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ABRF 2001 ABSTRACTS P25-S Truncated midkine (tMK): its molecular cloning and expression in Escherichia coli, and the detection of native tMK in Wilms’ tumor by an anti-tMK monoclonal antibody. S. Paul1, W. Dansithong1, T. Mitsumoto1, Y. Asano1, M. Kato2, M. Kato2, T. shinozawa1; 1Gunma Univ., Japan, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 Japan, 2Tottori Univ., Japan Midkine (MK) is a heparin binding growth factor identified as a product of a retinoic acid-responsive gene; it is frequently expressed at high levels in many human carcinomas. Although the expression of the mRNA encoding truncated MK (tMK) in unique human cancer cells has been reported, the tMK polypeptide itself has not yet been identified. In order to clarify the biological role of tMK, recombinant tMK was expressed in Escherichia coli and purified. Purified recombinant tMK showed the same extent of proliferative activity for the Wilms’ tumor (G401) cells as full length human MK. A mouse hybridoma cell line producing an IgG2b monoclonal antibody (mab) against this purified recombinant tMK was also established. This anti-tMK mab (MiStMK-V3) did not crossreact with synthetic full length (or c-half) human MK. A native tMK, showing the same apparent molecular mass as the recombinant tMK in SDS-PAGE, was identified in G401 cells using this mab. These results suggest that the structure of the recombinant tMK is same as that of native tMK. The expression of tMK in Wilms’ tumor patient specimens was also detected in an immunohistochemical study using the anti-tMK mab. The usefulness of this mab (MiStMK-V3) for the detection of tMK in Wilms’ tumor was demonstrated. P27-T Performance evaluation of a novel application for comparative DNA sequencing analysis and mutation detection. C.A. Kosman, L. Johnston-Dow, H. Breu, G. Chappell, S. Kumar, B. Iasnopolski, B. Kshirsagar, P.A. Suri, R. Paul, G. Mason, D. Siu, E. Sword, M. Schoppe, V. Bawge; Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404 Various software tools are available for the analysis and interpretation of denovo DNA sequencing data. While such applications work well for sequencing for discovery many of the underlying assumptions in these tools are not valid when applied to comparative sequencing data. We have developed a new practical application for comparative DNA sequencing analysis. The focus of this new application is the discovery or identification of sequence variants in a dataset of sequence derived from a single locus in various individuals. This tool allows for the rapid and accurate analysis and alignment of multiple sequence comparisons containing mixed base positions against a reference sequence. While NT-based, this software is compatible with data generated from all of Applied Biosystems sequencing platforms (ABI PRISM ® 373, 377, 310, 3100 and 3700). This application uses procedures and algorithms better suited to the unique nature of comparative sequencing data. Specifically, the prior knowledge of a reference sequence available in many comparative sequencing projects is used to streamline and automate portions of the analysis. The software performs sequence analysis and aligns the analyzed sequence to a reference sequence. It also aligns imported sequences that contain variants. It will then analyze the compared sequences, provide protein translation and report the analysis in a convenient format. Here, we present the results from the performance evaluation of this new software tool on several types of comparative sequencing datasets. For these analyses, this new application produced results concordant with those previously obtained using standard DNA analysis tools while providing substantial performance advantages over these tools. In all cases the analyses were completed in significantly less time and required less manual manipulation of the data. The innovative approaches employed in this tool result in less user intervention needed to achieve a better level of mutation detection. POSTER ABSTRACTS 194 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 P26-M Information management systems for molecular biology. T.M. Smith, D. Campbell, R. Connelly, A. Leonard, S.G. Porter, J. Slagel; Geospiza, Inc., Seattle, 2442 NW Market St. #344, Seattle, WA 98107 Technological advances in molecular biology have made it possible for laboratories to generate unprecedented amounts of data. DNA sequencing, for example, has seen an increase in throughput of over 400-fold in recent years. The accelerated production of data has produced a corresponding need for better data management. The increased need for data management is partly due to institutional changes. Many institutions have been able to economize by establishing core facilities which offer laboratory services to researchers on a per sample or project basis. Core facilities are cost-effective because they provide several researchers with access to expensive equipment and skilled personnel. Because these core facilities handle a variety of data types and a large number of samples from multiple sources, their information management needs provide a worthwhile model for laboratory information management systems (LIMS). LIMS are collections of