CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
AN ANALYSIS OF SPONTANEOUS REVERSION
IN CONIDIAL SPORES AND ASCOSPORES
OF NEUROSPORA
CF~SA
A thesis submitted in partial satisfaction of the
r~quirements for the degree of Master of Science in
Biology
by
Edward Vincent Koprowski
/~
January, 1977
,---------~---·------~--------------------~~-~--~----~----~-"
!
Koprowski is approved:
The
t
,
eth
}
~B.
c. J~
Maxwell, Committee Chairman
California State University, Northridge
l
!
~----.--~----. --.~------..-~-------------~-------------~--------~----·----------····-~·------··-·--·J
ii
------ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - · - - - -
ACKNOWLEDGEMENTS
I would like to express my sincere appreciation
1
to Dr. Joyce B. Maxwell for the guidance and insight that
I
I
;she has provided during this thesis project, and to Dr.
Donald E. Bianchi and Dr. Kenneth
c.
Jones for serving as
!members of my graduate committee.
I would especially like to thank my entire family
for their help throughout my scholastic career.
i
I
L_________________ ------------------------------------------------------------~
iii
TABLE OF CONTENTS
: ACKNO\ii7LEDGEIYIENTS
i
LIST OF TABLES
iii
.
vi
'LIST OF FIGURES.
vii
LIST OF PLATES
• viii
;
;
I
'ABSTRACT
ix
I. INTRODUCTION •
1
II. MATERIALS AND METHODS.
Conidial Studies
4
4
•
Strains.
4
Maintenance and Growth of Cultures
5
Reversion Studies.
5
Isolation of Revertants.
•
Conidial Survival.
6
Ascospore Studies.
Strains.
6
7
7
•
Maintenance of Spores.
8
Analysis of Reversion Frequency.
9
Freeze-Drying.
-·
10
11
III. RESULTS
Ser, Met Reversion Studies
11
Ascospore Studies.
25
'L--------~--~~-- -------- - - - - - - - - - - - - - - - - - - - - - -----------------~------
iv
'
-------~----------
--~-------~---··--------------~-------~------------------·-----~----·-·-~----~------·----------------------------~
Control Experiment •
•
•
The Drying Experiment.
·Ascospore
>
37
IV. DISCUSSION •
Met
-Ser, --
32
50
..
Reversion Studies
Studies~
50
53
The Drying Experiment.
•
59
The Status of Ser (JB!-1 4-13) •
..
60
V. BIBLIOGRAPHY
64
--·---···--- __ j
v
~--~
--·---------------------------------~---------------------------
------~---- -~
·-.
------------------·-·------ --···-·--------·;
I
Table
I. Colony counts for ~(JB.t-1 4-·13) containing
cultures from agar pla·tes, after fortyeight hours incubat~on at 32°C • • • • •
II. Reversion frequencies of eight ~(JBM 413) vegetative cultures • • • • • • • • •
13
15
III. Hemacytometer counts of conidial suspensions from cultures of ~(JBM 4-13)
18
IV. Conidial viability in ser(JBM 4-13) vegetative cultures • • • • • • • • • • • •
21
V. Conidial viability of ~- cultures versus ~+ cultures • • • • • • • • • •
..
23
VI. Ascospore counts from minimal medium plates
for the -l9°C, 4°C, 25°C, and 32°C storage
temperatures • • • • • • • • • • • • • • • •
27
VII. Ascospore counts from serine medium plates
for the -l9°C, 4°C, 25°C 1 and 32°C storage
temperatures • • • • • • • • • • • • • • • •
38
VIII. Ascospore counts from minimal medium plates
for room temperature and 32°C dried spores •
42
IX. Ascospore counts from serine medium plates
for room temperature and 32°C dried spores •
48
vi
--
--
~-------
---------------------------
--~------~-
----
-~-------------
- - - - - - - - - - - -·l
'
LIST OF FIGURES
i
!Figure
I. Germination frequencies on minimal medium
for ascospores stored at -l9°C, 4°C, 25°C 1
and 32°C • • • . • • • • • • • • • • •
....
II. Phenotypic frequencies for ascospores
stored at -l9°C • • • • • • • • • • •
III. Phenotypic frequencies for ascospores
stored at 4°C • • • • • - • • • • • • •
IV. Phenotypic frequencies for ascospores
stored at 25°C. • • • • • • • • • • •
V. Phenotypic frequencies for ascospores
stored at 32°C . • • • • • • • • •
....
.. .
....
....
e
31
33
34
35
36
VI. Germination frequencies on serine medium for
ascospores stored at -l9°C, 4°C, 25°C, and
32°C. • • • • • • • • •
• • • • • • •
40
VII. Germination frequencies on minimal medium
for ascospores dried at room temperature and
32°C • • . • • • • • • •
....
44
frequencies for ascospores dried
at room temperature • • • • • • • • • • • • •
46
VIII.
Phenotypi~
IX. Phenotypic frequencies for ascospores dried
at 32°C • • • • • • • • • • • • •
47
X. Germination frequencies on serine medium
for ascospores dried at room temperature and
32 ° c.
