The practical aspects of the Workshop will be divided into three Modules and a detailed protocol book will be made available to the participants. A brief description of the objectives and outline for the three modules follows.

Module 1: Shotgun cloning, sequence analysis and comparative genomics

Plan outline: Participants will be involved in the sequencing of clones derived from a shotgun cloning of a BAC from turnip (Brassica rapa). The subclones will be sequenced at the Plant-Microbe Genomics Facility (PMGF, http://www.biosci.ohio-state.edu/pmgf) and the sequences will be assembled and compared with those of the corresponding region of Arabidopsis. The BACs are in contig 234 of the IGF A genome physical map (http://brassica.bbsrc.ac.uk/IGF) and correspond to part of B. oleracea contig D (O'Neill et al, Plant J 23:233-243, 2000). The B. oleracea contig is being sequenced at the moment, so there would be some scientific interest in the comparison with B. oleracea and with Arabidopsis.

Specific techniques to be covered:

Participants will work with a variety of Bioinformatics programs. These include Phred-Phrap and DNAStar Seqman for contig assembly, Glimmer and Genmark for gene predictions, tRNA-ScanSE for tRNA predictions and BLAST searches for protein similarity searches. Participants will also use a variety of web-based tools for similarity searches; they will also develop custom databases for more specialized similarity searches. Metabolic reconstructions will be modeled using KEGG pathway maps and Go Ontology databases. Some familiarity with UNIX is helpful, but not required.

Module 2: Analysis of gene expression using Affymetrix microarrays

Plan: The students will isolate RNA form sugar treated and control Arabidopsis plants and Affymetrix ATH1 microarrays will be hybridized with the corresponding cRNA. Participants will become familiar with the isolation of microarray-quality Arabidopsis RNA, cDNA synthesis and fluorescently labeled cRNA production. Hybridizations and scanning will be carried out at one of the facilities at OSU. Systematic methods for the organization and visualization of microarray data will be illustrated using the Arabidopsis data sets generated during the workshop. Recent developments in algorithms for the analysis of large scale expression data, including statistical analysis, clustering tools, and methods for correlating data with other biological information will be discussed. In addition some of the available software for the various steps in microarray analysis, and considerations for their use, will also be presented.

Specific techniques to be covered:

• RNA extraction and quality check
• Labeling, hybridization, scanning, and data acquisition
• Data filtering, transformation, normalization, and statistical significance metrics
• Data analysis I: toolbox
• Data analysis II: pattern identification
• Data analysis III: plug in your own data
• Public data search, acquisition, analysis, and utilization

Module 3: Protein analysis using mass spectrometry

Plan: Participants will carry out the proteomic analysis of an Arabidopsis proteins. After cutting the desired band(s) from 1-D SDS-PAGE and 2-D gels at the PMGF, the MS analysis will be done at the CCIC (http://www.ccic.ohio-state.edu).

Specific techniques to be covered:

• One- and two-dimensional gel electrophoresis of a crude Arabidopsis protein extract lysate to isolate a specific protein
• Excision of a protein band and subjecting the same to tryptic digestion
• MALDI-TOF analysis of the tryptic peptide mixture
• MASCOT analysis of the resulting spectra to deduce the protein identity
• Development of a protein database using coiled-coil proteins as an example employing MultiCoil, ExtractProp and MySQL .