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Enrichment for Auxotrophic Mutants of Tetrahymena Using Density Gradient Centrifugation *
Author(s) -
HEPFER CAROL ELY,
KANEY ANTHONY R.
Publication year - 1980
Publication title -
the journal of protozoology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 0022-3921
DOI - 10.1111/j.1550-7408.1980.tb05391.x
Subject(s) - auxotrophy , tetrahymena , mutant , centrifugation , wild type , biology , density gradient , differential centrifugation , strain (injury) , biochemistry , microbiology and biotechnology , anatomy , gene , physics , quantum mechanics
SYNOPSIS. A protocol based on density differences between starved and fed cells and employing density gradient centrifugation has been devised to facilitate the isolation of auxotrophic mutants of cell lines derived from Tetrahymena thermophila strain B1868. First, a mass phenotype screening procedure was established whereby true auxotrophic mutants and slow‐growing wild‐type cells such as strain C* could readily be distinguished. Second, simulation experiments were performed in which wild‐type cells starved first in non‐nutritive buffer, then suspended in a defined medium lacking a single essential amino acid became significantly denser than the same cells when starved, then suspended in a complete defined medium. Finally, using the same protocol, a reconstruction experiment was carried out which resulted in effective separation of wild‐type cells from cells of a tyrosine auxotroph. The overall procedure resulted in a 9‐fold increase in the relative frequency of auxotrophic cells, while the density gradient centrifugation alone provided a 400‐fold enrichment.