GENETICAL AND BIOCHEMICAL CHARACTERIZATION OF QA-3 MUTANTS AND REVERTANTS I N THE Q A GENE CLUSTER OF NEUROSPORA CRASSA MARY E. CASE, CARMEN PUEYO,1 J. LOPEZ BAREA,1

AND

NORMAN H. GILES

Program in Genetics and Department of Zoology, University of Georgia, Athens, Georgia 30602 Manuscript received December 13, 1977 Revised copy received March 31, 1978 ABSTRACT

The qa-3 gene, one of the four genes in the qa gene cluster, encodes quinate (shikimate) dehydrogenase (quinate: NAD oxidoreductase, ER 1 . I .I .24), the first enzyme in the inducible quinic acid catabolic pathway in Neurospora crassa. Genetic analyses have localized 26 qa-3 mutants at 11 sites on the qa-3 genetic map on the basis of prototroph frequencies. Certain mutants, e.g., 3363-10 and 336-3-3, are located at opposite ends of the qa-3 gene. Data from four-point crosses (qa-2* mutant 124 x five different qa-3 mutants in triple mutants qa-3, qa-4, qa-2) indicate the following orientation of the qn-3 gene within the qa cluster: qa-2, qa-3 mutant 336-3-10 (“left” end) qa-3 mutant 336-3-3 (“right” end), qa-4, qa-2. Ultraviolet-induced revertants have been obtained from 14 of the qa-3 mutants. The revertable mutants fall into two major classes: those that revert by changes either at the same site or at a second site within the qa-3 gene, and those that revert by unlinked suppressor mutations. The intragenic revertants can be farther distinguished by quantative and/or qualitative differences in their quinate dehydrogenase activities. Some revertants with activities either equivalent to or less than wild type produce a thermostable enzyme, and others an enzyme which is thermolabile in vitro at 35”. A concentration of quinic acid or shikimic acid as low as 50 BM protects the enzyme markedly from heat inactivation. The genetic organization and the orientation of the qa-3 gene are discussed with respect to its direction of transcription and to the possible localization of a promoter (initiator) region(s) within the qa gene cluster.

e qa gene cluster of Neurospora crassa, the ga-3 gene encodes the inducible ‘ Y n t p e quinate dehydrogenase [quinate:NAD oxidoreductase ( QDHase) EC 1.1.1.241. This enzyme plus two other inducible enzymes encoded in two additional structural genes in the cluster catalyze the three initial reactions in the catabolism of quinic acid. The two additional genes are ga-2, which encodes catabolic dehydroquinase [S-dehydroquinate hydrolyase (C-DHQase) EC 4.2.1.IO], and ga-4, which encodes dehydroshikimate dehydrase (DHS-Dase). The fourth gene in the cluster encodes a regulatory protein that apparently exerts positive 1975). control over the synthesis of these three ga enzymes (CASEand GILES Present address: Laboratory of Environmental Mutagenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709. Genetics 90: 69-84 September, 1978.

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Efforts are underway to determine, at the molecular level, the organization and mode of regulation of the qa cluster. Genetic studies have established the gene order in the cluster (CASEand GILES1976). Biochemical studies have involved the purification and characterization of the three qa enzymes. Quinate dehydrogenase has been purified and extensively characterized (BAREA and GILES1978). The enzyme is a monomer with a molecular weight of ca. 41,000 daltons. Studies are underway to determine the N-terminal amino acid sequence of this enzyme. Utilizing an automatic sequencer, a preliminary sequence has been determined for the first twenty amino acids from the wild type QDHase (STRBMAN, REINERT and GILES,unpublished). The present studies are concerned with the genetical and biochemical characterization of qa-3 mutants and revertants. These studies have established a genetic map that localizes various qa-3 mutants within the ga-3 gene. In addition, the studies provide genetic evidence concerning the orientation of the ga-3 gene within the qa cluster. Numerous revertants have been characterized both genetically and biochemically. Most revertants appear to involve mutational changes within the qa-3 gene, although at least one results from an external suppressor mutation. Appropriate primary mutants and revertants should be useful in amino acid sequence studies designed to determine which end of the qa-3 gene encodes the N-terminal sequence of QDHase. MATERIALS .4ND METHODS

Strains: Most of the 26 qa-3 strains used in these studies were derived from or are very closely related to wild type 74A, and eight have been previously described (CHALEFF1974; CASEand GILES1976). An additional 13 qa-3 mutants were obtained in an arom-9 strain (Y335 isolates) which lacks biosynthetic dehydroquinase (RINES,CASEand GILES1969) and five were induced in a double qa-4 qa-2 strain (Y336 isolates) (CASEand GILES1976). Subsequently the latter five qa-3 mutants were recovered as recombinants from a cross of the resulting triple mutant, qa-3 qa-4 qa-2, with a qa-1 mutant (strain 140). [For details, consult Table 5 in CASEand GILES(1976)l. Additional strains employed in these studies were as follows: me-7 allele 4894, which is very rlosely linked to the qa gene cluster and is useful as a marker; pan-2 alleles B23 and B36, used as markers in the reversion experiments; tryp-3 allele td 140 and nrom-54, used in the suppressor experiments; the supersuppressor (ssu) strains ssu-I (WRN33), ssu-2 (WRU35), and ssu-3 (WRU118), all obtained from the Fungal Genetics Stock Center. These ssu strains contain the am 17 allele. Additional ssu strains were ssu-I (Y319-44), ssu-5 (Y319-45), ssu-6 (Y319-26) and ssu-8 (Y319-37) from our own stock collection. Qa-1 mutant 124 nrom-9 was used in the genetic analysis of the orientation of the qa-3 gene with respect to its adjacent genes qa-1 and qa-4. Allelic complementation tests: Allelic complementation tests were performed at both 25" and 35". Mixed conidial suspensior~susing all pairwise combinations of qa-3 mutants from experiments Y330 and Y335 (21 different mutants) were inoculated into Fries liqiud minimal medium supplemented with 0.3% quinic acid as a carbon source in 13 x 100 mm. test tubes. These tubes were examined for growth at suitable intervals up to four weeks. Control inoculations were used to determine the stability of the individual strains. Recombinition analyses: All crosses were made on Difco corn meal agar. In the tetrad analyses, crosses were made to me-7, a very closely linked marker (CHALEFF1974), because of better fertility. Asci were isolated from crosses of qa-3 revertants to obtain homocaryotic

