[4] genetic and microbiological research techniques - stanford

[4] GENETIC TECHNIQUES FOR N. crassa 79

[4] Genetic and Microbiological Research Techniques for Neurospora crassa

By ROWLAND H. DAVIS AND FREDERICK J. DE SERRES

I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 0

I. B i o l o g y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 0

I I . M e d i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4

S y n t h e t i c M e d i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

C o m p l e x M e d i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 6

So l id M e d i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

I I I . S i m p l e M i c r o b i o l o g i c a l T e c h n i q u e s a n d E q u i p m e n t . . . . . . . . . . . . . . . . . . . . . . . 8 9

T r a n s f e r s a n d S p o t - T e s t i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 9

C o l o n y I s o l a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 0

C o n i d i a l S u s p e n s i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 0

P l a t i n g T e c h n i q u e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2

C o n t a m i n a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3

IV. S tocks a n d S t o c k M a i n t e n a n c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Sil ica Ge l P r e s e r v a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

P u r i f i c a t i o n o f S tocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

G e n e t i c N o m e n c l a t u r e a n d S t o c k R e c o r d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5

T h e N e w s l e t t e r a n d t h e B i b l i o g r a p h y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

S t r a i n s of Neurospora, t h e S t o c k C e n t e r , a n d a G e n e t i c

M a p of Neurospora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 8

V. M e a s u r e m e n t o f G r o w t h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Myce l ia l E l o n g a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

S t a t i o n a r y a n d S h a k e n C u l t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

L o g a r i t h m i c C u l t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

VI . M u t a g e n e s i s a n d M u t a n t S e l e c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

G e n e r a l P r i n c i p l e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

M u t a g e n e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

M u t a n t E n r i c h m e n t a n d S e l e c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 V I I . H e t e r o k a r y o s i s , C o m p l e m e n t a t i o n , a n d D o m i n a n c e . . . . . . . . . . . . . . . . . . . . . . . . 113


H e t e r o k a r y o n T e s t s f o r C o m p l e m e n t a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

D o m i n a n c e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 N u c l e a r Ra t io s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

V I I I . G e n e t i c A n a l y s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122


P r o c e d u r e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 IX. G r o w t h , H a r v e s t i n g , a n d E x t r a c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

L a r g e - S c a l e C u l t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

H a r v e s t i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 E x t r a c t i o n a n d F r a c t i o n a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

80 MICROBIOLOGICAL TECHNIQUES [4]

I n t r o d u c t i o n

This is the first broad description of Neurospora research techniques since that of Ryan t in 1950, or the more general article of Emerson 2 in 1955. Many techniques have been devised or perfected in this time, and there has been an increasing need for a general treatment of the sort presented here. This article is designed as a self-sufficient manual for microbiological and genetic handling of Neurospora for biochemists and for beginning investigators. The article was not designed to review biochemical methods of Neurospora, although a brief treatment of generally applicable methods is included in the last section.

We have attempted to describe the simpler techniques first in each section, followed by more complex modifications applicable to careful and sustained research. One or both authors has had personal experience with almost all techniques described. Readers who use these techniques will find many points at which a procedure can be modified or simplified for a specific purpose.

We have departed somewhat from the usual style of the "Methods in Enzymology" series in that principles of another d i sc ip l ine-gene t ics - are summarized briefly at appropriate points. We hope this will increase the value of the article to biochemists and other workers outside the field of genetics. A full treatment of fungal genetics can be found in books by Fincham and Day, 3 and by Esser and Kuenen. 4 The Neurospora literature is collated in the "Neurospora Bibliography and Index, ''s with supplements in the Neurospora Newsletter (see Section IV).

I. B i o l o g y

Neurospora crassa is a eucaryotic organism, a member of the fungal class Ascomycetes. As an ascomycete, it is related to yeasts, and as a fungus, it is more distantly related to mushrooms. The species is found in tropical or subtropical areas growing on trees and cellulosic plant remains. 6 It has been known in the past as a serious contaminant

1F.J. Ryan, MethodsMed. Res. 3, 51 (1950). 2Emerson, in "Handbuch der physiologisch- und pathologischchemischen Analyse" (F. Hoppe-Seyler and H. Thierfelder, eds.), Vol. 2, part 2, p. 443. Springer, Berlin, 1966.

3.]. R. S. Fincham and P. R. Day, "Fungal Genetics," 2nd ed. Davis, Philadelphia, Pennsyl- vania 1965.

4K. Esser and R. Kuenen, "Genetics of Fungi." Springer, Berlin, 1967 (Engl. Transl. by E. Steiner).

5B. J. Bachmann and W. N. Strickland, "Neurospora Bibliography and Index." Yale Univ. Press, New Haven, Connecticut, 1965.

eC. L. Shear and B. O. Dodge,J. Agric. Res. 34, 1019 (1927).


of bakeries, hence its common name, the pink bread mold. More recently, it has found another habitat as a laboratory organism becaus~ of its simple nutrition ~ and its straightforward biochemistry and genetics. s,9 The primary value of Neurospora in research at present is that the fungus is eucaryotic, can be handled as easily as bacteria, and thus provides a valuable basis of comparison between procaryotes and eu- caryotes in molecular biology.

Neurospora, a heterotroph, can use acetate, succinate, glycerol, glucose, and other monosaccharides, and a number of oligo- and poly- saccharides as carbon sources. Nitrogen can be supplied as nitrate, nitrite, ammonium, and amino acids. Besides carbon and nitrogen, Neurospora requires only a few simple salts, trace elements, and a single vitamin, biotin, for vigorous growth. Despite its multicellular vegetative condition, Neurospora can be grown in logarithmic culture.

Neurospora cells contain normal mitochondria, an endoplasmic reticulum with ribosomes, nuclei with typical nuclear membranes and nucleoli, and various inclusions such as ergosterol crystals, oil droplets, and glycogen bodies? ° Cells are bounded by a double-layered plasma membrane, and a strong cell wall composed of glucose polymers, a peptide-polysaccharide complex, chitin, and polygalactosamine. T M

The vegetative system is composed of multinucleate, branched filaments, or hyphae. The hyphae are segmented by incomplete cross-walls, or septa, which have pores about 0.5/z in diameter in their centers. The pores allow cytoplasm to flow along the hyphae, carrying nuclei, mitochondria, and other inclusions for some distance, usually in the direction of growth. Growth proceeds by hyphal tip extension and by development of branches behind the tips. Considerable hyphal anastomosis takes place within and between strains. A hyphal system is called a mycelium. If hyphae are cut, the resulting drop in cytoplasmic pressure stimulates rapid formation of plugs in septal pores nearby, which prevents serious leakage of mycelial contents.13

Nuclei divide most actively near hyphai tips, and while there is some debate about the events of mitosis in Neurospora, it appears that chromosomes remain in groups comprising entire sets during the division cycle?* Such grouping may prevent chromosome loss during division in

~F. J. Ryan, G. W. Beadle, and E. L. Tatum, Am.J. Botany 30, 784 (1943). 8C. C. Lindegren, Iowa State CoU.J. Sci. 16, 271 (1942). 9G. W. Beadle and E. L. Taturn, Am.J. Botany 32,678 (1945). 1OA. J. Shatkin and E. L. Tatum,J. Biophys. Biochem. Cytol. 6, 423 (1959). 11p. R. Mahadevan and E. L. Tatum,J. Bacteriol. 90, 1073 (1965). I*F. M. Harold, Biochim. Biophys..4cta 57, 59 (1962). a3j. F. Wilson, Am.J. Botany 48, 46 (1961). X4A. N. Namboodiri and R.J. Lowry, .4m.J. Botany 54, 735 (1967).

8 2 MICROBIOLOGICAL TECHNIQUES [4]

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rapidly flowing cytoplasm. In most hyphae there is more than one nucleus in each "cell" (i.e., between successive septa), ranging from about three to as many as 100 in certain unusual strains. 14

Neurospora has three spore forms, two asexual (microconidia and macroconidia) and one sexual (ascospores). In agar slants, aerial hyphae grow upward as the culture matures, and these develop into large masses of orange macroconidia. As macroconidia mature, thick walls develop between them, and they become easily separated by light air movements. Macroconidia are multinucleate, with an average nuclear number of 2.5 in most media? 5 Macroconidia are used to inoculate vegetative cultures, and they serve as the fertilizing (male) parent in sexual crosses.

Microconidia are formed late in growth by a process quite distinct from macroconidial formationJ 6 Microconidia are smaller, almost always uninucleate, and have poor viability. Because they are uninucleate, however, they are useful for some types of mutation experiments.

Ascospores are formed at the end of the sexual process. The process takes place in conditions inimical to vegetative growth, and requires the participation of strains of both mating types, A and a. The ascospore is a long-lived, dormant stage at which genotypes may be isolated in un- ambiguously pure form.

The life cycle of Neurospora is shown in Fig. 1. Neurospora cultures are haploid and morphologically hermaphroditic: a normal strain of either mating type can produce male "gametes" (macro- or microconidia) and female gametes (ascogonia, enclosed in a protective sheath, the whole known as a protoperithecium). Gametic fusion is accomplished by spreading conidia of one mating type over the surface of a culture of the other mating type which has protoperithecia (Fig. 1, bottom). Prior to nuclear fusion, parental nuclei divide many times as a network of ascogenous hyphae develops in the growing perithecium. At the termini of this hyphal system, nuclei of the two mating types associate in pairs in the ascus initials, where nuclear fusion occurs. The resulting diploid nucleus is immediately resolved into haploid products by two meiotic

1~C. Huebschman, Mycologia 44,599 (1952). teR. J. Lowry, T. L. Durkee, and A. S. Sussman,J. Bacteriol. 94, 1757 (1967).

FIG. 1. Semidiagrammatic representation of the Neurospora crassa life cycle. While cultures of both mating types (A and a) can form conidia and protoperithecia (the two gametic cells) the sexual process can be completed only if parents of different mating types are used. The different size-scales used in the diagram are very roughly indicated by the size of the ascospores (30 tz long), and macroconidia (15-25 /.L in diameter), and by the bars next to the rightmost three figures, in each case about 30 # long. A diagram of the meiotic process and ascospore formation is shown in Fig. 6.

84 MICROBIOLOGICAL TECHNIQ.UES [4]

divisions and one mitotic division, which take place in the elongat ing ascus initial (see Section VIII) . T h e final p roduc t o f each meiotic event is an ascus, containing eight ascospores (Fig. 1-, top). From each ascospore, a haploid vegetative cul ture may be obtained. Individual asci may be dissected, or a r a n d o m sample of ascospores f rom many asci, ejected f rom the per i thecium at maturity, may be obtained. A convenient fea- ture o f the Neurospora sexual system is that do rm an t ascospores must be t reated at 60 ° to activate them. This allows crosses to be analyzed at any time af ter they are mature , and the activation t rea tment kills parental cells and o ther contaminants at the same time. T h e two mat ing types segregate in a 1:1 ratio among the ascospores, as two alleles o f a single g e n e .

II. Media

Synthetic Media

1. Vogel's Medium N. 17 This is the most widely used minimal med ium for vegetative cultures. T h e salt mixture can be stored indefinitely as a 50-strength solution, and is p repa red as follows:

T o 750 ml of distilled water, add the ingredients below in order , dissolving each one prior to the addition of the next. This is best done with the aid o f a magnetic stirrer.

Na3 citrate.5 H20 150 g KH2PO4 250 g NH4NO3 100 g MgSO4"7 HzO 10 g CAC12-2 H20 (predissolve in 20 ml H20; add slowly) 5 g Biotin solution (see below) 5 ml Trace element solution (see below) 5 ml

Predissolving the CaCI2 usually prevents precipitation. I f a precipitate forms, it will dissolve on stirring and standing, and biotin and trace elements may be added before it does so. T h e volume of the solution is adjusted to 1 liter. Ch lo ro fo rm (2-3 ml) is added as a preservative, and the solution is s toppered and stored at room tempera ture .

Single-strength medium (medium N) is made by adding 1 part stock solution to 49 parts distilled water, add ing 1.5% sucrose as a carbon source, and autoclaving. T h e final pH is approximate ly 5.8.

17H. J. Vogel, Am. Naturalist 98, 435 (1964). This medium was first described by Vogel in Microbial (;enet. Bull. 13, 42 (1956), but the publication is inaccessible to many workers.


Trace element stock solution is prepared by adding the following ingredients successively, with stirring, to 95 ml of distilled water:

Citric acid-I H~O 5.00 g ZnSO4-7 H~O 5.00 g Fe(NH4h(SO4h'6 H~O 1.00 g CUSO4-5 H~O 0.25 g MnSO4' 1 H20 0.05 g H3BO3 0.05 g Na2MoO4-2 H20 0.05 g

The final volume is adjusted to 100 ml. One milliliter of chloroform is added as a preservative, and the solution is stored at room temperature. The solution may also be used for other synthetic media.

Biotin solution is prepared by dissolving 5 mg of biotin in 100 mi of 50% (v/v) ethanol and is stored in the refrigerator. Cold, aqueous solutions undergo slow decomposition, and this may be true of ethanol solutions. Insufficient biotin causes serious metabolic changes before it interferes with growth, and therefore biotin solutions should be freshly prepared every 6 months. Some investigators freeze small portions in aqueous solution and use one for each new batch of Vogers stock solution. Excess biotin has not been reported to influence growth, metabolism, or crosses.

2. Nitrate, Fries', and Synthetic Crossing Media. These three media, the ingredients of which are given in Table I, differ mainly in nitrogen source. Fries' medium ~,9 is similar to Vogel's medium N, and is little used now because it cannot be made up in concentrated form. A de- hydrated medium similar to Fries' (containing NaNO3 in place of NH4NO3) is available from Difco Laboratories, Detroit, Michigan (No. 0817-01).

Nitrate minimal medium was devised as the basis of a "complete" medium TM (see below). It contains nitrate as the sole nitrogen source. Nitrate minimal medium supports slower growth than Vogel's, but induces better conidiation.

Synthetic crossing medium TM has a low nitrogen content (as nitrate), which encourages the development of protoperithecia and perithecia. The medium is used both for crosses and for certain conidial plating procedures.

All synthetic media,~including Vogel's medium N, may be used with 2%, rather than 1.5% sucrose. In some critical work, the sugar may have

lSN. H. Horowitz,.]. Biol. Chem. 171,255 (1947). lgM. Westergaard and H. K. Mitchell, Am.J. Botany 34,573 (1947).


TABLE I CONSTITUENTS OF FRIES', NITRATE MINIMAL, AND SYNTHETIC CROSSING

MEDIA, PER LITER FINAL VOLUME

Medium

Nitrate Synthetic Ingredient Fries' minimal crossing

(NH4)2 tartrate (g) 5.0 - - NH4NO3 (g) 1.0 -- -- K2 tartrate • 1/2 H20 (g) - 5.0 - KNOz (g) - -- 1.0 NaNOa (g) - 4.0 - KzHPO4 (g) - - 0.7 KH2PO4 (g) 1.0 1.0 0.5 MgSO4 • 7 H20 (g) 0.5 0.5 0.5 CaCI2 (g) 0.1 0.1 0.1 NaCI (g) 0.1 0.1 0.1 Biotin solution a (ml) 0.1 0.1 0.1 Trace element solution ~ (ml) 0.1 0.1 0.1 Sucrose (g) 15.0 15.0 15.0 Final pH 5.6 5.6 6.5

aUse solution described for Vogers Medium N. bUse solution described for Vogers Medium N. Any trace-element salts may be used if they give about the following amounts (milligrams per liter of medium): Zn, 2.0; Fe, 0.2; Cu, 0.1; Mn, 0.02; B, 0.01; and Mo, 0.02.

tO be a u t o c l a v e d s e p a r a t e l y , b u t this is u n n e c e s s a r y fo r r o u t i n e work . Glucose , g lycero l , a n d o t h e r c a r b o n sou rce s m a y be u s e d , t h o u g h t hey

m a y d i f f e r in t h e i r abi l i ty to s u p p o r t g r o w t h . F o r sol id m e d i a (see be low) , 1 . 5 - 2 . 0 % a g a r is a d d e d . F o r g e n e r a l

p u r p o s e s , B a c t o - A g a r (Difco L a b o r a t o r i e s , De t ro i t , M i c h i g a n ; No. 0140-01) is mos t c o m m o n l y used , t h o u g h m o r e h igh ly p u r i f i e d a g a r m a y be neces sa ry on occas ion . W i l d - t y p e Neurospora can g r o w to a ve ry

l i m i t ed e x t e n t on a g a r as a c a r b o n source .

Complex Media

1. Supplementation. Syn the t i c m e d i a m a y be s u p p l e m e n t e d to s u p p o r t the g r o w t h o f a u x o t r o p h i c s t ra ins . C o m m o n i n g r e d i e n t s in " c o m p l e t e " m e d i a a r e yeas t e x t r a c t (0 .5%) , m a l t e x t r a c t (0 .5%) , a n d case in h y d r o l y - za te (0 .5%). Case in h y d r o l y z a t e is a c o n v e n i e n t a m i n o ac id sou rc e , b u t lacks t r y p t o p h a n , wh ich m u s t be a d d e d specif ical ly . A d e h y d r a t e d m e d i u m m a d e by Di fco L a b o r a t o r i e s ( N e u r o s p o r a C u l t u r e A g a r , No. 0321-15) , c o n t a i n s p r o t e o s e p e p t o n e , yeas t ex t r ac t , a n d m a l t o s e a n d can be u sed to g row m o s t m u t a n t s . 2° C o m p l e t e m e d i a , a l t h o u g h u s e d by

2°W. N. Ogata, Neurospora Newsletter 1, 13 (1962).


many workers, may encourage growth of spontaneous mutants, including mutants with impaired assimilatory functions. Another complication of rich media is the inhibitory influence of one compound upon the uptake of another, which leads to a poorer growth of some auxotrophs on complete than on singly-supplemented medium. 21 For these reasons, specific supplementation is preferable to the addition of complex ingredients. The concentrations of supplements are, in general, 100-200 mg per liter for L-amino acids, uridine (for pyrimidine mutants), or adenine (for purine mutants); 50 mg per liter for inositol; and 10 mg per liter for vitamins. It should be noted here that the presence of a degradative pathway for many compounds may cause unusually high requirements for optimal growth. In addition, per- meability differences among derivatives of certain compounds make some forms of a supplement more desirable than others. For example, nicotinamide is used rather than nicotinic acid, and nucleosides are used rather than free bases or nucleotides.

2. Corn Meal Agar. Corn meal agar supports good sexual development, without an abundance of conidia. It may be obtained as a prepared medium from Difco Laboratories in two forms: with glucose (No. 0114-01) and without glucose (No. 0386-01). These are easier to prepare than synthetic crossing medium in small-scale work. Oc-

casional crosses which do well on synthetic medium, however, may do poorly on corn meal agar and vice versa. Moreover, the presence or absence of glucose in corn meal agar may be critical in rare cases. Most strains cross adequately on all three media, but nutritional supplements appropriate to both parents should be added for optimal development and germinability of ascospores (see Section VIII).

