Copyright 0 1982 by the Genetics Society of America
A CIS-DOMINANT MUTATION IN ASPERGILLUS NIDULANS
AFFECTING THE EXPRESSION OF THE amdS GENE IN THE
PRESENCE O F MUTATIONS IN THE UNLINKED GENE, amdA
MICHAEL J. HYNES’
Department of Genetics, La Trobe University, Bundooro, Victoria, Australia
Manuscript received February 8, 1982
Revised copy accepted June 1,1982
ABSTRACT
A mutant producing very high levels of the acetamidase enzyme encoded by
the amdS gene has been isolated in a strain containing the amdA7 mutation,
which itself causes high levels of this enzyme. Genetic analysis has shown that
this mutation, designated amd166, is adjacent to the amdS gene and is cisdominant in its effect. The amd166 mutation has little effect on amdS expression
when present in strains not containing the amdA7 mutation. Two other omdA
mutations investigated also interact with the amd166 mutation to result in high
acetamidase levels. No interaction between amd166 and any of the other putative
regulatory genes affecting amdS expression has been observed. The amd166
mutation has been located by fine structure mapping at the extreme end of the
controlling region, which has previously been defined by genetic mapping
(HYNES1979). Analysis of this region has been extended by mapping new
mutations resulting in loss of amdS expression. One of these defines the most
extreme site capable of mutation to loss of gene function found so far.
N lower eukaryotes many apparent regulatory genes have been defined that
Igenes.
appear to act in trans to regulate the expression of one
more structural
Genetic methods can provide evidence for a direct regulatory action of
or
a proposed regulatory gene if cis-acting mutations, adjacent to the controlled
structural gene, affect regulation of gene expression and show specific interactions with mutations in regulatory genes. Such specific mutations are particularly valuable because, in a number of cases in fungi, apparent cis-acting
mutations have been found to arise by fusions to new controlling regions
(promoters) resulting from chromosomal rearrangements (ARST, RAND and
BAILEY1979; MCKNIGHT,CARDILLO
and SHERMAN
1981; SHERMAN
and HELMS
1978) or insertions (ERREDE
et al. 1980).
A new cis-dominant mutation affecting expression of the amdS gene coding
for the acetamidase enzyme of Aspergillus nidulans is described here. This
mutation interacts with mutations in the unlinked amdA gene to cause high
levels of amdS expression.
The complex control of the amdS gene has been reviewed previously (HYNES
1978a). Briefly, the acetamidase enzyme is inducible by sources of acetylcoenzyme A such as acetate, L-threonine and acetamide by a mechanism sharing
at least some control elements with the regulation of acetate metabolism via the
glyoxylate by-pass. Benzoate also results in enzyme induction but no genes
Present address and address for correspondence Department of Genetics, Melbourne Unlversity, Parkville,
Victoria 3052, Australia
Genetics 102 139-147 October. 1982
140
M.
I.
HYNES
specifically involved in this control are known. The acetamidase is highly
inducible by w-amino acids (e.g., p-alanine), and the amdR gene, unlinked to the
amdS gene, appears to act as a positive regulatory gene in this process. Nitrogen
metabolites such as ammonium and L-glutamine result in strong repression of
the acetamidase. The areA gene has been implicated in this control mechanism,
which also affects most nitrogen catabolic pathways (ARSTand COVE1973).
The amdA gene on linkage group VI1 is of prime interest in this work.
Mutations (e.g., amdA7) in this gene have been found to cause elevated acetamidase levels compared with wild type (HYNES1978a). Mutations in the amdA
gene do not interact with other regulatory mutations, and induction by all
inducers is observed in amdA mutants. Thus the effect of amdA mutations is to
elevate the overall level of amdS expression. No mutations in amdA have been
observed to result in lowered levels of amdS expression. Mutations in the amdA
gene have no observed effect on growth on a variety of carbon and nitrogen
sources. Thus the effect of the amdA gene has been thought to be specific for
amdS expression. However, recent observations of labeled polypeptides separated on polyacrylamide gels show that amdA mutants synthesize high levels
of a polypeptide of unknown function (M. J. HYNESand J. A. KING,unpublished).
A cis-dominant mutation, amdI66, adjacent to amdS, is described here. This
mutation interacts with amdA lesions resulting in very high levels of acetamidase in both the presence and absence of inducers. By itself amdI66 has only a
small effect.