QA-3 M U T A N T S A N D REVERTANTS

71

isolates and to determine whether the revertants are mutations within the qa gene cluster or are due to unlinked suppressor mutations. In the genetic analysis of the two-point crosses, ascospores from each cross were suspended in distilled water and heat shocked f o r one h r at 60". The ascospore suspensions from each cross were added to 300 ml Fries minimal medium with 0.3% quinic acid as sole carbon source. Three ml aliquots were dispensed into 13 x 100 mm test tubes at a spore concentration of no more than 4000 spores/tube. These tubes were incubated at 25" for two weeks. Prototrophs were detected as growth in individual tubes. At this spore concentration, no more than one prototroph/tube would he expected. All prototroph frequencies used to establish the relative positions of alleles on the 4a-3 genetic map are given as the percent of the total ascospore population tested. The genetic analyses of all four-point crosses, qa-1s (124 arom-9) with various qa-3 qa-4 qa-2 arom-9 strains were done in the usual manner by plating ascospores on sorbose-fructose-glucoseagar and the characterization of all prototrophs used to determine the orientation of the qa-3 gene within the qa cluster have been described in detail (CASEand GILES1976). Reversion experiments: Reversions in qa-3 strains (with or without a pan-2 mutant also present) that had regained the ability to grow on quinic acid were isolated following ultraviolet irradiation and dispersion of macrocondia into liquid Fries minimal medium supplemented with 0.3% quinic acid in 13 x 100 mm test tubes at 3 ml/tube. These tubes were incubated at 25" for two weeks. Initial concentrations were approximately 2.5 x 104 conidia per tube, assuming 50% o r less survival following irradiation. A total of ca. 50 x 106 conidia were treated for each strain. Enzyme assays: Growth conditions and enzyme assay procedures have been described previously (CHALEFF1974). Enzyme heat inactivation experiments, with or without added shikimic or quinic acid, were performed at 35" for the times indicated, utilizing a dialyzed enzyme extract as described by BAREAand GILES(1978). Specific activities are given as the change in nanomoles of substrate per minute per mg protein at 37". RESULTS

Location of the centromere relative to the qa gene cluster and to me-7: Random ascospore data have established that the qa gene cluster is proximal to and very closely linked to me-7 in linkage group VI1 ( CHALEFF1974). The position of the centromere with respect to the qa gene cluster and to me-7 has been of continuing interest. From various qa crosses involving 221 asci, no second division segregation asci have been obtained. However, from 288 tetrads isolated from crosses involving various qa mutants and revertants with me-7, one second division segregation was obtained. [These data include the qa-l constitutive revertant asci described by VALONE, CASEand GILES (1971)l. Thus, the map distance between the centromere and me-7 has been estimated as ca. 0.2 map units. Since the single ascus giving a second division segregation came from a cross of a qa-3 revertant to me-7, it is not possible to determine the position of the qa gene cluster with respect to me-7. However, these data suggest that the qa gene cluster is closer to the centromere than is me-7. Genetic map of the qa-3 gene: T h e fine-structure genetic map derived from the analyses of crosses is indicated in Figure 1. The relative positions of the 26 mutants on the genetic map are based on prototroph frequencies for the various crosses. Representative data are given in Table 1. A minimum of 11 separable mutational sites within the qa-3 gene have been detected. The cluster of mutations located at the right end of the genetic map would probably be resolved into

72

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E. CASE et al.

45

16

22 23

34 0 41

54

1

I

1

27 I

1

33 I

13

5

160

2

I

l05l

1

I

I 3

26 I

I

1

1.9

I

1

I

I

I

I

,

z 1

1

I

l

8 II

‘\

3.4.7

18.21,38

1

I

14

16.7

3-3 3-7

29 3-28 3-13

9

24

L 1

I

59

14

22

I

I

FIGURE 1.-The genetic map of the qrr-3 gene based on data presented in Table 1. All mutants shown above the line have been crossed in all paimise combinations. All mutants shown below the line in the cluster at the right end, “homoallelic” to M16, have been crossed in all pairwise combinations and also to all mutants above the line. Based on prototroph frequencies, the map distance between mutant sites in strains M45 and M16 is 0.049%, or as given on the map, 49/105 ascospores.