Solid Media

1. Tubes ("Slants"). Neurospora grows well in slants of solidified medium. For general purposes, 18 × 150 mm culture tubes (without lip) with about 7 ml of medium are used for stocks, and 10 x 75 mm culture tubes (without lip) with about 1.5 ml medium are used for isolation of many colonies in mutation or formal genetic work. Nonabsorbent cotton should be used for plugs. Many workers use food colors (McCormick Food Colors, Baltimore and San Francisco), available in most markets, to identify different solid media. The colors are used in a ratio of 0.05 ml per 100 ml of medium, and withstand autoclaving.

Ten-by-ten racks for the small tubes may be made out of galvanized or stainless steel wire mesh, or may be ordered from Marlboro Wire Goods Co., Marlboro, Massachusetts. Wire racks of this type are rust-

21H. E. Brockman,J. Gen. Microbiol. 34, 31 (1964).


free and permit gross inspection of tubes for growth without removing them from the rack. Aluminum racks, made by riveting together a solid bottom and two upper plates with 10 × 10 arrays of holes, can be made for 10 × 75 mm tubes by a machine shop at a reasonable cost.

2. Plates: Plating Medium. Neurospora has diffuse, unrestricted growth on ordinary agar media. Tight, colonial growth with little or no conidiation for the first 2 days may be induced by adding L-sorbose to the medium and reducing the concentrat ion of sucrose or other carbon source. 2z In cases where a high percentage of germinat ion is required (e.g., in ascospore platings designed to measure recombination frequencies), glucose and fructose are substituted for sucrose. This circum- vents the inhibition of sorbose upon the initial utilization of sucrose ("sorbose toxicity")/3

In media for plating, 0.05% fructose + 0.05% glucose are used, and sorbose is added to at least 1.0%. This concentrat ion of sorbose re- stricts growth sufficiently in all media so that colonies may be isolated easily under the dissection microscope 6 to 18 hours after plating. For longer incubations with the intent of count ing macroscopic colonies, 1.0% sorbose is suitable if the salt mixture is that of synthetic crossing medium. For Fries' salts, 2.0% sorbose is added, and for Vogel's salts, 3.0% sorbose is added. Extremely compact, small colonies are obtained on synthetic crossing medium lacking sucrose and supplemented with 0.01% glucose and 1.0% sorbose; it is a useful modification where large numbers of colonies are expected/4 In all plating media, 2% agar is used because it forms a dryer surface. Plates are stored or incubated in an inverted position so that condensation will not run onto the agar from the lid. Plastic, 100 × 15 mm petri dishes (Falcon Plastics, Los Angeles, California) are recommended.

3. Plates: Spot-Testing Media. For spot-testing strains for nutrit ional requirements, conidial transfers are made to solidified media in petri dishes (see Section III). Many strains may be tested on a single plate (a square plate with a 6 × 6 grid, Falcon No. 1012, is obtainable). The medium used is made by adding 0.8% sorbose and 0.4% sucrose to Vogel's salts, with 1.5% agar. While growth is restricted by the sorbose, after 1 or 2 days at 25 ° in this medium, vigorous, slow-growing, and nongrowing inocula can be distinguished.

22E. L. Tatum, R. W. Barratt, and V. M. Cutter, Jr., Science 109, 509 (1949). ~H. E. Brockman and F. J. de Serres, Am.J. Botany 50, 709 (1963). ~T. H. Pittenger, Neurospora Newsletter 6, 23 (1964).

[4] GENETIC TECHNIQUES FOR N. crassa ,89

III. Simple Microbiological Techniques and Equipment

Neurospora has a well-deserved reputation as a contaminant. Con- tamination can be minimized by transferring conidia with wetted inoculating loops or applicators, and by performing transfer operations in a chemical hood or a transfer room. Cultures in flasks, tubes, and petri dishes should be kept no longer than necessary. Mycelia should not be allowed to conidiate in petri dishes, and if disposable plates are used, they should be autoclaved soon after use or discarded in a plastic bag that can be sealed. Glassware should be autoclaved before being unplugged or disassembled for washing. Surfaces should be wiped down frequently with Lysol or 70% ethanol. The level of contamination may be checked by exposing petri dishes containing complete medium to the laboratory air. This reveals contamination from an investigator's own work or other sources, and is a way of evaluating different personnel. An "open-plate" test can provide evidence and incentive required for improvement of technique.

Transfers and Spot-Testing

Most investigators transfer Neurospora with flamed inoculation loops or wires (e.g., Nichrome wire inoculating loop, Clay-Adams No. A-95; platinum inoculating wire, Clay-Adams No. A98-A), cooled and wetted in the medium to which the transfer is to be made. Only barely visible inocula need be transferred, and dusty clumps of conidia should be avoided.

Conidial transfers may also be made with the wetted tip of sterile, 6 x 1/12 inch, disposable wood applicators, plain or cotton-tipped. (Plain applicators, made by Hardwood Products Co., Guilford, Maine, can be bought for 75 cents per thousand at most pharmacies.) Appli- cators are useful where Bunsen burners are undesirable, and are sterilized in glass cylinders described below for Pasteur pipettes. The applicators are wetted in sterile water before use, and are discarded afterward into a beaker of Lysol or 70% ethanol.

Spot-testing in most genetic work is done by transferring conidia from many 10 x 75 mm slants to one or more plates of spot-testing medium. A flamed loop, cooled in sterile agar or water, is touched lightly to the conidial mass in the slant and is then touched to the surface of spot-test plates, held inverted. Several plates can be inoculated in succession without returning to the slant. With reference marks and grids, plates can be compared in the manner of bacterial replica plates.


Nutritional tests of leaky strains, or others difficult to classify according to spot-tests, are made in 10 x 75 mm slants of appropriately supplemented medium; with sucrose and no sorbose.

Colony Isolation

Individual colonies may be transferred from plates to tubes in several ways. All or parts of colonies may be taken from the agar under a dissection microscope (20-80x, with substage illumination) with a spear-point needle (teasing needle, Clay-Adams: holder No. A-96; replaceable spea~ point, No. A-94/D), flamed in 70% ethanol. (The needle should not be heated to incandescence.) Hyphae from nearby colonies which lie out of focus in the agar should be avoided. Efficient single-colony isolations are also made simply by coring a plug out of a colony with the tip of a sterile, 6-inch, disposable Pasteur pipette. The agar plug is then blown out into a tube of the appropriate medium. Pasteur pipettes and wood applicators may be sterilized and stored in glass cylinders, 18 inches long and 2 inches in diameter, and sealed off at one end. These tubes are cotton-plugged, or covered at the open end with aluminum foil held fast with tape.

Isolation of single ascospores or of ascospore colonies is described in Section VIII.

Conidial Suspensions

Small conidial suspensions may be made with an inoculation loop if done carefully. Wetted, cotton-tipped applicators may also be used, and if one returns several times to the inoculum tube, dense suspensions may be obtained. Conidial suspensions may be cleared of clumps and mycelia by filtering them through a weft of fine glass wool held in the tapered tip of a segment of glass tubing, sterilized prior to use. Sieve funnels may also be used to filter conidial suspensions. They are made from 20-mm Pyrex tubing with a 20-mm diameter disk of 250- mesh stainless gauze fused into the glass just above a tapered tip, which has a 5-ram opening. The capacity of the funnel should be no less than 10 ml.

An aspirator for preparing conidial suspensions is illustrated in Fig. 2. 25 The upper end is connected to a vacuum, and sterile water is drawn into the bulb, followed by air to dry the tube somewhat. The tip is then held over conidia in a culture tube, which are drawn up and trapped in the water. The vacuum is removed and the suspension is

~SF. J. de Serres, Neurospora Newsletter 1, 10 (1962).


Ftc,. 2. Aspirator for preparing conidial suspensions. The overall length is about 12 inches. The upper segment is made of 8-mm glass tubing. The hole in the upper portion (right) is placed 2.5 inches from the top for suction control with the forefinger. In the lower portion, fused into the bulb, the hook in the bulb is in the same plane as the bend of the tip, and perpendicular to the suction control opening. The opening of the fine tube in the bulb should be near the base of the bulb so that solutions can be blown out of the bulb completely.

blown into sterile tubes. The aspirator may be flushed, and, between uses, sterilized by drawing boiling water into it and standing it on its upper end to drain.

Conidia may be grown conveniently in large numbers in 125-ml Erlenmeyer flasks, containing 25 ml of medium solidified with 2% agar. Conidiation is best if the cultures are grown at 30 ° for 2 days, and then moved to 25 ° in well-lighted conditions~for at least 3 more days. 26 Conidia are suspended by carefully introducing 10-20 ml of sterile distilled water into the flask, which is plugged and swirled. After airborne conidia are allowed to settle, the suspension is poured off through 1 or 2 layers of cheesecloth, held as a bag with a rubber band in the mouth of a 125-ml flask. (These may be sterilized while capped with aluminum foil.) The suspension is centrifuged in sterile centrifuge tube~ with tight cotton plugs, and resuspended in sterile water for use. The method described unavoidably carries the danger of conidial release, and should be performed with care throughout. For conidial production on an even larger scale, see Section IX.

Conidial densities may be measured directly with a hemacytometer or, more conveniently, the turbidity of suspensions may be measured with a Klett-Summerson colorimeter (No. 42 filter) or a spectropho- tometer. Either determination may be related to the viable count determined by plating on sorbose medium (see below). For fresh, wild-type material, 10 Klett units is equivalent to approximately 10 n viable conidia per milliliter. While viability is excellent for as long as 10 or 20 days after conidia form, z7 inocula for physiological experiments should be standardized precisely with respect to medium, temperature, and age. Five-day or week-old cultures are most frequently used as a source of inoculum.

2SM. Zalokar, Am.J. Botany 46, 555 (1959). 2~K. Haard, Neurospora Newsletter 11, 12 (1967).


Plating Techniques

Plating of conidia for counting or for isolation may be done in several ways. For small-scale work, a filtered conidial suspension is prepared and diluted in 10- or 100-fold steps in sterile water to a level of 300- 3000 viable conidia .per milliliter. Samples of 0.1 ml are placed on plating medium (see Section II) and are spread evenly with a bent glass rod. Dilution should be done with a fresh, unplugged, sterile pipette at each step, and suspensions should be well mixed. Dry plates with 2% agar are used so that the suspending medium will be absorbed quickly. Spreading should be done within a minute or less after the suspension is added to the plate. Bent glass rods (3 mm soft glass, about 6 inches long, bent 70 degrees 2 inches from one end) should be fire-polished, but not beaded at the working end, since they must make good contact with the agar. They are flamed in 70% ethanol before use.

Ascospores may be handled with the same methods, with the following ~ additions: First, a hemacytometer count, rather than a turbidimetric measurement, is needed for dilutions. Second,.as explained in Section VIII, a 60 ° heat-shock must be applied for 30-60 minutes to activate ascospores. Third, suspension and dilution of ascospores is best done in 0.1% sterile agar (in water) to prevent rapid settling, and pipetting should be done slowly to prevent accumulation of ascospores at the tip of the pipette.

An alternative plating method, preferred for large-scale work, is to suspend the cells in medium, kept molten at 45 ° in a water bath, prior to pouring it into empty plates. Media are prepared With 75 ml in 123-ml Erlenmeyer flasks (distributed to three plates) or 125 ml in 250-ml flasks (distributed to five plates). Three to 3 replicate flasks are used for each plating. Bubbles which form whenflasks are swirled to suspend the cells may be removed before the medium solidifies in plates by direct flaming with a Bunsen burner.

Colonies should be counted only after they all have made their appearance, usually 3-4 days after plating. One problem is that crowding of cells may lead to competitive interactions which supwess colony formation. 2s This often occurs in platings designed to select rare members of big populations, and is recognized by the appearance of proportion- ately more colonies than expected at successively lower concentrations. The overcrowding, or '~Gi~igg," effect should be tested for by recon- struction experiments if possible, in platings of more than l0 s cells per plate.

28G. W. Grigg, AustralianJ. Biol. Sci. l l , 69 (1957).

[4] CENETIC TECHNIQUES VOR N. crassa 93

Contamination

Contaminated cultures can be purified by single-colony isolation. This is accomplished by streaking a small drop of conidia over the surface of a plate with a loop, and isolating single colonies appearing at the dilute end of the streak. Because conidia are multinucleate, and in any case may stick together, single-colony isolation does not assure genetic homogeneity. Several isolations in series, however, make homogeneity probable unless selection works against it.

A mite infestation is one of the most inconvenient occurrences in a Neurospora laboratory. Mites (often originating from soil) are capable of entering flasks and tubes through cotton plugs, or plates under their lids, then eating, breeding, and fanning out over an entire laboratory. The end result is the presence of mites and cross-contaminants in all cultures. Cultures should therefore be disposed of soon after use, so that they will not attract mites in the first place. It is difficult to free cultures of mites through serial transfer, and the attempt to do so may magnify the problem. Since mites find it difficult to enter or to enjoy silica gel stocks in screw-capped tubes (see Section IV), all strains should be kept in this form. When mites are seen, infected or potentially infected materials are removed and autoclaved, and all surfaces (bench tops, shelves, incubators, racks, containers, the outsides of culture tubes, etc.) are painted with a concentrated solution, in 95% ethanol, of Lin- dane (Gammexane; 1,2,3,4,5,6-hexachlorocyclohexane). 29 This should be handled with rubber gloves, as it is said to be carcinogenic. A safer material is reported 3° to be Kehhane AP (l,l-bis(chlorophenyl)-2,2,2- trichloroethanol, Rohm and Haas, Philadelphia, Pennsylvania). In addition, it is reported that mites and mite eggs can be killed by freezing at - 18 ° for 24 hours, 3° which may permit rescue of unique cultures. Conidia survive this treatment well, ascospores poorly. Neither author of the present review has had experience with mite control or indeed. with mite infestation.

IV. Stocks and Stock Maintenance

Stocks should be maintained with as little growth as possible, to avoid selection of mutations during growth. Strains carrying a mutation in a biosynthetic pathway, for instance, sometimes acquire a second muta-

29Stan|'ord Neurospora Methods, Neurospora Newsletter 4, 21 (1963). Cited with permission. 3°R. E. Subden and S. F. H. Threlkeld, Neurospora Newsletter 10, 14 (1956).


tion in the same pathway. 3t,32 The resulting double-mutant nucleus is strongly selected for in some cases, 32 and in any event is not easily recognized. For these reasons, stocks should not be maintained by frequent vegetative transfer, and one should return to a standard inoculum for each experiment. If stocks are kept as agar cultures, they should be transferred at 3- to 6-month intervals and kept in the refrigerator except for periods of growth. This method has the disadvantage that as mycelia age, differential survival may select for abnormal nuclear types.

Silica Gel Preservation

Maintaining stocks in an inert state was previously done by lyophiliza- tion, but the ease, effectiveness, and low cost of silica-gel preservation has made the latter the preferred method. In this method, conidia are added to anhydrous silica gel. The stocks, which remain inert but viable for years, may be sampled repeatedly by shaking a grain of the gel onto fresh medium. Some workers initiate all experiments with silica gel stocks, while others maintain agar cultures, renewed frequently from silica gel, for routine work. The following description of the preservation method is taken from Perkins az and from Ogata, 2° with minor modifications.

Screw-capped tubes (13 × 100 mm, preferably with Teflon-lined caps), are half filled with silica gel granules (6-12 mesh, grade 40, desiccant, activated, without indicator, Davison Chemical Corp., Baltimore, Maryland). Since humidity indicators included in some gels may be toxic, material containing such dyes should be avoided. With the caps loosened, the tubes are heated in an oven at 180 ° for 1.5 hours to sterilize and activate the gel, and then are allowed to cool with the caps tight. Conidia from 7- to 10-day slants in 18 × 150 mm tubes are used for preservation. The conidia are suspended in reconstituted non- fat dry milk (any common brand will do) which has been sterilized. The suspension is made by introducing 1.0 ml of milk into the slant with a plugged, dry-sterilized Pasteur pipetteL The conidia are worked into suspension either with the pipette or, after plugging the tube, with a Vortex mixer. With the same pipette, the suspension is withdrawn from the culture tube and introduced into prelabeled silica gel tubes. The suspension is exposed as fully as possible to the surface of the gel grains by putting the tip of the pipette all the way to the bottom of the silica gel tube, and allowing the suspension to run out as the

31N. E. Murray, (,enetics 52,801 (1965). 32M. B. Mitchell and H. K. Mitchell, Proc. Natl. Acad. Sci. U. S. 36, 115 (1950). anD. D. Perkins, Can.J. M#robiol. 8, 591 (1962).


tip of the pipette is raised. No more than 0.5 ml should be put into each tube. Heat generated by absorption of water by the silica gel is minimized by cooling the tubes in ice water before and for 10 minutes after the suspension is added. Aconidial strains may be grown on silica gel saturated with medium. When such cultures mature, a substantial amount of activated gel is added and the tube is capped, shaken, and used as a stock thereafter. Since silica gel preservation of normal strains may lead to conidial release, the procedure should be confined to a hood or transfer room.

Preparations are tested for viability and for humidity (grains in the tubes should be "surface-dry") after 5-7 days at room temperature. The tubes are labeled permanently with a glass-etching stylus or with Time tape (Professional Tape Co., Inc., Riverside, Illinois); common gummed paper labels are not satisfactory in the long run. Long-term storage of stocks is done in racks contained in air-tight bread boxes (Tupperware) or lying flat in 12-cm square sandwich boxes. More silica gel, with indicator (Tel-Tale indicating gel, 6-16 mesh, grade 42, Davison Chemical Corp.), is placed in the boxes, and the boxes are taped shut with plastic tape. The indicating gel is regenerated as necessary. Because cold silica gel absorbs atmospheric water vapor rapidly, stocks frequently used should be warmed before opening or be kept at room temperature.

Purification of Stocks

Purification of stocks by serial, single-colony isolation has been described in Section III. In purifying a stock, one should start with the oldest viable culture. The phenotype of the final isolate should be verified, in the case of nutritional mutants, by tests in liquid, stationary cultures (see Section V). Refractory situations may require mating and reisolation of the desired genotype from the progeny. The parent mated to the contaminated stock is chosen with care, and the parentage of reisolated progeny should be recorded (see below). Serious contamination problems can be avoided by having silica gel stocks of all strains.

Genetic Nomencla ture and Stock Records

The rules of nomenclature for Neurospora genetics have been set forth by Barratt and Perkins 34 and by Barratt? 5 While their description should be referred to for details, the basic conventions are summarized here.

34R. W. Barratt and D. D. Perkins, Neurospora Newsletter 8, 23 (1965). 35R. W. B arratt, Neurospora Newsletter 12, 11 ( 1967); ibid., 14, 13 (1969).


1. Every independent mutation must be uniquely designated. The designation includes letters to signify the name of the investigator, the place, or the university, followed by a number, e.g., H263 or UM-728. (A check of the stock lists in the Neurospora Newsletter should be made to avoid prefix duplication.) The designation is used to distinguish a new mutation from all preexisting alleles.

