Copyright 0 1992 by the Genetics Society of America

Perspectives Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove

Neurospora: The Organism Behind the Molecular Revolution David D. Perkins Department of Biological Sciences, Stanford University, Stanford, Calgornia 94305-5020

NDER the title “Fifty Years Ago: The Neuro(1 991) has celespora Revolution,” HOROWITZ brated an anniversary of the epochal 1941 paper of BEADLE and TATUM, which reported the first mutants with biochemically defined nutritional requirements. HOROWITZ’S account and others (HOROWITZ1973, 1990) have focused on the 1985, 1990; LEDERBERC people who were involved, the genesis of their ideas, and therole of the 1941 results in transforming biology. The present essay will be concerned mainly with the research organism that was so important to the success of the initial experiments.Neurospora possesses a combination of features that made it an ideal choice not only for accomplishing the original objectives set by BEADLEand TATUM but also for a continuing succession of contributions,including many in areas that transgress the bounds of biochemical genetics and molecular biology. 1 shall begin by outlining the story of Neurospora prior to BEADLE and TATUM and then go on to sketch its subsequent history. The previous accounts have stressed biochemical genetics and molecular biology. I shall consider other aspects as well, focusing first on genetics, continuing with a summary of research accomplishments of all sorts, and concluding with a consideration of the potential usefulness of Neurospora for population studies. The vegetative phase of Neurospora was described and used for experiments by French microbiologists nearly 100 years before BEADLE and TATUM (PAYEN 1843; MONTAGNE1843). In thewarm, humid summer of 1842, bread from bakeries in Paris was spoiled by massive growth of an orangemold. A commission was set up by the minister of war to investigate the cause of the infestation and to make recommendations. T h e commission’s report (PAYEN1843) includes a colored plate whichshows colonies, mycelia, conidiophores and conidia of the “champignons rougesdu pain.” An experiment in photobiology is described. Colonies

U

Genetics 1 3 0 687-701 (April, 1992)

grown in the light quickly became brightorange. Colonies grown in the dark,however, remained white for more than8 days, but thewhite colonies developed orange pigment within 2 hr when they were brought into thelight. Thermal tolerance was also studied (see PAYEN1848, 1859). These results were cited by PASTEUR (1 862)in reporting his own experiments on the survival of mold spores, which helped to refute theories of spontaneous generation. The next experimental study of Neurospora began in Indonesia during Dutch colonial times. In marketplaces of East Java, bright orangecakes are displayed. These consist of Neurospora grown on soybean or peanut solids from whichoil and proteinforcurd have been pressed. The Javanese inoculate the solids with conidia to create an appetizing and highly nutritious food called oncham, which has a mushroom-like taste(WENT 1901a; SHURTLEFF and AOYACI1979; H o 1986). Producing oncham is a cottage industry which has probably gone on for centuries and which continues today. A Dutch plant physiologist, F. A. F. C. WENT,was stationed at the famous Buitzenjorg (now Bogor) Botanic Gardens in Java at the turn of the century. WENT was attracted by theorangeonchamfungusand started experimenting with it. He was frustrated because humidity in Java is sa great that the organism grew through the cotton plugs of his culture tubes. WENT (1904) also foundNeurospora in Surinam, where he noted that the fungus was used to process cassava meal in preparation of an indigenousalcoholic beverage. Back home in Utrecht, he described the onchamfungus and its culture (WENT 1901a) and used it for a series of studies on the effects of various substrates on enzymes such as trehalase, invertase and tyrosinase (WENT 1901b). WENT(1 904)also studied the effect of light on carotenoid production. With knowledge of WENT’Swork, PRINGSHEIM (1909) included Neurosporain a study of oxidases, and KUNKEL

688

D.

D.

Perkins

(1 9 13, 19 14)used it in studies of chemical toxicity. All these observations were made using the vegetative phase of the organism and the asexually produced powdery conidia (vegetative spores). The association of Neurospora with heat and fire must have been known from the earliest times. We nowknow that the sexually produced heat-tolerant ascospores remain dormant until exposed toheat. Heat activation of ascospores explains the occurrence of Neurosporaboth in bakery infestations and on burned vegetation. Numerousrecordsgoing back over a century describe large orange areas following volcanic eruptions in tropical areas. In New Guinea, tribesmen traditionally set hillsides on fire to flush game, and Neurospora bloomed fobowing the burns. In Brazil, MOLLER(190 1) described an orangefungus growing on burned vegetation (and on maize bread). A typically ascomycete sexual phase appeared in his cultures, and the sexual fruiting bodies (perithecia) and ascospores were later identified as Neurospora. In Japan, Neurospora made a dramatic appearance following thegreatTokyoearthquakeandfire of 1923. Within a few days, burned and scorched trees became festooned with orange. Mycologistsin two laboratories cultured the organism. KITAZIMA(1 925) observed perithecia in his cultures, and going back to the source, discovered that perithecia were present underthe bark of trees in theTemple of Shiba. Orange progeny were obtainedfrom single ascospores. TOKUGAWA and EMOTO(1924) studied survival of the fungus following exposure to moist and dryheat,andidentifiedtheorangepigment as a carotenoid. Neurospora is commonly seen following agriculturalburning in warm, moist climates. Sugarcane appears to be an ideal substrate. Ascospores are no doubt activated by burning in the fields and by heating in the mill. Bales of bagasse (fiber from which the sap has been pressed) become orange. In Australia,solids from refinery filters are spread on fields as fertilizer. Largeorange colonies develop on this filtermud. Honey bees can be seen visiting the colonies and filling their pollen baskets with the brightly colored conidia (SHAWand ROBERTSON 1980). The modern history of Neurospora begins in the mid-1920s with materialfromsugarcane bagasse. DODGE.Like BEADLE, T h e key person was BERNARD DODGEhad grown upon a farm. He worked for years asa school teacher and managed to complete his bachelor’s degree only at age 39. He published his first paper at the age of 40 and was already past 50 when he began to work with Neurospora (ROBBINS 1962). Prior to the Neurospora work, DODGEwas the first to discover heat activation of ascospores (1912) and to describe mating types in ascomycetes (1 920), both in Ascobolus.

About the time KITAZIMAwas examining his orange a colleague of fungus in Japan, CHARLESTHOM, DODGE’S at the Department of Agriculture mycology and pathology laboratory in Arlington, Virginia, was studying cultures of orange mold from sugar cane was of the opinion that bagasse in Louisiana. THOM theorange fungus, then called Moniliasitophila, lacked a sexual stage. However, C. L. SHEAR, the head of the laboratory, found perithecia in one of THOM’S plates. The material was given to DODGEfor analysis. T h e success of DODGE’S experimental crosses kindled his enthusiasm, and Neurospora became hismain lifelong interest. DODGE’SfirstNeurosporapaper, with SHEARin 1927, goes far beyond the conventional taxonomic descriptions of genus and species. Cultures of the orange fungus had been obtained from many sources. Isolates were assigned to the new genus Neurospora on the basisof their grooved ascospores. (Prior to 1927, thevegetative stage hadsuccessively been called Oidiumaurantiacum,Penicilliumsitophilum and Moniliasitophila.) DODGEshowed that the cultures included three species which were set off fromone another by their crossing behavior. Hybrid perithecia from crosses between different species developed slowly and were unproductive or poorly fertile. Although a conventional morphologically based taxonomic species description was provided for each species, crossing behavior was implicitly taken into consideration and used to assign strains to the designated species. This innovation contrasted with the purely morphological criteria then used by mycologists and clearly anticipated the idea of biological species long before the concept was formalized. Two species with eight-spored asci, Neurospora crassa and Neurosporasitophila, were shown to be heterothallic: individual haploid cultures from single ascospores were unable to enter the sexual cycle. They fell into two mating types, defined because crosses could occur only between strains of opposite mating t YPea DODGEcarried out the first tetrad analysis with N . crassa, showing that the mating types segregated 4:4 in individual asci. The asci were obtained as groups of eight ascospores thathad been spontaneously ejected from the perithecia. The Neurospora ascospores were activated by heat, as inAscobolus. In contrast to the eight-spored species, isolates of Neurosporatetrasperma, with four-spored asci, appeared to be homothallic. Culturesfrom single ascospores were usually self-fertile. A few self-sterile progeny were produced, however, that behaved as though they were heterothallic. DODGE(1927) was shortly to describe the cytological basis ofthis “pseudohomothallic” behavior of N . tetrasperma, showing that individual ascospores were usually (but not always) heterokar-

Perspectives

yons that contained haploid nuclei of opposite mating types. DODGElost no time in communicating his enthusia s m . A t Columbia University, he urged T. H. MORGAN and the Drosophila group to use Neurospora. He traveled to Cornell for a seminar. Among the graduate students in the audience were GEORGE BEADLE and BARBARA MCCLINTOCK. BEADLE( 1 966) later recalled how thestudents, familiar with then-recent results of E. G. ANDERSON using Drosophila attachedX half-tetrads, were able to point out to DODGEhow the second-division segregations he described in Neurospora could be explained by crossing over between chromatids at the four-strand stage. DODGEsoon moved to a job as plant pathologist at the New York Botanical Garden. In addition to his official duties, he managed to continue experiments with Neurospora. These included pioneeringwork on interspecies crosses and on mutations affecting ascus development. He was intrigued by heterokaryons and obtained combinations of strains that showed heterokaryotic vigor (DODGE1942). In the next 30 years he published nearly 50 papers on the genetics, cytology, morphology and life cycle of Neurospora. DODGE’S enthusiasm resulted indirectly in the recruitment of CARLLINDEGREN,who did the most significant genetic workwith Neurospora priorto moved to BEADLE and TATUM. In 1928, LINDEGREN California from Wisconsin, where he had obtained a Master’s degree in Plant Pathology. He visited T . H. MORGANto inquire about continuing graduate work at the California Institute of Technology, whereMORGAN had come with his Drosophila group to head the newBiologyDivision. LINDEGRENfound MORGAN using dissecting needles in an attempt to isolate Neurospora ascospores from an agarplate (see LINDEGREN 1973). MORGANsuggested that LINDEGREN work with Neurospora. Following a visit to DODGE,LINDEGREN chose the species N. crassa as best suited for genetic work. He developed highly fertile wild-type strains,

689

(:;\HI. (;. 1,lSl>b:GREN

identified mutants that could be used as markers and discovered the first linkages. T h e linked genes provided confirming proof thatcrossing over occurred at the four-strand stage. Genetic maps were constructed for two linkage groups using matingtype,centromeres, and morphological mutants. But at about the time BEADLEand TATUM were turning from Drosophila to Neurospora,LINDEGRENabandonedNeurospora to begin work on Saccharomyces. His last Neurosporapaper (LINDEGRENandLINDEGREN 1942), written with his life-long collaborator GERTRUDE LINDEGREN, was submitted just as the Stanford workers were about to obtain their first biochemical mutant. LINDEGREN’S Neurospora papers are listed in BACHMANN and STRICKLAND ( 1 965). Neurospora was used for several other investigations during the decade before 194 1 . I first heard of it in a plant physiology course taught by DAVIDGODDARD, who used N. tetrasperma in studies of ascospore activation and dormancy(GODDARD 1935,1939). BUTLER,ROBBINS and DODGE(1941) demonstrated that biotin was the sole growth factor requirement. In England, WHITEHOUSE ( 1 942) subjected LINDEGREN’S tetraddatato a detailed analysis and went on to produce hisown five-point map of the mating-type chromosome of N. sitophila. I t was DODGEand LINDEGREN, however, who developed the genetics of Neurospora duringthe 1930s and made the organism known to geneticists. The accumulated information enabled DODGE( 1 939) to assert: “The fungi in their reproduction and inheritance follow exactly the same laws that govern these activities in higher plants and animals.” Additional information regarding early history can be found in the introductory sections of SHEAR and DODGE(1 927), MOREAU-FROMENT (1956), and PERKINS and BARRY(1977), and in essays by RYANand OLIVE( 1 96 l ) , TATUM ( 1 96 1 ), LEDERBERG (1 990). SRB ( 1 973), CATCHESIDE (1 973) and LINDEGREN ( 1 973).

