zy zyxw zyxwvut zyxwvu GENETIC CONTROL OF PHOSPHATE-METABOLIZING ENZYMES IN NEUROSPORA CRASSA: RELATIONSHIPS AMONG REGULATORY MUTATIONS BARBARA S. LITTLEWOOD, WILLIAM CHIA of zyxw ROBERT L. METZENBERG Physiological Chemistry, Uniuersity of Wisconsin, Madison, Wisconsin 53706 Manuscript received September 12, 19.74 ABSTRACT In Neurospora crassa, the pho'sphate-metabolizing enzymes are made during phosphate starvation, but not under phosphate sufficiency. The synthesis of these enzymes is controlled by three regulatory genes: pcon-nuc-2, p e g and nuc-I. pcon-nuc-2 and preg are closely linked. A model of the hierarchical relationships among these regulatory genes is presented. Studies of double mutants and revertants confirm several predictions of the model. It has been found that nuc-2 (null) and pconc (constitutive) mutations reside i n the same cistron. p r e p (constitutive) mutations are epistatic to nuc-2 mutations. nuc-l (null) mutations are epistatic to all others. I N Neurospora crassa, phosphate starvation causes the derepression of enzymes needed for efficient scarenging of phosphorus from the environment: an alkaline phosphatase (LEHMAN et al. 1973; BURTON and METZENBERG 1974), an acid phosphatase (NYC1967), a high pH, high affinity phosphate permease (LOWENDORF and SLAYMAN 1970; LEHMANet al. 1973), 0-phosphorylethanolamine permease (METZENBERG, unpublished results) and one or more nucleases (HASUNUMA 1973). These enzymes are repressed when phosphate levels are high. Four types of mutations are kEown which affect the ability of Neurospora to repress and derepress these enzymes ( TOH-Eand ISHIKAWA 1971;LEHMAN et al. 1973; METZENBERG, GLEASON and LITTLEWOOD 1974). Two of these mutations (pcon" and pregc) lead to constitutive production of these enzymes and two others (nuc-l and nuc-2) render the cells "null", i.e., incapable of making the enzymes even during phosphate starvation. The mechanism of control in a system involving multiple elements is sure to be complex. From studies of dominance afid epistasis reported in previous papers (LEHMANet al. 1973; METZENBERG, GLEASON and LITTLEWOOD 1974) we developed a model that describes the hierarchic relationship among these genes. The model makes several critical predictions which have been borne out by subsequent experiments. Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 Department AND zyxwvutsrqp This work was supported by an NIH Grant, GM-08995. Genetlcs 79: 419434 March, 1975 420 zyxwvuts zyxwvutsrqpo B. S. LITTLEWOOD, W. C H I A A N D R. L. METZENBERG STRUCTURAL GENES ALK. PHOSPHATASE -- ‘ I nuc-2+ pcon+ PW+ nuc- I+ HIGH pH PERMEASE I I I etc. Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 zyxwvuts zyxw zy zy Because the experiments are complicated in design, we take the unusual approach of presenting the model in advance of the supporting data to provide a conceptual framework for understanding the experiments. Our working model is presented in Figure 1. The experiments reported here confirm the following four predictions of this model. ( 1 ) We have obtained a revertant of a pcon“ mutant which behaves like a nuc-2 mutant. (2) If this cascading sequence of events is correct, nuc-2 pregc double mutants should be constitutive. Among nuc-2 revertants able to make the phosphate permease, some should contain new constitutive mutations at the preg locus. (3) If p e g c and nuc-2 mutations are in different cistrons, then preg“ nuc-Z/preg+ nuc-2+ and pregf nuc-2/prege nuc-2+ partial diploids should behave identically with respect to alkaline phosphatase production. (4) Partial diploids of the constitution pregC nuc-2/preg+ nuc-2 should be null. Combinations of positive and negative control of the synthesis of functionally related enzymes are known in other fungal systems. In Saccharomyces cereuisiae, a number of regulatory genes coordinate the synthesis of repressible acid phosphatase and repressible alkaline phosphatase (ToH-E et aZ. 1973; TOH-E,UEDA and OSHIMA1973). Both positive and negative control elements have been identified and these act in a sequential manner to control enzyme production. At the zyxwv zy FIGURE 1.-Model of the hierarchy of regulatory genes. Our working model of the sequential interactions between pcon+-nuc-2+, preg+ and nuc-If functional units gives nuc-I+ the positive role of “turning on” the (unlinked) structural genes for the phosphate-metabolizing enzymes. The pregf product inactivates or represses the nuc-l+ product. The pconf-nuc-2f product inactivates or represses the p e g + product. Phosphate o r a corepressor derived from it inactivates or represses the pconf-nuc-2+ product. The phenotypes of strains carrying mutations in these genes are described in MATERIALS A N D METHODS. This model illustrates the hierarchic genetic interactions among these regulatory loci and is not intended t o describe the physical nature of the gene products o r their molecular interactions. Enzyme production in a strain carrying a mutation in a given control gene can be “calculated” by multiplying the positive or negative signs of the regulatory products. Only those regulatory products that are connected to the structural genes by a sequence unbroken b y mutation can be included in the calculation. For example, in nuc-2 strains, only the final two regulatory steps are operative (“-” times “+”) and such strains are “-”, i.e. null. Regardless of the phosphate concentration, strains carrying pconc mutations have three regulatory steps working (“-” times “-” times “+”) and are i.e. constitutive. Similarly, wild-type cells grown under conditions of phosphate starvation are “-” times “-” times which multiplies to “+”-the strains produce alkaline phosphatase and its congeners. “+”, “+”, zy zyxwvuts zyxwvuts zy REGULATORY M U T A N T S IN NEUROSPORA 421 MATERIALS .4ND M E T H O D S Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 presefit time, it is not possible to draw exact analogies between the regulatory genes in S. cereuisiae and N . crassa, although it is clear that many similarities exist. Also in S. cereuisiue, synthesis of the three galactose-metabolizing enzymes and HAWTHORNE 1972). is controlled by at least two regulatory genes (DOUGLAS I n that system, the G A L 4 locus acts to turn on the unlinked structural genes. In the absence of