For large-scale mutant selection, a more elaborate system may be required. A common system records the origin of mutants with numbers which identify the starting material, the experiment, and the particular mutant. The three numbers are set off by hyphens; for example, 74A- Y196-M2 designates mutant M2 from experiment Y196, which used strain 74A from which to select mutants.

2. If the mutations are at a new locus, a locus name and corresponding symbol is given, based upon a quite obvious phenotypic attribute of mutant alleles. Auxotrophs are named after the end product for which they are deficient. Where this is not adequate, more fundamental at- tributes such as an enzyme deficiency may be used to name the locus. Symbols based upon loose presumption about gene action (e.g., regu- lators or permeases) should be avoided in consideration of posterity, and confusion with preexisting symbols should be avoided as a tribute to history. Published locus symbols which have been vacated for reasons of synonymy are not to be reused for new mutants.

The locus symbol should be descriptive, distinctive, and short, e.g., arg (arginine), aga (arginase), arom (aromatic amino acids). A number distinguishing different loci of the same name follows the locus symbol, but not as a subscript (i.e., arg-5 or arg5, not args). Closely linked loci once considered alleles of the same gene may be differentiated by a letter while retaining the original number (e.g., ad-3A and ad-3B). New loci are best defined with evidence from both recombination and complementation tests; the former is more critical in practice except in cases of close linkage, and questions of allelism of closely linked mutations may require sophisticated evidence. Unusual nomenclatural problems should be resolved with the Fungal Genetics Stock Center (see below).

3. Superscripts may modify locus names. The wild-type allele is designated with a plus superscript on the locus name or, if context warrants it, on the designation of the particular allele used. Mutant alleles may be given a minus superscript if context warrants it, but this is not necessary as a general rule. Alleles of loci governing response to an antimetabolite are designated by lower-case superscripts s (sensitive), r (resistant), and d (dependent).

Suppressor loci are designated su, and the locus suppressed follows


in parentheses; thus, su(me-2) is the suppressor of methionine-2 mutants. Locus numbers can be used if more than one suppressor locus with the same specificity is found. Where a suppressor shows allele-specificity, the allele of the locus suppressed, rather than the locus name, is used in the parentheses. The wild-type (i.e., inactive) allele of the suppressor locus is designated with a plus superscript; the active allele carries no superscript or a minus superscript. All scxppressor mutations should have allele numbers. Rules governing the renaming of su loci when they are recognized as muitilocus suppressors ("supersuppressors") and, more rarely, when supersuppressors are definitely known to be nonsense or missense suppressors will probably be formalized soon.

4. Strain designations should be distinguishable from designations of individual mutations, because of possible differences in genetic background. (For the same reason, accurate records should be kept on the parentage and mutational origin of each strain.) Multiple mutants should be described in terms of locus names and the alleles present, beginning with those on linkage group I. Each mutation is set off from the next by a comma (for those on the same linkage group) or a semi- colon (for those on different linkage groups), but never by a colon or a hyphen. In publications, locus names rather than allele numbers may be used in most of the text, but the alleles used should always be identi- fied in the materials section. By convention, mating type is given last and is not set off by a punctuation mark.

Stock records should contain locus names, allele numbers, and mutagenic origirt (mutagen and strain) of the component mutation~,~.and the mating type, parentage, enzyme deficiency, phenotypic character (e.g., nutritional requirement), and date of preparation of the stock. The system of maintaining accurate pedigrees used by N. H. Giles at Yale University has been widely adopted. In a cross book, all essential data for every cross is recorded. Each ci-oss is assigned a cross number, and then strain numbers and genotypes are given. The reason for making the cross is given. The cross number provides the basis for the new numbers for progeny that are saved. All progeny saved shoui~be recorded so, that pedigrees can later be followed through successive generations. An example:

CROSS NUMBER: 319-OR2 PARENTS (Number) : 74A-Y152-M4 x J H 3 1 9 - O R I - l a Parental Genotypes: ad-3A A x al-2; inos; pan-2 a Purpose: To extract ad-3A, al-2; inos; pan-2 A Progeny saved: 319-OR2-1A : ad-3A, al-2; inos; pan-2 A

-14a : ad-3A, al-2; inos; pan-2 a -18a : ad-3A; inos; pan-2 a -20A : ad-3A; inos; pan-2 A


Designations of unusual strains such as extrachromosomal mutants, chromosomal aberrations, and heterokaryons also obey established conventions, and may be found in the sources cited. 34,~s

The Newsletter and the Bibliography

The Neurospora Newsletter is published twice a year in cooperation with the Fungal Genetics Stock Center. Besides stock lists, the News- letter publishes new linkage data, an annual Neurospora bibliography, research notes, and technical improvements, to all of which this review owes a great deal. The Newsletter is edited by Dr. BarbaraJ. Bachmann, Department of Microbiology, Yale University School of Medicine, 310 Cedar St., New Haven, Connecticut, 06510. The Newsletter is sent free of charge to bona fide investigators working with Neurospora or closely related organisms who maintain their names on the mailing list.

The Neurospora Bibliography and Index, 5 a definitive bibliography through December, 1963 includes a list of publications arranged by author, a subject index, and a mutant index. An annual supplement to the Bibliography is published in the Newsletter.

Strains of Neurospora, the Stock Center, and a Genetic Map of Neurospora

Most Neurospora strains described in the literature are available from the Fungal Genetics Stock Center, Dartmouth College, Hanover, New Hampshire, 03755. The Center, administered by R. W. Barratt and W. N. Ogata, and supported by the National Science Foundation, provides cultures in numbers less than l0 to research workers and teachers free of charge. Stock lists, with references, are published approximately every two years in the Newsletter, with recent additions in each issue. Investigators are asked to deposit stocks of interest in the Center, but a deposition sheet should be requested before doing so.

Many wild-type strains used in the early Neurospora work were collected independently from several natural sources. Many stocks cur- rently used are therefore heterogeneous with respect to genetic background. A chart of the known relationships among common wild types is given by Barratt. 36 Neurospora work initiated in the future should begin with standard wild types. The most widely used are the St. Lawrence wild types, ST 74A (or a vegetative reisolate, STA-4) and ST 73a. These strains differ in a heterokaryon-incompatibility factor

~R. W. Barratt , Neurospora Newsletter 2, 24 (1962). In this article, strain 74-OR23-1A is incorrectly given as 74A23-1A.


in addition to mating type. The Oak Ridge wild types 74-OR8-1a and 74-OR23-1A, derived from ST 74A and ST 73a by extensive inbreeding, are desirable for reasons of isogenicity and vigor.

Many genetic loci are represented by mutant strains in the Stock Center collection, and different alleles of loci in which allelic diversity is known are available. Classes of Neurospora mutants, with locus symbols and selected general references to gene-enzyme relations, are listed below. A genetic map of Neurospora is presented in Fig. 3, based on recent and original sources.

1. Amino Acid Auxotrophs. Arginine (arg) 4"37 aromatic amino acids (arom), 3s asparagine (asp), 39 aspartate (aspt), cysteine (cys), 31'40-43 glutamic dehydrogenase, TPN-specific (am), 44 glutamine (glm), 45 histidine (hist), 46 homoserine (hs), 4° isoleucine plus valine (iv and val), 47"48 leucine (leu), 49 lysine (/ys), 5° methionine (me), 31"4°-43'51 phenylalanine and/or tyrosine (phen, pt, and tyr), 52 proline (prol-1, arg-8, and arg-9), 4"3~ serine (set), threonine (thr), 53"54 tryptophan (tryp). ~'56

2. Nucleic Acid Auxotrophs. Pyrimidines (pyr), 4"3~ purines (ad). 57 3. Vitamin Auxotrophs. p-Aminobenzoic acid (pab), 42"5s inositol (inos), 59

3rR. H. Davis, in "Organizational Biosynthesis" (H. J. Vogel, J. O. Lampen, and V. Bryson, eds.), p. 302. Academic Press, New York, 1967.

aSN. H. Giles, M. E. Case, C. W. H. Partridge, and S. I. Ahmed, Proc. Natl. Acad. Sci. U. S. 58, 1453 (1967).

8as. W. Tanenbaum, L. Garnjobst, and E. L. Tatum, Am.J. Botany 41,484 (1954). 4°H.J. Teas, N. H. Horowitz, and M. Fling, J. Biol. Chem. 172,651 (1948). 41.]. L. Wiebers and H. R. Garner,J. Biol. Chem. 242, 12 (1967). 4~M. Zalokar,J. Bacteriol. 60, 191 (1950). 4aG. A. Marzluf and R. L. Metzenberg,J. Mol. Biol. 33,423 (1968). 44j. R. S. Fincham and A. Coddington, Cold Spring Harbor Symp. Quant. Biol. 28, 517 (1963). 45E. Reich and S. Silagi, Proc. 11 th Intern. Congr. Genetics 1, 49 (1963). 46A. Ahmed, M. E. Case, and N. H. Giles, Brookhaven Symp. Biol. 17, 53 (1964). 4~R. P. Wagner, A. Bergquist, T. Barbee, and K. Kiritani, Genetics 49, 865 (1964). 4aR. P. Wagner, A. Bergquist, B. Brotzman, E. A. Eakin, C. H. Clarke, and R. N. LePage,

in "Organizational Biosynthesis" (H. J. Vogel, J. O. Lampen, and V. Bryson, eds.), p. 267. Academic Press, New York, 1967.

4aS. R. Gross, Proc. Natl. Acad. Sci. U. S. 54, 1538 (1965). 50j. S. Trupin and H. P. Broquist,J. Biol. Chem. 240, 2524 (1965). 51N. E. Murray, Heredity 15,199 (1960). 52T. I. Baker, Genetics 58,351 (1968). ~M. Flavin and C. Slaughter,J. Biol. Chem. 235, 1103 (1960). 54M. M. Kaplan and M. Flavin,J. Biol. Chem. 240, 3928 (1965). 5SR. Htitter andJ. A. DeMoss,J. Bacteriol. 94, 1896 (1967). ~ej. A. DeMoss, R. W. Jackson, andJ. H. Chalmers, Jr., Genetics 56, 413 (1967). S~H. Bernstein,J. Gen. Microbiol. 25, 41 (1961). 5aM. Zalokar, Proc. Natl. Acad. Sci. U. S. 34, 32 (1948). 5gE. Pifia and E. L. Tatum, Biochim. Biophys. Acta 136, 265 (1967).


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t a t e (ac), 66"67 s u c c i n a t e (suc), 88 a n d f o r m a t e (for), r e s p i r a t i o n d e f i c i e n t

(cyt [ n u c l e a r ] a n d mi a n d poky [ e x t r a c h r o m o s o m a l ] ) , 69 r e q u i r e m e n t f o r

m e t h i o n i n e p l u s a d e n i n e p l u s c y s t e i n e (mac), r e q u i r e m e n t f o r a r g i n i n e

p l u s p y r i m i d i n e p l u s p u r i n e ( a r g - l l ) , p h o s p h o g l u c o m u t a s e ( r a g g e d ,

nOD. Newmeyer and E. L. Tatum, Am.J. Botany 40, 393 (1953). nlR. P. Wagner and C. H. Haddox, Am. Naturalist 85, 319 ( 1951 ). 62L. Garnjobst, Genetics 47, 281 (1962). noB. M. Eberhart and E. L. Tatum, Am.J. Botany 48, 702 ( 1961 ). 64R. B. Flavell andJ. R. S. Fincham,J. Bacteriol. 95, 1056 (1968); ibid. p. 1063. noK. D. Munkres, N. H. Giles and M. E. Case, Arch. Biochem. Biophys. 109, 397 (1965). nOB. S. Strauss and S. Pierog,J. Gen. Microbiol. 10, 221 (1954). e~D. Stone, Arch. Biochem. Biophys. 97, t 99 (1962). nOB. S. Strauss,J. Biol. Chem. 225,535 (1957). nOD. O. Woodward and K. D. Munkres, Proc. Natl. Acad. Sci. U. S. 55,872 (1966).

FIG. 3. Genetic map of Neurospora crassa. The seven linkage groups, I-VII, are aligned at their centromeres (open circles) with the left arm above and the right arm below. The order has been firmly established for genes with lines crossing the verticals, while thepositions of other genes are indicated in a less definite way on the other side of the vertical. A scale of map distance is shown, but it is emphasized that distance varies as much as twofold from cross to cross. The linkage groups have been drawn long enough to accommodate distal markers; the lengths do not imply known limits to the genetic or cyto- logical maps. The T (tyrosinase) and acr-1 (acriflavine) loci of linkage group I are too poorly mapped to include; many other loci, mainly morphological genes, have been omitted, even though well mapped. Locus symbols are given in the text. *Synonyms: arg-8 = prol-3; arg-9 = prol-4; cys-10 = me-4.

This map is based on the following literature: D. D. Perkins, Genetics 44, 1185 (1959); D. D. Perkins and C. Ishitani, ibid., p. 1209; B. Maling, ibid., p. 1215; W. N. Strickland, D. D. Perkins, and C. C. Veatch, ibid., p. 1221; D. D. Perkins, M. Glassey, and B. A. Bloom, Can.J. Genet. Cytol. 4, 187 (1962); D. R. Stadler, Genetics 41,528 (1956); D. Newmeyer and C. W. Taylor, ibid. 56, 771 (1967); K. S. Hsu,J. Gen. Microbiol. 32,341 (1963); D. D. Perkins and N. Murray, Neurospora Newsletter4, 26 (1963); N. Murray, ibid. 1.3, 19 (1968); D. New- meyer, C. W. Taylor, and D. C. Bennett, ibid. 13, 21 (1968); N. H. Giles, M. E. Case, C. W. H. Partridge, and S. I. Ahmed, Proc. Natl. Acad. Sci. U. S. 58, 1453 (1967); M. Ahmad and S. H. Mirda, Neurospora Newsletter 13, 22 (1968); A. A. EI-Eryani, ibid., p. 21; P, A. Walker, ibid. 3, 15 (1963); M. B. Mitchell and H. K. Mitchell, Proc. Natl. Acad. Sci. U. S. 40, 436 (1954); N. E. Murray, Genetics 52, 801 (1965); B. R. Smith, Neurospora Newsletter 1, 16 (1962); H. G. Kolmark, ibid. 13, 23 (1968); D. B. Printz and S. R. Gross, Genetics 55, 451 (1967); M. E. Case and N. H. Giles, Cold Spring Harbor Symp. Quant. Biol. 23, 119 (1958); H. B. Howe, Jr., Microbial Genet. Bull. 18, 12 (1962); K.J. McDougali and V. W. Woodward, Genetics 52,397 (1965).


rg), TM glucose-6-phosphate dehydrogenase (colonial, col-2), 71 requirement for nicotinic acid or tryptophan (nt), oleic acid (ol), choline (chol), TM

nitrate nonutilizing (nit), TM tyrosinase (ty and T), TM ornithine transaminase (ota), TM arginase (aga), TM urease (ure), 77 invertase (inv), D-amino acid oxidase (ox-D), TM /3-glucosidase (gluc), TM cellobiase (cell), TM and several unknown (un) which behave as temperature-sensitive lethals.

5. Antimetabolite Resistance. Acriflavine (acr), actidione (act), ethionine (eth), methyltryptophan (mtr), p-fluorophenylalanine ([pa), fluoroleucine (leu-4), canavanine (can), and sulfonamides (sjb).

6. Morphological and Visible Variants. Albino (al), amycelial (amyc), aurescent (aur), balloon (bal), biscuit (bis), colonial (col and cot, the latter temperature-sensitive), fluffy (fl), frost ([0, osmotic (os), peach (pe), ragged (rg, see above), scumbo (sc), skin (sk), and hyaline ascospores (asco=lys-5). On the genetic map, mating type is designated A/a, linkage group I.

V. Measurement of Growth

There are several methods for the measurement of growth in Neuro- spora. These are (1) measurement of the rate of mycelial elongation in "race tubes," (2) measurement of the amount of growth in stationary or shaken liquid culture after selected time intervals, and (3) measurement of the doubling time in logarithmically growing culture. A discussion of the kinetics of growth in systems (1) and (2) has appeared recently. 80,81

Mycelial Elongation

Mycelial elongation is a convenient parameter because it is a linear function of time and measurable with a meter stick. Race tubes 7 are made by making 45-degree bends, in the same plane, in 400-500 mm

7°S. Brody and E. L. Tatum, Proc. Natl. Acad. Sci. U. S. 58, 923 (1967). 71S. Brody and E. L. Tatum, Proc. NatI. Acad. Sci. U. S. 56, 1290 (1966). raG. A. Scarborough andJ. F. Nyc,J. Biol. Chem. 242,238 (1967). ~3G. J. Sorger, Biochim. Biophys. Acta 118,484 (1966). ~4N. H. Horowitz, M. Fling, H. Macleod, and Y. Watanabe, Cold Spring Harbor Syrup.

Quant. Biol. 26, 233 (1961). ~SR. H. Davis, andJ. Mora,J. Bacteriol. 96, 383 (1968). 7"R. H. Davis, M. B. Lawless, and L. A. Port,J. Bacteriol., in press. 77H. G. K¢lmark, Mutation Res. 8, 51 (1969). 7SE. Ohnishi, H. Macleod, and N. H. Horowitz,J. Biol. Chem. 237,138 (1962). ~M. G. Myers and B. Eberhart, Biochem. Biophys. Res. Commun. 24, 782 (1966). 8°0. J. Gillie,J. Gen. Microbiol. 51, 179 (1968). slO.J. Giltie,J. Gen. Microbiol. 51,185 (1968),

[4] C, ENETIC TECHNIQUES FOR N . crassa 103

lengths of Pyrex tubing (o.d., 15 mm; i.d., 13 mm) about 50 mm from each end. With the ends pointing upward, and the tubes held in position with spring clothespins, the horizontal part of the tube is half-filled with molten agar. The tubes are plugged and autoclaved, and are allowed to cool so that the agar solidifies in a long lane for growth. The tubes should be relatively dry before inoculation. After inoculation and initiation of growth at one end, growth rates are usually constant. The position of the frontier is marked twice a day with a glass marking pen, and the corresponding time is recorded. When growth is complete, measurements are taken with a meter stick and the growth rate may be determined. Wild-type growth rates in minimal media are about 3-4 mm per hour at 25 ° .

Large numbers of short-term determinations of linear growth can be made in modified 18 × 150 mm test tubes. The tubes are made into horizontal growth tubes by heating the glass about an inch from the lip and indenting it to make a dam about 10 mm high, parallel to the lip. The tubes are filled with 10 ml of agar medium, plugged, and autoclaved in a vertical position. The tubes are then laid horizontally, with their indentations straddling a length of glass rod or tubing, and the agar is allowed to solidify.

