CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
A GENETIC STUDY OF REVERTANTS OF AN UNSTABLE
'I
SERINE-REQUIRING MUTANT OF NEUROSPORA CRASSA
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Marc Joseph Sievers
August, 1975
I
------------~----~-
.-,
'-
The thesis of Marc Joseph Sievers is approved:
California State University, Northridge
August, 1975
f
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ACKNOWLEDGEMENTS
I would like to express deep appreciation to Dr. Joyce B.
Maxwell for introducing me to the techniques and attitudes involved
in research, and for her patient and dedicated guidance in the
preparation of this thesis.
I would also like to thank Dr. Mary Corcoran and
Dr. Richard Potter for
serv~ng
as members of my graduate committee.
My sincere appreciation goes to my wife, Dale, for not
hassling me too much during this entire project.
iii
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TABLE OF CONTENTS
Page
•(
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ACKNOWLEDGEMENTS
iii
LIST OF TABLES •
v
.....
ABSTRACT • • .
I.
. II.
INTRODUCTION
1
MATERIALS AND METHODS • •
5
..
Strains • •
III.
IV.
v.
vi
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t·
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5
J;
Maintenance and Growth of Cultures . •
5
f
Isolation of Revertants .
6
Genetic Analysis of Revertants • .
7
•·
RESULTS
11
Analysis of Revertant #9·X 25a.
11
Ser, Met Reversion Studies • . • .
13
Genetic Analysis of Revertants.
16
DISCUSSION. .
22
Properties of Ser(JBM 4-13)
22
Models for Gene Instability
27
BIBLIOGRAPHY . .
33
iv
\'·
LIST OF TABLES
Table
I.
II.
III.
IV.
Distribution of markers from revertant
#9 X 25a • . • • . • • • • . . . . . •
14
Distribution of markers from ser, met
X 25a
14
Reversion Data .
17
Distribution of markers from revertants
X ser, met
20
...
v.
. ·,
. .· ,.·. . . . .. . . . . . . . .
.
Distribution of markers from revertants
...
.
X wild-type
. . . .. . . . . . . . .. .
I
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1-
v
20
ABSTRACT
A GENETIC STUDY OF REVERTANTS OF AN UNSTABLE
SERINE-REQUIRING MUTANT OF NEUROSPORA CRASSA
by
Marc Joseph Sievers
Master of Science in Biology
August, 1975
A serine auxotroph of Neurospora crassa, ser(JBM 4-13),
exhibits genetic instability, reverting to prototrophy at high
frequency.
This studyexamines reversion in a mutant stock and
the genetic behavior of serine-independent cultures derived from
that reversion.
Individually isolated
.ascospores of the mutant,
as well as subcultures of individual progeny, exhibited spontaneous
reversion frequencies ranging from
8
X
10-7 to 3.7 x 10 -2 .
The
great variability in reversion frequency and variability in both
mutant and revertant colony size were notable properties.
All
revertants tested demonstrated qualitatively similar growth
characteristics and identical genetic behavior.
Three revertants were studied genetically.
Revertant
#9 had behaved in earlier crosses as if it were a heterocaryon.
In this study, heterocaryosis was verified and the revertant was
reisolated and crossed to wild-type.
Newly isolated revertants
116Rl and 123R6 were made homocaryotic by backcrossing to·a mutant
vi
stock; they were then outcrossed to wild-type.
No serine-requiring
progeny were isolated from all spores examined for the crosses of
the revertants to wild-type.
These results indicated that the
reversion of ser(JBM 4-13) involves either back mutation or a
closely linked suppressor.
The behavior of ser(JBM 4-13) is examined in the light
of several models proposed to account for genetic instability in
Neurospora and other orga~isms~
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INTRODUCTION
The phenomenon of high frequency reversion of spontaneous
and induced mutants of various systems has been examined by a
number of investigators in order to determine more precisely the
molecular nature of the mutational event involved.
Emerson (1914)
suggested that gene instability might account for pericarp
variegation in maize.
Demerec (1925, 1926, 1927, 1928, 1941)
initiated fifteen years work on unstable genes with the discovery
.
.
.
of the frequently mutating gene, reddish, in Drosophilia virilis.
In general, these unstable mutants demonstrated a change from a
mutant allele to wild-type with frequencies higher than that
expected for spontaneous mutation.
In all cases, the revertants
never changed back to the mutant form.
The explanation for such reversion phenomena has been
varied.
The instability of revertible genes may be inherent in
the structure of the specific locus in question.
Barnett and de
Serres (1963) explained the variable, high frequency reversion and
forward mutation rates of an unstable adenine auxotroph of
Neurospora crassa by a model in which the gene in question
originally mutated via a transversion with the subsequent
instability being due to complementary transitional changes.
Other cases of instability have been associated with
mechanical alteration of the chromosome.
1
Laughnan (I949) pointed
.[
2
··-------
1~
1
I
II
I
out that mutation at the unstable
~
locus in maize may be associated
1
with crossing-over suggesting therefore that mutations arise from
Recombination was also suggested
structural changes at that locus.
for the revertibility of f 3n in Drosophila in which reversion is
at least one-third as frequent as forward mutation (Lefevre & Green,
1959).
