Macromolecule Synthesis and Germination of Conidia in Temperature Sensitive Mutants of Neurospora Crassa

JAPAN. J. GENETICS Vol. 45, No. 3: 357-369 (1970)

MACROMOLECULE SYNTHESIS AND GERMINATION OF CONIDIA IN TEMPERATURE SENSITIVE MUTANTS OF NEUROSPORA CRASSA

HIROKAZU INOUE AND TATSUO ISHIKAWA

Department of Botany, Faculty of Science, University of Tokyo, and Institute of Applied Microbiology, University of Tokyo Bunkyo-ku, Tokyo 113

Received July 3, 1970

Studies on auxotrophic mutants contributed to the analyses of the structure and function of the gene and many metabolic pathways. However, the isolation of auxotrophic mutants has a limit, because mutants of indispensable genes may not grow by supplementation of nutrients. Beadle and Tatum (1945) first isolated temperature sensitive mutants in Neurospora cyassa, and Horowitz and Leupold (1951) suggested that the isolation of temperature sensitive mutants might provide a special technique for detecting mutations in indispensable genes including genes responsible for macromolecule synthesis. The temperature sensitive mutants have since been studied in other micro- organisms such as phages (Edgar and Lielausis 1964; Nishihara and Roming 1964), Escherichia coli (Horowitz and Leupold 1951; Kohiyama et al. 1966), yeast (Hartwell 1967) and Ascobolus (Yu-Sun 1969), and contributed to the elucidation of functions of indispensable genes. This paper reports the isolation and preliminary characterization of temperature sensitive mutants in Neurospora cyassa which grow at 25°C but are unable to grow at 35°C on enriched media. The mutants were particularly studied to elucidate a relationship between the ability to synthesize protein and nucleic acids and the germination of conidia.

MATERIALS AND METHODS

Strains : The wild type strain 74 A of Neurospora cyassa was used as the standard strain. Temperature sensitive (ts) mutants were selected from the wild type 74 A following ultraviolet irradiation of conidia. The is mutants of the opposite mating type were obtained from crosses with the wild type 3.1a. Media : Fries minimal medium (Beadle and Tatum 1945) was used in general. Enriched medium means a medium containing vitamin mixture, casamino acids and yeast extract. Glycerol complete medium which is an enriched medium containing glycerol was used for preparing conidial samples. Sorbose minimal medium containing 1.0% sorbose and 0.2% sucrose was used for plating medium to form colonies. Phosphate free medium used for induction of acid phosphatase was prepared by substituting KC1 in Fries minimal medium for KHZPO4. Westergaard and Mitchell's crossing medium (Westergaard 358 H. INOUE ANDT. ISHIKAWA

and Mitchell 1947) was used for crossing. Growth measurement : To measure growth in various media, conidial suspensions were inoculated into 20 ml liquid media in 100 ml Erlenmeyer flasks. Mycelial mats were harvested after a designated time, dried and weighed. Preparation of conidial suspension : Conidia were inoculated into 20 ml glycerol complete medium in 100 ml flasks. After 7 days conidia produced were harvested by pouring chilled water and filtered'through 2 sheets of gauze to remove hyphal segments. Conidia were washed with chilled water by centrifugation. A conidial suspension thus prepared was able to store at 4°C for at least 7 days keeping a constant ability of germination. The number of conidia was counted by a hemacytometer. Measurement of germination : Washed conidia were suspended in chilled water and diluted to 107 conidia per ml. 1 ml of conidial suspension was pipetted into 10 ml of Fries minimal medium in 100 ml Erlenmeyer flask and shaken in an incubator at the designated temperature. Shaking speed was about 100 strokes per minute. At appropriate intervals, an aliquot was taken out and fixed with 10% neutral formalin. The germination was identified by the emergence of germ-tube from conidial cells. A maximum ability of germination was observed for the culture containing 1 x 106 conidia per ml or less. The germination was interfered in the culture containing more than 106 conidia per ml. Incorporation of 14C02: Na214CO3 was used as the source of 14C02. 108 conidia were inoculated into 100 ml of Fries minimal medium (pH 5.4) in 500 ml Erlenmeyer flask with a special stopper made of rubber. 10 iCi of Na214CO3 was injected and the flask was shaken immediately. To stop 14CO2incorporation, the flask was chilled and 1 ml of 1 N NaOH was injected. Conidia were collected by centrifugation, washed 3 times and fractionated into RNA and DNA fractions according to the convenient method described by Tsay et al. (1965). Incorporation of 14C-amino acids : Conidia and 0.5 iCi 14C-amino acid mixture were pipetted into 100 ml flask containing 20 ml of Fries minimal medium and incubated at 25°C or 35°C with shaking. At appropriate intervals, an aliquot was taken out and 1 ml of 10% perchloric acid (PCA) was added to stop incorporation. 107 conidia were added tothe suspension as a carrier, washed with 5% PCA 3 times, heated with 5% PCA at 90°C for 15 minutes, filtered through a membrane filter and washed well with 5% PCA. The radioactivity of the membrane filter thus prepared was counted. Incorporation of uridine-2-14C : Conidia and 0.5 iCi uridine-2-14C were pipetted into 50 ml flask containing 5 ml Fries minimal medium and shaken at 25°C. At the indicated time 1 ml of 30% PCA was added to stop incorporation. Conidia were washed with Z ml of 5% PCA 2 times and then with water. After mixing 1 ml of 1 N KOH, the conidial suspension was incubated at 35°C overnight. The mixture was neutralized by adding 6 N HCI. After adding 1 ml of 5% PCA, the mixture was centrifuged. The supernatant was taken as an RNA fraction and the radioactivity was counted. Determination of acid phosphatase activity : Acid phosphatase activity was measured by a colorimetric method as described by Nyc (1967). The reaction mixture (total volume 1.0 ml) contained 0.05 ml of culture filtrate. Incubation was made at 37°C for 20 minutes and the reaction was stopped by adding 1 ml of 10% trichloroacetic acid (TCA). Then TEMPERATURE SENSITIVE NEUROSPORA MUTANTS 359