galactose, GAL-4 activity is inhibited by the galactose-sensitive i+ gene product, which is thought to repress GAL-4 activity by interacting with an operator region adjacent to the G A L 4 region. WIAME(1971) has proposed a model for the control of arginase and ornithine transaminase, in which a regulatory protein (or complex of several proteins) represses the formation of arginase and ornithine transaminase. In the absence of a careful analysis, this “double negative” control would have been categorized as positive control. zyxwvuts zyxw Strains and nomenclature: All strains of N . crassa were made heterokaryon-compatible with the two Oak Ridge wild types, 74-OR8-la and 74-0R23-1A, and as nearly isogenic with them as practical by several sequential crosses to these standard strains. Most of the auxotrophic strains used in this study were obtained from the Fungal Genetics Stock Center (FGSC), Arcata, California. pconC, “phosphate-controller-constltutiye”mutants, first isolated by LEHMAN et al. (1973), are constitutive for repressible alkaline phosphatase and for a high affinity, high pH phosphate permease. pconC alleles are roughly codominant with the wild-type (peon+) allele in heterakaryons and in heterozygous partial diploids (LEHMAN et al. 1973; METZENBERG, GLEASON and LITTLEWOOD 1974). pregc, “phosphate-regulator-constitutive”(pronounced “pee-reg”) , mutants (METZENBERG, GLEASONand LITTLEWOOD 1974) are also constitutive for the above enzymes. pregc alleles are recessive to the wild-type ( p e g + ) allele in heterokaryons and in heterozygous partial diploids (METZENBERG, GLEASON and LITTLEWOOD 1974). nuc-2 mutants are null (no detectable activity) for the repressible alkaline phosphatase and for the high a f f i t y , high pH phosphate permease when they are grown on either high o r low concentrations of inorganic phosphate. Because they lack this permease, they do not grow on high pH, low Pi medium. nuc-2 alleles are recessive to the wild-type ( n u c - 2 f ) allele in heterozygous partial diploids (METZENBERG, GLEASON and LITTLEWOOD 1974). Our standard nuc-2 allele is T28-M2 (from FGSC strain # 1W8). nuc-1 mutants are phenotypically indistinguishable from nuc-2 mutants. Our standard nuc-l allele is T28-MI (from FGSC strain # 1994). pconc, nuc-2, and pregc mutations map o n Linkage Group 11. nuc-l is unlinked to these three genes; it maps to the right of the centromere on LG I. A genetic map showing those pwtions of LG I and LG I1 relevant to the present study is presented in Figure 2. The figure compares Normal Sequence euploid, Translocation euploid, and diploid strains. TramZocation strains: The euploid translocation strains used here, T(II-+I)NM177 ( A , FGSC #I610 and a, FGSC #2003), have a segment of the right arm of LG I1 moved into LG I (PERKINS1972). Euploid strains carrying this translocation are designated by a “ ( T ) ”preceding the genotype. Partid diploids: Strains diploid for a segment of LG I1 were used for complementation and dominance studies. Diploids carrying T(II+I)NM177 are extremely stable; in one vegetative GLEASON and cycle, fewer than 2% of the nuclei lose the translocated segment (METZENBERG, LIITLEWOOD 1974). Partial diploids were prepared by crossing strains carrying T(II+I)NM177 to Normal Sequence strains. Putative diploids were isolated from nonparental ditype asci (containing f o u r white deficiency spores and four black partial diploid spores) and were then further identified by 422 zyxw zy zyxwvu zyxwvutsrq zyxwvut zyx B. S . LITTLEWOOD, W. CHIA AND R . L. METZENBERG LG II LG I leu-3 cys-ll nuc-l NORMAL SEQUENCE V arg-5 nuc-2 pcon A leu-3 nuc-l arg-5 orom-l -*J--x---S- leu-3 PARTIAL DIPLOID cys-ll P W cys-ll + ’ nuc-2-pconq\ A ,.+/ \)A arg-I2 nuc-l nuc-2 pcon _ - _ _L-,_ arg-5 -*J zy org-I2 orom-l _ _ _ _ L--J- prep preg FIGURE 2.-A Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 TRANSLOCATION EUPLOID (TI arg-12 orom-I -+J ____ J-,---- - J---l- partial genetic map of euploid and diploid strains of N . crassa used in the present study. pconc is about 2 centimorgans from prege; no crossing over has been observed between pconc and the standard nuc-2 allele (METZENBERG, GLEASON and LITTLEWOOD 1974). Genes on the other five linkage groups are in the Normal Sequence (see, for example, DAVISand DESERRES 1970). The map of the translocation and partial diploid strains is a composite of data from PERKINS (1972) and METZENBERG, GLEASON and LITTLEWOOD (1974). their Barren phenotype and the presence of the mxting type allele from the translocation parent (METZENBERG, GLEASON and LITTLEWOOD 1974). The nomenclature for partial diploids is as follows: the alleles to the left of the slash are those on the Normal Sequence chromosome (LG 11), those to the right of the slash are on the translocated segment (in LG I). Media. “High Pi” medium is unmodified Fries minimal medium containing 7.35 mM phos1945); this phosphate level prevents formation of repressible alkaline phste (BEADLE and TATUM phosphatase in wild-type strains. “Low P i ” medium is Fries minimal with the phosphate concentration lowered to 0.05 mM, and with an equivalent amount of KC1 added to make up the deficit of KH,PO,. Wild-type strains are derepressed for repressible alkaline phosphatase on this medium. “High pH, low P i ” medium is “low P i ” medium adjusted to pH 7 with 0.1 M Na-MOPS (morpholinopropane sulfonic acid) buffer. In some experiments, 2.0 mM O-phosphorylethanolamine, “PE’, replaced KH,PO, as the phosphate source in “low Pi” medium (METZENBERG, GLEASON and LITFLEWOOD 1974). Liquid media cmtained 1.5% sucrose as the carbon source. For colonial growth on solid media, 1.5% Bacto-agar was added and sucrose was replaced by 1% sorbose, O.