Reversion models may also be based on mechanisms such as
that proposed by Grigg and Sergeant (1961) in which back-mutation
in Neurospora is attributed to unequal sister-strand exchange in
multi-repeat loci.
Other hypotheses have seen instability at one site to be
due to mutator genes at a different site.in the genome.
The Mu
gene in Drosophila is known to affect mutation pre-meiotically
causing X-linked
~ to revert to
frequency (Green, 1970).
Y: at
an inordinately high
Bacon and Treffers (1961) demonstrated
that the·presence of a mutator gene increases reversion 3200-fold
in an ornithine
mutant~:
of E. coli.
Controlling factors are also prcposed to account for
genetic instability.
McClintock (1951) suggested that instability
in maize is due to the integration of a controlling element at the
site of the mutable gene.
This is exemplified by the Ds and Ac
loci which are capable of moving about in the genome and inserting
at diverse chromosomal locations where they cause unstable
suppression of contiguous genes, the Ac gene
the suppressive effect of Ds.
functi~ning
to activate
3
Mutation rates can also be affected by episomes,
integrated or autonomous genetic elements most often associated
with bacterial systems.
Hill (1963) and Dawson and Smith-Keary
(1963) worked with unstable systems in which changes from
prototrophy to auxotrophy and vice versa were associated with the
attachment and detachment of an episome at a suppressor site.
In
Neurospora~
Giles (1951) studied a multiple allelic
series of inositol-dependent mutants, one of which demonstrated
'
a spontaneous reversion frequency of 7.26 x 10
~
Barnett and
de Serres (1963) noted a reversion frequency of 4.29 x 10- 6 for
the unstable ad-3B mutant 137.
These have been the only studies
to date of high-frequency spontaneous reversion in Neurospora.
The present study involves the genetic analysis of
a recently isolated Neurospora serine auxotroph, ser(JBM 4-13),
I
i
which maps on linkage group V, 3.5 map units to the right of
met~
(Kline, 1973).
While mapping the location of ser(JBM 4-13)
in the genome, Kline noted the number of wild-type progeny to be
disproportionately high compared to the value expected by
recombination.
Spontaneous reversion was also noted in vegetative
cultures of ser(JBM 4-13).
Bengtson (1975) further characterized
the properties of the mutant and the associated reversion
phenomenon.
As part of her study, a genetic analysis of three
revertants isolated from ser(JBM 4-13) was carried out.
Two of
the three revertants behaved like back mutations at the ser locus,
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or like closely linked suppressors.
However, the third, revertant
#9, segregated a high number of serine-requiring progeny.
The
work reported here involves a re-examination of revertant #9 and
a controlled genetic analysis of two newly-isolated revertants in
an effort to resolve the discrepancy of the previous mating
experiments.
The results are discussed in the light of various
models which have been suggested to account for such reversion
phenomena.
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MATERIALS AND METHODS
Strains
The original source of the serine auxotroph used in
these studies was a nutritionally wild-type strain: A, al-2(15300);
cot-l(Cl02t) from which five serine-requiring mutants were selected
after ultraviolet irradiation, as described by Bengtson (1975).
Because the serine auxotroph used in this study is unstable, mutant
stocks were frequently reisolated by plating conidia.
The conidia
were grown under conditions which yielded a single colony from
each
Separate colonies were isolated and tested for their
conidium~
growth requirements.
A conidial reisolate of one of the five
auxotrophs isolated, A, al-2(15300); cot-l(Cl02t);
was crossed to FGSCf/780:
al-2;
~(JBM
~(JBM
a; his-l(K744), met-3(361-4).
The latter cross was designated number
N3 and two progeny, N3-12A; ser(JBM 4-13), met-3 and
II
J
Culture A,
4-13), met-3 was obtained from this cross and was
crossed to wild-type 25a.
~(JBM
4-13),
4-13), met-3 were used in this study.
of N3-12 was crossed to 25a.
N3-10~;.
A conidial reisolate
Five month-old spores from this
cross were assayed yielding fifty-seven double auxotrophs which
were designated
as~·
met(M.S.#l, M.S.#2, etc.)
Maintenance and Growth of Cultures
Vegetative cultures were maintained on agar slants of
Vogel's minimal medium N (Vogel, 1956) supplemented with 0.3mg/ml
L-serine and 0.4 mg/ml L-methionine, a ratio of these amino acids
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t
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t
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r
5
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sterile nine milliliter water blanks to yield further suspensions
of 10
-1 , 10 -2 , 10 -3 , and 10 -4 dilutions.
All plates were
inoculated with one-tenth milliliter of the appropriate dilution.
Duplicate plates of Vogel's supplemented with methionine were
2
3
inoculated with concentrated, 10-1 , 10- and 10- dilutions while
plates of Vogel's supplemented with both methionine and serine were
2
4
inoculated with 10- , 10- 3 , and 10- dilutions.
All plates were
incubated at 32"C and routinely examined for revertants at 48 hours.
~ .(
:_
The cultures were tested for their growth requirements upon plating
by subculturing as described previously and were also transferred
to large (18xl50mm) tubes of doubly supplemented medium to preserve
them for later study.