additional sites if larger numbers of ascospores were tested from appropriate crosses. The particular procedure used limits the population size that can be easily analyzed. For example, mutants 336-3-3 and 336-3-7 are separable by a very low prototroph frequency, but their relationship to M I 6 still remains to be determined. It should be noted that mutant 336-3-7 (of particular interest in the reversion experiments) maps near one end of the qa-3 gene and is a “homoallele” of mutant M16. On the basis of prototroph frequencies in the cross of M I 6 to M45, the qa-3 gene (0.049% prototrophs) appears to be considerably larger than the qa-2 gene (0.0047% prototrophs) [CASE,HAUTALA and GILES(1977) ; as the result of a typographical error, this value is incorrect as originally published]. Orientation of the qa-3 gene with respect to the qa gene cluster: The order of the four genes within the qa cluster has already been determined utilizing fourpoint crosses (CASEand GILES1976). The order of certain qa-3 mutations within the qa-3 gene with respect to the two adjacent genes has been determined by utilizing similar four-point crosses. A qa-1” mutant (strain 124) was crossed to various triple mutants, qa-3 qa-4 qa-2. [Far the origin of the triple mutants, cf., CASEand GILES(1976) 1. The triple mutants used in these experiments contained five different qa-3 alleles located at different positions within the qa-3 gene. These different qa-3 mutants were originally induced in the double-mutant strain qa-4 qa-2 and were subsequently obtained as single qa-3 isolates, which were used in the previously described mapping studies. The five qa-3 alleles, as qa-3 qa-4

73

QA-3 M U T A N T S A N D REVERTANTS

TABLE 1 Representative data for crosses between qa-3 mutants Cross

No. ascospores tested

16 X 3-7 16 X 3-3 3-13 X 59 46 x 3-10 16 X 59* 16 x 3-28* 16 x 26+ 16 x 29 16 x 22 16 x 2+ 16 x 160 16 x 33* 16 x 54 16 x 45* 16 x 41 3-3 x 59 3-3 x 22 3-3 x 33 3-3 x 45 3-7 x 59 3-7 x 22 3-7 x 33 3-13 X 29* 3-13 X 22 3-13 X 33 45 x 3 23 x 3-10 29 x 160 33 x 29 3-10 X 33 3-28 X 59 3-28 X 22 3-28 X 33 45 x 26* 46 x 54* 45 x 33* 45 X 160 160 x 2* 33 x 26' 22 X 3-28* 160 x 33* 160 x 29 33 x 54

418,000 282,300 92,000 172,500 465,200 148,200 66,250 257,150 429,600 75,200 112,000 583,300 76,8010 342,600 72,850 151,000 109,650 134,400 161,950 133,500 179,100 127,200 87,400 214,200 128,400 75,000 82,150 63,000 207,200 147,000 133,500 160,660 123,000 132,000 74,250 316,700 267,000 76,500 51,300 160,600 207,000 63,000 156,950

NO.

frequency

0

0 0 0 0

0

0 0 38 17 9 4 57 17 20 2M 26 170 29 13 31 33 70 11

40 40 2 10 26 35 14 9 50 7 3 16 34 22 2 3 3 1

1 16 1 9 2

* These data were used to construct the qa-3 genetic map (Figure 1).

i- Prototroph frequencies given as percent

Prototroph+

prototrophs

0.008 1 0.01 15 0.0136 0.0015 0.0133 0.0226 0.0178 0.0349 0.0338

0.0496 0.0398 0.0086 0.qb3

0.0246 0.0432 O.(F082 0.W 0.0614 0.0023 0.0047 0.0202 0.0466 0.01 70 0.0143 0.0241 0.0048 0.01022

0.0099 0.0276 0.0167 0.0027 0.0009 0.0011 0.0013 0.0019 0.0099 0.0005 0.0143 0.0013

of the total ascospore population tested.

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+

QA- I

+

J 3-10 t

+

\

3-3 3-7

3-28 3-13 t

I

I

FIGURE 2.-Diagram of the cross qa-I 124 arom-9 to qa-3 qa-4 qa-2 strains (cf., Table 1 for data). Roman numerals indicate the various regions and the arrows indicate the direction of the crossover events. Isolates recovered in Region I are wild type, in Region I1 are qa-3 isolates, and in Region I11 qa-3 qa-4 isolates. Because o f the mom-9 background in both strains, no isolates with qa-1 or qa-2 would be recovered. I

qa-2 arom-9 isolates, were crossed to a strain carrying ga-lg allele 124 and the arom-9 mutation. A diagram of the type of cross is sholwn in Figure 2. These crosses were plated on a minimal sorbose-fructose-glucosemedium. This procedure can be utilized since both parents carry the same arom-9 mutation (and hence lack biosynthetic dehydroquinase activity) and both also lack catabolic dehydroquinase activity, thus requiring an aromatic amino acid supplement for growth. All prototrophs were first characterized by their ability to grow on quinic acid. Those prototrophs unable to grow on quinic acid were further characTABLE 2 Prototroph frequencies (on minimal medium) observed in ascospores plated from crosses of five qa-3 (arom 9) mutants induced in the double mutant strain qa-4 qa-2 with qa-1s mutant 124 (arom 9)

a-3 mutant in cross

336-3-10 336-3-28 336-3-1 3 336-3-7 336-3 -3

No. of ascospores tested

98,820 338,200 262,350 512,450 232,900

Prototroph frequencies of recombinant types on miniial medium Region I* Region 11' Region 111' qa+ 9a-3 9a-3 9a-4

____

0.0101 0.0169 0.0187 0.0215 0.0253

( IO) ( 57) ( 49)

(110) ( 59)

._

0.0044l ( 4)

0.0033 0.0030 0.0029 0.0034

(11) ( 8) (15) ( 8)

0.0101 0.0056 0.0129 0.0125 0.0103

The numbers in parentheses represent the actual numbers of prototrophs obtained. * See figure 2 for a diagram of this cross.

