CALIFORNIA S'l"'}\.TE UNIVERSITY,
,,Part
I
1,
NORTH:R.IfJGE
THE A.i'rALYSIS OF' SFONTANECUS
REVERSION IN A SERINE AUXOTROPH OJ:i'
NEIJRO.SPOJ:~A
-··-------
CRASS.,!\. ,
·-·--·---·-·
)
Pa.rt I I
REQUIRING l\'lUTANT : ~I-~F-..;~.
A thesis submitted in parTial satisfaction of the
requirements .L\..-.:r th<c-' de9ree of· 1\l.::~.stc:<~' ~_yf Sc.Lencr::: in
Biol.o~~y
Dy
.Ramon::-. S1..1.nd.hcrn Bengt ::.->on
i
\
The t.hesi:3 of Ramona. Sundbom Bengt.son is
approved~
California S·tai.:e Un.ivt-.::rsity, Northridge
Dec~nnber,
J..l.
197 4
ACKNOWLEDGEMENTS
I
would like to acknowledge Dr. Joyce MaxvJell for
the constant guidance and encouragement she has provided
during the preparation o£ this thesis, and Dr. Daisy Kuhn
a.nd Dr. Georg·e Lefevre .Jr. who gave me invalua.ble editoI.'ial advice as members o£ my graduate comrni ttee.
~/.
sincere appreciation goes to my family,
the
Sundborns, and to my husband William, who have helped to
make this thesis a
I
rea.~i ty.
would also like to extend a special thank you to
RosaJ.yn Kutchin.s, Kathy Gallaher, Ron Koller; an.d Ka.thy
Wiedenheft for their technical assistance.
iii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS •
iii
*
LIST OF TABLES .
vii
LISl'----QF· FIGURES.
viii
ABSTRACT . •
ix
PART I
·r
J...
LL
INTRODUCTION.
1
MATERIALS AND METHODS •
6
Strains •
0
6
Maintenance of Cu.l turcs .
7
Reversion Studies •
8-
Control E:xperiment for Reversion
Studies •
11
Forwa:•:d Mutation o£ Revertants.
11
CE-~llu1ar
Growth Requirr,~ments
for Revers:Lon- •
12
Growi:J-·, Comnax:Lson of Revertants
versus ~'Vi1d~·Type ~
Genetic: Analysis o:f Revertants
III.
Q
•
•
14
...
16
RESULTS •
The Vegetative Reve:rtibility o£
the Ser(JBM 4-13) Locus •
..
16
Reversion Studies •
16
Coritrol Expm:iment.
23
:i.v
Forward Mutation of the
Revertants •
24
Independence of Reversion
from Cell Growth ~
24
Analysis of Revertants .
IV.
26
Comparative Growth Studies •
28
Genetic Analysis of the
Revertants •
31
DISCUSSION .
35
Discussion of the Results.
35
Apparent Meiotic Stabi1i.ty of ·
Ser ( JBM 4-13).
--
35
'·
High Reversion Frequencies
•
36
Variations in Reve:csion Freauencies ve~l..'sus Size of
Conidi<:...l Populations •
36
Time Dependence o:f Mt1.t.:..tion.
37
Reversion versus Cell Growth •
38
Colony Size Variation.
39
Evidence Ruling Out the
P:resence of Gross Chr:onK1SOtn6.l
RearrD.r:.gcmeilts .
40
Prop~~r.ti.es
·of the .Ser;(JBN 4-·13)
Models to E:>..."Plain R(wersion.
Recombination or Sister
Strand Recombination
~
Extrachromosomal Determinants.
Suppressors
v
Iv"IUtant •
40
41
41
Models to E:>...rplain Gene Instabili·ty.
43
1. Single Base-pair Alterations •
43
2. Controlling Factors.
45
A) Mutator Genes .
45
B) Episomes.
46
C) Controlling Elements.
47
D) Viruses •
49
F't1.rther Investigation of the Ser
(JBM 4-13) i~tant •
49
PART II
I~
II.
INTRODUCTION
54
lv'LJi.TEE IALS ftj\fD JVIETHODS •
5.5
Strains •
55
Maintenance o£ Cultures •
55
Ser-5
Ser-5
Ser-5
._..,.... ... .,.,
X
Ser-1
----
Ser-.5
X
trn-l
~' .__..___
__
---
~-·
III.
.
X
56
56
.,
57
RESULTS .1\ND DISCUSSION.
59
I~
66
BIBLI()(3RAPHY FOR PARTS I AND I
vi
LIS'I' OF TABLES
Table
Part I
1.
2~
Reversion frequencies of three separate
subcultures ot' each J2C~fl; .~;s_x:._(JBM
4-13); .£!:--~ isolate showing reversion
during the three week testing period.
.l'tucroconidial counts to test forward
mutation in one
revertant .
3a,
3b.
18
~(.JBM
4-13)
...
25
Distribution o£, markers £'rom ser
(JB.l\1 4-13) x 12.§;_, £1; cot--1 •
32
Distribution of !;:arkers from
revertants ~ 25a.
33
Part II
Linkage data on random spores from
the cross ~5 x s£_, !rE.-~ . . ~
vii
~
64
LIST OF FIGURES
--------Paae
_......_
Part I
1..
Comparative growth curves of al-2,
cot-1 and two revertants as a
function of time •
...
29
Distribution of the germination frequencies shown from the cross a;
_ser-5 (JBlVI 9); _:::2t-l (Cl02t) to FGSC
#190: A; ~(5801), .!E.E.Ml(l057.5) as
a function of the age of the ascospores tested.
62
3.
Ca.lculatecl m.a.p ot: linkage group III.
65
4.
Published map of 1inkag?. group III •
65
2.
viii
ABSTRACT
Part I
THE ANALYSIS OF SPONTANEOUS
REVERSION IN A SERINE AUXOTROPH
OF NEUROSPORA CRA.SSA
----
Part II
LINKAGE DATA ON A NEW SERINE
REQUIRING MUTANT
SER-5
---
by
Ramona Sundbom Bengtson
Master of. Science in Biology
..:ranu2.ry 7
1975
Part I
A newly isolated serine auxotroph o:f
Neur:o~~-
crassa, ser (JBM 4-13), exhibited a high rate of reversion
in mapping e);:per.iments.
Thi.s study sov.gh t to determine
whether it was time dependent.
Individually isolated asco-
spore cultures of the mutant, as well as subcultures o£
individual progeny, showed spontaneous reversion £requen . -
cies ranging frora 1.0 x 10
~5
to 1.0.
Other properties of
the 5..~E.(JBM 4-13) mutant demonstrated by this study inM
elude:
1) the apparent meiotic stabili t.y of the ser (JBM
---
-
ix
4"'-13) gene, 2) the mitotically unstable and highly revertible state o£ ser (JBl\1
4-~13)
which occurs sometime during
vegetative development, 3) the time dependence of thereversion phenomenon, 4) the independence of the reversion
phenomenon from conidial population size, and 5) the independence of' reversion i'rom cell division or growth.
Three
s~(JBM
4-13) J:evertants were analyzed by
comparing_ their growth curves with the strain in which
2.££(JBM 4-13) was originally induced,
a.nd the revertants
were crossed to the wild-type·strain
25a
to check :for
?.
.
. serine requiring recombinants.
Two of the
reve~ctants
be-
•haved like back mutations or closely linked suppressors.
The third revertant gctve restll ts that could be explained by
.a cytoplasmic or environmental factor causing the apparent
reversion~
The properties o£ the
-~~(JBM
4-13) mutant a.re com-
pared to the proper:t:i.e:-; of· models proposed to explain
high
£requer.~y
rc~ve-rsior:,
found in other. organisms.
Part I I
A new serine requiring mutant, known to be located
on linkage group III, was crossed to FGSC #116 A,
(H605) to test for allelism.
Genetic analysis showed that
th:i...s mutant is not an allele o.f
_2_e1~·-J;
bu"t maps 12.1 map
units to its right.
To further locate the gene, the
mutant was crossed to FGSC #190 A; .E.£(5801), }:rp-1..(10575).
The results placed the mutant 1.2 map units to the left
of !_r-e:-1.:.·
It is now officially designated
the Fungal Genetics Stock Center,
xi
.§.~(JBM
9) by
(Humboldt, California).
INTRODUCTION
In order to gain kno·wledge about molecular gene
structure, investigators studied sponi;aneous and induced
mutation in various systems.
be highly
u.nstabl·.-~,
Senne mutants vJ"eJ:e found to
reverting to a phenotypically wild-
type state, sometimes with £J:equencies higher than the
observed spontaneous fo_rv-1ard mutation :frequencies for the
particular gene in question.
g;9dis~
was
r!'i~ported. by
.muta:ting character."
by Demerec in.
As ea:r:ly as 1926 the mutant
J)eme:rec
( 1926)
This and two other mutants described
DX:£§9Phi~~ viril:_;.~§..
of unstable mutants.
to be a "£:r:equently
were the first examples
Since that time, spontaneous and
induced genetic reversion has been studied in many organisms, and investigators havt:::! sug9ested various models to
explain hi9h £r:e:c··ruency :r:eves: si.on.
Several contrasting ways of· explaining reversion can
One explanation for a highly
be found in the literature.
revertible locus is that the locus has come under the control of a second gene or element.
For E!Xatnple, in E. coli
mutator genes have been studied extensively which induce
reversion at various loci.