2 ml of saturated sodium carbonate was added into the mixture. Optical density at 405 mp of this mixture was measured by a spectrophotometer. One unit of activity was defined as 1 ml of the culture filtrate producing a change of absorbance of 1.0 at 405 mj. Chemicals : Na214CO3 and uridine-2-14C were the products of Radiochemical Center, England. 14C-amino acid mixture (14C-Chlorella hydrolysate) was a generous gift from Prof. B. Maruo of this University. All other chemicals were obtained commercially.

RESULTS

Isolation and characterization of temperature sensitive mutants Conidia of the wild type strain 74 A were suspended in 250 ml Fries minimal medium in 500 ml Erlenmeyer flasks and incubated at 35°C on a low speed reciprocating shaker. Filtration and subsequent plating were made in accordance with the filtration-concentration technique of Woodward et al. (1954) as revised by Case (1963). Thus, 257 is mutants which form colonies at 25°C on Fries minimal medium but are unable to form colonies at 35°C were isolated. These is mutants include is auxotrophic mutants which form colonies at 35°C on an enriched medium containing small molecules such as amino acids, vitamins, purines or pyrimidines, since the mutants were identified by the inability to grow on the minimal medium at 35°C. Therefore, the mutants were tested for growth at 35°C on media containing casamino acids, yeast extract, or vitamin supplement. Two hundred and eleven of the mutants were able to form colonies with one or com- bination of these supplements, but 46 mutants were unable to form colonies with any supplements. The former group of the is mutants was designated as reparable is mutants and latter as irreparable is mutants. The irreparable is mutants which were assigned numbers from 1 to 46 were further characterized by testing the growth at 25°C and 35°C and the viablity of conidia at 35°C. Most of these is mutants showed no growth at 35°C and reduced growth at 25°C comparing with that of the wild type as shown in Table 1. The viability of conidia at 35°C was investigated as follows. Approximately 150 conidia were plated on sorbose minimal medium and the plates were divided into two groups. One group was incubated at 25°C and another at 35°C. Plates incubated at 35°C were transferred to 25°C after 20 hours. After 4 days, the number of colonies appeared on two kinds of plates was counted and compared. As shown in Table 1, conidia of most irreparable is mutants showed a reduced viability after incubation at 35°C for 20 hours. Among them, mutant 25 showed 95% reduction of viability of conidia after incubation at 35°C for 20 hours. The morphology of hyphae of irreparable is mutants was observed by incubating conidia on a thin layer of minimal agar medium at 25°C or 35°C. Hyphae of four mutants, mutant 28, 29, 44 and 46, showed morphological abnormality. Mutant 44 and 46 were colonial at 25°C and produced extremely condensed hyphae at 35°C, and mutant 28 and 29 were colonial at the early stage of growth at 25°C and later grew producing hyphae with a small number of conidia, but these two mutants did not grow at 35°C. Although the irreparable is mutants fail to grow on supplemented media at the nonpermissive temperature, 6 of them were able to form colonies at 35°C when 3% 360 H. INOUE AND T. ISHIKAWA

