Since the early days of molecular biology, the search for the minimal genome has been the Holy Grail

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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
"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 Venter Science 286, pp. 2088
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Todayone for Philosophers and Genomicists.
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The Problem Minimal Genome?
What is the minimal set of essential genes for
basic metabolism and replication?
the smallest autonomous self-replicating
entity.
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Determining the Minimal Set of Genesexperimental
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).

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Determining the Minimal Set of Genestheoretical
I

• Genomic method
• Assumption Essential genes are conserved across
  large phylogenetic distance.

G / G- 3,200 Myr
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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).

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Mycoplasma genetalium
Todays Answer
Pass through 0.45 µm filters

• 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.
  33387-396.

• 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.

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Mycoplasma genetaliumsmallest 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)
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Mycoplasma pneumoniae Comparative Genomics Again
816 kb All 480 M. genetalium 197 more genes
- 65 AA similarity between othologous genes -

• Genbank

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Orthologsspeciation

• similar genes sharing common ancestry, but found
  in the genomes of different species,
• often perform the same function in the various
  organisms.

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Reverse GeneticsRandom Mutagenesis Approach
Random Gene Disruption
Sequence Live Cells
Function
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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.

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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 gt1000
  events for M. pneumoniae.

How did they know that the cells growing on the
plates were mutants?
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Sequencing Flankinginverse PCR (iPCR)
Ligate to pUC18
Sequencing
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(No Transcript)
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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.

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(No Transcript)
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Minimal Genome(calculated)

• 108-121 non-essential genes,
• 180 - 215 non essential genes.
• I. Estimated from M. pneumoniae data (Table 2),
• 93/197 (47) Mp specific genes are
  non-essential,
• their logicthus, the 57 orthologous genes
  represent 47 of the non-essential orthologs.
• II. Saturated (statistically) with tags yields
  180 - 215.

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Minimal Genome(data driven)

• Non-essential M. genetalium genes, plus
  othologous genes tagged in M. pneunomiae,
• 129 (supplemental data-set),
• 480 - 129 351

Estimated Range 265 - 351 essential genes in M.
genitalium.
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Fig.1
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Intergenic region enriched in tags.
Genes of different functional specificity
enriched in tags.
Some surprises.
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Non-Essential Genes

• What types of genes turned out to be dispensable?

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Lipoproteins 4 of the genome

• 13 of 19 are disruptions (i.e. non-essential),
• Why?
• How would you test your hypothesis?

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Testing for Conditional Mutants
Fructose Permease (MG062), MP077) viable.
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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?

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Biosynthesis vs. Transport

• Biosynthesis genes, who cares?
• Transport, hands-off?
• Why?

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Untouchables

• Glycolysis, 0 of 10,
• whats the output of glycolysis?
• Proton-motive-force, 0 of 8,
• somebody tell me what this means?

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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)?

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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?

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Really Disrupted?

• Sequenced DNA from dead cells,
• Functional duplicate copies,
• Absorbed enzymes,
• Uptake of intermediate compounds from the medium,
  bypassing biosynthetic stop points,
• etc.

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Essential Minimal
Huh?
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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?