Because mycelia first spread rapidly on agar media, and then branch and increase in mass behind the frontier, there is no necessary relation between overall extension and dry weight increase, s~ Certain nutritional regimes will limit dry weight greatly while having no effect on the rate of hyphal extension. Amino acids have to be added in very low concentrations to limit linear growth of the corresponding auxotrophs. Agar has sufficient usable carbohydrate so that tests of sugar utilization by this method are not feasible. However, the race tube method is well adapted for studying (1) differences between mycelia, such as complementing heterokaryons, which differ in their metabolic capabilities on minimal medium, and (2) the influences of inhibitory compounds.

Stationary and Shaken Culture

Growth in stationary culture is measured in 10 or 25 ml of medium, contained in 50- or 125-ml Erlenmeyer flasks, respectively. Duplicate or triplicate flasks are inoculated with a drop of conidial suspension for each harvest to be made. Since number and age of conidia strongly influence the onset of visible growth, the conidia should be fresh, and suspensions should be adjusted roughly to a standard density. The inoculum should not yield appreciable turbidity in the growth flask. Growth usually appears at the surface of the medium within a day, and growth up the walls of the flask should be submerged by swirling the


flasks twice each day. If conidia form, dry weights are variable. Growth in the form of a mat is usually complete within 3 days for 10-ml cultures, and within 4 days for 25-ml cultures, at 25 ° or 30 °.

Mycelia are harvested from stationary cultures by rolling up the mycelium, together with the bits that become detached during growth, with a narrow spatula. The mycelium is squeezed firmly with the fingers and laid on a marked towel or filter disk in a pan. Wet mycelium should not be allowed to cling to the paper. Mycelia are dried at 90 ° for at least 4 hours or to constant weight, and weighed to the nearest 0.1 mg on an analytical balance. Some investigators collect relatively large mycelia by filtration so that mycelia can be washed.

A modification of the method is to place cultures, in Erlenmeyer or Florence flasks, on a rotary or reciprocal shaker. The motion keeps aerial elements from forming and aerates the cultures more efficiently. A silicone coating (Siliclad, Clay-Adams; Desicote, Beckman Instru- ments) on the inside of flasks reduces the tendency of mycelium to stick to the glass above the level of the medium. Lightly inoculated cultures of this type do not grow logarithmically. Harvesting must be done by filtration.

Some strains require longer than 3-4 days to reach maximal dry weights, and for most strains, a slow loss of weight follows the attain- ment of maximal weight. Moreover, the rate of growth changes with time. Thus, while no difference in dry weight will be noted between a wild type and a leaky mutant if they are incubated long enough, the leaky mutant may not have initiated visible growth by the time that of wild type is maximal. Both comparisons give a false impression. Despite the drawbacks, stationary cultures are useful. They are interesting "de- velopmental" systems, 26 and certain catabolic enzymes are seen best under these conditions late in growth. The method is adapted to tests of complete auxotrophs for the efficiency with which they use nutritional supplements, if maximal weight is used as a criterion. Finally, while it does not clearly discriminate leakiness from contamination or reversion, the method is the most critical qualitative test of growth because aseptic conditions can be maintained for a long time.

Logarithmic Culture

The last method of measuring growth is the determination of doubling time in logarithmic cultures. Logarithmic growth takes place in vigorously aerated cultures, heavily inoculated with conidia (about 10 e per milliliter). The large number of conidia generally assures a well dispersed culture, and each viable unit remains small enough to double at a constant rate for some time.


Cultures used most by one of us 82 are 200 ml in volume, contained in flat-bottomed Florence flasks (250 ml, Pyrex No. 4060) and fitted with a 22-cm glass aeration tube (o.d., 5 mm; i.d., 3 mm). The aeration tube almost reaches the bottom of the flask, and has a right-angle bend 5 cm from the upper end. The tube is held in the center of the cotton plug of the flask. To retain sterility, one should plug lightly the outer end of the aeration tube. Batches of medium may be made up single strength, inoculated with 1-2 ml of a dense conidial suspension, and 200-ml lots then distributed to dry, sterile culture flasks. Alternatively, 100 ml of medium can be made up double-strength in the culture flask (aeration tubes should clear the surface of the medium during autoclaving) and 100 ml of a double-strength inoculum, in water, may be added later. With the latter method, a single, standard inoculum can be distributed with a sterile, calibrated flask to different types of media. For the preparation of conidial suspensions from 125-ml flask cultures, see Section III.

Cultures are both aerated and stirred with forced air which has been hydrated to prevent the medium from evaporating. Compressed air from a laboratory outlet is bubbled through a liter bottle half full of water (two such bottles in series may be necessary). Water should not be allowed to splash into the outlet of the hydration system. The air is led to a number (6-18) of three-way, brass needle valves (Halvin Co., Brook- lyn, New York; available as aquarium accessories in pet stores) in series, screwed to narrow aluminum plates with vertical saddles which clip onto the edge of a constant-temperature bath. The bath contains the cultures. Each needle valve is fitted with slender rubber tubing, leading to the aeration tube of a culture flask. The air flow is adjusted with the main outlet valve and the needle valves to achieve vigorous bubbling for each culture. Above a certain low aeration rate, growth is not greatly in- fluenced by variation in air supply. At 25 °, wild types double every 2.5 hours in Vogel's Medium N.

Luck describes logarithmic cultures s3 in which aeration is accomplished by shaking. Conidia (106 per milliliter) are introduced into 1000-ml Florence flasks containing 160 ml of medium. The cultures are incubated on a reciprocal shaker set at 160 strokes per minute in a constant temperature. At 25 °, the doubling time is 2.5 hours, s4

Growth is monitored in several ways. First, very early in growth, before harvests are convenient, readings on 5-ml samples are taken with a Klett-Summerson colorimeter, with No. 42 filter. (A No. 54 filter may

S2R. H. Davis, Neurospora Newsletter 8, 12 (1965). SSD.J.L. Luck, Proc. Natl. Acad. Sci. U. S. 49, 233 (1963). S4D. J. L. Luck,J. CellBiol. 24,445 (1965).


also be used. sS) Samples taken aseptically may be returned to the culture. The increase in turbidity corresponds in many cases to the increase in dry weight, but this should always be verified because of possible changes in the optical properties of cells or medium.

Later in growth, small samples (7-30 mg dry weight) of mycelium may be removed with a pipette or a graduate and collected on a 1-inch suction filter with filter funnel (Gelman Instruments Co., Ann Arbor, Michigan, No. 1112). The filter may be used with 2.5-cm Whatman No. 54 or No. 540 filter circles, with identifying marks pencilled on the lower surface. After the volume of medium is gone and the mycelium is washed briefly, the filter funnel is removed. As aspiration continues, acetone from a wash bottle is passed gently and continuously through the moist pad of mycelium to dry it. The mycelium is acetone-free and dry soon after removal of the filter disk from the filter base. In humid climates, a period in a desiccator may be necessary to dry the disk thoroughly. The filter paper grades above permit easy removal of the pad with a fine spatula for weighing. With this method, dry weight determinations are available within minutes of sampling, and appear to be, if anything, less variable than determinations made on oven-dried samples. Many enzymes survive this treatment well, and if an enzyme is detectable in small samples, the same pad may be used both for dry- weight determination and for enzyme assay.

Both optical density and dry-weight determinations on samples are suitable for cultures that remain well dispersed. This state persists, as a rule, to about 1.3 mg dry weight per milliliter of medium, and in some conditions, it persists to 2.0 mg/ml. Once hyphal filaments become long and extensively branched, however, they become entangled and clumped. While growth of the culture may remain logarithmic for considerably longer, s° the entire contents of flasks must be harvested to define the growth curve adequately.

Logarithmic growth can be achieved in 1000 ml or larger cultures without change in character. Large-scale culture techniques are given in Section IX.

VI. Mutagenesis and Mutant Selection

General Principles

In theory, all heritable characteristics of an organism are mutable. Viable mutants for the majority of functions controlled by the genes of

85C. Bowman and R. F.Jones, Neurospora Newsletter9, 17 (1966). sac. W. Slayman and E. L. Tatum, Biochim. Biophys. Acta 88, 578 (1964).

[4] GENETIC TECHNIQ,UES FOR N. crassa 107

Neurospora are difficult or impossible to recover; only about 5 -10% of all mutat ions will be compensa ted for by consti tuents of the most complex m e d i u m Y Mutations o f "indispensable" genes can be recovered only in conditional-lethal forms, such as temperature-sensi t ive or osmotic- and pH-remediab le alleles. Most induced mutat ions impair or eliminate a gene product , and are the re fo re recessive (see Section VII). Because Neurospora is haploid, recessive mutat ions are not masked by dominan t alleles in the same nucleus and may the re fo re be recovered f rom uninucleate cells if they are recognizable and if a med ium can be found to suppor t their growth.

T h e r e are several basic types o f mutat ion. First, single base-pair alterations in the DNA may lead to missense, nonsense, or frameshif ts (one-base delet ion or addition). These types have the c o m m o n proper ty o f revertibility. Second, several to many base-pairs may be lost. Such mutat ions are called multisite mutations, and are nonrever t ib le (except by intergenic suppression, Section VIII) . Th i rd , delet ion o f two or more consecutive genes on a ch romosome may occur; such mutat ions are of ten lethal. Four th , chromosomal r ea r r angemen t s such as transloca- tions or inversions may occur. These may or may not be associated with phenotypic change, depend ing upon whe ther detectable mutat ions occur at the break-points.

T h e spect rum of mutat ional types varies with the mutagen. Spon- taneous mutat ions, and those induced by ultraviolet light and X-rays are o f all types listed above. Most chemical mutagens induce only point mutations. N - M e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNNG, "nitrosoguanidine") and O-methylhydroxylamine (OMHA) p roduce base-pair substitutions, the f o r m e r predominant ly A T to GC, and the latter GC to AT. 88 Ethylmethanesul fonate (EMS) causes p redominant ly base-pair substitutions with two times as many GC to A T than A T to GC transitions, as well as a low f requency o f f rameshif t muta t ions? 9 Nitrous acid (NA) is like EMS, though it p roduces more A T to GC than GC to A T transitions. ~'91 ICR-170, an acridine half-mustard, p roduces predominant ly f rameshif t mutat ions at low doses but base-pair transitions also occur at h igher doses? ~ T h e choice o f mutagen will be critical for some studies. For instance, tempera ture-condi t iona l alleles are for the most part those which specify a complete protein which functions at

8~K. C. Atwood and F. Mukai, Proc. Natl. Acad. Sci. U. S. 39, 1027 (1953). 88H. V. Mailing, personal communication. 8gH. V. Mailing and F.J. de Serres, Mutation Res. 6, 181 (1968). ~H. V. Mailing and F.J. de Serres, Mutation Res. 4, 425 (1967). ~IH. V. Mailing and F.J. de Serres, Mutation Res. 5,359 (1968). ~H. V. Mailing and F.J. de Serres, unpublished, 1968.


25 °, but not at 35 °. The mutagen used to obtain such mutants must be one which induces missense mutations rather than frameshift mutations, since the latter type lead to premature polypeptide chain termination. On the other hand, if "leaky" mutants are to be avoided, a mutagen inducing frameshifts is preferred.

Induction of mutations by many mutagens is accompanied by inactivation of other nuclei in treated conidia. This is useful, since the average number of viable nuclei soon drops to a level at which surviving conidia are effectively uninucleate, and thus homokaryotic for mutations they may carry. (Even without inactivation, there is a sufficiently large uninucleate conidial population to make mutant isolation efficient.) Exces- sive mutagenic doses, however, may induce more than one mutation per surviving nucleus, and in fact, most selected mutants are backcrossed to remove any additional mutations, whether recognizable or not, that may have been induced.

The recovery of specific auxotrophic mutants is greatly enhanced by the filtration-enrichment and inositol-less death methods, described below. Direct selection may be used for resistance mutations and for reversions of auxotrophic strains, merely by plating treated populations of conidia on selective media. More complex rationales based upon duplicate enzymatic functions, 03 requirements for enzyme constitutivity, 94 and enzyme competition TM are also possible. If efficient selective methods for an enzyme deficiency are not available, the genetic control of an enzyme may be approached by searching among exotic wild-type strains for electrophoretic variants. The Fungal Genetics Stock Center has a small collection of such strains from various parts of the world.

Mutagenesis

For all procedures, filtered conidial suspensions, harvested from 7- to 10-day-old slants are used. Both the growth medium and the conidial age should be standardized for regular work. The specific information given below is based on conidia grown on specifically supplemented Fries' minimal medium. The effectiveness of chemical mutagens was judged by reversion of specific auxotrophic strains.

1. Ultraviolet Light (UV). UV is probably the most convenient, all- purpose mutagen. Suspensions (5-20 ml) of conidia (2 × 106 to 107 per ml) in water or 0.067 M phosphate buffer are irradiated at 25 ° in petri dishes. The plates are swirled during irradiation to keep conidia in suspension. A germicidal lamp (15-watt, No. G 15 T 8,

93j. L. Reissig,J. Gen. Microbiol. 30, 327 (1963). ~P. Weglenski, Genet. Res. 8, 311 (1966).

[4] GENETIC TECHNIOUES FOR N. crassa 109

General Electric Co.) is used, calibrated if desired with a Jagger-type dose-rate meter, a5 The lamp housing and the condition of conidia vary greatly from one laboratory to the next, and preliminary survival vs. dose curves should be obtained. In most cases, irradiation at 10 cm distance for intervals varying from 0.5 to 6 minutes will allow a choice of dose for later work. The dose chosen should yield between 20 and 50% survivors, a range which is optimal for recovery of induced mutants. ~ Careful work should take account of photoreactivation by visible light, although rather high light intensities are required, and the spectrum of mutations is not altered, a7

2. X-Rays. Suspensions of conidia (106 per milliliter) in water or 0.067 M phosphate buffer are irradiated at 25 ° in Erlenmeyer flasks. A survival curve is determined for doses between 0 and 35,000 R, applied at a rate of 500-1000 R per minute. Mutagenic treatments used should yield between 20% and 50% survivors. During irradiation, suspensions are stirred with a magnetic stirrer. High concentrations of conidia {over 107 per milliliter) should be avoided, since anaerobiosis and a resulting decrease in mutant frequency is encountered, as

3. Chemical Mutagens. Too few critical experiments with chemical mutagens have been done in Neurospora to reveal whether all conditions used in presently published work, and given below, are crucial. One general observation is that the yield of mutants for a given level of survival is usually greater for chemical mutagens than for X-ray or UV. Perhaps the greatest complication encountered-part icular ly with "ni trosoguanidine"-is the occurrence of multiply mutant nuclei. The resulting danger of falsely assigning several metabolic changes to a single mutational event can be avoided by backcrossing the mutants isolated to wild type one or more times, or by isolating independently and repeatedly mutations having the same constellation of phenotypic characters.

For all chemical mutagens, the conidial concentration during treatment should be about 2 x 107 per milliliter. Solutions used for mutagenesis should be sterile-filtered, rather than autoclaved. Treatments are carried out with shaking in a water bath at 25 °. In all cases, treatment may be stopped by washing the treated conidia with medium or water. However, washing with cold Fries' minimal medium adjusted to pH 8.0 immediately stops the action of alkylating agents and nitrous acid. This method should be used for critical work. Washing may be done by cen-

95j. Jagger, Radiation Res. 14, 394 ( 1961). 96F. J. de Serres and H. Brockman, unpublished, 1963. ~TB. J. Kilbey and F.J. de Serres, Mutation Res. 4, 21 (1967). 9SF. J. de Serres, Radiation Res., 35,524 (1968).


trifugation or on a membrane filter (Millipore or Gelman, 1.2 ~ pore size).

a. NITROUS ACID (NA). To conidia suspended in 3 ml of 0.05 M sodium acetate, pH 4.5, 1 ml of 0.02 M sodium nitrite is added. Treat- ment continues at 25 ° for 40 minutes. 99 The pH is critical, both for survival and for the effectiveness of nitrous acid as a mutagen. While survival of conidia is reported to be 80% under these conditions, 99 inactivation may be rapid under others (e.g., at 37 ° at pH 4.4). The investigator should choose a dose which causes no more than 50% inactivation, though data justifying this choice are admittedly scarce.

b. ETHYLMETHANE SULFONATE (EMS; Eastman Chemical Co., Roches- ter, New York). To conidia, suspended in 0.067 M phosphate buffer, pH 7.0, EMS is added to a final concentration of 0.1 M. Treatment continues for 300 minutes at 25 ° . Under these conditions, survival is reported to be approximately 70% .99

c. O-METHYLHYDROXYLAMINE (OMHA, Sapon Laboratories, Ocean- side, New York). To one milliliter of conidia in B M NaCI is added B ml of the following solution: 10 ml of H20, 17 ml of 4 M NaCI, and 1.59 g of OMHA.HCI; the latter solution is titrated slowly to pH 5.0 with 10 N NaOH before addition. Conidia are treated for 9-16 hours for a survival of 80-90%. l°°

d. METHOXY - 6 - CHLORO - 9- (3- [ ETHYL - 2 - CHLORETHYL] AMINOPROPYL - AMINO)ACRIDINE OIHYI~ROCHLORmE (ICR-170; Dr. Hugh J. Creech, In- stitute of Cancer Research, Fox Chase, Pennsylvania). (Caution: ICR-170 is a very potent mutagen and may be considered a hazard to health. Accordingly, it should be handled with care, and glassware exposed to the compound should be rinsed with dilute ammonium hydroxide before washing.) To 4.9 ml of a conidial suspension in 0.067 M phosphate buffer, pH 7.0, is added 0.1 ml of a solution containing 250/zg ICR-170 per milliliter (in water). Treatment continues at 25 ° for 130 minutes for a survival of 75%. ICR-170 should be used under red light or in the dark to avoid photodynamic effects involving the acridine ring, and conidia should be allowed to grow in the dark for 25 hours after treatment. 99

e. N-METHYL-N'-NITRO-N-NITROSOGUANIDINE (MNNG; Aldrich Chem- ical Co., Milwaukee, Wisconsin). (Caution: MNNG is a potent carcinogen and must be handled with great care.) To 9.5 ml of a conidial suspension in 0.067 M phosphate buffer, pH 7.0, is added 0.5 ml of a fresh, 0.4 mM MNNG solution (58.8/~g per milliliter of H~O). After 1 hour at 25 °, survival is approximately 90%. 88 The final concentration of MNNG

~H. V. Mailing, Mutation Res. 3,470 (1966). t°°H. V. Mailing, Mutation Res. 4,559 (1967).


in this procedure is about 3 /zg/ml. Some investigations have used as much as 50 /~g MNNG per milliliter, in minimal medium, for short times (e.g., 5-10 minutes). While mutants are simple to obtain by this procedure, considerable killing occurs, and probably many multiple mutants are induced. We prefer the low "dose rate," under conditions where growth is minimized.

Mutant Enrichment and Selection

1. Filtration Concentration. The most useful general method of mutant enrichment is by successive filtration, through cheesecloth or glass wool, of a minimal culture inoculated with conidia which have received mutagenic treatment. 1°1,1°2 This process removes a high proportion of non- mutant, germinating conidia, while the mutant, ungerminated conidia pass through the filter and are retained in the medium. At the end, the final conidial suspension is plated on a medium on which the desired type of mutant can grow. The method is not efficient for "leaky" mutants, nor for those which cannot tolerate starvation.

