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, Calgornia 94305-5020

NDER the title “Fifty Years Ago: The Neuro- grown in the light quickly became brightorange. U spora Revolution,” HOROWITZ(1 991) has cele- Colonies grown in the dark,however, remained white brated an anniversary of the epochal 1941 paper of for more than8 days, but thewhite colonies developed BEADLEand TATUM,which reported the first mutants orange pigment within 2 hr when they were brought with biochemically defined nutritional requirements. into thelight. Thermal tolerance was also studied (see HOROWITZ’Saccount andothers (HOROWITZ1973, PAYEN1848, 1859). These results were cited by PAS- 1985, 1990; LEDERBERC1990) have focused on the TEUR (1 862)in reporting his own experiments on the people who were involved, the genesis of their ideas, survival of spores, which helped to refute theo- and therole of the 1941 results in transforming ries of spontaneous generation. . The present essay will be concerned mainly The next experimental study of began with the research organism that was so important to in Indonesia during Dutch colonial times. In market- the success of the initial experiments.Neurospora places of East Java, bright orangecakes are displayed. possesses a combination of features that made it an These consist of Neurospora grown on soybean or ideal choice not only for accomplishing the original peanut solids from whichoil and proteinfor curd objectives set by BEADLEand TATUMbut also for a have been pressed. The Javanese inoculate the solids continuing succession of contributions,including with conidia to create an appetizing and highly nutri- many in areas that transgress the boundsof biochem- tious food called oncham, which has a mushroom-like ical genetics and molecular biology. 1 shall begin by taste(WENT 1901a; SHURTLEFFand AOYACI1979; outlining the story of Neurospora prior to BEADLE Ho 1986). Producing oncham is a cottage industry and TATUMand then go on to sketch its subsequent which has probably gone on for centuries and which history. The previous accounts have stressed biochem- continues today. ical genetics and molecular biology. I shall consider A Dutch plant physiologist, F. A. F. C. WENT,was other aspects as well, focusing first on genetics, con- stationed at the famous Buitzenjorg (now Bogor) Bo- tinuing with a summary of research accomplishments tanic Gardens in Java at the turnof the century. WENT of all sorts, and concluding with a consideration of was attracted by theorange oncham and the potential usefulness of Neurospora for population started experimenting with it. He was frustrated be- studies. cause humidity in Java is sa great that the organism The vegetative phase of Neurospora was described grew through the cotton plugs of his culture tubes. and used for experiments by French microbiologists WENT (1904) also foundNeurospora in Surinam, nearly 100 years before BEADLEand TATUM(PAYEN where he noted that the fungus was used to process 1843; MONTAGNE1843). In thewarm, humid summer cassava meal in preparation of an indigenousalcoholic of 1842, bread from bakeries in Paris was spoiled by beverage. Back home in Utrecht, he described the massive growth of an orangemold. A commission was onchamfungus and its culture (WENT 1901a) and set up by the minister of war to investigate the cause used it for a series of studies on the effects of various of the infestation and to make recommendations. The substrates on enzymes such as trehalase, invertase and commission’s report (PAYEN1843) includes a colored tyrosinase (WENT 1901b). WENT(1 904)also studied plate whichshows colonies, mycelia, conidiophores the effect of light on carotenoid production. With and conidia of the “champignons rougesdu pain.” An knowledge of WENT’Swork, PRINGSHEIM(1 909) in- experiment in photobiology is described. Colonies cluded Neurosporain a study of oxidases, and KUNKEL

Genetics 130 687-701 (April, 1992) 688 D. D. Perkins (1 9 13, 19 14)used it in studies of chemical toxicity. About the time KITAZIMAwas examining his orange All these observations were made using the vegetative fungus in Japan, CHARLESTHOM, a colleague of phase of the organism and the asexually produced DODGE’Sat the Department of Agriculture mycology powdery conidia (vegetative spores). and pathology laboratory in Arlington, Virginia, was The association of Neurospora with heat and fire studying cultures of orange mold from sugar cane must have been known from the earliest times. We bagasse in Louisiana. THOMwas of the opinion that now know that the sexually produced heat-tolerant theorange fungus, then called Moniliasitophila, remain dormant until exposed toheat. lacked a sexual stage. However, C. L. SHEAR, the head Heat activation of ascospores explains the occurrence of the laboratory, found perithecia in one of THOM’S of Neurosporaboth in bakery infestations andon plates. The material was given to DODGEfor analysis. burned vegetation. Numerousrecords going back The success of DODGE’Sexperimental crosses kindled over a century describe large orange areas following his enthusiasm, and Neurospora became hismain volcanic eruptions in tropical areas. In New Guinea, lifelong interest. tribesmen traditionally set hillsides on fire to flush DODGE’Sfirst Neurospora paper, with SHEARin game, and Neurospora bloomed fobowing the burns. 1927, goes far beyond the conventional taxonomic In Brazil, MOLLER(1 90 1) describedan orangefungus descriptions of genus and species. Cultures of the growing on burned vegetation (and on maize bread). orange fungus had been obtained frommany sources. A typically ascomycete sexual phase appeared in his Isolates were assigned to the new genus Neurospora cultures, and the sexual fruiting bodies (perithecia) on the basisof their grooved ascospores. (Prior to and ascospores were later identified as Neurospora. 