/0 Reprinted from the AMERICAN JOURNAL oF Borany, Vol. 35, No. 3, 150-157, Mareh, 1948 Printed in U.S. 4. INDUCED REVERSIONS OF BIOCHEMICAL MUTANTS IN NEUROSPORA CRASSA t Norman H., Giles, Jr., and Esther Zimmer Lederberg 2 One oF the problems connected with biochemical mutants requiring specific growth factors—such as have been induced in Neurospora and other micro- organisms (Beadle and Tatum, 1941; Beadle, 1946 )—-concerns the ability of these mutant strains to regain the power of growth in a medium lacking the substance originally required. “Adaptation,” as this phenomenon is usually designated, has been observed in a number of instances (Ryan, 1946). The question immediately arises whether adapta- tions represent some kind of non-genetic change presumably involving cytoplasmic components, or whether they are the result of genetic changes in- volving reverse-mutations of specific genes to their wild-type alleles, thus restoring the synthetic ca- pacity of the organism. It has been shown in an extensive study of a leucineless mutant of Neuro- spora crassa that spontaneous adaptations in this strain do involve reverse-mutation (Ryan and Lederberg, 1946; Ryan, 1946). Thus it seemed of interest to investigate the possibility of reverse- mutations in other Neurospora mutants. Further, it 1 Received for publication August 23, 1947. 2 Present address: Department of Genetics, University of Wisconsin, Madison 6, Wisconsin. appeared especially desirable to determine whether the rates of such reversions could be increased by various treatments. The techniques for detecting changes involving the restoration of synthetic ca- pacity for specific substances essential for growth are much simpler than those for changes involving losses of such capacities. Thus in instances where adaptations are found to result from reverse muta- tions, it should be possible to utilize these techniques to obtain data on the mutability of numerous genes with a variety of agents and under many different conditions. EXPERIMENTAL METHODs.—For most of the ex- periments multiple biochemical mutant stocks ob- tained by recombinations of single mutants were utilized. The use of such stocks makes it possible to compare the effect of a single uniform treatment on two or more genetic loci. Further, the additional mutant genes serve as useful genetic markers. In most stocks the marker gene for colorless conidia (albino-2) was also present, making possible the visible detection of possible contamination by wild type conidia. The procedure in a typical experiment (exp. 12. table 1) was as follows: a conidial suspension of Mar., 1948] the multiple mutant stock #G37a was prepared, filtered through absorbent cotton to remove myce- lial fragments, and the concentration of conidia determined with a haemocytometer (in this experi- ment the conidial concentration was 5.7 < 106/ml.). This stock, originally synthesized as a genetic tester stock, has the following mutant loci, all on different chromosomes: albino-2 15300 [al], inositolless 37401 [inos], pantothenicless 5531 [pan], trypto- phaneless 10575 [tpt], riboflavinless 51602 [rbl]. The numbers following the mutant name in this and all subsequent stocks used refer to the original Stanford culture numbers (Beadle and Tatum, 1945), while the abbreviations in brackets refer to the growth factor required. The normal (wild) type will be indicated by adding ‘*”. These symbols will be used throughout. Half of the suspension was retained for use in controls; the other half was sub- jected to ultra-violet radiation from a Hanovia quartz mercury-are lamp for 2 min, at 15 cm. from the lamp hood. Dilution platings from the control and treated suspensions were made to determine conidial viability (approximately 100 per cent) and percentage survival after treatment (approximately 50 per cent). The use of a limiting inositol concen- tration (0.3y/ml.) results in a colonial type of growth rather than the typical spreading type (Beadle, 1944), and facilitates colony counts in agar. Samples from the control and irradiated sus- pensions were then dispensed