A portion of the ser, met spore isolates were tested
for reversion at twelve days after germination.
Additional
isolates were tested over a twenty-two week period.
All suspected
revertant cultures were retested for requirements after isolation
from the plates and revertant stocks were maintained on Vogel's
medium supplemented with methionine.
During the eighteenth week
some cultures which showed no reversion during the previous tests
were plated again to search for later-appearing revertants.
Genetic Analysis of Revertants
Previously,microconidiating stocks had been utilized
by Bengtson (1975) in order to obtain conidia for reversion
8
studies.
One explanation for the results obtained with revertant
#9 was that it was heterocaryotic.
To test for the presence of unreverted, mutant nuclei,
subcultures of revertant #9 which appeared phenotypically revertant
in a growth test on slants were plated on Vogel's minimal medium
(2% sucrose) and on minimal medium supplemented with 0.4mg/ml
L-serine.
Individual colonies of variable size were isolated from
the plates and tested for their growth requirements.
Those colonies
which tested as revertants were crossed to 25a to reexamine the
segregation patterns.
The results indicated that the revertant #9 stock used
by Bengtson (1975) was heterocaryotic.
In light of these results
and reported evidence that microconidia can actually be
multinucleate (Pittenger, 1965), the suspected ser+, met
revertants in this study were made homocaryotic by crossing them
to either N3-12:
4-13), met-3.
A,
~(JBM
4-13), met-3 or N3-10:
a, ser (JBM
As the ser+, met progeny of this cross are all
single spore isolates, homocaryotic revertant cultures are thus
obtained.
The cross was carried out on large slants of
Westergaard-Mitchel! (1947) crossing medium supplemented with
0.2mg/ml L-methionine and 0.15mg/ml L-serine.
Dense conidial
suspensions made from revertant cultures were used to inoculate
duplicate slants of crossing medium.
three ways:
Crosses were initiated in
9
1.
Coinoculation
2.
Adding the revertant culture as a conidial (male)
parent one week after the protoperithecial (female)
ser, met parent.
3.
Using the ser, met culture as a conidial parent
and the revertant as the protoperithecial parent.
All crosses were incubated in the dark at 25"C.
The coinoculated crosses began shooting spores twenty
days after initiation of the cross.
Spores were isolated by
taking them off the wall of the crossing tube with a sterile
cotton-tipped applicator and spreading them on a plate of 4% agar.
Random, mature spores were then cut out on small agar blocks and
placed onto small (12x75mm) slants of medium containing both serine
and methionine.
Germination was induced by heat-shocking the
spores in a 60"C water-bath for forty-five minutes.
Germination
was recorded after 24 hours and 48 hours of incubation at 32"C
and the germination frequency was calculated.
Five days after
germination the cultures were tested for their requirements by
transferring each to a small slant of medium as described
previously.
Homocaryotic revertant progeny from these crosses were
crossed to a wild-type strain, either ST74A and ScottA or 25a.
These crosses were carried out on two different media:
Westergaard-Mitchell (1947) crossing medium supplemented with
both serine and methionine and crossing medium supplemented with
10
methionine alone.
Duplicate crosses were initiated by coinoculation
and by using either the revertant or the wild-type culture as
the protoperithecial parent in the same manner the first cross was
prepared.
At three weeks, the first crosses had shot spores, many
of which were light-colored or colorless.
A week later, mature-
appearing spores were selected and heat-shocked.
Four days after
germination, cultures were transferred to four types of slants:
Vogel's minimal medium and minimal medium with the following
supplements: .. serine alone' methionine alone' and serine and
methionine.
The growth test was scored after 24 hours at 32"C.
Spores from a mating of another revertant to wild-type
were isolated five weeks after initiation of the cross.
These
were heat-shocked and tested for their requirements by the
procedure described above.
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·-------:
RESULTS
Analysis of Revertant #9 X 25a
Analysis of three revertants of ser(JBM 4-13) indicated
;..
/_·
that two of the three revertants were due to back mutations at
the
~(JBM
4-13) locus or to forward mutation at a closely-
linked suppressor gene (Bengtson, 1975).
The third, revertant #9,
differed from these two in that the high number of serine-requiring
progeny recovered suggested that the revertant was genetically
~(JBM
4-13) and that a cytoplasmic or environmental factor was
responsible for the apparent reversion.
Alternatively, revertant
#9 may have been heterocaryotic for both revertant and mutant nuclei.
To test for heterocaryosis, revertant #9 subcultures
which were phenotypically revertant according to the standard
growth test were plated on minimal medium and on medium supplemented
with serine.
Colonies of various size were observed and more than
twice as many colonies appeared on minimal as appeared on serine.
The small colonies which appeared on serine medium grew up by
the end of two weeks while the small colonies on minimal remained
quite tiny.
Both large and small colonies were isolated from the
plates and their growth patterns on serine and minimal medium
slants were observed for two weeks.
The large colonies
demonstrated better growth on minimal medium than on serine while,
conversely, the small colonies grew better on serine-supplemented
medium.
11
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12
Large colonies were then isolated from the minimal plates
and were split into three portions.