(10) (19) (34)

(64.) (24)

QA-3M U T A N T S

A N D REVERTANTS

75

terized by complementation tests (CASEand GILES1976). The results of these five crosses are shown in Table 2. The frequency of recombinants in region I (cf., Figure 2) reflects the distance between a particular qa-3 allele and qa-I mutant 124. These data indicate that qa-3 allele 336-3-10 (0.010% prototrophs), which maps at one end of the qa-3 gene (Figure l ) , is closer to 124 than are alleles 336-3-7 and 336-3-3 (0.0215 and 0.0253% prototrophs, respectively) , which map at the opposite end of the qa-3 gene. A recombinant in region I1would be a qa-3 isolate and the frequency of recombination would reflect the distance between that qa-3 allele and the qa-4 allele (strain 228) in the double mutants. These data again suggest that qa-3 allele 336-3-10 (0.0040) is farther from the qa-4 allele than are the qa-3 alleles 336-3-3 and 336-3-7 (0.0034 and 0.0029). The events occurring in region I11 should be independent of events occurring in regions I and 11, and the frequencies obtained in this region can serve as “controls” for the five crosses. With the exception of the cross with 336-3-28, these frequencies are basically similar. From these data, then, the orientation of the qa-3 gene with respect to the other qa genes appears to be qa-I, qa-3 allele 3363-10 (located at the ‘left” end of the qa-3 gene) qa-3 alleles 336-3-3 and 336-3-7 (located at the ‘‘right” end of the qa-3 gene), 92-4, qa-2 (Figure 1). Allelic complementation tests with qa-3 mutants: Evidence for allelic complementation between qa-3 mutants would be of interest since biochemical data indicate that the enzyme consists of a single polypeptide chain (BAREAand GILES 1978). However, all allelic complementationtests with qa-3 mutants at both 25” and 35” were negative. In addition, no pseudowild-type formation was observed in the crossing analyses on quinic acid. Classification of qa-3 mutants by reversion analyses: All the qa-3 mutants have been tested for their ability to revert following ultraviolet irradiation. Revertants were obtained from 14 of the 26 mutants. On the basis of reversion frequencies and the genetic characterization of revertants, qa-3 mutants fall into three general classes (Table 3): (1) stable mutants in which no revertants have as yet been obtained; (2) mutants that revert either by a change or at the same site as the original mutation or at a second site within the qa-3 gene (these two types of changes are indistinguishable at this time); (3) one mutant that is phenotypically suppressed by a mutation outside the qa-3 gene. Revertants used in further tests were outcrossed to the closely linked marker gene me-7 to obtain homocaryotic isolates for further genetic and biochemical studies. The F1isolates were again backcrossed to me-7 to determine whether the revertants were within TABLE 3 Resulis of reuersion analyses of qa-3 mutants following ultraviolet irradiaiion Categories of mutants

Stable

Revertible

Suppressible

33O-M%M22,-M29 330-M16,-M26,-M33,-M45,-M59 335-M4,-M7,-M13,-M21,-M38,-M54 335-M3,-M16O,-M17,-M18,-M23,-M31 336-3-13,3-28 336-3-3-,-3-7,-3-10 3363-7

M. E . C A S E et

76

al.

the qa-3 gene. At the present time, only one mutant (336-3-7) is known to revert by suppressor mutation and this evidence will be discussed later. Biochemical and genetical characterization of qa-3 intragenic revertants: Prior to obtaining homocaryotic isolates, all revertants were tested f o r growth on quinic acid at 25" and 35" to determine whether any revertants were temperature sensitive. The only mutant that yielded temperature-sensitive revertants was strain 330-M33. For further studies, a sample of revertants was selected from each mutant (e.g.,revertants that grew on quinic acid at rates e i t h k equivalent to or less than wild type or temperature-sensitive revertants). All revertants could be classified into a minimum of three different categories on the basis of the quantitative and qualitative characterization of their QDHase activities (Table 4): (1) revertants with a level of thermostable activity equivalent to wild type; (2) revertants with a level of thermostable activity considerably less than wild type (between 25 and 75%); (3) revertants with a thermolabile activity (also between 25 and 75% of wild type). As indicated in Table 4,certain mutants give rise to more than one type of revertant. The first two revertants from M33 (RI and R2) are of particular interest. These revertants had high levels of QDHase activity in vitro. However, during zymogram assays on acrylamide gels, no electrophoretic band of activity was observed, suggesting that the revertant protein was more labile than that of wild unpublished). Running the gels in the presence of quinic acid or type. (PUEYO, shikimic acid did not protect the activity. Subsequent studies indicated that the QDHase extracted from the two revertants was thermolabile in vitro. Although these revertants are not temperature sensitive with respect to growth, the restored enzymic activity is thermolabile in vitro, having a half-life at 35" of ca. one minute .compared with a half-life of 90 minutes for the wild-type enzyme (Figure 3). In addition, at 35" the presence of 50 mM quinic acid or shikimic TABLE 4 Characterization of quinate dehydrogenase activities in revertants induced in uarious qa-3 mutants on the basis of leuels of enzyme activity in vitro' Classification of revertants

__

__

Thermostable activities Activity equal to wild type

Activity 25 to 75% of wild type

Thermolabile activities Activity 25 to 75% of wild type

Mutants yielding specified types of revertants

330LM45 335-M3 336-3-3, -3-7, -3-10 33@M16, -M26, -M%, -M59 335-M18, -MI60 336-3-3, -3-10 330~M33 335-Ml60

* Enzyme extracts were assayed before and after heating at 35" for ten min (Cf., BAREAand GILES1978).