690

D. D. Perkins

”

I hree genetics textbooks published in 1939 (STURand BEADLE,SINNOTT and DUNN, AND WADDINGTON) included accounts of Neurospora in the context of recombination and sex determination, with diagrams showing therelation of crossing over to second division segregation in the linear ascus. In February 194 1 , BEADLEwrote to DODGEregarding stocks. His letter begins “Dr. Tatum and I are interested in doing some work on thenutrition of Neurospora with the eventual aim of determining whethertherequirements might be dependent on genetic constitution.” Eight months later, their paper reporting success in obtaining nutritional mutantswas submitted to theProceedings of the National Academy of Science. Obtaining the first biochemical mutants and proposing the one-geneone-enzyme hypothesis were only two of many advances in which Neurospora played a pioneering role. T h e problems for which it has since been used are extremely diverse, often ranging far afield from the biochemical genetics that first made it famous. For example, Neurospora soon made fundamental contributions to understanding themechanism of recombination. It was also used to resolve the great confusion existing at that time about fungal chromosomes and their behavior in meiosis. In 1944, BARBARAMCCLINTOCK visited Stanford University at BEADLE’Sinvitation; KELLER (1983, pp. 1 13-1 18) describes the visit. MCCLINTOCK’S long experience with maize enabled her toshow convincingly that the chromosomes of Neurospora and their behavior in the ascus were typically eukaryotic. Using simple light microscopy, she went far beyond the original objective of determiningthechromosome number. She outlined the details of meiosis and described the seven chromosomes. T h e smallest Neurospora chromosomes are now known each to have a 1 C DNA content less than that of Escherichia coli. She showed that they were nevertheless individually recognizable by their distinctive morphology at pachytene (MCCLINTOCK 1945; for photographs comparing Neurospora and maize pachytene chromosomes see Figure 6 in PERKINS 1979). She went on to describe pachytene pairing in a translocation heterozygote and to record the ascus types that resulted from different modes of segregation when the translocation was heterozygous. At the end of her two-month stay in California there was no longer any question: it was clear that fungal chromosome cytology, like fungal genetics, is basically similar to thatof plants and animals. The 1941 paper of BEADLE and TATUM opened up exciting possibilities justat atime when war was divertingfundsfrom pure to appliedresearch and when young scientists were moving either intoapplied research or intothe military. BEADLE’Ssuccessin keeping his group intact and in obtainingsupport TEVANT

BARBARA MCCI.IN.I.OCK

testifies to his confidence in the importance of the research and his persuasiveness as to its value. (There was thenno National Science Foundation andno program of external research support by the National Institutes of Health. BEADLE turned to the Rockefeller Foundation andthe NutritionFoundation,andto pharmaceutical firms. See BEADLE1974; KAY 1989.) Noteveryone was persuaded.HOROWITZ (1979) describes how some geneticists continued to resist the idea that individual enzymes were specified by single genes. And BEADLE(1974) recalls a wartime visit by THOM. After being shown the mycologist, CHARLES some of the striking morphological mutants that were known to segregate as single-gene differences, THOM took BEADLEaside and advised him “What you need is a good mycologist. Those cultures you call mutants are not mutants at all. They arecontaminants!” At the war’s end in 1945, the Neurospora work had progressed substantially and was widely known. (When I returned to Columbia University from the army, DOBZHANSKY told me that thetwo highlights in biology duringthe war had been HUXLEY’S book Evolution the Modern Synthesis and BEADLE and TATUM’S Neurospora mutants.) Students were attracted to Neurospora. So also were established scientists who had previously been working on other organisms: D. EMERSON,NORMANGILES, G. CATCHESIDE, STERLING HERSCHEL MITCHELL,FRANCISRYAN and MOGENS WESTERGAARD. During this period, Neurospora also provided the first introduction to research for numerous individuals who were later to become known for their work with other organisms. Among those whose careers began in this way were EDWARD ADELBERG, BRUCEAMES,AUGUSTDOERMANN, NAOMIFRANKLIN, LEONARD HERZENBERG, DAVID HOGNESS,BRUCE HOLLOWAY, ESTHERLEDERBERG, JOSHUA LEDERBERG, NOREEN MURRAY, NORORU SUEOKAandCHARLES

Perspectives YANOFSKY; see, for example, RYAN and LEDERBERG (1 946). The Neurospora approach was soon extended to other fungi such as Ophiostoma and Ustilago. GUIDO PONTECORVO, who had previously worked on Drosophila with H. J. MULLER, began his program with Aspergillus nidulans. Genetic work flourished on Podospora, Sordaria, Ascobolus, Coprinus and Schizophyllum. Biochemical mutants were obtained in Schizosaccharomyces, Chlamydomonas and even in a flowering plant, Arabidopsis (LANGRIDGE 1955). Application of the Neurospora approach to bacteria was not long delayed. Auxotrophic mutants of E. coli were obtained byCHARLES GRAY(a Stanford under(1 944), and independently by graduate) and TATUM ROEPKE,LIBBYand SMALL( 1 944). These made possible the 1946 demonstration of recombination in E. coli and opened the way for theexplosive development of bacterial genetics. Saccharomyces was a relatively slow starter. Heterothallism with two mating types was discovered by CARL andGERTRUDE LINDEGREN only in 1943. T h e first induced auxotrophic mutationsand thefirst linkages were reported in 1949. Eleven workers attended the first yeast conference in 196 1 (VON BORSTEL 1963), compared to 92 participants at a Neurospora conferenceheld the same year (DE SERRES1962). (Attendance at international yeast meetings now exceeds 1 OOO!) Advantageous features of Neurospora that were recognized as novel and noteworthy in the 1940s are now largely taken for granted because the same features are shared in various combinations by many other organisms that have since come into common use. Neurospora differed in important ways from the animals and plants used by most geneticists in 194 1 . It was haploid. All four productsof individual meioses could be recovered, and in such a way that centromeres were readily mapped. Heterokaryons could be formed. Nutritional requirements were defined and simple. Stocks could be preserved in suspended animation, effectively conferring immortality on individual strains. In addition, growth was rapid, generation time short andfecundity high. Propagules suitable for plating were producedabundantly.Purecultures could readily be obtained and tested for auxotrophic mutations. Neurospora ascospores are large enough to permit manual isolation without a micromanipulator. Workers were initially intrigued by the ability to map centromeres, and geneticanalysis was mostly done atfirst by laboriously dissecting the spores from linearasci in serial order. With time, it was realized that ordered ascus analysis is rarely necessary and that for most purposes random ascospores provide the needed information with far less effort (see PERKINS1953).

69 1

When entire asci are needed for such purposes as studying interference, obtaining double mutants, or identifying chromosome rearrangements,it was found that unordered asci, shotfromtheperithecium as octets, can easily beobtained in largenumbers (STRICKLAND 1960). Addition of a centromere marker made these unordered groups essentially as informative as intact asci (e.g., PERKINS et al. 1986). Neurospora conidia are a boon fortransferring, plating, transforming, preservingstocks and sampling wild populations. These powdery vegetative spores are potentially hazardous as airborne contaminants, however. Laboratory practices were quickly developed that minimized the risk. It was found that if simple precautions are taken, there is no reason why Neurosporacannot coexist in the same laboratory with bacteria, yeast or slowly growingmicroorganisms. The rapid linear growth of Neurospora (which can exceed 4 mm/hr) is a great advantage for many purposes, butfor platings it was necessary to develop appropriate media containing colonializing agents such as sorbose (TATUM, BARRATT and CUTTER1949) or touse genetic variantswith restricted growth, such as the conditional colonial mutant cot-1. Reliable and economic methods were developed for maintaining permanentstocks in suspended animation in silica gel, by lyophilization, or by freezing. These methods (WILSON1986) enable the Fungal Genetics Stock Center ( 1 990) to carry over 7000 Neurospora strains, with no need for periodicserial transfers. Along with successes, Neurospora workers inevitably experienced frustrations and disappointments. It was initially hoped that new mutations might reveal previously undiscovered essential metabolites, but none were found. (A prospective new amino acid, tentatively named neurosporin, proved to be a crystalline mixture of isoleucine, valine and leucine; see KAY 1989). An elegant scheme to use heterokaryons for quantitative studies of dominance (BEADLEand 1944) proved impractical because many COONRADT laboratory stocks were heterokaryon incompatible, but this finding opened up the study of vegetative incompatibility and led to the finding that genes responsible for this incompatibility are numerous and are highly polymorphic in natural populations. Recombinant DNA research with Neurospora was initially impeded by regulatory guidelines that first denied permission to proceed, then required that a disabling mutant be built into recipient strains. After permission was granted, it was found that genes introduced by transformation were poorly recovered from crosses, although they remained stably integrated in the chromosomes during vegetative growth. The poor sexual transmission proved to be due to RIP (repeatinduced point mutation), a process that mutates du-

