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Mosaic analysis of autonomy of spore development in Neurospora
EXPEmMENTALMYCOLOGY
1, 253-258 (1977)
BRIEF NOTES Mosaic Analysis of Autonomy of Spore Development in Neurospora THOMAS E. JOHNSON *
Department of Genetics, University of Washington, Seattle, Washington 98195, and Genetics, Development & Physiology, Cornell University, Ithaca, New York 14853 Received June 6, 1977; revised August 1, 1977
JOHNSON, T. E. 1977. Mosaic analysis of autonomy of spore development in Neurospora. Experimental Mycology 1, 253-258. Genetically mosaic rosettes of asei have been constructed by using as the female parent heterokaryons containing nuclei of two distinct genotypes. One nucleus contains the ascospore-autonomous color mutant, per-1 (peritheeial-1). The other nucleus contains the ascus-autonomous dominant mutant tl (round spore). The per-1 mutation is used to unambiguously determine the genotype of each ascus within the mature rosette of asei. Most rosettes of asei originate exclusively from either the per-1 R + nucleus or the per-I + R nucleus. When both types occur within the same peritbeeium, individual asei are either one type or the other and asei of the same type are usually contiguous. INDEX DESG1RIPTORS: Neurospora crassa; spore development; mosaics; ascus; aseospore; peritheeium; mutant; rosette.
The existence of cell autonomous mutants in higher eukaryotes has permitted extensive clonal analysis of development using genetic mosaics. The per-1 locus of Neurospora crassa (Shear and Dodge) has been shown to be completely autonomous in the aseospore (Howe and Johnson, 1976) and at least partially autonomous in the outer peritheeial wall (Johnson, 1976). The color of the aseospore is determined completely by the genotype of the spore. Several other loci in Neurospora including asco ( Stadler, 1956), ts (Nakamura, 1961), cys-3 (Murray, 1965), and ws (Phillips and Srb, 1967) Current address: Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309. Address reprint requests to Cornell address. 253 Copyright © 1977 by AcademicPress, Inc. All rights of reproduction in any form reserved.
also show ascospore autonomous expression of color. Many examples of autonomous ascospore color mutants can be found in other related fungi including Sordaria (Heslot, 1958) and Podospora (Pieard, 1971). However, all mutants which alter spore development in Neurospora are not spore autonomous. Round spore, R (Mitchell, 1966), is a dominant mutant which causes all eight spores within a single aseus to be round instead of spindle-shaped, as is typical of wild-type, Other mutants such as peak (Pk-1) and indurated (I) also affect all of the spores within a particular aseus regardless of the genotype of the individual spore.
254
BRIEF NOTES
This phenomenon (all the spores within an ascus being of the same phenotype independently of their genotype) has been explained by assuming that: (1) the mutation is dominant, and (2) the phenotype is determined during the brief diplophase, or shortly thereafter, while effective cytoplasmic communication between wild-type and mutant nuclei within an ascus is still possible. At least two other alternative explanations exist: (1) the phenotype could be one which is determined by the protoperithecial (female) or more unlikely, the fertilizing (male) parent in a cross; and (2) the phenotype could be determined by both parental nuclei but another tissue --for instance, the paraphysal tissue as suggested by Barry (1972)--could control the phenotype of the ascospores. The model of zygote dominance predicts that the phenotype of an ascus should be determined solely by the genotype of the nuclei within that ascus, i.e., the phenotype should be ascus autonomous. The latter two models predict that under certain conditions the phenotype of an ascus need not correspond to the genotype of the nuclei within that ascus. A situation in which the two models make different predictions can be constructed by using mixtures of nuclei of two different genotypes as one component of a cross. If R per-1 + nuclei and 1t+ per-1 nuclei are mixed in a heterokaryon which functions as the protoperithecial parent in a cross, the perithecia that are produced are mosaic 1 (Johnson, 1976). Usually all the asci within a rosette of one of the perithecia are of the same genotype; however, on rare occasions rosettes result which must be derived from two (or more) nuclei from the female (Sansome, 1947; Rad-
