Title: Since the early days of molecular biology, the search for the minimal genome has been the Holy Grail
1- 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
2Todayone for Philosophers and Genomicists.
3The Problem Minimal Genome?
What is the minimal set of essential genes for
basic metabolism and replication?
the smallest autonomous self-replicating
entity.
4Determining 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).
5Determining the Minimal Set of Genestheoretical
I
- Genomic method
- Assumption Essential genes are conserved across
large phylogenetic distance.
G / G- 3,200 Myr
6Minimal 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).
7Mycoplasma 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.
8Mycoplasma 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)
9Mycoplasma pneumoniae Comparative Genomics Again
816 kb All 480 M. genetalium 197 more genes
- 65 AA similarity between othologous genes -
10Orthologsspeciation
- similar genes sharing common ancestry, but found
in the genomes of different species, - often perform the same function in the various
organisms.
11Reverse GeneticsRandom Mutagenesis Approach
Random Gene Disruption
Sequence Live Cells
Function
12Mutagenesis
- 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.
13Mutants?
- 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?
14Sequencing Flankinginverse PCR (iPCR)
Ligate to pUC18
Sequencing
15(No Transcript)
16Disrupted 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.
17(No Transcript)
18Minimal 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.
19Minimal 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.
20Fig.1
21Intergenic region enriched in tags.
Genes of different functional specificity
enriched in tags.
Some surprises.
22Non-Essential Genes
- What types of genes turned out to be dispensable?
23Lipoproteins 4 of the genome
- 13 of 19 are disruptions (i.e. non-essential),
- Why?
- How would you test your hypothesis?
24Testing for Conditional Mutants
Fructose Permease (MG062), MP077) viable.
25Mycoplasma 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?
26Biosynthesis vs. Transport
- Biosynthesis genes, who cares?
- Transport, hands-off?
- Why?
27Untouchables
- Glycolysis, 0 of 10,
- whats the output of glycolysis?
- Proton-motive-force, 0 of 8,
- somebody tell me what this means?
28Non-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)?
29Disrupt 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?
30Really Disrupted?
- Sequenced DNA from dead cells,
- Functional duplicate copies,
- Absorbed enzymes,
- Uptake of intermediate compounds from the medium,
bypassing biosynthetic stop points, - etc.
31Essential Minimal
Huh?
32Points 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?