and Cells cellular states
Unit Preview
What is a cell? Whats in a cell? How do cells function? What is a cellular state? How do we visualize cells? Can we make a synthetic cell?
From grapes to wine
Prokaryotic Cell
Eukaryotic animal cell
Plant cell
Plant and animal cells
Membranes
Lipid bilayer Proteins
Receptors Transporters
Membranes
Roles of membranes
Compartmentalization of functions
Polar lipid molecules
Polar lipid molecules
Lipid bilayer
Proteins in membrane
Secretion
Transporters in membrane
Cholesterol in membrane
Organelles
Other organelles
Peroxisomes Lysosomes Golgi Endoplasmic reticulum
Evolution of organelles in eukaryotes
Some eukaryotic organelles possess minimal genomes chloroplasts
Mitochondria Chloroplasts Plants
N
Animals Fungi
cyanobacteria
N
How did this come about?
Some endosymbiont genes mitochondria were transferred to the nucleus Endosymbiotic bacteria protobecame organelles mitochondria
N
N
-proteobacteria
N
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Gene transfer or loss
Comparison of mitochondrial and R. prowazekii genomes
Arrangement of genes similar in several cases Same set of genes involved in ATP synthesis Phylogenetic analysis of respiration genes
Divergence of R. prowazekii and mitochondria 1.52.0 billion years ago
mitochondria R. prowazekii
other bacteria
cyanobacteria chloroplast
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The Mimivirus
Giant double stranded DNA virus Discovered in amoebas Belongs to entirely new family of viruses
From Figure in Bruich, S. (2004) Giant virus sequenced in http://sciencenow.sciencemag.org/cgi/content/full/ 2004/1015/1 (ScienceNow October 14, 2004).
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Mimivirus genome
1.18 Mb
Larger than some bacterial genomes
1262 putative ORFs
More genes than some bacterial genomes
50 genes not found in any other viruses
From Figure 1 in Raoult, D. et al. (2004) The 1.2Megabase Genome Sequence of Mimivirus Sciencexpress/www.sciencexpress.org/14 October 2004/Page 1/10.1126/science.1101485. 431.
2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
A controversial hypothesis
Did giant viruses play a role in the evolution of the nucleus? Nucleus-virus similarities
Linear chromosomes No ribosomes Membrane surrounds DNA replication
From Figure 2 in Raoult, D. et al. (2004) The 1.2Megabase Genome Sequence of Mimivirus Sciencexpress/www.sciencexpress.org/14 October 2004/Page 1/10.1126/science.1101485. 431.
2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Macromolecules in cells
DNA RNA Lipids
Form membranes
Proteins
Enzymes Information molecules Structural molecules
Cytoskeleton
Cytoskeleton
http://video.google.com/videoplay? docid=-7936328272029405599&q=tubulin&total=6&st art=0&num=10&so=0&type=search&plindex=0
Summary
Two types of cells
Prokaryotes and Eukaryotes
Membranes
Lipids and proteins
Organelles
Nuclei, mitochondria, chloroplasts Origins
Macromolecules
Cytoskeleton
Metabolism
Glycolysis
Krebs Cycle
Fermentation
Metabolic networks
Describe chemical reactions for building blocks
Amino acids Sugars Lipids
Enzymes and energy required for synthesis and the processes by which the building blocks combine to make larger molecules High-throughput metabolite detection
HPLC and mass spectrometry
Metabolite profiling combined with gene expression analysis
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Metabolic network
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Cellular state
All cellular components All interactions Protein interaction networks Metabolic networks Gene regulatory networks
2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Imaging cells
Confocal microscopy
Confocal microscope
Confocal image
Automated image analysis
Fig. 2. Comparison of compound profiles
Z. E. Perlman et al., Science 306, 1194 -1198 (2004)
Published by AAAS
The RootArray
Synthetic cells
Rational network design
Problem: to understand design principles of biological networks Approach: Design and construct synthetic network Knowledge gained from the design of synthetic networks should help understand real networks Could be used to engineer new cellular behaviors
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The repressilator
Cellular clocks oscillate with defined periods
Circadian clocks oscillate with 24-hour period
Elowitz and Leibler set out to build oscillator with components not in found cellular clocks Used three transcription factors in mutualrepression network
LacI TetR cI from lambda phage
Readout: GFP controlled by Tet repressor
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Repressilator design I
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Repressilator design II
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Activity of repressilator
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Network behavior
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The minimal genome
What is the minimal set of genes required by a selfreplicating organism? Approaches for answering this question
Computational
Compare genes in unicellular organisms Common set of genes are deemed essential
Experimental
Knock out genes by using transposon mutagenesis Inactivate genes by using RNAi Knock out genes through plasmid insertion
2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
The computational approach
Compare bacteria from ancient lineages M. genitalium (gram positive)
468 protein-coding genes
H. influenzae (gram negative)
1,703 protein-coding genes
240 orthologous genes
Add 22 nonorthologous genes involved in intermediate biochemical steps Subtract 6 pathogenic genes Minimal genome: 256 genes
Most essential genes have eukaryotic and archaeal homologs
2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Using transposon mutagenesis
Examined M. genitalium
Smallest known genome at the time (517 genes)
Transposons used to knock out genes Genes free of insertions in surviving organisms were deemed essential
265350 essential genes Involved in DNA replication, lipid-membrane formation, protein synthesis, and energy production 111 genes had no known function
Nonessential genes involved in DNA repair and phosphate transport
2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Using RNAi to inactivate genes
Expression of antisense RNA (RNAi) inhibits gene expression RNAi was used to disrupt gene expression in human pathogen S. aureus
200- to 800-bp fragments of sheared genomic DNA used to make plasmids Antisense plasmids activated by chemical ATc
150 genes found to be essential
Essential genes involved in transcription, translation, and some metabolic functions 30% had no known function
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Inserting plasmids to disrupt genes
Examined B. subtilis Used plasmid insertion to disrupt genes
Plasmids containing B. subtilis genes recombined with genes on chromosome Genes on plasmids regulated by IPTGinducible promoter B. subtilis strains dependent on IPTG indicated essential genes
271 essential genes found
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Characterization of essential genes
~50% information processing ~20% cell envelope, shape, and division ~10% cell energy
Percent of genes
Essential gene function
100 66 33 0 0 2 4 6 8 Genome size (Mb) 31% 35% 20% 14%
~70% of essential genes have homologs in Archaea and Eukarya Number of bacterial homologs depends on genome size
All Domains BacteriaEukarya
Bacteria BacteriaArchaea
2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
A comparison of answers
Approach Computational Transposon mutagenesis RNAi Plasmid insertion Organism(s) M. genitalium H. influenzae M. genitalium Genes in genome 468 1,703 517 Essential genes 260
265350
S. aureus
2,731
150
B. subtilis
4,100
271
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Minimal genome caveats
Problems with methods
Underestimate of minimal gene number if critical gene functions are duplicated RNAi expression may not always be sufficient for disruption of expression
Is there a universal minimal genome?
Determination of minimal genome depends on growth conditions Probably different minimal genomes for different species
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Venters synthetic cell
Its not a slam dunk or we would be announcing it today, Dr. Venter told reporters. Still, he said, I will be equally surprised and disappointed if we cant do it in 2008.
2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458
Summary
Metabolism
Aerobic and anaerobic
Cellular state Imaging cells
Confocal microscopy High throughput microscopy
Synthetic biology
Repressilator Minimal genome
Cells