into a series of flasks (flasks 1-5 received 0.3 ml. and 6~15, 0.1 ml. each of suspension) containing 40 ml. of minimal medium supplemented with all but one of the substances required for growth (e.g., for tests of adaptations at the inositolless locus, flasks received minimal plus 2y/ml. Ca-pantothenate, 10-y/ml. (1-) trypto- phane, and 5y/ml. riboflavin). Tests for riboflavin- less adaptations were run at 35°C., since this is a temperature-sensitive mutant (Mitchell and Houla- han, 1946); all other flasks were kept at 25°C. For the mutants used little or no growth normally occurs in the absence of the required substance, and adapt- ations ate detected as cultures showing normal growth and conidiation in the deficient medium. The experiment was maintained for 15 days. It is evident from table 1 that the four mu- tants used show three different types of behavior when control and ultra-violet treated conidial sus- pensions are compared: (a) the inositolless and riboflavinless mutants do not grow in the controls, but exhibit a striking response to irradiation, with adaptations resulting in almost all flasks; (b) the pantothenicless mutant shows no adaptations in any flasks, whereas (c) the tryptophaneless mutant adapts in all flasks. It should be noted that in gen- eral the adaptations in the flasks of treated conidia appeared sooner than those from non-treated coni- dia; further, the adaptations also appeared earlier in flasks with the greater amount of inoculum. INVESTIGATIONS WITH THE INOSITOLLESS MUTANT (37401).—Since the inositolless mutant (87401) GILES AND LEDERBERG—-MUTANTS IN NEUROSPORA CRASSA Taste lL. Effect of ultra-violet irradiation of a conidial suspension of multiple mutant 2G37a in inducing adaptations at four loci. Control (no irradiation) Irradiated Number of Number of Mutant locus Number flasks Number flasks being tested of showing of showing for adaptation flasks adaptation flasks adaptation Inositoless . (37401) 15 0 15 15 Pantothenicless (5531) 15 0 15 0 Tryptophaneless (10575) 15 15 15 15 Riboflavinless (51602) 4 0 4 3 showed the most marked effect of the treatment and is easy to manipulate experimentally, particular attention was focussed on this mutant in subsequent experiments. Effects of ultra-violet irradiation on adaptation of the inositolless mutant.—The results of some additional experiments with ultra-violet on the ino- sitolless locus are shown in table 2. The effect of the irradiation in increasing the frequency of adapta- tions (inositol-independent strains) is obvious. In all controls to date, involving experiments with ultra-violet as well as other treatments, no adapta- tions of the inositolless mutant with a wild-type growth habit have been obtained in media lacking inositol. In exp. 13, in which irradiated conidia were dispersed into agar plates without inositol, it is possible to obtain an idea of the frequency of in- duced adaptations per conidium by counting the number of colonies which appear. The average num- ber of colonies per plate was approximately sixteen ; test platings on limiting inositol indicated control conidial viability of about 100 per cent, and sur- vival rate after ultra-violet irradiation of 60 per cent. Thus the frequency of adaptations is approxi- mately 1 per 130,000 surviving conidia. Because of the normally spreading growth habit of Neurospora, colony counts are not easily made. Consequently, it was decided to use a colonial mu- tant (YM2-9.2), obtained in an experiment with nitrogen mustard, combined with inositolless to facilitate such counts. With this mutant the effect of increasing doses of ultra-violet on the frequency of adaptations was also investigated (in experi- ments performed in the Biology Division, Clinton National Laboratory, Oak Ridge, Tenn.) The mu- tant stock used was colonial—albino-2—pantothe- nicless—inositolless. After treatment. platings were made into minimal agar supplemented with Ca-pan- tothenate. The results, shown in table 3, demon- strate that the frequency of adaptations increases with the ultra-violet dose. The fact that the macro- conidia are