The materials required for filtration-enrichment are (a) a low-speed, rotary or reciprocal shaker to keep cells in suspension in the flask of medium used. (b) A 500-ml Erlenmeyer flask with 250 ml of medium of a type which supports the growth of the starting genotype only. The procedure may be scaled up or down in the volume of flask and medium. (c) A set of dry, sterilized flasks of the same size as used for medium, which have two to four layers of cheesecloth in the form of a bag in the mouth of the flasks, secured with a rubber band. Each flask is covered prior to sterilization with a paper cup or beaker; the cover should permit enough gas exchange for growth. There should be as many fresh flasks as there are filtrations contemplated, although the supply can be renewed in the course of the enrichment procedure. (d) A supply of 250 ml of fresh medium to renew the enrichment medium in the course of the procedure. (e) Sterile centrifuge tubes, pipettes, dilution medium, and plates of minimal and specifically supplemented media for initial plate counts and plating of conidia after the final filtration.

Treated conidia are added in a concentration of 105 to 106 viable cells per milliliter in the flask of 250 ml of medium. A sample is diluted and plated to verify the viable count, if desired. The flask is incubated with shaking at 25 ° or, if temperature-conditional mutants are being selected, at 35 ° . The culture is filtered periodically by pouring it through the

l°aV. W. Woodward, J. R. DeZeeuw, and A. M. Srb, Proc. Natl. Acad. Sci. U. S. 40, 192 (1954). ~°2M. Case, Neurospora Newsletter 3, 7 (1963).


cheesecloth filters into fresh flasks. The cheesecloth is removed aseptically with flamed forceps and the new flask, covered with its cup or beaker, is placed on the shaker. Filtration is first done at 7 hours for chemical mutagens, and at 10-12 hours for UV- or X-irradiated conidia, if incubation is carried out at 25 ° . Thereaf ter , filtration is done every 2-4 hours for the next 24 hours, and every 6-12 hours for the next 24-48 hours. The medium will decrease in volume in the course of filtrations, and a volume equal to the remainder is added when little more growth takes place. The intervals between filtrations are chosen according to how much growth occurs. Any growth which appears to the naked eye should be removed soon, since ungermina ted conidia stick to hyphae, and since any growth will deplete nutrients in the medium. The filtration procedure is terminated when no fur ther growth occurs after the medium has been refreshed. This time may be as early as 48 hours or as late as 72 hours. At the end of the procedure, conidia are concentrated by centr ifugation (as brief as possible to prevent conidial fusion). They are plated on appropriately supplemented medium for isolation of colonies to 10 × 75 mm tubes after 24 hours ' growth. The remainder of the suspension, if any, can be saved in the cold for replating the next day if the first plating is inappropriate for isolation. The yield of mutants of a given class can be as high as 5%. For auxotrophs, the senior au thor usually isolates 1000 colonies for testing.

In many cases nonmutan t conidia survive the filtration procedure sufficiently to swamp out the mutan t population in the final plating. The starting material and the particular medium (Fries', Vogel's, nitrate minimal, etc.) should be chosen by preliminary trial to maximize a high frequency of germination of the wild type in the liquid medium. Germinability can be j udged sufficiently well by inspection of a d rop of culture under a dissection microscope.

2. Inositol-less Death. The second useful method of mutan t enr ichment is the "inositol-less death" method, l°z The starting material for mutan t selection is an inositol-requiring strain (inos, allele 89601), rather than wild type. The inos strain dies quickly if it is incubated in minimal me- d ium without inositol. Mutations may be selected efficiently if they interfere with germination and growth and thereby prevent or forestall the "suicidal" process. The method is analogous to the penicillin method for selecting bacterial mutants.

Conidia of the inos strain are treated with a mutagen and are plated at l0 s to 10 r per plate on 20 ml of minimal sorbose-fructose-glucose agar

1°3H. E. Lester and S. R. Gross, Science 129, 572 (1959).

[4] GENETIC TECHNIQUES FOR N. crassa 1 13

medium. They are incubated for 3-5 days, during which a majority of the population dies. Colonies which appear during this time are inos + revertants or contaminants. The plates are then overlayered with 10 ml of the same medium (molten, at 45 °) which contains 150/~g inositol per milliliter and the nutrient required by the desired class of mutant. Colonies which appear are isolated to tubes of regular medium supplemented with 50 /zg inositol per milliliter and the particular nutrient. Isolates are tested when mature for a requirement in addition to inositol. If the proportion of isolates having no additional mutation is too large, or if the plates, after overlayering, are too thick with growth, the inositoi- less condition should be maintained for a longer time.

Both the filtration-enrichment and the inositol-less death methods are susceptible to interference by "cross-feeding." In some cases, a nutrient may be released from wild-type cells which will support the growth of the desired mutant under the initial, restrictive conditions. In others, an exoenzyme on the surface of conidia of the starting material can act to make substrates available to mutant cells genetically incapable of producing the enzyme. If such mutants are desired, exo- enzymes must be inactivated prior to mutagenesis by incubation for 5-10 minutes in 0.1 N HC1 at 370.1°4 This treatment leaves as much as 30% of the population viable? °5

3. Direct Selection. In selecting reversions and resistance mutations, mutagen-treated conidia are plated heavily on selective media. One should remember that competitive interaction between viable cells and the nongrowing background may take place, which tends to suppress growth of viable cells (see Section III). Further, the isolates recovered should be purified by single-conidial isolation or backcrossing, since the mutants arise in densely inoculated plates.

VII. Heterokaryosis, Complementation, and Dominance

General Principles

In Neurospora, there is no vegetative diploid phase. To study the physiological interaction of homologous genes, different haploid nuclei must be associated in the same cell, where they function in a common cytoplasm. Under these conditions, dominance and complementation may be tested to determine, respectively, whether a mutation is active or passive in comparison to its normal counterpart, or whether nuclei

1°4B. M. Eberhart, personal communication. I~M. S. Sargent, Neurospora Newsletter 14, 11 (1969).


carrying independen t ly isolated mutat ions can compensa te for one an- others ' deficiencies. T h e semantic confusion su r round ing functional interactions merits discussion.

I. Nuclear Phase. A genome is one complete set o f genes; in Neurospora, this cor responds to a set o f seven different chromosomes. Diploid nuclei carry two complete genomes, while haploid nuclei, such as those found in Neurospora mycelia, carry only one. A coenocytic organism, like Neuro- spora, has more than one nucleus per cytoplasmic unit (hypha).

2. Allelic Relations. Alleles are a l ternate forms o f a single gene, coding for homologous forms o f a polypept ide chain. Alleles occupy the same position, in relation to o ther genes, on homologous chromosomes (see Section VIII) . T h e r e are as many possible alleles o f a gene as there are ways o f muta t ing it, including extensive deletions. A heterozygous nucleus is a diploid nucleus in which the members of an allelic pair are different, in contrast to a homozygous nucleus, where they are the same. In a similar sense, a heterokaryon is a coenocytic organism in which the const i tuent nuclei are genetically different , in contrast to a homokaryon, where the nuclear popula t ion is pu re for one type. In Neurospora, the nuclei of heterokaryons, like those of homokaryons, are all haploid.

3. Functional Interactions. Two forms o f a gene (alleles) which differ greatly in their effects on the pheno type may display dominant-recessive relations. T h e interact ion is j u d g e d in a he te rokaryon containing nuclei o f the two types, such as wild type and mutant for an enzyme. At a ratio o f roughly 1 : 1, the dominan t (active) allele de te rmines the pheno type o f the he te rokaryon , and masks or overcomes the effect o f the recessive (inactive) allele. Incomple te dominance is very common, and in fact dominance can be put on a quanti tat ive basis by varying the nuclear ratio.~°6

Epistasis is an interaction between non-allelic mutat ions in a single genome, and is t he re fo re best s tudied in a homokaryot ic system. An example of an epistatic relat ion is a suppressor mutat ion as it overcomes the action of a muta t ion at ano ther genetic locus (see Section VIII) .

Complementation, like dominance , is an interaction o f different nuclei in a he te rokaryon . Complementa t ion involves two recessive mutations, borne by different nuclei o f a he te rokaryon. Intergenic, or nonallelic complementation merely implies two simultaneous dominant-recessive relationships. For instance, a nucleus which is mutan t for orni th ine t ranscarbamylase (arg-12-) but normal in all o the r respects will be com- p lementary to a nucleus which is mutan t for any o ther enzyme, such as adenylosuccinase (ad-4-). Since each nucleus carries the normal , dominant allele o f the mutat ion borne by the o the r nucleus, it is said that

l°~T. H. Pittenger and K. C. Atwood, Genetics 41,227 (1956).

[4] GENETIC TEC.NIQUES VOR N. crassa 115

arg-12 and ad-4 complement. Nonallelic complementation may become quite intimate: mutants lacking different enzymes of one pathway, or even mutants lacking different polypeptides of a single, complex enzyme display nonallelic complementation, meaning that different genes are involved. Nonallelic complementation usually, but not always, involves genes which are easily separable by recombination (see Section VIII).

Failure of similar mutants to complement implies that they are allelic, affecting the same "gene function" (species of polypeptide). However, complementation has often been observed between mutant alleles of the same gene. This is called intragenic or allelic complementation. Allelic complementation reflects the restoration of biological activity of an enzyme when homologous polypeptides, altered in different ways by mutation, aggregate in the cytoplasm of a heterokaryon. The aggrega- tion leads to a mutually beneficial change of polypeptide configuration over that seen in molecules composed wholly of one or the other mutant polypeptide. I°7 Thus complementing alleles cannot be extensive deletions, but must specify inactive forms of the polypeptide, usually recognizable as immunologically cross-reacting material ("CRM"). Moreover, the enzyme affected must be a multimeric protein. It should be noted, finally, that both intra- and intergenic complementation can take place with complex heteromultimeric proteins, of the quaternary form t~ctO/3; a probable case has been described in Neurospora. '°s

4. Heterokaryon Incompatibility. Strains carrying nonallelic mutations may fail to complement because of differences in heterokaryon-compati- bility genes. Several such genes, which interfere with the formation or growth of heterokaryons, are known. '°9 In all cases, an allelic difference between nuclei for these genes is the condition which interferes with heterokaryosis. The mating type locus is one such factor. Heterokaryons of a + a or of A + A combinations can form, but those of the A + a com- bination are unstable or slow-growing. (Mating, as opposed to heterokaryon formation, involves a special cell in which the specificities are reversed.) Several genes are known which interfere with heterokaryosis but which have no role in sexual interaction.

Many workers use the ST 74A strain or its isogenic derivatives, 74-OR23-1A and 73-OR8-1a as basic stocks. All mutants are then selected from them or are introduced into them by several backcrosses. Doing so excludes heterokaryon-incompatibility differences from a set of mutants. In backcrossing, the final isolates to be saved are tested for complementation on a selective medium (see below) with a nonallelic mutant in the

~oTj. R. S. Fincham, "Genetic Complementation." Benjamin, New York, 1966. 1°8S. R. Gross, Proc. Natl. Acad. Sci. U. S. 4 8 , 9 2 2 (1962). l°gL. Garnjobst andJ. F. Wilson, Proc. Natl. Acad. Sci. U. S. 4 2 , 6 1 3 (1956).


standard background. This tests for the presence of heterokaryon- incompatibility factors which may have been retained. Progeny with a genetic background compatible with that of the standard will form heterokaryons within 12-24 hours which grow at a wild-type rate thereafter. Incompatible pairs will not form heterokaryons or, if they form, they will begin to grow considerably later.

Heterokaryon Tests for Complementation

1. Spot Tests. Simple complementation tests are made by mixing concentrated conidial suspensions of auxotrophic strains on the surface of minimal spot-test medium (i.e., with sorbose) in petri dishes. Approxi- mately 0.03 ml of each suspension is placed in spots near the edge of the plates. Each plate contains control spots of the two strains, and two spots where the conidia are mixed and where heterokaryon formation takes place. The spots of mixed conidia are diametrically opposed, so that heterokaryons do not rapidly overgrow one another. Plates are incubated at 25 ° , and results can usually be recorded after 24-36 hours. With complementing allelic mutants, growth may be delayed up to 7-10 days or even longer. For leaky mutants and for large-scale tests, tube methods, below, are preferable to the plate method.

2. Tube Methods. Where a number of auxotrophic mutants are to be tested for complementation in pairwise combinations, grids (or "half- grids") of 10 × 75 mm or 13 × 100 mm tubes with minimal liquid medium, containing sucrose and no sorbose, are used. In addition to the large number of tests than can be run, the method permits a long period of observation.

Grids are set up in a 10 x 10 or other convenient array, with the mutants arranged in the same order on perpendicular axes of a test tube rack. In either size of tube, 1.5-2.0 ml of medium is used. One drop of a conidial suspension of each mutant is added to all corresponding tubes along both axes of the grid. The diagonal thus becomes the homokaryotic control (see Fig. 4). With a full grid, each mutant pair is tested twice; as a rule, the redundancy may be eliminated. The strain from which the mutants were selected should also be tested to provide a comparison for complementing heterokaryons.

Drops of suspensions containing 106 to 107 fresh conidia per milliliter are added carefully with a pipette. This will yield 105 to 106 conidia per milliliter of medium. Metal tube closures are preferable to cotton plugs because they are easily removed and replaced during inoculation. Growth is recorded each day after inoculation, and the pattern is usually clear after 2-3 days. The pattern is interpreted according to the general rules in the next part of this section.


I I

2 2 ( 0 )

3 3

4 + 0 + 0 4 * 0 + 0

5 0 * *- * 0 5 ÷ + * + 0

6 + 0 + 0 + 0 6 0 + 0 + + 0

7 + 0 + 0 * 0 0 7 + + + *

8 * + + + ÷ ÷ + 0 8 0 * -* *

9 * + * * + + * * 0 9 * + * +

I0 + * * + + + + 0 + 0 I0 + + + +

2 3 4 5 6 7 8 9 I 0 I 2 3 4

( b )

÷ ÷ 0

+ 0 + 0 ~[ 0 + + + 0 + + + 0

5 6 7 8 9 1 0

Complementot ion group Mutonts Comp lemen ta t i on group Mutants

I I , 5 I (complementers) : ( 0 ) 5; ( b ) 1 ,8

TT 2 , 4 , 6 , 7 I (noncomplementer ) : 6

] ] I 3 ] I 5 , 9 ,10

]]Z 8 ,10 g I 2 , 4

V 9 I ] l 7

FIG. 4. Complementation grids. Growth is indicated by +, weak growth by +, and no growth by 0. Two entirely different sets of mutants, each numbered 1-10, are shown. (a) Left. A half-grid of mutant-mutant pairings (intersections of vertical and horizontal rows) in which only intergenic complementation takes place. The homokaryotic control is the diagonal. The groups of mutants corresponding to different genes (complementation groups I-V) is given below the grid. (b) Right. A half-grid in which allelic complementation occurs in pairings 1 + 3 and 3 + 8. The interpretation of the map is given below the grid. Note that even if the intragenic complementation had been vigorous, rather than weak, mutant 6, a noncomplementer, would still have defined the members of complementation group I as presumptive alleles. For further details, see text.

Large-scale complementation tests are done in grids of 10 × 75 tubes. Ten-by-ten racks of tubes are sterilized without plugs or medium in the tubes. They are wrapped in a double layer of Saran Wrap when cool enough, covered with sterile blotting paper, and put in heavy paper bags. They are then heated in a drying oven at 60 ° for 60-72 hours to ensure sterility and to tighten the Saran Wrap. Inoculations with 1 ml of each conidial suspension (105 to 106 in medium) are made with hypo- dermic syringes, with 21-gauge needles, through the Saran Wrap. Syringes are boiled between uses, and the two l-ml injections of each tube are made vertically and at opposite sides of the lip. The holes made in the wrapping permit little evaporation of the medium but insure adequate aeration.ll°

With either tube method, by using filtered conidial suspensions of a

"°F. J. de Serres, Neurospora Newsletter 1, 9 (1962).


standard density, quite good data on complementation involving leaky mutants can be obtained. While some leaky mutants initiate growth on minimal medium rapidly regardless of conidial concentration, the lag period of others is lengthened appreciably at low conidial concentration. By using final conidial concentrations of 10 3 per milliliter medium rather than 10 5 to 10 e per milliliter, complementing heterokaryons are recognized as growing sooner than the leaky homokaryons.

3. Analysis of Results. It is not difficult to interpret positive complementation between mutations imposing different nutritional requirements. However, mutants having the same requirement may be selected in large numbers, and complementation tests are usually the first means of distinguishing genes (i.e., those determining different enzymes of a biosynthetic pathway) within the general class.

Fast and vigorous growth of a mixed inoculum usually indicates that intergenic complementation is taking place between the components. The speed and vigor may be judged by its similarity to the behavior of a homokaryon of the strain from which the mutants were selected. A complementation grid, when read at 3 or 4 days, usually reveals clear and self-consistent groups (see Fig. 4a). Groups are defined as sets within which there is no pairwise complementation; the members are considered presumptively allelic. Any mutants which are members of different (nonallelic) groups should complement vigorously. Some groups may be represented by a single isolate, and some, of course, may remain to be selected in further mutant hunts. The relation between complementation tests and tests for recombination (see Section VIII) is that mutants which complement vigorously, because they affect different genes, can usually be expected to recombine easily in crosses. Con- versely, little or no recombination will take place between noncomplementing mutations.

Once a representative member of each group is chosen as a tester, complementation tests may be made between testers and any additional mutants that may be available or later isolated. New groups appear in the form of mutants which complement with all of the testers, and if there is more than one which does so, they must be tested against one another.

Allelic complementation may complicate this pattern (Fig. 4b), but it is not difficult to recognize. First, allelic complementation is almost always weaker and later than nonallelic complementation. Second, many mutations of a given gene are unable to complement with any of their alleles. Strains carrying such mutations are called "noncomplementing" mutants. Noncomplementing mutations are unable to provide mutant polypeptides suited to "successful" protein-protein interactions. (Since

[4] GENETIC TECHNIQUES FOR N. crassa 1 19

nonallelic complementation does not depend upon the presence of mutant polypeptides, a "noncomplementing" mutant is noncomplementing only with respect to its alleles.) The value of a noncomplementing mutant is that it defines as alleles any strains which fail to complement with i t - even strains which complement with one another. Mutants which complement with one another but fail to complement with a third can therefore be interpreted as displaying allelic complementation. It is not unusual to see allelic and nonallelic complementation in the same grid, as illustrated in Fig. 4b.