Table 1. Growth, viability and germination of conidia, and macromolecule synthesis in the irreparable is mutants TEMPERATURE SENSITIVE NEUROSPORA MUTANTS 361

NaCI was supplemented to the minimal medium (Table 1). Supplementation with 3% KCI, 20% lactose or 10% mannitol resulted in the same effect as observed by supplementing 3 % NaCI. The irreparable is mutants were tested for dominance or recessiveness of their is mutation and for allelic relationship. All possible combinations among 39 mutants were made to test for heterocaryon complementation by mixing conidia suspensions of the two mutants at 35°C. Seven irreparable is mutants (mutant 3, 6, 32, 33, 42, 44 and 46) scarcely produced conidia and were not subjected to the heterocaryon test. As the result, most mutant combinations showed a positive heterccaryon response, indicating that these is mutations are recessive and not allelic. Nineteen mutant combinations showing no complementation at 35°C due to the failure of conidia germination were further tested for the allelic relationship by crossing. Ascospores obtained from crosses of noncomplementing combinations were plated on sorbose minimal medium and incubated at 25°C and 35°C. Eight crosses involving two particular mutants, mutant 5 and 37, failed to produce fertile ascospores. Among the fertile crosses most combinations of the mutants prcduced wild type colonies indicating that these mutants may not be allelic, but two combinations of the mutants, 19 and 24, and 20 and 30, failed to produce wild type colonies, indicating that these two groups of mutants may be allelic. 362 H. INOUE ANDT. ISHIKAWA

Germination ability of conidia of irreparable is mutants Wild type conidia began to produce germ-tube after 2 hours of incubation and most conidia produced germ-tube after 4 hours at 25°C and 35°C (Fig. 1). The germination ability of conidia of most irreparable is mutants was examined after shaking at 35°C for 5 hours (Table 1). Six mutants appear to be almost completely deficient in produc- tion of germ-tube at the nonpermissive temperature. These mutants are designated in this paper as germination deficient is mutants. However, it should be pointed out that

Fig. 1. Germination of conidia of wild type and three irreparable is mutants. Conidia were inoculated into 100 ml Erlenmeyer flasks containing 10 ml of minimal medium and incubated at 25°C or 35°C with shaking. An aliquot was taken out at the indicated time and germinating conidia were counted. 25°C; ..., 35°C. 0, wild type; •, mutant 14; •, mutant 25: A, mutant 31. these is mutations are responsible not only for the defective germination but also for the failure of vegetative growth of mycelia at the nonpermissive temperature. The germination ability of conidia of three germination deficient is mutants, mutant 14, 25 and 31, were further investigated for 7 hours, and it was found that conidia of these mutants show the maximum germination at 25°C after 5 hours and almost no germination at 35°C even after 7 hours (Fig. 1).

Nucleic acid synthesis at the nonpermissive temperature It was reported by Yanagita (1963) in Aspergillus niger that germinating conidia incorporate 14C02 into nucleic acids. The same experiment was tried for conidia of N. crassa. The significant amount of radioactivity appeared in the RNA and DNA fractions of wild type conidia after 2 hours of incubations. To test the ability to synthesize nucleic acids in some irreparable is mutants, the incorporation of 14C02 into nucleic acids was measured for first 2 hours of incubation of conidia. Table 1 shows the ability to incorporate 14C02 into the nucleic acid fractions in some irreparable is mutants in comparison with the wild type. Mutant 14 and 17 showed particularly low ability to incorporate 14C02 into the RNA and DNA fractions at 35°C. No further incorporation of 14C02 into the RNA and DNA fractions in these mutants was observed even after 3 hours. The low ability to incorporate 14C02 into nucleic acids of conidia may not be related directly with germination of conidia, since mutant 17 was not a germination deficient is mutant.