software, communication devices, and computers that acquire, store, analyze, and present data and information about laboratory samples and their processing. LIMS centralize data storage, automate data analysis, and provide quality assurance reports for process monitoring. The central component of a LIMS is a database with software interfaces for entering, viewing, and processing information. The level of complexity needed in a LIMS depends on factors such as the mission of the laboratory, the number of samples to be processed, analysis requirements, and workflow complexity. LIMS development presents many challenges and may take years to complete. Most labs have no need for a custom LIMS and lack the wherewithal to create one, and thus will benefit from commercial LIMS The general features of LIMS will be presented along with case studies of Geospiza’s Finch-Suite LIMS solution illustrating how a commercially available software system can be used to meet the information management needs of DNA sequencing facilities. P28-S A Web interface for BioLIMS System—data and user management for sequencing core facility. L. Liu, L. Roinishvili; Univ. of Illinois, Urbana, 330 ERML, 1201 W. Gregory Dr., Urbana, IL 61801 Sequencing core facility serves many customers. Samples in a sequencing core facility are very heterogeneous although the volume of samples from each customer is relatively small. Data and user management can be a challenge task. The BioLIMS database system from PE Bioinformatics provides a smooth data flow from sequencer to database. However, it really lacks of friendly user interface for both core facility and customers. To provide an easy management system for the core facility at the W. M. Keck Center for Comparative and Functional Genomics, we implemented a web interface for BioLIMS system. This interface serves two purposes in the core facility. One is for core facility staff to manage customers and their data. The other is for customers to view and retrieve their own data from the web. In customer management, we allow super user and sub user relationship. Super user will have access to all the data of his or her sub users. In data management, a customer can only view and retrieve his or her own data unless others give him or her privilege to view their data. The most convenient feature is web based. Customers can access their data from anywhere at anytime.
P29-M Protein/DNA technology DSL version 2000 is a set of shareware protocols for sample and data management. J. Medalle, M. Randesi, B. Imai; Rockefeller Univ., 1230 York Ave., Box 105, New York, NY 10021 Physical sample and data management are major concerns for a DNA sequencing core facility. Especially one with a small staff and limited space are affected the most. Samples can overtake the limited freezer space. Paper trail can overfill office and lab spaces. Both can cause a quick turnover of DNA sequencing staff. DNA Sequencing Lab’s (DSL) version 2000 objectives are to alleviate repetitive computer tasks for DNA Sequencing staff and eliminate physical paper trail. DSL 2000 is a set of protocols for sample and data management. Its major components are comprised of online submittal forms at http://protein13-pc.rockefeller.edu/, scripts from Apple Script, and standardized forms and macros from Microsoft Excel 98, and Outlook Express 5. The DSL funnels the online sample submissions into sample statements for each investigator. This is a file containing sample IDs, names, account numbers, primer info, template info, etc. DSL also transforms the web submissions into logs for prep and for slab or capillary sequencing reactions. These logs direct the staff on how to process incoming samples. The set of protocols streamlines the sample flow from the investigator to the machine as well as the data flow from machine back to the investigator. DSL’s protocols are “hands on” to help the staff create e-mails for investigator notifications and “a click of a button” to compress and to transfer investigator’s sample statements or data files into their web account. DSL 2000 is a freeware application and can be integrated into other small core facilities with a limited budget. P31-S The analysis of complex tryptic peptide mixtures by multidimensional LC-MS/MS on a hybrid quadrupole orthogonal acceleration time-of flight (Q-TOF) mass spectrometer. A. Millar1, C. Hughes1, T. Andresson2, T. Hemesath2, J.I. Langridge1; 1Micromass UK Ltd., Floats Road, Wythenshawe, Manchester M23 9LZ, United Kingdom, 2deCODE genetics Inc., Reykjavik Advances in both HPLC and mass spectrometry instrumentation have allowed the analysis of protein complexes which have not been separated on a two dimensional gel. These experiments involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data directed switching between the MS and MS/MS modes. Protein identification is then achieved via databank searching of the ESI-MS/MS, providing qualitative information on the proteins that are present. Hundreds of MS/MS spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single LC-MS/MS experiment1. If, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity is advantageous. We have, therefore, adopted a 2D LC-MS/MS approach using a capillary LC system (CapLC) operating at nanoliter per min flow