A MICROBIOLOGICAL METHOD FOR THE DETERMINATION OF CHOLINE BY USE OF A MUTANT OF NEUROSPORA

Abstract

Previous communications from this laboratory have described the production of biochemical mutants in the mold Neurospora by means of ultraviolet and x-rays (1, 2). Such mutants are characterized by the inability to carry out specific chemical syntheses which normally occur in the unmutated, or wild type, strain. In each case which has been genetically analyzed the failure of the synthesis has been found to be related to the mutation of a single gene. The strain to be described, known as No. 34486, or cholineless, arose from a culture of wild type Neurospora crassa which had been irradiated with ultraviolet light. It was found to be unable to grow in a medium containing only salts, sugar, and biotin, but it grew normally on the addition of a mixture of water-soluble vitamins. When the components of the mixture were tested singly, it was found that the addition of choline alone permitted normal growth. Up to the present, no completely satisfactory method for the determination of choline in natural products and tissue extracts has been described. Chemical methods, such as precipitation of the reineckate, lack specificity, while the biological method of Fletcher, Best, and Solandt (3) is time-consuming and difficult, and “possesses many dangerous pitfalls for the chemist” (4). The whole subject has been critically reviewed by Best and Lucas (4). It was therefore of interest to determine whether the Neurospora mutant is a suitable test organism in a quantitative assay for choline. The experiments to be described show that this is the case and form the basis of a simple, sensitive, and specific method for the determination of choline in natural products. By this procedure it is possible to determine choline in a concentration of 0.02 mg. per liter; routine analyses can be run on 100 mg. samples of material.