multinucleate and also difficult to sepa- rate completely in conidial suspensions, makes the AMERICAN JOURNAL OF BOTANY [Vol 35. TasLe 2. Effect of ultra-violet irradiation of conidial suspensions in inducing adaptations of the inos®olless mutant (87401). (40 ml. of liquid minimal plus necessary supplements in each flask; 15 ml. supplemented minimal agar (1.8%) per plate.) Control Irradiated No. of conidia No. flasks No. flasks Experiment added per flask (or plates) No. showing (or plates) No. showing number (or plate) tested adaptation tested adaptation 108 3.9 x 106 4 0 6 6 16” 3.3 X 105 10 0 10 10 13* 2.2 X 106 20 (plates) 0 20 (plates) 20 * Mutant stock used = #G37a (15300 — 37401 — 5531 — 10575 — 51602). * Mutant stock used = inositolless (37401) —- methionineless (4894) — a. determination from these data of survival and muta- tion frequencies in terms of individual inositolless nuclei very difficult. Consequently, it is not yet pos- sible to state whether the adaptation frequency in- creases directly with dose, or whether some other relationship holds, as the present data suggest. Attempts are being made to obtain more informa- tion on this point by utilizing a stock (Y-8743; Tatum, Barratt, and Garnjobst, unpublished) in which inositolless is combined with a double mutant which has a colonial type of growth and produces microconidia. In recent plating experiments with certain stocks of 37401 (in which greater dilutions of conidial suspensions have been used) small. colonies have appeared on minimal agar in both treated and con- trol plates. At least some of these appear to be inositol-independent when tested on fresh media and all apparently retain their colonial growth habit on either minimal or inositol-supplemented media. The problem of the origin of these unexpected types is being investigated further. Proof that the induced adaptations result from genetic reversions at the inositolless locus.—It re- mains to be determined whether the adaptations with wild-type growth habit induced by ultra-violet are non-genetic in nature or actually represent re- verse mutations of the inositolless gene. Crosses of adapted cultures were made to inositolless strains of the opposite mating type. Random ascospore iso- lations from F, gave both inositolless and inositol- independent cultures, but simple 1:1 ratios were not obtained, the inositolless nuclei being considerably in excess in most instances in which macroconidia were irradiated. Such results indicated that the adapted cultures were probably heterocaryons (Beadle and Coonradt, 1944) with inositolless nu- clei predominant. This situation might well be ex- pected, since the multinucleate condition of the treated macroconidia makes it probable that any induced change to inositol-independence, if nuclear in character, will affect only one of the nuclei, re- sulting in a mixture of nuclear types. However, if such is the case, random spore isolations fail to give adequate data on the ratio of the two nuclear types. nor do they furnish a direct demonstration of seg- regation ratios. To demonstrate segregation ratios directly, all the spores of an individual ascus must be iselated and the resulting progeny tested. Fur- ther, since present evidence (Grant, 1945) indicates that ordinarily all the asci in a single perithecium are derived from the descendants of a single pair of haploid nuelei, it is therefore necessary to ana- lyze a complete single ascus per perithecium from several perithecia to determine the ratio of nuclear types present in a heterocaryon. Table + gives the results of such an analysis with an adapted strain, R-3, crossed to inositolless (37401). The regular 1:1 ratios of inositolless and inositol- independent cultures obtained from asci III, VII. and VIII indicate that inositol-independence in the adapted strain is due to a chromosomal and not a non-Mendelian cytoplasmic factor. Further, the ra- tio of whole asci yielding only inositolless cultures (22) to those yielding both types (8), indicate that the adapted strain R-3 is a heterocaryon with a ratio of inositolless:inositol-independent