rates coupled to a Q-Tof 2 mass spectrometer. By placing a strong cation exchange (SCX) cartridge followed by a C18 trap cartridge it is possible to pre-fractionate the peptides before separation on an analytical C18 column. After loading the sample, discreet fractions are sequentially eluted from the cation exchange cartridge using a salt step gradient; the eluted peptides are then retained on the trapping C18 cartridge whilst they are desalted. Finally the peptides are eluted from the C18 pre-column, at 200 nL/min, onto a 75 �M ID � 10 cm Waters Symmetry analytical column for separation and elution into the mass spectrometer. This analytical approach will be discussed with examples where this methodology has been used for the analysis of standard protein mixtures and for the analysis of cell lysates and sub-cellular fractions. 1. Yates et al., Nature Biotechnology (1999);17, (7), 676–682. POSTER ABSTRACTS ABRF 2001 ABSTRACTS P30-T A laboratory information management system for a small DNA sequencing core facility. M.J. Miller; NCI, NIH, Bldg 37, Rm 3C28, MSC 4255, Bethesda, MD 20817-4255 The DNA Sequencing MiniCore facility of the NCI’s Division of Basic Sciences currently services over 300 investigators. The facility is designed to support small scale, short-term sequencing. In the past year we ran over 25,000 samples with an average turnover time of less than one day. While this represents a 60% increase in samples over the year before, it is still a relatively modest operation. Keeping all these users and their data organized, as well as minimizing the amount of paperwork involved is a major concern. Although several Laboratory Information Management (LIM) System software packages exist, they are often too expensive or too inflexible for a small operation such as ours. I describe here a LIM system built using components of the Microsoft Office software package. Users enter sample information data into an Internet form and this data is automatically entered into the database after inspection by the MiniCore staff. Programs built into the database determine which sample sets can be run together on the same gel, and what parameters (such as which virtual filter and which dye/primer set settings) should be used with each sample. Samples are assigned to a particular gel and the “sample sheet” for that gel is automatically generated by the database. By keeping track of when samples are submitted, how many samples there are, and how many basepairs of read the user requests, the database aids the facility’s operators in determining sample priority. An email message is automatically sent when samples have been processed and data deposited in the user’s data-destination directory. The database also keeps track of charges and automatically sends this data to the NIH’s accounting system. The facility thus runs in a virtually “paperless” environment. There are no hardcopy forms to fill out. All information is maintained by the computer system. P32-M Identification of in vivo phosphorylation sites in Drosophila armadillo by tandem mass spectrometry. C.S. Raska, R.M. Pope, D. Rubenstein; Univ. of North Carolina at Chapel Hill, 4 Casabelle Ct, Durham, NC 27713 Phosphorylation is one of the most important reversible modifications of eukaryotic proteins. Often, proteins which are associated with uncontrolled cell growth, and ultimately cancer show an anomaly in their phosphorylation/dephosphorylation pathways. In humans, a protein, �-catenin, plays a central role in the development, organization, and regulation of epithelial tissues. Aberrant regulation of �-catenin is associated with malignancies. For example, alterations in �-catenin gene structure have been identified in colorectal and breast carcinoma, and in a large number of melanoma cell lines. Specifically, �-catenin signaling function is constitutively active in many melanomas due to mutations that remove phosphoresidues at the amino terminus of the protein. Armadillo, a protein found in Drosophila melanogaster, is 71% identical to �-catenin at the amino acid level. �-catenin from Drosophila and from human keratinocytes binds to armadillo and �-catenin, respectively, and this binding has been found to be influenced by phosphorylation. Thus, we have been using armadillo as a model system to study �-catenin regulation. To further define the regulatory function of phosphorylation, we are using mass spectrometry to map post-translationally modified amino acids in armadillo. Armadillo was isolated by immuno-affinity and ion exchange chromatography. 1-D SDS-PAGE, and in-gel tryptic digestion were performed to produce a mass spectral fingerprint on a triple quadrupole instrument using nanoelectrospray. MS scans confirmed peptides matching both armadillo and �-catenin from one gel spot. Neutral loss scans identified potentially phosphorylated peptides. MS/MS generated enough sequence coverage to specifically identify phosphorylated residues. We note that phosphoresidues are located within the region where binding sites for cadherin, dTCF, and dAPC are located. We plan mutation studies to further elucidate the role of phosphorylation in these systems. JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 195
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