692

D. D. Perkins

plicate genes during the sexual phase (see below and SELKER 1990). RIP was then shown to provide an effective means of achieving targeted gene mutation, an asset which more than compensated for the inconvenience of poor transmission. Inevitably, other organisms sometimes proved to be superior to Neurospora for particular purposes. For example, bioassays using inducedauxotrophs were first developed in Neurospora during the war years, b u t bacteria proved to be so much faster for bioassay that Neurospora was not used to any extent. Beginning in the 195Os, Neurospora played a central role in studies of recombination, providing the first proof of gene conversion (MITCHELL1955) and revealing its main features (see below). Targeted regulation oflocal recombinationfrequencies by rec genes thatare unlinked or nonadjacent was discovered in Neurospora (CATCHESIDE,~ESSUP and SMITH1964). Random ascospores of Neurospora continue to be the main source of information on recombination control of this type. Other fungi proved to be superior for recombination studies that required tetrads,however. Conversion frequencies were found to be much higher in yeast and Ascobolus than in Neurospora, while Sordaria and Ascobolus both had the advantage of numerous viable, readily scorable, autonomously expressed ascospore mutants.Neurospora was still a major source of information on gene conversion in the early 1960s when molecular models for eukaryotic conversion and crossing over were proposed by HOLLIDAY and by WHITEHOUSE and HASTINGS. However, by the mid-I970s, when the more detailed Aviemore model was put forward by MESELSONand RADDING, the most extensive and most critical data came from asci of Saccharomyces, Sordaria and Ascobolus. As a result of this trend,one geneticist whose interests focused almost exclusively on recombination models asked me bluntly in 1984 what I had been doingsince the demise of Neurospora! In fact, the change of emphasis away from recombination may have beena blessing in disguise for Neurospora genetics. In my own laboratory, it resulted in attentionbeing given toother problems which might otherwise have beenneglected. Chief among these was the study of chromosomerearrangements (see PERKINS1979). Because deficiency ascospores remain unpigmented while nondeficiency spores are black, Neurospora proved ideal for detecting and diagnosing rearrangements. Meiotic mutants were examined cytologically and genetically, together with other mutants affected in development of the sexual phase. I also began to collect and analyze Neurospora from natural populations. This led, among other things, to the discovery of Spore killer elements, which bearastrikingformal resemblance in their behavior to Segregation distorter in Drosophila, the

t-complex of mice, and gamete eliminator in tomato. Far from being defunct, Neurospora continues to be a superb research organism. At the present time, it is used as the primary research object in about 70 laboratories in North America and 25 laboratories in 16 countries abroad. It remains the microorganism of choice for numerous specific problems. The knowledge and the geneticresourcesthat have been acquired during 65 years are invaluable assets. But the most important factor responsible for its wide use is probably an exceptionally happy combination of traits that makes it suitable for research on problems spanning the entire range frommolecules to populations. The versatility of the organism is illustrated by the examples gathered below. Many of the contributions that will be cited were pioneered using Neurospora. Some of the advances were the first for filamentous fungi, others for the fungal kingdom, and others for all eukaryotes. However, the object in citing them is not to stress priority but to illustrate the variety of research areas to which Neurospora has contributed significantly. The list is far from complete. For example, no attempt has been made to cover the extensive work on specific enzymes or pathways, oron novel biochemical mutants;fordocumentation of many of these see PERKINS et al. ( 1 982). Nutritional mutants were usedfor many purposes. Intermediate stepsin biosynthetic pathways were determined. By 1944, at least sevendifferent genes had been identified that were involvedin the synthesisof arginine (SRBand HOROWITZ 1944). [For early work on biosynthesis, see the reviews by HOROWITZ (1950) and by VOCELand BONNER(1959).] In contrast to what is often the situation in bacteria, genes concerned with successivesteps of the samebiosynthetic pathway were shown not to be clustered butto be scattered through the genome (for review see HOROWITZ 1950). An apparent exception, the aro cluster-gene (GROSSand FEIN 1960), proved to make a single protein product with seg(GAERTments that specify five separate enzymatic activities NER and COLE 1977). The first conditional biochemical mutants were identified (STOKES,FOSTERand WOODWARD1943; MITCHELL and HOULAHAN 1946). Temperature-sensitive mutants were used for testing the one-gene one-enzyme hypothesis (HoROWITZ and LEUPOLD1951). Differentalleles at a locus were shown to produce forms of an enzyme with qualitatively different properties (HOROWITZ and FLING1953). Some mutant strains that lackeda specific enzymatic activity were shown to produce a proteinthatcross-reacted dith antibody againstthe enzyme (SUSKIND, YANOFSKYand BONNER 1955). Complementation of allelic mutationswas demonstrated, first between nuclei in heterokaryons (FINCHAM and PATEMAN 1957; GILES,PARTRIDGE and NELSON 1957), then between the protein products in vitro (WOODWARD 1959; for review see FINCHAM 1966). Translational suppression was analyzed for the first time at the molecular level (YANOFSKY1956). Perhaps the best understoodmechanism of metabolicsuppression was de-

Perspectives

693

prepare plasma membrane vesicles and to demonstrate that scribed, involving suppression of arg-2 by pyr-3d and pyr-3a membrane ATPase is a proton pump (SCARBOROUGH 1975; by ~ r g - 1 (DAVIS 2~ 1967; REISSIG, ISSALY and ISSALY1967). for review see SCARBOROUGH 1978). These studies entailed the discovery of two genes for one Kinetic and genetic studies of amino acid transport (by enzyme, one gene for two enzyme activities, and duplicate GABRIELLESTER,DAVIDSTADLER, MARTIN PALL, GIB enzyme activities for two pathways. THWAITES and LAKSHMI PENDYALA) DEBUSK, and WILLIAM Proof was obtained for thechanneling of pathway-specific identified several transport systemswith broad substrate enzymesin separate pools (WILLIAMS,BERNHARDT and specificities, unlike the highly specific "permeases" of bacDAVIS1971). Compartmentation of metabolic pools and teria but resembling the broad-specificity systems of mampathwayswithinvacuoles,cytosol and mitochondria was maliancells,which had been based on kinetic evidence established and studied in detail (WEISS1973) (for reviews alone. Four major amino acid uptake systems were characsee DAVIS1986; DAVISand WEISS1988). terized, distinct from those inyeast (for review see PALL Unlinked genes concerned with the same pathway were 1970). shown to be coordinately controlled (GROSS1965). CrosMaternal transmission was demonstrated for a class of spathway (general) control of amino acid biosynthetic enMITCHELL non-Mendelian respiratory defects (MITCHELL, zymes was discovered (CARSIOTIS and LACY1965; CARSIand TISSI~RES 1953). A non-Mendelian cytochrome defect 1974). Convincing evidence for OTIS,JONES and WESSELINC was transferred between vegetative strains by injecting mipositive control was provided for thefirst time in eukaryotes 1965). tochondria (DIACUMAKOS, GARNJOBST and TATUM in a pioneering study of the regulation of sulfur metabolism Mitochondria were shown to increase in number by division (MARZLUF and METZENBERG1968); sulfur regulation in of preexisting mitochondria rather than being formed de eukaryotes has sincebeen analyzed most fully in Neurospora novo (LUCK1963). DNA was isolated from mitochondria for (for review see FU et al. 1990). A hierarchy of regulatory the first time (LUCKand REICH1964). Progeny were shown elements involved in phosphate metabolism was identified to receive mitochondrial DNA only from the maternal parand, on the basis of genetic evidence, the novel concept was ent, in both interspecific and intraspecific crosses (REICH proposed that regulation is not limited to interactions beand LUCK1966; MANNELLA, PITTENCER and LAMBOWITZ tween regulatory complexes and the DNA sequences of 1979). A cyanide-insensitive alternative oxidase was identisucceeding elements in the regulatory cascade, but that it fied for the first time in fungi (LAMBOWITZ and SLAYMAN also involves direct interaction between the protein products 197 1). of regulatory genes (METZENBERGand CHIA 1979) (for The first sequencing of a nucleic acidfrom mitochondria review see METZENBERG1979). et al. 1978) revealed unique features of initiator (HECKMAN Mutants with a wide spectrum of altered vegetative mortRNA that foreshadowed the discovery of numerous unexand phologies were obtained and analyzed (e.g., GARNJOBST TATUM 1967) and biochemical defects were identified in pected features of mitochondrial genomes (for review see some of them (for review see MISHRA 1977). The morphoBREITENBERGER and RAJBHANDARY 1985). A protein-codlogical mutant crisp-1 (one of LINDEGREN'S first markers) ing gene was shown to be located withinan intronof another mitochondrial gene (BURKE and RAJBHANDARY 1982), exFLAwas shown to lack adenylate cyclase activity(TERENZI, tending a discovery in yeast to the filamentous fungi. SelfWIA and TORRES 1974). (The important regulatory signal splicing of a mitochondrial intron was first demonstrated cyclic AMP is therefore absent and must be dispensable in Neurospora, unlike Saccharomyces.) The process of coni(GARRIGA and LAMBOWITZ 1984) and thefirst mutants were diation was studied in wild type and in mutants (SPRINGER found that are affected in the splicingof mitochondrial and YANOFSKY1989). Genes with greatly elevated expresRNA (MANNELLAet al. 1979). Reverse transcriptase was sion during conidial differentiation, identified by BERLIN first shown to be present in mitochondria (AKINS,KELLEY and YANOFSKY(1985), were usedin studying regulation and LAMBOWITZ 1986; KUIPERand LAMBOWITZ 1988). Ty(e.g., ROBERTSand YANOFSKY1989). rosyl-tRNA synthetase was shown to play an essential role Mutants affected in development of the sexual cyclewere in splicing (AKINSand LAMBOWITZ 1987). examined (for review see RAJU1992b). One of these (RAJU Several key discoveries concern the mechanisms respon1986) appears to be the Neurospora counterpart of the sible for the import into mitochondria of polypeptides that polpitotic mutant in maize, which BEADLEdescribed and are synthesized on cytoplasmic ribosomes. Pools of comstudied early in his career. pleted polypeptides were shown to be present in the cytoElectrodes weresuccessfully inserted into Neurospora ZIMMERMANand NEUPERT 1977). plasm (HALLERMAYER, hyphal cells (SLAYMAN and SLAYMAN 1962). ElectrophysioDifferent mitochondrial receptors were indicated to be relogical studies showed that glucose transport is driven by a sponsible for theimport of different precursor polypeptides transmembrane proton gradient(SLAYMAN 1970; forreview (ZIMMERMAN, HENNIG and NEUPERT1981). The first sesee SLAYMAN 1987); unlike animal cells, the plasma memquence was obtained for the precursor of a nuclear-coded brane potential is maintained primarily by protonflux protein of the mitochondrial inner membrane or matrix rather than by potassium and sodiumfluxes (SLAYMAN (VIEBROCK, PERZ andSEBALD 1982). Contact sites function1965). Other transport systems were shown to be driven by ing in import were demonstrated between inner and outer a proton-cotransport mechanism (SLAYMAN and SLAYMAN mitochondrial membranes (SCHLEYER and NEUPERT 1985). 1974). Mutant strains were obtained that canbe grown A processing protease responsible for cleaving targeting indefinitely as protoplasts, without a cellwall (EMERSON sequences in the mitochondrial matrix was purified (HAW1963; SELITRENNIKOFF, LILLEYand ZUCKER 1981). These LITSCHEK et al. 1988). were used to isolate and characterize plasma membranes, to Mitochondrial plasmids were found, with sequences un-