1927, thevegetative stage hadsuccessively been called In Japan, Neurospora made a dramatic appearance Oidiumaurantiacum, Penicillium sitophilum and Mo- following thegreat Tokyo earthquake and fire of niliasitophila.) DODGEshowed that the cultures in- 1923. Within a few days, burned and scorched trees cluded three species which were set off fromone became festooned with orange. Mycologistsin two another by their crossing behavior. Hybrid perithecia laboratories cultured the organism. KITAZIMA(1 925) from crosses between different species developed observed perithecia in his cultures, and going back to slowly and were unproductive or poorly fertile. Al- the source, discovered that perithecia were present though a conventional morphologically based taxo- underthe bark of trees in theTemple of Shiba. nomic species description was provided for each spe- Orange progeny were obtainedfrom single asco- cies, crossing behavior was implicitly taken into con- spores. TOKUGAWAand EMOTO (1924) studied sur- sideration and used to assign strains to the designated vival of the fungus following exposure to moist and species. This innovation contrasted with the purely dryheat, and identified the orange pigment as a morphological criteria then used by mycologists and carotenoid. clearly anticipated the idea of biological species long Neurospora is commonly seen following agricul- before the concept was formalized. turalburning in warm, moist climates. Sugarcane Two species with eight-spored asci, Neurospora appears to be an ideal substrate. Ascospores are no crassa and Neurosporasitophila, were shown to be doubt activated by burning in the fields and by heating heterothallic: individual haploid cultures from single in the mill. Bales of bagasse (fiber from which the sap ascospores were unable to enter thesexual cycle. They has been pressed) become orange. In Australia,solids fell into two mating types, defined because crosses from refinery filters are spread on fields as fertilizer. could occur only between strains of opposite mating Largeorange colonies develop on this filtermud. tYPea Honey bees can be seen visiting the colonies and filling DODGEcarried out the first tetrad analysis with N. their pollen baskets with the brightly colored conidia crassa, showing that the mating types segregated 4:4 (SHAWand ROBERTSON1980). in individual asci. The asci were obtained as groups The modern history of Neurospora begins in the of eight ascospores thathad been spontaneously mid-1920s with materialfrom sugar cane bagasse. ejected from the perithecia. The Neurospora asco- The key person was BERNARDDODGE. Like BEADLE, spores were activated by heat, as inAscobolus. In DODGEhad grown upon a farm. He worked for years contrast to the eight-spored species, isolates of Neu- asa school teacher and managed to complete his rosporatetrasperma, with four-spored asci, appeared bachelor’s degree only at age 39. He published his to be homothallic. Culturesfrom single ascospores first paper at the age of 40 and was already past 50 were usually self-fertile. A few self-sterile progeny when he began to work with Neurospora (ROBBINS were produced, however, thatbehaved as though they 1962). Prior to the Neurospora work,DODGE was the were heterothallic. DODGE(1927) was shortly to de- first to discover heat activation of ascospores (1912) scribe the cytological basis ofthis “pseudohomothallic” and to describe mating types in ascomycetes (1 920), behavior of N. tetrasperma, showing that individual both in Ascobolus. ascospores were usually (but not always) heterokar- Perspectives 689

yons thatcontained haploid nuclei of oppositemating (:;\HI. (;. 1,lSl>b:GREN types. identified mutants that could be used as markers and DODGElost no time in communicating his enthusi- discovered the first linkages. The linked pro- asm. At Columbia University, he urged T. H. MOR- vided confirming proof thatcrossing over occurred at GAN and the Drosophila group to use Neurospora. He the four-strand stage. Genetic maps were constructed traveled to Cornell for a seminar. Among the gradu- for two linkage groups using matingtype, centro- ate students in the audience were meres, and morphological mutants. But at about the and BARBARAMCCLINTOCK. BEADLE (1 966) later re- time BEADLEand TATUMwere turning from Drosoph- called how thestudents, familiar with then-recent ila to Neurospora,LINDEGREN abandoned Neuro- results of E. G. ANDERSONusing Drosophila attached- spora to begin work on Saccharomyces. His last Neu- X half-tetrads, were able to point out to DODGEhow rosporapaper (LINDEGRENand LINDEGREN 1942), the second-division segregations he described in Neu- written with his life-long collaborator GERTRUDELIN- rospora could be explained by crossing over between DEGREN, was submitted just as the Stanford workers chromatids at the four-strand stage. were about to obtain their first biochemical mutant. DODGEsoon moved to a job as plant pathologist at LINDEGREN’SNeurospora papers are listed in BACH- the New York Botanical Garden. In addition to his MANN and STRICKLAND(1 965). official duties, he managed to continue experiments Neurospora was used for several other investiga- with Neurospora. These included pioneeringwork on tions during the decade before 194 1. I first heard of interspecies crosses and on mutations affecting it in a plant physiology course taught by DAVIDGOD- development. He was intrigued by heterokaryons and DARD, who used N. tetrasperma in studies of obtained combinations of strains that showed hetero- activation and dormancy(GODDARD 1935,1939). karyotic vigor (DODGE1942). In the next 30 years he BUTLER,ROBBINS and DODGE(1941) demonstrated published nearly 50 papers on the genetics, cytology, that biotin was the sole growth factor requirement. In morphology and life cycle of Neurospora. England, WHITEHOUSE(1 942) subjected LINDEGREN’S DODGE’Senthusiasm resulted indirectly in the re- tetraddata to a detailed analysis and went on to cruitment of CARLLINDEGREN, who did the most produce hisown five-point map of the mating-type significant genetic workwith Neurospora priorto chromosome of N. sitophila. It was DODGEand LIN- BEADLE and TATUM.In 1928, LINDEGRENmoved to DEGREN, however, who developed the genetics of Neu- California from Wisconsin, where he had obtained a rospora duringthe 1930s and made the organism Master’s degree in Plant Pathology. He visited T. H. known to geneticists. The accumulated information MORGANto inquire about continuing graduate work enabled DODGE(1 939) to assert: “The fungi in their at the California Institute of Technology, whereMOR- reproduction and inheritance follow exactly the same GAN had come with his Drosophila group to head the laws that govern these activities in higher plants and newBiology Division. LINDEGRENfound MORGAN animals.” using dissecting needles in an attempt to isolate Neu- Additional information regarding early history can rospora ascospores from an agarplate (see LINDEGREN be found in the introductory sections of SHEAR and 1973). MORGANsuggested that LINDEGRENwork with DODGE(1 927), MOREAU-FROMENT(1956), and PER- Neurospora. Following a visit to DODGE,LINDEGREN KINS and BARRY(1977), and in essays by RYANand chose the species N. crassa as best suited for genetic OLIVE(1 96 l), TATUM(1 96 1 ), LEDERBERG(1 990). SRB work. He developed highly fertile wild-type strains, (1 973), CATCHESIDE(1 973) and LINDEGREN(1 973). 