lens or CRL) to refract an x-ray beam many times in small increments. The material of the lens affects its ability to deflect and absorb x-rays, the best lens deflects x-rays with minimal absorption. Compounds that have these properties tend to also have low Z-numbers (atomic numbers). Refractive lenses fabricated from various materials, such as aluminum, beryllium, and to some extent lithium have met with some success in focusing x-rays. Our goal is to design a manufacturing process for a CRL fabricated from lithium, a soft, malleable substance that sticks to most metal surfaces, and even to slippery surfaces such as Teflon. Despite the difficulties of working with lithium, it remains the best material for a CRL in the low to medium (2-30 keV) x-ray energy range. To design the lens, consideration is given to the physical properties of lithium, the handling of lithium and the process for fabricating the lens. The fabrication process that works best must be investigated and refined in future research. Injection molding, extrusion, pressing and stamping were considered in designing the lens. Extrusion was chosen as the first design process because of relatively low costs to manufacture components needed for this process, interchangeable die plates for single and multiple lens extrusion, no components that need to be released from lithium that could cause sticking, and previous experience in extruding an aluminum CRL.

SubBase: Protein Subunit Database. RICHARD LUSK (University of Chicago, Chicago, IL 60637) DR. NATALIA MALTSEV (Argonne National Laboratory, Argonne, IL 60439).
The subunit database is a collection of all known and hypothesized protein subunits indexed to facilitate convenient and accurate comparisons of protein composition across phylogenetic groupings. The data are drawn from all fully sequenced genomes in Swiss-Prot and TrEMBL and presently supported with annotations from the same. The dataset was expanded beyond the incomplete annotation available with a combination of loose gathering parameters and computational prediction of each possible subunit's enzymatic function and other properties necessary for sorting. This data expansion and the integration of the complete set of completed genomes into the database allows for an elsewhere unavailable accurate analysis of protein subunits over wide ranges of species.

Cryocrystallography of Rhodobacter sphaeroides Membrane Proteins. JOY MAYFIELD (University of Iowa, Iowa City, IA 52245) PHILIP LAIBLE (Argonne National Laboratory, Argonne, IL 60439).
Deciphering membrane protein structures is integral to rational drug design and understanding life processes. The Reaction Center (RC) and Cytochrome cy (Cyt cy) membrane proteins from Rhodobacter sphaeroides were studied. There were two different aspects of this project: crystallizing Cyt cy, and finding a suitable cryoprotectant for RC protein crystals. It was necessary to grow the bacteria and purify the proteins before they were used in crystal screens. The Cyt cy screens did not produce crystals, but they are useful in narrowing the protein and precipitant conditions for future trials. Trials at polyethylene glycol 4000 concentrations of 0 ? 6 w/v %, 0 ? 500mM NaCl, and 30 mg/ml Cyt cy could be tested in the future. The optimum protein concentration may lie between 30 and 50 mg/ml. Many cryoprotectants were tested for 'ice rings? at cryogenic temperatures. Polyglycines are not suitable cryoprotectants, but 28.6 w/v % ethylene glycol gave positive results. Ice rings were not visible in the X-ray diffraction patterns, and resolution was 3.5 ? 3.6 Å. When comparing data from two RC crystals, the best method of introducing the cryoprotectant is by soaking it in an increasing gradient of ethylene glycol.

The Development of a Biochip for the Detection of Plant Viruses. . RAKISHA NICHOLASON (North Carolina Agricultural and Technical State , Greensboro, nc 27405) DARRELL CHANDLER (Argonne National Laboratory, Argonne, IL 60439).
Through the use of a biochip, viral pathogens of the plant family Solanaceae including species of pepper, tobacco, tomato, and potato will be detected. Oligonucleotide sequences specific for Cucumber Mosaic Virus (CMV), were placed on the biochip. Using the National Center for Biotechnology Information website, primers for the seven viruses were selected and were used to generate probes. Capsid protein regions or replicase protein regions of each virus were blasted against those in the database. Those regions found in the database were placed into ClustalW software to align the nucleotide bases. Information was gathered using the Serpntn software to detect similarities amongst the viruses and provide the primers, which will serve as the template on the biochip. Perfectly aligned oligonucleotides with fifty base pairs were blasted into the NCBI software to ensure that the desired virus was the only plant virus obtained from that blast. Group and specific 20mers were obtained by a blasting procedure that was specific for a particular isolate or group of isolates. All 20mers were then placed in the ClustalW window and aligned to make certain that each 20mer was specific for a particular group, strain, or virus. The 20mers in the 5? to 3? direction were translated using Chargaff's Rule and reversed. These probes were placed into a table that consisted of accession number, type of protein (capsid or replicase), strain/isolate, location, melting temperature, genomic 20mer sequence and probe.

Design, Construction, and Implementation of New Vectors for Expression of Foreign Membrane Proteins in Rhodobacter. KATIE OLSON (University of Idaho, Moscow, ID 83843) DEBBIE K. HANSON (Argonne National Laboratory, Argonne, IL 60439).
In order to study membrane proteins it is important to have an efficient method to express and purify them. At the present time the lab is using its own modified expression vector with a c-terminal jistidine tag built in. This has worked for some proteins but we would like a vector with an n-terminal histidine tag as well. Another helpful addition to the vector is a protease site to cleave off the tag after purification. The construction of these vectors required taking pieces from previously existing vectors and ordering newly designed oligonucleotides (oligos) as well. Four proteins were all successfully inserted into the new vector using NheI and BglII restriction sites but we are yet to have results that the proteins were properly expressed with the histidine tail.