Bacon and
1
Tr.e££t=~rs
(1961)
showed that their mutator increased the frequency o£ spontaneous reversion at the ornithine l6cus from 1.16 x 10
-10
. -7
per bacterium per generation to 3.76.x 10
per bacterium
per generation when the mutator gene was introduced into
the genome.
Episorrl?-S are another type of controJ ling factor
which may modify mutation :rates.
E0__<-:ol~,
The iVIu factor, found in
has the properties of an episome and increases the
:rates o£ mutation to Sm resistance, while the reversion
·rates of auxo·trophic mutants J.n the presence o£ l'viu remain
unchanged (Gundersen, et al., 1962).
Episome models are
also used to explain 9en?.ticaJ.ly unsta,ble revertants, in
which a suppressor gene:! is activated o:r inactivated by the
detachment ox attachment of an episome (Schwartz, 1965;
Hill, 1963; Dawson, et al., 1963).
Controlling element systems have been shown to exist
in maize by Barb;:.. ra JV1cClintock ( 1965).
are the Ac-Ds sy s-eem,
sy s·t.em.,
Here
ar1
operator-like element is inserted at or next to the gene
locus, and a regulator element controls the gene by sending
messages to the operator.
These messages can signal the
release of the tJene from the system, change the mutation
rate, and/or alter the activity of the
genfc~.
3
The second approach in e::.,.rplaining revertible genes
is to study the molecular make-up of the gE-me itself.
genes seem to be inherently unstable.
Ernst
FreesE~
Some
(1959)
developed a model of mutation based on the observation that
spontaneous and mutagen induced mutations differed in their:
susceptibilities to mutagen induced reversion.
Mutants
whicn,were induced by base analogues and could o..lso be
induced to revert by the same base analogues were suggested
to be due to transitional changes.
Spontaneous or pro-.
:flavin induced mutants were not susceptible to base analogue induced reversion and were suggested to be due to
trarisversional changes.
gestc~d
B2crnett and de .Serres ( 1963) sug-
that an unstablf: f!Cne in
;~eu_:~~PO..:£:~ ~-~ssa
to undergo frequent transitional· changes.
is able
Aui to and Brown
(1973) have characterized the original lesions causing
the mutant phenotype.
By using various
mutagens~
which
have specific modes of action to induce rr2versions ~ it is
possible to dete.:r:mins what kind
originally occurred.
(~£
base·-pe.ir alteration
Perhaps in the future it will be
possible to describe in molecular detc.;.il the events ini tiated within the affected gene by controlling factors,
episomes or mutators.
Reversion can also be caused by physical changes in
the gerwme.
Recombination was proposed as a mechanism to
3n
account for the high reversion frequencies of the f _ gene
in
Droso~
by Lefevre and Green
(1959).
Unequal cross-
ing over causing frame-shift mutations was suggested to
I3., an
account for the high spontc:meous reversion rate of
rii mutant in bacteriophage T4 (Strigini, 1965)e
involving siste::c strand recombination in
Models
12E£2.~ohi:_la
have
been postulated by Bowman {1965) and by Grigg and Sergeant
{1961) to ex-tJlain reversion in Neurosnora.
In Neurospora there are only two studies where high
spontaneuus reversion frequencies have been noted.
The
normal range for spontaneous reversion frequencies is 10
to 10
-8
-7
, but when Norman Giles (1951) studied reversion o:f
inositolless
mut2~IY"cs,
one inositol allele called JH5202
-d
reverted at a frequency of 7.26 x 10 -
Barnett and de
~B
Serres (1963), while examining the unstable
mutant
;:
.
.
-4
137, a 1 so :...ound reversJ.on :t:requencles of 1.0
•
This thesis descritres the analysis of a newly isolated serine r:euuiri:1g m.utant; ;:;er.:f,JBM 4-13'l, which ma:os
--:J.
----· \
,
on linkage group V, 3.5 map unit2 to the right of
(Kline, 1974)
~
...
m~
During t.he mating experiments carried out
by Kline se;_(.JBM 4-13) showed an exceedingly high .rmmber
of wiJ..d type recombina.nts compared to the reciprocal class
o£ recombinants.
The p:n?sent study sought to determine
th..:?- cause of the une:x:pect.ed asymmetry in the progeny o£
....
~·
erosses involving the mutant.
that
~(JBi\'I
The rc:!sults demonstrate
4-13) exhibits a high frequency of spontaneous
reversion to a phenotypically normal. state.
Thf~
properties
of this highly revertible mutant are compar.ed with those
properties found in other systems where various models have
been suggested to e},.'Plain the :reversion phenomenon.
MATERIALS AND METHODS
Strains
The ser.inr>. a.uxotroph used during these studies was
derived from the nutritionally wild-type strain A, al-2
(15300);
cot~l(Cl02t)
::received from Mary B. Mitchell.
Conidia from this strain were irradiated to twenty percent
survival
(two minutes) under an ultraviolet Westinghouse
'·
. StfJrilamp, model 7821-30.
No precautions were taken to
preclude photoreactivation.
Dr. ..Joyce B ~
Maxwell and Mr..
Paul West selected for ser:I.ne auxot:rop:hs using the fil tration enrichment method (VJoodvvard, 1954).
One of the £i VE:~
serine auxotrophs which were isolated .from. conidict plated
on Vogel 1 s minimal medium (Vogel, 1956) supplemented with
Oo2 mg/ml L-ser:ine was used in these (:'!Xperiments~ ~~E._(JBi\1
4·-13).
Because the stoe;k
culturE~
of this mutant d:i.Sf>l.<:~.y<:~d
an unusual frequency of sponta.ncous reversion in early
studies 1 a serine-dependent derivative was isola trJ.d from
plated conidia £or the present study.
to as
~(JBM 4-·13)
The muta.nt referred
in this report aJ so car:r:ies the markers
A, al-2_(15300), and so!.:l..(Cl02t).
6
7
Other stock cultures were wild-type 25a, and
E£(Y8743m),
B. Mitchell,.
f.l.
~;
(L); _£2t-l_(Cl02t), also received from Mary
These cultures are currently available fr:om
FGSC~
The phenotypic characters of the loci in the strains
are described as follows;
Locus
al-2
1
albino conidia
cot-1
4
stock grows colonially
at temperatures above
30°C
pe
2
no macroconidia; forms
microconidia
£1
2
no mac:r:oconidi.a; few
or no microconidia;
P.~-' f~ qenoty~e forms
abundant microconidia
ser (JBl\1 4-13)
5
reouires 0,4ma/ml
"'
ser inf:: .for optimal
growth
l\la.interiance of' Cul tu,res
All vegetative cultures were maintained either on
slants of Vogel 1 s minimal m.edium N, or Vogel 1 s medium
{Vogel, 1956) supplemented with 0.4 mg/ml L-serine.
The
L-serine was obtained from Ca.lbiochem, ·San Diego,
California.
All chemicals were reagent.grade with the
exception that table sugar was used in some 8XJ=.'eriments.
8
Solid media were used unless otherwise specifi.ed.
Reversion Studies
In order to obtain a microconidiating stock of
~(JBM
4-13), the auxotroph seE_(JBM 4-13) was·crossed to
a; re(Y8743m), fl(L); cot-l(Cl02t).
Microconidia were used
to obtain reversion frequencies because they are almost
always uninucleate, whereas macroconidia have on the aver_age 2.5 nuclei per conidium (Davis and de Serres, 1970).
The cross v.ras carried out on VJestergaard-Mitchell (1947)
crossing medium supplemented with 0.2 mg/ml L-serine.
The
conidial suspensions were obtained £rom one-week-old
cultures.
In some ca.ses parents were coi.nocula.ted whereas
in other cases the conidial (male) parent was added one
week after the protoperithecial (female) parent.
All
plates were incubated in the dark at room temperature.
Twenty days a£·cer the initiation of the crosses
ascospores were :first seRn on the three plates vihere S(::r
(JBM 4-13) w2.. s the fema.le parent
:fl (L);
_E~t-1
(C102t) was the male.
and~;
pe(Y874Jm),
Random dehisced ·asco-
spores from these plates were used in.the experiment.
The
coinoculated crosses as well as the crosses where ser
(JBr•I 4-13) was used as the male parent dehisced .very few
spores and these crosses were not used.
9
The spores were isolated £rom the three crosses
starting ten days after the first spores were seen.
spores were spread onto four percent
agar~
The
and individually
transferred to l2x75 mm tubes o£ serine containing medium.
In order to induce germination the spores were heat shocked
0
in a water bath at 60 C for one hour.
Germination was re-
corded after 24 hours and again after 48 hours o£ incubaSix days after germination cultures were examined
tion •
. for the
12~
fl character by inverting the tubes and tapping
them on the desk.
This treatment releases macroconidia
and they are readily observed on the wall of the tube.
'Those cultures where there was no evidence o£ macroconidial
formation were
transferre~
to four small test tubes
. { 12 x 7 Smm) containing serine medium and one sma.ll tube
o£ Vogel 7 s minimal.
0
~32 C;
On<-:! of the serine tubes was placed at
all other.' tubes were incubated at room temperature.
After 48 hours each culture was
istic markers.
obser~ed
The albino character,
for its character-
.a~~'
was not re-
corded because in combination with pe £1 its phenotype was
masked.
All P.e £1, .£~_!_,
the other cultures were
and
-~~
cultures were kept and
discarded~
A colonial temoerature sensitive mutant
(~)
was
used so that direct Ieversion frequencies could be computed
from the reve:::-tant colonies on minimal plates compared to·
10
revertant and auxotrophic colonies on serine plates.