ford and Threlkeld, 1970; Johnson, 1976). These will be called mosaic rosettes. Mosaic rosettes have also been generated using mixtures of conidia of two different genotypes 2 (Nakamura and Egashira, 1961) as the male parent. Mosaic rosettes can be used to directly distinguish the two types of models. The former model (zygote determination of ascospore shape) predicts a complete correspondence of phenotype within each ascus with the genotype of the nuclei within an ascus. The alternative models predict frequent noncorrespondence of phenotype and genotype. Since per-1 is spore autonomous, the genotype of any mature ascus in these mosaic rosettes can be immediately determined by visual examination of the rosette; these asci are also examined for the round spore (R) phenotype. The spatial distribution of the two types of asei within the rosette is also presented. This analysis allows me to estimate the amount of nuclear migration which occurs in the ascogonium during the premeiotic mitoses. Traditional cytological studies suggest that the rosette arises from a number of dikaryotic cells which undergo crozier formation and meiosis (Fincham and Day, 1971). The results of the clonal analysis are interpreted in relation to this model. Mutant genes used in this study included per-1 (PBJ-1) and per-1 (ABI-1) (Howe and Johnson, 1976), ad-2 (STL2), pyr-3 (KS43), inl (89601), col-4 (70007c), and R (Mitchell, 1966); tl is female sterile when used as a homokaryon but this sterility can be overcome if an R ÷ nucleus is also present in heterokaryotie condition (Johnson, 1976). Two heterokaryons were used in these studies: (per-1 (ABI-1) pyr-3 ÷ ad-2 R ÷A) + (per-1 ÷ pyr-3 ad-2 ÷ R A) (abbre-
1 Johnson, T. E. 1975. Perithecial development and pattern formation in Neurospora crassa. Ph.D. thesis, University of Washington, Seattle, Wash.
2 Grant, H. 1945. A genetic analysis of the life cycle of Neurospora crassa. M.A. thesis, Stanford University, Stanford, Calif.
BRIEF NOTES
255
Fro. 1. Rosettes of ascospores from the heterokaryon (per pyr q-ad R)A crossed with per+ col-4 a. (30×). (A) All mature asci contain eight round dark spores. (B) All mature asci contain four spindle-shaped light spores and four spindle-shaped dark spores. (C, D) Some asci contain eight round dark spores and others four light spindle-shaped spores and four dark spindle-shaped spores. viated
per ad + pyr R); and (per-1 (PB]-I) pyr-3 ad-2 + R ÷ A ) + (per-1 ÷ pyr-3 + ad-2 R A ) (abbreviated per pyr + ad R ) . T h e heterokaryons were formed as
previously described (Johnson, 1976) and crossed with per-1 ÷ col-4 R ÷a. After 12 days at 25°C, individual perithecia were picked from the plate and fixed in Carnoy's fixa-
rive (60% ethanol, 30% chloroform, 10% acetic acid) for a minimum of 5 days. T h e contents of individual perithecia were squeezed into a drop of water and examined under a coverglass using a compound microscope. Photographs of wet mounts of all mosaic rosettes which remained intact were taken. These photo-
256
BRIEF NOTES
FIG. 2. Some asei derived from mosaic rosettes. "tt" indicates an aseus containing eight round dark spores (from an ascogonial nucleus of constitution per+ R), and "q-'" indicates an ascus containing four light spindle-shaped spores and four dark spindle-shaped spores (from an ascogonial nucleus of genotype per R+). The two oval spores in the former ascus are often seen in homokaryotic crosses of R (Fig. 1A) and are clearly distinguishable from the spindleshaped spores in R÷ crosses. graphs were used to substantiate the visual observations. A u t o n o m y of spore shape. Most of the perithecia contained rosettes in which the asei were of uniform phenotype. These rosettes were composed either of asci which contained eight round black spores (Fig. IA) or four spindle-shaped light spores and four spindle-shaped pigmented spores (Fig. 1B). In both types of rosettes, there can be seen some additional asci in which all the spores are light; these rosettes arise as a result of immaturity of the aseospores. Since per-1 has been shown to be completely spore autonomous for pigmentation of the ascospores, this gene can be used as a phenotypie marker to determine whether the per-1 + R or the per-1 R + nucleus has served as the protoperithecial component of the meiotic products which are represented within a single ascus. Thus, asci which contain all black spores must have arisen from the per-l + R component while