Mapping of mutations in the amdS region has resulted in the definition of an
adjacent controlling region defined by cis-acting mutations (HYNES1979). Analysis of this region has been extended by the mapping of the amdI66 mutation
and derived secondary mutations resulting in loss of amdS expression. One of
these new mutations defines the most extreme site on the genetic map capable
of mutating to loss of gene function found so far.
MATERIALS AND METHODS
Strains and genetic techniques: All strains were derived from those previously described (HYNES
1978a, 1978b, 1979, 1980). Where necessary, the genotypes of double mutant strains were checked
by appropriate crosses. Genetic techniques were standard for A. nidulans (CLUTTERBUCK
1974).
Methods used for the isolation of mutants and for fine structure mapping of the amdS region were
those previously described (HYNES1979).
Media and growth conditions: The standard minimal medium used was that of COVE(1966) with
1%glucose, 1%sucrose or no carbon source added. Ammonium was added as ammonium tartrate.
Growth tests were performed with solid medium (1%agar) and incubation at 37' for 2-3 days.
Mycelium for enzyme determinations was grown at 30' in a Gallenkamp orbital incubator and
harvested as described (HYNES1972).
Preparation of crude extracts and enzyme ussays: Crude extracts of freshly harvested mycelium
were made as described (HYNES1972) using 100 mM sodium orthophosphate buffer, pH 7.2.
Acetamidase (E.C. 3.5.1.4) was assayed as described (HYNES1972). All enzyme activities are
expressed as milli-units per milligram of soluble protein where one unit is the amount of enzyme
that catalyzes the production of 1pmol of ammonium/min. Results given are the average of two or
more independent determinations.
RESULTS
Only strains producing high acetamidase levels can grow strongly on acrylamide as the sole nitrogen source (HYNESand PATEMAN
1970a). Strains of
141
CONTROL-REGION MUTATIONS IN A . NIDULANS
genotype umdR4b; amdA7 are unable to use acrylamide as a nitrogen source
(HYNES1978a). The amdI66 mutation was found in a N-methyl-N'-nitro-nitrosoguanidine induced mutant by selecting for acrylamide utilization in a strain
of genotype amdM4; amdA7. Initial outcrosses showed that the mutant retained
both amdR4h and amdA7 lesions. Crosses to amdS-; amdA7 strains showed
that the amdI66 mutation responsible for allowing acrylamide utilization segregated in a 1:l ratio with the amdS mutation. A cross between a strain of
genotype amdI66; amdA7 to an amdSl2 mutant gave one-quarter progeny
capable of strong growth on acrylamide medium (genotype, amdI66; amdA7),
one-quarter progeny with a similar amide utilization phenotype to wild type
(genotype, amdI66; amdA +) and one-half of the progeny showed the acetamide
nonutilization phenotype of amdS12. These results indicated that the amdI66
mutation was close to the amdS gene and increased acetamidase levels only in
the presence of the amdA7 mutation.
Growth properties of double mutants: A wide variety of double and triple
mutants containing amdI66; amdA7 and other mutations affecting umdS expression were constructed. When necessary these were outcrossed to appropriate
strains to check for the presence of the amdI66 mutation. Results of growth
tests on amide media for these strains are shown in Table 1. Provided both
amdI66 and amdA7 mutations were present, growth on amide media was very
strong. By itself the amdI66 mutation had no detectable effect compared with
the wild-type strains. No interactions between the amdI66 mutation and mutations in the regulatory genes amdR and areA were observed.
That the effect of the amdI66 lesion was not specific for the amdA7 allele
TABLE 1
Effects of the amdI66 mutation on growth on amide media"
Growth medium
1%glucose
Relevant genotype
amd/R44; amdA7
amdRh; amdI66; amdA7
amdA7
amdI66; amdA7
Wild type
amdI66
amdRh
amdR44; amdI66
areA102
amdI66 areA102
amdI66 areA102; amdA7
areA217
amdI66 areA217
amdI66 areA217; amdA7
oreA217 amdA7
amdA301
amd166; amdA301
amdA4
amd166: amdA4
~~
-
mM
+ 10
acrylamide
-
+++
+
+++
-
-
++
++
+++
-
-
++
+
++
+
++
1%sucrose
+ 10 mM
acetamide
++
++++
+++
++++
+
+
*
*
++++
++++
++++
-
+++
++
+++
++++
+++
++++
50 mM acetamide
+
++++
+++
++++
+
+
++-
+
+
++++
+
+
++++
++
++
+++
++
+++
~
"Growth represented by symbols in order of increasing growth: -, f,+,
Symbols on different media are not equivalent.
++, +++, ++++.
