Student Abstracts: Biology at ANL

Development of Sentra - A Database of Signal Transduction Proteins for 45 Prokaryotic Organisms. SAURABHA BHATNAGAR (Illinois Institute of Technology, Chicago, IL 60616) DR. NATALIA MALTSEV (Argonne National Laboratory, Argonne, IL 60439) .
Advances in biology and bioinformatics have made a significant contribution to our understanding of biological systems, especially in identification of genes and the functions of their products. Identification of the components of biological systems has progressed significantly, such that new methodologies and techniques can be developed to aid in the understanding of the system as a whole. Reconstructing the sensory process requires understanding the nature of the transmitted signal as well as mechanisms involved in its transduction. Cellular responses to variety of environmental and internal cellular signals were identified in prokaryotic organisms by experimental studies. However, predicting the nature of a transmitted signal by computational analysis is problematic and should take into account all available information that could assist such functional assignments. We have performed identification of five classes of signal transduction proteins in 45 completely sequenced genomes. In order to provide conjectures about possible mechanisms of their signal transduction processes as well as the nature of transmitted signal it is necessary to analyze the domain composition of the components of the signal transduction proteins and their participation in conserved chromosomal gene clusters. This can be done within the environment of the new Sentra at: http://www-wit.mcs.anl.gov/sentra. Sentra provides flexible querying capabilities, as well as visualization of not only protein functional domains and similarity searches, but allows the user to examine the contig in which the gene encoding for the protein resides, as well as the genes clusters with the gene in question.

BN-350 Spent Fuel Disposition Storage Project Environmental Assessment. SHARON FELTS (University of Idaho, Moscow, ID 83843) MAUREEN FINNERTY (Argonne National Laboratory, Argonne, IL 60439) .
In an attempt to curtail proliferation risks, the United States agreed to assist Kazakhstan with the decommissioning of the BN-350 nuclear reactor and with the disposition of the spent fuel from the reactor. The US government is providing Kazakh officials with templates and guidelines based on current United States procedures to help build Kazakhstan's infrastructure as a newly independent country. Specifically, the spent fuel disposition program required characterization and packaging of the fuel within the BN-350 reactor as well as the development of a plan for the interim 50-year storage of this fuel. A dry well interim storage facility to be built at the Baikal-1 nuclear testing site in Kazakhstan was proposed for the second stage of the disposition program. In the United States, an environmental assessment (EA) is used to determine the environmental impacts of a proposed action on the surrounding environment. A template of an environmental assessment for the interim storage of the spent fuel in Kazakhstan was prepared considering the impact of the facility and its operation on the environment surrounding the Baikal-1 site. Due to lack of historical data from Kazakhstan and the Baikal-1 site specifically, some sections of the interim storage facility EA could not be completed but general information about the required data and computations necessary was compiled and included in the template.

Biochip Manufacturing Quality Control Research (Preparation of Acrylamide Micro-Matrices by Photo-polymerizationi). KELLY HAMMAN (Richard J. Daley , Chicago, IL. 60629) DR. GENNADIY YERSHOV (Argonne National Laboratory, Argonne, IL 60439) .
Creating a biochip involves a 7-step procedure, beginning with a cleaning step and ending with hybridization of oligonucleotides. Throughout these procedures, background fluctuates thus creating variations in fluorescent intensity. The fluorescent intensity can interfere with the readability of biochips. We have created a research design, using a Bio Imager (aka scanner), to read the Digital Luminescent Units[DLU]and to monitor the variations of background throughout the procedure of manufacturing Biochips. Our goal is to find specific background limits in which Biochips can successfully be manufactured. With our data and the rate of successfully produced Biochips, we can apply standard acceptable background limits to the quality control aspect of the manufacturing of Biochips. Further research is necessary to adjust the parameters in which standard acceptable background limits range and can be applied to the manufacturing protocol of Biochips. Data collection regarding contamination can also be appli ed to a manufacturing protocol 'problem-solving' design. The use of the ANL biochip will revolutionize the world of science. Our biochips are cost-effective, minimize chemical use, time-efficient and will provide an accurate testing medium for all bioscience areas. The Biochip Manufacturing Quality Control Research is imperative to the further development of quality ANL Biochips. While current methods successfully produce ANL Biochips, we desire to create a more cost and time effective protocol. The Quality Control design is vital to producing an ANL Biochip Manufacture method easily obtained and successfully executed by business.

Optimization of the Protocol for Nucleic Acid Sample Preparation. ANA JUAREZ (Richard J. Daley College, Chicago, IL 60652) SERGEI BAVYKIN (Argonne National Laboratory, Argonne, IL 60439) .
Many recent advances in genetics have provided a wealth of information that has facilitated the development of new technologies such as DNA microarray technology at a rapid rate. Because accuracy of the produced results is perhaps the most important goal, the method for sample preparation and labeling of the microarrays must provide quantitative results of the highest quality. Scientists have devised eight separate experiments where the results will be used to integrate any possible changes in the protocol in order to help miniaturize and eventually automate the procedure. We were able to perform two of the eight experiments, "Minimization of cell disruption time with lysozyme" and "Minimization of silica column volume". Nucleic acid yields after 1-hour of lysozyme treatment were not significantly higher than 5-minute treatment. Different silica column volumes do not greatly effect the yields of nucleic acids. Experiments will have to be repeated in order to ensure the integrity of results.