Full discussions of complementation and allelic complementation maps are available) °7,111

Dominance

A test of dominance asks whether a mutation, often of some non- auxotrophic character such as enzyme constitutivity or antimetabolite resistance, is dominant or recessive to its wild-type allele in a heterokaryon. Unfortunately, the heterokaryon in which the alleles are associated will not usually be distinguishable from a homokaryon carrying the dominant allele. Heterokaryotic association must therefore be en- forced and/or verified. To do this, the alleles to be tested are combined with one of a pair of complementary, auxotrophic markers by crosses. As an example, the allele determining resistance to an antimetabolite (ant r) is associated with a tyrosine requirement (tyr), and its allele (antimetabolite sensitivity, ant ~) with an arginine requirement (arg). Conidia of the two strains, arg, ant ~, tyr + and arg +, a n f , tyr, are then mixed in minimal medium containing the antimetabolite. Only heterokaryotic cells will grow, and then only if the ant r allele can exert its effect. A qualitative result is not useful, however, since disparate nuclear ratios may prevail where roughly equal proportions are desired.

A method for heterokaryon formation which allows some control of nuclear ratio is known. 112 Fresh conidial suspensions of the homokaryons above are mixed in 1:10, 1:1, and 10:1 proportions in centrifuge tubes. The mixtures are centrifuged and decanted. The dense pellets are transferred, undiluted, with Pasteur pipettes and placed as spots on plating medium (with sorbose), containing unlimiting concentrations of arginine and tyrosine, and lacking the antimetabolite. After 24 hours' germination at B0 °, widespread cytoplasmic fusion has occurred. The heterokaryotic spots are relatively coherent, restricted in radial growth by sorbose. The nuclear ratio of the mycelia which grow from sectors of

tltO. J. Gillie, Genet. Res. 8, 9 (1966). ~I2T. H. Pittenger, A. W. Kimball, and K. C. Atwood, Am.J. Botany 42,954 (1955).


the spots upon transfer is related to (but not identical with) the input ratio. Three input ratios are used to accommodate possible disparities in the contributions of conidial types to the initial heterokaryon. Growth and testing of the heterokaryon may be done in two ways:

1. Race Tubes. Sectors of the spots are transferred, with a flattened or spear point needle, to race tubes. The tubes contain minimal medium, lacking arginine and tyrosine, to which the antimetabolite has been added. A control of minimal medium without the antimetabolite is also used. Where growth in the presence of the antimetabolite occurs, it is necessary to know whether, at least in some cases, the nuclear ratios are roughly equal or are greatly biased in favor of ant r nuclei. The nuclear ratio, and its stability during growth, is measured by plating conidia from each end of the tubes in which growth occurs (see below). In heterokaryons, nuclear ratios may be stable, 1°6 they may change in one direction, or they may fluctuate. 113 In some cases, growth may stop and start, and conclusions about dominance may not be possible.

2. Logarithmic Cultures. The formation of multinucleate conidia re- solves a heterokaryon into one heterokaryotic and two homokaryotic conidial classes, with almost random distribution of the nuclear types among conidia. Conidia of a subcultured heterokaryon, made by the method given above, may be used to inoculate minimal logarithmic cultures containing antimetabolite. Under these conditions, any growth must originate with heterokaryotic conidia; vigorous stirring prevents widespread fusion among other classes. Since the average nuclear number per conidium is 2 to 3, and since heterokaryotic conidia must be at least binucleate, the nuclear ratio of heterokaryotic conidia is always close to 1:1. The nuclear ratio of the inoculum and its heterokaryotic component may be inferred by plating a sample of the inoculum (see below). Logarithmic growth from a heavy inoculum involves less opportunity for nuclear selection than race tubes.

Nuclear Ratios

Conidial platings are made to measure nuclear ratio. In the heterokaryon of arg ÷, tyr and arg, tyr ÷, disregarding now the antimetabolite- resistance locus, three classes of conidia are distinguishable. Their frequencies may be inferred by platings on three types of media: (a)' minimal, on which only heterokaryons grow; (b) minimal + tyrosine, on which heterokaryons and arg +, tyr homokaryons grow; and (c) minimal + arginine, on which heterokaryons and arg, tyr + homokaryons grow. A doubly supplemented plate may be used to verify the total inferred from

1taR. H. Davis, Am..]. Botany 47,648 (1960).


the other plates. Often the supplements stimulate or interfere with conidial germination, andAmtial, trials of doubly-supplemented plates should be made. Colony counts are used to infer the frequencies a, b, and r for arg +, t~,r homokaryons, arg, (vr + homokaryons, and heterokaryons, respectively. The calculation of nuclear frequency also requires a value4br the average nuclear number per conidium, ~. While this can be de termined by staining and count ing conidial samples, t5 it is usually safe to assume a figure of 2.5 (see below). With the knowledge of a, b, r, and ~, the frequency, p, of arg +, tyr nuclei among the total conidial population, may be approximated: ~4

r (1 -- r) + a (n-- 2r) P "~ ~ (] -- r) (1)

The frequency of arg, tyr + nuclei, 1 -- p, is given by substituting b for a in Eq. (1). A plot of a, b, and r as a function of p, assuming a typical distribution of nuclei per conidium, with fi = 2.46, is shown in Fig. 5.

hO

0.8

~,- 0.6

'~ 0.4

0.2

I I I I I

, , "---- 0.2 0.4 0.6 0.8 1.0

p, frequency of one nucleor type

FIG. 5. Rela t ionship o f conidial f i equenc ies o f two homokaryn t i c (a and b) and the he terokaryot ic (r) conidial classes with the f r equency o f one nuc lear type in a two-com- p o n e n t he te rokaryon . T h e f requenc ies o f conidia con ta in ing I. 2, 3, etc., nuclei were typical, and as follows: 0.15 (I), 0.46 (2). 0.25 (3), 0.09 (4), 0 .03 'C0. 0.01 (6), 0.01 (7);

= 2.46.

1NK. C. Atwood and F. Mukai, (h,m,tics 40, .138 (1955).


The figure is based on nuclear number distributions A and B of Pit- tenger 115 and the random distribution hypothesis of Prout et al. 11°

The proportion, p', of arg +, tyr nuclei in the heterokaryotic class of conidia, as called for by the second method for testing dominance, is approximated:t,7

p, .~ (1-- r) + a (ff-- 2) (1 - r) (2 )

The effect of misestimation of ~ may be tested by substituting different values in Eqs. (1) and (2). The senior author has rarely encountered values outside the range of 2 to 3.

A recent review of heterokaryosis is available, ns

VIII. Genetic Analysis

General Principles

Some basic concepts and definitions of genetics have been presented in Section VII. Principles of formal genetic analysis are summarized below, sufficient for most biochemical-genetic work in Neurospora. The reader is referred to texts on fungal genetics a'4 or to Barratt et al. no for details about the formal genetics of Neurospora.

Neurospora is a haploid organism, having seven cytologically distinct chromosomes. The genetic counterparts of these are referred to as linkage groups I-VII, represented as formal maps (Fig. 3), and having left (L) and right (R) arms connected through the centromere (see below). In a mating, following fusion of gametic cells and the entry of the male nucleus into the ascogonial cell, numerous divisions of the haploid nuclei of both parents take place. The events which follow are diagrammed in Fig. 6. Nuclei of each mating type associate in pairs (A + a) in the many ascus initials of each perithecium. The members of each pair fuse, yielding a diploid nucleus, or zygote, containing seven pairs of chromosomes. Any given genetic locus is represented twice, and may be heterozygous or, with the exception of mating type, homozygous, depending upon the parents.

The diploid nucleus proceeds immediately into meiosis, which takes

roT. H. Pittenger, Neurospora Newsletter 7, 4 (1965). 1leT. Prout, C. Huebschman, H. Levine, and F.J. Ryan, Genetics 38, 518 (1953). 11¢R. H. Davis, (;enetics44, 1291 (1959). naR. H. Davis, in "The Fungi: An Advanced Treatise" (G. C. Ainsworth and A. S. Suss-

man, eds.), Vol. 2, p. 567. Academic Press, New York, 1966. ~19R. W. Barratt, D. Newmeyer, D. D. Perkins, and l.. Garnjobst, Adwm. Genetics 6, 1 (1954).

[4] GENETIC TECHNIO..UES FOR N. crassa 123

place as the ascus initial matures into an ascus (Fig. 6). Meiosis consists of (1) pairing of homologous chromosomes lengthwise; (2) one division of each chromosome longitudinally into two chromatids, which remain attached at first at the undivided centromere; and (3) two nuclear divisions, which distribute the four strands representing each type of chromosome to four haploid meiotic products. The two divisions are designated Meiosis I and Meiosis II. In Neurospora, each product of meiosis is duplicated in a third, mitotic division ("Meiosis III"), and the eight resulting haploid nuclei are enclosed in ascospore walls. After the walls are formed, a second postmeiotic division takes place, rendering the spores binucleate. The net result of ascus development is an ordered tetrad, comprising four pairs of "sister spores." The ascospores are ordered so that all derivatives of each first-division product are in one or the other half of the ascus, and products of the third (mitotic) division remain adjacent. The spores of a single ascus may be isolated, or ascospores may be collected after ejection from many asci and analyzed as a random sample.

1. Segrregation. By virtue of the separation of homologous chromosomal strands in meiosis, any allelic differences associated with them undergo segwegation. The equal input, duplication, and recovery of parental genetic material leads to a precise 4:4 segregation of any pair of alleles in a single ascus. By the same token, a population of ascospores derived from many asci will display a 1:1 ratio of alleles.

In meiosis, chromatid exchange, or crossing-over, takes place. This is the exact exchange of homologous parts of homologous chromatids after each chromosome has divided, but prior to strand separation in Meiosis I. Exchange takes place between homologous strands attached to different, but homologous, centromeres, and the position of exchanges in the genome vary from one meiotic cell to the next. In an exchange event, entire segments of the participating chromatids, from the exchange point to the distal end (i.e., away from the centromere), are transposed. This is diagrammed for the chromosomes marked A or a in sequence 3 of Fig. 6. If the cell is heterozygous for a gene lying distal to an exchange, both nuclei derived from the first division remain heterozygous, and segregation of alleles takes place in the second division. This will be reflected in the order of ascospores in the mature ascus. A 4:4 arrangement of two alleles (AAAAaaaa) indicates first- division se~egation, while 2:2:2:2 or 2:4:2 arrangements (AAaaAAaa or AAaaaaAA) indicate second-division segregation. The proportion of the latter depends upon the probability of an exchange between gene and centromere.

2. Independent Assortment. If two pairs of alleles lying on different


m

_m

_.1

o

,',,t4~t,,',t4444

°1.,1°1.?L°1°1°1 I/ I/ I/ I/

,.,t ,.,t ot ot

°1 °t °[ °1 \ / \ / ®h,- .41-~

? 1 ° \ /

'~ I~ o .....

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z

[4] GENERIC TECHNIQUES FOR N. crassa 125

chromosomes are considered, the meiotic distribution of each pair will be independent of the other. This is because the polarity of chromosome alignments prior to chromosome movement in the first division is random with respect to alignments of other chromosomes. To this independent assortment may be added the choice of first- or second-division segregation for each gene pair. For any two pairs of alleles, the net result is a characteristic ratio of three ascus types: parental ditypes (PD), nonparental ditypes (NPD) or tetratype (T), as shown in Fig. 6. If the two loci lie on different chromosomes, the number of PD asci is equal to the number of NPD asci. 12° The frequency of T asci is determined by the second-division segregation frequencies of the two genes. Given equal numbers of PD and NPD asci and the equality of all genotypes in T asci, independent assortment yields equal numbers of the four genotypes among ascospores derived randomly from a large number of asci. The result is 50% parental, and 50% recombinant products. Recombination by independent assortment can be used to construct many multiply mutant strains, or, in a cross to wild type, to resolve many multiply mutant strains into their components. It must be borne in mind that genes on the same chromosome may yield 50% recombination if they are distant enough from one another for crossing over to randomize parental gene associations (see below).

3. Linkage and Mapping. Linked genes are close enough to one another on a single chromosome so that crossing over does not entirely randomize parental associations of their alleles. As distances along the chromosome increase, the probabilities of chromosome exchange in-

l~°D. D. Perkins, Genetics 38, 187 (1953).

FIG. 6. Diagram of meiosis in Neurospora, in which only two of the seven types of chromosomes are pictured. The nuclear events in the development of the ascus, starting with the ascus initial, are shown at the bottom of the figure, with lines connecting the products of the nuclear division which is occurring. After the formation of the diploid nucleus, in which homologous pairs of chromosomes (marked with allele pairs A/a and B/b) reside, three different alignments (1, 2, and 3) of chromosomes are shown and followed through three nuclear divisions, Meiosis I, II, and III. (1) This alignment leads to first- division segregation of both A/a and B/b, and the formation of a parental ditype (PD) ascus~ (2) This alignment leads to first-division segregation of both A/a and B/b, but the resulting ascus is a nonparental ditype (NPD). (3) This al ignment is similar to the first, but chromatids of the longer chromosome pair have undergone crossing-over. Since chromosome movement occurs by means of the centromere, the first-division products remain heterozygous for A and a. T he final result is an ascus showing second-division segregation for the A/a pair (note the 2:2:2:2 order for A/a among ascospores). Because the Bib pair underwent first-division segregation, four genotypes are present among the ascospores, and, the~ascus is a tetratype (T) with respect to the A/a and B/b genes.


crease, but frequencies o f recombinant meiotic products tend toward 50%. This limit is de t e rmined by the occur rence o f multiple crossovers, by which an exchange between a pair o f chromat ids can break up genetic associations created by a previous one.

Simple linkage studies are best p e r f o r m e d with r a n d o m ascospores f rom a cross. A genetic map unit in Neurospora, as in any organism, cor responds to 1% recombinant progeny. It is understood that both reciprocal classes of recombinants are considered, and that the distance measured is not so great that a significant n u m b e r o f undetec ted , multiple exchanges occurs. Crosses in which the latter occurs will yield underes t imated map distances; genes yielding 15% recombinant progeny may be significantly more than 15 map units apart . 119,121 This may be checked with a third marke r between the two first used, in a three- point cross.

T h e analysis of a three-point cross, where all genes are linked, allows de terminat ion o f gene o rde r as well as map distance. Mapping a mutan t may be illustrated by the following example. A new muta t ion (ota) 7~ was found to be linked to the gene ad-4: in a cross between ota (= ad-4 +, ota-) and ad-4 (= ad-4-, ota+), the p rogeny were 88 ad-4 +, ota-; 82 ad-4-, ota+; 16 ad-4-, ota-; and 13 ad-4 +, ota +. T h e recombinants (the last two classes) were 14.6% of the 199 isolates tested. T h u s ota could lie e i ther about 15 units to the right or about 15 units to the left ofad-4. T o decide this, the double mutant , ad-4-, ota-, was mated to tyr-l-, known to be linked to ad-4. T h e results are shown in Table II. It is clear that similar numbers o f each genetically reciprocal class are obtained. T h e p rogeny are first a r ranged in reciprocal pairs, which are then a r r anged in de- scending o rde r of f requency. T h e most f r equen t pair has, as expected, the same genotypes as the parents. T h e pairs occurr ing in in te rmedia te frequencies are single crossover classes, represen t ing crossovers in one or the o ther o f the two intervals def ined by the three mutations. T h e least f requen t pair, often entirely absent in the progeny, is the double- crossover class. T h e gene o rde r may be de t e rmined simply by a compar i - son of parental and double crossover classes. T h e terminal markers are those which remain in parental conf igurat ion in the double crossover class. In this example, ad-4 + remains associated with tvr-1-; ad-4- remains associated with t~,r-I +. T h e medial marker , ota, is the one which reverses its relation to the others in the double -mutan t class.

Next, the map distances co r re spond ing to the two adjacent regions are calculated according to the percent recombinat ion observed. Region I, bounde d by ad-4 and ota, has 10 + 7 + 1 + 1 = 29 rec0mbinants (the

1211). D. Perkins, Genetics 47, 1253 (1962).


TABLE II DESCRIPTION AND TABULATION OF RESULTS FROM A THREE-POINT CROSS:

ad-4, ota × t y r - P

Chromosomal arrangement of alleles in the diploid

ad-4 ÷ ota + tyr-1

ad-4 ota tyr-I +

Progeny Number

Parental genotypes: ad-4 + ota + tyr-1 75 ad-4 ota tyr-1 + 68

Single crossovers, Region I: ad-4 + ota tyr-1 + 10 ad-4 ota + tyr-I 17

Single crossovers, Region II: ad-4 + ota + tyr-I ÷ 10

ad-4 ota tyr-I 15

Double crossovers: ad-4 + ota tyr-1 1

ad-4 ota + tyr-1 + 1

197

Genetic map ad-4 Reg I ota Reg II tyr-I

14.7 13.7 units units

aData from R. H. Davis and J. Mora, J. Bacteriol. 97,383 (1968). The correct gene order is determined from the relative frequencies of the reciprocal classes of progeny (see text) and is used in the presentation of genotypes.

double crossover class, if present, should always be considered) for a frequency of 14.7%. Similarly, 13.7% recombination occurs in Region II. The cross allows us to place ota near to tryp-i on linkage group III (see Fig. 3). Because recombination values vary considerably f rom one cross to the next (the ad-4-tyr-1 distance, 28.4 units here, is 39 units on the map), the question of whether ota is proximal or distal to tryp-1 must be resolved with a three-point cross involving ota, tryp-1 and some other linked marker . Another format of present ing the same results, shown in Table III, is the s tandard form in which three-point linkage data are published.

The initial localization o f a new mutat ion to a particular linkage group has become quite efficient th rough the use of a strain, "alcoy, ''122"I2s which contains three chromosomal aberrations. With this strain, several chromosome arms may be tested conveniently and simultaneously in mutan t localization. The aberrations create c o m p o u n d chromosomes

n2D. D. Perkins, Neurospora Newsletter 6, 22 (1964). n3D. D. Perkins, Neurospora Newsletter 9, 11 (1966).


TABLE I11 STANDARD FORMAT FOR PUBLICATION OF THREE-POINT LINKAGE DATA, a

USING DATA OF TABLE 11

Total; Zygote genotype Recombinations percent

and percent Parental Singles Sir/gles Doubles germination; recombination combinations Reg. I Reg. II linkagegrot~p

Allele numbers

+ + tyr-1

ad-4 ota +

14.7 13.7

75 68 10 17 10 15 1 1 197 F6

82% UM-728

IIIR Y6994

aD. D. Perkins, Genetics 44, 1185 (1959).

such that IR and IIR are joined; IVR and VR are joined; and I I IR and VIL are joined. The first compound is marked with a mutat ion albino (al-1); the second with colonial (cot-l, a temperature-sensitive colonial); and the third with yellow (ylo-1). The alcoy strain is perfectly viable at 25 °, but in matings the formation of viable recombinants by crossing over between the compound chromosomes and the corresponding portions of a normal genomes is severely suppressed. Independen t assortment, however, takes place normally. The net effect is that linkage (less than 50% recombination) between a new mutat ion and one of the alcoy markers shows that the new mutat ion lies on one of the two chromosome arms marked by that alcov marker. Independen t assortment with all alcov markers excludes the six components of the alcoy system from fur ther consideration, and leads immediately to tests with markers on other chromosome arms.

