Cell lines

Wild type haploid Saccharomyces cerevisiae strain Y166 and the spontaneous CRY6 mutant

Preparation Method

Logarithmically growing yeast cells (about 2- 107/ml) were converted into spheroplasts by treatment with glusulase. The spheroplasts were recovered by incubation at 30℃ for 90 min in YEPD medium plus I M sorbitol. Then the culture was divided in three aliquots and each received 10 μCi/ml of [3H]leucine (54 Ci/mmol). One aliquot served as control, the other two received 250 and 500 μg/ml hygromycin B, respectively, and incubation was continued at 30℃.

Reaction Conditions

50 and 500 μg/ml,30℃,20-120 min.

Applications

Protein synthesis by yeast spheroplasts is blocked by hygromycin B as determined by the uptake of [3H]leucine into trichloroacetic acid-precipitable material

Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus. Widely used in veterinary medicine and in cell culture selections, it kills bacteria, fungi, and higher eukaryotic cells, including mammalian cells^[1].

The sensitivity of various cultured cell lines to Hygromycin B was assessed by plating cells at low density in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum and Hygromycin B to give final drug concentrations ranging between 50 and 400 ug/ml of medium. Usually one or two cycles of replication still occurred before the onset of cytotoxicity; cell death commenced ca.3 days after the beginning of drug treatment and was generally complete after 8days (CV1 and HeLa cells sometimes required 10 to 12 days before cell killing was complete). Sofar, no cellline has been found that is naturally resistant to Hygromycin B^[2].

The E.coli bacterial Hygromycin B resistance gene provides a basis for testing the usefulness of Hygromycin B as adominant selectable marker in Hygromycin B -susceptible cells. It may also be possible to use promoter less coding sequences of the Hygromycin B resistance gene as a promoter probe^[3].

Hygromycin B provides a generally applicable selection system for DNA transfer experiments between both procaryotic and eukaryotic cells^[2].

References:
[1]. Borovinskaya MA, Shoji S, Fredrick K, Cate JH. 2008. Structural basis for hygromycin B inhibition of protein biosynthesis. RNA 14: 1590–99
[2]. KAREN BLOCHLINGER，HEIDI DIGGELMANN. Hygromycin B Phosphotransferase as a Selectable Markerfor DNA Transfer Experiments with Higher Eucaryotic Cells. MOLECULAR AND CELLULAR BIOLOGY, Dec.1984, p.2929-2931
[3]. R. N. Rao, N. E. Allen, J. N. J. Hobbs, W. E. J. Alborn, H. A. Kirst & J. W. Paschal: Genetic and enzymatic basis of hygromycin B resistance in Escherichia coli. Antimicrob. Agents Chemother. 24, 689-695 (1983)