No other auxotrophic marker was used in conjunction
~(JBM
with
4-13) because Corran (1969) and Auerbach and
Ramsey (1971) found that reversions were prevented at one
locus when growth of a strain with multiple requirements
was limited by an insufficient amount o£ supplement needed
by a second locus.
Eight, :fifteen,
.o~nd
twenty--two days after the
original spore isolates were transferred to test for their
markers, each individual culture was tested £or revertants.
On each of the three days one serin€-: culture t:ube was used.
A conidial suspension w·as made by adding sterile water into
the
tub~:
·wire.
and scraping thf";
~:urface
wit·L a sterile platinum·
The suspension was then aseptically filtered through
. Pyrex glass wool into a test. tube.
The £iltP.red micro-
conidial suspension was then serially diluted into nine
milliliter sterile distilled watex blanh:s with plastic
disposable one n•illili ter graduate pipettes.
milliliter of the 10
-1
'
10
-2
and 10
onto Vogel's minimal agar medium.
·-4
dilutions were plated
Depending on the turbid-
ity of the conidial suspension either 10or 10
-3
-4
, 10
and 10
-5
One-ten1:h
3
4~
,
10--
and 10
-4
dilutions were plated onto Vogel's
minimal agar plates supplemented with 0.4 mg/ml L-serine.
An alcohol flamed glass hockey stick was used to spread the
l1
inoculum evenly across the surface o£ the plates.
All
plates were incubated at 32°C for six days before the nura·ber of colonies was counted to obtain the reversion frequencies.
The growth habits of each culture were noted on
both minimal and serin(-:: supplemented media after 48 hours
of incubation.
Cultures on minimal medium were observed
again on the tenth and the twentieth days.
Co-ntx·ol Exneriment
'---..-·--. -·-...--~-,_-
for Reversion Studies
One culture was 'arbitrarily selected from the group
which showed no signs of reversion during the testing
period.
Ten subcultures were made from the original tube
which contained the spore.
After eight days, the sub·-
cultures we:r:e analyzed for reversion.
Forward Mutation of r<.evertants
On,~ colony :fro:cn ea.cf1 of three cultures showing re-
version was transFerred .from the minimal medium plate to a
small Vogel's minimal medium t.ube.
The tubes were incu-
bated at room temperature :for e:i.gh'i:. days.
i\Jicroconidial
suspensions o£ these subcultures were filtered and plated
onto serine and minimal media using 10
dilutions.
-3
10
-4
and 10
-.5
Af'ter six days results were rec:ordeds and a
single colony was again selected from a niinimal plate from
th2 culture which showed equal numbers of colonies on both
serine and minimal medium plates.
This was done as a
precaution to assure that the culture tested would be
homocaryotic for the reversion.
Four days later ten sub-
cultures were made of this culture.
After eight days ea.ch
of these subcultures was assayed for colony :formation
on plates of minimal medium and minimal containing serine.
Celh\lar Growth
R~uirr=:m(-:mts
f?r Revcrsior.!_
A sterilized pi~ce of filter paper was placed into
a liter Erlenmeyer flask containing 300 milliliters of
·solid Vogel's supplemented with 0.4 mg/ml serine.
One
milliliter o£ a culture known to be heterocaryotic for
~e;:_(JBM
4·-13) and the revertant marked with pe fl_,
cot -J.,
and possibly _::.1-2_, was used as an inoculum and the culture
was incubated at
25°c.
AftB:r eight days of grov;th appnv<.:imat£:?ly one-third
of the culture wa.s transferred as<2'"pti.caJ.J.y to a ster:ile
petri dish.
Sterile water was added and the surface of
the filter paper scraped to make a microconidial suspension.
This was filtered, serially diluted and plated onto
minimal and minimal
10
··5
dilutions.
w~th
serine media at 10
-3
10
-4
· and
The plates were stored at 32°c for six
days.
Another third of the culture was also removed at
eight days and transferred to another sterile petri dish.
This culture was put into a desiccator with
lid over caso to collect moisture.
4
into a refrigerator at 5°C to
a
vacuum sealed
The desiccator was put
7°c.
On the fifteenth day the one-third kept in a desic-
cator was_plated as above.
Also a microconidia.l suspension
of the culture in the flask was made, filtered, diluted
and plated.
Three one l i tE!r Erlenmeyer flasks, each containing
,300 mil.lili ters o:f mir.ima1 medium, vrere inoculated with
one of two revertants or with fi,
a~-2_(15300);
s:_ot-::l-JC102t).
The cultures were grown at 25°c for eight days.
Micro-
conidial suspensions were made and were filtered through
glass wool into 50 ml test tub2s.
Ec-.. c:b. suspension \Vas then
diluted with sterile distilled water to give appr.oximatel.y
the same concentration of
Each culture
WC\S
con~dia.
used to inoculate thirty-·three
125 ml Erlenmeyer flasks containing twenty milliliters of
Vog~~l
's liquid minimal medium ·with two percent (w/v)
sucrose.
Tb.c~
inoculum was 0. 2 ml o£ the m:i.c:r:oconidial
suspension per flask.
The flasks were swirled once each
day to prevent the adherence o£ mycelium to the sides o£
the :flask.
The three microconidial suspensions were also tested
to see whether they gave approximately equal numbers of
colonies on minimal plates.
As soon as there was su.f:ficient mycelium to harvest,
and on each of the :following eleven days three pads o:f each
cultu:r:e were harvested, blotted dry with paper: towels, and
dried at 90°C for two hours (Davis and de Serres, 1970).
All pads were weighed to the nearest 0.1 mg.
Genetic_
Anal..x~is
of
Revert§!2!..:..~
The revertants were eros sGd to A, al-2 ( 15300);
_9ot-l (Cl02t) and wild-type 25.§:. on Westergaard-:rvii tchell·
crossing medium (1947) supplemented with 0.2 mg/ml L-serine.
It should be noted that the reverta.nts had been passed
·through the :fi.l tra tion,
plating~
2.nd
rci~;olation
procedure
several times to guarantee that they were homocaryotic.
The ascospores were spread onto :four percent agar
plates.
Using a dissection microscope to view the asco-
spores, they were isolated on small cubes cut :from the agar
plates using a platinum 1-vire spatula.
The cubes bearing
the spores were transferred to 0.4 rng/ml L-serine medium
15
tubes.
The ascospores were heat shocked at 60°C for one
hour, and germination was recorded at 48 hoursA
After six
days of growth the cultures were examined for pe £1,
cot-1, and ser by the procedures already described.
.
----
~_l-~,
RESULTS
The -;z:__e,.9etativ-e Rf~vertibili.tz_o£ the Ser (JB_M 4--13) Locus
.
St u d.1es.
R evers1on
Kl.1ne ( 10~4'
}-, ·1 e ana 1 yz1ng
.
~'
) M.l
a cross to determine where
observed a high ratio o£
~_{JBl\1 4-13)
se~+
was located,
versus ser progeny when spore
isolates were tested later than five days after germination.
Spores isolated from the same cross analyzed earlier
·than £i ve days after germin(:l-t ion did not show as great a
deviation £rom the expected 1:1 frequency.
experiment was done to determine whether
The following
~(JBM 4-13)
.reverts at a specific frequency) whether reversion occurs
only during the vegetative sta.te, and whether the rE:version
phenomenon is time dependent.
Se£,(JBM 4--13) was c:cossed :o th•2 nut:r itionally wild-
type strain
~;
~_(Y87 43m),
_£1 (L);
~~( Cl02t)
•
eros ses
with large numbers o£ dehised a.scospores gave germination
frequencies ranging from 66 percent to 88 percent.
Fev.r
brown or colorless spores were observed on the mating
plates.
The results of this
16
cro~;s
showed no devia·t:ions
17
from the
e>...~ec.ted
progeny.
ratio of
~,f:J.:.;.§...~
versus
ser +
~,£1;
In young crosses it seems that .the ser phenotype
is stable in the meiotic state.
The revertibili ty of three separate subcultures of
each of 102 E.§.,fl ;~;s_ot isolates was tested on the
eighth, fifteenth or twenty-second ctay.
During the th:ree
week period, 67 cultures showed reversion in at least one
of the subcultures.
Table l gives the reversion fre-
quencies shown by the 67 spore isolates over the three
week period.
Reversion frequencies were calculated by comparing
the.number of colonies on serine and minimal medium plated
from serial dilutions, taking into account that the inoculum size was 0.1 milliliter of the original suspension.
For example:
Minimal
Dilu·Li..on:
10
-1
-
-·- . -.......
10
-2
--~.,.,..,.-
Il
Serine
10
-4
---·
0
-~
10 -
--- ----62
10
--4
·-_.._...
6
This particular subculture had a reversion frequency o£
11 x 10 x 10 ,
~~
or 1.8 :x 10 ...
600xl02xl0
Colony size varied on both minimal and serine plates
but no additional prototrophic or auxotroph colonies ap·peared on the plates after four to six days incubation at
18
Table 1.
Reversion frequencies of three separa·te subcultures of
each pe, f1i ser(JBM 4-13) i cot-1 isolate showing
reversionduring the three week testing period.
Two weeks ,
-.L
One week
1.
*2.9
X
10
2.
6.5
X
10
-3
-3
*5.0
X
10
3.0
X
10
9.0
X
10
9.6
X
10
3.0
X
10
*1.6
X
1.0
7.9
X
10
11.
1.4
X
10
13.
5.0
X
10
*7.4
X
10
3.
-.3
4.
1.6
X
10
5.