690 D. D. Perkins ”I hree genetics textbooks published in 1939 (STUR- TEVANT and BEADLE,SINNOTT and DUNN,AND WAD- DINGTON) 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 DODGEregard- ing stocks. His letter begins “Dr. Tatum and I are interested in doing some work on thenutrition of Neurospora with the eventual aim of determining whetherthe requirements 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 . Obtaining the first biochemical mutants and pro- posing the one-geneone-enzyme hypothesis were only two of many advances in which Neurospora played a BARBARAMCCI.IN.I.OCK pioneering role. The problems for which it has since been used are extremely diverse, often ranging far testifies to his confidence in the importance of the afield from the biochemical genetics that first made it research and his persuasiveness as to its value. (There famous. For example, Neurospora soon made funda- was thenno National Science Foundation andno mental contributions to understanding themechanism program of external research support by the National of recombination. It was also used to resolve the great Institutes of Health. BEADLEturned to the Rockefeller confusion existing at that time about fungal chromo- Foundation andthe NutritionFoundation, and to somes and their behavior in meiosis. pharmaceutical firms. See BEADLE1974; KAY 1989.) In 1944, BARBARAMCCLINTOCK visited Stanford Noteveryone was persuaded.HOROWITZ (1979) University at BEADLE’Sinvitation; KELLER (1983, pp. describes how some geneticists continued to resist the 1 13-1 18) describes the visit. MCCLINTOCK’Slong ex- idea that individual enzymes were specified by single perience with maize enabled her toshow convincingly genes. And BEADLE(1974) recalls a wartime visit by that the chromosomes of Neurospora and their be- the mycologist, CHARLESTHOM. After being shown havior in the ascus were typically eukaryotic. Using some of the striking morphological mutants that were simple light microscopy, she went far beyond the known to segregate as single- differences, THOM original objective of determiningthe chromosome took BEADLEaside and advised him “What you need number. She outlined the details of meiosis and de- is a good mycologist. Those cultures you call mutants scribed the seven chromosomes. The smallest Neu- are not mutants at all. They arecontaminants!” rospora chromosomes are now known each to have a At the war’s end in 1945, the Neurospora work had 1 C DNA content less than that of . She progressed substantially and waswidely known. showed that they were nevertheless individually rec- (When I returned to Columbia University from the ognizable by their distinctive morphology at pachy- army, DOBZHANSKYtold me that thetwo highlights in tene (MCCLINTOCK1945; for photographs comparing biology duringthe war had been HUXLEY’Sbook Neurospora and maize pachytene chromosomes see Evolution the Modern Synthesis and BEADLEand TA- Figure 6 in PERKINS1979). She went on to describe TUM’S Neurospora mutants.) Students were attracted pachytene pairing in a translocation heterozygote and to Neurospora. So also were established scientists who to record the ascus types that resulted from different had previously been working on other organisms: D. modes of segregation when the translocation was het- G. CATCHESIDE,STERLING EMERSON, NORMAN GILES, erozygous. At the end of her two-month stay in Cali- HERSCHELMITCHELL, FRANCIS RYAN and MOGENS fornia there was no longer any question: it was clear WESTERGAARD. Duringthis period, Neurospora also that fungal chromosome cytology, like fungal ge- provided the first introduction to research for numer- netics, is basically similar to thatof plants and animals. ous individuals who were later to become known for The 1941 paper of BEADLEand TATUMopened up their work with other organisms. Among those whose exciting possibilities justat atime when war was careers began in this way were EDWARDADELBERG, divertingfunds from pure to appliedresearch and BRUCEAMES, AUGUST DOERMANN, NAOMI FRANKLIN, when young scientists were moving either intoapplied LEONARDHERZENBERG, DAVID HOGNESS,BRUCE research or intothe military. BEADLE’Ssuccess in HOLLOWAY,ESTHER LEDERBERG, , keeping his group intact and in obtainingsupport NOREEN MURRAY,NORORU SUEOKAand CHARLES Perspectives 69 1

YANOFSKY;see, for example, RYAN and LEDERBERG When entire asci are needed for such purposes as (1 946). studying interference, obtaining double mutants, or The Neurospora approach was soon extended to identifying chromosome rearrangements,it was found other fungi such as Ophiostoma and Ustilago. GUIDO thatunordered asci, shotfrom the perithecium as PONTECORVO,who had previously worked on Dro- octets, can easily beobtained in largenumbers sophila with H. J. MULLER, began his program with (STRICKLAND1960). Addition of a centromere marker Aspergillus nidulans. Genetic work flourished on Po- made these unordered groups essentially as informa- dospora, Sordaria, Ascobolus, Coprinus and Schizo- tive as intact asci (e.g., PERKINSet al. 1986). phyllum. Biochemical mutants were obtained in Schi- Neurospora conidia are a boon fortransferring, zosaccharomyces, Chlamydomonas and even in a flow- plating, transforming, preservingstocks and sampling ering plant, Arabidopsis (LANGRIDGE1955). wild populations. These powdery vegetative spores Application of the Neurospora approach to bacteria are potentially hazardous as airborne contaminants, was not long delayed. Auxotrophic mutants of E. coli however. Laboratory practices were quickly devel- were obtained by CHARLES GRAY(a Stanford under- oped that minimized the risk. It was found that if graduate) and TATUM(1 944), and independently by simple precautions are taken, there is no reason why ROEPKE,LIBBY and SMALL(1 944). These made pos- Neurosporacannot coexist in the same laboratory sible the 1946 demonstration of recombination in E. with bacteria, yeast or slowly growingmicroorga- coli and opened theway for theexplosive development nisms. of bacterial genetics. The rapid linear growth of Neurospora (which can Saccharomyces was a relatively slow starter. Heter- exceed 4 mm/hr) is a great advantage for many pur- othallism with two mating types was discovered by poses, butfor platings it was necessary to develop CARL andGERTRUDE LINDEGREN only in 1943. The appropriate media containing colonializing agents first induced auxotrophic mutationsand thefirst link- such as sorbose (TATUM,BARRATT and CUTTER1949) ages were reported in 1949. Eleven workers attended or touse genetic variantswith restricted growth, such the first yeast conference in 196 1 (VON BORSTEL as the conditional colonial mutant cot-1. 