Transcript Lecture
Central Dogma Information storage in biological molecules replication DNA transcription RNA translation Protein DNA---deoxyribonucleic acid phosphate sugar (deoxyribose) backbone 4 nitrogen bases Purines Pyrimidines Blackburn and Gait, Nucleic acids in chemistry and biology, Oxford University Press New York 1996. T-A base pair 2 H bonds C-G base pair 3 H bonds Central Dogma Information storage in molecules replication DNA transcription RNA translation Protein RNA—ribonucleic acid phosphate sugar (ribose) backbone There’s an OH here instead of an H! 4 bases, A,G,C but U instead of T Single stranded 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 RNA secondary structure especially important for: rRNA tRNA Chastain, M. and Tinoco Jr., I., (1991) Prog. Nucleic Acid Res. Mol. Biol. 41, 131-177. Central Dogma Information storage in molecules replication DNA transcription RNA translation Protein How do you sequence an entire genome? genomic DNA finishing and closure using PCR to close gaps and verify assembly sheared to 3kb computer assembly of sequence reads clone library insert ends sequenced to 8X coverage First complete genome sequence of a free-living organism: 1995 Haemophilus influenzae 1,830,137 base pairs (1.8 Mbp), 1743 genes Since 1995 there has been an explosion in the number of completed genomes http://www.genomesonline.org/ 2004 Bacteria: 405 completed, 994 ongoing 147, 463 Archaea: 31 Completed, 64 ongoing 18, 26 Eukaryotes: 44 completed, 631 ongoing 27, 414 Meta genome projects: 62 Why? Advances in sequencing technology—major sequencing centers have enough capacity to complete a bacterial genome in a day! http://www.genomesonline.org/ 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 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. Fosmid Library Construction 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 Products of an environmental BAC library from California coastal waters Beja et al 2000 Environmental Microbiology 2: 516-529 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 Expression and activity of rhodopsin from environmental BAC Beja et al 2000 Science Comparison of environmental BACs to genomes of cultured organisms Beja et al 2002 Nature 415: 630-633 Genomics in the Environment: a shotgun approach Science, April 2, 2004 http://www.sorcerer2expedition.org/main.htm 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 Whole genome sequencing genomic DNA finishing and closure using PCR to close gaps and verify assembly sheared to 3kb computer assembly of sequence reads clone library insert ends sequenced to 8X coverage GP2 MIT9302 75M 09 75M 08 75M 15 75M 18 MIT9201 MIT9312 MIT9321 75M 06 MIT9107 II Low B/A high light adapted Prochlorococcus NS_000023 SB MIT9314 AS9601 MIT9301 175M 16 MIT9215 RS810 75M 02 75M 20 75M 19 MB11E08 MB11F02 I MED4 MIT9515 NATL2A PAC1 NATL1A MIT9211 MIT9303 SS120 MIT9313 High B/A low light adapted Prochlorococcus WH6501 WH8102 WH7805 WH8101 0.1 marine Synechococcus Comparison of MED4 with environmental scaffolds Venter et al 2004, Science High degree of synteny between MED4 and environmental Prochlorococcus scaffolds MED4 Pro. SAR-1 Variation at the nt and aa level between MED4 and environmental Prochlorococcus scaffolds nt aa rbcL 87 100 glnA 83 96 idiA 91 91 % identity