. . . . . . . . .
..... ......
49
-- -----·------ ·-. ___ _;
vii
:-~---------------------~----------~--,c:-·---~---~-----
·-------------·---·--------------------··---·-·-·-------)
.LIST OF PLATES
I
[Plate
I.
II.
III.
IV.
A nonfiltered conidial suspension showing
conidia., mycelial fragments, and other
debris • • • • • • • • • • • • • • • • • • •
19
A filtered conidial suspension showing
individual conidia and conidial chains
20
A filtered conidial suspension using a
new technique which resulted in individual
conidia only .
• • • • • • • • • • • • •
24
Comparisons of two germinated ascospores,
one scored as wild-type and the other scored
as serine. • • • • • • • • • • • • • • • • •
29
l
I!
t_ -- ------------- --------------~------------------- - - - - - - - - - ------- -------1
viii
!
.,
;
ABSTRACT
AN ANALYSIS OF SPONTANEOUS REVERSION
IN CONIDIAL SPORES AND ASCOSPORES
OF NEUROSPORA CRASSA
by
Edward Vincent Koprowski
Master of Science in Biology
Cultures of a serine auxotroph of Neurospora
;
crassa, ser(JBM 4-13), demonstrate a high frequency of spon.:taneous reversion to prototrophy.
questions:
This study examines two
First, whether the phenomenon of spontaneous re-
version occurs in all vegetative cultures and, second,
whether reversion occurs in stored ascospores.
I
Eight vegetative cultures, which had not shown re~ersion
from ser(JBM 4-13) to wild-type in a previous study,
were repeatedly tested for the presence of revertant nuclei.,
I
Each of the eight stocks ultimately displayed a high frequency of revertant nuclei during a study of serial subcul'
;tures of the mutants.
l .. ___ ,_____ --· -------------------·-----------··------ --------------
ix
Reversion of ascospores stored in water at different emperatures was examined in the second part of this
study.
Results obt.ained over a twenty-week period indica-
ted that reversion of
~(JBM
4-13) does not occur at any
temperature in stored ascospores.
During the course of
;this study freeze-drying was to be used to exclude the pos-,
isibility of DNA synthesis during storage.
This treatment
'
1
was found to result in total inviability of ascospores.
'Separate factors ivolved in the freeze-drying technique
•\<Jere examined to determine the cause of lethality.
Results reported in this thesis indicate that reversion of ser(JBM 4•13) is a property of all vegetative
cultures of the mutant and' does not occur in stored ascospores.
X
INTRODUCTION
Genes that have been modified by mutation create
material for the study of basic genetic processes.
Of
special interest are spontaneous and induced mutations
that demonstrate instability by reverting to a phenotypically wild-·type state, in exceptional cases with hig·her
frequency than forward mutations of the gene in question.
Spontaneous reversions in general are rare
eve~ts,
but
sonte cases of high freq\lency of spontaneous reverse mutation have been reported.
ported in Neurospora.
Two such studies have been re-
The normal frequency for sponta-
neous reversion in Neurospora is one revertant per 107 or
10 8 cells.
Giles (1951), investigating the reversion of
inositolless mutants, observed the inositol mutant JH-5202
to revert at a greater than normal frequency of 7.26 x 10-~
and Barnett and de Serres (1963) reported a reversion fre- ·
quency of 4.29 x 10- 6 for the unstable ad-3B mutant 137.
In cultures which are genetically unstable,
there is a possibility that desired properties will be
lost by mutation and selection.
The long term storage of
various strains without change in the organism is of interest to many workers, from geneticists to curators of
1
2
has been used is freeze-drying (lyophilization).
Rhoades
(1970) reported that various cultures have been stored by
freeze-drying for extended periods of time.
However, mu-
tations have been reported to take place even in the absence of nuclear division.
Ryan et al.
(1961) reported
spontaneous reverse mutation from a his- to a his+ condition in nondividing E. coli.
Other investigators have re-
ported mutations accumulating in dormant systems.
Auerbach (1959) working with Neurospora conidia stored dry,
reported that mutations accumulated in strict linear proportion to time.
Drake (1966) observed a slow accumulation
jof mutations in bacteriophage T4 in nutrient broth.
I
! bacteriophage,
I inert
l that
These
lacking cellular hosts, were metabolically
and therefore were not replicating DNA.
a chemical event may be involved.
Genetic
Both Auerbach
j
l
(1959) and Drake (1966) observed mutations accumulating at
j a faster rate with increased temperature.
!
I
The present study involves continued genetic
analysis of a recently isolated Neurospora serine auxotroph,
~(JBM
4-13), which maps on linkage group V, 3.5
map units to the right of met-3
(Kline, 1973).
