What is the bioinformatic approach to determine the minimum number of protein-coding genes, required for life?
By whom was this approach primarly used?
identifying genes conserved across all sequenced bacterial genomes
—> determine minimal core set
Mushegian and Koonin in 1996
At what three bacterial genomes did Mushegian and Koonin look? And how many genes were in the minimal set?
H. influenzae
M. genitalium
E. coli
—> 256 genes in minimal set
What were some limitations to the estimate if Mushegian and Koonin?
with distantly-related species —> hard to identify orthologs
NOD (Non-Orthologous gene Displacement) —> some functions performed by non-orthologous genes
results change as new genome sequences become available
no evidence that minimal set is sufficient to life
What is a more recent check of the COG database of miminmal sets?
COG = Clusters of Orthologous Genes
63 common to all life (Archaea, Bacteria, Eukaryotes)
91 common to Archaea, Bacteria
217 common to Bacteria
Describe the experimental approach used by Craig Venter in 1999 to estimate the minimal genome of Mycoplasma genitalium.
at TIGR
randomly knock-out genes using transposable element (TE) insertional mutagenesis
M. genitalium and M. pneunomiae
then sequenced DNA flanking the TE to determine where it is inserted and which gene is knocked out
When is the transposable element (TE) a ‘hit’?
How many hits had the two bacteria?
TE inserted within first 80% of protein-encoding region
beyond nucleotide 9 of coding sequence
—> gene disruption / hit
M. genitalium —> 93 genes
M. pneumoniae —> 150 genes (57 M. genitalium orthologs)
How many genes were discovered as essential by Craig Venter?
What were possible problems with the minimal genome estimate?
265-350 essential genes
Possible problems:
not all 197 M. pneumoniae specific genes may be non-essential (‘new’ genes can become essential)
genes were knocked-out individually
—> What about synthetic lethals? (2 genes knocked out individually with no effect, but cell dies if both knocked out together)
Which scientiests made (bioinformatic) experiments about minimum number of protein-coding genes, required for life?
bioinformatic - Mushegian and Koonin 1996
experimental - Craig Venter 1999 at TIGR
experimental - Kobayashi 2003
Describe the experimental approach used by Kobayashi to estimate the minimal genome with Bacillus subtilis?
How many were essential/non-essential?
individually knocked-out genes using targeted, homologous recombination approach
Bacillus subtilis (4100 genes)
essential: 271 (6%)
non-essential: 3830 (94%)
How were the essential genes classified by Kobayashi?
classified into functional categories
about 50% involved in DNA/RNA metabolism or protein synthesis
over half encode ribosomal proteins
What are the limitations of the experimental approach by Kobayashi?
genes knocked-out individually —> synthetic lethal?? Can an organism with only 271 genes be engineered?
Is the number of essential genes correlated to the total numbers of genes in the genome?
not strongly, BUT a negative correlation
species with few genes (M. genitalium) —> high proportion essential
species with many genes (B. subtilis) —> low proportion essential
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