694

D. D. Perkins

related to those of mitochondrial DNA (COLLINS et al. 1981). Strains were discovered that became senescent following integration of plasmids into themitochondrial DNA (RIECK, GRIFFITHS and BERTRAND 1982; for review see BERTRAND and GRIFFIrHS 1989). Horizontal transfer of mitochondrial plasmids was shown to occur, independently of mitochon1989; GRIFFITHS et al. 1990; drial DNA (MAYand TAYLOR COLLINS and SAVILLE 1990). The first circadian rhythm in fungi was discovered, manifested as periodic conidiation that provided a permanent record as bands were formed along a growth continuum et al. 1959). Mutations were obtained that (PITTENDRIGH affect the free-running period length of the circadian clock (FELDMAN and HOYLE1973). Clock-controlled genes were identified that are transcribed only at specific times in the circadian day (LOROS,DENOMEand DUNLAP1989). For reviews see FELDMAN and DUNLAP (1983), LAKIN-THOMAS, COT$and BRODY (1990) andDUNLAP (1990). Resetting the circadian clock was shown to be mediated by a blue-light photoreceptor (SARGENT and BRICCS1967). Other effectsof blue light were studied, including the induction of carotenogenesis, formation of protoperithecia, and phototropism of perithecial beaks (for review see DEGLIINNWENTI and RUSSO1984). Mutants were identified that 1981). are blind to photoinduction (HARDING and TURNER Heterokaryons (for review see DAVIS 1966) were used, first to study dominance and complementation, then to transfer mitochondria, plasmids and transposable elements, to rescue and maintain lethal or deleterious mutations, to map deficiencies, and to determinemutation frequencies. Use of Neurospora heterokaryons led to thediscovery of vegetative (heterokaryon) incompatibility (for reviewsee PERKINS1988). It was found that the mating type locus functions vegetatively as a heterokaryon incompatibility locus (BEADLE and COONRADT 1944; SANSOME 1945). Other genes controlling the formation of stable heterokaryons (het genes) were identified and mapped (GARNJOBST 1953). Microinjection of incompatible cytoplasm or extracts was shown to be lethal to recipient cells (WILSON, GARNJOBST 1961). Partial diploids heterozygous for a het and TATUM locus were obtained and shown to behighly abnormal (NEWMEYERand TAYLOR 1967; PERKINS1975). An unlinked suppressor was discovered that neutralizes the vegetative incompatibility function of the mating type genes but not their mating function (NEWMEYER1970). Polymorphic het genes were found to be so numerous that they effectively preclude formation of heterokaryons in natural populations of N. crassa (MYLYK1976). The mating type genes A and a were cloned, sequenced, and shown to be present in a single copy per genome, with characteristics quite unlike those of the mating type genes of yeast. Although A and a occupy preciselythe same locus, their DNA sequenceswere found tocontain no recognizable et al. 1988) (for reviews see METZENBERC homology (GLASS and GLASS1990; GLASSand STABEN1990). Mutations had earlier been obtained that inactivate the mating type genes (GRIFFITHS and DELANGE 1978). Genes were identified that are transcribed preferentially during sexual development, and cloned sequences of these mating-specific genes were used to obtain, by RIP and gene disruption, mutant strains in which sexual development is impaired (M. A. NELSON

and R. L. METZENBERG, unpublished results). Attraction of trichogynes to cells of opposite mating type was shown to be mediated by a diffusible mating-type specific pheromone (BISTIS1983). Cytological techniques were perfected and details of meiosis and ascus development and of chromosome morphology and behavior were examined by light microscopy, both in wild typeand in mutants (for reviews see RAJU1980, 1992b). Synaptonemal-complex karyotypes were obtained by reconstructing meiotic prophase nuclei from thin sections (GILLIES1972). Recombination nodules were shown to be correlated with reciprocal crossing over events at pachytene and to exhibit positive interference (GILLIES1972, 1979; BOJKO1989). Synaptic adjustment of the synaptonemal complex was shown to occur in inversion heterozygotes (BOJKO 1990). Neurospora was the first filamentous fungus for which intact DNA molecules from entire individual chromosomes were separated electrophoretically, extending the maximum chromosome length that was then physically resolvable (ORBACH et al. 1988). Tetrad analysis using a long multiply marked chromosome arm showed that meiotic crossing over and interference closely resemble those inDrosophila and Zea mays (PERKINS 1962). The first definitive proof of gene conversion was accomplished in Neurospora (MITCHELL 1955). Important characteristics of conversion were delineated, especially by MARY MITCHELL,MARYCASE,NOREEN MURRAYand DAVID STADLER;see FINCHAM, DAYand RADFORD (1979). Genes were discovered that dramatically control the frequency of recombination at unlinked or nonadjacent target sites (CATCHESIDE, JESSUP and SMITH1964), with recombination reduced by dominant alleles at the controllinp loci (for review see CATCHESIDE 1975). N . crassa was the first fungus to have all linkage groups mapped genetically (BARRATT et al. 1954) and assigned to cytologically distinguished chromosomes (forreview see PERKINS and BARRY1977). Tester strains that incorporate translocations were devised and greatly speeded linkage detection and mapping (PERKINS et al. 1969). Genes at nearly 700 loci and breakpoints of more than 300 rearrangements have been mapped (PERKINS et al. 1982; PERKINS1990; unpublished PERKINS and BARRY 1977; and D. D. PERKINS, results). The conventional maps have been complemented using restriction fragment length polymorphisms (METZENBERG et al. 1984, 1985; METZENBERG and GROTELUESCHEN 1990) and random amplified polymorphic DNA markers (RAPD mapping) (WILLIAMSet al. 1991). Genes specifying 5 s RNA were shown to be dispersed through the genome in single copies(FREE,RICE and METZENBERG 1979; SELKER et al. 1981). Telomeres were cloned and shown to have a DNA sequence identical to that in Homo sapiens (SCHECHTMAN 1987, 1990). Random breaks inribosomal DNA sequences of the nucleolus organizer region were shown to acquire telomere sequences de novo (BUTLER 1991). Following MCCLINTOCK (1945), chromosome rearrangements of various types were identified and put to many uses (for review see PERKINS and BARRY1977). Becauseascospores that contain deficienciesfail to darken, frequencies of ejected asci with different numbers of black

Perspectives and nonblack spores could be used to distinguish different rearrangement types (PERKINS1974). Genetic analysisof insertional translocations has been more thorough than in other organisms (see, for example, PERKINS 1972). Numerous quasiterminal rearrangements with chromosome segments translocated to telomeres or subtelomere regions were also studied. Insertional and terminal rearrangements were shown to generate partial-diploid progeny (DE SERRES 1957; ST. LAWRENCE 1959; NEWMEYER and TAYLOR 1967). The duplications obtained as segmental aneuploids from insertional and terminal rearrangements proved useful for mapping (PERKINS 1975) andstudying for vegetative incompatibility (NEWMEYER 1970; PERKINS1975), instability (NEWMEYER and GALEAZZI 1977), anddominance and dosand CHIA1979). age of regulatory genes (e.g., METZENBERG Partial-diploid progeny from crosses heterozygous for terminal rearrangements were found to revert frequently to euploid condition, usually by loss of the translocated segment. Heterokaryons were used to recover and characterize recessive lethal mutations (ATWOOD and MUKAI 1953; DE SERRES and OSTERBIND 1962), to determine the frequency of recessive mutation for loci throughout the genome (DE SERRESand MALLING 1971; STADLER andCRANE1979), and tostudy mutagenesis, DNA repair, anddose-rate effects 1983; STADLER and (STADLER and MOYER 1981; STADLER MACLEOD 1984). The spectra of mutational lesions were examined for different mutagens and genotypes (e.g., DE SERRES and BROCKMAN 199 ; KINSEY 1 and HUNG 198 1). An excision-repair mutant was obtained that shows increased sensitivity solelyto UV, the first example of its type and INOUE 1991). subset A in eukaryotes (ISHII,NAKAMURA of mutagen-sensitive mutants was shown to be abnormally sensitive to histidine and hydroxyurea, and to cause chromosome instability (SCHROEDER 1986); this includes members of two epistasisgroups (KAFER 1983). Many mutants of this subset were found to have abnormal deoxyribonucleotide triphosphate pools (SRIVASTAVA and SCHROEDER 1989). Histidine and hydroxyurea were shown to cause chromosome instabilityin the absence of any mutation causing mutagen sensitivity (NEWMEYER, SCHROEDER and GALEAZZI 1978; SCHROEDER 1986), and histidine was found to cause breaks or nicks in DNA (HOWARD and BAKER1988). An endo-exonuclease of Neurospora was characterized and shown to beimmunochemically related both to the RecC polypeptide of E. coli and toan endo-exonuclease that is deficient in the rad52 mutant of Saccharomyces (FRASER, KOA and CHOW1990). The first DNA-mediated transformation in a sexual fungus was achieved in Neurospora (N. C. MISHRA,SZABO and TATUM 1973). [Aspergillus niger had been transformed earlier by SEN,NANDIand A. K. MISHRA (1969).] The prototrophic character, putatively dueto transformation, was poorly transmitted through crosses (N. C. MISHRAand TAT U M 1973), behavior now attributable to RIP but then a cause for skepticism. With the advent of DNA technology and efficient transformation methods (CASEet al. 1979), integration of transforming DNA was found to be primarily nonhomologous. [For a review see FINCHAM (1989).] Inactivation of duplicated DNA sequences was found to occur premeiotically during the period of proliferation be-

695

tween fertilization and fusion of nuclei (SELKER et al. 1987; for review see SELKER1990). This phenomenon, termed repeat-induced point mutation (RIP), was shown to involve methylation and C to T mutation in both copies of duplicated sequences. RIP can be used to achieve targeted gene inactivation following transformation. [Premeiotic inactivation of duplicated segments has since been shown to occur in other fungi; for review see SELKER(1990).] Independently, BUTLERand METZENBERG(1989) found thatthe number of ribosomal DNA repeats in the nucleolus organizer region undergoes change during the same premeiotic period that is subject to RIP. An active transposable element was identified, the first to be characterized molecularly in filamentous fungi (KINSEY and HELBER1989). This LINE-like element was shown to be transmitted from one nucleus to another in heterokaryons (KINSEY 1990). Methods were devised for sampling natural populations, and wild-collected strains were analyzed (PERKINS, TURNER and BARRY 1976;for reviewsee PERKINSand TURNER 1988). Discrete orange colonies found on burnedvegetation in warm, moist climates were shown usually to represent pure haploidclones of Neurospora from different ascospores. Fertility in crosses to standard reference strains was shown to be a convenient and reliable criterion for determining the species of wild-collected isolates. New heterothallic specieswere described. Homothallic Neurospora species, devoid of conidia, were also discovered (see FREDERICK, UECKER and BENJAMIN 1969). Genetic polymorphisms at the protein level were shown to be abundant in natural populations of heterothallic species (SPIETH1975), not a foregone conclusion for a haploid organism. Numerous vegetative incompatibilityloci were identified and shown tobe polymorphic (MYLYK 1975, 1976). Wild populations were shown to carry a loadof phase-specificrecessive mutations that adversely affect meiosis and the sexual diplophase (LESLIEand RAJU1985). The nonselective abortion of asci in crosses betweeninbred strains of a normally outbreeding species provided an exand ample of inbreeding depression in fungi (RAJU,PERKINS NEWMEYER 1987). Chromosomal elements (“Spore killers”) were discovered that show meiotic drive, resulting in the death of meiotic products that do not contain the element. Recombination was shown to be blocked in the chromosomal region that contains the killer element, reminiscent of SD in Drosophila and PERKINS 1979; for and the t-complex in mice (TURNER review see TURNER and PERKINS 1991). Length mutations in mitochondrial DNA were studied in different N . crassa populations (TAYLOR, SMOLICH and MAY 1986) and were used to construct a phylogenetic tree for four different species (TAYLOR and NATVIC 1989). Mitochondrial plasmid DNAs were also compared in different MAYand TAYLOR 1984). populations and species (NATVIG, Clearly, Neurospora research hastill now been concerned mostlywithgenetic,cellular and molecular mechanisms. Relatively little attention has been paid toevolutionary biology, or topopulationgenetics, which has been based since its beginnings almost exclusively on plants and animalswhile the fungal king-