4. Allelism. Perhaps the most important thing to establish initially about new mutations is their possible allelism with o ther mutat ions having similar phenotypes.

Simple tests of allelism are made with crosses. I f two similarly auxotrophic strains are mated in a test of" allelism, one of the two recombinant classes expected if they are not allelic (the a+b ÷ class in the cross a+b - × a-b +) is prototrophic. These will range in percentage f rom low values up to a maximum of 25%, depending upon linkage relations. Where recombination occurs, the percentage of prototrophs equals half" the map distance, since only one of the two recombinant classes is recognized. Ascospores from the mating may simply be plated on minimal medium for the detection of" prototrophic recombinants. No prototrophs, or at most, 0.1% of the population plated, are expected if the mutations in the parents are truly allelic.

Where very low recombination frequencies are found, there will be doubt about whether or not the two mutat ions in the parents affect the

[4] GENETXC TECHNIQUES FOR N. crassa 129

same gene product, or whether they control products of adjacent genes. Where this doubt prevails, the detection of pseudowild types (see below) usually gives presumptive evidence of nonallelism by complementation. A deliberate test for complementation, of course, can be made to test allelism if heterokaryon-compatible strains carrying the two mutations are available (see Section VII).

It has been found r'4"125 that any of the seven pairs of" homologous chromosomes may, very rarely, fail to segregate properly in meiosis. The pair of chromosomes, which need not be recombinant, is accordingly included in a single ascospore. Upon spore germination and nuclear division, the abnormal nucleus (with eight chromosomes, one represented twice) breaks down into two types of normal, seven-chromosome nuclei, one having one of the abnormal pair, and the other its homolog. The resulting mycelium is thus heterokaryotic for any factors by which the abnormally distributed chromosomes differ. If linked, complementing mutations are being tested for recombination, these heterokaryons will resemble wild-type ascospore colonies. Such colonies are called "pseudowild types" (PWT's). While valuable as a test for complementation, the occurrence of PWT's can greatly impair the accuracy of low recombination values. Critical analyses should include a check for PWT's, which involves plating conidia of prototrophic isolates. PWT's will yield a substantial proportion of mutant conidial colonies, while true wild types (true recombinants), being homocaryotic, will not.

5. E x t r a c h r o m o s o m a l M u t a n t s . In any organism, a variant phenotype may be controlled by a "cytoplasmic" or extrachromosomal determinant. Identification of such a factor is accomplished according to one or more of the following criteria: (1) A cytoplasmic trait usually fails to display segregation in crosses. (Phenotypes determined by a unique combina- tion of many unlinked genes will be superficially similar in this respect.) (2) Progeny of a cross of a cytoplasmic mutant to a wild-type strain usually resemble the parent which furnishes the protoperithecium, the exclusive donor of cytoplasm to a mating. Crosses which are reciprocal for sex (i.e., c~? x c+d vs. c+9 X cd, where c and c + are the cytoplasmic determinant and its normal counterpart) yield different results. (3) A cytoplasmic mutant does not display linkage to any chromosomal marker, even if it segregates in crosses. (4) A cytoplasmic determinant or its wild-type counterpart may be transferred to cells of" another nuclear type by formation and resolution of a heterokaryon. While cytoplasmic inheritance does not deserve extended comment here, i t

124M. B. Mitchell, T. H. Piuenger, and H. K. Mitchell, Proc. Natl. Acad. Sci. U. S. 38, 569 (1952).

lZS"F. H. Piltenger, (;enetics 39,326 (1954).


should at least be cons idered if simple hypotheses fail. A useful review of the subject is available. '2~

T o summarize up to this point: In biochemical genetic work, r andom- spore analysis can reveal whe ther and how many chromosomal factors are involved in a mutan t phenotype ; it can be used to purify and to backcross mutants; it can be used to map mutat ions genetically and to test for allelism between i n d e p e n d e n t mutants .

6. Reversion, Suppression, and the Use o f Tetrads. Reversion includes two operat ional ly distinguishable phenomena : (1) Intragenic reversion, or back-mutat ion, e i ther restores the wild-type nucleotide sequence or makes a compensa to ry change at a second site in the same gene. (2) In- tergenic suppression restores a normal phenot~,pe by the occur rence o f a muta t ion at a second gene which overcomes the phenotypic effects o f the first mutat ion.

T h e distinction between reversion within the gene and the occur rence o f a suppressor muta t ion at a distinct locus is made by formal genetic analysis. A cross o f an intragenic rever tan t with wild type usually yields only phenotypical ly wild-type progeny, because the cross involves strict segregation o f wild-type and rever tan t alleles o f a single gene. ( I f the rever tan t pheno type is distinguishable f rom wild type in a subtle way, such as in specific activity o f an enzyme, the ratio o f the two alleles among the p rogeny will be 1:1.) On the o ther hand, a rever tan t at- tr ibutable to a distinct suppressor mutat ion, when mated to wild type, yields recombinant p rogeny which display the original mutant phenotype. In most cases o f suppressor mutat ions, the suppressor has little or no gross effect in the otherwise wild-type genome. T h u s in the rever tan t × wild-type cross, m su × m + su +, the p rogeny will be dis- t r ibuted, if the de te rminan ts are unl inked, in the ratio o f 75% wild-type or quasi-wild-type phenotypes (m + su+; m + su; and m su) to 25% mutant phenotypes (m su+). I f the suppressor is l inked to the suppressible mutat ion, the f requency of m su + will be less, sometimes to the point o f uncer ta inty about whe ther the reversion is an inter- or an intragenic event. (Note: In Neurospora, as in most h igher organisms, the "normal" forms o f suppressor loci are designated by the plus sign, to signify the wild-type condit ion. This is exactly cont ra ry to the more recent convention in bacterial genetics, where su + is the "active" allele with respect to suppression.)

T h e recovery o f a suppressor muta t ion in an otherwise wild-type genome is done easily by dissecting asci o f wild-type × rever tan t crosses,

126J. L. Jinks, "Extrachromosomal Inheritance." Prentice-Hall, Englewood Cliffs, New Jersey, 1964.


in which such genotypes (m + su) may be inferred, even though un- recognizable phenotypically. In tetrads of the cross between m + su + and m su, tetratype and nonparental ditype asci will contain one or two m + su spore-pairs, respectively. The m + su spores will be associated with phenotypically mutant spore-pairs (m su +) in the same ascus. In the case of the nonparental ditype, both phenotypically wild-type spore pairs are automatically known to be of the desired genotype. The m + su genotype may be verified by a diagnostic cross.

Tetrads may be used to recover double mutants (ml , m2), which may resemble one or both corresponding single mutant strains. The double mutant is efficiently isolated in asci of an ml + m2 × ml , m2 + cross in which wild type (ml +, m2 +) spore-pairs appear.

Tetrads also reveal most simply chromosomal aberrations which exist in heterozygous conditions in a cross. Crossing-over in such matings may lead to duplications and deficiencies in genomes of the ascospores. A deficiency usually leads to spore abortion, and the presence of two or more aborted (white or hyaline) spores in many asci is presumptive evidence of a chromosomal aberration in one of the parents? 27

Procedures

1. Mating. Crossing media have been described in Section II; 18 × 150 mm slants are usually used. If the female parent is auxotrophic, the required metabolite is added to the crossing medium. Although supplementation of crossing medium is not always required for nutritional deficiencies carried by the male parent, it occasionally improves greatly the maturation rate or viability of mutant ascospores. Special conditions may be necessary in crosses of mutants having similar requirements, particularly allelic mutants. The maturing perithecium is relatively autonomous after a certain point, and matings of allelic mutants may become starved for their requirement and fail to develop fully. This is overcome in some cases by adding the required supplement in a much higher concentration than is used for optimal vegetative growth. In other cases, this does not work, and the genetic analysis of several interesting loci has been abandoned for this reason. Unfortunately, some supplements, such as amino acids, are good nitrogen sources and tend to reverse the nitrogen-starved condition which the sexual process requires. They should be added sparingly if crosses seem feeble on supplemented crossing medium. Another alteration of the medium which may improve fertility is a decrease in carbohydrate concentration. Neurospora crosses will proceed on 0.1% sucrose or filter paper as a sole

12~D. D. Perkins, Neurospora Newsletter 9, 10 (1966).


carbon source. Many investigators use a strip o f filter paper in synthetic crossing medium, with both sucrose and agar omitted. Perithecia form on the paper strip above the surface o f the liquid. Large numbers o f ascospores are obta ined on the walls o f 125- or 250-ml flasks one-fif th full of liquid crossing med ium and having filter paper cones s tanding tip upward.

Crosses may be made by inoculating crossing med ium with both parents. In many cases, however, it is desirable to control which strain will be the female parent . T h e strain chosen as female is allowed to grow 4-6 days or longer at 25 °. (The formation of protoperithecia is inhibited at higher temperatures.) A normal female paren t will have consumed enough o f the nutr ients in the med ium to prevent significant growth o f the male paren t when it is in t roduced. Fertilization is done by spreading conidia o f the male parent over the surface o f the female culture. T o avoid contamination, the conidia of the male parent are best added in suspension; excess water should be poured off af ter fertilization. (Excess conidia o f the female may be r emoved with a loop or by aspiration with a sterile Pasteur pipette.) Fertile matings, which should remain at 25 ° until mature , show significant deve lopment and darken ing of perithecia as soon as 12 hours af ter fertilization. I f black peri thecia have not appeared within 2 or 3 days, the cross is likely to be infertile. In such cases, the appearance o f rare perithecia could be (but is not necessarily) due to contaminat ion with more active male elements.

2. Random Spore Isolation. At approximate ly 10 days af ter fertilization, asci begin to mature and to shoot ascospores. T h e latter will appea r on the wall o f the tube opposite the agar surface, particularly if" crosses are b rough t into the light. Because matura t ion of ejected spores continues to t a day or two af ter spores are shot, it is best to wait t o t at least 6 days af ter the first ones appea r be tore collections are made tor analysis. In some cases, 2-6 weeks may be necessary to achieve satisfactory germinat ion and allele ratios; critical studies may require periodic tests o f germinabili ty to de te rmine the best t ime to analyze a cross. Germina- tion percentages o f at least 90% can usually be expected in matings involving nonallelic mutations, even within a week af ter spores first appear . Ascospores remain viable for long periods, and declining germination rates may be reversed by soaking the spores in water for 24 hours before activation, v'8

Ascospores may be collected and isolated in several ways. For small- scale work, a wet loop may be touched to the wall o f the tube to pick up ejected ascospores. T h e y are spread with the loop or a bent glass rod

1~8W. N. Strickland,J. (;en. Microbiol. 22,583 (1960).


on 4% agar (in water), and then isolated directly into 10 × 75 mm tubes, with a flat-ended or spear-point needle. This is followed by a 60 ° heat- shock in a covered water bath for 35-60 minutes. Ascospores are black and ellipsoid, about 10 × 25 t~, and are easily detected under 40x magnification.

An ascospore suspension may be made by introducing sterile water into the tube, scraping the wall of the tube with a loop to dislodge the spores and decanting the suspension. Filter paper, agar, and mycelial debris should be left in the cross tube, and conidia may be removed from the suspension by decanting it after ascospores have settled out or are centrifuged down. A small, wet, cotton-tipped applicator may also be used to withdraw ascospores from flasks or tubes and make the suspension. At this point, several procedures, parts of which can be re- combined, are possible: (a) The spores may be spread, isolated, and shocked as above. (b) The spores may be spread on minimal sorbose- fructose-glucose plating medium with 4% agar, shocked in a 60 ° oven for an hour, and scored after incubation at 34 ° for 4-6 hours or more. For some mutants, this is sufficient time for unambiguous differences in growth or morphology to be expressed. (c) The ascospore suspension may be heat-shocked in a covered water bath, and may then be plated on regular plating medium at a dilution roughly adjusted to minimize colony overlap after 18-20 hours' (overnight) growth at 25 ° . Colonies demonstrably arising from single, germinated ascospores are isolated under the dissection microscope into tubes at this point, after germination percentage is determined on at least 100 spores. Care should be taken in the isolation procedure to avoid hyphae of adjacent colonies which lie out of focus within the agar. The virtue of this method is the opportunity to distinguish failure to germinate from failure to grow into a mature culture after germination. In several cases, this has led one of us (R. H. D.) to recognize quickly the segregation of membrane transport mutations among the amino acid auxotrophs of crosses. While such double mutants germinate, their requirements cannot be fulfilled on normally supplemented media. (d) Ascospore suspensions may be heat-shocked and then plated fbr plate-counts, as in the case of conidia. Before plating, hemacytometer counts are made on the suspension to determine the proper dilution. The ascospores are suspended in sterile 0.1% agar (in water) to prevent settling, and they are then diluted appropriately in the same medium. Pipetting of suspensions should be done slowly to keep ascospores uniformly suspended. Further direc- tions about plating are given in Section III.

3. Phenotypic Testing. Spot-tests for nutritional phenotypes have been described in Section III. For certain characters, particularly leaky


auxotrophs, it may be necessary to test phenotypes in selective media in 10 × 75 mm tubes. In some cases, large, single-ascospore colonies on plates may be repeatedly sampled by taking plugs with a Pasteur pipette (see Section III) and distributing them directly to several media in tubes. Mating-type tests may be made by placing a drop of conidial suspension on cultures offl a andf l A, grown 6 days at 25 ° in petri dishes on crossing medium. The fl (fluffy) strains are safe to use this way because they do not conidiate, and a 5 × 5 grid of spot tests may be made on a single plate. The fl cultures, representing the standard genome, may also be used to score for chromosomal aberrations by noting pat- terns of spore abortion in asci? 29

4. The Use of Alcoy. Crosses with alcoy are made in the normal manner, with alcoy as the female parent. Tubes with ascospore isolates are initially incubated at 34 °. After 3 days, the tubes are separated according to growth; cot genotypes will have grown little, while cot + will have grown well. Further incubation at 25 ° allows cot isolates to grow. At this time all colonies may be classified with respect to color. The isolates carrying al or al + can be classified accordingly after a day in the light, but only those carrying the latter will be classifiable with respect to ylo (the distinction between ylo and ylo + improves with time in the light). The data are tabulated as follows, where mut/mut + is the determinant of the character being studied:

mut

rout +

ol al + cot cot + ylo ylo +

Linkage or independence of the new mutation can be done separately for each alcoy marker in this format. 122

A series of crosses to other markers, available from FGSC, may be made if no linkage to alcoy is seen? 2a A test of mating type in the progeny of the alcoy cross is good for IL; a mating to acr-2 is useful for IIIL, and a mating to nic-3, me-7 A (FGSC #152) or a (FGSC #153) serves for VII. I f linkage to alcoy markers is found, double-mutant, follow-up stocks, available from FGSC, are used to define further the linkage

~a*C. W. Taylor, Neurospora Newsletter 8, 21 (1965).

[4] GENETIC TECI-INIQUES FOR N. crassa 135

with one or the other chromosome arms involved in the first cross. These are (with FGSC numbers):

I vs. II : aur, pe A (#1203) or a (#1204)

or aur, arg-5 A (#1205) or a (#1206)

IV vs. V: cot, inos A (#1243) or a (#1244)

I I I vs. VI: tryp-1, ylo A (#1207) or a (#1208) .

Alternatively, any convenient markers (see Fig. 3) may be used, as single-mutant strains, for further matings.

5. Ascus Dissection. The procedure generally used requires the following materials.

Dissection microscope, stereoscopic, 20-60× with substage illumination, good depth of field, and a fully open working area toward the user. Working distances of at least 3 cm are required.

Watchmaker's forceps (e.g., Clay-Adams micro dissecting forceps, sharp, straight points, No. C-975)

Spear point needle (see Section III) Either 1% NaC10 (reagent) or a 20% solution of a commercial

bleach, such as Clorox. Make fresh each day. Agar, 4% (in water) in petri dishes. This should be poured free of

bubbles if possible. The surface may be freed of bubbles prior to solidification by a brief, direct flaming with a Bunsen burner.

Slants in 10 × 75 mm tubes for colony isolation. Glass needles or other implements for spore manipulation. Needles

are easily made from Kimax capillary tubing (i.d., 0.7 mm, o.d., 1.0 mm; Kimble Glass Co. No. 46485). A 6-inch segment is pulled sharply, as it melts in a narrow flame from a microburner, to make a pair of stiff, quickly tapering fibers. To make the fibers into hooks, they are held downward at a 45-degree angle and are passed rapidly through the flame, which turns the fiber upward. t3° The fiber beyond the bend is trimmed with scissors. The fibers should be fine enough (maximum diameter: 40/z) to ma- nipulate spores and strong enough to exert force on asci against 4% agar. Hooked needles are used because they do not have sharp points to catch in the agar.

Alternative or additional instruments often used: Fine insect pins, sharpened on an Arkansas stone, may be set in

wood or glass.

~a°We are indeb ted to Dr. W. T. Ebersold for this technique.


Fine tungsten needles may be sharpened by dipping in molten NaNO~. 131

A fiber of fine Pyrex glass wool (about 25/z in diameter) is doubled and inserted into a short segment of melting-point capillary. A small loop for spore manipulation is allowed to protrude, and is sealed in place with epoxy cement. The capillary is then cemented into a larger tube, which serves as a handle. 132

The procedure for ascus dissection starts with a mating made in the usual manner.. Nine days after fertilization and each day until the cross is judged ready, one or two perithecia are withdrawn and crushed in a drop of water on a slide so that asci are extruded. The cross is sufficiently mature for ascus dissection when 10% or more of the asci have eight dark spores. Although asci do not mature synchronously, a cross, if once appropriate for ascus dissection, should be put in the refrigerator and used within a few weeks.

For dissection of asci, a sharp-edged block of 4% agar is cut from the plate and placed as a working surface on a microscope slide under the microscope. A perithecium is crushed with watchmaker's forceps in the center of the block, and a drop of water is placed upon the extruded rosette of asci. The asci spread out, with the tips pointed outward. Manipulations thereafter are performed with needles, which are used to tease out individual asci. (A recent report 133 simplifies this part of the method. It was found that drying perithecia induces expulsion of intact asci. This is accomplished by transferring 1 1- to 13-day-old perithecia to 4.5% agar blocks and leaving them unwetted and uncovered for 30-60 minutes. During this period, perithecia are squeezed gently with forceps and intact asci are extruded on the agar.) Asci may tend to break easily; therefore, when a small clump of asci remains to be resolved, it should be pulled to an area free of extraneous spores. A single ascus is isolated, the area is cleared, and the dissection is begun. A needle is placed between the terminal and penultimate spore and is pressed to "pop" the former from the ascus. The spore is moved a short distance away, and, by a similar procedure, the other spores are removed; the ascus having been broken, the remaining spores will come out easily. The spores are treated with a small drop of NaCIO to kill contaminants and are transferred directly to slants with a straight, fine needle, sterilized in NaCIO or alcohol. Alternatively, the spores may be moved into a row and treated with Clorox at the edge of the block of agar, distant enough from one another so that the agar, once dry, may be cut safely between and

IaIV. Prakash, Neurospora Newsletter 3, 11 (1963). ~n2R. E. Totten and H. B. Howe, Neurospora Newsletter 7, 23 (1965). ~SaM. P. Morgan, L. Garnjobst, and E. L. Tatum, Genetics 47,605 (1967).