%% glucose and O.C5% fructose (BROCKMANN and DESERRES 1963). For growth of auxotrophs, the required supplements were added to give 1 mM, except for inositol, which was added to 50 pg/ml. Crosses were made on the medium of WESTERGAARD and MITCHELL(1947), with 1.5% sucrose as the carbon source. General methodology: Replica plating was done by the method of LITTLEWOOD and MUNKRES (1972). Standard methods were used for other genetic manipulations (DAVISand DESERRES 1970). zy zyx zy zyxwvuts zy zyxwvu zyxwv 423 REGULATORY MUTANTS I N NEUROSPORA Enzyme assays: Assays in cell-free extracts were performed as described previously (LEHMAN et al. 1973). Colonies on solid media were stained for alkaline phosphatase by the method of ToH-E and ISHIKAWA (1971), modified as described by LEHMANet al. (1973). RESULTS TABLE 1 Alkaline phosphatase in partial diploids carrying nuc-2ts-35 Alkaline phosphatase, specific activity Euploids wild type pconc-6 nuc-2 pregc-2 Temperature duringgrowth ~ HighPi 33” 25 33 33 33” 33” 1.6 1.1 2.5 243 1.0 478 33” 33” 33 33” 3.0 1.6 3.8 LowP, 5.4 1580 PE 5.4’ 161 111 34.6 null repressible repressible coastitutive null 2.5 235 constitutive 114 repressible null repressible constitutive Partial diploids peon+ pcon + pcon + pconc-6 nuc-2+ pregf/nuc-2t8-35 nuc-2 preg+/nuc-2ts-g5 nuc-2 + pregc-z/nuc-2ts-35 nuc-2 + preg+/nuc-2ts-s5 235 3.1 87.0 92.0 All strains were grown i n 20 ml medium at 33” for 2 days without shaking. “High P i ” contains 7.35 mM phosphate, “low P,” contains 0.03 mRI phosphate, and “PE” has 2.0 mM 0-phosphoryl ethanolamine as the sole phosphate source. Strains with the “nuc” phenotype grew too poorly in “PE” medium to be used; these strains were therefore grown i n the alternate derepression medium, “low P,”. Since this medium gave rapid growth but a very small final yield of mycelium due to the exhaustion of phosphorus, triplicate flasks were grown and the mycelia were pooled for assay. * Grew very poorly, but yield was sufficient for assay. Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 Rmersion of pconc-2to a temperature-sensitiue nuc-2: pconc-2 is a dominant mutation which causes constitutive production of alkaline phosphatase. It was isolated in a translocation euploid and maps on the translocated segment (METZENBERG. GLEASON and LITTLEWOOD 1974). In a search for revertants of this allele that are no longer constitutive, ( T)pconC-ZA conidia were mutagenized and plated to high Pi medium to give about 250 colonies per plate. The plates were incubated for two days a t 33” and the colonies were stained for alkaline phosphatase. One unstained colony was seen among the 1590 examined. This was picked, allowed to conidiate and backcrossed to ( T ) p c o r F a to get an assured homokaryotic culture. The revertant is phenotypically indistinguishable from nuc-l and nuc-2 mutants at 33’: at this temperature, it does not grow on high pH, low Pi medium and fails to make alkaline phosphatase on low P, plates or in low P, liquid medium (Table 1 ) . At 25 O and below, the revertant behaves very much like wild type: it grows, albeit rather slowly, on high pH, low P, solid medium and is repressible for alkaline phosphatase (Table 1 ) . zyxwvuts zyxwvu zyxw 4248 B. S. LITTLEWOOD, W. C H I A A N D R. L. METZENBERG We tested to see whether the event which gave rise to the nuctS phenotype was linked to the original pconc-2mutation. 'The nuct8strain was crossed to (T)pcon+. Spores were plated to high P, minimal medium and the resulting colonies were stained to detect pconc-2 segregants, if any. No constitutives were seen among about 1800 progeny. This eliminates the possibility that the nucts phenotype is due to a mutation at the nuc-1 locus and suggests that there has been a mutational event at the nuc-2 locus. (nuc-2 is very closely linked to, or part of, the pcon region-see Figure 2). We will provisionally designate this new mutation Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 zyx zyxwv Behavior of n ~ c - 2 ~in ~ -heterozygous ?~ partial diploids: Four partial diploids carrying n ~ ~ - in2 trans ~ ~ configuration - ~ ~ to wildtype, nuc-2, preg" and pconc respectively (Table 1) were isolated from crosses between ( T ) n ~ c - 2 ' ~and -~~ appropriate Normal Sequence strains. The specific activity of alkaline phosphatase in these partial diploids grown on repression and derepression media at 33" is presented in Table 1. At 33", n ~ ~ - is2recessive ~ ~ - to~ the ~ wildtype allele. as is the standard nuc-2 allele. At 33". a partial diploid of the constitution n ~ c - 2 / n u c - 2produces ~ ~ - ~ ~ no significant amounts of repressible alkaline phosphatase under any conditions (Table 1). It is also unable to grow on high pH, low P, medium. The failure of n ~ ~ -to2 ~ ~ - ~ complement with the standard nuc-2 allele further supports our hypothesis that the nuc-2 locus has been altered in n ~ c - 2 ~ * - ~ ~ . Diploids of the genetic constitution nuc-2+ p r e g c - z / n ~ ~ - 2 t sare - 3 5fully repressible, i.e. phenotypically wildtype, for alkaline phosphatase (Table 1). For such complementation to occur, this diploid must contain functional preg+ product, a condition which is met only if the n ~ ~ - chromosome 2 ~ ~ - carries ~ ~ a preg+ allele. Given the mapping and complementation. data, there remain two explanations for the phenotype of n ~ c - 2 (1 ~ )~A. reversion event has occurred at the exact site of the original pconC-*mutation, converting it to n ~ c - 2 or ~ ~(2) ; this strain is, in fact, a pconc-2n ~ ~ -double 2 ~ mutant. ~ - ~ ~ If the genetic alteration occurred at the pcone-2site, then, by definition, pconC and nuc-2ts-35 are within the same genetic locus. If the second explanation is ccrrect, and n ~ ~ - contains 2 ~ ~ two - ~mutations, ~ these two mutations could, in theory, be in the same cistroll or in different cistrons. Let us assume for the moment that the revertant is a double mutant and that pconc-2 and nuc-2ts-s5are 2 ~ ~pcon+ within two independent cistrons. The pcon+ nuc-2+/pconr n ~ c - and. n u ~ - 2 ~ ~ / p nuc-2+ c o n ~ partial diploids should then behave identically with respect to the control of alkaline phosphatase production. They do not. The pcon+ nuc-2/ pconCnuc-2+ diploids are constitutive for alkaline phosphatase production (METZENBERG, GLEASON and LITTLEWOOD 1974). while the putative pcon+ riu~-2/pcon~ n -~~ c - strain 2 ~ ~ is repressible for this enzyme (Table 1) . We therefore cordude, that, whatever the precise genetic comtitution of n u ~ - 2 ' ~the -~~, mutational event which converted pconc-L to n ~ r -s -21 5 ~ occurred within the cistron already containing pconc-2. zyxwv zyxwv zy zyxw REGULATORY M U T A N T S I N NEUROSPORA 425 Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 to nuc-2 in the nuc-2 pregc-2 double mutant: pregc--2is a Epistasis of recessive mutation leading to the constitutive production of alkaline