those asci which segregate four light and four black spores must have arisen from the per-1 R + component. In addition to rosettes containing only one type of ascus, there are a few rosettes which contain some asci with eight pigmented spores and some asci with four light and four pigmented spores (Figs. 1C and 1D). If the shape of the spore is controlled not by the diploid nucleus within the ascus, but rather by nuclei in some other perithecial tissue, we would expect that the shapes of the spores in the asci of these mosaic rosettes would not always correspond to those found in homokaryotic crosses, as illustrated in Figs. 1A and B. However, there is complete correspondence of spore shape with the shape that would be predicted by the model of aseus autonomous control of spore shape (Fig.' 2). Analysis of pattern. It is also apparent that the two distinct types of asci (those with 4:4 patterns versus those with 8:0)
BRIEF NOTES tend to be clustered together within distinct regions of the rosette (Figs. 1C and 1D). In an unselected sample of 25 rosettes which r e m a i n e d intact during examination and photography, 16 clearly showed complete clumping of all asci with similar phenotypes within distinct regions of the rosette. Seven of the remaining eight were also compatible with this finding although one or two asci of one genotype were mixed in with the group of asci of the second genotype; this p r o b a b l y was an artifact induced by the fact that the rosettes are three-dimensional structures which are squashed u n d e r a coverslip for examination. H o w e v e r , two rosettes contained m o r e than two distinct clusters of genetically different asci; these must b e real and m a y p r e s u m a b l y arise from instances where two distinct maternal nuclei of the same genot y p e give rise to the rosette. T h e spatial distribution of the two distinct ascus types within the rosette suggest that for the most part each mosaic rosette arises from the fusion of t w o female nuclear components ( a n d two m a l e nuclei). If this were not true and instead several different maternal nuclei w e n t into the m a k e - u p of each mosaic rosette, we would not expect to observe the tendency for clumping of asci with similar phenotypes. These observations also suggest that extensive cell a n d / o r nuclear m o v e m e n t does not occur within the ascogonial tissue from which these rosettes are derived. Since the p h e n o t y p e of asci within these mosaic rosettes is determined completely b y the genotype of the individual nuclear components which go into making the zygotic nucleus of the ascus, I conclude t h a t R is an ascus autonomous mutant. T h e r e is no evidence from the examination of these asci for the function of the R gene in any perithecial tissue outside of the ascus containing this gene. R has also b e e n successfully used as an autonomous ascus marker (Turner, 1977) to follow the break-
257
down of a R / R + heterozygous duplication. It would be interesting to see if several other mutants which affect spore and aseus m o r p h o l o g y in N e u r o s p o r a (Srb et al., 1973) are also ascus autonomous. ACKNOWLEDGMENTS I would like to thank Adrian Srb, Mary Basl, and David Perkins for discussions concerning this work, and Debi Ferguson for help with the manuscript. This work was supported by USPHS Predoctoral Fellowship GM-47532 and USPHS Training Grant GM-01035. REFERENCES BARRY, E. G. 1972. Meiotic chromosome behavior of an inverted insertional translocation in Neurospora. Genetics 71: 53-62. FINCHAM, J. R. S., AND DAY, P. R. 1971. Fungal Genetics, pp. 2-8. Blackwell Scientific, Oxford/ Edinburgh. HESLOT, H. 1958. Contribution a l'6tude cytog6n6tique et g6n6tique des Sordariacdes. Rev. Cytol. Biol. Veg. 19 (Suppl. 2): 1-209. HowE, H. B., JR., AND JOHNSON, T. E. 1976. Phenotypic diversity among alleles at the per-1 locus of Neurospora crassa. Genetics 82: 595603. Join
MtraRAY, N. E. 1965. Cystine mutant strains of Neurospora. Genetics 52: 801-808. NAKAMURA,K. 1961. An ascospore color mutant of Neurospora crassa. Bot. Mag. (Tokyo) 74: 104109. NAKAMURA, K., AND EGASHIRA, T. 1961. Genetically mixed perithecia in Neurospora. Nature (London) 190: 1129-1130. PHILLIPS, R. L., AND SBB, A. M. 1967. A new white ascospore mutant of Neurospora crassa. Canad. J. Genet. Cytol. 9: 766-775. PICARD, M. 1971. Genetic evidence for a polycistronic unit of transcription in the complex locus "14" in Podospora anserina, I: Genetic and complementation maps. Mol. Gen. Genet. 111: 35--50. RADFORD, A., AND THRELKELD, 8. F. H. 1970.
