1. Central Dogma Information storage in biological molecules DNA RNA Protein transcription translation replication

2. DNA--- deoxyribonucleic acid phosphate sugar (deoxyribose) backbone 4 nitrogen bases Blackburn and Gait, Nucleic acids in chemistry and biology, Oxford University Press New York 1996. PyrimidinesPurines

3. T-A base pair 2 H bonds C-G base pair 3 H bonds

4. Central Dogma Information storage in molecules DNA RNA Protein transcription translation replication

5. RNA—ribonucleic acid phosphate sugar (ribose) backbone 4 bases, A,G,C but U instead of T Single stranded There’s an OH here instead of an H!

6. Types of RNA- mRNA holds the Message transcribed from DNA will be translated into a protein rRNA -- a component of the Ribosome tRNA —helps Transfer the message from base pairs to protein Note that rRNA and tRNA function in the cell as RNA molecules and are never themselves translated into proteins

7. RNA secondary structure especially important for: rRNA tRNA Chastain, M. and Tinoco Jr., I., (1991) Prog. Nucleic Acid Res. Mol. Biol. 41, 131-177.

8. Central Dogma Information storage in molecules DNA RNA Protein transcription translation replication

9. genomic DNAsheared to 3kb clone library insert ends sequenced to 8X coverage computer assembly of sequence reads finishing and closure using PCR to close gaps and verify assembly How do you sequence an entire genome?

10. First complete genome sequence of a free-living organism: 1995 Haemophilus influenzae 1,830,137 base pairs (1.8 Mbp), 1743 genes

11. Since 1995 there has been an explosion in the number of completed genomes http://www.genomesonline.org/

12. 147, 463 18, 26 27, 414 Why? Advances in sequencing technology—major sequencing centers have enough capacity to complete a bacterial genome in a day! Bacteria: 405 completed, 994 ongoing Archaea: 31 Completed, 64 ongoing Eukaryotes: 44 completed, 631 ongoing Meta genome projects: 62 2004 http://www.genomesonline.org/

13. Environmental Genomics 100s of liters of water Concentrate on filter Extract HMW DNA Clone into BAC or fosmid Idea: to look at DNA directly from the environment One way: clone really large pieces

14. Large insert vectors BAC—bacterial artificial chromosome Can clone DNA fragments 100- to 300-kb insert size (average, 150 kb) in Escherichia coli cells. Based on naturally occurring F-factor plasmid found in the bacterium E. coli. Fosmid/Cosmid---- Artificially constructed cloning vector containing the cos gene of phage lambda. Cosmids can be packaged in lambda phage particles for infection into E. coli; this permits cloning of larger DNA fragments (up to 45kb) than can be introduced into bacterial hosts in plasmid vectors. YAC—yeast artificial chromosome Can clone DNA fragments up to 1000 kb insert size (average, 150 kb) in yeast cells. Issues with insert stability, high rates of chimerism, and difficulty in purifyiing vector DNA.

15. Fosmid Library Construction

16. CopyControl TM System (Epicentre Technologies) Allows maintenace of cell stock at low vector copy number, and inducibility to high copy number when needed Can be used for any vector type— plasmid, BAC, fosmid

17. Beja et al 2000 Environmental Microbiology 2: 516-529 Products of an environmental BAC library from California coastal waters

18. Can screen BAC/fosmid libraries multiple ways: Sequence ends of each BAC/fosmid Probe with gene of interest (rRNA or functional gene) Sequence entire fosmid to see what else is there PCR pooled library with primers for gene of interest Narrow down which fosmid gave positive band Sequence entire fosmid to see what else is there

19. Expression and activity of rhodopsin from environmental BAC Beja et al 2000 Science

20. Comparison of environmental BACs to genomes of cultured organisms Beja et al 2002 Nature 415: 630-633

21. Genomics in the Environment: a shotgun approach Science, April 2, 2004 http://www.sorcerer2expedition.org/main.htm

22. Genomics in the Environment Applied whole genome shotgun sequencing technique to 200 l of surface seawater  1.045 billion bases sequenced  1800 microbial species estimated to exist in sample, including 148 novel phylotypes  1.2 million previously unknown genes  12 microbial genomes partially assembled

23. Whole genome sequencing genomic DNA sheared to 3kb clone library insert ends sequenced to 8X coverage computer assembly of sequence reads finishing and closure using PCR to close gaps and verify assembly

24. marine Synechococcus High B/A low light adapted Prochlorococcus Low B/A high light adapted Prochlorococcus I II

25. Venter et al 2004, Science Comparison of MED4 with environmental scaffolds

26. High degree of synteny between MED4 and environmental Prochlorococcus scaffolds MED4 Pro. SAR-1

30. aa rbcL glnA idiA 9683 91 100 Variation at the nt and aa level between MED4 and environmental Prochlorococcus scaffolds nt % identity 87