696

D. D. Perkins

dom has been largely ignored. Yet the fungioffer certain advantages for studying populations, not least of which is haploidy during thevegetative stage. Imagine what could be done if it were possible to sample animal or plant populations by obtaining individual sperm or pollen grains and growingthem up into immortal haploid or homozygous individuals. T h e equivalent of this is accomplished routinely in Neurospora,whereorange haploid colonies thatoriginated from single ascospores are readily sampled in the wild, propagated in thelaboratory,and maintained as permanent viable stocks in suspended animation. Some 4000 strains are already available that have been obtained in this way from populations in many parts of the world. What has been learned from them so far suggests that Neurospora can perhaps become for the population genetics of haploid organisms what Drosophila has been for diploids (for review see PERKINS and TURNER 1988). As with Drosophila, attention in the laboratory has been focussed primarily on one Neurospora species, N . crassa, but other species have also come into use. T h e known Neurospora species range from highly outbred to highly inbred. Some are heterothallic and cross-fertilizing, others are homothallic and self-fertilizing. One species, N . tetrasperma, does not fall into either category and has been termed pseudohomothallic. Like its counterparts in other genera, it represents a breeding system that is based on heterokaryosis and is therefore unique to the fungi. N . tetrasperma normally perpetuates itself as a self-fertile heterokaryon containing haploid nuclei of both mating types. Most conidia and ascospores are heterokaryotic, producing self-fertile cultures that behave as though they were homothallic. Aminority of the spores are homokaryotic, however, resulting in selfsterile, functionally heterothallic cultures.The species is therefore predominantly inbred, but it retains the capacity foroutbreedingasareadyoption (RAJU 1992a). This diversity oflifestylesin the various Neurospora species shows promise for comparative studies. After many years of asking “how” questions about the way that Neurospora functions,we should now be in astrong position to ask “why” questions about adaptations, populationsand evolutionary origins.Research on molecular,cellular and genetic mechanisms is certain to continue. It remains to be seen whether the promise of Neurospora for population genetics will be fulfilled. Sixty-five years have passed since SHEAR and DODGE named and described Neurospora, and 50 years since BEADLEand TATUM thrust it into prominence. In 1952, DODGEfelt that hewould soon be ableto assert, “The old red bread-mold has at last comeinto its own.” Developments since then have taken his favorite

organism far beyond what he could have imagined. Neurospora continues to be a source of innovations and surprises. This essay is dedicated to the memory of B. 0. DODGEon the 120th anniversary of his birth. The summary of research contributions benefited from discussion with numerous colleagues. Their comments are much appreciated. I am indebted to the Library of the New York Botanical Garden, Bronx, New York, for the photograph of DODGE,to HERSCHEL ROMAN for that of LINDEGREN, for the 1947 photograph of MCand to MARJORIEM. BHAVNANI CLINTOCK. Work on Neurospora in my laboratory has been supported since 1956 by grant AI 01462 from the National Institutes of Health.

LITERATURE CITED AKINS, R. A., R. L. KELLEY and A. M. LAMBOWITZ, 1986 Mitochondrial plasmids of Neurospora: integration into mitochondrial DNA and evidence for reverse transcription in mitochondria. Cell 47: 505-516. 1987 A protein required AKINS,R. A,, and A. M. LAMBOWITZ, for splicing group I introns in Neurospora mitochondria is mitochondrial tyrosyl-tRNA synthetase or a derivative thereof. Cell 5 0 331-345. ATWOOD, K. C., and F. MUKAI,1953 Indispensable gene functions in Neurospora. Proc. Natl. Acad. Sci. USA 39: 1027-1035. BACHMANN, B. J., and N. W. STRICKLAND, 1965 Neurospora Bibliography and Index. Yale University Press, New Haven, Conn. BARRATT, R. W., D. NEWMEYER, D. D. PERK INS^^^ L. GARNJOBST, 1954 Map construction in Neurospora crassa. Adv. Genet. 6: 1-93. BEADLE,G. W., 1966 Biochemical genetics: some recollections, pp. 23-32 in Phage and the Origins of Molecular Biology, edited by J. CAIRNS,G. S. STENTand J. D.WATSON. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. BEADLE, G. W., 1974 Recollections. Annu. Rev. Bioch. 43: 1-13. BEADLE,G. W., and V. L. COONRADT, 1944 Heterocaryosis in Neurospora crassa. Genetics 2 9 291-308. BEADLE, G. W., and E. L. TATUM,1941 Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci. USA 27: 499-506. BERLIN, V., and C. YANOFSKY, 1985 Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa. Mol. Cell. Biol. 5 849-855. 1989 Linear plasmids that BERTRAND, N., andA. J. F. GRIFFITHS, integrate into mitochondrial DNA in Neurospora. Genome 31: 155-159. BISTIS,G. N., 1983 Evidence for diffusible, mating-type-specific trichogyne attractants in Neurosporacrassa. Exp. Mycol. 7: 292-295. BOJKO,M.,1989 Two kinds of “recombination nodules” in Neurospora crassa. Genome 32: 309-317. BOJKO, M., 1990 Synaptic adjustment of inversion loops in Neurospora crassa. Genetics 124: 593-598. BREITENBERGER, C. A,,and U. L. RAJBHANDARY, 1985 Some highlights of mitochondrial research based on analyses of Neurospora crassa mitochondrial DNA. Trends Biochem. Sci. 10: 478-483. BURKE, J. M., and U. L. RAJBHANDARY, 1982 Intronwithin the large rRNA gene of N . crassa mitochondria: a long open reading frame and a consensus sequence possibly important in splicing. Cell 31: 509-520. BUTLER, D. K . , 1991 Recombination and chromosome breakage in the nucleolus organizer region of Neurospora crassa. Ph.D. Thesis, University of Wisconsin, Madison. BUTLER,D. K . , and R. L. METZENBERG, 1989 Premeiotic change

Perspectives of nucleolus organizer size in Neurospora. Genetics 122: 783791. BUTLER,E. T., W. J. ROBBINS and B. 0. DODGE,1941 Biotin and the growth of Neurospora. Science 9 4 262-263. CARSIOTIS, M., R. F. JONES and A. C.WFSSELING,1974 Crosspathway regulation: histidine-mediated control of histidine, tryptophan, and arginine biosynthetic enzymes in Neurospora crassa. J. Bacteriol. 1 1 9 893-898. CARSIOTIS, M., and A. M. LACY,1965 Increased activity of tryptophan biosynthetic enzymes in histidine mutants of Neurospora crassa. J. Bacteriol. 89: 1472-1477. CASE,M. E., M. SCHWEIZER, S. R. KUSHNER and N. H. GILES, 1979 Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA.Proc. Natl. Acad. Sci. USA 7 6 52595263. CATCHESIDE, D. G., 1973 Neurospora crassa and genetics. Neurospora Newsl. 20: 6-8. CATGHESIDE, D. G., 1975 Regulation of genetic recombination in Neurospora crassa, pp. 301-3 12 in The Eukaryote Chromosome, edited by W. J. PEACOCK and R. D. BROCK. Australian National University Press, Canberra. CATCHESIDE, D. G., A.P. JESSUP and B. R. SMITH,1964 Genetic controls of allelic recombination in Neurospora. Nature 202 1242-1243. COLLINS, R. A., and B. J. SAVILLE, 1990 Independent transfer of mitochondrial chromosomes and plasmids during unstable vegetative fusion in Neurospora. Nature 3 4 5 177-179. COLLINS, R. A,, L. L. STOHL,M. D. COLEand A. M. LAMBOWITZ, 1981 Characterization of a novelplasmidDNA found in mitochondria of N . crassa. Cell 24: 443-452. DAVIS,R. H., 1966 Mechanisms of inheritance. 2. Heterokaryosis, pp. 567-588 in The Fungi: An Advanced Treatise, Vol. 2, edited by G. C. AINSWORTHand A. S. SUSSMAN. Academic Press, New York. DAVIS,R. H., 1967 Channeling in Neurospora metabolism, pp. 303-322 in Organizational Biosynthesis, edited by H. J. VOGEL, J. 0. LAMPEN and V. BRYSON. Academic Press, New York. DAVIS,R. H., 1986 Compartmental and regulatory mechanisms in the arginine pathways of Neurospora crassa and Saccharomyces cerevisiae. Microbiol. Rev. 5 0 280-3 13. DAVIS,R. H., and R. L. WEIS, 1988 Novel mechanisms controlling arginine metabolism in Neurospora. Trends Biochem. Sci. 31: 101-104. DEGLI-INNOCENTI, F., and V. E. A. RUSSO, 1984 Genetic analysis of the blue light-induced responses in Neurospora crassa, pp. 2 13-219 in Blue Light E f f t s i nBiological Systems, edited by H. SENGER. Springer Verlag, Berlin. DE SERRES, F. J., 1957 A genetic analysis of an insertional translocation involving the ad-3 region in Neurospora crassa. Genetics 42: 366-367 (Abstr.) DE SERRES, F. J. (Editor), 1962 Neurospora Information Conference (National Research Council Publication 950). National Academy of Science, Washington, D.C. DE SERRES, F. J., and H. E. BROCKMAN, 1991 Qualitative differences in the spectra of genetic damage in P-aminopurineinduced ad-3 mutants between nucleotide excision-repair-proficient and -deficient strains of Neurospora crassa. Mutat. Res. 251: 41-58. DE SERRES, F. J.,and H. V. MALLING,1971 Measurement of recessive lethal damage over the entire genome and at two specific loci in the a d - 3 region of a two-component heterokaryon of Neurospora crassa, pp. 3 11-342 in Chemical Mutagens, Vol. 2, edited by A. HOLLAENDER. Plenum, New York. DE SERRES, F. J., and R. S. OSTERBIND, 1962 Estimation of the relative frequencies of the X-ray-induced viable and recessive lethal mutations in the ad-3 region of Neurospora crassa. Genetics 47: 793-796. DIACUMAKOS, E. G., L. GARNJOBSTand E. L. TATUM, 1965 A

