Purine auxotrophs

A similar collection of purine auxotrophs, with corresponding linkage groups and map positions, has been found in Escherichia coli [18]. Direct determination of corresponding enzyme deficiencies has not been made in most cases, but by phenotypic inference and map positions, a unified nomenclature of the E. coli genes has been made to correspond to that of the Salmonella data of Table I [19]. Essentially, there is a close correspondence between the two systems. The major difference is an apparent absence of purl mutants in E. coli and a closer linkage of purG to the gua operon. 

Pigment formation can be used to visually detect mutants constitutive for enzymes of the purine pathway (Dorfman, 1969). The technique uses an auxotroph blocked early in the pathway which forms red colonies because of the polymerization into a red pigment of accumulated S-amino-4-imidazole ribonucleotide. Normally, high levels of adenine in the medium inhibit pigment formation because AMP represses the pathway. After mutation of the auxotroph, the population is plated on agar containing adenine derepressed mutant colonies are red. 

Yeast strains that carry a mutation at the adel2 locus are adenine-specific auxotrophs and accumulate inosine and h3T>oxanthine 114). In addition, the normal regulation of purine biosynthetic activity is modified toward constitutive synthesis, even in the presence of high levels of exogenous AMP 114, 115). 

Cohen et al. [61] reported that inducible strains of S. aureus were derepressed by 10 minutes of treatment at 42°C, producing a burst of penicillinase synthesis on subsequent growth at 37°C. The increase in rate started immediately but had stopped after 10 minutes. In the presence of puromycin no protein was formed, but, when the puromycin was removed, the same burst of penicillinase synthesis occurred. Cohen et al. conclude that this behavior could be produced by the thermal inactivation of a repressor and that the restoration of the repressed state required protein synthesis. This thermal derepression is sensitive to certain metabolic conditions, for one particular auxotrophic mutant requiring both adenine and guanine was derepressed only if optimal concentrations of the purines were present at lower concentrations derepression was not observed. Constitutive mutants were not stimulated by the heat treatment. 

In nucleotide-depleted yeast, protein synthesis and amino acid incorporation is dependent on and is proportional to the amount of added purines and pyrimidines, which serve as precursors for RNA (379). Conversely, in amino acid-depleted S. aureus, RNA and protein synthesis both are dependent on and proportional to the amount of added amino acids (411). Furthermore, as we have already seen, no synthesis of protein takes place in pyrimidineless mutants in the absence of the missing RNA precursors and, conversely, no synthesis of RNA takes place in amino acid auxotrophs in the absence of the missing amino acid (412-416). The dependence of RNA synthesis on the presence of amino acids was also established for animal systems (liver) by Munro and co-workers (4l7, 418). 

The enzymic propanoylation of 2-deoxy-D-ribose at 0-5 is the first step in a new one-pot chemicoenzymatic synthesis of 2 -deoxyribonucleosides. Alginate gel-entrapped cells of an auxotrophic thymine-dependent strain of E. coli have been used to catalyse the transfer of the 2-deoxy-D-ribofuranosyl unit from T-deoxyuridine to purine and pyrimidine bases, as well as to their aza- and deaza-analogues. Enzymic transglycosylation was also used as a stereoselective alternative to chemical synthesis in the preparation of the imidazole deoxynucleo-side 22.36... 

In 1948, Fries and Kihlman in Sweden reported the production of auxotrophic mutations in the ascomycete Ophiostoma multiannulatum by prolonged immersion of the conidia in solutions of caffeine (0.2%) or theophylline (0.6%). Either treatment yielded about 1% auxotrophs at survival levels of about 0.2% control mutation frequencies were very low. Qualitatively, the results were especially interesting because they showed a difference between the mutational spectra induced by these two purines on the one hand and X-irradiation on the other. This was most striking for inositol requirers, which formed less than 5% of radiation-induced auxotrophs but more than 40% of purine-induced ones. This is one of the rare cases of mutagen specificity for a whole locus or biochemical pathway. Like similar cases that were subsequently reported from the same laboratory, it may rest on selective effects of the plating medium, amplified by the previous treatment of the plated conidia. 

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