Protein synthesis at the non permissive temperature When wild type mycelia of Neurospora are transferred to phosphate free medium, a kind of acid phosphatase is produced into the culture medium (Nyc 1967). The process TEMPERATURE SENSITIVE NEUROSPORA MUTANTS 363

Fig. 2. Incorporation of 14C-amino acids into conidia of wild type and three irreparable is mutants. 106 conidia and 0.5 iCi 14C-amino acid mixture were added into 100 ml flask containing 20 ml minimal medium and incubated at 25°C or 35°C with shaking. An aliquot was taken out at the indicated time, washed, heated at 90°C for 15 minutes with 5% PCA and filtered through a membrane filter. Radioactivity on the membrane filter was counted. 25°C; ..., 35°C. 0, wild type; •, mutant 14; ®, mutant 25; s, mutant 31.

of this enzyme induction involves de novo protein synthesis (Toh-e, unpublished data). Therefore, it is expected that mutants which have a defect in protein synthesis are unable to produce this acid phosphatase at 35°C. However, it should be pointed out that mutants which involve any defect in one of the processes leading to the induction of active enzyme during a prolonged incubation (3 days) at the nonpermissive temperature may also fail to produce this enzyme. To measure the inducibility of this acid phosphatase for the irreparable is mutants, mycelia grown in liquid minimal medium for 3 days at 25°C were transferred to phosphate free medium after washing with sterilized water, and further incubated at 25°C or 35°C. The activity of acid phosphatase in the culture medium was measured day by day after transferring to phosphate free medium. The wild type strain produced a significant amount of phosphatase after 3 days both at 25°C and 35°C. As shown in Table 1, the irreparable is mutants tested were able to produce the acid phosphatase at 25°C, although most of them produced a reduced amount of the enzyme at 25°C, comparing with the wild type. Some is mutants tested produced no significant amount of phosphatase at 35°C. However, 21 of 39 mutants tested involving one germniation deficient mutant (mutant 31) were able to produce this acid phosphatase at 35°C, indicating that protein synthesis of these mutants may not be defective. Three germination deficient is mutants, mutant 14, 25 and 31, were further tested for the incorporation of 14C-amino acids into the protein fraction. As shown in Fig. 2, the radioactivity incorporated into protein in mutant 14 conidia during incubation at 35°C for 4 hours was approximately one half of that incorporated at 25°C. In the wild 364 H. INOUE ANDT. ISHIKAWA type and mutant 31, the radioactivity incorporated at 35°C was higher than that incorporated at 25°C. Mutant 25 conidia appear to have higher ability to incorporate protein precursors at 25°C and 35°C than wild type conidia.

Comparison between effects of inhibitors of RNA and protein synthesis and nonpermissive temperature on germination of conidia It has been reported in Peronospora tabacina that proflavin inhibits RNA synthesis (Hollomon 1969). To confirm this in Neurospora, the incorporation of uridine-2-14C and 14C-amino acids into germinating conidia were investigated under the presence of various concentrations of proflavin. Wild type conidia were inoculated into 5 ml liquid minimal medium containing various amounts of proflavin and 0.5 pCi uridine-2-14C or 14C-amino acid mixture. As shown in Fig. 3, 30 pg per ml proflavin inhibited 80% of uridine-2-14C incorporation into RNA and 60% of 14C-amino acid incorporation into protein. The result indicates that proflavin primarily inhibits RNA synthesis in Neurospora. On the other hand, the effect of proflavin on germination of wild type conidia was tested as

Fig. 3. Effect of proflavin on germination Fig. 4. Effect of cycloheximide on germi- of conidia and on incorporation of nation of conidia and on incorpo- uridine-2-14C and 14C-amino acids. ration of 14C-amino acids. The Wild type conidia were inoculated experiment was carried out as into 100 ml flask containing 10 ml in Fig. 4, except that various of minimal medium supplemented with various amounts of proflavin amounts of cycloheximide were and incubated at 25°C with shak- used. , germination; •, in- ing. After 5 hours, germinating corporation of 14C-amino acids. conidia were counted. 0.5 pCi uridine-2-14C or 0.5 pCi 14C-amino acid mixture was added into 5 ml of minimal medium to test incorporation. After 2 hours, conidia were collected and fractionated by the method described in Ma- terials and Methods. Radioactivi- ty of each fraction was counted and percent of incorporation was calculated. , germination; -•-.- , incorporation of uridine-2-14C; ..•, incorporation of 14C-amino acids . TEMPERATURE SENSITIVE NEUROSPORA MUTANTS 365 follows. Wild type conidia were inoculated in 10 ml liquid minimal medium containing various amounts of proflavin. After shaking 25°C for 5 hours, germinating conidia were counted. As shown in Fig. 3, 30 pg per m1 of proflavin showed a slight effect on germination, indicating that RNA synthesis may not be a limiting factor in germination of conidia. Cycloheximide is known as an inhibitor of protein synthesis in Neurospora (Pall 1966; Hitchcock and Cochrane 1969). As shown in Fig. 4, 3 pg per ml of cycloheximide inhibited approximately 90% of incorporation of precursors into hot PCA precipitable material. On the other hand, 4 beg per ml of cycloheximide inhibited approximately