1963), compared to 92 participants at a Neurospora Reliable and economic methods were developed for conferenceheld the same year (DE SERRES1962). maintaining permanentstocks in suspended animation (Attendance at international yeast meetings now ex- in silica gel, by lyophilization, or by freezing. These ceeds 1 OOO!) methods (WILSON1986) enable the Fungal Genetics Advantageous features of Neurospora that were Stock Center (1990) to carry over 7000 Neurospora recognized as novel and noteworthy in the 1940s are strains, with no need for periodicserial transfers. now largely taken for granted because the same fea- Along with successes, Neurospora workers inevita- turesare shared in various combinations by many bly experienced frustrations and disappointments. It other organisms that have since come into common was initially hoped that new mutations might reveal use. Neurospora differed in important ways from the previously undiscovered essential metabolites, but animals and plants used by most geneticists in 194 1. none were found. (A prospective new amino acid, It was haploid. All four productsof individual meioses tentatively named neurosporin, proved to be a crys- could be recovered, and in such a way that centro- talline mixture of isoleucine, valine and leucine; see meres were readily mapped. Heterokaryons could be KAY 1989). An elegant scheme to use heterokaryons formed. Nutritional requirements were defined and for quantitative studies of dominance (BEADLEand simple. Stocks could be preserved in suspended ani- COONRADT1944) proved impractical because many mation, effectively conferring immortality on individ- laboratory stocks were heterokaryon incompatible, ual strains. In addition, growth was rapid, generation but this finding opened up the study of vegetative time short andfecundity high. Propagules suitable for incompatibility and led to the finding that genes re- plating were producedabundantly. Pure cultures sponsible for this incompatibility are numerous and could readily be obtained and tested for auxotrophic are highly polymorphic in natural populations. mutations. Recombinant DNA research with Neurospora was Neurospora ascospores are large enough to permit initially impeded by regulatory guidelines that first manual isolation without a micromanipulator. Work- denied permission to proceed, then required that a ers were initially intrigued by the ability to map cen- disabling mutant be built into recipient strains. After tromeres, and geneticanalysis was mostly done atfirst permission was granted, it was found that genes intro- by laboriously dissecting the spores from linearasci in duced by transformation were poorly recovered from serial order. With time, it was realized that ordered crosses, although they remained stably integrated in ascus analysis is rarely necessary and that for most the chromosomes during vegetative growth. The poor purposes random ascospores provide the needed in- sexual transmission proved to be due to RIP (repeat- formation with far less effort (see PERKINS1953). induced point mutation), a process that mutates du- 692 D. D. Perkins

plicate genes during the sexual phase (see below and t-complex of mice, and gamete eliminator in tomato. SELKER1990). RIP was then shown to provide an Far from being defunct, Neurospora continues to effective means of achieving targeted gene mutation, be a superb research organism. At the present time, an asset which more than compensated for the incon- it is used as the primary research object in about 70 venience of poor transmission. laboratories in North America and 25 laboratories in Inevitably, other organisms sometimes proved to be 16 countries abroad. It remains the microorganism of superior to Neurospora for particular purposes. For choice for numerous specific problems. The knowl- example, bioassays using inducedauxotrophs were edge and the geneticresources that have been ac- first developed in Neurospora during the war years, quired during 65 years are invaluable assets. But the but bacteria proved to be so much faster for bioassay most important factor responsible for its wide use is that Neurospora was not used to any extent. probably an exceptionally happy combination of traits Beginning in the 195Os, Neurospora played a cen- that makes it suitable for research on problems span- tral role in studies of recombination, providing the ning the entire range frommolecules to populations. first proof of gene conversion (MITCHELL1955) and The versatility of the organism is illustrated by the revealing its main features (see below). Targeted reg- examples gathered below. Many of the contributions ulation oflocal recombinationfrequencies by rec that will be cited were pioneered using Neurospora. genes thatare unlinked or nonadjacent was discovered Some of the advances were the first for filamentous in Neurospora (CATCHESIDE,~ESSUPand SMITH 1964). fungi, others for the fungal kingdom, and others for Random ascospores of Neurospora continue to be the all eukaryotes. However, the object in citing them is main source of information on recombination control not to stress priority but to illustrate the variety of of this type. Other fungi proved to be superior for research areas to which Neurospora has contributed recombination studies that required tetrads,however. significantly. The list is far from complete. For ex- Conversion frequencies were found to be much higher ample, no attempt has been made to cover the exten- in yeast and Ascobolus than in Neurospora, while sive work on specific enzymes or pathways, oron Sordaria and Ascobolus both had the advantage of novel biochemical mutants;for documentation of numerous viable, readily scorable, autonomously ex- many of these see PERKINSet al. (1 982). pressed ascospore mutants.Neurospora was still a Nutritional mutants were usedfor many purposes. Inter- major source of information on gene conversion in mediate stepsin biosynthetic pathways were determined. By the early 1960s when molecular models for eukaryotic 1944, at least sevendifferent genes had been identified that conversion and crossing over were proposed by HOL- were involvedin the synthesisof arginine (SRBand HOROW- LIDAY and by WHITEHOUSEand HASTINGS.However, ITZ 1944). [For early work on biosynthesis, see the reviews by the mid-I970s, when the more detailed Aviemore by HOROWITZ(1950) and by VOCELand BONNER(1959).] In contrast to what is often the situation in bacteria, genes model was put forward by MESELSONand RADDING, concerned with successivesteps of the samebiosynthetic the most extensive and most critical data came from pathway were shown not to be clustered butto be scattered asci of Saccharomyces, Sordaria and Ascobolus. As a