697

cytoplasmic character in Neurospora crassa: the role of nuclei and mitochondria. J. Cell Biol. 2 6 427-443. DODGE,B. O., 1927 Nuclear phenomena associated with heterothallism and homothallism in the ascomycete Neurospora. J. Agric. Res. 3 5 289-305. DODGE,B. O., 1939 Some problems in the genetics of fungi. Science 9 0 379-385. DODGE,B. O., 1942 Heterocaryotic vigor in Neurospora. Bull. Torrey Bot. Club 6 9 1-74. DODGE,B. O., 1952 The fungi come into their own. Mycologia 44: 273-291. DUNLAP, J. C., 1990 Closely watched clocks. Trends Genet. 6 159-165. EMERSON, S., 1963 Slime, a plasmodioid variant in Neurospora crassa. Genetica 3 4 162-182. FELDMAN, J. F., and J. C. DUNLAP,1983 Neurospora crassa: a unique system for studying circadian rhythms. Photochem. Photobiol. Rev. 7: 319-368. FELDMAN, J. F., and M. N. HOYLE,1973 Isolation of circadian clock mutants of Neurospora crassa. Genetics 75: 605-6 13. FINCHAM, J. R. S., 1966 Genetic Complementation. W. A. Benjamin, New York. FINCHAM, J. R. S., 1989 Transformation in fungi. Microbiol. Rev. 53: 148-1 70. J. R. S., P. R. DAYand A. RADFORD, 1979 Fungal FINCHAM, Genetics, Ed. 4. Blackwell, Oxford. FINCHAM, J. R. S., and J. A. PATEMAN, 1957 Formation of an enzyme through complementary action of mutant “alleles” in separate nuclei in a heterocaryon. Nature 1 7 9 741-742. FRASER,M. J., H. KOA and T. Y.-K. CHOW,1990 Neurospora endo-exonuclease is immunochemicallyrelated to therecC gene product of Escherichia coli. J. Bacteriol. 172: 507-510. FREDERICK, L., F. A. UECKER and C.R. BENJAMIN, 1969 A new species of Neurospora from the soil of West Pakistan. Mycologia 61: 1077-1084. FREE, S. J., P. W. RICE and R. L. METZENBERG, 1979 Arrangement of the genes coding for ribosomal ribonucleic acid in Neurospora crassa. J. Bacteriol. 137: 1219-1226. Fu, Y.-H., H.-J. LEE,J. L. YOUNG,G. JARAI and G. A. MARZLUF, 1990 Nitrogen and sulfur regulatory circuits of Neurospora, pp. 319-335 in Developmental Biology, edited byE. H. DAVIDSON, J. V. RUDERMAN and J. W. POSAKONY. Wiley-Liss, New York. Fungal Genetics Stock Center, 1990 Catalog of Strains. (Supplement to Fungal Genet. Newsl. 37.) GAERTNER, F. H., and K. W. COLE,1977 A cluster-gene: evidence for onegene, one polypeptide, five enzymes. Biochem. Biophys. Res. Commun. 75: 259-264. GARNJOBST, L.,1953 Genetic control of heterocaryosis in Neurospora crassa. Am. J. Bot. 40: 607-614. GARNJOBST, L., and E. L. TATUM,1967 A survey of new morphological mutants in Neurospora crassa. Genetics 57: 579-604. GARRIGA,G.,and A. M. LAMBOWITZ, 1984 RNAsplicingin Neurospora mitochondria: self-splicing of a mitochondrial intron in vitro. Cell 39: 631-641. GILES,N. H., C.W. H. PARTRIDGE and N. J. NELSON,1957 The genetic control of adenylosuccinase in Neurospora crassa. Proc. Natl. Acad. Sci. USA 43: 305-31 7. GILLIES,C.B., 1972 Reconstruction of the Neurospora crassa pachytene karyotype from serial sections of synaptonemal complexes. Chromosoma 3 6 1 19-1 30. GILLIES, C. B., 1979 The relationship between synaptinemal complexes, recombination nodules and crossing over in Neurospora crassa bivalents and translocation quadrivalents. Genetics 91: 1-17. GLASS, N. L., and C. STABEN,1990 Genetic control of mating in Neurospora crassa. Semin. Dev. Biol. 1: 177-184 C. STABEN, J. GROTELUESCHEN, R. L. GLASS, N.L., S. J. VOLLMER,

698

D.

D.

METZENBERCand C. YANOFSKY, 1988 DNAs of the two mating-type alleles of Neurospora crassa are highly dissimilar. Science 241: 570-573. GODDARD, D. R., 1935 The reversible heat activation inducing germination and increased respiration in the ascospores of Neurospora tetrasperma. J. Gen. Physiol. 19: 45-60. GODDARD, D.R., 1939 The reversible heat activation of respiration in Neurospora. Cold Spring Harbor Symp. Quant. Biol. 7: 362-376. GRAY,C. H.,and E. L. TATUM,1944 X-ray induced growth factor requirements in bacteria. Proc. Natl. Acad. Sci. USA 30: 404-410. GRIFFITHS, A. J. F., and A. M. DELANGE, 1978 Mutations of the a mating-type gene in Neurosporacrassa. Genetics 88: 239254. GRIFFITHS, A. J. F., S. R. KRAUS,R. BARTON,D. A. COURT,C. J. MEYERSand H. BERTRAND, 1990 Hetrokaryotic transmission of senescence plasmid DNA in Neurospora. Curr. Genet. 17: 139-145. GROSS,S. R., 1965 The regulation of synthesis of leucine biosynthetic enzymes in Neurospora. Proc. Nat. Acad. Sci. USA 5 4 1538-1546. GROSS,S. R., and A. FEIN,1960 Linkage and function in Neurospora. Genetics 45: 885-904. HALLERMAYER, G., R. ZIMMERMAN and W. NEUPERT, 1977 Kinetic studies on the transport of cytoplasmically synthesized proteins into the mitochondria in intact cells of Neurospora crassa. Eur. J. Biochem. 81: 523-532. HARDING, R. W., and R. V. TURNER, 1981 Photoregulation of the carotenoid biosynthetic pathway in albino and white collar mutants of Neurospora crassa. Plant Physiol. 68: 745-749. HAWLITSHECK, G., H. SCHNEIDER, B. SCHMIDT, M. TROPSCHUG, F.U. HARTLand W. NEUPERT,1988 Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein. Cell 53: 795-806. HECKMAN, J. E., L. I. HECKER, S. D. SCHWARTZBACH, W. E. BARNETT, B. BAUMSTARKand U. L. RAJBHANDARY, 1978 Structure and function of initiator methionine tRNA from the mitochondria of Neurospora crassa. Cell 13: 83-95. Ho, C. C., 1986 Identity‘and characteristics of Neurosporaintermedia responsible for oncom fermentation in Indonesia. Food Microbiol. 3: 115-132. HOROWITZ, N. H., 1950 Biochemical genetics of Neurospora. Adv. Genet. 3: 33-71. HOROWITZ, N. H., 1973 Neurospora and the beginnings ofmolecular genetics. Neurospora Newsl. 2 0 4-6. HOROWITZ, N. H., 1979 Genetics and the synthesisof proteins. Ann. N Y Acad. Sci. 325: 253-266. HOROWITZ, N. H., 1985 The origins of molecular genetics: one gene, one enzyme. BioEssays 3: 37-39. HOROWITZ, N. H., 1990 George Wells Beadle. Biogr. Mem. Natl. Acad. Sci. 5 9 26-52. HOROWITZ, N. H., 1991 Fifty years ago: the Neurospora revolution. Genetics 127: 631-635. HOROWITZ, N. H., and M. FLING,1953 Genetic determination of tyrosinase thermostability in Neurospora. Genetics 38: 360374. HOROWITZ, N. H., and U . LEUPOLD, 1951 Some recent studies bearing on the one gene-one enzyme hypothesis. Cold Spring Harbor Symp. Quant. Biol. 16 65-74. HOWARD, C. A,, and T . I. BAKER,1988 Relationship of histidine to DNA damage and stress induced responses in mutagen sensitive mutants of Neurospora crassa. Curr. Genet. 13: 391399. ISHII, C., K. NAKAMURA and H. INOUE,1991 A novel phenotype of an excision-repair mutant in Neurosporacrassa: mutagen sensitivity of the mus-18 mutant is specific to UV. Mol. Gen. Genet. 228: 33-39.

Perkins KAFER,E., 1983 Epistatic grouping of DNA repair-deficient mutants in Neurospora: comparative analysis of two uus-3 alleles and uus-6, and their mus double mutant strains. Genetics 105: 19-33. KAY,L. E., 1989 Selling pure science in wartime: the biochemical genetics of G. W. Beadle. J. Hist. Biol. 22: 73-101. KELLER, E. F., 1983 A Feeling for the Organism. The Lqe and Work of Barbara McClintock. Freeman, San Francisco. KINSEY, J. A., 1990 T a d , a LINE-like transposable element of Neurospora, can transpose between nuclei in heterokaryons. Genetics 126: 317-323. KINSEY,J. A., and J. HELBER,1989 Isolation of a transposable element from Neurosporacrassa. Proc. Natl. Acad. Sci. USA 8 6 1929-1933. J. A., and B.-S. T . HUNG,1981 Mutation at the am locus KINSEY, of Neurospora crassa. Genetics 9 9 405-414. KITAZIMA,K., 1925 On the fungus luxuriantly grown on the bark of the trees injured by the great fire of Tokyo on Sept. 1, 1923. Ann. Phytopathol. Soc. Jpn. 1: 15-19. KUIPER, M. T. R., and A. M. LAMBOWITZ, 1988 A novel reverse transcriptase activity associated with mitochondrial plasmids of Neurospora. Cell 55: 693-704. KUNKEL, L. 0..19 13 The influence of starch, peptone,and sugars on the toxicity of various nitrates to Monilia sitophila (Mont.) Sacc. Bull. Torrey Bot. Club 40: 625-639. KUNKEL, L. O., 1914 Physical and chemical factors influencing the toxicity of inorganic salts to Monilia sitophila (Mont.) Sacc. Bull. Torrey Bot. Club 41: 265-293. LAKIN-THOMAS, P. L., G. G. COTEand S. BRODY,1990 Circadian rhythms in Neurospora crassa: biochemistry and genetics. Crit. Rev. Microbiol. 17: 365-416. LAMBOWITZ, A.M., and C.W. SLAYMAN, 1971 Cyanide-resistant respiration in Neurospora crassa. J. Bacteriol. 1 0 8 1087-1096. LANGRIDGE, J., 1955 Biochemical mutations in the crucifer Arabidopsis thaliana (L.) Heynh. Nature 1 7 6 260. LEDERBERG, J., 1990 Edward Lawrie Tatum. Biogr. Mem. Natl. Acad. Sci. 5 9 356-386. LESLIE, J. F., and N.B. RAJU, 1985 Recessive mutants from natural populations of Neurospora crassa that are expressed in the sexual diplophase. Genetics 111: 759-777. LINDEGREN, C. C., 1973 Reminiscences ofB. 0. Dodge and the beginnings of Neurospora genetics. Neurospora Newsl. 20: 1314. LINDEGREN, C. C., and G. LINDEGREN, 1942 Locally specific patterns of chromatid and chromosome interference in Neurospora. Genetics 27: 1-24. LOROS,J. J., S. A. DENOME and J. C. DUNLAP,1989 Molecular cloning of genes under control of the circadian clock in Neurospora. Science 243: 385-388. LUCK,D. J. L., 1963 Genesis of mitochondria in Neurospora crassa. Proc. Natl. Acad. Sci. USA 49: 233-240. LUCK,D. J. L., and E. REICH,1964 DNAin mitochondria of Neurospora crassa. Proc. Natl. Acad. Sci. USA 52: 931-938. MANNELLA, C. A., T . H. PITTENGER and A.M. LAMBOWITZ, 1979 Transmission of mitochondrial deoxyribonucleic acid in Neurosporacrassa sexual crosses. J. Bacteriol. 137: 14491451. MANNELLA,C. A,, R. A. COLLINS, M. R. GREENand A.M. LAMBOWITZ, 1979 Defectivesplicing of mitochondrial rRNA in cytochrome-deficient nuclear mutants of Neurospora crassa. Proc. Natl. Acad. Sci. USA 7 6 2635-2639. MARZLUF,G. A., and R. L. METZENBERG, 1968 Positive control by the c y - 3 locus in regulation of sulfur metabolism in Neurospora. J. Mol. Biol. 33: 423-437. MAY, G., and J. W. TAYLOR, 1989 Independent transfer of mitochondrial plasmids in Neurospora crassa. Nature 3 3 9 320322. MCCLINTOCK, B., 1945 Neurospora. I. Preliminary observations

