Central Dogma Information storage in biological molecules

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

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. Purines Pyrimidines

3. T-A base pair2 H bonds C-G base pair3 H bonds

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

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

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 DogmaInformation storage in molecules replication DNA transcription RNA translation Protein

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

10. First complete genome sequence of a free-living organism: 1995 Haemophilusinfluenzae 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. 2004 Bacteria: 405 completed, 994 ongoing Archaea: 31 Completed, 64 ongoing Eukaryotes: 44 completed, 631 ongoing Meta genome projects: 62 147, 463 18, 26 27, 414 Why? Advances in sequencing technology—major sequencing centers have enough capacity to complete a bacterial genome in a day! http://www.genomesonline.org/

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

14. Large insert 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. 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.

15. Fosmid Library Construction

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

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

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

24. GP2 MIT9302 75M 09 75M 08 75M 15 75M 18 MIT9201 MIT9312 MIT9321 75M 06 MIT9107 NS_000023 SB MIT9314 AS9601 MIT9301 175M 16 MIT9215 RS810 75M 02 75M 20 75M 19 MB11E08 MB11F02 MED4 MIT9515 NATL2A PAC1 NATL1A MIT9211 SS120 MIT9303 MIT9313 WH6501 WH8102 WH7805 WH8101 0.1 II Low B/A high light adapted Prochlorococcus I High B/A low light adapted Prochlorococcus marine Synechococcus

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

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

30. Variation at the nt and aa level between MED4 and environmental Prochlorococcus scaffolds • aa • % identity • nt rbcL 100 87 glnA 83 96 idiA 91 91