through the genome (for review see HOROWITZ1950). An result of this trend,one geneticist whose interests apparent exception, the aro cluster-gene (GROSSand FEIN focused almost exclusively on recombination models 1960), proved to make a single protein product with seg- asked me bluntly in 1984 what I had been doing since ments that specify five separate enzymatic activities(GAERT- the demise of Neurospora! NER and COLE 1977). In fact, the change of emphasis away from recom- The first conditional biochemical mutants wereidentified bination may have beena blessing in disguise for (STOKES,FOSTER and WOODWARD1943; MITCHELL and HOULAHAN1946). Temperature-sensitivemutants were Neurospora genetics. In my own laboratory, it re- used for testing the one-gene one-enzyme hypothesis (Ho- sulted in attentionbeing given toother problems ROWITZ and LEUPOLD1951). Differentalleles at a locus which might otherwise have beenneglected. Chief were shown to produce forms of an enzyme with qualita- among these was the study of chromosomere- tively different properties (HOROWITZand FLING1953). arrangements (see PERKINS1979). Because deficiency Some mutant strains that lackeda specific enzymatic activity ascospores remain unpigmented while nondeficiency were shown to produce a protein that cross-reacted dith spores are black, Neurospora proved ideal for detect- antibody against the enzyme (SUSKIND,YANOFSKY and BON- ing and diagnosing rearrangements. Meiotic mutants NER 1955). were examined cytologically and genetically, together Complementation of allelic mutationswas demonstrated, first between nuclei in heterokaryons (FINCHAMand PATE- with other mutants affected in development of the MAN 1957; GILES,PARTRIDGE and NELSON 1957), then sexual phase. I also began to collect and analyze Neu- between the protein products in vitro (WOODWARD1959; rospora from natural populations. This led, among for review see FINCHAM1966). other things, to the discovery of Spore killer elements, Translational suppression was analyzed for the first time which beara striking formal resemblance in their at the molecular level (YANOFSKY1956). Perhaps the best behavior to Segregation distorter in Drosophila, the understoodmechanism of metabolicsuppression was de- Perspectives 693 scribed, involving suppression of arg-2 by pyr-3d and pyr-3a prepare plasma membrane vesicles and to demonstrate that by ~rg-12~(DAVIS 1967; REISSIG, ISSALYand ISSALY1967). membrane ATPase is a proton pump (SCARBOROUGH1975; These studies entailed the discovery of two genes for one for review see SCARBOROUGH1978). enzyme, one gene for two enzyme activities, and duplicate Kinetic and genetic studies of amino acid transport (by enzyme activities for two pathways. GABRIELLESTER, DAVID STADLER, MARTIN PALL,GIB Proof was obtained for thechanneling of pathway-specific DEBUSK,and WILLIAMTHWAITES and LAKSHMIPENDYALA) enzymesin separate pools (WILLIAMS,BERNHARDT and identified several transport systemswith broad substrate DAVIS1971). Compartmentation of metabolic pools and specificities, unlike the highly specific "permeases" of bac- pathwayswithin vacuoles, cytosol and mitochondria was teria but resembling the broad-specificity systems of mam- established and studied in detail (WEISS1973) (for reviews maliancells, which had been based on kinetic evidence see DAVIS1986; DAVISand WEISS 1988). alone. Four major amino acid uptake systems were charac- Unlinked genes concerned with the same pathway were terized, distinct from those in yeast (for review see PALL shown to be coordinately controlled (GROSS 1965). Cros- 1970). spathway (general) control of amino acid biosynthetic en- Maternal transmission was demonstrated for a class of zymes was discovered (CARSIOTISand LACY1965; CARSI- non-Mendelian respiratory defects (MITCHELL,MITCHELL OTIS,JONES and WESSELINC1974). Convincing evidence for and TISSI~RES1953). A non-Mendelian cytochrome defect positive control was provided for thefirst time in eukaryotes was transferred between vegetative strains by injecting mi- in a pioneering study of the regulation of sulfur metabolism tochondria (DIACUMAKOS,GARNJOBST and TATUM1965). (MARZLUFand METZENBERG1968); sulfur regulation in Mitochondria were shown to increase in number by division eukaryotes has sincebeen analyzed most fully inNeurospora of preexisting mitochondria rather than being formed de (for review see FU et al. 1990). A hierarchy of regulatory novo (LUCK1963). DNA was isolated from mitochondria for elements involved in phosphate metabolism was identified the first time (LUCKand REICH1964). Progeny were shown and, on the basis of genetic evidence, the novel concept was to receive mitochondrial DNA only from the maternal par- proposed that regulation is not limited to interactions be- ent, in both interspecific and intraspecific crosses (REICH tween regulatory complexes and the DNA sequences of and LUCK1966; MANNELLA,PITTENCER and LAMBOWITZ succeeding elements in the regulatory cascade, but that it 1979). A cyanide-insensitive alternative oxidase was identi- also involves direct interaction between the protein products fied for the first time in fungi (LAMBOWITZand SLAYMAN of regulatory genes (METZENBERGand CHIA 1979) (for 197 1). review see METZENBERG1979). The first sequencing of a nucleic acidfrom mitochondria Mutants with a wide spectrum of altered vegetative mor- (HECKMANet al. 1978) revealed unique features of initiator phologies were obtained and analyzed (e.g., GARNJOBSTand tRNA that foreshadowed the discovery of numerous unex- TATUM1967) and biochemical defects were identified in pected features of mitochondrial genomes (for review see some of them (for review see MISHRA 1977). The morpho- BREITENBERGERand RAJBHANDARY1985). A protein-cod- logical mutant crisp-1 (one of LINDEGREN'Sfirst markers) ing gene was shown to be located withinan intronof another was shown to lack adenylate cyclase activity (TERENZI,FLA- mitochondrial gene (BURKEand RAJBHANDARY1982), ex- WIA and TORRES1974). (The important regulatory signal tending a discovery in yeast to the filamentous fungi. Self- cyclic AMP is therefore absent and must be dispensable in splicing of a mitochondrial intron was first demonstrated Neurospora, unlike Saccharomyces.) The process of coni- (GARRIGAand LAMBOWITZ1984) 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 expres- RNA (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 LAMBOWITZ1986; KUIPER and LAMBOWITZ1988). 