Genetically mixed perithecia and somatic re-
258
BRIEF NOTES
combination in a pseudo-wild type strain of Neurospora crassa. Y. Genet. Cytol. 12: 547-552. SANSOME, E. R. 1949. The use of heterokaryons to determine the origin of the ascogeneous nuclei in Neurospora crassa. Genetics ( The Hague ) 24: 59--64. SRB, A. M., NASRALLAH,J. B., AND BASL, M. 1973. Genetic control of the development of the sex-
ual reproductive apparatus of Neurospora (from Basic Mechanisms in Plant Morphogenesis). Brookhaven Syrup. Biol. 25: 40-50. STADLEn, D. R. 1956. A map of linkage group VI of Neurospora crassa. Genetics 41: 528--543. TUnN~R, B. C. 1977. Euploid derivatives of duplications from a translocation in Neurospora. Genetics 85: 439-460.
1, 253-258 (1977)
BRIEF NOTES Mosaic Analysis of Autonomy of Spore Development in Neurospora THOMAS E. JOHNSON *
Department of Genetics, University of Washington, Seattle, Washington 98195, and Genetics, Development & Physiology, Cornell University, Ithaca, New York 14853 Received June 6, 1977; revised August 1, 1977
JOHNSON, T. E. 1977. Mosaic analysis of autonomy of spore development in Neurospora. Experimental Mycology 1, 253-258. Genetically mosaic rosettes of asei have been constructed by using as the female parent heterokaryons containing nuclei of two distinct genotypes. One nucleus contains the ascospore-autonomous color mutant, per-1 (peritheeial-1). The other nucleus contains the ascus-autonomous dominant mutant tl (round spore). The per-1 mutation is used to unambiguously determine the genotype of each ascus within the mature rosette of asei. Most rosettes of asei originate exclusively from either the per-1 R + nucleus or the per-I + R nucleus. When both types occur within the same peritbeeium, individual asei are either one type or the other and asei of the same type are usually contiguous. INDEX DESG1RIPTORS: Neurospora crassa; spore development; mosaics; ascus; aseospore; peritheeium; mutant; rosette.
The existence of cell autonomous mutants in higher eukaryotes has permitted extensive clonal analysis of development using genetic mosaics. The per-1 locus of Neurospora crassa (Shear and Dodge) has been shown to be completely autonomous in the aseospore (Howe and Johnson, 1976) and at least partially autonomous in the outer peritheeial wall (Johnson, 1976). The color of the aseospore is determined completely by the genotype of the spore. Several other loci in Neurospora including asco ( Stadler, 1956), ts (Nakamura, 1961), cys-3 (Murray, 1965), and ws (Phillips and Srb, 1967) Current address: Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309. Address reprint requests to Cornell address. 253 Copyright © 1977 by AcademicPress, Inc. All rights of reproduction in any form reserved.
also show ascospore autonomous expression of color. Many examples of autonomous ascospore color mutants can be found in other related fungi including Sordaria (Heslot, 1958) and Podospora (Pieard, 1971). However, all mutants which alter spore development in Neurospora are not spore autonomous. Round spore, R (Mitchell, 1966), is a dominant mutant which causes all eight spores within a single aseus to be round instead of spindle-shaped, as is typical of wild-type, Other mutants such as peak (Pk-1) and indurated (I) also affect all of the spores within a particular aseus regardless of the genotype of the individual spore.
