Table 1 Effect of hydroxylamine treatment on T7
Exposure time (hr)SurvivalaMuts/genomebC → T/genome
01.02.30.7
210.05, 0.05, 0.0256.94.9
450.0005, 0.001, 0.000211.18.1
  • Methods. Virions of T7Hi from Heineman and Bull (2007) were suspended in 0.1 M sodium phosphate buffer (pH 6.0) with 1 mM EDTA and 3.5 μM phenol red, incubated with or without hydroxylamine (HA) at 0.3M for the time indicated (Tessman 1968). At the end of exposure, HA-treated cultures were neutralized with acetone at 1.5% and NaOH at 0.1 M, the latter to maintain the pH near 7.0. DNA was extracted from all exposed virions regardless of viability and subjected to 454 pyrosequencing; reads were mapped with breseq (Barrick et al. 2009) and mutations enumerated on a per-read basis; bases were counted only if the quality score was at least 25. Our inference of the in vivo mutation spectrum assumes that the in vitro sequencing method accurately mimics the in vivo conversion of lesions into base changes.

  • a survival range across three separate trials, each standardized to the HA-free sample.

  • b Mutations observed in the control may be due to partly or entirely to sequencing error.