Summtary. A new strain of Neuirospora crassa which exhibits a rhythm of conidiation when growing along an agar surface in a growth ttube is described. The rhythm has been shown to be circadian for it meets the following criteria: A) the period under constant environmental conditions in the dark is abolut 24 hours (22.7 hours at 250) ; B) the period is relativcly temperatuire-independent (Q, We have developed a system for the biochemical stuldy of a circadian rhythm employing a new strain of Neurospora crassa (timex)2 which satisfies both criteria mentioned above. Pittendrigh et al. (16) were the first to point ouit the advantages of working with Neutrospora, and Sussman, Dturkee, and Lowry (21), Berliner and Neuirath (4), and Bianchi (5) have uised Neuirospora to stuidy the rhythm expressed in the clock strains described by Sussman, Lowry, and Duirkee (11,22

One approach to an understanding of the nature of extrachromosomal heredity, subeellular morphology, and nucleocytoplasmic relationships is through the investigation of the genetics of enzymes and proteins of mitochondria. Genetic information for the primary structure of some mitochondrial-localized enzymes resides in nuclear DNA;' 2 no direct demonstration of a gene-protein relationship involving mitochondrial DNA has been previously reported.Concepts developed in several areas of independent investigation led to the working hypothesis of this study. First, although concepts of extrachromosomal heredity have been evolving for many years, only one class of extrachromosomal mutations affecting mitochondria are well known; these are the respiratory-deficient mutants of yeast and Neurospora.3 Extensive analyses of such mutants have not produced results interpretable in terms of either the classical one gene-one enzyme hypothesis or related corollaries regarding pathways of intermediary metabolism. Rather, several of the components of the electron transport chain are found to occur in either excess or deficit in those mutants. In addition, Green and associates4 have proposed that polymacromolecular assemblies called elementary particles, with a fixed set of proteins in invariant proportions, are associated with the mitochondrial inner membrane. Among the principal components of those particles are the enzymes of the electron transport system and the mitochondrial structural protein. The importance of the structural protein is emphasized not only by its quantitative preponderance in the mitochondria, but also by its critical role in the organization and assembly of the enzymes of the particles. Moreover, recent investigations indicate that mitochrondria from all organisms contain DNA.' That this DNA may play a hereditary role is indicated by observations that Neurospora mitochondria replicate by division of pre-existing Inlitochondria6 and exhibit genetic continuity after interhyphal transplantation.7In this communication, the results of experiments stimulated by the foregoing concepts support the hypothesis that two different extrachromosomal mutations of (presumably) mitochondrial DNA lead to alterations of the primary structure

Three cases of complementation between allelic mutants in heterocaryons of Neurospora crassa have been reported in which the individual homocaryons lack a specific enzymatic activity.'-3 More recent and apparently similar phenomena have been observed in which data relating to the involvement of a single enzyme are still lacking.4 6The present paper presents a detailed analysis of heterocaryon complementation between ad-4 mutants, each of which has impaired adenylosuccinase activity. The results suggest that the linear structure of both a gene and its products may be revealed by the pattern of interallelic heterocaryon complementation. The enzyme adenylosuccinase, found in wild-type extracts, catalyzes the splitting of adenosine monophosphate succinate (AMP-S) to adenosine monophosphate (AMP) and fumarate, as well as the splitting of the analogous purine precursor SAICAR to AICAR6 and fumarate." I Adenylosuccinase activity has not been found in appreciable amounts (less than 1 per cent of wild-type activity) in any of the ad-4 mutants tested, and partial restoration of activity has been detected in all cases examined in which complementation occurs. The degree of complernentation varies widely among the different mutant combinations; i. e., growth rates of the heterocaryons on minimal medium range from less than 0.2 to 4 mm/hr (wild-type rate), and enzyme activities range from less than 1 to 25 per cent of wild-type activity. Brief reports of certain of these results have been presented previously.7 8

Krebs cycle enzymes were repressed in Neurospora during growth on sucrose as carbon source compared with growth on acetate. In mitochondrial, cytochrome‐deficient strains grown on sucrose, this glucose or catabolite repression was reduced. In mutant strains in which protein synthesis and mitochondrial respiration were inhibited, catabolite repression was enhanced. These data support the hypothesis that ATP or the cellular energy level is involved in catabolite repression. The interrelationships between energy metabolism, the energy distribution in the cell and catabolite repression are discussed. The addition of casamino acids to a culture growing on sucrose or acetate, did not cause repression of any of the Krebs cycle enzymes measured, in spite of the fact that many of the constituent amino acids can be considered as biosynthetic products of the Krebs cycle.

Timex, a strain of Neurospora crassa which exhibits a circadian rhythm of conidia formation in growth-tube cultures, has been found to differ from wild-type strains by two genes. One gene, inv, is responsible for an invertase deficiency, whereas the second gene, bd, is of unknown function. Both genes map independently from other genes known to induce Neurospora rhythmicity. The inv gene is not essential for the timex phenotype because bd strains express that phenotype on certain media. Although inv strains do exhibit some rhythmicity of their own, the rhythmicity apparently is not a direct result of the invertase deficiency, since there is no correlation between invertase level and rhymicity in 29 strains tested. Of the 29 strains tested, 20 exhibited some rhythmicity in growth-tube cultures, suggesting that morphological manifestations of rhythmicity in Neurospora may result from the function or the loss of function of numerous genes, or both. There was no correlation in these strains between rhythmicity and (i) genetic background; (ii) geographical origin; or (iii) nutritional requirements. ' Taken in part from a thesis submitted to Stanford University by M. L. S. in partial fulfillment of the requirements for the Ph.D. degree.