Perspectives of the chromosomes of Neurospora crassa. Am. J. Bot. 32: 67 1678. METZENBERG, R. L., 1979 Implications of some genetic control mechanisms in Neurospora. Microbiol. Rev. 43: 361-383. METZENBERG, R. L., and W. CHIA,1979 Genetic control of phosphorus assimilation in Neurospora crassa: dose-dependent dominance and recessiveness in constitutive mutants. Genetics 93: 625-643. METZENBERG, R. L., and N. L. GLASS,1990 Mating type and mating strategies in Neurospora. BioEssays 12: 53-59. METZENBERG, R. L., and J. GROTELUESCHEN, 1990 Neurospora crassa restriction polymorphism map, pp. 3.22-3.29 in Genetic Maps, Ed. 5, edited by S. J. O’BRIEN.Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. METZENBERG, R. L., J. N. STEVENS, E. U. SELKER and E. MORZYCKAWROBLEWSKA, 1984 A method for finding the genetic map position of cloned DNA fragments. Neurospora Newsl. 31: 3539. METZENBERG,R. L.,J. N. STEVENS, E. U. SELKER and E. MORZYCKAWROBLEWSKA, 1985 Identification and chromosomal distribution of 5 s R N A genes in Neurospora crassa. Proc. Natl. Acad. Sci. USA 82: 2067-2071. MISHRA,N. C., 1977 Geneticsand biochemistry of morphogenesis in Neurospora. Adv. Genet. 1 9 341-405. MISHRA, N. C.,G. SZABOand E. L. TATUM, 1973 Nucleic acid induced genetic changes in Neurospora, pp. 259-268 in The Role of RNA in Reproduction and Development, edited by M. C. NIUand S. J. SEGAL.Elsevier/North Holland, Amsterdam. MISHRA, N. C., and E. L. TATUM,1973 Non-mendelian inheritance of DNA-induced inositol independence in Neurospora. Proc. Natl. Acad. Sci. USA 70: 3875-3879. MITCHELL, H. K . , and M.B. HOULAHAN, 1946 Neurospora. IV. A temperature-senstivie, riboflavinless mutant. Am. J. Bot. 33: 31-35. MITCHELL, M. B., 1955 Aberrant recombination of pyridoxine mutants of Neurospora. Proc. Natl. Acad. Sci. USA 41: 215220. MITCHELL, M. B., H. K. MITCHELL and A. TISSI~RES, 1953 Mendelian and nowMendelian factors affecting the cytochrome system in Neurospora crassa. Proc. Natl. Acad. Sci. USA 3 9 601-613. MOLLER,A., 1901 Phycomyceten und Ascomyceten. Untersuchungen aus Brasilien, pp. 1-319 in BotanischeMittheilungen Fischer, aus den Tropen, Vol. 9, edited by A. F. A. SCHIMPER. Jena. MONTAGNE,C., 1843 QuatriPme centurie de plantes cellulaires exotiques nouvelles. Ann. Sci. Nat. Bot. 2e Ser. 20: 352-379 (+ one plate). MOREAU-FROMENT, M., 1956 Les Neurospora. Bull. SOC.Bot. Fr. 103: 678-738. MYLYK,0. M., 1975 Heterokaryon incompatibility genes in Neurospora crassa detected using duplication-producing chromosome rearrangements. Genetics 80: 107-124. MYLYK,0. M., 1976 Heteromorphism for heterokaryon incompatibility genes in natural populations of Neurospora crassa. Genetics 83: 275-284. NATVIG,D. O., G. MAY and J. W. TAYLOR,1984 Distribution and evolutionary significance of mitochondrial plasmidsin Neurospora spp. J. Bacteriol. 159: 288-293. NEWMEYER, D., 1970 A suppressor of the heterokaryon-incompatibility associated with mating type in Neurospora crassa. Can. J. Genet. Cytol. 12: 914-926. NEWMEYER, D., and D. R. GALEAZZI, 1977 The instability of Neurospora duplication Dp(IL+IR)H4250 and its genetic control. Genetics 85: 461-487. NEWMEYER,D., A. L. SCHROEDER and D. R. GALEAZZI, 1978 An apparent connection between histidine, recombination and repair in Neurospora. Genetics 8 9 271-279.

699

NEWMEYER, D., and C. W. TAYLOR, 1967 A pericentric inversion in Neurospora, with unstable duplication progeny. Genetics 5 6 771-791. R. W. DAVISand C. YANOFSKY, ORBACH, M. J., D. VOLLRATH, 1988 An electrophoretic karyotype ofNeurospora crassa. Mol. Cell. Biol. 8: 1469-1473. PALL,M. L., 1970 Amino acid transport in Neurospora crassa. Ill. Acidic amino acid transport. Biochim. Biophys. Acta211: 51 3520. PASTEUR, L., 1862 L’influence de la tempirature sur la ficunditi des spores de Muci.dinies. Compt. Rend. Acad.Sci. 52: 1619. PAYEN, A. (rapporteur), 1843 Extrait d’un rapport adressi i M. Le Marichal Duc de Dalmatie, Ministre de la Guerre, Prisident du Conseil, sur une altiration extraordinaire du pain de munition. Ann. Chim. Phys. 3‘ Ser. 9 5-2 1 (+ one plate). PAYEN, A,, 1848 Tempiratures qui peuvent supporter les sporules de I’Oidiumaurantiacum sans perdre leur faculti. vigitative. Compt. Rend. Acad. Sci. 27: 4-5. PAYEN,A,, 1859 Untitled discussionfollowing remarks of M. Milne Edwards rejecting spontaneous generation of animalcules. Compt. Rend. Acad. Sci. 48: 29-30. PERKINS, D. D., 1953 The detection of linkage in tetrad analysis. Genetics 38: 187-197. PERKINS, D. D., 1962 Crossing-over and interference in a multiply-marked chromosome arm of Neurospora. Genetics 47: 1253-1274. PERKINS, D. D., 1972 An insertional translocation in Neurospora that generates duplications heterozygous for mating type. Genetics 71: 25-51. PERKINS,D. D., 1974 The manifestation of chromosome rearrangements in unordered asci of Neurospora. Genetics 77: 459-489. PERKINS,D. D., 1975 The use of duplication-generating rearrangements for studying heterokaryon incompatibility genes in Neurospora. Genetics 80: 87-105. PERKINS,D. D., 1979 Neurospora as an object for cytogenetic research. Stadler Genet. Symp. 11: 145-164. PERKINS, D.D., 1988 Main features of vegetative incompatibility in Neurospora. Fungal Genet. Newsl. 35: 44-46. PERKINS, D. D., 1990 Neurospora crassa genetic maps, June 1989. Genet. Maps 5: 3.9-3.18. PERKINS, D.D., and E. G. BARRY,1977 The cytogenetics of Neurospora. Adv. Genet. 19: 133-285. PERKINS, D. D., and B. C. TURNER, 1988 Neurospora from natural populations: toward the population biology of a haploid eukaryote. Exp. Mycol. 12: 91-131. PERKINS, D.D., B. C. TURNER and E.G. BARRY, 1976 Strains of Neurospora collected from nature. Evolution 30: 28 1-3 13. PERKINS, D. D., D. NEWMEYER,C. W. TAYLOR and D. C. BENNETT, 1969 New markers and map sequences in Neurospora crassa, with a description of mapping by duplication coverage and of multiple translocation stocks for testing linkage. Genetica 40: 247-278. PERKINS, D. D., A. RADFORD, D. NEWMEYERand M. BJORKMAN, 1982 Chromosomal loci ofNeurospora crassa. Microbiol. Rev. 46: 426-570. PERKINS, D. D., N. B. RAJU,V. C. POLLARD, J. L. CAMPBELL and A. M. RICHMAN, 1986 Use of Neurospora Spore killer strains to obtain centromere linkage data without dissecting asci. Can. J. Genet. Cytol. 28: 971-981. PITTENDRIGH, C. S., V.G. BRUCE,N. S. ROSENSWEIG and M. L. RUBIN, 1959 A biologicalclock in Neurospora. Nature 184: 169-170. PRINGSHEIM, H., 1909 Studien uberden Gehalt verschiedener Pilzpresssafte an Oxydasen. Hoppe-Seylor’s Z. Physiol. Chem. 62: 386-389.

700

D. D. Perkins

RAJU,N. B., 1980 Meiosis and ascospore genesis in Neurospora. Eur. J. Cell Biol. 23: 208-223. RAJU,N. B., 1986 Postmeiotic mitoses without chromosome replication in a mutagen-sensitiveNeurospora mutant. Exp. Mycol. 1 0 243-251.

RAJU,N. B., 1992a Functional heterothallism resulting from homokaryotic conidia and ascospores in Neurosporatetrasperma. Mycol. Res. 96: (in press). RAJU,N. B., 1992b Genetic control of the sexual cycle in Neurospora. Mycol. Res. 9 6 (in press). RAJU,N. B.,D.D. PERKINS and D. NEWMEYER, 1987 Genetically determined nonselective abortion of entire asci in Neurospora crassa. Can. J. Bot. 65: 1539-1549. REICH,E., and D. J. L. LUCK,1966 Replication and inheritance of mitochondrial DNA. Proc. Nat. Acad. Sci. USA 55: 16001608.