Do Rare Codons Influence the Expression of Heterologous Proteins in Rhodobacter Sphaeroides?. MATTHEW KELLER (Vanderbilt University, Nashville, TN 37235) DR. PHILIP LAIBLE (Argonne National Laboratory, Argonne, IL 60439) .
Knowledge of soluble proteins far exceeds that of membrane proteins, largely due to the difficulty in purifying and crystallizing membrane-bound proteins. The physiology of Rhodobacter sphaeroides makes it an excellent choice for expression of important membrane proteins from almost any organism through a system currently under development at Argonne. To test the expression of foreign proteins whose genes include codons rare to R. sphaeroides, Quantum Biotechnology's red-shifted Green Fluorescent Protein was used as a reporter gene. An existing vector made in the lab, pBSrsGFP, was used as the template for site-directed mutagenesis in creating a silent mutation near the N-terminus of the rsGFP gene. Another silent mutation had to be made at the adjacent position, however, to create a unique restriction site. A control mutant was also made harboring the restriction site-creating mutation only. These constructs were transformed into Escherichia coli, then cloned into an R. sphaeroides expression vector, and eventually conjugated into R. sphaeroides. The expression of rsGFP was characterized by fluorescence in E. coli, and by absorption after affinity chromatography in R. sphaeroides. Unfortunately, it was discovered late that the original "correct" mutant candidates contained an insertion in the gene that inhibited rsGFP expression. Fortunately, a correct single mutant candidate was discovered on a plate, and a correct double mutant was created by recombining portions of the vector and gene. Results are not final yet, but preliminary findings seem to indicate that the rare codon investigated severely inhibits expression of heterologous proteins in R. sphaeroides.

Isolation of two unknown genes potentially involved in differentiation of the hematopoietic pathway, and studies of spermidine/spermine acetyltransferase regulation.. CATHRYN KUBERA (Cornell University, Ithaca, NY 14853) ELIEZER HUBERMAN (Argonne National Laboratory, Argonne, IL 60439) .
Differential display identified a number of candidate genes involved with growth and differentiation in the human leukemia cell lines HL-60 and HL-525. Two of these genes were previously unknown, and one is the gene for the enzyme spermidine/spermine acetyltransferase (SSAT). One of our objectives is to isolate and sequence the unknown genes, 631A1 and 510C1, in order to characterize them and determine their functions. The other is to determine how SSAT is regulated, and look at how the polyamines that SSAT regulates effect macrophage differentiation. By screening the CEM T-cell DNA library and the fetal brain library, we were able to identify clones that had inserts with homology to the 631A1 cDNA probe sequence. The insert was amplified using the polymerase chain reaction (PCR) and is currently being sent to the University of Chicago for automated sequencing. The library screens for 510C1 are currently underway, but hybridization of the 510C1 cDNA probe with nylon membranes containing CEM library lambda-phage DNA produced strong signal, indicating the gene is there. SSAT experiments identified that the rate-limiting enzyme that marks the polyamines spermidine and spermine for degradation is regulated by PKC-beta and a transcription factor called Nrf2. The knowledge of regulation and function of these genes involved in macrophage differentiation will provide new insight into this cellular process, potentially making it possible to discover the roots of the problems that cause cancerous diseases.

Characterization of the Morphology of the Inter-Cytoplasmic Membrane Found in Photosynthetic Mutants of Rhodobacter sphaeroides. DAVID METS (university of Rochester, Rochester, NY 14627) PHIL LIABLE (Argonne National Laboratory, Argonne, IL 60439) .
Understanding the membrane morphology of R. sphaeroides is an important part in the assessment of a given strain, and its ability to be used in the expression of heterologous protein. R. sphaeroides expresses it's photosynthetic aparatus in a specific, easily purifyable membrane invaginations. Therefore understanding the membrane structure in different strains will allow insight into the regulation of this membrane formation and, perhaps, allow heterologous protein expression levels to be increased. The use of transmission electron microscopy is particularly suited to this situation. It allows a visualization of the internal membrane structure of the bacterium. This technique coupled with a range of strains with known phenotypes and genotypes will allow a greater understanding for what forms the intercytoplasmic membranes (ICMs) that house the photosynthetic protein.

Expression and Structural Analysis of Membrane Proteins. ZACHARY MORRIS (Ripon College, Ripon, WI 54971) DR. PHILIP LAIBLE (Argonne National Laboratory, Argonne, IL 60439) .
Membrane proteins, while tremendously active in biological processes, are under-represented in scientific understanding as a result of difficulties in completing functional and structural analysis by traditional methods. These difficulties arise from the amphiphilic character of membrane proteins, which provides a tremendous challenge to the maintenance of a native environment and the production of suitable crystal structures for x-ray crystallography. In this paper I discuss research I have conducted this past summer aimed at further developing and understanding a high throughput expression system for the production, purification, and crystallization of membrane proteins from Rhodobacter (R.) sphaeroides. Such research has entailed attempts at such expression and purification, development of protein crystallization techniques from the cubic phase of a lipid solution, and investigation of protease activity in R. sphaeroides.

. NANCY REESE (Benedictine University, Lisle, IL 60532) KIRK E. LAGORY (Argonne National Laboratory, Argonne, IL 60439) .