254
BRIEF NOTES
This phenomenon (all the spores within an ascus being of the same phenotype independently of their genotype) has been explained by assuming that: (1) the mutation is dominant, and (2) the phenotype is determined during the brief diplophase, or shortly thereafter, while effective cytoplasmic communication between wild-type and mutant nuclei within an ascus is still possible. At least two other alternative explanations exist: (1) the phenotype could be one which is determined by the protoperithecial (female) or more unlikely, the fertilizing (male) parent in a cross; and (2) the phenotype could be determined by both parental nuclei but another tissue --for instance, the paraphysal tissue as suggested by Barry (1972)--could control the phenotype of the ascospores. The model of zygote dominance predicts that the phenotype of an ascus should be determined solely by the genotype of the nuclei within that ascus, i.e., the phenotype should be ascus autonomous. The latter two models predict that under certain conditions the phenotype of an ascus need not correspond to the genotype of the nuclei within that ascus. A situation in which the two models make different predictions can be constructed by using mixtures of nuclei of two different genotypes as one component of a cross. If R per-1 + nuclei and 1t+ per-1 nuclei are mixed in a heterokaryon which functions as the protoperithecial parent in a cross, the perithecia that are produced are mosaic 1 (Johnson, 1976). Usually all the asci within a rosette of one of the perithecia are of the same genotype; however, on rare occasions rosettes result which must be derived from two (or more) nuclei from the female (Sansome, 1947; Rad-
ford and Threlkeld, 1970; Johnson, 1976). These will be called mosaic rosettes. Mosaic rosettes have also been generated using mixtures of conidia of two different genotypes 2 (Nakamura and Egashira, 1961) as the male parent. Mosaic rosettes can be used to directly distinguish the two types of models. The former model (zygote determination of ascospore shape) predicts a complete correspondence of phenotype within each ascus with the genotype of the nuclei within an ascus. The alternative models predict frequent noncorrespondence of phenotype and genotype. Since per-1 is spore autonomous, the genotype of any mature ascus in these mosaic rosettes can be immediately determined by visual examination of the rosette; these asci are also examined for the round spore (R) phenotype. The spatial distribution of the two types of asei within the rosette is also presented. This analysis allows me to estimate the amount of nuclear migration which occurs in the ascogonium during the premeiotic mitoses. Traditional cytological studies suggest that the rosette arises from a number of dikaryotic cells which undergo crozier formation and meiosis (Fincham and Day, 1971). The results of the clonal analysis are interpreted in relation to this model. Mutant genes used in this study included per-1 (PBJ-1) and per-1 (ABI-1) (Howe and Johnson, 1976), ad-2 (STL2), pyr-3 (KS43), inl (89601), col-4 (70007c), and R (Mitchell, 1966); tl is female sterile when used as a homokaryon but this sterility can be overcome if an R ÷ nucleus is also present in heterokaryotie condition (Johnson, 1976). Two heterokaryons were used in these studies: (per-1 (ABI-1) pyr-3 ÷ ad-2 R ÷A) + (per-1 ÷ pyr-3 ad-2 ÷ R A) (abbre-
1 Johnson, T. E. 1975. Perithecial development and pattern formation in Neurospora crassa. Ph.D. thesis, University of Washington, Seattle, Wash.
2 Grant, H. 1945. A genetic analysis of the life cycle of Neurospora crassa. M.A. thesis, Stanford University, Stanford, Calif.
