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- J. Maniloff PNAS 93, pp. 10006

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.

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- J. Maniloff PNAS 93, pp. 10006

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  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

  2. Today

  3. The ProblemMinimal Genome? What is the minimal set of essential genes for basic metabolism and replication? ‘‘…the smallest autonomous self-replicating entity.’’

  4. 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).

  5. A minimal set of 256 genes has been proposed by comparison of Haemophilus influenzae (G-) with M. genitalium (G+) complete genomes. Arcady et. al., 1996 Determining the Minimal Set of Genes“theoretical I” Genomic method: Assumption: Essential genes are conserved across large phylogenetic distance. G+ / G-: 3,200 Myr

  6. 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).

  7. Today’s Answer 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. 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.

  8. 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)

  9. Mycoplasma pneumoniaeComparative Genomics Again Genbank • 816 kb • All 480 M. genetalium + 197 more genes • - 65% AA similarity between othologous genes -

  10. Paralogsduplications • …similar genes located at different loci in the genome of a single species, • result from gene duplications (usually), • often evolve specific functions. Orthologsspeciation …similar genes sharing common ancestry, but found in the genomes of different species, • often perform the same function in the various organisms.

  11. Living Reverse GeneticsRandom Mutagenesis Approach Identify KO (disrupted gene) Random Gene Disruption Function Phenotype Sequence Non-essential gene

  12. Tn4001 IS256 IS256 Gentamicin selection 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.

  13. 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?

  14. Genomic DNA was isolated from mid-log cultures. Primers DraI DraI Ligase PCR DraI aaattt Sequencing Flankinginverse PCR (iPCR) Ligate to pUC18 Sequencing

  15. 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.

  16. 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 logic…thus, the 57 orthologous genes represent 47% of the non-essential orthologs. II. Saturated (statistically) with tags yields ~180 - ~215.

  17. 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.

  18. Fig.1

  19. Intergenic region enriched in tags. Genes of different functional specificity enriched in tags. Some surprises.

  20. Non-Essential Genes What types of genes turned out to be dispensable?

  21. Lipoproteins4% of the genome • 13 of 19 are disruptions (i.e. non-essential), • Why? • How would you test your hypothesis?

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

  23. 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?

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

  25. Untouchables • Glycolysis, 0 of 10, • what’s the output of glycolysis? • Proton-motive-force, 0 of 8, • somebody tell me what this means?

  26. MP232, 233 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)?

  27. 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?

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

  29. Essential = Minimal Huh?

  30. 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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