7.1
X
10
1.6
X
10
8.0
X
10
*6.8
X
10
5.0
X
10
-5
-2
-4
-5
6.
5.0
X
10
7.
7.6
X
10
8.
188
X
10
9.
*3.7
X
10
-5
-4
-3
-·3
-5
-2
-5
-1
-·2
-5
-3
-·2
-5
-3
3.1 .......
"" 10
14.
-4
·-3
15~
*8.3
X
3Q5
X
10
--2
11"'\
.t.."-..>
17.
,
·-.L
*3~9 X
18.
10
-·3
19~
5e0
X
10
... 2
:w.
2.0
X
10
-5
21.
2.0
-2
22.
'*3. 7
X
1.0
-2
*6.1
X
10
X
1.0
J.9
Table 1 cont.
On~k
23.
24~
-1
25.
*1.1
X
10
26.
*3.7
X
10
27.
*7.4 X ·10
Two weeks
-2
10
X
*3.5
-3
3.0 X 10
-1
1.3 X 10
'l'hree weeks
-2
-2
-4
*'2 .7
X
1.0
1.8
X
10
1.3
X
10
2.5
X
10
5.0
X
10
1.2
X
10
-3
28.
-4
-3
29.
3.1
X
10
-4
30.
3.0
X
10
-3
31.
4~2
X
-3
10
-5
-5
32.
2.0
X
10
1.7
X
10
-4
33.
6.6
X
10
34.
6.0
X
10
-3
-4
-·4
~
-1
-J..
10
*2~0
X
10
1.,1
X
10
3.8
X
10
39~
*2..7
X
10
40 ..
*9~1
~·
.II"\.
10
2.0
X
10
2o3
X
10
1~8
X
10
*:L8
X
10
35.
*1.2
X
-4
-4
36~
1.0
X
10
1.5
X
10
*3.6
X
10
4.5
X
10
-2
-2
37.
2.0
X
10
-·4
-5
-5
38~
-1
-2
-1
4lu
*1 .. 0
X
10
-2
-3
42.
4o0
X
10
2«9
X
10
_.,,:t
·-2
-3
43.
-1
*1.4
44.
X
10
-3
45 ..
-4
-4
4:6.
5.0
X
10
2.0 :{ 10
20
Table 1 cont.
-5
-5
47.
8.0
X
10
48.
*3.7
X
10
Three weeks
-5
6.0 X 10
-1
*2.9 X 10
'Ivlo weeks
One week
2.0
X
10
X
10
-1
*1.0
-2
49.
3.5
X
10
-3
-5
1.0
X
10
*1.1
X
10
1.2
X
10
8.7
X
10
57.
2.3
X
10
58.
1.7
X
10
*1.7
X
10
*2.5
X
10
4.0
50.
-1
51.
*4.0
X
10
-1
-2
*1.3 x·lO
52.
-5
53.
2.0
X
10
54.
*1.4
X
10
55.
1.0
X
10
56.
2.0
X
10
-1
-5
-2
-4
-4
-3
-3
:::> ....
1.4
X
1
r.
-~U
6Qo
*9.7
X
10
61.
6.0
X
10
62.
*1.6
X
10
63.
1.6
X
10
3.6
X
10
6.0
X
10
,..Q
-2
-2
-1
-5
-1
-4
-4
-5
64.
6.0
·v
~
10
-·2
65.,
-2
66.
*1.1
X
10
X
10
-1
-2
67.
*4.0
5
3
*Subculture suspens-ions with 10
microconidia per millili·ter
to 10
21
32°C.
This is unlike Bacon and Tre££ers (1961) experiments
with an unstable revertant of E. coli where colonies appeared after 24 hours and continued to appear until seven
days had elapsed.
Twenty-four cultures showed reversion at one week
compared to 42 and 39 cultures after tvm and three weeks
respectively (Table l).
The majority of the subcultures
showed approximately
same number of colonies on serine
th•~
plates at one week compared to weeks two and three, indicating that growth and conidiation have ceased by the
end of the first week.
These results agree with the hy-
pothesis that reversion increases with time and suggests
thc..t it does not
requir(~
cell division.
The :results shown in Table 1 indicate that
.§..~(JBM
4-13) is mitotically unstable, and that it does not revert
at a specific frequency.
from 1.0 x 10
-5
Reversion frequencies varied
to 1.0 in 'different spore isolates.
The
subcultures o£ a given isolate va::.:ied as to whetl1er rever·sion occurred, and as to the frequency at which they
reverted.
Thirty o£ the cultures showed reversion
either two or in all three of the
subcultures~
~n
One hun-
dred five o£ the 306 subcultures showed reversion.
3
Subcultures showing reversion that had 10 to 10 5
microconidia per milliliter in their test-solution are
marked with an asterisk in Table 1.
All 6thers had 106
to 107 microconidia per milliliter.
Those subcultures
tested where the microconidial suspension had 10
3
to 10
5
microconidia per milliliter showed higher frequencies of
.
.
1()6 to 107 m1crocon1
.
"d"1a per
revers1on
than those w1th
milliliter.
The average :reversion frequency for the thirty-
seven subcultures with 10
3
to 10
5
microconidia per milli-·
1
liter of suspension was 1.8 x 10- , while the average fre-
quency of.those with suspensions of 10
3
per milliliter was 7.6 x 10- •
6
7
to 10 microconidia
The average frequency of
2
all subcultures showing reversion is 6.8 x 10- •
However,
3
it should be noted that those subcultures with 10
to 10
5
microconidia per milliliter of suspension did not show a
greater tendency for reversion than those subcultures with
a higher concentration of microconidia.
Only 30 percent of
.
. d.1a per
. .
103 to 10 5 m1crocon1
t h e sub cu 1 tures conta1n1nq
rnillili ter of suspension
rew:~rted
the subcultures containing 10
milliliter.
6
compared to 37 percent of
or 10
7
microconidia per
Thus, the number o:f microconidia in
:::1
culture
has no effect on v?h>=ther or not reversion occurs.
The growth pattern of each culture was observed on
serine compared to minimal slants during the experiment.
On the serine slants the amount of growth did not change
a:fter six days for any of the 102 cultures, but 40 cultures
23
on minimal slants changed from having sparse growth at
48 hours to having heavy growth after either ten or twenty
days.
Twenty-seven cultures o£ this type displayed wild
type colonies within the test system described above.
Thirteen of the cultures which showed this leakiness or
reversion upon standing were cultures which did not show
any rt:Nertants on minimal plates during the three week
Control Experiment.
Thirty-five of the 102 cultures
tested showed no signs 'of reversion during the three week
period.
Even i£ the thirteen cultures which seemed to
revert upon standing were included, there would still be 22
cultures which appeared to be stable.
Ten subcultures of
one of these cul tun:!s were examined for reversion after
.
:f::t.fteen
days of growth.
.
' .
d
A1 1 suspens::t.ons
conta::t.ne
1~ o 6
microconidia per
milliliter,
.
. and three of the subcultures
reverted at frequencies of 1.0 x 10
4.5
X
10
-4
, 2.0 x 10
-4
and
-2
Another apparently stable mutant isolate :reverted
later during an experiment carried out by Elaine Leboff and
Dr. Maxwell (unpublished).
These two results suggest that
all ser(JBM 4-13) cultures might revert if the cultures
were examined exhaustively.
The remaining twenty cultures
24
which did not revert during the experiment were not
tested.
Forward Mutation of the Revertants_.
determine whether the
both directions of
~(JBM
In order to
4-13) locus is unstable in
one colony was isolated from
mutation~
a revertant culture which produced approximately equal
numbers of colonies on serine and minimal plates.
Ten sub-
cultures 6f this culture were then tested for forward
mutation.
The calculated microconidial counts o.f each
subculture tested on m±nimal and serine plates are given
in Table 2.
No instance of total forward mutation to ser
(JBM 4-13) or of extreme ratios on serine versus minimal
plates were observed.
Howe•Jer, the po:::;sibili ty remains
that small percentages o£ forward mutation may occur in
this and/or other revertant cultures.
reversion at ·the ---~·ser(JBM
4-1~
'
locus, seemed to increase
with time apparently without cell division.
The number of
cultures showing reversion increased between the first and
second week after inoculation, whereas the number of viable_
conidia per culture remained constant.
To determine whether reversion depends on cell
growth~
a culture known to be reverting, in a hetero-
25
Table 2. N.icroconidial count.s to test for forv.rard
mutation in one ser (.JBM 4-13) revertan·t.
Minimal
Serine
6
6.0
X
10
8.0
X
10
5.2
x
10
4.6
X
10
6
6.0
X
10
7.0
X
10
5.4
X
10
3.6
X
10
5.6
X
10
6
6
6
6
6
6
6
6.4
X
10
7.8
X
10
2.9
X
10
6.2
X
10
6.0
X
10
6.0
X
10
6
6
6
6.0 ...'" 10
~
6
6
3.4
X
10
5.2
X
10
7.8
X
10
5.0
X
10
6
6
6
6
6
6
26
caryotic state, was grown on minimal medium supplemented
with 0.4 mg/ml serine.
After one week, part of the culture
showed a revertant frequency of 8.5 x 10
-4
.
Another part
was removed from the culture flask and put into a desiccator without nutrients.
Its revertant frequency w<:\.s 2. 8
x 10-3 after a week of· storage.
The frequency of a third
portion of the culture was 2.1 x 10-
3
after two weeks of
incubation on growth medium at room temperature.
These results indicate that reversion is a time
dependent process, which is not dependent on cell division.