BRIEF NOTES
255
Fro. 1. Rosettes of ascospores from the heterokaryon (per pyr q-ad R)A crossed with per+ col-4 a. (30×). (A) All mature asci contain eight round dark spores. (B) All mature asci contain four spindle-shaped light spores and four spindle-shaped dark spores. (C, D) Some asci contain eight round dark spores and others four light spindle-shaped spores and four dark spindle-shaped spores. viated
per ad + pyr R); and (per-1 (PB]-I) pyr-3 ad-2 + R ÷ A ) + (per-1 ÷ pyr-3 + ad-2 R A ) (abbreviated per pyr + ad R ) . T h e heterokaryons were formed as
previously described (Johnson, 1976) and crossed with per-1 ÷ col-4 R ÷a. After 12 days at 25°C, individual perithecia were picked from the plate and fixed in Carnoy's fixa-
rive (60% ethanol, 30% chloroform, 10% acetic acid) for a minimum of 5 days. T h e contents of individual perithecia were squeezed into a drop of water and examined under a coverglass using a compound microscope. Photographs of wet mounts of all mosaic rosettes which remained intact were taken. These photo-
256
BRIEF NOTES
FIG. 2. Some asei derived from mosaic rosettes. "tt" indicates an aseus containing eight round dark spores (from an ascogonial nucleus of constitution per+ R), and "q-'" indicates an ascus containing four light spindle-shaped spores and four dark spindle-shaped spores (from an ascogonial nucleus of genotype per R+). The two oval spores in the former ascus are often seen in homokaryotic crosses of R (Fig. 1A) and are clearly distinguishable from the spindleshaped spores in R÷ crosses. graphs were used to substantiate the visual observations. A u t o n o m y of spore shape. Most of the perithecia contained rosettes in which the asei were of uniform phenotype. These rosettes were composed either of asci which contained eight round black spores (Fig. IA) or four spindle-shaped light spores and four spindle-shaped pigmented spores (Fig. 1B). In both types of rosettes, there can be seen some additional asci in which all the spores are light; these rosettes arise as a result of immaturity of the aseospores. Since per-1 has been shown to be completely spore autonomous for pigmentation of the ascospores, this gene can be used as a phenotypie marker to determine whether the per-1 + R or the per-1 R + nucleus has served as the protoperithecial component of the meiotic products which are represented within a single ascus. Thus, asci which contain all black spores must have arisen from the per-l + R component while
those asci which segregate four light and four black spores must have arisen from the per-1 R + component. In addition to rosettes containing only one type of ascus, there are a few rosettes which contain some asci with eight pigmented spores and some asci with four light and four pigmented spores (Figs. 1C and 1D). If the shape of the spore is controlled not by the diploid nucleus within the ascus, but rather by nuclei in some other perithecial tissue, we would expect that the shapes of the spores in the asci of these mosaic rosettes would not always correspond to those found in homokaryotic crosses, as illustrated in Figs. 1A and B. However, there is complete correspondence of spore shape with the shape that would be predicted by the model of aseus autonomous control of spore shape (Fig.' 2). Analysis of pattern. It is also apparent that the two distinct types of asci (those with 4:4 patterns versus those with 8:0)