nuclei of approximately 3:1. Segregation of the other charac- Tanre 3. Frequency of induced adaptations of inositoliess (37401) in relation to ultra-violet dosage. Platings of colonial (YM2-9.2)—albino-2—pantothenic less-inositolless into minimal agar supplemented with Ca-pantothenate. Ultra-violet No. of macro- Per cent Total No. of adaptations irradiation time conidia tested macroconidial number of per 106 surviving (in seconds) after treatment survival adaptations macroconidia 0 48.0 x 106 100 0 0.0 15 45.6 X 106 53.8 216 8.8 30 60.0 X 106 53.8 584 18.3 45° 224 XK 106 4.5 63 63.0 60 134.4 X 106 2.0 376 139.4 * Data for 43 sec. treatment obtained in an independent experiment. Mar.. » = 2 oo -independent) strain, B-3 (derived from ultra-violet irradiation of multiple ed from a cross of an adapted (inositol Tanus 4. Characteristics of Fy cultures obtain er from whole asci, one per perithecium, tolless, 87401-a. Aseospores isolated in ord. mutant, 15300-5531-87401-A) with inosi VIT VII? Vip Vv Ascus number IV Wil II no. Spore al inos inos al+ al+ al+ al al inos pant al inos pan al+ al+ ali inos pan inos pan inos pan inos pan inost pant al+ inos+ pan+ al+ inos pant al+ inos pant alt inos pant al inos pant alt inos pant al+ inos+ als inos pan inos pan inos pan 2 inus+ alt al+ al+ inos inos inos al inos al inos+ pan® al al inos pan inos pan inos pan inos pan inos pan- GILES AND LEDERBERG—~MUTANTS IN NEUROSPORA CRASSA inost+ al+ al+ inos+ pant al inos pant al al al al ali al+ inos+ al inos inos inos al+ al pan inos al al inos pan inos pan al inos+ al inos alt inos inos pant al inos pant al 6 inos al inos pant al+ inos pant alt inos pant al al+ al+ inos pan pan inos alt al+ ines pan inos pan al+ inos pan- al+ 7 8 inost+ al inos+ al “Spore did not germinate. » Pantothenic requirement not determined in resulting spores. 8 30 OD ae and inositol-independent cultures inositolless cultures g both inositolless i yielding only i yieldin Number ase Total whole asei analyzed—one per perithecium (including those in order) Number asc ters, albino and pantothenicless, are as would be expected from a cross of the indicated parentage, and show that these genes are present and un- changed in both the inositolless and inositol-inde- pendent nuclei of the heterocaryon. Finally, it is necessary to demonstrate that the inositol-independence of the adapted strain is due Tass 5. Origin and number of single-ascospore cultures from crosses of three inositol-independent Fy cultures with wild-type. All cultures were found to be inositol- independent. No. No. of spores . x ofasci isolated at Cross of wild-type analyzed random R-3.23 (al+ inos+ pan)..... 15300-a 10 128 R-3.21 (al inost pan)...... 15300-a 0 167 R-1l.l (alt inos+ pan)..... 15300-A 0 84 to an induced reversion of the original inositolless gene, and not to a mutation at some other locus. If another locus has mutated to give inositol-indepen- dence, crossing an extracted F, adapted strain to the original wild-type strain should yield a recom- bination class which is inositolless, whereas if the adapted strain is actually a reverse mutation at the original inositolless locus, only wild-type cultures are expected from such a cross. In table 5 are tabu- lated the ascospores isolated from whole asci or at random from crosses involving three different inosi- tol-independent cultures—obtained by single asco- spore isolations from the F, of crosses of adapted strains R-1 (from ultra-violet irradiation of strain 5531-87401-A) and R-8 with inositolless. When the cultures produced from these ascospores were tested, they all proved to be inositol-independent. As in previous crosses, regular expected segrega- tions for other genetic markers present, such as albino and pantothenicless, were observed. Since no recombinations were found in any of the cultures tested, it follows that the genes involved are prob- ably alleles. Physiological studies with induced reversions of inositolless—Growth rate comparisons were made of the reverted inositol-independent strains and wild-type (table 6). The procedure was essentially