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 LAMBOWITZ1987). examined (for review see RAJU 1992b). One of these (RAJU Several key discoveries concern the mechanisms respon- 1986) 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 com- studied early in his career. pleted polypeptides were shown to be present in the cyto- Electrodes weresuccessfully inserted into Neurospora plasm (HALLERMAYER,ZIMMERMAN and NEUPERT 1977). hyphal cells (SLAYMAN and SLAYMAN 1962).Electrophysio- Different mitochondrial receptors were indicated to be re- logical studies showed that glucose transport is driven by a sponsible for theimport of different precursor polypeptides transmembrane proton gradient(SLAYMAN 1970; forreview (ZIMMERMAN, HENNIG andNEUPERT 1981). The first se- see SLAYMAN1987); unlike animal cells, the plasma mem- quence 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 function- 1965). 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 (SCHLEYERand 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 (HAW- 1963; SELITRENNIKOFF,LILLEY and ZUCKER 1981). These LITSCHEK et al. 1988). were used to isolate and characterize plasma membranes, to Mitochondrial were found, with sequences un- 694 D. D. Perkins related to those of mitochondrial DNA (COLLINSet al. 1981). and R. L. METZENBERG,unpublished results). Attraction of Strains were discovered that became senescent following trichogynes to cells of opposite was shown to integration of plasmids into themitochondrial DNA (RIECK, be mediated by a diffusible mating-type specific pheromone GRIFFITHSand BERTRAND 1982; forreview see BERTRAND (BISTIS1983). and GRIFFIrHS 1989). Horizontal transfer of mitochondrial Cytological techniques were perfected and details of plasmids was shown to occur, independently of mitochon- meiosis and ascus development and of chromosome mor- drial DNA (MAYand TAYLOR1989; GRIFFITHSet al. 1990; phology and behavior were examined by light microscopy, COLLINSand SAVILLE1990). both in wild typeand in mutants (for reviews see RAJU1980, The first circadian rhythm in fungi was discovered, man- 1992b). Synaptonemal-complex karyotypes were obtained ifested as periodic conidiation that provided a permanent by reconstructing meiotic prophase nuclei from thin sections record as bands were formed along a growth continuum (GILLIES1972). Recombination nodules were shown to be (PITTENDRIGHet al. 1959). Mutations were obtained that correlated with reciprocal crossing over events at pachytene affect the free-running period length of the circadian clock and to exhibit positive interference (GILLIES1972, 1979; (FELDMAN andHOYLE 1973). Clock-controlled genes were BOJKO1989). Synaptic adjustment of the synaptonemal com- identified that are transcribed only at specific times in the plex was shown to occur in inversion heterozygotes (BOJKO circadian day (LOROS,DENOME and DUNLAP1989). For 1990). Neurospora was the first filamentous fungus for reviews see FELDMANand DUNLAP(1983), LAKIN-THOMAS, which intact DNA molecules from entire individual chro- COT$and BRODY(1 990) andDUNLAP (1 990). mosomes were separated electrophoretically, extending the Resetting the circadian clock was shown to be mediated maximum chromosome length that was then physically re- by a blue-light photoreceptor (SARGENTand BRICCS1967). solvable (ORBACHet al. 1988). Other effectsof blue light were studied, including the Tetrad analysis using a long multiply marked chromo- induction of carotenogenesis, formation of protoperithecia, some arm showed that meiotic crossing over and interfer- and phototropism of perithecial beaks (for review see DEGLI- ence closely resemble those inDrosophila and Zea mays INNWENTI andRUSSO 1984). Mutants were identified that (PERKINS1962). are blind to photoinduction (HARDINGand TURNER1981). The first definitive proof of gene conversion was accom- Heterokaryons (for review see DAVIS 1966) were used, plished in Neurospora (MITCHELL 1955). Important char- first to study dominance and complementation, then to acteristics of conversion were delineated, especially by MARY transfer mitochondria, plasmids and transposable elements, MITCHELL,MARY CASE, NOREEN MURRAY and DAVIDSTAD- to rescue and maintain lethal or deleterious mutations, to LER;see FINCHAM,DAY and RADFORD(1 979). map deficiencies, and to determinemutation frequencies. Genes were discovered that dramatically control the fre- Use of Neurospora heterokaryons led to thediscovery of quency of recombination at unlinked or nonadjacent target vegetative (heterokaryon) incompatibility (for reviewsee sites (CATCHESIDE,JESSUP and SMITH1964), with recombi- PERKINS1988). It was found that the mating type locus nation reduced by dominant alleles at the controllinp loci functions vegetatively as a heterokaryon incompatibility lo- (for review see CATCHESIDE1975). cus (BEADLEand COONRADT1944; SANSOME1945). Other N. crassa was the first fungus to have all linkage groups genes controlling the formation of stable heterokaryons (het mapped genetically (BARRATTet al. 1954) and assigned to genes) were identified and mapped (GARNJOBST1953). Mi- cytologically distinguished chromosomes (forreview see croinjection of incompatible cytoplasm or extracts was PERKINSand BARRY1977). Tester strains that incorporate shown to be lethal to recipient cells (WILSON, GARNJOBST translocations were devised and greatly speeded linkage and TATUM1961). Partial diploids heterozygous for a het detection and mapping (PERKINSet al. 1969). Genes at nearly locus were obtained and shown to behighly abnormal 700 loci and breakpoints of more than 300 rearrangements (NEWMEYERand TAYLOR1967; PERKINS1975). An un- have been mapped (PERKINSet al. 1982; PERKINS1990; linked suppressor was discovered that neutralizes the vege- PERKINSand BARRY 1977;and D. D. PERKINS,unpublished tative incompatibility function of the mating type genes but results). The conventional maps have been complemented not their mating function (NEWMEYER1970). Polymorphic using restriction fragment length polymorphisms (METZEN- het genes were found to be so numerous that they effectively BERG et al. 1984, 1985; METZENBERGand GROTELUESCHEN preclude formation of heterokaryons in natural populations 1990) and random amplified polymorphic DNA markers of N. crassa (MYLYK1976). (RAPD mapping) (WILLIAMSet al. 1991). The mating type genes A and a were cloned, sequenced, Genes specifying 5s RNA were shown to be dispersed and shown to be present in a single copy per genome, with through the genome in single copies(FREE, RICE and METZ- characteristics quite unlike those of the mating type genes ENBERG 1979; SELKERet al. 1981). Telomeres were cloned of yeast. Although A and a occupy preciselythe same locus, and shown to have a DNA sequence identical to that in their DNA sequences were found tocontain no recognizable Homo sapiens (SCHECHTMAN1987, 1990). Random breaks