One was transferred to crossing
medium to be used as a protoperithecial parent while the others
were transferred to a slant of minimal medium and a slant of
minimal medium with serine.
ser
When these split colonies grew as
+ revertants, they were mated to wild-type
To
25~
(conidial parent).
observe the characteristics of presumptive homocaryons
on plates, conidia of
+ and ser colony isolates were plated
the~
on minimal medium and on serine-supplemented medium at the time
this cross was prepared.
~er+
isolates demonstrated much better
growth on minimal than on serine in regards to both number of
colonies and colony size.
Conidia from ser isolates did not
grow on minimal while good growth and colonies of varied size were
noted on serine.
Shot spores were observed twenty-five days after the
initiation of the cross, revertant
#9~ X 25a
209 spores were isolated and heat-shocked.
was 31.4%.
Germination frequency
All of the spores isolated were wild-type; no serineTwo months later, 192 spores
from the same cross were isolated.
Germination frequency was
These isolates were analyzed for the markers cot-1, al-2,
and pe,fl as well as
I
Ten days later,
requiring progeny were recovered.
35.4%.
.i
cf.
for the serine requirement.
serine-requiring progeny were recovered.
demonstrated the expected ratios.
Again, no
Cot-1 and al-2
Pe,fl versus pe+ ,fl+ (Table I),
13
2
on the other hand, gave widely divergent ratios (x =18, f=l,
p)O.Ol).
This was also observed by Bengtson (1975) who noted that
there had been no divergence from the expected ratios of markers
when ser(JBM 4-13) was originally crossed to pe,fl; cot-1.
Revertant 119 thus appears to have been heterocaryotic in
the initial microconidial stock while the reisolate behaved as a
.,.
;
homocaryon when crossed to wild-type.
~-
Ser, Met Reversion Studies
To isolate new, homocaryotic
revert~nts
of ser(JBM
4~13)
for further genetic analysis, 206 spores of a five month old cross
of ser, met X 25a were isolated and heat-shocked.
The met marker
was included in the analysis to rule out contamination by wild-type
as a source of revertants.
The germination frequency was 90.8%.
The spore isolates were tested for growth requirements by
transferring each to medium with serine and methionine, medium with
serine alone, medium with methionine alone, and minimal medium.
A deviation from the expected ratios was noted with the appearance
+ met progeny than would be expected based on
reciprocal progeny (ser, met+ ) or on previous mapping. As the
of far more
number of
~·
~.
met parental types was much less than expected, it
appears that reversion at the ser locus occurred during storage
of the spores and accounted for the divergent ratios (Table II).
'i
67 (35.4%)
192
64
65
~,fl
206
,
187 (91%)
II of spores
germin. (frequ)
177
cultures tested
85
±,±
,
+
or~
57
~,met.
. +
~,fl
.
+
. 49:15
+
,fl.
~,Q_:~,flx
Distribution of markers from ser, met X 25a
isolates were not scored for al-2
II spores
isolated
Table II.
* -
0
0
ser
~,!l
cultures tested
x - 3 possible genotypes for microconidia -
65 (31.1%)
209
II of spores
germin. (frequ)
Distribution of markers from Revertant #9 X 25a
II spores
isolated
Table I.
34
ser+,met
34:30
cot+ :cot
+
2
~,~
27:22*
al+ :al
To examine the question of whether the ser, met stocks
which had not reverted during storage would revert vegetatively,
the ser, met isolates obtained
from~·
reversion on a random, continual basis.
met X 25a were tested for
During a twenty-two week
period, 34 of the 57 cultures were tested.
have shown reversion.
Of these, twenty-one
Eight of the twenty-one showed reversion
only upon subsequent testing after demonstrating no reversion ten
to twelve weeks earlier.
Reversion frequencies were calculated by comparing the
number of colonies on serine and methionine to the number appearing
on methionine alone, plated from serial dilutions, taking into
account that 0.1 milliliter of each dilution was plated.
For example:
Serine + Methionine
Methionine
Dilution:
cone.
Count:
26.5*
86
3
8
This particular culture had a reversion frequency of
26.5 X 10
8.6 X 10:3
X
10
=
3.8 X 10- 3
*(The counts for methionine plates were an average of
the number of colonies appearing on duplicate plates)
Colony size varied on both methionine plates and
methionine/serine plates.
All possible revertants were tested
until a good morphological criterion was established for
distinguishing revertant from non-revertant, leaky (limited growth
----·-,
on methionine) colonies.
Revertant colonies demonstrated the same
morphology, in regards to depth and density, as the colonies which
appeared on doubly-supplemented medium.
Colonies which appeared on
methionine plates but which did not have this characteristic
morphology, proved to be mutant when tested.
Table III indicates the mitotic instability of
and the random variation of reversion frequencies.
·varied from 3. 7 x 10-
2
to
~er(JBM
4-13)
Frequencies
8 x 10- 7 in separate spore isolates.
If 28 of the 34 ser+ , met progeny recovered from
~·
met x 25a
· are considered to be due to reversion (determined from the
difference between 85 expected ser, met parental types and the 57
observed), then, including the twenty-one revertants .:isolated to date
57.6% of the total ser, met progeny from that cross have shown
reversion thus far.
Genetic Analysis of Revertants .