phosphatase. nuc-2 is also recessive and leads to a “null” phenotype. The two mutations are linked on LG I1 (Figure 2). To further our understanding of the functional relationships between genes containircg these mutations, we prepared and examined a pregc--2nuc-2 double mutant in the Normal Sequence background. The double mutant was constructed using a semi-selective procedure. At the outset, we did not know the phenotype OI such a double mutant with respect to alkaline phosphatase production and therefore this property could not be incorporated into the selection procedure. However, since we did know that the gene order on LG I1 of the Normal Sequence chromosome is arg-5 nuc-2 preg arom-1 (Figure 2), it was possible to enrich for nuc-2 pregc-2 recombinants by selecting prototrophic segregants from a cross between arg-5 pregc-2-aand nuc-2 arom-1-A. Spores from this cross were plated on high PI minimal medium and 220 prototrophic germinants (recombinants between arg-5 and arom-1 ) were picked. The prototrophic progeny were tested for their ability to produce alkaline phosphatase on high PI and low P, media. Forty-seven were “null” (“nuc-2like”), 164 were constitutive ( “pregc-Zike”) and nine were repressible (wildtype). All of the “nuc-2-like” progeny and 50 of the “preg“-like” progeny were analyzed for the presence of the mutation not indicated by their phenotype. The analysis of the “nuc-2-like” isolates for the presence of the pieg“-2 mutation will not be described as none of these strains proved to be the desired double mutant. If a particular “pregc-like” segregant is actually the nuc-2 pregc-z double mutant, a cross to nuc-2f pregf will produce a small percentage of nuc-2 segregants. To look for such segregants, the “pregC-like”isolates were crossed to inos strains of the opposite mating type. Spores were plated to low P, inositol medium and the resulting colonies stained for alkaline phosphatase. The cross between “pregC-like” #32 and inos-a produced about 2% unstained colonies, i.e. nuc-2 segregants. Hence, “preg“-like” #32 is the nuc-2 pregc--2double mutant. The production of alkaline phosphatase by nuc-2 pregC-$grown on repression and derepression medium is shown in Table 2. The strain grows on high pH, low P, medium, indicating that the high affinity, high pH phosphate permease is also under pregC-% control. These data show that pregc is epistatic to nuc-2. The simplest interpretation of this fact is that the pmgf product acts between the nuc-2+ product and the structural genes in the series of events required to “turn on’’ alkaline phosphatase production. (See the model presented in the beginning of this paper). Such a conclusion is unwarranted, however, if pregc-z and nuc-2 represent mutational events within a single cistron. The rather large map distance (1-2 centimorgans) between these mutations argues against this. Epistasis of nuc-1 to nuc-2 pregc-2: We previously reported that both preg“; nuc-l and pconc; nuc-l double mutants have the “nuc” phenotype (METZENBERG, GLEASON and LITTLEWOOD 1974), as do nuc-1; nuc-2 strains. By examin- zy 426 zyxwvut zyxwvuts zyxwv zyxw zyxwvu zyxwv B. S. LITTLEWOOD, W. CHIA A N D R. L. METZENBERG TABLE 2 Alkaline phosphatase in nuc-2 pregc-2 and nuc-2 pregc-2; nuc-1 euploids Alkaline phosphatase, specific activity ~~ HighPi 2.2 1.0 PE 1580 2.5 10'0 478 Y35 744 445 0.96 1.3 7.8 2.1 repressible null constitutive constitutive null null Strains were grown as described in the legend of Table 1. ing the nuc-2 preg"; nuc-I triple mutant, it was possible to show similar epistasis of nuc-1 over nuc-2 pregc. The triple mutant was constructed as follows. nuc-2 prege+a was crossed to arg-12; nuc-1-A and 15 prototrophic sporelings were isolated. Of these, seven were mating type A and had the "nuc" phenotype. On the basis of linkage relationships (Figure 2), these seven were presumed to be the desired triple mutants. The nuc-2 pregc-2; nuc-1 genotype of two such "nuc" strains was confirmed by the isolation of constitutive progeny when these strains were crossed to wild type. Both of the testcrosses (nuc-2pregc-2; nuc-2-A by nuc-2+ preg+; nuc-I+-a) gave approximately 25 % constitutive ( n u c - 2 preg"-') progeny. Assays of alkaline phosphatase in the nuc-2 pregC-'; nuc-1 triple mutant grown on repression and derepression media (Table 2) confirm its "nuc" phenotype. nuc-2 mutations can effectively eliminate the constitutive production of alkaline phosphatase resulting from nuc-2 pregc, preg" or pconC mutations. Hence, the action of the nuc-I+ product must be exerted between that of the pcon' or p r e g f products and the structural genes. Our data suggest that, under repressing conditions, the "turn on" function of the n u c - l f product is cancelled by the p r e g f product, and that preg" mutants lack this product. Behavior of nuc-2 pregc-2 in heterozygous partial diploids: Partial diploids carrying nuc-2 pregc--"in trans configuration to wild type, to pregc-', and to nuc-2 respectively (Table 3) were isolated from crosses between fiuc-2 preg"-'-A and the appropriate ( T ) a strains. Unselected progeny (50-100) from each cross were tested for diploidy and for their ability to produce alkaline phosphatase on high P, and low P, media. In each cross, the phenotype of the diploid segregants was uniform and a small number of them were arbitrarily chosen for further study. The production of alkaline phosphatase by these diploids grown on repression and derepression media at 33" is shown in Table 3. nuc-2 preg"-* is recessive to nuc-2+ preg+, as are both nuc-2 and preg"--"alleles individually. Partial diploids of the constitution nuc-2 ~ r e g ~ - ~ / n u c -pregc-' 2+ are constitutive; there is no complementation between nuc-2 pregc--" and pregc-'. From these results, we conclude that the pregc-2 allele is the direct cause of the Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 wild type nuc-2 pregc-2 nuc-2 pregc-8 nuc-I nuc-2 pregc-2 ; nuc-2 hwPi zyxw zyx zy zyxw zyxwvu 42 7 REGULATORY M U T A N T S I N NEUROSPORA TABLE 3 Alkaline phosphatase in partial diploids carrying nuc-2 pregc Alkaline phosphatase, specific activity Euploids wild type HighPi LowP, PE 2.2 1580 1GQ null 2.5 1.8 Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 1.o 1.o repressible zyxwvutsr nuc-2 pregc-2 (T)nuc-2t8-95 pregc-1 478 869 235 506 constitutive 744. 