homology (GLASSet al. 1988) (for reviews see METZENBERC inribosomal DNA sequences of the nucleolus organizer and GLASS 1990; GLASSand STABEN1990). Mutations had region were shown to acquire telomere sequences de novo earlier been obtained that inactivate the mating type genes (BUTLER1991). (GRIFFITHSand DELANGE1978). Genes were identified that FollowingMCCLINTOCK (1945), chromosome re- are transcribed preferentially during sexual development, arrangements of various types were identified and put to and cloned sequences of these mating-specific genes were many uses (for review see PERKINSand BARRY1977). Be- used to obtain, by RIP and gene disruption, mutant strains causeascospores that contain deficienciesfail to darken, in which sexual development is impaired (M. A. NELSON frequencies of ejected asci with different numbers of black Perspectives 695 and nonblack spores could be used to distinguish different tween fertilization and fusion of nuclei (SELKERet al. 1987; rearrangement types (PERKINS1974). Genetic analysisof for review see SELKER1990). This phenomenon, termed insertional translocations has been more thorough than in repeat-induced point mutation (RIP), was shown to involve other organisms (see, for example, PERKINS1972). Numer- methylation and C to T mutation in both copies of duplica- ous quasiterminal rearrangements with chromosome seg- ted sequences. RIP can be used to achieve targeted gene ments translocated to telomeres or subtelomere regions inactivation following transformation. [Premeiotic inactiva- were also studied. Insertional and terminal rearrangements tion of duplicated segments has since been shown to occur were shown to generate partial-diploid progeny (DE SERRES in other fungi; for review see SELKER(1 990).] Independ- 1957; ST. LAWRENCE1959; NEWMEYERand TAYLOR1967). ently, BUTLERand METZENBERG(1989) found thatthe The duplications obtained as segmental aneuploids from number of ribosomal DNA repeats in the nucleolus organ- insertional and terminal rearrangements proved useful for izer region undergoes change during the same premeiotic mapping (PERKINS 1975) andstudying for vegetative incom- period that is subject to RIP. patibility (NEWMEYER 1970; PERKINS1975), instability An active was identified, the first to (NEWMEYER andGALEAZZI 1977), and dominance and dos- be characterized molecularly in filamentous fungi (KINSEY age of regulatory genes (e.g., METZENBERGand CHIA1979). and HELBER1989). This LINE-like element was shown to Partial-diploid progeny from crosses heterozygous for ter- be transmitted from one nucleus to another in heterokar- minal rearrangements were found to revert frequently to yons (KINSEY 1990). euploid condition, usually by loss of the translocated seg- Methods were devised for sampling natural populations, ment. and wild-collected strains were analyzed (PERKINS,TURNER Heterokaryons were used to recover and characterize and BARRY 1976;for reviewsee PERKINSand TURNER recessive lethal mutations (ATWOODand MUKAI 1953; DE 1988). Discrete orange colonies found on burnedvegetation SERRESand OSTERBIND1962), to determine the frequency in warm, moist climates were shown usually to represent of recessive mutation for loci throughout the genome (DE pure haploidclones of Neurospora from different asco- SERRESand MALLING 1971; STADLERand CRANE 1979), spores. Fertility in crosses to standard reference strains was and tostudy mutagenesis, DNA repair, anddose-rate effects shown to be a convenient and reliable criterion for deter- (STADLERand MOYER 1981; STADLER1983; STADLERand mining the species of wild-collected isolates. New hetero- MACLEOD 1984). The spectra of mutational lesions were thallic specieswere described. Homothallic Neurospora spe- examined for different and genotypes (e.g., DE cies, devoid of conidia, were also discovered (see FREDERICK, SERRESand BROCKMAN 199; KINSEY 1 and HUNG 1981). UECKERand BENJAMIN1969). An excision-repair mutant was obtained that shows in- Genetic polymorphisms at the protein level were shown creased sensitivity solelyto UV, the first example of its type to be abundant in natural populations of heterothallic spe- in eukaryotes (ISHII,NAKAMURA and INOUE 1991). subset A cies (SPIETH1975), not a foregone conclusion for a haploid of -sensitive mutants was shown to be abnormally organism. Numerous vegetative incompatibilityloci were sensitive to histidine and hydroxyurea, and to cause chro- identified and shown tobe polymorphic (MYLYK 1975, mosome instability (SCHROEDER1986); this includes mem- 1976). Wild populations were shown to carry a loadof bers of two epistasisgroups (KAFER 1983). Many mutants of phase-specificrecessive mutations that adversely affect this subset were found to have abnormal deoxyribonucleo- meiosis and the sexual diplophase (LESLIEand RAJU 1985). tide triphosphate pools (SRIVASTAVAand SCHROEDER1989). The nonselective abortion of asci in crosses betweeninbred Histidine and hydroxyurea were shown to cause chromo- strains of a normally outbreeding species provided an ex- some instabilityin the absence of any mutation causing ample of inbreeding depression in fungi (RAJU,PERKINS and mutagen sensitivity (NEWMEYER,SCHROEDER and GALEAZZI NEWMEYER1987). 1978; SCHROEDER1986), and histidine was found to cause Chromosomal elements (“Spore killers”) were discovered breaks or nicks in DNA (HOWARD andBAKER 1988). that show meiotic drive, resulting in the death of meiotic An endo-exonuclease of Neurospora was characterized products that do not contain the element. Recombination and shown to beimmunochemically related both to the was shown to be blocked in the chromosomal region that RecC polypeptide of E. coli and toan endo-exonuclease that contains the killer element, reminiscent of SD in Drosophila is deficient in the rad52 mutant of Saccharomyces (FRASER, and the t-complex in mice (TURNERand PERKINS1979; for KOA and CHOW1990). review see TURNERand PERKINS199 1). The first DNA-mediated transformation in a sexual fun- Length mutations in mitochondrial DNA were studied in gus was achieved in Neurospora (N. C. MISHRA,SZABO and different N. crassa populations (TAYLOR,SMOLICH and MAY TATUM1973). [Aspergillus niger had been transformed ear- 1986) and were used to construct a phylogenetic tree for lier by SEN,NANDI and A. K. MISHRA (1969).] The proto- four different species (TAYLORand NATVIC 1989). Mito- trophic character, putatively dueto transformation, was chondrial DNAs were also compared in different poorly transmitted through crosses (N. C. MISHRAand TA- populations and species (NATVIG,MAY and TAYLOR1984). TUM 1973), behavior now attributable to RIP but then a cause for skepticism. With