To determine whether all
+ met revertants are due to
~.
back mutation or whether some may arise by unlinked suppressor
mutations, two of
+ met revertants, 116Rl and 123R6, were
the~.
first back-crossed to
~·
met to produce homocaryotic revertant
stocks, and these were then outcrossed to wild-type and the progeny
examined for ser, met segregants.
In each case, it was noted that the most vigorous crosses
were those in which the revertant was the protoperithecial parent.
All spores analyzed were therefore derived from revertant females.
17
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Table III.
Reversion Data ( R.F. = reversion frequency)
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.l
culture
M.S. II
time tested
(da:ls after germ.)
12
30
0
2B
13
+
0
151
117
0
143
3
.-·1-
.3
123
29
3.8
6
1.58
.130
29
1
0
138
29
1
0
116
31
1.6
28
118
31
117
153
0
0
123
3
121
43
1.6
98
43
0
95
43
0
113
58
50.5*
2
53
26.5
9.8
0
133
49
46
123
61
26.5
0 .
34
197
6B
0
49
49
0.5
·0
49
129
1.28
0
122
9B
16.8
0
48
123
;'
60
43
123
f
17.5
0
31
151
~
R.F.
xl0- 3
0
14
151
t'
II
revertants
lB
8B
i
II cells
Elated
3
x10
0
0
0.434
5.7
Table III (cont).
culture
M.s. IF
II cells
time tested
(days after germ.)
Elated
II
revertants
3
xlO ·
151
167
0
4B
50
0
5B
50
0
lOB
71
31B
71
',_,,
.... 143
R.F.
xl0- 3
1260
1
.0008
8.1
48.5
6
0
·-· ,,
.
.,8.1
300
37
.-_: ~.
32B
78
48
8
.167
57
78
490
2
.0041
88
126
··15.3
0
137
92
0
96
126
42
23
. 5.47
i43
126
8.6
26.5
3.8
74
130
56
106
1.89
112
130
76
1365
18
145
137
5.1
37
7.26
108
137
1170
0
78
137
1.1
10.5
9.55
79
137
36
23
0.65
99
137
2.2
7.5
3.4
$
+- specific figures were not reco3ded 5 for some early tests showing
no reversion; an average of 10 -10 conidia were plated
*-
revertant counts are an average of the number of colonies
appearing on duplicate plates
------------------------
In the initial cross of both revertants to ser,
~,
the majority
of the spores shot were either colorless or very lightly colored.
Germination frequencies tended to be rather low although the most
Ser+ , met
mature-appearing spores were selected (Tables IV, V).
116Rl gave approximately 50% germination both in the initial cross
to ser, met and in the subsequent cross to 25!!_.
Because soaking
increases germination frequency in old spores (Davis and de Serres,
1970), a second group of spores from the latter cross were soaked
in sterile water for two weeks and then isolated and heat-shocked,
yielding an improved germination frequency of-70.4%.
As this
appeared to improve the germination frequency in the fresh spores
-
-
+
of the above cross, a second group of spores from ser , met 123R6 X
ScottA were soaked for a week after spores initially isolated gave
a frequency of 55%.
In this case, the frequency was not improved.
When 123R6 was first crossed to ser, met, none of the first group
of spores isolated germinated.
The cross was then allowed to mature
one more week and dark spores were again isolated; this second
group gave a germination frequency of 24.2%.
Unlike 116Rl which
gave similar frequencies through two crosses, 123R6 crossed to
wild-type gave a germination frequency which was much greater than
in the earlier cross to ser, met.
It should be noted that light and colorless spores were
produced in great quantity in the crosses of the two revertants
to wild-type as well as to ser, met.
Bengtson (1975) noted
immature spores and low germination frequencies in her initial set
-----·--------·---------·
Table IV.
Distribution of markers from revertants X ser, met
Cross
II spores
isolated
II of spores
germin. (freq.)
cultures tested-
ser,met
ser.+ ,met
116Rl X N3-12A
57
28 (49. 2%)
27
16
11
123R6 X N3-10a
75
18 (24.2%)
18
7
11
Table V.
Distribution of markers for revertants X wild-type*
II spores
isolated
rev til
II of spores
germin. (freq.)
cultures tested
+
+
~,met.
ser,met+
ser+ ,met
ser,met
116Rl
89
45 (50.5%)
44
22
22
0
0
116Rl
108
___1§_ (70. 4%)
68
27
41
0
0
Total
116Rl
197
12l
112
49
63
0
0
123R6
108
60 (55.5%)
60
29
31
0
0
123R6
108
57 (52.8%)
56
28-
28
0
0
59
0
0
I Total-
123R6
.
216
I
* - crosses analyzed:
116Rl ~ X 25a Q'
123R6 ~ X Sc-;;tt!
··. 57
116
117
I.
cf
---·-·· -····
N
0
···.-·
0,"·':""""':-:;'---c.T.~
':·':~:.'"-:o·~~
=~~_T:::;-o=.::::~=:~--":
_.,-oo.....,_...,., ·"""·-
·.o
---
·-·-
~-:·o
--
···----------·-.
...... -·--:.;::;::;:••• __ -;-;_;~:""·~·:;.< ..~;,;z;::::::;·:·;·
--------------
of mating experiments between revertants and wild-type.