Analysis of Revertants
Because only one revertant, B-2, was saved f:com the
.
. .,::
exper1ment
to £'.1n d ou t · 1.1.
I T
2_E.:£,\~
BM
A
"J:-
1?)
-=>
rever t e d at a
.specific frequency and whether reversion was time dependent, more ascospores were isolated from the original eros·ses which had been stored :for approximately eight months.
The plates containing the crosses b.ad been
f'rigerator at 7°C.
l~ept
in the re-
Because of their age, the spores were
soaked in sterile distilled water for one week, a procedure
which has been shown to increase germination frequency of
old spores (Davis and de Serres, 1970).
The germination
frequency of these ascospores was 85.6 percent.
of pe,f'l
+
V<:~rsus p_~
+
£1
The ratio
stocks was as expected ( l: 1) as was
27
the ratio of
~l-2
+
versus al-2_ in the non Re,£l stocks.
However, out of 89 Qe,£1 cultures tested only 29 required
2
serine (x =10.78, £=1,
p<
0.01).
Whether or not this di-
vergence in the serine ratio was caused by the soaking of
the spores or whether reversion had occurred in the spores
upon standing for eight months has not yet been determined.
Of the 29 serine stocks, 16 were tested for rever-
tants after fifteen days.
Only one showed reversion, while
the other thirteen were tested at 22 days with four showing
reversion.
These frequencies are considerably lower than
those observed when the ascospores were young.
servation combined with the unexvected ser + : ser
This ob.
rat~o
obtained with these old spores suggest that reversion may
occur in aged spores as well as in conidia.
Three revertants, 9, 81, 65, were selected and were
repeatedly reisolated from plated mic:x:ocon:Ld:ia to insure
that each one was a homocaryotic culture.
Revertant 65 showed a
g~eater
proportion of coloni.es
on serine compared to minimal plates eve.:-1 after a series o:f
reisolation attempts.
For this reason, it was not used in
the experiments designHd to evaluate the type of revertants
arising from
~E.(.JBM
4-13).
28
Comparative Growth
St~ie#s.
Giles (1951) compared
the growth of revertants of inositolless and methionineless
mutants on unsupplemented medium with the growth of the
wild type culture in which the mutants had been induced.
Revertants due to suppressors showed retarded growth curves
compared 'INi th wild-type, although the mutants eventually
attained dry weights equal to the wild-type; while back
mutation revertants gave the same growth curve as the wildtype culture.
Assuming that such differences would characterize
the different classes of revertants derived from the
serineless mutant, the growth of reverta.nts 9 c\nd R-2 ·was
compared to that of a}:_£; cot-l as a f"unction of t:ime.
Revertant 81 was not used because it produced few microconidia.
The results of this study are shown in Figure l.
Revertant 9 grows at the same rate as revertant B-2 after
.a. five day lag period.
Neither displays the growth curve
of a,~~; co!..:l. on minimal medium.
The growth curve o£
revertant 9 resembles that of a leaky serine auxotroph.
It should be noted that
_?~(JBM
4···13) does not grow in
liquid Vogel's minimal medium even after six days of incubation (Kline, 1974).
Based on Giles' work (1951) neither
revertant appears to be due to a. back mutation.
29
Figure 1.
Comparative growth curves of _al-?,; sot-1 and
two revertants as a function of time.
Cultures were
grown at 25°C in 125 ml Erlenmeyer flasks containing 20 ml
of Vcgei's minimal medium with two percent (w/v) sucrose.
Each point on the graph represents the average dry weight
of three mycelial pads.
30
ai-2 cot -1
revertant B -2
revertant .9
6
llo,.,...--:Lt--==c-..,.J&..--==---••-'gjl,..,-...ll,i,-_.....,n•~-=~
1
2
3
4
5
6
DAYS
7
8
9
10
31
revertants were crossed to both A,
(Cl02t) and 25a.
~(15300);
cot~~
Culture 25a was used because the a-mating
type of al-2; cot-1 could not be obtained from the Fungal
Genetics Stock Center (Humboldt, California).
The first set o:f mating experiments were incubated
at
zs°C.
Only a few ascospores were dehised from one of·
·the plates where revertant 81 was the protoperithecial
parent and
25~
was the conidial parent.
Most o:f these
spores were immature, clear or brown in color, and had low
. germination f·requencies, ranging from 16 to 43 ~4 percent.
·The· results o.f these ascospore cultures are marked by an
asterisk in Table 38.
None of the other crosses matured.
When the crosses were rep(;')ated at room temperature
in the dark, all the crosses were successful.
Revertant
9 crossed to 25a dehised no spores on the coinoculated
plates and very few spores were dehised on the plates
where they
>.'v·e:ce
lE;ed
as
separate parents.
Spores
were
0
analyzed from the following crosses: revertant 81 + x
25a
d' ,
B-2 rJ'
0
25~ + x revertant 9 ~}
and
25~
0
+ x revertant
Only an occasional immature, colorless or b,rown
ascopore was seen on the plates.
According to the anal-
ysis shown in Table 3B both revertant B-2 and 81 seem to
be due to back m.u tat ions at the
,.;?_~::::_(
JBM 4·-13) locus or
Table 3Ac
Plate
Number
Distribution of 1'4arkers from Ser (J~! 4-13) x
# of spores
isolated
# of spores
Total ~~ fl
. germi!l. ( fr~q)
cultures
~~
.E§_,_,
fl·
_fl: cot-):.•
__
ser
cultures
al-2+ : al-2
A
150
99 (66. 0%}
45
21
26 : 27
A
~50
117 (78. 0%)
52
32
36 : 29
B
150
.l.
.. 2·-2. {81 ~ .j'"'%'
o)
46
18
44 : 32
B
150
104 (69.3%)
44
24
25 : 34
c
150
129 (86.0%}
63
32
32 : 34
c
150
132 (88. 0%)
71
34
30 : 31
C*
15Q.
__99 J±6. 0%)
13
__§_
23 : 33
1050
772 (73. 5%)
'334
167
Totals
*
216 : 220
heat shocked too long
w
t.J
Table 3B.
Distribution of Markers from Revertants x 25a
# of spores
Revt #
isolated
# of spores
_?,Ermin. ( fre_g)
cultures
.:tes_te(j__
+
+
se.r
p_eLf_l__~p§:_.
fl
cot+ :cot
+
a1 : al
81*
288
47 (16. 3%)
47
0
41:6
22:25
18:23
81*
189
82 (43.4%)
82
0
66:16
40:42
36:30
81_
108
§7 {80. 5%)
83
0
29:54
47:36
11:18
Total 81
585
216
212
0
136:76
109:103
65:71
9
216
75
(34~7%)
73
38
42:.31
43:30
25:17
9
135
_:1.5 (33.3%}
36
ll
30:6
---
18:18
15:15
Total 9
351
:t20 (34. 2%)
109
59
72:37
61:48
40:32
To·t:al B-2
216
174
0
89:85
81:93
41:48
*
189 (87.5%)
Results from the first experiment
L0
t,IJ
due to a closely linked suppressor gene.
Revertant 9 on
the other hand shows a ratio very similar to what is
expec·ted if a cytoplasmic or enviromnent2.1 factor is causing the apparent reversion.
ThE=re is some discrepancy
between the results of this experiment and the results
obtained from the growth comparison study between the revertants tested and
~l.::_~;
co!.=..!_•
The growth curve of
revertant B-2 looks like that o£ a suppressed mutant;
however, its genetic analysis revealed no serine requiring
progeny.
· d ely
W~
·
d~vergent
·
rat~os
nPe+, f]_·tof ee,fl versus .t:..::..
were observed in cultures tested from revertants 81 and 9.
There was no divergence from the expected ratios of
ers when
cot-1.
~(JBM
mark~·
4·-13) was originally crossed to E.§., fl 1
The difference in the results can be seen by com-
pa:r·ing parts A and B of Table 3.
None of the other
phenotypic characters showed abnormal segiegz.tion patterns
in any o£ the .r.ever·tant crosses.
DISCUSSION
Discussion of the Results
Apparer.!_
Meioti~
5_!.:-:!?..i.l.i:J:Y
~£
Ser
(~1BM
4-13).
Ser
(JBM 4-13) appeared to be meiotically stable as shown by
its normal segregation in ma. ting experiments, but became
Ini·totically unstable in vegetative cultures.
Sometime
·during .the development ,of the cultures and stable state of
~(JBM
4-13) and/or its environment chan9ed so that re-
version could occur.
known.
The cause of this change is not
A similar phenomenon was described by.Drew Schwartz
(1960) in his study of- the mutable ~rn gene in maize.
This
gene is stabilized after fertilization, and not until
sometime during the life cycle does it change to the un_stable state.
The idt--:a that "st.3.tes" of a gene can
deterrnine v,rhat e.ffects certain controllin9 elements have
on the expression of a locus has been advanced by
McClintock {1965).
Cellular environments may also play a
role in mutational states of a gene, as has been suggested
by Lefevre and Green (1959) with respect to the forked
locus in Dro2_9phila.
35
36
High Reversion Frequencies.
Other investigators
have not reported reversion frequencies as high as the ones
shown in this study.
Perhaps in the past mutants exhibit-
ing such high frequency reversion were discarded as being
too unstable to study.
in several
sy~tems
Frequencies of 10-
4
have been noted
by various investigators including
Giles (1951), and Barnett and de Serres (1963) in Neurospora: Gundersen et al.