QA-3 MUTANTS A N D REVERTANTS

77

TIME IN MINUTES AT 35OC FIGURE 3.-Comparative heat inactivation experiments with extracts of quinate dehydrogenase from strains 330-M33-R1, 330-M33-R2, 335-Ml60-Rl3, and wild type 74A. Dialized extracts were heated for five and ten minutes at 35". Strains 330-33-R1 and 330-33-R2 were also tested in the presence of 50 mM quinic acid (QA). (Cf., BAREAand GILES1978).

acid has a marked protective effect. Only the curve for the quinic acid is shown in Figure 3. The data with shikimic acid are similar, The thermolabile protein produced by R2 has the same sedimentation properties on a sucrose density gradient as doss the wild type enzyme. Genetic data indicate that a single gene difference determines the thermolability of the QDHase produced by each of the revertants. Representative data from ten tetrads isolated from each cross of the t w o revertants with a wild-type strain carrying a methionine mutant (me-7),which is very closely linked to the qa cluster, are presented in Table 5. The overall genetic evidence, based on both tetrad and random ascospore isolations, utilizing the me-7 marker, indicates that both revertants can be considered alleles of the qa-3 gene. Thirty-seven additional revertants have been induced in mutant 330-M33. To date I 2 additional revertants have been examined as homocaryotic isolates, and all produce a thermolabile enzyme in vitro when induced on quinic acid at either 25" or 35". The QDHase from all of these revertants is protected by quinic acid from inactivation during heating at 35 O. Six revertants are basically similar

78

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TABLE 5 Evidence for segregation in tetrads of quinate dehydrogenase thermolability in two revertants of mutant 330-M33 crossed to wild type carrying an me-7 mutation Revertant and ascus no ___--

33lLM33-R1-9.1

Quinate dehydrogenase activity. Unheated Heated -

me-7 genotype

~ _ _ _ _ _ ~ ~ _ _ ~ 88 68 me-7

9.5 9.7

120 23 27

136 0 0

me-7 me-7 f me-7f

330-M33-R2-10.1 10.4 10.5 10.7

84 132 36 27

79 82

me-7 me-7 me-7+ me-7+

9.4

0 4

* Each isolate was grown at 25' on minimal supplemented with methione and induced for six hrs on 0.3% quinic acid. Dialyzed enzyme extracts were incubated at 35" for ten min and then assayed. to the first two studied. The other six revertants are temperature sensitive and can not be induced on quinic acid at 35". As indicated in Table 4, the only other mutant that has yielded revertants having a thermolabile QDHase in vitro is 335-M160. In this instance, revertant 335-MI60-Rl3 appears to differ from revertants of mutant 330-M33 in producing a QDHase that is relatively less thermolabile, having a half-life of ca. 10 minutes (Figure 2). The QDHase from these revertants is also protected by quinic acid on heating. Furthermore, this revertant differs from the revertants obtained in M33 in that the initial level of enzyme activity is equivalent to wild type, while that of 33-R1 and of 33-R2 is about 25%-35% of wild type (Table 5 ) . It is of interest that this mutant appears to map in the same general region of the qa-3 gene as does mutant 330-M33. Genetic evidence that revertant R4 from qa-3 mutant 336-3-7 contains a suppressor mutation: When conidia of qa-3 mutant 336-3-7 were irradiated with ultraviolet and dispensed into tubes of liquid quinic acid medium, 16 revertants able to grow on quinic acid as a carbon source were obtained. Four of these revertants proved to be intragenic ga-3 revertants with restored enzymic activities equivalent to wild type. The other 12 revertants were due to unlinked suppressor mutations. Growth tests on quinic acid at 25" and 35" indicated that these suppressed mutations were temperature sensitive and unable to grow on quinic acid at 35". One of these suppressed strains, R4, was selected for further detailed study because it exhibited the best growth on quinic acid. In a cross between homocaryotic isolate 3-7-Rkla and me-7, two asci of the following phenotype were obtained, based on growth tests. In one of the two asci, ascospores 6.1 and 6.4 were methionine independent and quinate nonutilizing, while ascospores 6.5 and 6.7 were methionine requiring and quinate utilizing. The recovery of quinate nonutilizing recombinants suggests very strongly that strain R4 is not a true reverse mutation, but carries a suppressor mutation for qa-3 mutant 336-3-7. The second complete ascus had the following phenotype: ascospores 10.1 and 10.3