514 445 44.3 coastitutive 92.7 154 repressible 381 559 constitutive Partial diploids nuc-2 pregc-z/nuc-2+ preg+ nuc-2f preg+/nuc-2ts-35 pregc-1 nuc-2 pregc-2/nuc-2+ pregc-1 nuc-2+ pregc-z/nuc-2ts-s5 pregc-1 nuc-2 pregc-2/nuc-2ts-35 p e g + nuc-2 preg+/nuc-2ts-35 pregc-1 nuc-2 pregc-2/nuc-2ts-s5 pregc-1 1.6 2.9 666 701 1.o 1.5 null 8.8 1.6 924 879 constitutive zyxwvu zyxwvu Strains were grown as described in the legend of Table 1. constitutivity of the euploid double mutant, and the pregcw2has not been altered by virtue of being cis to nuc-2. Partial diploids of the constitution nuc-2 p r e g C - 2 / n ~ ~ - 2 t S p -e9g5+ have the "nuc" phenotype at 33", i.e., they are unable to produce alkaline phosphatase on either repression or derepression media. The only simple explanation of this is that (a) the wild-type preg product made by the n ~ ~ -pregf 2 ~ segment ~ - ~ is~ exerting its expected dominance over pregC-2,and (b) the restoration of normal preg function unmasks the cryptic nuc-2 allele on the Normal Sequence chromosome. Because the nuc-2 alleles are noncomplementing (Table I ) , the nuc-2 p r e g c - z / n ~ ~ - 2 t sp-e3g5+ diploid is "null" for alkaline phosphatase production. The results show that the cis configuration of the nuc-2 and pregc-z mutations lias not changed the properties of either pregc-2 or nuc-2. Both alleles remain recessive and non-functional when they are adjacent to one another. We conclude, therefore, that p e g e and nuc-2 must represent lesions in separate and distinct cistrons. (As nuc-2 and pconC mutations have been shown to be within the same cistron, it follows that preg" and pconCmutations must be in different cistrons.) Construction of n ~ c - 2 ~preg-l " ~ ~ and its behavior in partial diploids: A procedure analogous to that used to prepare n u ~ - 2 p r e g ' - was ~ used to construct ( T ) n u ~ - 2 preg"'. ~ ~ - ~ Because ~ the translxated segment is inserted in LG I, re- 428 zyxwvu zyxwv B. S. LITTLEWOOD, W. C H I A A N D R. L. METZENBERG Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 combination between the flanking markers Zeu-3 and cys-22 (Figure 1) was used to enrich for recombinants between the pregc and nuc-2 loci. In addition, since nuc-2 pregc-zhad proven to be constitutive, it was assumed that n ~ ~ -preg"' 2 ~ ~ - ~ ~ would also have this phenotype. Spores from a cross between (T)leu-3 preg"'-A and ( T ) n ~ c - 2 ~ ~ - ~ ~ c y swere - 2 1 -plated a on high P, minimal medium and the resulting prototrophic colonies stained for alkaline phosphatase. Twenty-three constitutive segregants were selected for further analysis. The genotype of the constitutive prototrophic recombinants ( (T)preg"' us. I T ) ~ u c -preg"') ~ ~ ~ -could ~ ~ be tentatively assigned by examining their behavior in partial diploids with nuc-2 preg+. nuc-2 preg+/pregc diploids are repressible GLEASONand LITTLEWOOD 1974), for alkaline phosphatase (METZENBERG, whereas nuc-2 p r e g + / n u ~ - 2preg"' ~ ~ - ~ ~diploids were expected to be null since they are analogous to the nuc-2 pregc-2/riuc-2ts-35 preg+ diploids described above. Each of the 23 constitutive isolates was crossed to Normal Sequence nuc-2 preg+ arg-12 and spores were plated to low P, minimal medium at 33". The resultipg prototrophic colonies ( ( T ) n ~ c - 2preg"' ~ ~ - ~ or ~ nuc-2 prcg+ a r g - 2 2 / n ~ c - 2 ~ ~ - ~ ~ prep'-') were stained for alkaline phosphatase. Of the 23 isolates so crossed, 21 produced 100% stained colonies and two gave some unstained "nuc-2-like" colonies. The latter two isolates were considered to have the genotype ( T ) ~ u c - ~ ~ ~ - ~ ~ pregc-'. Confirmation of this genotype was obtained by outcrossing one of the putative n ~ ~ -pregc-'-A 2 ~ ~ strains - ~ ~and looking for T I U C - ~ ~segregants. ~ - ~ ~ The strain in question was crossed to (T)nuc-2f preg+-a and ascospores were examined as above. As predicted, 1-2% of the resulting colonies had the "nuc" phenotype, indicating that the constitutive parent is indeed ( T ) ~ u c -pregc-'-A. ~ ~ ~ - ~ ~ Partial diploids carrying n ~ ~ -preg"' 2 ~ trans ~ - to ~ wild ~ type, pregc-2,nuc-2 and nuc-2 pregc-2were isolated from crosses between ( T ) n ~ c - 2preg"-'-A ~ ~ - ~ ~and the appropriate Normal Sequence strains. Alkaline phosphatase production in these strains grown on repression and derepression media at 33" is shown in Table 3. Diploids of the constitution LLX7'/nuc-2ts-35 pregc-' always behaved identically with the nuc-2 pregc-2/,cX77 strains described above. Our previous conclusion that nuc-2 and pregc occupy separate cistrons is strengthened by the fact that all results obtained with nuc-2 pregc-2"X" diploids have been confirmed with strains carrying n ~ ~ -pregc-' 2 ~ on ~ the - ~translocated ~ segment. nuc-2 pregc-2/nuc-2t"-s5 preg"' diploids (Table 3) produce alkaline phosphatase constitutively. This was expected, since pregc-eand pregC-' are non-complementing alleles (METZENBERG, GLEASON and LITTLEWOOD 1974). Characterization and mapping of a temperature-sensitive nuc-2 mutant in the Normal Sequence background: n u ~ - 2 ~ ~ previously -~ss, designated n ~ c - (MKG2 ~ ~ 139), was isolated from a wild-type strain as an unstained colony during a search for pho-2 mutants (GLEASON and METZENBERG 1974). On high pH, low P, medium, the strain grew slowly at 25", but did not grow at all at 33" on this medium. The strain was spotted on low Pi medium and grown at 25" and 33". At 25", the mutant stained weakly for alkaline phosphatase; at 33", no alkaline phosphatase was detectable. I n a heterokaryon test, nuc-2ts-1ssfailed to comple- zy zyxwvu zy zyxwvu zy REGULATORY MUTANTS I N NEUROSPORA 429 Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 ment with the standard nuc-2 allele at the restrictive temperature. Partial diploids of the constitution n u ~ - 2 ~ ~ - - ' ~ ~ / n u had c - 2 ~the " - "nuc~' "~ phenotype at 33". n ~ c - 2 ~was " ~previously ~ ~ reported to map on LG I1 near pconC (GLEASON and METZENBERG 1974). To locate its map positioc with respect to pcone more precisely. nuc-2ts-139 arg-12-A was crossed to pconc-6 arg-5-a. Spores were plated to high pH, low PI arginine medium and grown at 33". Under these conditions, I L U C - ~ spores ~ ~ - ~ will ~ ~ not form colonies. Colonies