(1962) and Allen and Yanofsky
(1963) in E. Coli; Lefevre and Green (1959) and Sheldon
et al.
(1969) in Drosophila; and Strigini (1965) in
bacteriophage.
of Conidial
~£Eulations.
In the reversion experiment sub3
cultures which contained 10
to 10
5
.
.
.
m:Lcrocon:.tdJ..a. per
milliliter showed higher frequencies of revertants than
subcultures containing 10
liter.
6
I... ietJ
7
to 10 microconidia per mill.ia similar
~elationship
between
the increase in culture size and decrease in spontaneous
reversion rate when histidine mutants in E. coli were
grown on excess histidine.
Examination of the data shows that the number of
colonies found on minimal plates is not proportional to
the conidial population size within the range
.,-:~xamined
37
( 103 - 107 conidia per ml o£ suspension).
On th~ contrary,
the average number o£ colonies found on plates inoculated
3
5
with cultures having 10 to 10 microconidia per milliliter
was of the same order of magnitude as cultures with 10
7
10 microconidia per milliliter.
6
to
Thus, the chance that
revertant nuclei will be found in a culture is independent
of conidial population size.
This result and the observa-
tion that whether or not reversion occurs
.
lS
.,
a.1.SO
indepen-
dent of the conidial population size leads to the conclusion that reversion is independent of population size.
!_i.me Dependence of MUtation.
Several observations
made during this study suggest that the high frequency
reversion of ser(JBfvJ 4-13) is time dependent.
First, dur-
ing the three week testing period, 24 cultures showed
reversion at one week compared to 42 and 39 cultures which
included revertants at weeks two and three respectively.
Second, the results obtained from the cell growth. study
show that reversion increases with time.
Also, upon exam-
ination of growth characteristics on minimal slants, 40
cultures changed from having sparse growth at 48 hours to
having heavy growth either ten or twenty days later.
Accumulation of recessive 1 etha 1 s i:tn1----rd'h1-.y..-..c..,-onn...-x+·-r'dt-;i~a:r---------of Neuroseora (Auerbach, 1959) and accumulation of
38
revertants o£ certain rii mutants during storage in
bacteriophage T4 (Drake, 1970) were observed to increase
linearly with time.
in the
Although reversion is time dependent
~(JBM 4-13)
increases
line~rly
mutant, it is not known whether it
with time.
Whatever factor may cause the instability at the
~(JBM 4-13)
locus, is reminiscent of time dependent
phenomena associated with McClintock's (1965) controlling
elements.
Reversion versus Cell Growth.
It is well known
that microconidia, when suspended in water without nutrients, die relatively r.;.pidly at room temperature.
How-
ever, in order to determine whether reversion could occur
without cell growth, part of the culture had to be stored
without growth nutrients.
this study.
A new procedure was used during
Part o£ the culture was kept in a sterile
0
petri dish at 5 C t o 7 °~
·- ~n
... a
· t ure ....~ ree
mo~s
h ere~
+
a~.mospJ
No adverse effects were noted using ·tb.is procedure.
Reversion was found to be independent of cell
growth.
The part of the culture which was grown on sup-
plemented media for two weeks gave a reversion frequency of
2~1
x 10
-3
, while the part which was grown for one
.~eek
and then transferred to storage showed a frequency o£
2.8
X
-3
10 . •
The initial culture tested after one week of
growth had a reversion frequency of 8.5 x 10
Other investigators have, in
-4
addition~
tested
whether DNA replication is necessary for mutations to occur.
Drake (1970) working with bacteriophage T4, Auerbach
(1959) working with NeurosDora, and Ryan and Nakada (1961)
studying E. coli came to the conclusion that during storage
or starvation, mutations do occur without gene replication.
Ryan and Nakada {1961) proposed that minute amounts
of DNA may replicate, or minor and selective base sub-
stitution in DNA may take place in non-dividing cells.
Another possibility that can be considered was first suggesi:ed by Muller (1954) and reite.·rated by Auerbach (1959).
Perhaps mutation does not occur in non-dividing cells, but
storage increases the chances that mutation will occur at
the first cell division after storage.
Colonv .Size Variation.
Demerec and Cahn (1953)
suggested that the difference in colony size observed on
plates in mutation experiments might mean that reversions
are a very heterogeneous group.
This has been proven by
Dawson and Smith-Keary (1963), Bacon and Treffers (1961),
and Helinski and Yanofsky (1963).
This possibility exists
in this study because variations in colony size were noted,
40
and revertant 9 gave results unlike those obtained with
B-2 and 81.
However, all colonies appeared on the plates
0
between four to six days at 32 C, which does differ from
some investigations carried out in other systems.
Evidence Rulinq Out the Presence of Gross Chromosomal Rearrangem.ents.
The £ollow·ing points suggest that
no gross chromosomal rearrangements are present
cultures bf
1.
~(JBM
When
~n
the
4-13).
~(JBM
4-13) was mapped, the linkage
relationships among other markers in the
region were within an acceptable range (Kline,
197 4).
2.
There was no ev:5.dence o£ spore abortion.
Very
few brown or colorless ascospores were observed.
3.
The germination frequencies
ra..nge~d
from 66
to 88 percent.
4.
The ser_(JP.M 4-13) marker showed normal seg::-:-ega·tion.
Properties o£ the Ser _{JBN 4-13) Mu.tan_!_
Any model proposed to explain the reversion phenom··
enon in the
~(JBJ\1
4-13) culture must take into account
the following properties:
41
1.
§er (JBM 4-13) is apparently meiotically
stable.
2.
Sometime during development
~(JBM
4-13)
becomes mitotically unstable and highly
revertible.
3.
The reversion phenomenon is time dependent.
4.
Reversion when it occurs is not related to
conidial population size.
5.
Reversion is not dependent on cell division •.
6.
There is no evidence £or the presence o£
gross chromosomal rearrangements.
7.
§..~(J"BM
4-1.3) does not revert at any specific
frequency.
Individual progeny (and sub-
cultures o£ individual progeny) carrying this
allele show different reversion frequencies.
8.
Ser(JBM 4-13) maps at a specific site like
a genetic factor.
9.
Two of the three revertants (81 and B-2)
studied behave like ba.ck muta.tions or closely linked suppressors.
Models to
S~lain
Reversion
Recombination or Sister Strand Recombination.
----'0·
"
. ---------
Di:f-
£erent types o£ recombination have been propos·ed as models
42
to explain the mechanism of back mutation.
Normal re-
combination rather than a mutational model, was proposed
by Lefevre and Green (1959) in Drosoohila, and shift mutations caused by unequal crossing over were proposed by
Strigini ( 1965) to e)q)lain the high reversion in bacteriophage.
Recombination as a mechanism to explain the ser
(JBM 4-13) system would be x·uled out i:f ser_(JBM 4-13) is
demonstrated to be meiotically stable, and the reversion
occurs only in the vegetative (haploid) state o.f the
organism.
Other investigators like Bo\vman ( 1965) and Grigg
and Sergeant (1961) have suggested that revertible loci
may consist o£ multiple duplicated segments, and unequal
sister strand recombination causes the reversion.
So far
no one has demonstrated that sister strand exchanges can
take place in Neurospora, but this possibility exists.
Extrachromosomal Determinants¢
that
~(JBM
The possibility
4-13) is an extrachromosomal mutant can be
discarded since it maps like a genetic factor, and it shows
normal segregation in crosses.
If the factor causing the
revertibili ty can be separated from the
~er
(.JBM 4-13) gene,
the possibility remains that it is a cytoplasmic trait,
since the ascospore cultures analyzed came only from
crosses where
~(JBM
4-13) was the protoperithecial
parent.
Suppressors.
No evidence for suppressors was ob-
tained from genetic studies of the revertants.
Possibly
in the future revertants from ser: (JBl\1 4-·13) may be found
which are due to unlinked suppressor mutations, or a closely linked or intragenic suppressor may be found upon
further examination of the revertants already analyzed.
A word of caution is offered by Allen and Yanofsky
Helinski and Yanofsky (1963) and Brink et al.
studied second site reversion.
(1963),
(1969) who
Complex rever.·sion patterns
are sometimes present in revertants thought to be strictly due to back mut&tions.
Revertants which appear to be
identical by genetic ·analysis may be very diverse in their
properties depending upon the kinds o£ tests used
~n
their
classification.
Mod~·0!2.....E){}>_}air.2_
1
s
Gene _IE.staJ:?J:.l i.!J::··
Single Base-pair Altera_'f:io!22..•
Several investi-
gators working at the molecular level have suggested that
reversions yielding either stable or unstable products are
brought about by base incorporation mistakes in the DNA.
Freese (1959) proposed a model of mutation based on the
observation that mutants
induct~d
by base analogues could
44
also be induced to revert by the same base analogues whereas spontaneous or pro£lavin induced mutants were not
a:f:fected by the base analogues but they could revert spontaneously.
The type o:f mutation which is unstable in the
presence o:f base analogues is due to a transitional change
whereby a purine is substituted :for a purine, and a
pyrimidine by another pyrim.idine.
The second type where
base analo9'Ues have no effect in producing reversions is
caused by a tra.nsversional change, the substitution o:f a
purine :for a pyrimidine and vice versa.
Freese also sug-
gested that the pairing mistakes of the second
t}~e
may be
increased by an unbalanced nucleotide pool caused by certain kinds of starva·tion.
forth by Ryan et al.
This hypothesis was also put
(1961) in their paper about the inde-
pendence o:f spontaneous reversion :from DNA replication.