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were methionine requiring and quinate utilizing; ascospores 10.6 and 10.8 were methionine independent and quinate utilizing. This result suggested that spores 10.6 and 10.8 carried the original qa-3 mutant (336-3-7) combined with a suppressor mutation. Support for this conclusion came from an additional cross to wild type utilizing the homocaryotic F, isolate 10.6. From a plating of random ascospores, 200 colonies were isolated and tested for their ability to utilize quinic acid. A total of 158 isolates were able to utilize quinic acid for growth, while 42 could not. When tested further, two of the quinate utilizing cultures were shown to be highly inducible for QDHase, while two nonutilizing cultures were noninducible. The recovery of approximately 25 % qa-3 isolates from the homocaryotic R4 strain 10.6 provides clear evidence for the presence of an unlinked suppressor mutation in this strain. Genetic analysis of the other 11 suppressed strains was done by crossing homocaryotic isolates to me-7 and analyzing the me+ isolates for their ability to grow on quinic acid. As in the case with R4, approximately 25 % qa- isolates were obtained in each cross. Experiments to determine whether qa-3 mutant 336-3-7 contains a nonsense mutation: The genetic evidence just discussed indicates clearly that 336-3-7 is a suppressible mutant. It was of interest to determine whether 336-3-7 is suppressible by any of the known supersuppressor (ssu) strains. Consequently, crosses were made between qa-3 mutant 336-3-7 and each of the following strains, ssu-1 (WRN33), ssu-2 (WRU35), ssu-3 (WRUl18), ssu-l (319-44), ssu-5 (319-45), ssu-6 (319-26), and ssu-8 (319-37), to determine whether any of the known ssu strains would suppress the qa-3 mutant. Results of these crosses indicated that the qa-3 mutant was not suppressed since an approximately 50:50 ratio of qa+ and qa- isolates was obtained from each cross. Experiments to determine whether the qa-3 suppressoir suppresses known nonsense mutations td 140 and arom 54: Crosses were made with the qa-3 suppressor strain 336-3-37-R4-10.6 (qa-3,su) to two nonsense mutations, td 140 and arom-54, to determine whether the qa-3 suppressor would suppress these mutations. Random ascospore isolates were made, and the data indicated that although the qa-3 allele was suppressed (a typical 3 qa+ to 1 qa- ratio was observed) , no suppression was observed for td 140 and arom-54. Reversions of double-mutant strains qa-3 (336-3-7) TD 140 and qa-3 (3363-7) arom 54: Since none of the known ssu strains suppressed 336-3-7, a double reversion experiment was undertaken to determine if suppressors could be induced in a double-mutant strain containing the qa-3 mutant and one of the strains known to revert freely by supersuppressors. A wide variety of ssu mutations have been recovered in both td 140 and urom-54 strains. (SEALE1976; CASE and GILES1974). A cross was made between qa-3 336-3-7 and each of these two strains, and a double-mutant isolate recovered. If a ssu mutant occurs that can suppress both genes simultaneously, then revertants induced by UV should be able to grow on a minimal medium supplemented with quinic acid as a carbon source. A large number of reversions induced by W in both strains were recovered initially on a sucrose medium. These revertants were isolated and

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TABLE 6 Specific activities of the qa enzymes in two qa-3 mutants, 336-3-7-la and M i 6 , in the revertant (suppressor) of qa-3 mutant (336-3-7-la) strain R4-10.6, and in wild type 74A Strains

336-3-7-la MI6 R4-10.6 74A

(qa-3su+) (qa-3su+) (41-3su) (qa-3+su +)

Specific activities in enzyme extracts' QDHase C-DHQase DHS-Dase

~______

0.4 0.4

49.0

.____

4.0

46.0 25.0

19.0 20.0 4.0

15.0

20.0

3.0

* Mycelia were grown on 1.5% sucrose medium in shake flasks for 24 hours at 25" and then shifted for six hours to medium containing 0.3% quinic acid. Extraction was by procedure I1 (CHALEFF1974).

subsequently tested for their ability to grow on quinic acid. The results of these tests indicated that no suppressor mutations had been obtained that could simultaneously suppress both td 140 and the qa-3 mutant or arom-54 and the qa-3 mutant. Biochemical studies of suppressor mutant R4: Assays have been performed for the three qa enzymes utilizing an extract of suppressor mutant R4. These results are presented in Table 6, together with assay data from additional strains for comparative purposes. The data indicate that qa-3 mutant 336-3-7 carrying the suppressor mutation (R4-10.6) has a significantly elevated level of QDHase activity which is approximately 25% of wild type. The levels of the other two qa activities are similar in the two qa-3 mutants, 336-3-7 and M16, as well as in the case of suppressed 336-3-7 and wild type. In a sucrose density gradient, the QDHase activity from R4-10.6 sedimented at a position identical to that of wild type QDHase. DISCUSSION

The order of the four genes in the qa gene cluster has been previously established to be qa-I, qa-3, qa-4, qa-2 (CASEand GILES1976). The centromere distance has been determined for me-7, but no second division segregations were observed for the qa gene cluster. I n the present studies the relative positions of 11 different mutational sites within the qa-3 gene have been determined. It is difficult to determine unequivocally from our data the precise order of the various mutational sites. However, the data appear to provide clear evidence that the extreme sites can be determined on the basis of the consistently high frequencies of prototrophs observed in crosses between certain mutants. One of our main objectives has been to decide which mutants represent these sites since they should carry mutations at the amino- and carboxyl-terminal ends of the QDHase protein, respectively. Our second major objective has been to determine the orientation of the extreme sites within the qa-3 gene relative to a reference mutant in the adjacent qa-2 gene. Our data indicate that qa-Zs mutant 124 is closer to qa-3 mutant 336-3-10 (a homoallele of M45) than to qa-3 mutant 336-3-3 (a homoallele of M16),