When the
crosses were repeated at room temperature, few immature spores were
seen.
In the present study, all crosses were incubated at 25"C and
were all apparently vigorous, yet a great number of immature spores
were produced.
As indicated by the data in Table V, revertants 116Rl and
123R6 yielded only expected parental-type progeny when crossed to
wild-type (x
2
.
2
= 1.75, f=l, p)0.05; x =0.34, f=l, p)0.80).
Thus,
reversion appears to be due tbbackmutation at the ser(JBM 4-l3)
!ocus or to a closely linked supp~essor gene.
As noted above; data
for revertant /f9 are now also consistent with this conclusion.
'i
DISCUSSION
Properties of Ser(JBM 4-13)
The instability of the ser(JBM 4-13) locus is evident in
its unusually high frequencies of spontaneous reversion.
In general,
previous reversion studies have not seen frequencies as high as
those obtained_ in this study.
described in Neurospora.
Two notable exceptions have been
Giles (1951) reported a maximum reversion
. -4· .
frequency of 7.2 x 10
..
for separate isolate~ of an inositol-·
dependent mutant, ·JH5202, while Barnett and de Serres (1963)
reported the unstable
of 4.29 x 10
-6 .
ad-3~
mutant 137 to revert at a frequency
High frequency reversion has also been reported
for Drosophila (Lefevre & Green, 1959; Sheldon, et al., 1969) and
E. coli (Allen & Yanofsky, 1963).
Ser(JBM 4-13) exhibits certain specific properties as
I
l
il
well as demonstrating high reversion frequencies.
Foremost among
these is the variation in reversion frequency observed among
individual spore isolates, which gave values from 8 x 10 -7 to
3.7 x 10
-2
.
This is in agreement with the variation in frequency
noted by Bengtson (1975).
Giles (1951) also observed a high
degree of variability in the reversion of his unstable mutant,
JH5202, which varied from 15 to 726 revertings per 10
tested.
6
microconidia
Barnett and de Serres (1963) reported up to"a 50-fold
variability in the reversion frequency of forward mutants derived
from revertant strains of the ad-38 mutant 137. These investigators
22
suggested that variation in reversion frequency may be due to the
variation in time at which back-mutation occurs during the growth
of a given mutant culture.
Different degrees of instability in
independently isolated revertants of leu-151 in Salmonella
typhimurium was noted by Dawson and Smith-Keary (1963).
(1963) observed that subclones of
~
Ruth Hill
revertants of E. coli did
not always show the same pattern of instability as the colony from
which they were isolated.
It is possible that very low frequency reversion of
ser(JBM 4-13) inay be undetected within the limits of the testing
procedure utilized.
8 x 10
-7
Thus~
the lowest reversion frequency obtained,
, may represent the minimum frequency observable within the
limits of the test.
ranged from 10
3
to 10
i
The number of cells examined for each culture
6
with 10
for the majority of the
3
to 10
cultures~
4
!~
conidia having been plated
The results obtained by
Bengtson (1975) indicated that the chance that revertants would
be observed in a given test was not dependent upon the number of
conidia produced by a culture.
cultures with 10
6
- 10
7
Approximately one-third of the
conidia/ml of suspension contained revertant
cells; the same fraction of cultures with 10
3
- 10
5
conidia/ml
contained revertants.
As the presence of ungerminated conidia was routinely
observed during the plating experiments, reversion frequency
reflects the percentage of revertants appearing among surviving
conidia.
It is possible that conidial death may be due to a toxic
.~~ .;~j~,.,O\¥·.:::~".?"
2
'
~~':::f. ~~f . s
4:M.:,
effect of sorbose in the medium.
This toxicity may be non-specific
or it may find revertant conidia to be less vulnerable than mutant,
in which case reversion frequencies would be over-estimated.
However, the range of frequencies is similar to that obtained by
Bengtson (1975) in the absence of sorbose.
Previous studies have suggested a heterogenous nature of
revertants indicated by the variation of colony size and growth
rates observed in
~utation exp~riments.
Bacon and Treffers (1961)
noted four different growth rates for revertant colonies of their
ornithine mutant of E. coli.
They suggested that fast and slow-type
colonies represented different mechanisms of reversion, either
back mutation or suppression, respectively.
Hill (1963) observed
variation in colony size among slow-growing E. coli tryp
revertants on minimal medium as well as the appearance of
t~yptophan-dependent
colonies.
She concluded this variation in
colony size was due to mutation during the growth of the
prototrophic clones on minimal agar.
Forward mutation at the serine locus was not detected
I
I
!
in ser(JBM 4-13) revertants during the present investigation or by
Bengtson (1975).
All revertant colonies in this study appeared
between 48 and 72 hours; revertant colonies were quite variable in
size, however.
Colony size and reversion frequency are the only
examples of variation in ser(JBM 4-13) revertants as·all those
tested to date are similar in regard to qualitative growth
characteristics and genetic behavior.
Growth on slants in the
absence of serine is indistinguishable from that of serineindependent cultures.
- t
All revertants show normal 1 : 1 Mendelian
segregation of serine-independent and serineless progeny when
backcrossed to
~'
met.