which arose were replicated to high P, arginine plates at 33" and stained for alkaline phosphatase. Unstained colonies were picked and tested for their ability to produce alkaline phosphatase on high P, and low Pi medium and for their arginine requirement. Among 501 nuc-2+ colonies examined, two wild types (repressible for alkaline phosphatase) were found; both were prototrophic for arginine. This shows that the gene order on LG I1 Normal Sequence is arg-5 pconC n ~ ~ - arg-12, 2 ~ with ~ - the ~ ~n ~~ c - 2 ~ ~ mutation being about 0.4 centimorgans from pcone. Since no recombinants among 465 nuc-2+ progeny were found in an analogous cross between the standard nuc-2 allele and pconc-6 (METZENBERG, GLEASON and LITTLEWOOD 1974), nuc2ts-139 is presumably also to the right of nuc-2. In a confirmatory experiment, the cross was repeated with the outside auxotrophic markers in the opposite configuration. arg-12 pconc-6; inos-a was crossed and - 'spores ~ ~ - were A examined as above. Among 598 nuc-2+ to arg-5 ~ u c - ~ ~ ~ spores examined, one repressible recombinant was found, and this strain required arginine. It did not grow on ornithine, which supports the growth of arg-5 but not of arg-12. Hence the recombinant did carry arg-12. Whether it also carried arg-5 was not determined. Constitutive reuertants of n ~ c - 2 ~ ~We - * ~found ~ : that both the nuc-2 preg"-' and nuc-2ts-ss preg"" double mutants have the pregc phenotype; that is, they grow on high pH, IOWPI medium and are highly constitutive for alkaline phosphatase. This finding predicts that amoFg revertants of nuc-2 that are able to grow on high pH, low Pi medium, one class should result from new constitutive mutations at the preg locus, rather than reversion in the nuc-2 gene. A test of this prediction seemed critical since, when nuc-2 revertants are selected with RNA as the sole phosphorus source, no highly constitutive revertants are found (ToH-Eand ISHIKAWA 1971). To obtain revertants, conidia of arg-12 n u ~ - 2 ~ ~ - were - ' ~ ~irradiated -A with UV light as usual and plated on high pH, low Pi medium at 33". One hundred fiftysix revertant colonies arose from 1.5 x IOF survivors. These revertants, designated nuc-2t8-13grev-l through nuc-2ts-rsgrev-156, were grown on high Pi and low P , plates at 33" and stained to test for alkaline phosphatase production. Among the 156 revertants, eleven were highly constitutive for alkaline phosphatase. The constitutive revertanls were crossed to (T)fL-a. Assuming that pconC and nuc-2ts-139are within a single cistron, at least five different mutational events could convert the n ~ c - 2strain ~ ~ to a constitutive phenotype: (1) a reversion at the n ~ c - site, 2 ~ which ~ converts n ~ c - to 2 ~pconc, ~ (2) the induction of an unlinked suppressor which imparts to n ~ c - the 2 ~pheno~ type of pconC, ( 3 ) the induction of a linked suppressor which imparts to n ~ c - 2 ~ ~ zyxwv 430 zyxwvutsr zyxwvutsrqpo zyxwvut B. S. LITTLEWOOD, W. CHIA A N D R. L. METZENBERG jg TABLE 4 zyx Possibls origin of constitutiue revertants of nuc-2ts and their distinguishing characteristics %gin of constitutive revertants (1) (2) (3) (4) (5) site reversion unlinked suppressor linked suppressor constitutive mutation in preg constitutive mutation not linked to nuc-2 Genotype of revertant peonC nuc-2ts; Su nuc-2ts Su nuc-2ts pregc nuc-2ts; X c Linkage to nuc-2'8 linked unlinked linked linked unlinked Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 zyxwv zy zy zyxwvu the phenotype of pcon", (4) the induction of a new constitutive mutation linked to n ~ c - 2(such ~ ~ as a new preg") and ( 5 ) the induction of a new constitutive mutation unlinked to n u ~ - 2and ~ ~in , a gene in which constitutive mutations have not been previously identified. These alternatives are outlined in Table 4. Alternatives ( l ) , ( 3 ) and (4)involve a reversion event that is linked to nuc2tS-'3g.To test for such linkage, each of the arg-12-A constitutive revertants was crossed to arom-1-.* and spores were plated on high Pi arginine plates at 33". The resulting arom+ colonies, the majority of which will carry n ~ c - 2 ~ ~ (see - ' Figure 2), were stained for alkaline phosphatase. In eight of the crosses. over 90% of the arom+ colonies were constitutive for alkaline phosphatase, indicating that the genetic event leading to the constitutivity of the revertants is linked to arom-1 and hence to In three of the crosses, the constitutivity of the revertants was found to be unlinked to n ~ ~ - 2these ~ ~three - ~ revertants ~ ~ ; which fall under alternatives (2) and ( 5 ) . will be discussed in a future publication. Alternatives (1) ad ( 3 ) predict that the constitutivity of the n u ~ revertants 2 ~ ~ will be co-dominant in partial diploids, as are pcun" mutations. To test this, partial diploids were prepared between each of the eight constitutive revertants mapping on LG I1 and wild type. As an example, arq-12 n u ~ - 2 ~ ~ rev-58-A - - ' ~ ~ was crossed to (T)fZ-a and spores were plated on high P, minimal medium at 33". (Only the translocation euploid and the heterozygous partial diploid are prototrophic.) The resulting colonies were stained for alkaline phosphatase. No stained colonies were observed among the seventy tested. In a control experiment, spores from this cross were plated on arginine high P,, where all progeny are viable. About one-half of the resulting colonies stained. Similar data were obtained for diploids heterozygous for I ~ u c - rev-27, ~ ~ ~ n-~ ~ c - ~2 ~~s -rev-35, 1 ~ ~ nuc-2tfi-1s9rev-54, nuc-2ts-1sg rev-59, nuc2ts-1sgrev-136 and rev-146. Hence the genetic event leading to the constitutivity of these eight revertants of nuc-2ts-1sgis recessive in diploids, has been converted to pcorr" (alternaeliminating the possibility that tives (1) and ( 3 ) ) . Having eliminated alternatives ( 1) , ( 2 ) , ( 3 ) and ( 5 ) , the most likely explanation for the constitutive production of alkaline phosphatase in the eight 1 r u c - 2 ~ ~ revertants mapping on LG I1 is alternative (4): that they carry new constitutive mutations outside the nuc-2 pcon cistron. T o test if the new mutations are in the preg locus, diploids between the n ~ c - constitutive 2 ~ ~ revertants and pregc were Behavior in diploids with wild type ccr-dominant co-dominant co-dominant