Charting 2D Gels making use of the different Chlamydomonas databases available

1. Charting 2D Gels making use of the different Chlamydomonas databases available Christine Markert,Universität Jena

2. Outline • Project: Mapping Thylakoid Proteins • 2D-PAGE • Databases • Types • Limitations

3. Thesis Project Comparison of different mutant strains • Isolation of Thylakoids • 2D-PAGE • Mass Spectrometry • Database Analysis  PROTEIN INFORMATION

4. Information of Interest • Amino acid sequence • Database Entry • Annotation • possible Modifications • New Database Information

5. Types of Databases • Assembled Lhc Proteins • EST Databases • Genomic Database (first release February 2003)

6. Functional Proteomics of Transmembrane Multiprotein Complexes • Protein dynamics of photosystem I and its light-harvesting antenna proteins in assembly and adaptation processes • Variation of physiological conditions • Mutant analysis (PSI defective)

7. Data Mining • Annotation of Database • Genome  Introns • Modifications (e.g. Phosphorylation) • Homologues

8. Chlamydomonas reinhardtii is a unicellular green alga which swims with two flagella and has chloroplasts for photosynthesis. • The genome is ~100 Mbp and is distributed in 17 chromosomes. • The initial goal of the Chlamydomonas reinhardtii genome project is to generate EST sequences, and to date there are over 55,860 Chlamydomonas ESTs in GenBank. • Because Chlamydomonas reinhardtii is quite GC rich (~65% G+C), we have begun a pilot project to sequence several BACs in collaboration with Dr. Susan K. Dutcher in the Department of Genetics at Washington University School of Medicine in St. Louis, Missouri. These BAC clones cover a region of ~600 kbp in the Chlamydomonas reinhardtii genome. • The BAC library prepared by Pete Lefebvre in collaboration with Genome Systems (now IncyteGenomics) is available from IncyteGenomics. • Requests for these BACs should be directed to Incyte Genomics • A table viewing the BAC clones being sequenced can be viewed at URL: http://www.genome.ou.edu/chlamy_totals.html • In accord with the Bermuda Agreement and NHGRI policy, we are depositing all our human genomic sequence data into the High-Throughput Genomic Sequences (HTGS) division of GenBank as soon as a target large insert clone sequencing project can be assembled into contigs greater than 2 kb. The following BAC clones are being sequenced. This sequence data has been deposited into GenBank, and given accession numbers:

9. Status The current draft release, version 2.0 of the Chlamydomonas reinhardtii genome, was generated using the whole genome shotgun strategy, using only data generated here at the JGI. Gene models for this release are currently undergoing manual curation by members of the Chlamydomonas community.This massive effort began during the Chlamydomonas Jamboree, held at the JGI during the week of Dec 8th, 2003. Annotations can be viewed immediately from the protein pages as they become available. To search for genes that have manual annotations, please read the directions on our advanced search page. ReleaseThis assembly was constructed with JAZZ, the JGI assembler, capitalizing on paired-end sequencing reads. After trimming for vector and quality, 1.8 Million reads assembled into 3211 scaffolds totaling 125 Mbp. Roughly half of the genome is contained in 72 scaffolds, all of at least 504 Kb in length. Gene models have been updated using the depth of EST/cDNA information publicly available for C. reinhardtii. Future plansManual review of gene models will continue through the spring, and results will be discussed at a second Jamboree due to be scheduled this coming summer. Stanford will begin finishing the genome later this year. We will update this site once finishing efforts are complete. FundingThis work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program and the by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Laboratory under contract No. DE-AC03-76SF00098 and Los Alamos National Laboratory under contract No. W-7405-ENG-36.

10. AssemblyC. reinhardtii release 2.0 assembled scaffolds (unmasked): chlre2.fasta.gz (29054 Kb)  C. reinhardtii release 2.0 assembled scaffolds (masked): chlre2.allmasked.gz (28364 Kb)  C. reinhardtii release 2.0 predicted proteins: proteins.finalModelsV2.fasta.gz (5009 Kb)

11. SEARCH BY KOG IDSEARCH BY KOG KEYWORDCELLULAR PROCESSES AND SIGNALING • MCell wall/membrane/envelope biogenesis    80 gene modelsNCell motility     6 gene modelsOPosttranslational modification, protein turnover, chaperones   712 gene modelsTSignal transduction mechanisms   666 gene modelsUIntracellular trafficking, secretion, and vesicular transport   308 gene modelsVDefense mechanisms    55 gene modelsWExtracellular structures    28 gene modelsYNuclear structure    33 gene modelsZCytoskeleton   170 gene models Total Gene Count2058INFORMATION STORAGE AND PROCESSING • ARNA processing and modification   319 gene modelsBChromatin structure and dynamics   239 gene modelsJTranslation, ribosomal structure and biogenesis   369 gene modelsKTranscription   304 gene modelsLReplication, recombination and repair   219 gene models Total Gene Count1450METABOLISM • CEnergy production and conversion   255 gene modelsDCell cycle control, cell division, chromosome partitioning   199 gene modelsEAmino acid transport and metabolism   278 gene modelsFNucleotide transport and metabolism   111 gene modelsGCarbohydrate transport and metabolism   265 gene modelsHCoenzyme transport and metabolism   100 gene modelsILipid transport and metabolism   232 gene modelsPInorganic ion transport and metabolism   203 gene modelsQSecondary metabolites biosynthesis, transport and catabolism   172 gene models Total Gene Count1815POORLY CHARACTERIZED • RGeneral function prediction only  1057 gene modelsSFunction unknown   381 gene models Total Gene Count1438

12. Summary

13. 2D-map of Lhca proteins from C. reinhardtii 9