Characterization of the Cobalamin and Fep Operons in Methylobium petrolphilum PM1. JANE EWING (Merced College Merced, CA 95340) ANU CHAKICHERLA (Lawrence Livermore National Laboratory, Livermore, CA, 94550)

The bacterium Methylobium petroleophilum PM1 is economically important due to its ability to degrade methyl tert-butyl ether (MTBE), a fuel additive. Because PM1 is a representative of all MTBE degraders, it is important to understand the transport pathways critical for the organism to survive in its particular environment. In this study, the cobalamin pathway and select iron transport genes will be characterized to help further understand all metabolic pathways in PM1. PM1 contains a total of four cobalamin operons. A single operon is located on the chromosome. Located on the megaplasmid are two tandem repeats of cob operons and a very close representative of the cob operon located on the chromosome. The fep operon, an iron transport mechanism, lies within the multiple copies of the cob operon. The cob operon and the fep operon appear to be unrelated except for a shared need for the TonB-dependent energy transduction complex to assist the operons in moving large molecules across the outer membrane of the cell. A genomic study of the cob and the fep operons with that of phylogenetically related organisms helped to confirm the identity of the cob and fep operons and to represent the pathways. More study of the pathways should be done to find the relationship that positions the two seemingly unrelated cob and fep genes together in what appears to be one operon.

Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA) in Bacillus anthracis. CHAD SMITH (Merced College Merced, CA 95348) SHARON L. MESSENGER (Lawrence Livermore National Laboratory, Livermore, CA, 94550)

Anthrax, caused by the bacterium Bacillus anthracis, is a serious disease that if used in a biocrime or bioterrorism incident could cause widespread illness in any community. The anthrax letters of 2001 awoke the United States to the realization that a bioterrorism attack could occur in our country and demonstrated the need to rapidly identify the source of production and route of distribution for biothreat agents. Historically, B. anthracis strains have been difficult to differentiate due to the overall similarity within the genome. Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA), however, differentiates among most B. anthracis strains. Within B. anthracis strains there are unique Variable-Number Tandem Repeats (VNTRs) that are short sequences repeated numerous times within genomic regions. The varying number of these repeats enables differentiation among strains. In order to type these VNTRs within the DNA of each B. anthracis strain PCR is used to amplify eight loci (vrrA, vrrB1, vrrB2, vrrC1, vrrC2, CG3, pX01, pX02) containing these VNTRs. Agarose gel electrophoresis is used to qualitatively analyze the amplicons created by each locus. Fragment analysis by acrylamide gel electrophoresis is used to accurately size the VNTRs fragments at each of the eight loci based upon the fact that each B. anthracis strain will exhibit variation in the number of tandem repeats present at each locus. By analyzing the pattern of fragment sizes at the eight loci enables a genetic profile or "fingerprint" of each known B. anthracis strain to be made. This genetic typing system allows investigators to potentially trace the source of the B. anthracis. This technique provides a useful tool for forensic investigation and has been employed in the ongoing investigation of the anthrax letters sent out in 2001.

Production of Arrayed and Rearrayed cDNA Libraries for Public Use. KRISTEN RASMUSSEN (Merced College Merced, CA 95340) CHRISTA PRANGE (Lawrence Livermore National Laboratory, Livermore, CA, 94550)

Researchers studying genes and their protein products need an easily available source for that gene. The I.M.A.G.E. Consortium at Lawrence Livermore National Laboratory is an important source of such genes in the form of arrayed cDNA libraries. The arrayed clones and associated data are available to the public, free of restriction. Libraries are transformed and titered into 384-well master plates, from which 2-8 copies are made. One copy plate is stored by LLNL while others are sent to sequencing groups, plate distributors, and to the group which contributed the library. Clones found to be unique and/or full-length are rearrayed and also made publicly available. Bioinformatics tools supporting the use of I.M.A.G.E. clones are accessible via the World Wide Web.