recessive (untestable) zy zyxwvu REGULATORY M U T A N T S IN NEUROSPORA 43 1 DISCUSSION The repression and derepression of the enzymes needed by Neurospora crassa for the liberation and uptake of inorganic phosphate from the environment is controlled by the extracellular concentration of inorganic phosphate and by at least three regulatory genes: pcon-nuc-2, preg and nuc-1. Studies of the phenotypes of strains carrying mutations in two o r more of these genes have shown that their actions are exerted in a definite sequence. It is this cascade of molecular events that the model presmted in our introduction is intended to illustrate. I n this discussion, we use the term gene L ' p r o d ~ ~when t ~ ' ' describing the activities of the control loci, but we must admit at the outset that we have only circumstantial evidence that all three are indeed making products. The fact that all three control loci can harbor mutations which are recessive in both partial diploids and Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 prepared and analyzed for the ability to produce alkaline phosphatase on high P, medium. For example, arg-12 n ~ c - 2 ~rev-58-A " ~ ~ ~ was crossed to (T)preg"-'-a and spores were plated to high P, minimal medium at 33". Twenty of the resulting prototrophic colonies were tested for diploidy and for their ability to produce alkaline phosphatase constitutively. Eight of the prototrophs were arg-12 nuc-2ts-13g rev-58/pregC-l diploids and all produced alkaline phosphatase on high P, medium. As a control, spores from this cross were plated to arginine high P, and stained for alkaline phosphatase; all the resulting colonies produced the enzyme constitutively. Similarly data were obtained for diploids between preg"-l and six of the other recessive constitutive revertants. (One has not been classified.) Since the constitutivity of both pregc and the nuc-2ts-139revertants is recessive, the finding that diploids between these two are constitutive for alkaline phosphatase indicates that n ~ c - revertants 2 ~ ~ of this class contain a new preg" mutation. Constitutive revertants of n ~ c - 2 ~ ~Experiments -~;: were carried out to deter2 ~ ~ this one on the translocated segment. could also mine if another n ~ c - mutation, revert via the acquisition of a new preg" mutation. Constitutive revertants were selected essentially as above. The reversion of n ~ ~ - to2a ~constitutive ~ - ~ ~phenotype can theoretically occur by the same five genetic mechanisms considered for reversion of (Table 4). Two factors not present in the case of n u ~ - 2 ~ revertants ~ - - ' ~ ~ complicate the analyses of nuc-2ts-s5constitutive revertants. First, n ~ ~ -may 2 ~contain ~ - an ~ ~ unexpressed pconC mutation, in which case, a site reversion or suppression of to wild type would produce a constitutive phenotype, namely the original pconc-2.The second complicating factor is that, in the tramlocation strains, nuc-2 is linked (about 10 centimorgans) to nuc-1 (Figure 1). If constitutive mutations can arise at the nuc-1 locus, linkage analysis would no longer be a definitive test of whether the reversion event has occurred within one of the LG I1 control loci. Despite these complications, a genetic analysis of the revertants revealed that n ~ c - on 2 ~the ~ translocated segment can give rise to constitutives by the same sort of processes as occurred with Normal Sequence nuc-2. 432 B. S. LITTLEWOOD, W. zyxwvu zyx CHIA AND R. L. METZENBERG heterokaryons is evidence that each wild-type allele makes a diffusible product which is necessary for repression and derepression to occur. Since nuc-1 is on a different chromosome from the other two control loci, at least one diffusible product is necessary for the loci to interact. The isolation of temperature-sensitive rzuc-2 mutants reported here and by TOH-Eand ISHIKAWA (1971) suggests that the nuc-2+ allele makes a macromolecular product. HASUNUMA and ISHIKAWA 1972) have tentatively proposed that the nuc-l + allele codes for a protein with nuclease activity and that nuc-2f produces a protein inhibitor of this nuclease. These proteins are present and active in both nuc-1 and nuc-2 mutants, though it appears they are qualitatively altered. Their activity has not been examined in strains carrying constitutive mutations. For these reasons, it is not safe to speculate on the molecular nature of the products of these control loci. Most of the interactions discussed below could be accounted for by protein-protein, proteinDNA or other macromolecule-macromolecule binding. nuc-1 mutations are recessive to the wild-type allele in both heterokaryons and partial diploids (unpublished results), so we conclude that the nuc-1 + allele makes a product which is necessary to “turn on” the structural gene for alkaline phosphatase. The finding that nuc-l + is essential, even in low phosphate medium, for production of alkaline phosphatase, even in strains carrying pconc, preg“ or nuc-2 pregc, further indicates that the action of the nuc-l+ product is the final and indispensable step needed to “turn on” the structural genes. This leads to the prediction that constitutive mutations should exist at the nuc-1 locus in which the nuc-1 product always “turns on” the structural genes, even in the presence of preg+ and pconf. Indeed, Constitutive mutations mapping in or near nuc-l have now been found (CHIA,unpublished). Studies of nuc-2 pregc double mutants have shown that nuc-2 and pregc mutations are ir?different cistrons and that preg“ is epistatic to nuc-2. Regardless of the allele at the nuc-2 locus, pregc;nuc-l+ produces alkaline phosphatase constitutively. Therefore, in wild type, p m g + product must exert its effect between nuc-2+ and nuc-1+ . Let us proceed from the conclusion that nuc-l+ turns on the structural genes. Then in repressible strains (e.g., wild type and pregc/preg+) grown on high phosphate, the pregf product must inactivate or repress the nuc-l+ product. Conversely, when the pieg product is absent or is unable to neutralize the “turn-on’’ role of the nuc-I+ product, alkaline phosphatase and its congeners will be made. If the nuc-l+ product interacts with the structural genes, the interaction between preg+ and nuc-l+ can be explained by three models. (1) nuc-l+ and preg+ products combine, thus preventing the nuc-l+ product from “turning on” the structural genes (see introduction) o r (2) preg+ product represses transcription at nuc-l+ o r (3) both the nuc-I+ and preg+ products act at control sites adjacent to the structural genes, with the binding of the pregf product causing repression of enzyme synthesis. The third model seems implausible, since it predicts that constitutive mutations mapping at nuc-l would not occur. Such mutations have, however, been found (see above). Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 zyxw zyxw zyxwvut REGULATORY M U T A N T S IN NEUROSPORA 433 If the role of the pregf product is to inactivate the nuc-Z+ product, then, for derepression of alkaline phosphatase to occur, some other element must render the preg+ product inactive (or, conceivably, repress its formation). This element appears to be the nuc-2+ product. Since nuc-2 mutations are recessive to wild type in both heterokaryons and partial diploids, the nuc-2+ product must be necessary to “turn on” alkaline phosphatase production in wild-type strains. Yet nuc-2 preg“ double mutants are constitutive. The nuc-2 mutation can block alkaline phosphatase production if pregf product is present, but it cannot override the constitutivity caused by preeg“ mutations. This indicates that the nuc-2+ product acts prior to the preg+ product. It also indicates that nuc-2 mutants lack something which. in nuc-2+ strains, prevents the preg+ product from inactivating the nuc-Z+ product. In nuc-2f preg+ strains or in partial diploids carrying at least one wild type allele of each, the nuc-2+ product must, under derepressing con&tions, cancel out the pregf product. This allows the nuc-I+ product to activate the structural gepes. Since the reversion of pconCto n ~ c - has 2 ~ proven ~ to be an intragenic event, p o n C mutations must alter the same gene product that is affected by nuc-2 mutations. According to our model, pconCmutations should make a product which always eliminates p e g + function, regardless of the phosphate concentration. One explanation of the phenotype of pconC mutants is that they make a pcon (nuc-2) product which has lost its sensitivity to phosphate or to a corepressor derived from it. Such a mutation should be dominant, since this P,-insensitive product would always be available to inactivate preg+ product, even if a P,-sensilive pcon+ product was present in the cell. In agreement with this prediction, pconCmutants are dominant. Although we know that peonCand nuc-2 mutations lie within the same cistron, it remains to be determined what kinds of genetic lesions lead to these opposing phenotypes. One can speculate that the two phenotypes result from mutations in different regions of the gene or that different types of mutations (i.e. nonsense, missense) are needed to produce the two phenotypes. We have not done sufficient intragenic mapping to know if constitutive and null mutations are interspersed along the map. We are grateful for the fine technical help of DAVIDBESWICK during part of this work, and for the valuable aid of PETERD A V I who ~ participated in this work as part of his high-schd science program. LITERATURE CITED Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 zyxwv zyx zyxwvut BEADLE, G. W. and E. L. TATUM, 1945 Neurospora. 11: Methods of producing and detecting mutations concerned with nutritional requirements. Am. J. Botany 32 : 678-686. BROCKM~NN,H. E. and F. J. DESERRES, 1963 “Sorbose toxicity” in Neurospora. Am. J. Botany 50: 709-714. BURTON, E. G. and R. L. METZENBERG, 1974 Properties of repressible alkaline phosphatase from wild type and a wall-less mutant of Neurospora crassa. J . Biol. Chem. 249 : 4 6 7 9 4 8 8 . DAVIS,R. H. and F. J. DESERRES, 1970 “Genetic and microbiological techniques for Neurospora crassa,” pp. 79-143. In: Methods in Enzymology, Vol. 17A. Edited by H. TABOR and C. W. TABOR. Academic Press, New York. 434 zyxwvut zyxwvut zyxw zyxwvutsr zyxwv B. S. LITTLEWOOD, W. CHIA AND R. L. METZENBERG zyxwvuts zyxwvuts DOUGLAS, H. C. and D. C. HAWTHORNE, 1972 Uninducible mutants i n the gal i locus of Saccharomyccs cereuisiae. J. Bacterial. 109 : 1139-1 143. GLEASON, M. K. and R. L. METZENBERG, 1974 Regulation of phosphate metabolism in Neurospora crassa: isolation of mutants deficient in the repressible alkaline phosphatase. Genetics 78: 645-659. HASUNUMA, K., 1973 Repressible extracellular nucleases in Neurospora crassa. Biochim. Biophys. Acta 319: 288-293. LEHMAN,J. F., M. K. GLEASON, S. K. AHLGRENand R. L. METZENBERG, 1973 Regulation of phosphate metabolism in Neurospora crassz. Characterization of regulatory mutants. Genetics 75: 61-73. 1972 Simple and reliable method for replica plating in LITTLEWOOD, R. K. and K. D. MUNKRES, Neurospora crassa J. Bacteriol. 110: 1017-1021. LowENnoRF, H. s. and c. W. SLAYMAN, 1970 Phosphate transport in Neurospora crassa. Bacteriol. Proc. 1970: 130 (abstr.). 1974 Genetic control of alkaline METZENBERG, R. L., M. K. GLEASON and B. S. LITTLEWOOD, phosphatase synthesis in Neurospora crassa: the use of partial diploids in dominance studies. Genetics 77: 25-43. NYC,J. F., 1967 A repressible acid phosphatase in Neurospora crassa. Biochem. Biophys. Res. Commun. 27: 183-188. PERKINS, D. D., 1972 An insertional translocation in Neurospora that generates duplications heterozygous for mating type.Genetics 71: 25-51. TOH-E, A. and T. ISHIKAWA, 1971 Genetic control of the synthesis of repressible phosphatase in Neurospora crassa.Genetics 69 :339-35 1. TOH-E, A., Y. UEDAand Y. OSHIMA, 1973 Genetic regulatory system for acid phosphatase formation in Saccharomyces. Genetics 74: s277. Downloaded from https://academic.oup.com/genetics/article/79/3/419/5991269 by guest on 01 April 2022 HASUNUMA, K. and T. ISIIIKAWA, 1972 Properties of two nuclease genes in Neurospcra crassu. Genetics 70: 371-384. zyxwvut TOH-E, A., Y. UEDA,S. KAKIMOTO and Y. OSHIMA,1973 Isolation and characterization of acid phosphatase mutants in Saccharomyces cereuisiae. J. Bacteriol. 113 : 727-738. WESTERGAARD, M. and H. K. MITCHELL,1947 Neurospora. V. A synthetic medium favoring sexual reproduction. Am. J. Botany 34: 573-577. WIAME, J. M., 1971 The regulation of arginine metabolism in Saccharomyces cereuisiae: Exclusion mechanism. Cum. Top. Cell. Reg. 4: 1-38. Corresponding editor. D. R. STADLER