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GENETIC CONTROL OF PHOSPHATE-METABOLIZING ENZYMES IN
NEUROSPORA CRASSA: RELATIONSHIPS AMONG
REGULATORY MUTATIONS
BARBARA S. LITTLEWOOD, WILLIAM CHIA
of
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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unpublished results) and one or more nucleases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.
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Department
AND
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This work was supported by an NIH Grant, GM-08995.
Genetlcs 79: 419434 March, 1975
420
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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.
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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
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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.
“+”,
“+”,
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REGULATORY M U T A N T S IN NEUROSPORA
421
MATERIALS .4ND M E T H O D S
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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.
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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
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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
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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).
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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.
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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 ) .
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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
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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.
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REGULATORY M U T A N T S I N NEUROSPORA
425
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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-
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426
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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
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wild type
nuc-2
pregc-2
nuc-2 pregc-8
nuc-I
nuc-2 pregc-2 ; nuc-2
hwPi
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zy
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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
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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
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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-
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REGULATORY MUTANTS I N NEUROSPORA
429
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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
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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)
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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
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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
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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).
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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
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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
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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
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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.
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M. K. and R. L. METZENBERG,
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78: 645-659.
HASUNUMA,
K., 1973 Repressible extracellular nucleases in Neurospora crassa. Biochim.
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LEHMAN,J. F., M. K. GLEASON,
S. K. AHLGRENand R. L. METZENBERG,
1973 Regulation of
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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
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R. L., M. K. GLEASON
and B. S. LITTLEWOOD,
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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
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Corresponding editor. D. R. STADLER