When crossed to wild-type, no serine-
requiring recombinants have been recovered among all spores analyzed ..
1
'l
!
Both revertants examined in this study, 116Rl and 123R6, produced
'
rJ
many lightly-colored and colorless spores in the crosses to ser, met
.i -.
and wild-type •. This was not observed, however, in revertant 119 X 25a
or in previous
l
l
studies~
(Giles, 1951; Bengtson, 1975).
Since the mutant ser, met cultures tested for reversion
l
were derived from relatively old spores of which at least one-third
apparently underwent reversion within the stored ascospores, the
revertants observed in this· study could be considered late-appearing.
Of the twenty-one revertants isolated, only six were found in the
first three months of the study, the remainder appeared during
the last two months.
At the beginning of this study, cultures were
selected for plating at random from the 57 ser·, met progeny.
Because the presence of revertants was found to be associated with
growth of subcultures on methionine, later studies involved
selection of cultures for plating which looked "revertant" by
growth test.
Thus, the majority of the revertants were isolated
during the latter part of the study.
One principal question regarding ser(JBM 4-13) is
whether all cultures will eventually undergo reversion or whether
"
..:.u
. j
;j
.I
f
l
··I
.
}
'
i
i
:I
j
4
the gene is ever stabilized.
During a three-week period, Bengtson
(1975) noted reversion in 67 out of 102 mutant cultures.
In
general, cultures though to be stable have always reverted upon
j
further testing.
i
34 ser+, met recombinants from ser, met X 25a are considered to be
:j
_f
.. f
i
Al
i
If, based on reciprocal parental types, 28 of the
due to reversion, then, including those isolated during this study,
49 of 85 ser(JBM 4-13) progeny (latter figure calculated from
wild-type progeny of ser, met X 25a) have reverted in ten months.
Will the remainder eventually show reversion~ if tested
extensively?
ll
The 34 cultures examined here are presently being
examined continually in an effort to answer this question.
The normal segregation patterns seen in the mating
experiments suggest that
~(JBM
4-13) is meiotically stable
although low frequency reversion would be likely to be undetected
within the crosses.
The reversion observed in this study occured
either in stored spores or in vegetative cultures.
The results
cif the crosses of the homocaryotic revertants to wild-type
indicate that the reversion is due to a back mutation at the
~(JBM
4-13) locus or to a forward mutation at a closely linked
suppressor.
If an unlinked suppressor mutation were responsible
for the reversion phenomenon, a 3 : 1 segregation of revertant
versus mutant progeny would occur (Houlahan & Mitchell, 1947).
The discrepancy involving revertant #9 in a previous study
(Bengtson, 1975) is now explained by the presence of unreverted
nuclei in the heterocaryotic stock used in the initial crosses.
<.I
While the results of these studies contain no indication of the
presence of a suppressor mutation~ Jinks (1961) observed 129 h+
revertants of various T4h mutants to have resulted from suppressors
all of which mapped in the original mutated gene.
Possibly,
revertants of ser(JBM 4-13) may be due to such intracistronic or
very closely linked suppressors which have not been detected due
to the resolving limits of the crosses analyzed.
The identification
of intragenic suppressors is rather difficult in Neurospora
because a very large number of spores must be analyzed.
MOdels for Gene Instability
Models of varied perspective have been postulated to
account for gene instability and associated reversion phenomena.
Inherent instability of certain loci has been suggested to be due
to base-pairing errors in the DNA.
Barnett and de Serres (1963)
based their explanation of spontaneous gene instability at the
ad-3B locus in Neurospora on a mutational model involving
transversional,
AT~CG
(Freese, 1959), and transitional
changes in the nucleotide bases.
TA~CG,
It is possible that ser(JBM 4-13)
is an inherently unstable locus, although reversion has not yet
been observed in all spore isolates.
Instability may also be due to extrinsic factors. Rhoades
(1941) described such control in his work with the gene, a, in
maize, which becomes unstable in the presence of the unlinked gene,
Dt.
Mutator genes are factors which are located at specific sites
28
within the genome.
Mutators are known to increase spontaneous
mutation rates throughout the genome.
Sheldon~ ~
al. (1969) notes
that high reversion frequencies suggest the presence of such
mutators in Drosophila.
Three mutator genes in E. coli previously
known to induce transitional base changes significantly increase
the reversion frequency of lacZ frameshift mutations (Siegal & Kamel,
1974).
Mutator-induced reversion of an E. coli auxotroph was
suggested to indicate site specificity of the mutator, mutT, in
that its presence gave only one slow-growing type of prototrophic
colony where four growth types were observed in spontaneous
reversion (Bacon & Treffers, 1961).
The Treffers mutator gene
dramatically increases reversion rates as well.
(1962) described a specific mutator in E. coli
Gunderson~
et al.
which increased
mutation rate to streptomycin resistance but did not affect
mutation rates at several other sites tested.
If a factor responsible for the high revertibility of
ser(JBM 4-13) can be separated from
the~ locus~
it is still
possible that reversion is due to some cytoplasmic agent since the
revertant was the protoperithecial parent in all crosses analyzed
in this study.
