Todays News

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Todays News

• 2002 Nobel Prize (Medicine/Physiology)
• Sydney Brenner and John E. Sulston, Britain
• H. Robert Horvitz, United States
• for discoveries concerning how genes regulate
  organ development and a process of programmed
  cell death.

Dr. Carol Trents Ph.D. Advisor! Dr. Trents
work is a significant part of the body of
research that warranted the prize.
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Mycoplasma genetalium

• 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

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Mycoplasma genetalium

• http//www.ncbi.nlm.nih.gov80/entrez/query.fcgi?c
  mdRetrievedbnucleotidelist_uids12044850dopt
  GenBank

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Reverse Genetics

• Current Definition
• ...Genetic analysis that begins with DNA sequence
  information,
• - from sequence to function,
• - the first step is most often the
  molecular-genetic disruption of the sequence in
  question.

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Reverse Genetic Systems

• Humans?
• mice homologous recombination,
• bacteria homologous recombination,
• yeast homologous recombination,
• C. elegans random transposon mutagenesis, RNAi,
• Drosophila random transposons,
• Arabidopsis random transposons, random T-DNA,
  RNAi.

All have in common paucity of phenotypes!
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0/17(0/63)
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Mycoplasma genetalium
517 genes 480 protein coding genes How many are
required for Cell Growth?
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Reverse Genetic Strategy

• Randomly mutate large populations of Mycoplasma
  cells,
• Use a transposon with a selective marker,
• i.e. only cells with transposons in the genome
  will grow.
• Sequence the genomic region disrupted.
• Genes that are disrupted are not essential.

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Mycoplasma pneumoniae
All 480 M. genetalium 197 more genes - 65
similarity between othologous genes -

• http//www.ncbi.nlm.nih.gov80/entrez/query.fcgi?c
  mdRetrievedbnucleotidelist_uids13507739dopt
  GenBank

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Orthologs

• 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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Paralogs

• similar genes located at different loci in the
  genome of a single species,
• result from gene duplications (usually).

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Mutagenesis

• The transposon...
• Transposon Tn4001, originally from Staphylococcus
  aureus, was propagated in Escherichia coli
  plasmid pISM2062,
• pISM2062 contains the gene that codes for
  gentamycin (Gm) resistance.

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Mutagenesis

• The transformation...
• pISM2062 was introduced into the mycoplasma by
  electroporation.

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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Mutants?

• How did they know that the tagged cells were
  viable?
• And no non-tagged cells were sneaking by?
• The populations were allowed to recover in SP4
  medium overnight, followed by growth in the
  presence of Gm for 2 to 4 weeks, resulting in the
  expansion of cell number by a factor of gt109.
• This procedure was designed to make the
  subsequent cloning of transposition events from
  nonviable cells highly improbable.

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Sequencing?

• DNA (template)
• Isolated Genomic DNA was isolated from mid-log
  cultures
• Randomly cut 2 µg of DNA was digested with Dra I
  (AAATTT), 
• Circularized The genomic DNA...fragments were
  circularized using DNA ligase.
• Transposon junctions were amplified using inverse
  PCR (iPCR) with two primers specific for the end
  of the transposon Tn4001.

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iPCR
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More Work?

• Reaction products containing oligonucleotide-encod
  ed Eco RI and Hind III sites were digested with
  these enzymes and cloned into the corresponding
  sites in pUC18. DNA sequencing templates were
  prepared from selected colonies and sequences
  generated as described (2).
• Transposon junction sequences were aligned with
  the appropriate genomic sequence to establish the
  site in the genome of transposon insertion.

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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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Minimal Genome(calculated)

• Calculated 121 non-essential genes estimated
  from transposon tag data,
• estimating the number in a mutant population
  (Table 2),
• 93/197 (47) M. pneumoniae specific genes are
  non-essential,
• their logicthus, the 57 orthologous genes
  represent 47 of the non-essential genes in M.
  genitalium.
• saturated with transposon tags yields 180 -
  215,
• 480 genes - 215 non-essential genes 265
  essential genes,

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Minimal Genome(data driven)

• Non-essential M. genetalium genes, plus
  othologous genes tagged in M. pneunomiae,
• 129,
• 480 - 129 351

Estimated Range 265 - 351 essential genes in M.
genitalium.
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Non-Essential Genes

• What types of genes turned out to be dispensable?

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Fig.1
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Functional Specificity

• all functional classes of genes are not equally
  mutable under the selective growth conditions
  used in this study.

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Lipoproteins

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

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Orphans?

• no known orthologs,
• 69 of 180,
• What does this ratio tell us?
• What about the 111 essential genes?
• How would you test your hypothesis?

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Biosynthesis vs. Tranport

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

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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, and phosphate transport,
• ?

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Disrupt DNA pol III?

• Non-essential?
• ditto for recA and uvrA,
• How can this be?

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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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Point 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.

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And

• 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?

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Bigger Genomes

• What can we expect to find when we start reverse
  genetic projects on multicellular organisms?

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