Fig. 5. Effect of cycloheximide or temperature-shift from 35°C to 25°C on germination of conidia. (a) Wild type conidia were inoculated into 10 ml minimal medium containing 5 pg/ml cycloheximide and incubated at 25°C with shaking. At the indicated shift-time, conidia of each flask were filtered through a membrane filter, washed with sterilized water and transferred into fresh medium without cycloheximide. Germinating conidia were counted at various intervals. (b-d) Conidia of three germination deficient is mutants were incubated at 35°C with shaking. At the indicated shift-time, the flask was transferred to 25°C. Germinating conidia were counted at various intervals. (b), mutant 14; (c), mutant 25; (d), mutant 31. The shift-time: 0, 0 minutes; 0, 30 minutes; A, 60 minutes; A, 90 minutes; C], 120 minutes. 366 H. INOUE ANDT. ISHIKAWA

90% of germination at 35°C (Fig. 4) indicating that protein synthesis is necessary for germination of conidia. The time sensitive to the effect of cycloheximide during germination was investigated in the following way. Wild type conidia were incubated in 10 ml of liquid minimal medium cantaining 50 pg of cycloheximide at 25°C with shaking. At the designated time (30, 60, 90 and 120 minutes) the conidial suspension of each flask was filtered through a membrane filter. Conidia obtained on the membrane filter were washed with 20 ml of sterile water and inoculated again in 10 ml of fresh medium containing no cycloheximide. The further incubation was made at 25°C with shaking and at the designated time (150, 180, 210, 240, 270 and 300 minutes) 1 ml of conidial suspension was taken out and germinating conidia were counted. The result shown in Fig. 5(a) indicates that the increase of percent of germination depends on the incubation period with cycloheximide. The result suggests that protein synthesis necessary for germination is initiated at the beginning of incubation and lasts constantly for at least 2 hours. To compare the effects of cycloheximide on germination of wild type conidia studied above with effects of the nonpermissive temperature on germination of conidia of three germination deficient is mutants, the following experiments were performed. Conidia of mutant 14, 25 and 31, were inoculated into 100 ml Erlenmeyer flasks containing 10 ml liquid minimal medium. After incubating for 30, 60, 90 or 120 minutes at 35°C, each flask was transferred to 25°C and incubated continuously. An aliquot was taken out from each flask at the indicated time and germinating conidia at that time were counted. As shown in Fig. 5 (b, c), the germination of conidia in mutant 14 and 25 was influenced significantly by preincubation at 35°C. Comparing these results with those indicated in Fig. 5 (a), it is suggested that these mutations have a similar effect to that of cycloheximide on germination of wild type conidia. The preincubation of conidia of mutant 31 at 35°C for 2 hours from the initiation of germination had no effect on germination (Fig. 5, d). This result indicates that the metabolism in conidia of this mutant may be normal during incubation at the nonpermissive temperature for 2 hours but defective after incubating for a longer period than 2 hours, resulting in the germination deficiency.