that described by Ryan, Beadle and Tatum (1943). The use of unwashed Difco Noble (3 per cent) agar probably accounts for the somewhat lower average growth rates obtained. In exp. 1 the growth rates of the original adapted and F, extracted inositol- independent strains, R-1 and R-1.1, were essen- tially similar to those of wild-type both with or without added inositol. This is further evidence that a reverse mutation to the normal wild-type allele of inositolless had been induced by the ultra- violet treatment. Even though R-3, as demonstrated previously (table +), is genetically a heterocaryon, AMERICAN JOURNAL OF BOTANY [Vol]. 35, Tare 6. Comparison of growth rates (mm./hr. at 25°C.). Exp. I Exp. 2 Minimal agar supplemented with: A B Cc dD. E 2.07Ca asin A, 2,07Ca asin C, as in D, panto- plus 4.07 panto- plus 16y plus 0.5% Strain Origin thenate/ml. — inositol /ml. thenate/ml. inositol/ml. yeast extract I-A wild type 3.7" 3.7 3.7 i 3.7 15300-5531- al pan inos 37401 (parental) 0.0 3.5 0.0 3.8 3.6 R-1 U.V. induced 4.0 reversions of R-3 inositolless 3.5 3.7 3.3 3.7 3.4 R-L.1 extracted F, 3.5 7 . . . R-3.21 cultures 2.1 21 2.0 2.2 1.8 R-3.23 2.4 24 “Each rate given represents the average of measurements in two growth tubes. including a high proportion of the inositolless nu- clei, yet essentially wild-type growth rate is ob- tained both on minimal and inositol-supplemented media. The growth rates obtained with strains R-3,21 and R-3,23 were much less than those for wild-type. At first it was thought that these slow-growing types might represent new alleles at the inositol locus— controlling inositol synthesis, but in amounts less than those required for optimal growth. The evi- dence from the B part of exp. 1 as well as from exp. 2 rules out this hypothesis, however, since the addition of inositol, even in amounts well above those required for maximal growth of the inositol- less strain, has no effect on the reverted strains. It has further been shown that the restricted growth rate of R-3.21 is not due to diffusible growth factors other than inositol, since in part E of exp. 2, the addition of 0.5 per cent yeast extract has no stimu- latory effect. The problem of the relation of these slow-grow- Taner 7. Characteristics of cultures derived from random single ascospore isolations of a cross of the F, slow- growing inositol-independent (reverted) strain R-3.21 with inositolless (15300-37 401-5581), Growth rate (in mm. /hr.)” Inositol Spore no. requirement? 24 24 2.3 2.3 2.2 3.9 3.8 4.2 3.6 3.9 WW es “tO Or oe P+] btt++ own 1 * +. indicates inositol-independence, — inositol required. "One growth tube measurement on minimal 39% agar plus 4yCa-pantothenate and S8yinositol/ml. at 25°C. ing reversions to the inositol locus still remained. It was necessary to determine whether the slow-growth character segregated in a regular Mendelian fashion and, if so, what was the relation of this character to the inositol locus. In crosses of both R-3.21 and R-3.23 to strains with wild-type growth rates, ap- proximately 1:1 segregations for slow and normal growth rates were obtained. In addition, back- crosses of strain R-3.21 to the parental inositolless stock (15300-87401-5531) were made and sepre- gations for inositol-dependence and growth-rate type studied (table 7). Here also the slow-growth rate character shows a regular Mendelian segrega- tion, indicating that a single major gene is responsi- ble. Further, although most of the resulting cultures have the same character combinations as the par- ental types, it is possible to obtain inositol-inde- pendent strains with normal growth rates, as in spore No. 7. The present data thus suggest that a single gene, s, is responsible for the characteristic growth rate of R-3.21, and that this gene may be linked with the inositol locus, but can be separated from it by crossing over. There are at least two possibilities as to the origin of gene s. It may have been induced by the ultra-violet treatment at the same time as the inositolless reversion in strain R-3, where its effect would not be immediately detectable because of the heterocaryotic nature of this strain, or the gene may have been present in one of the wild-type stocks used in crossing, where its pres- ence would again be obscured if wild-type nuclei were also present. Spontaneous reversions of inositolless.