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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
Today
The Problem
Minimal Genome?
What is the minimal set of essential genes
for basic metabolism and replication?
‘‘…the smallest autonomous self-replicating entity.’’
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).
Determining the Minimal Set of Genes
“theoretical I”
Genomic method:
Assumption: Essential genes are conserved
across large phylogenetic distance.
• A minimal set of 256 genes has been proposed
by comparison of Haemophilus influenzae (G-)
with M. genitalium (G+) complete genomes.
G+ / G-: 3,200 Myr
Arcady et. al., 1996
Minimal Set of Genes
two “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).
Today’s Answer
Mycoplasma
genetalium
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. 33:387-396.
• 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.
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
816 kb
All 480 M. genetalium + 197 more genes
- 65% AA similarity between othologous genes Genbank
Orthologs
Paralogs
speciation
duplications
…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.
Reverse Genetics
Random Mutagenesis Approach
Identify KO (disrupted gene)
Random
Gene Disruption
Phenotype
Sequence
Function
Living
Non-essential gene
Mutagenesis
The transposon...
Tn4001
IS256
Gentamicin selection
IS256
• 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.
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 Flanking
inverse PCR (iPCR)
Genomic DNA was
isolated from mid-log
cultures.
DraI
DraI
Primers
DraI Ligase
PCR
Ligate to pUC18
aaattt
Sequencing
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.
II.
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 logic…thus, the 57 orthologous genes represent 47%
of the non-essential orthologs.
II. Saturated (statistically) with tags yields ~180 - ~215.
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.
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?
Testing for Conditional Mutants
Fructose Permease (MG062), MP077) viable.
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?