DISCUSSION

Neurospora is one of the eucaryotic organisms which has extremely small amount of genetic material, approximately 8 X 10 pg per single haploid nucleus if single conidium contains two nuclei in average (Minagawa et al. 1959). On an assumption that each protein molecule contains 500 amino acid residues in average, this DNA content corres- ponds to approximately 4,500 genes, each of which may produce a different protein. Since approximately 400 genes have been reported in Neurospora to date, there should be more than 4,000 remaining genes (more than 90% of total genes) to be identified. Horowitz and Leupold (1951) indicated in Neurospora that 46% of the is mutants were irreparable and suggested that all kinds of mutants may be obtained as ts-mutants. In the present experiment, about 20% of the is mutants isolated were characterized as the TEMPERATURE SENSITIVE NEUROSPORA MUTANTS 367 irreparable is mutants. If the is mutation occurs on all genes of Neurospora at the same frequency, approximately 90% of the is mutants should be irreparable. Lower frequency of appearance of the irreparable is mutants observed may suggest that a large part of the mutations are unable to be detected in the present experiment by unknown reasons. The fact that most of the irreparable mutants isolated were found to be not allelic may indicate the existence of really large number of unknown and probably indispensable genes. Among 46 irreparable is mutants studied, 4 mutants showed morphological abnormality. It would be expected that mutations in genes which function in the cell wall formation result in morphological changes at the nonpermissive temperature (Mahadevan and Tatum 1965; Brody and Tatum 1966, 1967) . On the other hand, six mutants restored the growth by high osmotic pressure at the nonpermissive temperature. This phenome- non is a kind of phenotypic reversion and similar mutants were reported by Kuwana (1961) and Metzenberg (1968). As suggested by them, the effect of these is mutations may be related with the cell membrane or transport system. It should be pointed out that a significant number of this kind of genes is expected to exist in Neurospora, since some of these mutants appear to be not allelic. Macromolecule synthesis during germination of conidia of the wild type and is mutants was investigated to find the process affected by the is mutation. In Bacillus cspeies, messenger RNA (mRNA) wass ynthesized de novo after the activation of spores and no stored mRNA appears to exist in spores (Halvorson et al. 1966; Rodenberg et al. 1968). Therefore, mRNA synthesis necessary for germination of bacterial spores should be induced after the initiation of germination, and then protein synthesis may be initiated. In Peronospora tabacina, however, it was reported that protein synthesis is necessary for germination of conidia, but RNA synthesis is not (Hollomon, 1969). Ono et al. (1966) indicated in Aspergillus oryzae that RNA synthesized at the early stage of germination is ribosomal RNA and transfer RNA but no mRNA. The results of the present experiment using inhibitors of RNA andprotein synteses showed that RNA synthesis at an early stage of germination may not have a direct relation to germination, but protein synthesis is essential. This protein synthesis necessary for germination starts at the beginning of inoculation of conidia and lasts constantly thereafter for at least 2 hours. This is different from the result obtained in Peronospora that protein synthesis necessary for germination is accomplished within 30 minutes of incubation (Hollomon 1969). Characteristics of three germination deficient is mutants which did not show the emergence of germ-tube at 35°C were investigated and compared with the effects of inhibitors of RNA and protein synthesis on germination of conidia. One of these mutants, mutant 14, showed poor incorporation of 14C02 into the RNA fraction and of '4C-amino acids into the protein fraction at 35°C. Effect of the elevated temperature on germination of conidia in this mutants was similar to the effect of cycloheximide on germination of wild type conidia (Fig. 5). The results indicate that the synthesis of a particular protein may be defective in this mutant and that the protein synthesis is necessary for germination of conidia. The ability to synthesize nucleic acids may be depressed in the mutants which lack the ability of protein synthesis (Hartwell 1967). 368 H. INOUE AND T. ISHIKAWA

Another mutant, mutant 31, had almost normal ability to synthesize RNA and protein. This mutant may have a defect on a material which is synthesized after 2 hours from the initiation of germination. The mutational defect in this mutant might be related with a process emerging the germ-tube from conidial cells, such as cell wall synthesis. The third mutant, mutant 25, appears to have the abnormality in a cellular metabolism at the nonpermissive temperature, since the incorporation rate of 14C-amino acids was higher than that of the wild type and the viability of conidia decreased abruptly during incubation in minimal medium at 35°C. Thus, there appears to be a number of defects leading to the loss of germinating ability of conidia in Neurospora. Further analyses of irreparable is mutants which fail to germinate may result in elucidation of the genetic regulation of germination of conidia in Neurospora. Most irreparable is mutants isolated in this work showed varying levels of the germinating ability and did not show a preferential defect in any of the parameters measured at the nonpermissive temperature. Studies of further variables, such as re- spiration, lipid metabolism and synthesis of cellular structure, will result in elucidation of the site of the defect in these mutants.

SUMMARY

Two hundred and fifty seven temperature sensitive mutants of Neurospora crassa which were unable to grow at 35°C but grew at 25°C were isolated. 46 of these (irreparable temperature sensitive mutants) were unable to restore growth at 35°C by supplementing various nutrients. The irreparable temperature sensitive mutations were found which resulted in loss of viability of conidia, abnormality in morphology, loss of growing ability which is reversible by high osmotic pressure, loss of germinating ability of conidia or reduced ability to synthesize nucleic acids or protein at 35°C. Possible defects in macromolecule synthesis leading to loss of germinating ability of conidia were analysed in some of these mutants. Significance to study these temperature sensitive mutants was discussed.

ACKNOWLEDGMENTS

The authors wish to express their appreciation to Misses T. Oguchi and H. Akiyama for their excellent technical assistance. This work was supported by U.S. Public Health Service Grant GA-10505 from the National Institute of General Medical Sciences and in part by a Research Grant from the Ministry of Education of Japan.

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