—Since re- verse mutations occurred only in the treated conidia in all previous experiments, the question naturally arose whether such mutations ever oceur sponta- neously. Consequently, experiments were set up utilizing larger pepulations of inositolless nuclei over longer periods of time than when conidial sus- pensions were used to test this point. Five series of flasks containing 30 ml. each of minimal medium Mar., 1948] plus 2.0 y Ca-pantothenate/ml. were supplemented with increasing amounts of inositol to permit in- creasing mycelial growth and thus larger popula- tions of inositolless nuclei. The highest concentra- tion of inositol used, 1.2y/ml., still gives much less than maximal growth, and thus any adaptations can be readily detected. The flasks were inoculated with the inositolless stock 15800—5531-37401-a; twenty flasks of each series were kept at 25°C, for 30 days, and ten at 30°C. for 57 days. The results of these experiments are shown in table 8. It is evident that spontaneous adaptations do occur, but at a very low rate. Genetic tests of the type discussed previously indicate that these adaptations also result from reverse mutations of the inositolless gene. The results of these experiments are of further interest in showing that the frequency of sponta- neous reverse mutations increases with inercased inositol concentration (and consequently with in- creasing populations of inositolless nuclei, as would be expected). This evidence suggests that selection against reverse-mutations to inositol-independence in the presence of increasing amounts of inositol (and thus increasing populations of inositolless nu- clei) does not occur. Thus the behavior of inositol- less reversions is different from that found in leu- cine reversions (Ryan and Lederberg, 1946), where such negative selection does occur. Further evidence against negative selection is obtained from observa- tions with adapted cultures which are heterocaryons containing both inositolless and reverted nuclei. ‘The nuclear ratios in such heterocarvons, as obtained by crossing tests, do not seem to be appreciably modi- fied when they are grown on inositol-supplemented as compared with minimal media. It thus appears that when a mutation to inositol-independence oc- curs it will be regularly selected for in the absence of inositol and an adaptation will result. This mu- tant should thus provide favorable material for a study of mutation rates and for a comparison of the effectiveness of various mutagenic agents, especially when uninucleate conidia are used. Effects of mutagenic agents other than ultra- violet in inducing reversions of inositolless.—Pre- liminary tests have been made of the effects of three agents in addition to ultra-violet in inducing rever- sion of the inositolless gene in macroconidia. These results are given in table 9. In the x-ray experiments separate conidial sus- pensions of multiple mutant stock #G87a were ex- posed to 25,000 and 50,000r respectively using a tube operating at 50 KV and 25 ma with a tungsten target at an intensity of 1200r/min. For the nitrogen mustard experiments (Auerbach et al., 1947; Tatum, 1946) conidia were exposed to the indicated concentrations of tris (8-chloroethyl) amine hydrochloride in phosphate buffer at pH 6.2, centrifuged and resuspended in Neurospora liquid minimal twice and then transferred to the test flasks of supplemented minimal medium. The radiophosphorus used was the separated iso- tope, p32 (half-life:14.3d. 8-radiation:1.69 Mev, GILES AND LEDERBERG-——_MUTANTS IN NEUROSPORA CRASSA obtained from the Clinton Laboratories at Oak Ridge). The concentration (activity) of the radio- phosphorus solution when obtained was approxi- mately 0.58 millicuries/ml. Appropriate amounts of this solution were added directly to suspensions of conidia in minimal medium to give