Barnett and de Serres (1963) also based their model of
spontaneous gene instabili-ty :found at the ad-3 locus in
---
-
Neurosnora on
Frr~ese
1
s hypothesis.
They proposed th?,t the
instability at this locus is due to complementary
transitional changes
(TA~--:;
CG)
*
It is possible to envision a model similar to
Freese's to explain reversion in 2£E.{JB!Vl 4-13), but :further
studies would have to be done.
45
Other investi9ators including Bridges, Dennis and
fvlunson {1967) have suggested that only mutants containing
chain terminating codons revert to prototrophy at high,
easily measureable rates.
The main premise behind this hy-
pothesis is that several chain terminating suppressors,
or the codon itself, can undergo reversion by base sub-·
stitution changes.
So far, there has been no indication
of suppressors affecting the ser(JBM 4-13) mutant.
2.
Controlling FactoS2.·
All of the following
factors have one property in common.
They modify the ex-
pression of the gene under their control and/or increase
·its rate of mutation.
Perha_ps they a.re all similar ele-
. mcnts with different capacities for control depending upon
the systems with which they are affiliated.
Mutator genes can be mapped
at specific loci unlike controlling elements or episomes.
,The mutator gt=mes desc:.cibed by Bacon and Trf:;f'fers (1961)
and Siegel and Kamel (1974) are very specific in their mode
of action upon the particular gene they cause to rever-t.
For example, the rout
the transversion of
T.~.
gene found in E. coli induces only
adenine~-thymine
(Siegel and Kamel 1974).
to cytosine-guanine
Mutator genes do not necessarily
direct their specificity to only one gene in the genome.
46
Many different types o£ loci may come under the control of
a mutator gene and in general,- mutators can increase both
directions o£
mutation~
Here again, this model cannot be ruled out at the
present time.
A closely linked mutator gene may be pres-
ent, controlling .2_er(JBM 4-13) reversion.
bility of
~_(JBM
B)
Also the sta-
4-13) revertants is not knownQ
§Eiso~_.2..·
Episomes are thought to be gen-
etic elements that can exist in either an autonomous or
integrated state.
Map locations may vary because even in
the integrated state many of them. do not have fixed positions.
Episomes are also subject to a high rate of loss
from the system. they are operating in, and the loss is
permanent.
Dawson and Smi th-Keary (1963), Hill (1963),
and Schwartz (1965) proposed that episomes modifying the
e}~ression
o£ a suppressor were responsible for the un-
stable revertants found in their studies of Salmonella and
E .. coli.
A. suppressor modifies the activity of the mutant
without coi·recting the original mutation.
In these cases
an episome either integrates at the suppressor site or
turns off suppressor activity in another way so that the
. mutant phenotype reappears.
Gundersen~
Jyssum and Lie (1962) have studied a
mutator factor possessing the prope:r.'ties of an episomeQ
47
The action of this :mutator is specific for the Sm locus,
unlike mutator genes in general.
An episome could explain the
if the episome acts directly on the
~(JBM
4-13) system
~~(JBM
4-13) gene,
inactivitating i t and thereby producing the mutant phenotypeo
If the_ episome became inactive or detached, normal
activity would then be restored.
C) Controlling Elements.
Controlling elements
have been studied mainly in maize by McClintock (1965) ..
Controlling elements move around (transpose themselves in)
the genome, thereby placing different loci under their
control.
The well-known !>-c-·Ds regulato:ry system works by
the insertion o£ the Ds (operator-like) element at a gene
locus which then comes under the control of the Ac (activator) regulator elemente
an active or inactive
The Ac element can be in either
state~
When Ac is inactive, it
produces no signals, so that the Ds element does not
initiate any change at the associated gene locus.
Both Ac
and Ds elements have been shown to become fixed at specific
genetic loci.
"Vv'hen the Ds element becomes fixed, it is
still under the control of the Ac element and produces both
forward and back 'mutations in adjacent genes in response to
Ac.
When the Ac element becomes fixed, the a.f.fected gene
48
is put under total control o£ the Ac system, and not until
the Ac element is removed does the restoration o£ the gene
locus result.
Another important feature of the Ac-Ds sys-
tem is that some unknown mechanism de·termines when the Ac
regulator element produces its signals during the development o£ the organism in question.
These signals can either
change the action o£ a gene, o:c remove the gene completely
from the system's influence.
It has also been noted that
an allele of a gene can exist in different "states" which
are more or less receptive to the regulator's instructions.
Other more complicated systems, like the Spm system are
also known in maizeo
Sheldon, Hendel and Finlay {1969) explained the
high revertibility of the sc locus in Drosophila on the
basis of McClintock's models
They suggested that the wild
type allele, or the operator of the allele, was being held
partially inactive by
som~
type of regulator particle.
A model where a re9ui a tor element is :fixed at the
~JJBM
4-13) locus, or controls it (like Dt at the A
locus) could
E~xplain
what is happening in this systemo
This model could account for
~(JBM
as the change in stability of the
4-13) properties such
~(Jl3M
4-13) gene, the
time dependence of the reversion process, and the independence of the reversion process from
po~ulation
size and
49
cell growth.
It is also possible that the abnoraml be-
havior seen at the
~
or £1 locus upon crossing revertants
9 and 81 to 25a
is due to a transnosition
of a regulator
c
element.
However, this behavior may stem from factors
introduced into the system when the revertants were crossed
to 25a for their genetic analysis.
D) yiruses.
Baumiller (1967) studied the in-
duction of sex-linked recessive lethal mutations in
Drosophila
by a Sigma virus.
me~c:-nogaster
Similar studies
involving bacteriophage induced mutation were carried out
by Taylor (1963).
prophage state
He found that when phage Mul l in its
inse~ted
itself into or next to a gene, the
gene's normal activities were completely abolished.
In both of these studies the recessive lethals or
mutations produced by the viruses showed no tendency to
revert to prototrophy.
I£ a virus can be specific in the
direction o£ forward mutation, perhaps there are viruses
which can induce revE:rsion.
Further Investigation of the Ser (JBM 4-ll..)_Mutant
Many questions remain in the study o£ the revertibili ty o£ the
~::.:.(JBJ.VI
4-13) locus.
The models to explain
tlH?. revertibili ty are numerous: and some models contain
variables which must be taken into consideration.
The
50
cell's DNA replication and errors in the same, the genetic
and cytoplasmic backgrounds, the different states of the
gene, and states of controlling factors (if they are
present) all should be examined.
Future investigation o£
~~(JBM 4-13)
should include
the following experiments:
1~
Is the reversion phenomenon characteristic of
all
~(JBM 4-13)
progeny?
An experiment of the same kind as was done in
this study should be carried out, and all progeny should be
tested extensively to see if there is any background where
the
~r_(JBM
4-13) gene is stabilized.
4-13) cultures can be isolated,
If stable ser(JBM
extragenic factors may be
the cause of reversion.
2.
_t\re
.§_~(JBM 4-~13)
revertants stable?
The three revertants obtained from these experiments and several other revertants should be tested to see
i£ they £onvard mutate to s er (JBM 4--13).
This can be done
by plating revertant cultures on 0.4 mg/ml L-serine plates
at a dilution where overcrowding does not occur.
Indi-
vidual colonies should then be tested for !2._er (J"BM 4-13)
mutants by transferring each colony culture to minimal and
minimal containing serine media.
New ser would then have
to be tested for allelism by crossing and/or complementa-
51
tion.
This is a long and tedious experiment, :for filtra-
tion enrichment procedures do not seem to work properly
for isolating serine mutants.
3.
Is the instability a.t the
~-(JBM 4-13)
locus
caused by an episomal factor?
A microconidial suspension of a homocaryotic
~E_(JBM 4-13)
culture grown on solid 0.4 :mg/ml L-·serine
should be inoculated into liquid growth medium containing
~~g
of acriflavin per milliliter, and into a control tube
containing only the liquid growth medium.
Acriflavin has
been observed to eliminate episomal factors from other
organisms (Gundersen £.!_
are incu.bated
overnight~
..§-.1.·
1962)
c
After these cultures
the microconidia should be
centrifuged, resuspended in sterile distilled water, diluted and plated on serine medium.
Individual colonies
from both the control and the experimental plates should
be tested for reversion.
4.
Is the reversior1 caused by a closely linked
or intragenic suppressor?
An analysis of the revertants crossed to a wild
type strain should be carried out, looking for
4-13) mutant recombinants.
~(JBM
For this study the revertants
should have closely linked markers on each side o£ the
2~r+(,JBM 4-13)
gene~
5.2
5.
What is the nature o£ the original lesion
induced by UV at the
~(JBM
4-13) locus?
The spot plate method used by Balbinder (1962)
in his reversion study of _!;ry C and trv D in Sa..lmonella
typhimuri'l!:.!!!. could easily be used in qualitative reversion
studies in Neur_222ora with few modifications.
Depending
on the different mutagens used, it should be possible to
determine whether the original lesion was a gene deletion,
f·rameshift,
transversion, or transition.
A conclusive
study of this type was done by Brown and Aiuto
met-1 alleles in
NeurosEo~a.
crassa.
(1973) on
LINKAGE DATA ON A NEW SERINE
REQUIRING MUTANT : SER-5
53
I
INTRODUCTION
This investigation was carried out to determine
the precise chromosomal location of a serine requiring
mutant isolated by Dr. Joyce B. Max-well.
Preliminary
results indicated that the new mutant, ser-S(JBl\1 9), was
located on linkage group III.