One of the more complex factors involved in the control
of gene expression is the controlling element, studied extensively
in maize by McClintock (1950, 1951, 1965).
Controlling elements
can be exemplified by the dissociator locus, Ds.
This element has
no fixed location in the genome as it is capable of transposition
to completely different sites.
The Ds element causes an unstable
suppression of the activity of contiguous genes.
manifested as a
var~egated
phenotype.
This effect is
Ds activity is dependent
on another transposable element, Ac, which is a dominant factor
required for Ds to exert its suppressive
effect.
Ds activity is usually associated with structural
alteration in the chromosome, including breaks, deficiencies, and
translocations.
l
l
Dicentric and .acentric chromatid formations are
indicative of .Ds activity. ,
,:
Other cases of ins.tability have also involved chromosomal
j
1
l
aberrations.
Green
(1967~
1969, 1973) has proposed controlling
elements to explain mutable white eye alleles in Drosophila, often
associated with chromosome aberrations, and notes that deficiencies
of mutable loci in Drosophila are quite unstable.
Transposition
of the we associated controlling element has been demonstrated
although the appearance of instability at another site has not been
observed.
The relationship of structural changes of the chromosomes
to instability and reversion is quite interesting in regard to the
observed spore abortion of ser(JBM 4-13) revertants in this study.
Instability at the A locus in maize has appeared in plants derived
1
from aged kernels (Rhoades, 1950) and McClintock (1951) points out
that such aging is known to give rise to chromosome aberrations as
well as mutations.
McClintock (1945) has used spore abortion
patterns in ordered asci to estimate the types of disjunction of
chromosomes heterozygous for a specific translocation.
Examination
of spore patterns in asci could therefore serve as cytological
evidence for the type of structural change involved in a mutational
event.
The instability of ser(JBM 4-13) could be due to a
controlling element although there is no indication of
transposability, always implicit evidence for the existence of
such factors.
It is possible that the controlling element has
.become fixed in the genome; both Ds and Ac are capable of such
:-~~---
·--~
:.
_:;,
behavior.
..
.
·,.:
:
.'
.
-
..
-.-
,.._
:
_;
.
'
As controlling elements are postulated to undergo
"changes of state", manifesting changes in the expression or
mutability of associated
genes~·
it is possible that the
characteristics of ser(JBM 4-13) instability are due to some such
heritable change.
Episomes, existing either autonomously or integrated in
the genome, have also been proposed to account for localized genetic
instabilities.
1:
. ll
Dawson and Smith-Keary (1963) suggested "controlling
episomes" to account for unstable reversion of the su-leu A locus in
Salmonella typhimurium.
In their model,
instability appears when
an episome attaches to a leucine suppressor locus inducing an
increased frequency of mutation to auxotrophy.
The episome
increases the probability that a mutated suppressor will backmutate
to its original inactive state.
This model therefore involves
increased mutation of the suppressor itself.
the instability of
~
Hill (1963) explained
revertants of E. coli by proposing a
_1}t~---~i~~; ~''"";;~"'
r::~.:;::',
slightly modified model in which an episome actually masks the
activity of the suppressor gene.
Reversion to prototrophy is due
to the detachment of the episome from this locus and the backmutation
t.o auxotrophy is the result of the attachment of the episome itself
to the suppressor locus.
It should be noted that viruses are capable of inducing
point mutations.
Baumiller (1967) studied the induction of sex-
linked recessive lethals in Drosophila by a Sigma virus.
Alteration
of gene activity due to prophage insertion has also been
demonstrated in E. coli (Taylor, 1963).
These studies have only
demonstrated induction of forward mutation although the existence
li'
of viruses capable of causing reversion remains a possibility.
Schwartz (1965), in his transductional analysis of
unstable lac- reversion in E. coli, proposed a suppressor carried on
an extrachromosomal element.
He suggested that observed differences
in stability are due to the differencesin "state" of the suppressorbearing element.
This is similar to the suggestion that a decrease
in reversion frequency might be due to the stable integration of
the episome into some unknown site in the
genome (Dawson & Smith-
Keary, 1963).
At the present time, it is impossible to decide which of
these models (if any) accounts for the unorthodox
ser(JBM 4-13).
behavior
of
The work reported here confirms the conclusions
reached by previous investigators (Kline, 1973; Bengtson, 1975)
that the site is highly revertible with variable frequency.
This
Ii
study has clarified the nature of the revertants which are all the
result of back mutation at the ser(JBM 4-13) site or forward
mutation at a closely linked suppressor gene.
The anomalous results
obtained with revertant #9 have been explained by heterocaryosis of
the original isolate.
·.,
i
.-
.i . .•.
BIBLIOGRAPHY
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study of reversion with the A-gene A-protein system of
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-· Barnett,' W. E. , and de Serres, F. J. , 1963 Fixed genetic
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Baumiller, R.C., 1967 Virus induced point
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33
I~
Reddish -
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34
Emerson, R.A., 1914 The inheritance of a recurring variation
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35
Pittenger, T.H., 1965 The distribution of nuclei in conidia
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4.
z:
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1950
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i'
I
Westergaard, M., and Mitchell, H.K., 1947 Neurospora V.
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AM. J. Bot • 34 : 57 3 •