Section 4— Gene Action 



to differ qualitatively from the wild enzymes. 



The phenoloxydases of three of these mutants, 

 belonging to different linkage groups and having 

 different morphogenetic blocks, have been 

 further investigated biochemically. Methods 

 applied were: behavior in ammonium sulfate 

 fractionations, substrate specificity and heat in- 

 activation. The experiments have established 

 both quantitative and qualitative alterations of 

 the two enzymes. 



The implications of these findings will be short- 

 ly discussed with respect to gene-enzyme relations 

 and to the genetic basis of morphogenesis. 



4.46. Purification and Properties of Tryptophan Syn- 

 thetase from Neurospora crassa. M. Carsiotis, 

 E. Appella and S. R. Suskind (Baltimore, 

 U.S.A.). 



A purification procedure has been developed 

 employing Sephadex G-100 and G-200 which 

 permits routine preparation of tryptophan syn- 

 thetase of N. crassa. The enzyme is about 95 per 

 cent pure by physical criteria. It has an S20 of 

 5.9 and a diffusion coefficient of 4.92 10 7 cm 2 

 sec -1 . The Svedberg molecular weight of about 

 122,000 is in good agreement with the value 

 obtained by Archibald equilibrium sedimen- 

 tation. These protein preparations have been 

 used for physical and chemical studies in an 

 effort to provide a structural basis for previous 

 genetic, enzymological and immunochemical 

 observations. The results from amino acid com- 

 position studies, fingerprint analysis of tryptic 

 peptides, and from ultracentrifugation in 5m 

 guanidine — HC1 are consistent with the existence 

 of a quaternary structure for tryptophan synthe- 

 tase, comprised of two or more subunits. Such a 

 structure could explain the phenomenon of inter- 

 allelic complementation in this system, as well 

 as the occurrence of CRM -negative mutants. 



This research was supported by Grant C-03080 

 of the National Institutes of Health. 



4.47. Genetic Control of Tryptophan Accumulation in 

 Neurospora. David R. Stadler (Washington, 

 U.S.A.). 



Wild-type Neurospora fails to grow on medium 

 supplemented with 4-methyltryptophan (4MT) 

 due to the inhibition of one or more enzymes 

 required for the synthesis of tryptophan. Resis- 

 tant mutants have been selected after u.v. or 



nitrous acid treatment, and the twenty-two 

 mutants examined to date are alike in the charac- 

 teristics which follow. Resistance results not 

 from any direct change in the regulation of tryp- 

 tophan synthesis, but from the loss of the ability 

 to accumulate 4MT (or tryptophan) from the 

 medium. Under conditions in which wild-type 

 accumulates tryptophan to about 15 times the 

 external concentration, the mutants only ap- 

 proximate the external concentration. The wild- 

 type accumulation is inhibited by sodium azide. 

 The mutants are all in the same genetic region of 

 linkage group IV, less than one map unit from 

 co-4. The mutants are recessive; heterocaryons 

 between sensitive and resistant strains are sen- 

 sitive. Preliminary studies of one mutant indicate 

 that the same gene is responsible for uptake of 

 several other amino acids. 



4.48. A Modifier Mutation affecting Tryptophan 

 utilization by Tryptophan Auxotrophs of Neu- 

 rospora crassa. B. Maling, P. St. Lawrence, 

 L. Altwerger and M. Rachmeler (Berkeley, 

 U.S.A.). 



A mutation (nwd-5) which allows growth of 

 tryptophan auxotrophs of Neurospora crassa on 

 media containing yeast extract (YE) or Bacto- 

 peptone (BP) without added tryptophan segre- 

 gates independently of the td locus and is neither 

 locus nor allele specific, td; mod-5 cultures 

 require tryptophan and show no detectable res- 

 toration of tryptophan synthetase activity, mod-5 

 activity is not specific for tryptophan auxotrophs; 

 it affects the growth of a lysine and of an aro- 

 matic mutant. 



The amount of free tryptophan in YE and BP 

 is sufficient for greater growth of td stocks than 

 is observed; mod-5 appears to permit utilization 

 of this tryptophan. Acid hydrolysis of BP and 

 YE removes growth activity for modified strains. 

 Sephadex chromatography of BP and YE yields 

 fractions with activity for both td and td; mod-5 

 cultures. The active substance in these fractions 

 has been tentatively identified as tryptophan. 



Differential growth on enriched media may be 

 due to greater sensitivity of unmodified strains to 

 inhibitors. Some BP sephadex fractions appear 

 to have a differential inhibitory effect on td and 

 td; mod-5 cultures. The nature of the inhibitor 

 and the mechanism of the increased resistance of 

 td; mod-5 strains to it is not known. However, 

 differential responses of modified and unmodified 

 strains to tryptic digests of (3-lactoglobulin in- 

 dicate that the inhibitor may be a protein con- 

 stituent. Equivalent growth of both strains is 

 obtained at low concentrations of digest but 



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