[4] GENETIC TECHNIQUES VOR N. crassa 137

behind the spores with a spear-point needle. Each block defined in this way is undercut with the spear-point needle, lifted out, and transferred to a slant.

There are several degrees of care in ascospore isolation. First, ascospores may be isolated individually and in order, a procedure which later permits determination of second-division segregation frequency. Second, both members of a sister-spore pair may be transferred to the same tube, though the danger of transferring a non-sister pair will quickly become clear to the novice. Third, the eight spores may be isolated without respect to order in separate tubes. The order of the four meiotic products may not be particularly important, and sister-spore pairs can usually be worked out by genotype for most asci. Useful tetrads may be obtained without complete germination, of course, if four demonstrably different and self-consistent products survive.

Spores are left in tubes to mature for 3 or 4 days at 25 ° or 30 ° prior to activation. Contaminants which initiate growth in this period can be observed, but should, with good technique, rarely be encountered. After activation at 60 ° for 35-60 minutes and 3-4 days' growth, cultures may be classified. Reshocking spores which have failed to germinate is occasionally productive.

IX. Growth, Harvesting, and Extraction

Large-Scale Culture

1. Mycelia. (a) 700- to 1000-ml cultures are grown in low-form, 2500-ml culture flasks (Pyrex No. 4422) on a reciprocal shaker. A conidial inoculum of 105 to 10 e per milliliter is used. A sturdy shaker, such as the new Brunswick Scientific Co. Reciproglide type, with plat- forms covered with Neoprene matting to prevent flask slippage, is used. With shaking speeds of 90 cycles per minute, and a 1.5-inch stroke, the medium moves in a rotary fashion, and little mycelium clings to the upper part of the flask. TM This method of culture is a compromise between elegance and convenience. The logarithmic phase is relatively short, but a large amount of material ( 1-2 g dry weight per flask) can be harvested after 18-24 hours. Growth may be monitored with samples harvested and acetone-dried in a Btichner funnel (see below). The system is suitable for transfer of mycelia from one medium to another after collection and washing in cheesecloth. Sterility during transfer is not vital if the inoculum is large enough and the incubation after transfer is short. Some characteristics of the mycelia, such as enzyme activities,

~34R. H. Davis and F. M. Harold, Neurospora Newsletter 2, 18 (1962).


change significantly with age in lightly inoculated cultures, but the pat- terns are quite reproducible.

(b) Another method involves logarithmic cultures of 200-1000 ml contained in flat-bottomed Florence flasks, as described in Section V, or of 2-10 liters of medium contained in bottles or carboys. For the larger cultures, Pyrex bottles or carboys (e.g., Pyrex No. 1595) are filled one-half to three-fourths full of medium. With large volumes, some time is necessary to reach sterilizing temperature and to cool the medium sufficiently for removal from the autoclave without sudden boiling. Cultures are aerated and stirred by forced air. The culture vessels are fitted with 2-hole stoppers with an inlet tube reaching nearly to the bottom of the vessel, and a short, cotton-plugged outlet. The inner end of the inlet should be open, not tipped with sintered glass. A second, small sterile vessel (suction flask or drying tube), packed with cotton so that air passes through it, is attached to the inlet to sterilize the entering air. Compressed air, humidified by passage through water, is forced into the flasks to stir and to aerate the medium. Conidia, mycelial macerates, or small logarithmic cultures are used as inocula. Cer- tain arrangements permit withdrawal of 90% of the culture at maturity, the introduction of more medium by gravity flow, and a second cycle of growth. 135 Some investigators grow mycelia in converted washing machines, sterilized with live steam. ~36

(c) Large batches of mycelium may be prepared by growth in a 100- liter Fermacell (New Brunswick Scientific Co., New Brunswick, New Jersey). ~37 The Fermacell is inoculated with a 15-liter culture grown from conidia for 24-30 hours with forced aeration as in method (b). The Fermaceli culture is grown at 30 ° with the maximum aeration rate and an agitation rate of 250 rpm. The time of incubation varies with the strain, but the disappearance of metabolites or increase in dry weight can be monitored by sampling through the exit port. The high rate of aeration and agitation keep the mycelium well fragmented and ensure passage of the mature culture through the exit port. The mycelial mass can be collected by gravity on cheesecloth (see below) and centrifuged in a basket centrifuge.

2. Conidia. Large-scale growth may require large-scale conidial production, and the physiology of dormant or germinating conidia may themselves be of interest. A medium for large-scale conidial production is the nitrate minimal medium (Table I), modified to contain 5.0 g of KNa tartrate, 3.0 g of NaNO3, 3.0 g of KH2PO4, 10 g of sucrose, 10 ml

135M. D. Garrick, Neurospora Newsletter 11, 5 (1967). la~M. Fling, N. H. Horowitz, and H. Macleod, Neuro.qmra Newsletter 3, 8 (1963). laTj. A. DeMoss, personal communication.


of glycerol, and 20 g of agar per liter, t38 Fernbach flasks (2500 ml) with 250 ml of medium are inoculated with fresh conidia and incubated up- right at 34 ° for 24 hours, then at room temperature for 24 hours more. The flasks are then inverted in a well-lit room at room temperature. A gentle stream of sterile, humidified air is introduced with a tube through the cotton plug, and growth continues for 3 to 4 days. The many conidia which form are harvested by suspension in water or in 0.1% (v/v), sterile Tween 80 with a sterile test-tube brush or with a magnetic stirrer and large stirring bar. The suspension is filtered through cheesecloth to remove clumped material. Conidia are collected by vacuum filtration on Whatman No. 1 filter paper. The yield is approximately 3 g wet weight per flask, or 3 to 6 × 101° conidia. 139

Harvesting

1. Centrifugation. Brief centrifugation in a clinical centrifuge is convenient for replicate, small harvests of well-dispersed mycelia prior to extraction. Centrifugation is also a gentle and precise method of harvesting and washing mycelia prior to transfer. Conical centrifuge tubes (12-50 ml) should be used, since packing of mycelia is poor in round- bottom tubes.

2. Membrane Filtration. A suction flask with a 1-inch filter holder and funnel (Gelman Instrument Co., Ann Arbor, Michigan, No. 1112; Milli- pore Filter Corp., Bedford, Massachusetts, No. XX10 025 00) is used with membrane filters of 0.45 /z (Millipore type HA or Gelman type GA-6) or 1.2/.* (Millipore type RA or Gelman GA-3) pore size. One- to 40-ml samples of whole cells or trichloroacetic acid-insoluble residues may be collected. Washing of material on the filter is efficient. For radio- active uptake studies, filters may be glued, after harvests have been made, to planchets with rubber cement, or immersed in scintillation fluid for counting. For extraction, moist mycelia may be scraped cleanly from the filters with a spatula and placed in centrifuge tubes. 14°

Harvests made for dry-weight determination on 1-inch filter circles have been described in Section V.

3. Filter Paper Harvests. Bulk harvests of over 50 mg dry weight may be made on a Btichner funnel, using Whatman No. 1 or Schleicher and Schuell Sharkskin filter circles. The material may be frozen, lyophilized, or, while still in the funnel, acetone-dried with reagent grade acetone. T M

Many enzymes withstand the latter treatment without loss of activity. For acetone-drying, thin pads of mycelium should be collected so that the

lasS. D. Wainwright, Can..]. Biochem. Plo'siol. 37, 1417 (I 959). 139R. W. Barratt,J. (;en. Microbiol. 33, 33 (1963). 14°R. H. Davis,]. Bacteriol. 96,389 (1968).


acetone passing through quickly removes all water. The stability of many enzymes in crude, acetone-dried material is excellent. Small molecular weight materials, however, are not completely retained during acetone- drying.

Mature or starved cultures may be difficult to collect on filter paper or membrane filters because of an accumulation of polysaccharide in the medium. Under these conditions, centrifugation and washing should be used as an initial step for small volumes, and cheesecloth harvesting (below) for large volumes.

4. Cheesecloth Harvests. One or two layers of cheesecloth lining a wire basket can be used to harvest large cultures. The material is washed in the basket with a stream of" distilled water. The mycelia are then gathered in the cheesecloth and wrung out forcibly. The mycelia may be frozen, lyophilized, or extracted directly.

Extraction and Fractionation

Because a review of purely biochemical techniques is beyond the scope of this article, various techniques are named, with citations to recent descriptions. Many techniques continue to improve rapidly, particularly the isolation of organelles, enzyme aggregates, and nucleic acids.

1. Uptake, Content, and Turnover of Cell Constituents. Methods for extraction and measurement of various classes of compounds, and their turnover, worked out for Escherichia coli by Roberts and co-workers TM

are suitable for Neurospora with minor modification. The publication of Roberts et al. has a short chapter on Neurospora metabolism. Many of these techniques have been refined for specific purposes. For instance, a widely applicable method for uptake and active transport studies in Neurospora is described by Wiley and Matchett. 142 The determination of phosphorus compounds and their turnover, based on those of Schneider, 143 is given by Harold. TM Harold explicitly recognizes inorganic polyphosphate accumulation in starved mycelia.

2. Extraction of Small Molecules. Amino acids and many other small molecules are extracted in cold, 5% trichloroacetic acid for 10-30 minutes, followed by removal of the extractant by shaking the extract with chloroform or ether. Nearly as effective for amino acids is 10 minutes' extraction in water at 90°, T M or 10 minutes' extraction in 80%

141R. B. Roberts, P. H. Abelson, D. B. Cowie, E. Bolton, and R. J. Britten, Studies of Bio- synthesis in Escherichia coli. Carnegie Inst. Washington Publ. 607, 3rd printing. Washing- ton, D. C., 1963.

roW. R. Wiley and W. H. Matchett,J. Bacteriol. 92, 1698 (1966). I~W. C. Schneider, Vol. III, p. 680. 144H. Aurich, Neurospora Newsletter 8, 11 (1965).


ethanol at 800.145 The last method is preferred for glycolytic inter- mediates? ~a4~ Lipids may be extracted in chloroform and methanol according to Crocken and Nyc. 1~ Cold, 5% HCIO4 can be used for extraction, and perchlorate may be removed as the insoluble potassium salt. Controls for hydrolysis of compounds should be used. Small-molecular- weight compounds are not reliably recovered from oven- or acetone- dried material.

3. Cell Disruption and Protein Extraction. Lyophilized or acetone- dried material is easily extractable by homogenization in buffer. Lyophilized mycelia are first powdered (dry) in a Wiley mill. Large, acetone-dried pads are powdered before extraction by blending in cold (-20°), reagent acetone with a spark-free blender (e.g., Omni- mixer, Ivan Sorvall Co., Norwalk, Connecticut)? 34 The slurry is collected by filtration in a Bt~chner funnel, using Whatman No. 1 filter paper, and is washed briefly with acetone at room temperature to prevent condensation of atmospheric water vapor in the powder. The powder is turned out and broken up on filter paper. The powder may be stored prior to extraction in a stoppered flask a t - 2 0 °. The use of acetone powders for enzyme preparation is highly favored by the senior author.

Mechanical disruption of intact mycelia can be accomplished with many devices. A gentle extraction method is grinding with sand in a mortar. Tissue homogenizers such as the VirTis 45 (VirTis Co., Gardiner, New York), blendors such as the Omnimixer mentioned above, and colloid mills of various sizes (Gifford-Wood, Inc., Hudson, New York) are widely used for small or large volumes. In most cases, glass beads (0.2 mm pavement-marking beads, type 100-5005, 3M Co., St. Paul, Min- nesota) are added to the homogenizing medium to occupy as much as one-half of the volume to be extracted. With the colloid mills, mycelia must first be blended with a coarse blendor (Waring or Omnimixer) so that clumps will not clog the grinding aperture. Material prepared by grinding with glass beads has been used successfully for isolation of mitochondria and nuclei, though the integrity of organelles should be checked to verify the point. Sonic oscillation is often used for fresh material, although it inactivates some enzymes and disrupts organelles. The Raper-Hyatt pressJ 49 designed specifically for disruption of large quantities of fungal material, may also be used. Frozen mycelia may be

145R. Fuerst and R. P. Wagner, Arch. Biochem. Biophys. 70, 311 (1957). 1~K. Budd, A. S. Sussman, and F. I. Eiters,J. Bacteriol. 91,551 (1966). 147S. Brody and E. L. Tatum, Proc. Natl. Acad. Sci. U. S. 56, 1290 (1966). 148B. J. Crocken and J. F. Nyc,J. Biol. Chem. 259, 1727 (1964). 149j. R. Raper and E. A. Hyatt,J. Bacteriol. 85, 7 ! 2 (1963).


disintegrated in a blendor with powdered Dry Ice (an inexpensive household seed grinder, "Rollmix," Coronation Brand, Universal Dis- tributors, Culver City, California, is reported 15° to be effective for small volumes) with good cell breakage, m Similarly, a motor-driven mortar and pestle can be used to fragment mycelia at liquid-nitrogen temperature. ~52 Certain protein extraction methods have been compared by Stine et a l l 5a and by Bates et al. TM

4. OrganeUes. For mitochondria, the methods most widely used to date are those of Luck, ~55 of Hall and Greenawalt, TM and of Hall and Baltscheffsky? s~ Kiintzel and NolP 5s have recently described a similar set of methods in work on the isolation of mitochondrial and cytoplasmic ribosomes. Nuclei may be isolated according to the method of Reich and Tsuda? 5a Munkres et al? e° have developed a method for isolating several cell fractions, including nuclei and mitochondria, from a single batch of mycelia.

Ribosomes may be isolated by the methods of Alberghina and Sus- kind; m their paper includes a study of ribosomal proteins of Neuro-

spora. Ki~ntzel and NolP ss have developed methods for isolating cytoplasmic and mitochondrial ribosomes.

Cell wall may be isolated and analyzed by the methods of Mahadevan and Tatum. n

5. Nucleic Acids. DNA can be isolated crudely by the general methods of Marmur? e~ More refined methods which distinguish mitochondrial and nuclear DNA can be found in the papers of Luck and his co- workers.~55'~

Ribosomal RNA extraction has been described by Dure et al. ~ and

~S°Arnold Bendich, personal communication. ~51j. Bucknell, personal communication. ~S2E. E. Brandt and A. G. DeBusk, Neurospora Newsletter 6, 17 (1964). ~5~G. J. Stine, W. N. Strickland, and R. W. Barratt, Can.J. Microbiol. 10, 29 (1964). ~s4W. K. Bates, R. S. Bach, and B. M. Eberhart, NeurosporaNewsletter 12, 16 (1967). t~SD.J.D. Luck,J. CellBiol. 16, 483 (1963). I~D. O. Hall and J. W. Greenawalt,J. Gen. Microbiol. 48, 419 (1967). ~STD. O. Hall and H. Baltscheffsky, Nature 219, 968 (1968). ~SSH. KiJntzel and H. Noll, Nature 215, 1340 (1967). ~SSE. Reich and S. Tsuda, Biochim. Biophys. Acta 53,574 ( 1961). lS°K. D. Munkres, M. N. Munkres, P. Greene, S. Hedman, B. J. Andrews, G. Holland, and

D. O. Woodward, Neurospora Newsletter 9, 14 (1966). ISlF. A. M. Alberghina and S. R. Suskind,J. Bacteriol. 94, 630 (1967). lszj. Marmur,J. Mol. Biol. 3,208 (1961). ~eaD. J. D. Luck and E. Reich, Proc. Natl. Acad. Sci. U. S. 52, 931 (1964). I~L. S. Dure, J. L. Epler, and W. E. Barnett, Proc. Natl. Acad. Sci. U. S. 58, 1883 (1967).


by Alberghina and Suskind. tnt Transfer RNA has been studied extensively by Barnett's g r o u p , t65 which makes use of extraction methods of Holley et al. t66 Barnett's work concentrates on specificity of mitochondrial and cytoplasmic tRNA's, as well as of similarly distributed, specific, aminoacyl-tRNA synthetases, t67 Little has been done on mRNA or polysomes, although a method of detecting and characterizing them has been described by Henney and Storck. a6s

6. Protoplasts. For wild-type cells, protoplasts are made by digestion of the cell wall with snail-gut enzyme in isotonic or hypertonic medium. The common method is that of Bachmann and Bonner; ls9 this has been modified by Trevithick and Metzenberg. 1~° An "osmotic" strain (os, allele M 16) can be induced to form protoplasts without added enzyme in media containing a high concentration of sugar, m There is some hope that the cell wall deficient strain, sl ("slime," FGSC No. 327 and 1 118) t~2 will be useful in place of prepared protoplasts, but the biochemical study of the strain has only begun, tT3

Acknowledgments We should like to thank the following persons for reviewing all or parts (as indicated)

of the manuscript as it was being prepared: Drs. J. A. De Moss (IX), R. W. Barratt (IV, IX; genetic map), D. D. Perkins (VIII; genetic map), A. Radford (VIII), D. Stadler (VIII), D. Newmeyer (VIII; genetic map), A. Schroeder (VIII), G.J. Stine (IX), L. G. Williams (all sections), M. S. Sargent (all sections). In addition, we would like to thank those who con- tributed details and unpublished information for inclusion. Finally, we would like to apologize for the inevitable omissions and errors in citing the originators of certain techniques. Our apologies come with a tribute of thanks: the early days of Neurospora work are the most obscure in terms of technical advances, yet most important to the development of this organism as a tool of biochemical and genetic research. Research of the authors, from which much of this review is derived, was supported by the National Science Foundation (RHD) and the U.S. Atomic Energy Commission under contract with Union Carbide Corporation (F.J. de S.).

l~W. E. Barnett and D. H. Brown, Proc. Natl. Acad. Sci. U. S. 57, 452 (1967). 166R. W. Holley, J. Apgar, B. P. Doctor, J. Farrow, M. A. Marini, and S. H. Merrill, J.

Biol. Chem. 236, 200 (1961). 16~W. E. Barnett, D. H. Brown, and J. L. Epler, Proc. Natl. Acad. Sci. U. S. 57, 1775 (1967). 16SH. R. Henney, Jr. and R. Storck, Proc. Natl. Acad. Sci. U. S. 5 l , 1050 (1964). ~eSB.J. Bachmann and D. M. Bonner,J. BacterioL 78, 550 (1959). ~0j. R. Trevithick and R. L. Metzenberg, Biochem. Biophys. Res. Commun. 16, 319 (1964). mj . G. Hamilton andJ. Calvet,J. BacterioL 88, 1084 (1964). 1r2S. Emerson, Genetica 34, 162 (1963). lr3V. W. Woodward and C. K. Woodward, Neurospora Newsletter 15, 18 (1968).

[4] genetic and microbiological research techniques - stanford

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