BRIEF NOTES tend to be clustered together within distinct regions of the rosette (Figs. 1C and 1D). In an unselected sample of 25 rosettes which r e m a i n e d intact during examination and photography, 16 clearly showed complete clumping of all asci with similar phenotypes within distinct regions of the rosette. Seven of the remaining eight were also compatible with this finding although one or two asci of one genotype were mixed in with the group of asci of the second genotype; this p r o b a b l y was an artifact induced by the fact that the rosettes are three-dimensional structures which are squashed u n d e r a coverslip for examination. H o w e v e r , two rosettes contained m o r e than two distinct clusters of genetically different asci; these must b e real and m a y p r e s u m a b l y arise from instances where two distinct maternal nuclei of the same genot y p e give rise to the rosette. T h e spatial distribution of the two distinct ascus types within the rosette suggest that for the most part each mosaic rosette arises from the fusion of t w o female nuclear components ( a n d two m a l e nuclei). If this were not true and instead several different maternal nuclei w e n t into the m a k e - u p of each mosaic rosette, we would not expect to observe the tendency for clumping of asci with similar phenotypes. These observations also suggest that extensive cell a n d / o r nuclear m o v e m e n t does not occur within the ascogonial tissue from which these rosettes are derived. Since the p h e n o t y p e of asci within these mosaic rosettes is determined completely b y the genotype of the individual nuclear components which go into making the zygotic nucleus of the ascus, I conclude t h a t R is an ascus autonomous mutant. T h e r e is no evidence from the examination of these asci for the function of the R gene in any perithecial tissue outside of the ascus containing this gene. R has also b e e n successfully used as an autonomous ascus marker (Turner, 1977) to follow the break-
257
down of a R / R + heterozygous duplication. It would be interesting to see if several other mutants which affect spore and aseus m o r p h o l o g y in N e u r o s p o r a (Srb et al., 1973) are also ascus autonomous. ACKNOWLEDGMENTS I would like to thank Adrian Srb, Mary Basl, and David Perkins for discussions concerning this work, and Debi Ferguson for help with the manuscript. This work was supported by USPHS Predoctoral Fellowship GM-47532 and USPHS Training Grant GM-01035. REFERENCES BARRY, E. G. 1972. Meiotic chromosome behavior of an inverted insertional translocation in Neurospora. Genetics 71: 53-62. FINCHAM, J. R. S., AND DAY, P. R. 1971. Fungal Genetics, pp. 2-8. Blackwell Scientific, Oxford/ Edinburgh. HESLOT, H. 1958. Contribution a l'6tude cytog6n6tique et g6n6tique des Sordariacdes. Rev. Cytol. Biol. Veg. 19 (Suppl. 2): 1-209. HowE, H. B., JR., AND JOHNSON, T. E. 1976. Phenotypic diversity among alleles at the per-1 locus of Neurospora crassa. Genetics 82: 595603. Join
MtraRAY, N. E. 1965. Cystine mutant strains of Neurospora. Genetics 52: 801-808. NAKAMURA,K. 1961. An ascospore color mutant of Neurospora crassa. Bot. Mag. (Tokyo) 74: 104109. NAKAMURA, K., AND EGASHIRA, T. 1961. Genetically mixed perithecia in Neurospora. Nature (London) 190: 1129-1130. PHILLIPS, R. L., AND SBB, A. M. 1967. A new white ascospore mutant of Neurospora crassa. Canad. J. Genet. Cytol. 9: 766-775. PICARD, M. 1971. Genetic evidence for a polycistronic unit of transcription in the complex locus "14" in Podospora anserina, I: Genetic and complementation maps. Mol. Gen. Genet. 111: 35--50. RADFORD, A., AND THRELKELD, 8. F. H. 1970.
Genetically mixed perithecia and somatic re-
258
BRIEF NOTES
combination in a pseudo-wild type strain of Neurospora crassa. Y. Genet. Cytol. 12: 547-552. SANSOME, E. R. 1949. The use of heterokaryons to determine the origin of the ascogeneous nuclei in Neurospora crassa. Genetics ( The Hague ) 24: 59--64. SRB, A. M., NASRALLAH,J. B., AND BASL, M. 1973. Genetic control of the development of the sex-
ual reproductive apparatus of Neurospora (from Basic Mechanisms in Plant Morphogenesis). Brookhaven Syrup. Biol. 25: 40-50. STADLEn, D. R. 1956. A map of linkage group VI of Neurospora crassa. Genetics 41: 528--543. TUnN~R, B. C. 1977. Euploid derivatives of duplications from a translocation in Neurospora. Genetics 85: 439-460.