Section 4 — Gene Action 



strains obtained to date. Concentrations of 500 

 mg/1. L-glutamine (D-glutamine is ineffective) are 

 required to sustain optimal growth of all strains 

 bearing the glm~ allele. Even at these glutamine 

 levels there is an abnormally long lag phase, and 

 the growth rate is less than wild-type at all stages, 

 although the final yield of cell mass may be 

 normal. 



Since glutamine functions as a nitrogen pre- 

 cursor for purines, amino acids, vitamins and 

 glucosamine, the potential sparing action of these 

 metabolites on the glutamine requirement has 

 been investigated. No significant sparing action 

 has been observed for any glutamine-related 

 metabolites, singly or in combination. 



Through appropriate crosses a number of 

 strains have been obtained containing combina- 

 tions of the glm - allele with other gene-con-trolled 

 deficiencies affecting biosynthetic steps in which 

 glutamine normally participates. The nutri- 

 tional properties of these strains will be described 

 and discussed. The level of glutamine synthetase, 

 the enzyme responsible for glutamine synthesis, 

 has been recorded in various strains containing 

 mutational alterations which might be expected 

 to affect intracellular glutamine concentration. 

 These results will also be described. 



The glm gene is located on linkage group V, 

 where it is very closely linked to inos (approxi- 

 mately 2 per cent recombination) and probably 

 distal to it. 



4.41. Some Characteristics of the Protein Determined 

 by the am locus in Neurospora crassa. R. R. 



Burk and J. A. Pateman (Cambridge, Great 

 Britain). 



Amination deficient {am) strains of Neurospora 

 crassa lack both NADP linked glutamate de- 

 hydrogenase and alanine dehydrogenase activi- 

 ties. The purified glutamate dehydrogenase pro- 

 tein has alanine dehydrogenase activity. This is 

 interpreted to mean the am locus determines a 

 protein which has both glutamate and alanine 

 dehydragenase activity. 



When the protein is dissolved in 0.05 m pH 7.4 

 o-phosphate buffer the glutamate dehydrogenase 

 reaction rate is initially low and subsequently 

 increases to a level dependent on the assay 

 system. In contrast the alanine dehydrogenase 

 activity is initially high and decreases. When the 

 protein is in pH 7.4 o-phosphate buffer of 0.1 or 

 greater molarity the initial rate of the glutamate 

 reaction is high. This suggests that at pH 7.4 in 

 a buffer of molarity 0. 1 or greater the protein is 

 in a form which favours glutamate dehydro- 



genase activity while in 0.05 or less the form of 

 the protein favours alanine dehydrogenase 

 activity. 



It is possible to distinguish three components 

 in purified glutamate dehydrogenase by inverse 

 filtration on a 5 per cent agar column. One of 

 these probably corresponds to the 28,000 mole- 

 cular weight sub-unit of Fincham and Codding- 

 ton. It does not possess enzyme activity. Another 

 component corresponds to their 220,000 m.w. 

 component, and the other behaves as if its 

 molecular weight is greater than 800,000. The 

 relative amounts of the two larger components 

 seem to be affected by the concentration of the 

 buffer in which they are applied to the column, 

 higher concentrations favouring the larger com- 

 ponent. 



4.42. Partial Revertants at the am locus in Neurospora 

 crassa. J. A. Pateman and R. R. Burk (Cam- 

 bridge, Great Britain) and J. R. S. Fincham 

 (Hertford, Great Britain). 



The amination deficient, am mutants N. crassa 

 do not possess normal NADP linked glutamic 

 dehydrogenase or alanine dehydrogenase activity. 

 Backmutant strains were produced from am- 3 

 by ultraviolet irradiation and then assayed for 

 glutamic dehydrogenase activity. A total of six- 

 teen strains were found to possess less than 20 

 per cent of normal enzyme activity. A genetic 

 analysis has shown that all the backmutations are 

 in or close to the am locus. A biochemical in- 

 vestigation of the glutamic dehydrogenase pro- 

 duced by each of the partial revertants showed 

 that the partial revertants can be classified into 

 six distinct groups. The members of each group 

 produce a unique variety of glutamic dehydro- 

 genase with respect to the following criteria: 

 thermostability, thermal activation, substrate 

 affinities and substrate activation. A study was 

 made of enzyme complementation between each 

 of the partial revertant groups and a number of 

 the original am mutants. In some cases the partial 

 revertants show different enzyme complementa- 

 tion to that shown by the am-3 mutant from which 

 they were derived. The partial revertants have 

 been assayed for alanine dehydrogenase activity 

 and none of them possess wild type alanine de- 

 hydrogenase activity. There are significant differ- 

 ences between some of the partial revertants 

 with respect to their alanine dehydrogenase 

 activity. The significance of the genetical en- 

 zymatic and complementation characteristics of 

 the partial revertants will be discussed. 



50 