REISSIG, J. L., A. S. ISSALYand I. M. ISSALY,1967 Argininepyrimidine pathways in microorganisms. Natl. Cancer Inst. Monogr. 27: 259-27 1. RIECK, A., A. J. F. GRIFFITHSand H. BERTRAND,1982 Mitochondrial variants of Neurospora intermedia from nature. Can. J. Genet. Cytol. 24: 741-759. ROBBINS, W. J., 1962 Bernard Ogilvie Dodge. Biogr. Mem. Natl. Acad. Sci. 3 6 85-124. ROBERTS, A. N., and C. YANOFSKY, 1989 Genes expressed during conidiation in Neurospora crassa: characterization of con-8. Nucleic Acids Res. 17: 197-2 14. ROEPKE, R. R., R. L. LIBBYand M. H. SMALL,1944 Mutation or variation of E. coli with respect to growth requirements. J. Bacteriol. 4 8 401-419. RYAN,F. J., and J. LEDERBERG, 1946 Reverse-mutation and adaptation in leucineless Neurospora. Proc. Natl. Acad. Sci. USA 32: 163-173.

RYAN,F. J., and L. S. OLIVE, 1961 The importance ofB. 0. Dodge’s workfor the genetics of fungi. Bull. Torrey Bot. Club 8 8 118:120.

SANSOME, E. R., 1945 Heterokaryosis and the mating-type factors in Neurospora. Nature 1 5 6 47. SARGENT, M. L., and W. R. BRIGGS,1967 The effects of light on a circadian rhythm of conidiation in Neurospora. Plant Physiol. 42: 1504-1510.

SCARBOROUGH, G. A., 1975 Isolation and characterization of Neurospora crassa plasma membranes. J. Biol. Chem. 2 5 0 11061111.

SCARBOROUGH, G. A., 1978 The Neurospora plasma membrane: a new experimental system for investigating eukaryote surface membrane structure and function. Methods Cell Biol. 20: 1 17133.

SCHECHTMAN, M. G., 1987 Isolation of telomere DNA from Neurospora crassa. Mol. Cell. Biol. 7: 3168-3177. SCHECHTMAN, M. G., 1990 Characterization of telomere DNA from Neurospora crassa. Gene 88: 159-165. SCHLEYER, M., and W. NEUPERT, 1985 Transport of proteins into mitochondria: translocational intermediates spanning contact sites between outer and inner membranes. Cell 43: 339-350. SCHROEDER, A. L., 1986 Chromosome instability in mutagen sensitive mutants of Neurospora. Curr. Genet. 1 0 381-387. SELITRENNIKOFF, C. P., B. L. LILLEYand R. ZUCKER, 1981 Formation andregeneration of protoplasts derived from a temperature-sensitive osmotic strain of Neurospora crassa. Exp. Mycol. 5: 155-161. SELKER, E. U., 1990 Premeiotic instability of repeated sequences in Neurospora crassa. Annu. Rev. Genet. 2 4 579-613. SELKER, E. U., C. YANOFSKY, K. DRIFTMIER, R. L. METZENBERG, B. ALLNER-DEWEERD and U. L. RAJBHANDARY, 1981 Dispersed 5s RNA genes in N . crassa: structure, expression and evolution. Cell 24: 819-828. SELKER, E. U., E. B. CAMBARERI, B. C. JENSEN and K. R. HAACK,

1987 Rearrangement of duplicated DNA in specialized cells of Neurospora. Cell 51: 741-752. SEN,K., P. NANDIand A. K. MISHRA,1969 Transformation of nutritionally deficient mutants of Aspergillusnzger. J. Gen. Microbiol 5 5 195-200. SHAW,D. E., and D. F. ROBERTSON, 1980 Collection of Neurospora by honeybees. Trans. Br. Mycol. SOC.7 4 459-464. SHEAR, C. L., and B. 0. DODGE,1927 Life histories and heterothallismof the red bread-mold fungi of the Moniliasitophila group. J. Agric. Res. 3 4 1019-1042. SHURTLEFF, W., and A. AOYAGI,1979 Oncham or ontjam. Appendix H, pp. 205-214 in The Book of Tempeh, Professional Edition. Harper & Row, New York. SLAYMAN, C. L., 1965 Electrical properties of Neurospora crassa: respiration and the intercellular potential. J. Gen. Physiol. 49: 93-1 16.

SLAYMAN, C.L., 1970 Movement ofions and electrogenesis in microorganisms. Am. Zool. 1 0 377-392. SLAYMAN, C.L., 1987 The plasma membrane ATPase ofNeurospora: a protein-pumping electroenzyme. J. Bioenerg. Biomembr. 19: 1-20. SLAYMAN, C. L., and C. W. SLAYMAN, 1962 Measurement of membrane potentials in Neurospora. Science 1 3 6 876-877. SLAYMAN, C. L., and C. W. SLAYMAN, 1974 Depolarization of the plasma membrane of Neurospora during active transport of glucose: evidence for a protondependent cotransport system. Proc. Natl. Acad. Sci. USA 71: 1935-1939. SPIETH,P. T., 1975 Population genetics of allozyme variation in Neurospora intermedia. Genetics 80: 785-805. SPRINGER, M. L., and C. YANOFSKY, 1989 A morphological and genetic analysisof conidiophore development in Neurospora crassa. Genes Dev. 3: 559-571. SRB, A.M., 1973 Beadle and Neurospora, some recollections. Neurospora Newsl. 2 0 8-9. SRB,A. M., and N. H. HOROWITZ, 1944 The ornithine cyclein Neurospora and its genetic control. J. Biol. Chem. 1 5 4 129139.

SRIVASTAVA, V., and A. L. SCHROEDER, 1989 Deoxyribonucleoside triphosphate pools in mutagen sensitive mutants of Neurospora crassa. Biochem. Biophys. Res.Commun. 162: 583590.

ST. LAWRENCE, P., 1959 Gene conversion at the nic-2 locusof Neurospora crassa in crosses between strains with normal chromosomes and a strain carrying a translocation at the locus. Genetics 44: 532. STADLER, D. R., 1983 Repair and mutation following UV damage in heterokaryons of Neurospora. Mol. Gen. Genet. 190: 227232.

STADLER, D. R., and E. CRANE,1979 Analysis of lethal events induced by ultraviolet in a heterokaryon of Neurospora. Mol. Gen. Genet. 171: 59-68. STADLER, D. R., and H. MACLEOD,1984 A dose-rate effect in Uv mutagenesis in Neurospora. Mutat. Res. 127: 39-47. STADLER, D.R., and R. MOYER,1981 Induced repair of genetic damage in Neurospora. Genetics 98: 763-774. STOKES,J. L., J. W. FOSTER and C . R. WOODWARD, JR., 1943 Synthesis of pyridoxin by a “pyridoxinless”X-ray mutant of Neurospora sitophila. Arch. Biochem. 2: 235-245. STRICKLAND, W. N., 1960 A rapid method for obtaining unordered Neurospora tetrads. J. Gen. Microbiol. 22: 583-588. SUSKIND, S. R., C. YANOFSKYand D. M. BONNER,1955 Allelic strains of Neurospora lacking tryptophan synthetase: a preliminary immunochemical characterization. Proc. Natl. Acad. Sci. USA 8: 577-582. TATUM,E.L., 1961 Contributions of Bernard 0. Dodge to biochemical genetics. Bull. Torrey Bot. Club 88: 1 15-1 18. TATUM, E. L., R. W. BARRATTand V. M.CUTTER, 1949 Chemical

Perspectives induction of colonial paramorphs in Neurospora and Syncephalastrum. Science 1 0 9 509-51 1. TAYLOR, J. W., and D. 0.NATVIG,1989 Mitochondrial DNA and evolution of heterothallic and pseudohomothallic Neurospora species. Mycol. Res. 93: 257-272. TAYLOR, J. W., B. D. SMOLICHand G. MAY,1986 Evolution and mitochondrial DNA in Neurospora crassa. Evolution 4 0 7 16739. TERENZI, H. F., M. M. FLAWIAand H. N. TORRES, 1974 A Neurospora crassa morphological mutant showing reduced adenylate cyclase activity. Biochem.Biophys.Res. Commun. 5 8 990-996. TOKUGAWA, Y., and Y. EMOTO,1924 Uber einen Kurz nach der letzten feuersbrunst plotzlich entwickelten Schimmelpilz.Jpn. J. Bot. 2: 175-88. TURNER, B. C., and D. D. PERKINS, 1979 Spore killer, a chromosomal factor in Neurospora that kills meiotic productsnot containing it. Genetics 93: 587-606. TURNER, B. C., and D.D. PERKINS,1991 Meiotic drive in Neurospora and other fungi. Am. Nat. 137: 416-429. VIEBROCK, A.,A. PERZand W. SEBALD,1982 The imported preprotein of the proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa. Molecular cloning and sequencing of the mRNA. EMBO J. 1: 565-57 1 . VOGEL,H. J., and D. M. BONNER,1959 The use of mutants in the study of metabolism, pp. 1-32 in Encyclopedia of Plant Physiology, Vol. 2, edited byW. RUHLAND. Springer,Heidelberg. VON BORSTEL, R. C., 1963 Carbondale yeast genetics conference. Microb. Genet. Bull. 19 (Suppl.): 1-21. WEISS,R. L., 1973 Intracellular localization of ornithine and arginine pools in Neurospora. J. Biol. Chem. 248: 5409-541 3.

70 1

WENT, F. A. F. C., 1901a Moniliasitophila (Mont.) Sacc., ein technischer Pilz Javas. Zentralbl. Bakteriol. Abt. 11, 7: 544550, 591-598. WENT,F. A. F. C., 190 1 b Uber den Einfluss der Nahrung auf die Enzymbildung durch Moniliasitophila (Mont.) Sacc. Jahrb. Wiss. Bot. 3 6 61 1-664. WENT,F.A. F. C., 1904 Uber den Einfluss des Lichtes auf die Entstehung des Carotins und auf die Zersetzung der Enzyme. Rec. Trav. Bot. Neerl. 1: 106-1 19. WHITEHOUSE, H. L. K., 1942 Crossing-over in Neurospora. New Phytol. 41: 23-62. WILLIAMS,L. G., S. A. BERNHARDT and R. H. DAVIS,1971 Evidence for two discrete carbamyl phosphate pools in Neurospora. J. Biol. Chem. 246: 973-978. WILLIAMS, J. G. K., A. R. KUBELIK, J. A. RAFALSKI and S. V. TINGEY, 1991 Genetic analysis with RAPD markers, pp. 43 1439 in MoreGeneManipulations in Fungi, edited by J. W. BENNETT and L. L. LASURE. Academic Press, Orlando, Fla. WILSON, C., 1986 FGSC culture preservation methods. Fungal Genet. Newsl. 33: 47-48. WILSON, J. F., L. GARNJOBST and E. L. TATUM, 196 1 Heterokaryon incompatibility in Neurospora crassa-micro-injection studies. Am. J. Bot. 48: 299-305. WOODWARD, D. O., 1959 Enzyme complementation invitro between adenylosuccinaselessmutants of Neurospora crassa. Proc. Natl. Acad. Sci. USA 45: 846-850. YANOFSKY,C., 1956 Gene interactions in enzyme synthesis, pp. 147-160 in Enzymes: Units of Biological Structure and Function, edited by 0. H. GAEBLER. Academic Press, New York. ZIMMERMAN, R., B. HENNIGand W. NEUPERT,1981 Different transport pathways of individual precursor proteins in mitochondria. Eur. J. Biochem. 1 1 6 455-460.