Central DogmaInformation storage in biological molecules

DNA

RNA

Protein

transcription

translation

replication

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

T-A base pair2 H bonds

C-G base pair3 H bonds

Central DogmaInformation storage in molecules

DNA

RNA

Protein

transcription

translation

replication

RNA—ribonucleic acidphosphate sugar (ribose) backbone

4 bases, A,G,Cbut U instead of T

Single stranded

There’s an OH here instead of an H!

Types of RNA-

mRNA holds the Message transcribed from DNAwill be translated into a protein

rRNArRNA----a component of the a component of the RRibosomeibosome

tRNAtRNA—helps —helps TTransfer the message from base ransfer the message from base pairs to proteinpairs to protein

Note that rRNA and tRNA function in the cell as RNA Note that rRNA and tRNA function in the cell as RNA molecules and are never themselves translated into proteins molecules and are never themselves translated into proteins

RNA secondary structure

especially important for:

rRNAtRNA

Chastain, M. and Tinoco Jr., I., (1991) Prog. Nucleic Acid Res. Mol. Biol. 41, 131-177.

Central DogmaInformation storage in molecules

DNA

RNA

Protein

transcription

translation

replication

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

How do you sequence an entire genome?

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/

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/

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

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.

Fosmid Library Construction

CopyControlTM

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

Beja et al 2000 Environmental Microbiology 2: 516-529

Products of an environmental BAC library from California coastal waters

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

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

0.1

GP2MIT930275M 09

75M 0875M 15

75M 18MIT9201MIT9312

MIT932175M 06MIT9107NS_000023SBMIT9314

AS9601MIT9301175M 16MIT9215RS810

75M 0275M 2075M 19

MB11E08MB11F02

MED4MIT9515

NATL2APAC1NATL1A

MIT9211SS120

MIT9303MIT9313

WH6501WH8102

WH7805WH8101

marine Synechococcus

High B/A low light adapted

Prochlorococcus

Low B/A high light adapted

Prochlorococcus

I

II

Venter et al 2004, Science

Comparison of MED4 with environmental scaffolds

High degree of synteny between MED4 and environmental Prochlorococcus scaffolds

MED4

Pro. SAR-1

aa

rbcL

glnA

idiA

9683

91 91

100

Variation at the nt and aa level between MED4 and environmental Prochlorococcus scaffolds

nt% identity

87