142
M. J. HYNES
was shown by the increased amide utilization of the double mutants amdI66;
amdA3OZ and amdI66; amdA4 compared to the amdI66, amdA301 or amdA4
single mutants.
Acetamidase activities: As expected from their growth properties, mutants
containing both amdI66 and amdA7 mutations were found to have very high
acetamidase activities in media lacking added inducers (Table 2). Responses of
the high constitutive enzyme level to repression by ammonium and induction
by p-alanine, benzoate or acetate were still observed, compatible with the
independence of amdA-mediated regulation from other regulatory mechanisms
(see HYNES1978a). By itself the amdI66 mutation led to only slightly higher
enzyme activities (particularly in glucose-containing media) compared to the
wild-type strain (Table 2). This effect was clearly too small to result in observable growth differences in the growth tests. When the amdl66 mutation was
combined with either the amdA302 or amdA4 mutations in double mutants high
acetamidase activities were observed, indicating that the interaction was not
specific for the amdA7 mutation. However, the effect of these amdA alleles
was not as great as for amdA7, indicating allele specific interaction with amdI66.
Cis-dominance of the amdI66 mutation: In order to construct diploids with
the amdI66 mutation in cis to amdS mutations, it was necessary to isolate amdS
mutations in strains of genotype amdI66; amdA7. Such mutants were isolated
by selecting for fluoroacetamide resistance (HYNESand PATEMAN,
1970b) and
were shown to contain amdS mutations by fine-structure mapping and complementation tests (see below), with known amdS alleles. One such strain of
genotype amdS401 amdI66; amdA7 was used to construct various heterozygous
diploids that were growth tested on amide media and assayed (Table 3). As
previously described (HYNES1978a) the amdA7 allele was semi-dominant to
wild type for its effects on amide utilization. The amdI66; amdA7 combination
was found to also be semi-dominant to the amdl' and amdA+ alleles, but only
when amdI66 was in the cis position relative to a wild-type amdS+ allele.
Similar results were found using amdS alleles other than amdS402 (data not
TABLE 2
Acetamidase activities of amdI66-containing strains
Acetamidase specific activity with
Relevant genotype
amdRz4; amdA7
amdRT4; amd166; amdA7
amdA7
amd166; amdA7
Wild type
amd166
amdI66; amdA301
amd166; amdA4
1% glucoseD+
1% glucasea
10mM
20mM
alanine
NH4+
24
341
41
295
13
21
133
187
+
3
82
8
102
0
11
48
42
10 mM SOdium
dium acebenzoateb
tate*
10 mM SO-
No addition*
10 mMP-
54
44
355
439
1002
186
127
ND
ND
364
75
448
25
35
ND'
ND
alanine*
127
587
210
619
96
82
ND
ND
95
672
115
725
60
89
ND
ND
"Mycelium grown for 16-18 hr at 30" on this medium before harvesting, extraction and assay.
Mycelium grown for 16 hr at 30° in 1%glucose + 20 mM NH,+ medium and then transferred to
minimal medium with the indicated additions for 4 hr before harvesting, extraction and assay.
ND = not determined.
'
143
CONTROL-REGION MUTATIONS IN A . NIDULANS
TABLE 3
Cis-dominance of amdI66
Growth on"
acetamide
Acetamidase specific activity with
1% glucose + 10
mM L-alanine*
+
++
ND"
amd166; amdA7
amdI+; amdA'
++
+++
ND
amdI66amdS'; amdA7
amdl+amdS17; amdA+
++
+++
173
amd166amdS401; amdA7
amdl+amdS+amdA'
+
+
12
amd166amdS401; amdA7
amdl+omdS+;amdA7
+
++
34
amdl66amdS+; amdA7
amdPamdS223; amdA7
+++
++++
Relevant genotype of diploid
amdA7
amdA7
1%glucose + 10
mM acrylamide
1%sucrose
+ 10 mM
223
Growth symbols as for Table 2.
* Mycelium grown for 16 hr before harvesting, extraction and assay.
e
ND
=
not determined.
shown). Therefore, the dominance properties of the amdZ66 mutation were
compatible with it defining a cis-acting site affecting amdS gene regulation.
Fluoroacetamide resistant mutants of amdI66; amdA7 strains: Seven spontaneous revertants showing reduced amide utilization were isolated from the
amdRq4; amdI66 amdA7 strain by selection for fluoroacetamide resistance.
Two distinct classes were observed.