Spore Disruption Analysis and Detection Limit Determination at Low Volume Amplifications (2-10 µL) Using eTags. LAUREN TRACY (University of California, Berkeley Berkeley, CA 94720) SHANAVAZ NASARABADI (Lawrence Livermore National Laboratory, Livermore, CA, 94550)

In the post 9/11 world the threat of bioterrorism attacks in public venues has ignited a demand to develop a cost effective autonomous pathogen detection system capable of detecting the multitude of biological agents that can pose a threat to public safety. The major cost of such a pathogen detection system is the large volume of reagents it must expend. With the goal of reducing the reagent consumption, and therefore cost, of a pathogen detection system, we used the bacteria Bacillus globigii (Bg) as a surrogate for the pathogen Bacillus anthracis (anthrax) to determine the lowest amplifiable volume and concentration of amplified sonicated and unsonicated Bg spores that would still be detectable using capillary electrophoresis. We created a serial dilution of unsonicated Bg spores ranging in concentration from 10^8 to 10^1 cfu/mL. From each of these unsonicated spore dilutions we formed three aliquots that were sonicated to disrupt the spores. These sonicated aliquots were analyzed alongside the unsonicated spore samples for each dilution at reaction volumes of 25, 10, and 2 µL. All samples were amplified through a polymerase chain reaction (PCR) in the presence of small fluorescent molecules known as electrophoretic tags (eTags), which were analyzed with capillary electrophoresis to detect the presence of certain nucleic acid signatures. Using this process, Bg samples with concentrations as low as 10^1 cfu/mL and total reaction volumes of amplification as small as 2 µL were readily detectable. Interestingly, detection was more consistent for Bg samples with initial spore concentrations between 10^6 and 10^3 cfu/mL, with the higher and lower concentrations yielding less compelling results. The volume of the sample also affected the efficacy of detection, with detection for 2 µL samples compromised in relation to 25 and 10 µL samples. Detection of sonicated Bg spores appeared to be just as efficient as detection of unsonicated Bg spores. This work is a small portion of a larger project being researched at LLNL and Sandia National Laboratory to develop a low cost briefcase size autonomous detector for small-scale 24/7 detection of a variety of common biothreat agents.

Utilizing DNA Microarray Technology for the Analysis of Gene Expression Changes Between Normal and Tumor Human Kidney Cells. NICOLE SADLER (University of California, Davis Davis, CA 95616) CHITRA F. MANOHAR (Lawrence Livermore National Laboratory, Livermore, CA, 94550)

DNA microarray technology is a remarkably powerful tool for the study of various physiological processes and cellular mechanisms. It is now possible to analyze the expression of over 25,000 genes using the information from the 2003 sequencing of the Human genome. The genetic blueprint, or genome, of a cell defines the patho-physiology of a cell and can provide insight into downstream health implications. With nearly 1.4 million new cancer cases and 500,000 cancer related deaths annually in the US alone, a greater understanding of the differences in genetic processes between normal and cancerous cells is in dire need. Tumor formation is the first stage in the progression of cancer. In this study, to better understand the differences between normal and tumor kidney cells, gene transcript profiles were analyzed using DNA microarray technology. Each gene can be correlated to gene transcripts (mRNA) in a cell. Gene transcript profiles are unique for different cell types. The quantity of each transcript in normal and tumor cells was measured using the mRNA from seven pairs of microarray experiments. Biotin reporter tags were incorporated into the processed mRNA molecules, and the tagged molecules bind to precise DNA spots that are complementary on the GeneChip®. Gene transcripts derived from normal and tumor samples from the Hoag Hospital were analyzed using DNA microarray technology. Microarray experiments were performed at the LMAC using in-house validated protocols. Quality control experiments were conducted on all RNA samples, and the RNA was linearly amplified, labeled, and fragmented. The samples were then hybridized onto GeneChip® Human Genome U133 Plus 2.0 arrays overnight, washed the following morning and scanned with a laser scanner. The data is currently being analyzed to identify gene transcript markers aberrantly regulated in kidney tumor cells using LMAC statistic and bioinformatics procedures. Scatter plots from the raw data comparing control versus experimental samples indicated differences in gene expression in many genes between the control and experimental samples, and those comparing two control groups indicated few gene expression differences. It is not hard to visualize that newer tools of biological measurement like DNA microarray Technology will enable personalized medicine in the future.