the final test concentrations of radiophosphorus indicated. The conidia remained in the radiophosphorus solutions throughout the course of the experiment. Additional control experiments in which inositol was added to conidial suspensions in flasks containing radiophos- phorus concentrations as high as 0.29 me./ml. indi- cated that normal growth occurs and is maintained for a considerable period even in the presence of continuous radiation of the intensity indicated. At least one adapted culture preduced by cach of these types of treatment has been tested to show that an actual genetic reversion is involved. These experiments thus demonstrate that reversions at the inositolless locus can be induced by other mu- tagens—hoth chemical and physical. Although none of the agents tested was as effective under the indi- cated experimental conditions as ultra-violet radia- tion, it is not vet possible to make adequate quanti- tative comparisons of the relative mutagenic cffi- ciencies of the various treatments. INVESTIGATIONS WITIE MUTANTS OTHER INosITOLLEss.—Attention has been centered princi- pally on the behavior of the inositolless mutant (87401), but data have been obtained for adapta- tions of several other mutants as well. In table 1. the results indicate no effect of ultra-violet treat- ment on pantothenicless (5581). This mutant ap- pears to be extremely stable, as no adaptations have been obtained either spontaneously or with any of the several treatments tried. This stability of the pantothenicless mutant may represent a case of an extremely low reverse mutation rate, or. perhaps more likely, may result from a chromosomal dele- tion—a genetic change which is presumably irre- versible. The riboflavinless mutant (51602) appears to behave more like inositolless in response to ultra- violet and other treatments. It is more difficult to THAN Taste 8 Numbers of spontaneous reversions of the inositolless mutant (37401) at different levels of inositel concentration, Mutant strain used: 15300- 5981-87 401-a, 30 ml. of liquid minimal medium plus 2.0y/ml. of Ca-pantothenate/flask, Figures in paren- theses indicate days after start of experiment on which reversions were first detected. 25°C 30°C.” + inositol No. of No. of No. of No. of per ml. flasks reversions flasks reversions 0.00 20 0 10 0 0.15 20 0 10 0 0,30 20 0 10 0 0.60 20 1 (16) 10 2 (24,41) 1.20 20 3 (22,24,25) 10 1 (17) 4 Experiment maintained for 30 days. » Experiment maintained for 57 days. AMERICAN JOURNAL OF BOTANY [Vol. 35, TaBle 9, Effect of various treatments of conidial suspensions on reversion of the inositelless mutant (37401). Mul- tiple mutant stock £G37a used 40 ml. liquid minimal plus necessary supplements per flask. 30°C. Control Treated Duration No. of conidia = Approx. No, of No.of re- No.of No.ofre- of exp. Treatment added per flask % killed flasks versions flasks versions in days X-ray 25,000r 3.3 X 106 60 6 I 10 9 21 50,000r 85 15 3 Nitrogen mustard 0.001% 10 min. 3.7 X 106 15 5 0 5 0 39 0.01% 30 min. 1.5 X 106 68 10 0 10 4 30 Radiophosphorus 5.8 10-5me./ml. 10 1 5.8X10-4me./ml. 7.2% 106 15 2 25 10 9 5.8 10-3me. /m). 10 3 work with, however, because it is a temperature- sensitive mutant (Mitchell and Houlahan, 1946) and requires more precisely controlled conditions for its expression. One of the ultra-violet induced riboflavin adaptations has been shown to be a re- verse mutation by the appropriate genetic tests. The tryptophane-requiring mutant (10575) adapts as frequently without treatment as with, both in ultra-violet and other experiments. Some tests have been made to determine whether this regular adap- tation is genetic. From crosses of three spontane- ously adapted cultures with the original trypto- phaneless, only tryptophaneless progeny have been recovered in 154 ascospore isolations made at ran- dom. From a similar cross involving an ultra-violet treated adapted culture, three tryptophane-inde- pendent strains have been recovered from a total of thirty-eight random ascospore isolations. These results suggest that the spontaneous adaptations are non-genetic in