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Since the early days of molecular biology, the search for the minimal genome has been the ‘‘Holy Grail’’ in an effort to define the necessary and sufficient components for a living system. - J. Maniloff PNAS 93, pp. 10006. - PowerPoint PPT Presentation
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Since the early days of molecular biology, the search for the minimal genome has been the ‘‘Holy Grail’’ in an effort to define the necessary and sufficient components
for a living system. - J. ManiloffPNAS 93, pp. 10006
"What is life? …We're...working on a reductionist view of trying to take the smallest genome that we have...and see if we can't understand how those...[genes] work together to create life...
- J. Craig VenterScience 286, pp. 2088
Today
The Problem Minimal Genome?
‘‘…the smallest autonomous self-replicating entity.’’
What is the minimal set of essential genes for basic metabolism and replication?
Determining the Minimal Set of Genes“experimental I”
Lethal Mutagenesis Methods:
Assumption: a small but significant data can be extrapolated,
• Saccharomyces: By single disruptions in 200 random loci, 12% of the disruptions were haploid-lethal (Goebl and Petes, 1984).
• Bacillus subtilis: By mutagenesis on 79 randomly selected chromosomal loci, 6 out of 79 ( 9%) are indispensable representing 542 kb (Itaya, 1995).
Genomic method:
Assumption: Essential genes are conserved across large phylogenetic distance.
Determining the Minimal Set of Genes“theoretical I”
G+ / G-: 3,200 Myr Arcady et. al., 1996
• A minimal set of 256 genes has been proposed by comparison of Haemophilus influenzae (G-) with M. genitalium (G+) complete genomes.
Minimal Set of Genestwo “ontologies”
Bottom-up approach:
• Evolution of a minimal genome (and organism) or the synthesis of a minimal set of genes and the establishment of an environment that allows metabolic activity and replication.
Top-down approach:
• Removal or inactivation of unnecessary genes from a larger genome (by evolution or technology).
Mycoplasma genetalium
• Fraser CM, Gocayne JD, White O, Adams MD, Clayton RA, Fleischmann RD, Bult CJ, Kerlavage AR, Sutton G, Kelley JM, et al. (1995) The minimal gene complement of Mycoplasma genitalium. Science 270(5235): 397-403.
– Genbank.
• Tully, J.G., Taylor-Robinson, D., Rose, D.L., Cole, R.M., and Bove,
J.M. (1983) Mycoplasma genitalium, a new species from the human urogenital tract. Int. J. Syst. Bacteriol. 33:387-396.
Pass through 0.45 µm filters0.45 µm filters
Today’s Answer
Mycoplasma genetalium…smallest characterized autonomous organism.
• Gram-positive parasitic bacterium,
• 517 genes, 480 code for proteins,
• 180 genes without functional assignment.
Top Down: Streptococcus lineage w/ 2,000 kb (570 myr)
Mycoplasma pneumoniae Comparative Genomics Again
Genbank
816 kb
All 480 M. genetalium + 197 more genes
- 65% AA similarity between othologous genes -
Orthologsspeciation
…similar genes sharing common ancestry, but found in the genomes of different species,
– often perform the same function in the various organisms.
…similar genes located at different loci in the genome of a single species,
– result from gene duplications (usually),
– often evolve specific functions.
Paralogsduplications
Reverse GeneticsRandom Mutagenesis Approach
Sequence Function
Non-essential gene
RandomGene Disruption
Phenotype
Living
Identify KO (disrupted gene)
Mutagenesis
The transposon...
• Transposon Tn4001, originally from Staphylococcus aureus,
was propagated in Escherichia coli plasmid pISM2062,
– Tn4001 contains the gene that codes for gentamycin (Gm) resistance.
The transformation...
• Tn4001 was introduced into the mycoplasma by “electroporation”,
– the cell is exposed to high intensity electric field pulses.
– temporarily destabilization of the cell membrane makes it highly permeable to exogenous molecules present in the surrounding media.
IS256 IS256
Tn4001
Gentamicin selection
Mutants?
• Cultures were split immediately after electroporation to
generate eight separate populations for each species.
• Each population harbored cells representing ~200 transposition
events for M. genitalium and >1000 events for M. pneumoniae.
How did they know that the cells growing on the plates were mutants?
Sequencing Flankinginverse PCR (iPCR)
DraI Ligase PCR
Ligate to pUC18
Sequencing
Primers
aaattt
DraIDraI
Genomic DNA was
isolated from mid-log
cultures.
Disrupted Genes?
• Within 80% of the 5’ end of the gene,
– excludes insertions in the 3’ end of the gene,…
• 9 bp downstream of the protein coding region,
– the transposon has “promoter” elements,
– non-essential amino acids may be derived from the insertion sequence.
Minimal Genome(calculated)
I. 108-121 non-essential genes,II. 180 - 215 non essential genes.
I. Estimated from M. pneumoniae data (Table 2),
• 93/197 (47%) Mp specific genes are “non-essential”,
– their logic…thus, the 57 orthologous genes represent 47% of the non-essential orthologs.
II. Saturated (statistically) with tags yields ~180 - ~215.
Non-essential M. genetalium genes, plus othologous genes tagged in M. pneunomiae,
• 129 (supplemental data-set),
• 480 - 129 = 351
Minimal Genome(data driven)
Estimated Range: 265 - 351 essential genes in M. genitalium.
Fig.1
Intergenic region enriched in tags.
Genes of different functional specificity enriched in tags.
Some surprises.
Non-Essential Genes
What types of genes turned out to be dispensable?
Lipoproteins 4% of the genome
• 13 of 19 are disruptions (i.e. non-essential),
– Why?
– How would you test your hypothesis?
Fructose Permease (MG062), MP077) viable.
Testing for Conditional Mutants
Mycoplasma Unique?“orphans”
…no known homologs,
• 69 of 180 can be disrupted,
– What does this ratio tell us?– What about the 111 “essential” genes?– How would you test your hypothesis?
Biosynthesis vs. Transport
• Biosynthesis genes, who cares?
• Transport, hands-off?
Why?
Untouchables
• Glycolysis, 0 of 10,
– what’s the output of glycolysis?
• Proton-motive-force, 0 of 8,
– somebody tell me what this means?
Non-Orthologous Complementation
• ABC transporters,
– “orphan” …a different definition, a protein that appears to lack 4o partners,
– 3/12 “orphans” have disruptions, suggesting that the “orphans” are functioning,
• and phosphate transport,
– should be knocked out (2/3)?
MP232, 233
Disrupt DNA pol III?Yes, viable.
• Non-essential?
– ditto for recA and uvrA (excission repair genes),
• How can this be?
• How would you test your hypothesis?
Really Disrupted?
• Sequenced DNA from dead cells,
• Functional duplicate copies,
• Absorbed enzymes,
• Uptake of intermediate compounds from the medium, bypassing biosynthetic stop points,
• etc.
Essential = Minimal
Huh?
Points to Ponder
…the presence of so many genes of unknown function, suggest that all of the molecular mechanisms underlying
cellular life have may not yet have been described.
…the data presented here suggest some specific experiments that could be carried out as a first step in the engineering of a cell with a minimum genome…
– like what?
…what can we expect to find when we start reverse genetic projects on multicellular organisms?
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