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which are located at opposite ends of the qa-3 genetic map. The procedure we used to obtain these data involved selection and characterization of prototrophs from four-point crosses within the qa gene cluster, employing primarily qa-3 alleles presumably located near opposite ends of the gene. In these four-point crosses, prototrophs carrying either qa-Y or qa-2 parental markers would be eliminated as aromatic amino acid requiring recombinants, and the overall prototroph frequencies would be reduced. Consequently, calculations of distances between mutant sites from these four-point data are not directly comparable to those obtained from two-point crosses involving different pairs of qa-3 mutants. As a consequence of this situation, the size of the qa-3 gene appears to be quite different, depending upon whether this value is based on two- or four-point crosses. For example, in the two-point data, the prototroph frequency observed between mutants M45 and M16 (located at opposite ends of the qa-3 gene) is 0.049. In the four-point data, the prototroph frequency between qa-l allele 124 (located to the left of M45)and M336-3-3 (a homoallele of MI6 located at the right end of the qa-3 gene) is 0.0253, approximately half the value determined from the two-point data. Additional complexities of recombination within this region in linkage group VI1 have been previously discussed with respect to crosses of me-7 and me-9 alleles (MURRAY 1970). I n the case of the adjacent qa region, previous mapping studies utilizing two-point crosses with the qa-l and qa-2 genes, as well as some of the present qa-3 data, suggest that negative interference is probably in effect within these genes (CASEand GILES 1976; CASE,HAUTALA, and GILES 1977; CASEand GILES,unpublished) , while positive interference apparently exists in two-point and four-point crosses between genes (CASEand GILES1976). Furthermore, the high frequency of pseudowild types occurring in crosses of mutants in two different qa genes prohibits the making of such pairwise crosses with the exception of crosses involving qa-ls mutants (CASEand GILES1976). Indirect methods utilizing three-point crosses are underway to provide additional information on the sizes and orientations of all four genes (CASEand GILES,unpublished). The present results, which localize certain qa-3 alleles at extreme sites within the gene and orient these sites with respect to the adjacent qa-l gene, are particularly important in relation to the amino acid sequencing studies now underway. At the present time, a preliminary sequence has been established f o r the first 20 amino acids at the N-terminal end of wild type QDHase. In addition, comparable sequencing studies are underway with QDHase proteins from qa-3 mutants M16 and M45, which map at either end of the qa-3 gene, and from revertants of these mutants (STR~MAN, REINERT,and GILES,unpublished). It is hoped that the amino acid sequences from these mutants and revertants, together with the genetic data, will make it possible to determine the direction of transcription of the qa-3 gene and suggest the possible location of a promoter (initiator) region(s). Since the qa-3 gene is located at one end of the three structural genes encoding the qa enzymes, a promoter region might be located between the qa-l gene and the

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qa-3 gene. In fact, based on the observed coordinate induction of the three qa enzymes, CHALEFF(1974) proposed that the structural genes for these enzymes might be transcribed as a single polycistronic mRNA. However, other data (CHALEFF1974; CASEand GILES,unpublished) suggest that there may be an internal promoter(s) between the qa-4 and ga-2 genes. Certain qa mutations that lack DHS-Dase activity are pleiotropic and affect not only the DHS-Dase activity encoded in the qa-4 gene, but also the levels of the enzymic activities encoded in both the qa-3 and qa-2 genes. These data appear to be inconsistent with the occurrence of a single polycistronic mRNA. Rather, they suggest that transcription may proceed in both directions from within the qa cluster. To date, attempts to define such a site (s) by inducing and characterizing initiator constitutive mutants (ENGLESBERG and WILCOX1974) have been unsuccessful. Additional evidence bearing on the occurrence of a polycistronic mRNA has come from attempts to detect and characterize suppressible qa-3 mutants carrying nonsense codons. Only one qa-3 mutant has been found that reverts by suppressor mutation, but the evidence is not conclusive that this mutant has a nonsense codon. This mutant maps at the right end of the qa-3 gene and does not lower the levels of enzymic activities in the two adjacent qa genes. The fact that no qa-3 mutants have been detected that have pleiotropic effects on the levels of enzymic activities encoded in the qa-4 and qa-2 genes lends support to the hypothesis of an internal qa promoter region(s). At present, most evidence (SHERMAN and STEWART 1975; BIGELIS,KEESEY and FINK1977) supports the view that the polycistronic mRNA does not occur in eukaryotes. The negative results of complementation tests with qz-3 mutants can be interpreted in either of two ways. First, the inability of qa heterocaryons to grow o n quinic acid as a sole carbon source has been reported previously with qa-2 mutants. Allelic complementation between qa-2 mutants was observed only on sucrose minimal medium, and not when quinic acid was used as a sole carbon source (CASE,HAUTALA and GILES1977). Since complementation tests with qa-3 mutants cannot be made on sucrose, growth, even if it could occur as a result of allelic complementation, may be so reduced on quinic acid that no complementation would be observed. Secondly, quinate dehydrogenase has been shown to be a single polypeptide (BAREAand GILES1978), and complementation is typically observed with multimetric proteins where defective subunits interact to form a partially active hybrid polymer. However, allelic complementation has been observed between Zeu-2 mutants in Neurospora ( REICHENBECHER, unpublished). This gene encodes isopropylmalate isomerase, which comprises a single polypeptide chain. On the basis of tests on sucrose density gradients, complementing heterocaryons between Zeu-2 mutants produced an isopropylmalate isomerase that has twice the molecular weight of the purified enzyme from wild type. These data suggest that complementation occurred by the formation of a dimeric enzyme aggregate composed of two differently defective single polypeptide chains. The possibility that this type of complementation might occur between qa-3 mutants cannot be excluded.

