Sunday, May 21, 2006

"Synthetic retrotransposons" - Jef Boeke

  • Retrotransposition is one of the two basic types of transposition we find in biology. It's sort of fun to survey where transposons are in the tree of life: in the case of more eukaryotes, most of the genome is transposons; little of microbial genomes are retrotransposons.

LINEs (L1) transposon sequences comprise ~ 21% of the human genome, and generally dominate mammalian genomes.
  • The RNA is oth mRNA and "genome" (template) RNA
  • Most of the ~500,000 L1 copies are severely 5' truncated and live 1) in introns or 2) between genes
  • normally inserts only in the germ line - not somatically.
  • The promoter is entirely embedded in the mRNA and hence is taken along with the transposon.
Synthetic biology of retrotransposons:
make synthetic ORF2 and ORF1. Properties:
  • 25% of the nucleotides altered - new species of retrotransposons
  • adenosine content reduced 40%.
Built "ORFeus" retrotransposon which is driven by an external promoter, so it should jump just once, because it doesn't "take its promoter with it." Average number of insertions/progeny ~0.42 (pretty good). These are germ line insertions; there are even more somatic insertions. These insertions are very random across genes and chromosomes.

Besides mutagenesis, another application might be cancer gene discovery: they've developed a method of activating retrotransposition in any tissue of interest.

Anyhow, they've built 7+ new transposons (thanks DNA 2.0). And now for something completely different: the systems biology of yeast is the best of any, os if we know so much, can we use that knowledge to refactor it and make a "synthetic" strain?

Boeke's lab is designing and synthesizing yeast chromosome 3. They're doing so in an iterative fashion, refactoring the chromosome in chunks of 30Kb at a time, so they can check their work as they go. It's a straightforward procedure; get thousands of colonies from ~ug of refactored DNA.

Changes to make; want to make a super-stable genome:
  • remove all transposons
  • remove all introns
  • downsize telomeres
  • relocate all tRNA genes
Unfortunately, fitness has a tendancy to evaporate with each recombination. Can this be overcome with "genome swarms" and conditional evolution? Hmm.

P.S. - Joel Bader is writing a Genome Revision control System!

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