the advent of DNA technology Clearly, Neurospora research hastill now been con- and efficient transformation methods (CASEet al. 1979), cerned mostlywith genetic, cellular andmolecular integration of transforming DNA was found to be primarily mechanisms. Relatively little attention has been paid nonhomologous. [For a review see FINCHAM(1989).] toevolutionary biology, or topopulation genetics, Inactivation of duplicated DNA sequences was found to which has been based since its beginnings almost ex- occur premeiotically during the period of proliferation be- clusively on plants and animalswhile the fungal king- 696 D. D. Perkins dom has been largely ignored. Yet the fungioffer organism far beyond what he could have imagined. certain advantages for studying populations, not least Neurospora continues to be a source of innovations of which is haploidy during thevegetative stage. Imag- and surprises. ine what could be done if it were possible to sample animal or plant populations by obtaining individual This essay is dedicated to the memory of B. 0. DODGEon the 120th anniversary of his birth. The summary of research contri- sperm or pollen grains and growingthem up into butions benefited from discussion with numerous colleagues. Their immortal haploid or homozygous individuals. The comments are much appreciated. I am indebted to the Library of equivalent of this is accomplished routinely in Neu- the New York Botanical Garden, Bronx, New York, for the pho- rospora,where orange haploid colonies thatorigi- tograph of DODGE,to HERSCHEL ROMANfor that of LINDEGREN, nated from single ascospores are readily sampled in and to MARJORIEM. BHAVNANIfor the 1947 photograph of MC- CLINTOCK.Work on Neurospora in my laboratory has been sup- the wild, propagated in thelaboratory, and main- ported since 1956 by grant AI 01462 from the National Institutes tained as permanent viable stocks in suspended ani- of Health. mation. Some 4000 strains are already available that have been obtained in this way from populations in LITERATURE CITED many parts of the world. What has been learned from them so far suggests that Neurospora can perhaps AKINS, R. A., R.L. KELLEY and A. M. LAMBOWITZ,1986 Mitochondrial plasmids of Neurospora: integration into mito- become for the population genetics of haploid orga- chondrial DNA and evidence for reverse transcription in mi- nisms what Drosophila has been for diploids (for re- tochondria. Cell 47: 505-516. view see PERKINSand TURNER1988). AKINS,R. A,, and A. M. LAMBOWITZ,1987 A protein required As with Drosophila, attention in the laboratory has for splicing group I introns in Neurospora mitochondria is been focussed primarily on one Neurospora species, mitochondrial tyrosyl-tRNA synthetase or a derivative thereof. Cell 50 331-345. N. crassa, but other species have also come into use. ATWOOD,K. C., and F. MUKAI,1953 Indispensable gene functions The known Neurospora species range from highly in Neurospora. Proc. Natl. Acad. Sci. USA 39: 1027-1035. outbred to highly inbred. Some are heterothallic and BACHMANN,B. J., and N. W. STRICKLAND,1965 Neurospora Bib- cross-fertilizing, others are homothallic and self-fertil- liography and Index. Yale University Press, New Haven, Conn. izing. One species, N. tetrasperma, does not fall into BARRATT,R. W., D. NEWMEYER,D. D. PERK INS^^^ L. GARNJOBST, 1954 Map construction in . Adv. Genet. 6: either category and has been termed pseudohomo- 1-93. thallic. Like its counterparts in other genera, it rep- BEADLE,G. W., 1966 Biochemical genetics: some recollections, resents a breeding system that is based on heterokar- pp. 23-32 in Phage and the Origins of Molecular Biology, edited yosis and is therefore unique to the fungi. N. tetra- by J. CAIRNS,G. S. STENTand J. D.WATSON. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. sperma normally perpetuates itself as a self-fertile BEADLE,G. W., 1974 Recollections. Annu. Rev. Bioch. 43: 1-13. heterokaryon containing haploid nuclei of both mat- BEADLE,G. W., and V. L. COONRADT,1944 Heterocaryosis in ing types. Most conidia and ascospores are heterokar- Neurospora crassa. Genetics 29 291-308. yotic, producing self-fertile cultures that behave as BEADLE,G. W., and E. L. TATUM,1941 Genetic control of bio- though they were homothallic. Aminority of the chemical reactions in Neurospora. Proc. Natl. Acad. Sci. USA 27: 499-506. spores are homokaryotic, however, resulting in self- BERLIN,V., and C. YANOFSKY,1985 Isolation and characterization sterile, functionally heterothallic cultures.The species of genes differentially expressed during conidiation of Neuro- is therefore predominantly inbred, but it retains the spora crassa. Mol. Cell. Biol. 5 849-855. capacity foroutbreeding as a ready option (RAJU BERTRAND,N., andA. J. F. GRIFFITHS,1989 Linear plasmids that integrate into mitochondrial DNA in Neurospora. Genome 31: 1992a). This diversity oflife styles in the various 155-159. Neurospora species shows promise for comparative BISTIS,G. N., 1983 Evidence for diffusible, mating-type-specific studies. trichogyne attractants in Neurosporacrassa. Exp. Mycol. 7: After many years of asking “how” questions about 292-295. the way that Neurospora functions, we should now be BOJKO,M., 1989 Two kinds of “recombination nodules” in Neu- rospora crassa. Genome 32: 309-317. in astrong position to ask “why” questions about BOJKO, M., 1990 Synaptic adjustment of inversion loops in Neu- adaptations, populationsand evolutionary origins.Re- rospora crassa. Genetics 124: 593-598. search on molecular,cellular and genetic mechanisms BREITENBERGER,C. A,, and U. L. RAJBHANDARY,1985 Some is certain to continue. It remains to be seen whether highlights of mitochondrial research based on analyses of Neu- rospora crassa mitochondrial DNA. Trends Biochem. Sci. 10: the promise of Neurospora for population genetics 478-483. will be fulfilled. BURKE,J. M., and U. L. RAJBHANDARY, 1982 Intronwithin the Sixty-five years have passed since SHEARand DODGE large rRNA gene of N. crassa mitochondria: a long open named and described Neurospora, and 50 years since reading frame and a consensus sequence possibly important in splicing. Cell 31: 509-520. BEADLEand TATUMthrust it into prominence. In BUTLER,D. K., 1991 Recombination and chromosome breakage 1952, DODGEfelt that hewould soon be ableto assert, in the nucleolus organizer region of Neurospora crassa. Ph.D. “The old red bread-mold has at last comeinto its Thesis, University of Wisconsin, Madison. own.” Developments since then have taken his favorite BUTLER,D. K., and R. L. METZENBERG,1989 Premeiotic change Perspectives 697

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