Wednesday, June 10, 2015
255 - Segregation characteristics of multiple chromosomes of Azotobacter vinelandii
Again on the subject of multiple chromosome copies in Azotobacter vinelandii, they wanted to see if the larger amount of DNA in the cells compared to E. coli actually meant that many more copies of each gene.
What They Saw
They mutagenized A. vinelandii UW (aka CA) with transposons and tried to see if these insertions were present in as many copies as they had observed for other genes (30-40). They tried to find auxotroph mutants by conjugating with E. coli carrying a transposon. They didn't find any A. vinelandii that failed to grow on plates lacking certain amino acids, but they did find some that didn't grow very well unless the amino acids were present. This poor growth got less poor over time in successive generations though, but the cells were still resistant to the selective marker (ampicillin). They interpret this as being related to the proportion of genome copies with an insertion vs. without.
They also saw that mutagenized cells mostly couldn't grow with ampicillin when plated directly, though they could grow on antibiotic-free Burk medium. Then when cells from each of these conditions were transferred again to plates with antibiotic, only some from selective plates grew, while all from non-selective plates grew. The idea is that growing on selective plates, the ability to grow without added amino acids would be lost (since the transposon knocked out that ability in some copies, and those copies would be higher in proportion because of the selection). They should've tried growing cells from selective medium on non-selective medium to see if they got the same result.
And gaining the ability to resist the antibiotic? Shouldn't they have had that from the beginning? This whole set of experiments is unclear.
They also mutagenized cells with a different transposon that conferred tetracycline resistance, isolated DNA from them after growing on different amounts of tet, and probed with radioactive probes, correlating radioactivity to number of copies. They observed more radioactivity in cells grown with greater selective pressure, implying that there were multiple alleles and selection increased the proportion of resistant allele in a population.
I'm still somewhat dubious, but it seems like the data might be fairly solid.
Phadnis, S. H., Dimri, G. P. & Das, H. K. Segregation characteristics of multiple chromosomes of Azotobacter vinelandii. J. Genet. 67, 37–42 (1988).