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Revertants of qa-3 mutants have been characterized as those which are reversions within the qa-3 gene and those which are due to unlinked suppressor mutations. Revertants within the qa-3 gene that are of particular interest are those obtained from mutants 330-M33 and 335-M160. These two strains are located in the same region on the genetic map. Both of these mutants revert by producing a QDHase enzyme that is thermolabile in vitro and is protected by the addition of quinic or shikimic acid during heating. This stabilization of the enzyme in the revertants suggests that these mutations may be located in the substrate binding region of the enzyme. Qnly one of the qa-3 mutants, 336-3-7, has been found to revert by suppressor mutation. The basis for defining a nonsense suppressor in Neurospora is to determine that the suppressor is allele specific, but not locus specific. Crossing analyses, employing known nonsense suppressor mutations with the qa-3 mutant, gave negative results. In addition, experiments to induce suppressor mutations in double mutants simultaneously were not successful. Furthermore, the qa-3 suppressor was also unable to suppress the known nonsense mutations, arom-54 and td 140. One difference between the qa-3 suppressor strains and the known ssu strains is the inability of the qa-3 suppressor strains to grow on quinic acid at 35". However, temperature-sensitive mutants encoding a tRNA are known in yeast (RASSE-MESSENGUY and FINK1973). At this time there is no direct evidence that the qa-3 suppressor is a nonsense suppressor, although this seems the most likely possibility. Consequently, the nature of the suppression of this qa-3 mutant remains to be determined. Recently it has been possible to clone at least part of the qa cluster (the qa-2+ gene for catabolic dehydroquinase) in an Escherichia coli plasmid (VAPNEK, et al. 1977). The evidence is not yet clear whether the other two qa genes are present in the plasmid, but continuing studies should establish whether the qa-3 gene is also present. We anticipate that this recombinant plasmid will be very useful in elucidating the molecular mechanisms involved in genetic regulation in Neurospora since the cloned DNA sequence may well contain both structural and regulatory genes, as well as regulatory sequences such as promoters. Note added in proof: Since this manuscript was completed, partial amino-terminal sequences have b:en obtained for two revertants, one induced in M45 and one induced in M I 6 (STRBMAN, REINERTand GILES,unpublished). As indicated in this paper, these two midant sites are located at opposite ends of the qa-3 genetic map. The sequence for the first ten amino acids for R45 (from M45) is identical to that of wild type. However, in the sequence for RI (from M16), an isoleucine is substituted for the proline of wild type at amino acid position three. These results permit the conclusion that MI6 is near the amino-terminal end of the qa-3 gene, whereas M46 is presumably near the carboxyl-terminal end. Consequently, it can be concluded that the direction of transcription of the qa-3 gene is from right to left on the basis of the indicated orientation of its mutant sites within the qa cluster (Figure 1). This research was supported in part by Research Contract E(38-1)-735 with the United States Energy Research and Development Administration and by Research Grants GM 22054 and GM 23051 from the Public Health Service. C. PUEYOand J. L. BAREAwere supported in part by funds from the Spanish Science Council. We would like to acknowledge the efficient technical assistance of JOANNLAY.

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LITERATURE CITED

BAREA, J. L. and N. H. GILES, 1978 Purification and characterization of quinate (shikimate) dehydrogenase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa. Biochem. Biophys. Acta 524: 1-14. BIGELIS,R., J. KEESEYand G. R. FINK,1977 The his-4 fungal gene cluster is not polycistronic. In: ICN-UCLA Symposiz on Molecular and Cellular Biology, Vol. VIII: 179-188. Edited by G. WILCOX, J. ABELSON and C. F. Fox. Academic Press, New York. CASE,M. E. and N. H. GILES,1974 Revertants and secondary arom-2 mutants induced in noncomplementing mutants in the arom gene cluster of Neurospora crassa. Genetics 7 7 : 613-626. --, 1975 Genetic evidence on the organization and action of the qa-I gene product-a protein regulating the induction of three enzymes in quinate catabolism in Neurospora crassa. Proc. Nat. Acad. Sci. US. 72: 553-557. -, 1976 Gene order in the ga gene cluster of Neurospora crassa. Molec. Gen. Genet. 147: 83-89. CASE,M. E., J. A. HAUTALA and N. H. GILES, 1977 Characterization of ga-2 mutants of Neurospora crassa by genetic, enzymatic, and immunological techniques. J. Bacteriol. 129 : 166-172. CHALEFF,R. S., 1974 The inducible quinate-shikimate catabolic pathway in Neurospora crassa: genetic organization. J. Gen. Microbiol. 81 : 337-355.

ENGLESBERG, E. and G. WILCOX,1974 Regulation: Positive control. Ann. Rev. Genetics 8: 219-242.

MURRAY, N. E., 1970 Recombination events that span sites within neighboring gene loci in Neurospora. Genet. Res. Camb. 15: 109-121. F. and G. R. FINE, 1973 Temperature-sensitive nonsense suppressors in RASSE-MESSENGUY, yeast. Genetics 7 5 : 459-464. RINES,H. W., M. E. CASEand N. H. GILES,1969 Mutants in the arom gene cluster specific for biosynthetic dehydroquinase. Genetics 61 : 789-2300,

SFALE,T. W., 1976 Supersuppressor action spectrum in Neurospora. Molec. Gen. Genet. 148: 105-108. SHERMAN, F. and J. W. STEWART, 1975 The use of iso-I cytochrome c mutants of yeast for elucidating the nucleotide sequences that govern initiation and translation. In: Organization Proc. 10th FEBS and Expression of the Eucaryotic Genome. Edited by G. BERNARDI. Meeting, 38 : 175-1 99. VALONE,J. A., M. E. CASEand N. H. GILES,1971 Constitutive mutants in a regulatory gene exerting positive control of quinic acid catabolism in Neurospora crassa. Proc. Nat. Acad. Sci. US. 48: 1555-1559. VAPNEK,D., J. A. HAUTALA, J. W. JACOBSON, N. H. GILESand S. R. KUSHNER,1977 Expression in Escherichia coli K-12 of the structural gene for catabolic dehydroquinase of Neurospora crassa. Proc. Nat. Acad. Sci. U.S. 74: 3508-3512. Corresponding editor: C. W. SLAYMAN