Goodson, H. | bioRxiv Science

bioRxiv2020

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of weeks. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methylmethane sulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to MMS demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain but with lower sensitivity than the plate-based assay. In conclusion, we propose yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.View Full Text

pharmacology and toxicology↗

bioRxiv2018

Behaviors of microtubules depend on two critical concentrations

The concept of critical concentration (CC) is central to understanding behaviors of microtubules and other cytoskeletal polymers. Traditionally, these polymers are understood to have one CC, measured multiple ways and assumed to be the subunit concentration necessary for polymer assembly. However, this framework does not incorporate dynamic instability (DI), and there is work indicating that microtubules have two CCs. We use our previously established simulations to confirm that microtubules have (at least) two experimentally relevant CCs and to clarify the behaviors of individuals and populations relative to the CCs. At free subunit concentrations above the lower CC (CCIndGrow), growth phases of individual filaments can occur transiently; above the higher CC (CCPopGrow), the populations polymer mass will increase persistently. Our results demonstrate that most experimental CC measurements correspond to CCPopGrow, meaning \"typical\" DI occurs below the concentration traditionally considered necessary for polymer assembly. We report that [free tubulin] at steady state does not equal CCPopGrow, but instead approaches CCPopGrow asymptotically as [total tubulin] increases and depends on the number of stable microtubule seeds. We show that the degree of separation between CCIndGrow and CCPopGrow depends on the rate of nucleotide hydrolysis. This clarified framework helps explain and unify many experimental observations.

cell biology↗