nature, whereas those adaptations re- sulting after ultra-violet treatment may represent a combination of genetic and non-genetic effects. Other multiple mutant strains have been utilized in similar preliminary experiments te determine the effect of irradiation on adaptation of additional bio- chemical mutants (table 10). In the two instances cited, the treatment greatly increases the freqnency of adaptations, though these also occur spontaneous- Iy, but appear much later than in the flasks of treated conidia. Genetic analyses have not yet been made of these adapted strains, but the evidence obtained in the previously described experiments with other mutants, strongly suggests that they rep- resent reverse mutations. SUMMARY Studies have been made of the effects of various mutagenic agents in inducing adaptations of a num- ber of biochemical mutants in Neurospora crassa. The frequency of adaptation of certain mutants, such as inositolless (Stanford mutant number 37401), cholineless (34486), methionineless (4894), and riboflavinless (51602) can be greatly increased by ultra-violet radiation; for other mutants, such as pantothenicless (5531), no adaptations have been produced by any treatment yet attempted. Genetic and physiological tests, particularly with the inosi- tolless mutant, indicate that these induced adapta- tions represent reverse mutations to the original wild-type allele. X-rays, radiophosphorus, and ni- trogen mustard have also proved effective in induc- ing reversions of inositolless. The frequency of re- verse mutation has been demonstrated to increase with increasing radiation dose (for ultra-violet treatment) for the inositolless mutant in experi- ments utilizing a plating colony-count technique with a colonial-inositolless mutant, Ossorx Boranican J.anporarory. YArLe UNIVERSITY, New Haven. Connecriccr Tarte 10. Effect of ultra-violet irradiation of conidial suspensions in inducing adaptations of two biochemical mu- tants. In all experiments four flasks received 0.5 ml. of conidial suspensions, three, 0.2 ml.. and three, 0.05 ml. Figures in parentheses indicate days after start of experiment when adaptations were first detected, Other ex- perimental conditions as described for exp, 12 (table 1), Control (no irradiation) Irradiated Original conidial Mutant locus being concentra- No. of No, of No. of No. of tested tion /ml. flasks adaptations flasks adaptations* Cholineless (34486) ............0.... 45.0 X 106 19 1(9) 10 9 Methionineless (4894) ............... 26.6 X 106 10 2(10) 10 8 4 All adaptations in irradiated flasks appeared within 4 days after treatment. Mar. 1943] EMERSON AND CANTINO—-BLASTO CLADIA LITERATURE CITED Avernaci, C., J. M. Rosson, anp J. G. Carr. 1947. Chemical production of mutations. Science 105: 243- Q47. Beanie, G. W. 1944. Neurospora and its use in bioassays. Chem. 156; 683-689. 1946. Biochemical genetics. 15-96. , anp L. V. Coonranr. 1944, Heterocaryosis in Neurospora crassa. Genetics 29; 291-308. ,anp E. L. Tarom. 1941. Genetic control of bio- chemical reactions in Neurospora. Proc. National Acad. Sci. (U.S.) 27: 499-506. , AND — 1945. Neurospora. II. Methods of producing and detecting mutations concerned with nutritional requirements. Amer. Jour. Bot. 32: 678- 686. An inositolless mutant strain of Jour, Biol. Chem. Rev. 37: Grant, H. Neurospora crassa. ford University. Mircuewt, H. K., ann M. B. Hounanax. 1946. Newro- spora. IV. A temperature-sensitive riboflavinless mutant. Amer. Jour, Bot. 33:31-35. Ryan, F. J. G. W. Beane, anp FE. L. Tatum. 1943. ‘The tube method of measuring the growth rate of Neurospora. Amer. Jour. Bot. 30: 784-799. , AND Josiiva LEDERBERG. 1946. Reverse mutation and adaptation in leucineless Neurospora. Proc. National Acad. Sci. (U.S.) 32: 163-173. 1946. Back-mutation and adaptation of nutri- tional mutants. Cold Spring Harbor Symp. Quant. Biol. 11: 215-227. Tarum, E. L. Experiments discussed in Bonner, D. M. 1946. Biochemical mutations in Neurospora. Cold Spring Harbor Symp. Quant. Biol. 11: 14-24. 1945. A genetic analysis of the life evcle of Unpublished M.A. Thesis, Stan-