Three of the revertants grew as well as amdR44 and amdR44; amdI66 strains
on amide media and did not segregate the amdA7 mutation in outcrosses to
wild-type strains. They retained the amdI66 mutation because in crosses to
amdS; amdA7 strains they segregated one-quarter progeny capable of strong
growth on acrylamide medium. Apparently these mutants arose as a result of
reversion events at the amdA locus. For two of these mutants scoring of large
numbers of progeny from outcrosses to a wild-type strain showed a low
proportion of amdA7 segregants. This indicated that mutation at a second site
within the amdA locus could reverse the amdA7 phenotype. Further revertants
of amdA7 have been obtained in subsequent experiments. So far no amdA7
revertant isolated in this way has been shown to have an observable effect on
wild-type amide utilization or to have any other phenotypic effect.
The remaining four mutants showed a complete loss of ability to utilize
amides. Segregation of low frequencies of progeny capable of strong growth on
acrylamide medium in outcrosses to wild-type or amdA7 strains showed that
they retained the amdI66 and amdA7 mutations. Complementation tests with
amdS alleles and fine structure mapping (see below) showed that these mutants
contain mutations in the amdS region leading to loss of enzyme activity.
144
M . J. HYNES
Fine structure mapping of amdI66 in relation to the amdS gene and its
controlling region: Using the methods and strains described previously (HYNES
1979) three of the new amdS region mutants isolated in the amdI66; amdA7
background were investigated in detail in order to map the amdI66 mutation
relative to amdS. Figure 1 gives the orientation of the genetic map. Mapping
experiments with defined amdS deletion mutants showed that one of the
mutants had a lesion (designated amdS401) in the left hand region of the amdS
gene, and clearly mapped to the left of amdI66 (cross 1 in Table 4). The other
two strains were found to have lesions in the right hand end of the amdS region.
Because it was not clear whether these mutations were inside or outside the
amdS coding region they were designated amd-406 and amd-407 in accordance
with previous practice (HYNES1979). The marker mutation cbxA17, which is on
the left-hand centromere proximal side of the amdS gene, was used to order the
mutations relative to defined point mutations and to amdI66 (Table 4). The
amdS224 mutation is the most rightward confirmed structural gene mutation
(HYNES1979). Both amd-406 and amd-407 were mapped to the right of this site
(crosses 6 and 7 in Table 4 ). The amd-406 mutation yielded a low frequency of
amdS+ recombinants with the amd-205 mutation (an apparent promoter-down
mutation-see HYNES1979) but has not been observed to recombine with amd407. Recent physical mapping studies employing Southern blot analysis with
amdS DNA probes have shown that amd-406 and amd-407 are small deletions
(M. J. HYNESand C. M. CORRICK,
unpublished). amd-407 clearly was well to the
right of the amd-205 site (crosses 4 and 5 in Table 4). The amd-407 mutation
therefore has defined the most rightward site capable of mutation to loss of
amdS function so far isolated. The amd166 mutation was found to be located to
the right of amd-407 and amd-406 (crosses 2 and 3 in Table 4). Subsequent
mapping of further amdS region mutations has so far failed to show any new
mutations mapping to the right of either the amd-407 or amdI66 mutations (M.
J. HYNES,unpublished data).
Frequencies of amdS+ recombinants in crosses between several of the mutants were accurately calculated by determining total numbers of ascospores
plated. Assuming equal frequencies of undetected double mutant recombinants
the following map distances were obtained: amd-406-andS224 = 0.03 map
units; amd-407-amdS224 = 0.15 map units and amd-407-amdS222 = 0.65 map
units. These results were comparable with those obtained previously (HYNES
1979).
The amdI93 mutation, which specifically affects a-amino acid induction
s222
cbxA
I
I
Y
sun
205
h
5224
1
l!B
I18
407
166
I
1
406
---
FIGURE1.-Map order of mutations at the right hand end of the amdS gene. No recombination
has been observed between amd-205 and amd-I18 (HYNES1979). The amd193 mutation is to the right
of amdll8, but its position with respect to amd-407 and amdI66 is unknown. The amd-406 mutation
appears to be a small deletion. The amdS401 mutation has not been ordered with respect to
amdS222.
