Thermostability of 5-Fluoroorotic Acid (FOA):

Researchers using the budding yeast, Saccharomyces cerevisiae, have a great number of genetic tools at their disposal. Among these are the plasmids of many types and copy number that have been developed for use in S. cerevisiae. The selection for transformants with these plasmids often relies on the complementation of a genomic mutation in the genes required to synthesize various amino acids (leucine, histidine, tryptophan, etc.) or nucleotide bases (generally adenine or uracil). Recently the use of aurine resistance has been utilized.

Most laboratory strains lack a functional URA3 gene and can be complemented to uracil auxotrophy by supplying URA3 on a plasmid or by integration into the genome. URA3 strains can also be selected against by including 5-Fluoroorotic Acid (5-FOA) in the growth media. Cells with wild-type URA5 and URA3 genes convert the 5-FOA into the toxic substance 5’ Fluorouridine monophosphate, severely limiting growth of the cell. The ability to carry out positive and negative selection with one marker has allowed yeast researchers to devise ingenious screens and genetic schemes.

The use of 5-FOA is not always as simple as theory might dictate. Many researchers have a difficult time getting reproducible results from experiments using 5-FOA. The reasons for this are not always apparent. The anecdotes have lead to researchers to question the stability of 5-FOA in light, heat, solution, etc. Some protocols even recommend boiling to completely dissolve 5-FOA.

The following experiment was designed to demonstrate the effect of these variable conditions on the end use of 5-FOA, growth inhibition of uracil auxotrophs in the laboratory.

5-FOA was obtained from US Biological frozen stocks and aliquots incubated at -20° (control) or 45° C for 16 hours (to simulate an unprotected ground shipment in an overheated UPS truck). To investigate the effects of boiling, we also added 5ml of water to an aliquot of 5-FOA and incubated in boiling water for 10 minutes. We then added the 5-FOA at 1g/liter to synthetic complete media (26.7 g/liter Drop-out Base, 2g/liter SC Drop-out Amino Acid Supplement, stirred until no particulate matter could be observed (generally 10-30 minutes). The media was filtered through a 0.2um filter to sterilize.

We inoculated the 5-FOA media with an overnight culture of W303a pRS316 grown in SC-ura (26.7g/liter Drop-out Base, 2g/liter Drop-out mix minus uracil. Growth was followed by measuring the OD600.

All 5-FOA samples, regardless of the thermal abuse heaped upon them, severely limited the growth of the URA3 culture relative to the SC control. In fact, the boiled sample consistently performed better than the non-boiled control. This might be due to the complete solublization of the 5-FOA.

We conclude that 5-FOA is very thermotolerant for at least short periods of 1-2 weeks. It can be safely shipped via ground without temperature protection.

Zymolase

Zymolyase, produced by a submerged culture of Arthrobacter luteus (1), is an enzyme preparation which effectively lyses cell walls of viable yeast cells. An essential enzyme responsible for lysis of viable yeast cells in this preparation is b-1, 3-glucan laminaripentaohydrolase. It hydrolyzes linear glucose polymers with b-1,3-linkages and releases laminaripentaose specifically as the main and minimum product unit (4,5,10,11). This lytic activity releases spheroplasts and protoplasts in the preparation of yeast DNA prior to restriction enzyme digestion and Southern Blot analysis. The extent of lysis of yeast cells by Zymolyase varies with yeast strain, growth stage of yeast and cultural condition (6-8).

Drop-out Media

Drop-out Mix Complete (DOC) is used as a supplement to Drop-out Base for making synthetic, complete media formulations in accordance with Methods in Yeast Genetics.
All lots are quality control tested using the appropriate yeast strains and transformants.

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United States Biological References

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