The question remained as
to whether it was allelic to ser-l(H605), a mutant isolated
by Hungate (1946), on chromosome III, or whether it is
located elsewhere on the chromosome.
54
MATERIALS AND METHODS
-------~--~~~~~~~
Strains
_?er·-S(JBM 9) was one of f'ive serine requiring mutants induced in the nutritionally wild-type strain A,
al~(l5300);
cot-l(Cl02t).
The mutants derived from this
strain were isolated by filtration enrichment (Woodward
et
~·
1954) following ultraviolet irradiation to twenty
percent survival (two minutes) under an ultraviolet
Westinghouse Sterilamp, model 7821-30.
~;
A sexual reisolate,
_2er -5 (JBM 9); cot -1 {Cl02t) was used in the mapping ex-·
peri.ments and was obtained by crossing A,al-2(15300);
ser-5(JBM 9); cot-l(Cl02t) to FGSC #333;
in1J37401);
~!_(Y30539y);
~;
cot-l(Cl02t);
nt_(C86).
The two stocks used- to localize
link:a.ge grcup ITT ·were FGSC #190: A;
_;:;~_F-5
(JBM 9) on
~(5801) ~
trp-1(10575)
and FGSC #116: A; ser-l(H605).
Maintenance o£ Cultures
All ser-5 and ser-1 vegetative cultures were maintained on solid slants of Vogel's minimal m.edium N (1956)
supplemented with Oe4 mg/ml L-serine.
55
The.§_£, tx:p-1_
56
culture was maintained on Vogel's medium supplemented
with 0.1 mg/ml L- tr.yptophane.
All
cbemi_cals_u_s_ed~·_..,w._..e..._..r"-'e-=-_ __
reagen·t grade quality, and the amino acids were obtained
from Calbiochem, San Diego, California.
Ser-5 x Ser-5
A control experiment was done to see i£ the ser-5
stock was homocaryotic.
{15300);
_§_~.E..:.:?_( 3BM
sexual reisolate
The original isola.te A, al-2
9); cot -Jj Cl02t) was crossed to the
~; -~~r-S(JBM
9); cot=l(Cl02t) on
Westert;Jaard-M:i.tchell (1947) crossing medium supplemented
. '
with 0.2 mg/ml L-·serJ..ne"
Random ascospores were isolated
£rom one plate two months after the initiation o:f the
cross.
The ascospores were spread onto four percent aga1=,
and individually transferred to small tubes (12 x 75Il1I11)
containing 0.4 mg/ml serine medium.
In order to induce
germination the spores were heat shocked in a waterbath at
0
60 C for one hour.
Germination
24 and 48 hours later.
£requenc~es
vvere recorded
F'henotypes among the spore isolates
were determined six days after the ascospores had
ger:min~·
ated, by transferring each culture onto minimal and minimal
containing serine media.
Growth.was recorded after 48
hours.
Ser-5 x Ser-1
In order to find out i:f
~.r.:J?~
was allelic to _§,er-1,
~;
ser-S(JBM 9); cot-l(Cl02t) was crossed with FGSC #116
A; ser-l(H605) using both cultures as separate parents.
The procedures used for isolating and testing the progeny
are described above.
Ser-5 x sc,tro-1
More precise mappin9 of ser-5 was carried out by
crossing~;
ser-S(JBM 9); s_ot-l(Cl02t) to FGSC #190:
!£2~1(10575).
{5801),
:th;~
The first time the cross was set up
each culture was used as the protoperithecial (female)
parent and as the conidial (male) parent on different
plates, and other plates were coinoculated.
The Wester-
gaard-·f....U tchell crossing medium was supplemented with
mg/ml L-serine and 0.1 mg/ml DL-t:ryptophane.
0~2
The second
time the cross was set up all pl2.tes were coinoculated and
were supplemented with 0.1 mg/ml L-serine and 0.1 mg/ml
L-trYPtophane.
All crossing plates were kept at room
temperature in the dark.
Ascospores were
spre~d
onto four percent agar plates
from crossing plates which varied in age from 26 days to
79 days.
The spores were then transferred individually
to small tubes containing Vogel's medium supplemented with
0.4 mg/ml L-serine and 0.1 mg/ml L-trYPtophane.
To induce
germination some ascospores were soaked in sterile
58
distilled water before spreading onto four percent agar
and all spores were heat shocked in a water bath at 60°C
for one hour after isolation into individual tubes.
Ger-
mination was checked after 24 to 48 hours.
After two weeks each culture was transferred to
four separate media: minimal alone, minimal containing 0.4
mg/ml serine, minimal containing Del rng/ml L-trYPtophane,
and doubly supplemented serine-try·topha.ne media.
The
characteristic markers of each culture were recorded after
48 hours of incubation.
cot-1.
The cultures were not tested £or
RESULTS AND DISCUSSION
The :following crosses were done in order to localize
the position of
~-.5
with respect to other markers on
linkage group III in l:Jeurospora
~~·
A control experi-
ment was set up to see if the ser-5 culture was homo··
caryotic and stable.
The control experiment between the two ser-5 cultures gave an average germination :frequency o:f 4.5 percent.
This low :frequency is not unexpected :from an allelic cross.
All o£ the 56 germinated ascospores tested showed the
auxotrophic character"
~5
Although there is inconclusive
evidence :for the absence o:f wild-·type nuclei, the cultures
were presumed to contain only
~r-.5
nuclei.
Once it was established that ser-5 was located on
linkage -group IJI ,, the possibility v,ras exa:r.;_ined 'IJvhe·ther it
was an allele o£ ser-1.
To test this nossibility a; ser-5
'
--
(JBIVI 9); cot-l(Cl02t) was crossed to FGSC #116~ A; -~~
(H605)
e
The germination :frequency _o:f isolated ascospores
ranged :from 23.7 to 86.0 percent.
O:f the 348 spore cul-
tures tested, 23 were prototrophic with respect to the
serine requirement, giving a map distance o£ 13.2 units
59
60
between ser-5 and ser-1.
I f the data from the small group
of spores showing the germination frequency of 23.7 percent
were ignored, leaving those with frequencies of 53 to 86
percent, 20 cultures were wild-type out of 330 cultures
tested.
This gives a map distance to 12.1 units between
----
.ser-5.... and ser-1.
Further localization of the ser-5 gene was carried
out by
crossing~;
~:..~(JBM
A; ss(S801), trp-1(10575).
9); cot-l(Cl02t) to FGSC #190:
The~'
trp-1 stock was chosen
based on the map distance established between
~!..::2.
and
Since there are only ten to twelve map units to the
ser-1.
left .of .§_er-1, it was considered much more likely that
sAr-5 was located to the right of ser-1 than to its left.
The crossP.s weie set up using each culture as
separate parents on some plates and coinoculating the other
plates.
where
The coinoculated crosses as well as the crosses
_2er-~
wa.s the protoper:i thecial parent dehised spores
ver:;l well, a.nd nc immature. spores were seen.
where
~'
~trp-1
W'.iS
The crosses
used as the protoperithecial parent
never matured and very few ascospores were ever dehised.
Perhaps the morphological mutant sc cannot :form £"unctional
peri thecia.
The germination :frequencies o:f this cross showed
a bell,-shaped distribution with regard to the age of the
6l
plates as seen in Figure 2.
Random ascospores were iso-
lated £rom the crossing·plates which varied in age £rom 26
to 79 days.·
Spores isolated :from coinoculated cultures at
26 days gave a 68 percent germination frequency compared
to spores isolated between 32 and 39 days where the frequencies ranged from 80.6 to 92.3 percent.
Even when the
spores were soaked, spores over 39 days old showed decreasing germination frequencies declining to 11.8 to 18
percent after day 64.
The average germination frequency
of the 1041 germinated spores tested was 63.5 percent.
However, the majority o£ the data used .for mapping· came
.from 905 germinated spores which were 39 days old or
younger and showed an average germination frequency of
80.1 percent.
The results o£ the crossover data are shown in
Table 4.
The calculated map of linkage group III based
on the values shown in Table 4 is illustrated in Figure
3.
Figure 4 sliOws the published map distance (Radford,
1972).
The experiraental linkage data on
~er-5
and linkage
data on ser(JBM 4-13) has recently been published in the
Neurospora
Newsl~tter
(rvraxwell et
a~~
1974).
62
Figure 2.
Distribution o£ the germination frequencies
shown from the cross ~; s~r-S.(JBM 9); cot~·l (Cl02t) to
FGSC #190: A; ES,_(580l), trJ2...:1:.(10575), as a £'unction of
the age of the ascospores tested.
63
.o~
\
:z
C)
t-
cc
\
z
:E
"
<l>
1M
~
\
....
z
1.11.1
y
"'
Idol
A.
\
20
0
~·
G
30
10
AGE
0 F SPORES
so
(DAYS)
70
90
Table 4.
Linkage data.on random ascospores from the cross ser-5 x sc, gp-1
recombination
zygo·te
geno·type
sc
+
+
ser-5
25.4
parental
trp-1
cornbina tion
single
region I
374
131
393
130
+
single
region II
double
.region I & II
% germination
7
0
1041
3
3
total
63.5
1.2
·------------·--··
()\
~~
65
Figure 3. Calculated map of linkage group III based
on data shown in Table 4.
sc
ser-5
trp-1
-I
~~-----:--- 25 "4 ------~~-·~-}
l-le2----l
Figure 4. Published map of linkage group III
{Radford 1972).
trp-1
-:iww
l
19.0 ------~---·---1
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66
6'7
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.•