145
CONTROL-REGION MUTATIONS IN A. NIDULANS
TABLE 4
Fine structure mapping of amdI66
No. of recombinantsa
cross
Relevant genotypes in cross
cbxA17
cbxA+
1
2
amdS401; amd166; amdA7 x cbxA17
omd-407 omd166; amdA7 X cbxA17; amdA7
amd-406 omdI66; amdA7 X cbxA17; amdA7
cbxA17amd-407 amdI66; amdA7 X amd-205; amdA7
amd-407 amdI66; amdA7 X cbxA17omd-205; amdA7
cbxA17amd-407 amdI66; amdA7 x amdS224 amdlld; omdA7
cbxA17amd-406 amd166 X amdS224 amd118; amdA7
amdI18 amd193; omdA7 x cbxA17amd-205; amdA7
22
13
14
21
0
45
23
1
2
0
3
3
4
5
6
7
8
8
6
5
3
38
Recombinants were selected an either glucose-acrylamide medium (to select the genotype
amdS+amdI66; amdA7 in crosses 1, 2 and 3 or amdS+amdI18; amdA7 in cross 8) or sucroseacetamide medium (to select amdS+ recombinants in crosses 4, 5, 6 and 7). Recombinants were
purified and tested for the presence of the cbxA17 allele by determining carboxin resistance. The
principles and details of amdS mapping were as in HYNES1979.
mediated by the amdR gene product, has been described (HYNES1980). This
mutation has been observed to recombine at a low frequency with amd-407. A
cross with a strain of genotype cbxAZ7 amd-207 allowed further mapping of
amdI93 within the controlling region (cross 8 in Table 4). It is clear that the
amdI93 mutation was to the right of amdI28 and amd-205. Because of their
different phenotypes it has not been possible to map amdI93 with respect to
amdI66.
The map order of mutations at the right-hand end of the amdS gene determined in this study and the previous one (HYNES1979) is shown in Figure 1.
DISCUSSION
The amdI66 mutation described here represents an especially interesting
addition to the variety of cis-acting control mutations adjacent to the amdS
structural gene. This mutation only exerts a strong effect on gene expression if
a mutation in the unlinked amdA gene is present. No obvious interactions with
other proposed trans-acting regulatory genes affecting amdS expression have
been observed. Therefore, it appears that the amdI66 mutation alters a receptor
site for the control signal generated or modified by amdA mutations without
detectably affecting responses to the regulation mediated by other regulatory
genes.
The role of the amdA gene in regulating amdS expression is not clear. There
is no evidence for the wild-type amdA gene product affecting amdS because no
amdA mutations resulting in reduced expression have been isolated. This may
result from amdA effects being weak in comparison to other control signals.
Alternatively, loss of function mutations in amdA could be lethal. The phenotype of deletion mutations in amdA would be of great interest. It is possible
that the wild type amdA gene normally does not play a role in amdS regulation,
but is involved in regulation of other genes. Evidence for an unknown polypeptide under amdA control has been obtained recently (see Introduction). The
amdA mutations that lead to increased acetamidase synthesis could alter the
146
M. J. HYNES
specificity of the amdA gene product so that it now recognizes a DNA sequence
adjacent to the amdS gene and stimulates gene expression. On this hypothesis
amdI66 would alter this sequence so that the amdA gene product has a greater
effect. Until more information is available on the role of the amdA gene it is not
possible to distinguish between these possibilities.
The interaction between the amdI66 mutation and mutations in the amdA
gene is the third case of specificity between controlling region mutations and
one of the control mechanisms affecting amdS expression. The amdI93 mutation
specifically affects a-amino acid induction and interacts with mutations in the
amdR gene (HYNES
1980), and the amdI9 mutation causes increased inducibility
by acetate that is probably mediated by the facB gene product (HYNES
1977; M.
J. HYNESand J. M. KELLY,unpublished data). These mutations provide strong
evidence for independent control mechanisms operating by a n interaction
between the product of a regulatory gene and a specific site adjacent to the
structural gene.
No data regarding the molecular level at which gene expression is affected by
the amdI66-amdA interaction is presented in this paper. It is formally possible
that the amdI66 mutation affects the primary structure of the amdS-coded
polypeptide and the altered product of mutant amdA genes interacts with the
altered amdS product to increase enzyme activity. However, data based on
separation of labeled polypeptides on polyacrylamide gels, immunological titration of the amdS product and determinations of translatable messenger RNA
levels indicate that regulation is at the level of gene activity (M. J. HYNES,
unpublished data). amdS DNA has been recently cloned, using recombinant
DNA techniques, and is being used to directly measure mRNA levels and in
physical mapping of the controlling region (M. J. HYNESand C. M. CORRICK,
unpublished data). The results of these studies on the molecular nature of
regulation of the amdS gene will be the subject of future communications.
This research was supported by the Australian Research Grants Committee
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Corresponding editor: R. C. ULLRICH