GENETIC ANALYSIS OF THYMINELESS(thy) MUTANTS IN SALMONELLA TYPHIMURIUM

Abstract

MINOPTERIN may be used to select readily thymineless ( t h y ) mutants in A Escherichia coli ( OKADA, HOMMA, and SONOHARA 1962) ; however, this selection is more difficult in Salmonella typhimuriun strain LT2. Since this may be due to the rarity of thy mutation in this organism, the mutagen N-methy1-N'nitro-N-nitrosoguanidine (NG) was combined with aminopterin to successfully mutate and select t h y mutants (NISHIOKA and EISENSTARK 1964).Approximately 300 t h y mutants of S. typhimurium were collected (Table 1 ) and many of these were analyzed, seeking answers to these questions: ( 1 ) What is the precise position of t h y on the chromosome map of S. typhimurium (see SANDERSON and DEMEREC 1965 for detailed map)? (2) What loci are included in the same phage P22 transducing fragment with thy? (3) What is the clockwise map orientation of the thy locus and its neighbors? (4) Do all thy fall into a single locus? (5) Are all thy physiologically identical? (6) By reversion tests with a series of mutagens, which thy appear to be single-site and which multisite mutants? In the course of analyses, a number of observations led to additional probing of the degree of heterogeneity of transducing particles, sensitivity of cells to deoxyribosides, and the mapping of neighbors of thy.While this investigation was in progress, a report appeared (ALIKHANIAN et al. 1966) of similar experiments with E. coli. MATERIALS A N D METHODSConjugation methods for S. typhimurium were presented by SANDERSON and DEMEREC (1965). Most other materials and methods were described by DEMEREC et al. (1956), and special materials and techniques are presznted in RESULTS.Induction and selection of thy mutants: A series of liquid cultures was inoculated with a small number (ca. 103-104) of S. typhimurium-LT2 cells. The medium contained thymine (200 pg/ml), aminopterin (200 pg/ml), NG ( 3 @g/ml), K,HPO, ( I O pg/ml), KH,PO, (4.5 pg/ml), (NH,), SO, (1 pg/ml), sodium citrate (0.5 pg/ml), MgSO, (0.05 Pg/ml) and glucose (4 mg/ml). The salts were prepared in one batch, and each of the other components was prepared separately; all ingredients were mixed prior to use. NG and aminopterin w x e not sterilized.T w o days of incubation were required before turbidity appeared in the broth cultures. A loopful of grown culture was taken from each of the tubes and passed through single colony isolation on minimal f thymine (0.5 to 5 pg/ml) agar plates. Aminopterin and NG were omitted from the agar. Plates were incubated overnight. About 1 to 10% of the colonies were t h y and ' Slipported by National Science Foundation Grant GB4702. Contribution No. 976,

GENETIC ANALYSES OF NONMOTILE DOUBLE MUTANTS IN SALMONELLA TYPHIMURIUM: A NEW MAPPING METHOD BY ABORTIVE TRANSDUCTION

“…From these data the length of the transducing fragments is inferred to be determinate. As to the manner of cutting, whether it is random or not, several data indicate that some degree of heterogeneity exists as to the termini of the fragments carrying a given genetic marker (ROTH and HARTMAN 1965;PEARCE and STOCKER 1965;ENOMOTO 1967;EISENSTARK, EISENSTARK and CUNNINGHAM 1968), though these data do not deny the termination of most of the fragments at a certain preferential point (OZEKI 1959;PATTEE et al 1968). Accordingly, at present the following two possibilities should be considered as to the formation of the transducing fragments: fragments with a standard length produced by cutting either at preferred positions or at random position(s).…”