•
•
,_
' • ·• ''•• •' •
~-
•··" • ,_ " ' • •
·~·
• •
•-~- ~-·
·• ·-·
•".<~· ~-
• •-·
,_,._,
~~
--•
-~--·.,..•~ .o.-... ~-·~-·---...-.-«·----=·~-~~•~•
••...,_•""-.>>...Ox-<>.-<>«~~_.._..- .• --__,_~.,,...=•~
Previous
•-<•~h,,._,._,..,,~~-n~~·---.•·• ·~···
3
!investigations of
~(JBM
4-13) by Bengston (1975) and
!Sievers (1975} have described some of the properties of
this mutant, including high frequency of spontaneous reI
version and variation in reversion frequency among separate'
spore isolates.
Revertants behave genetically like back
mutants, that is, crosses to wild-type yield no mutants,
as would be expected if the revertants arose from suppres'
sion of the serine requirement by mutation at a genetically
separable site.
In addition, Bengston observed that re-
version occurred in the absence of cell growth in both vegetative cultures and stored ascospores.
The work reported
here examines whether or not the spontaneous reversion
phenomenon is a property of all ser(JBM 4-13} progenyi
v1hether or not reversion occurs in stored ascospores in the
absence of cellular divisioni and whether or not reversion
1in stored ascospores is affected by the temperature of
l
!storage.
l
j
I
i
L, ... ~---·-----~"""''"''""'-·~--··-··-·-·--··-·-·--·-··~-~--~··------·~-·--··--------·-·--~.-··-·--··-····- .... -.~·····--·'
MATERIALS AND METHODS
iI
!Conidia Studies
i
~=~~_;:,_=
!strains
iI
!
The original source of the serine auxotrophic
:culture
used in these studies was a nutritionally wild-type
i
'
!strain A, al-2(15300); cot-l(Cl02t).
Following ultraviolet
irradiation, five serine-requiring mutants were isolated
from this strain, as described by Bengston (1975).
One of
the five serine auxotrophs, referred to in this report as
~(JBM
4-13), also carries the markers A, al-2(15300),
and cot-l(Cl02t).
The double auxotrophic cultures used in these
studies and designated
~'
met, were produced from a cross
of N3-12A: ser(JBM 4-13), met-3(361-4) and wild-type
described by Sievers (1975).
Fifty-seven
~'
25~,
met progeny ·
from this cross were previously studied in an investigation
of ser (JBM 4-13) revertibility, (Sievers, 1975).
Eight of ·
the fifty-seven cultures showed no reversion from ser to
{
;
ser+ during that study and were thus chosen for the search'
for a nonreverting strain described below.
~,
These eight
met cultures are referred to in this report as M.S.2B,
4
5
!teen day old stock cultures.
Ii
!Maintenance and Growth of Cultures
j
I
Vegetative cultures were maintained on agar
I
!slants of Vogel's minimal medium N (Vogel, 1956) supple-
I1
mented with 0.3 mg/ml L-serine and 0.4 mg/ml L-methionine.
I!All
I
chemicals were reagent grade.
Cultures were kept at
room temperature.
Reversion Studies
In order to test for the presence of revertants,
conidial suspensions of the M.S. cultures were plated onto
selective media.
Conidial suspensions were made by adding
sterile distilled water to culture tubes and scraping the
surface with a sterile inoculating loop.
The suspension
was aseptically filtered through a Pyrex glass wool lined
funnel into a test tube.
This filtered conidial suspension
labelled 10°, was then serially diluted into sterile nine
milliliter water blanks to make dilutions of 10 -1, 10-2 ,
10
-3
, and 10
-4
All plates were inoculated with one tenth
milliliter of the appropriate dilution.
Each culture was
plated onto Vogel's minimal medium supplemented with both
0.3 mg/ml L-serine and 0.4 mg/ml L-methionine, and Vogel's
minimal medium supplemented with 0.4 mg/ml L-methionine
only.
The plating medium contained 1.5% (w/v) sorbose and
0.5% (w/v) sucrose in order to produce colonial growth.
6
r··<>·-~--
...
.....
='-~'-"""'"""'"''-"·~~--~
,,.,_~........,,~=~~·:•c.-·~.-,~-·~---
...
...
,~-..,
,,,'-"'~-,...,.,,..,...-..,,....,_,.._~~"'"'"'""'--·"'__,.._
.
.,..,,...,_~_.._-_.,...__.._.
......,.,_______
.....................,.-="""·.»<.."v...-.c ... ,_ .........
-""'--"~•>'"_...
..
..,..,__._.~,._,.,_ ._~"-_
!face of the plate with an alcohol-flamed glass spreader.
!
:Plated cultures were incubated at 32°C for forty-eight
hours and then examined for growth.
Reversion frequencies
were calculated from the number of presumptive revertant
colonies on the plates supplemented with methionine only
compared to the number of colonies on the plate supplemented with both serine and methionine.
Isolation of Revertants
Presumptive revertant colonies were isolated from
the plates containing medium supplemented with methionine,
and retested for requirements.
Retesting consisted of cut-
!'
ting the colony into two halves using a flamed platinum
'
iwire, and placing one half onto a large (18xl50mm) slant of
!
;vogel's minimal medium supplemented with methionine and the
!other half onto a large slant of Vogel'~ minimal medium
/supplemented with serine and methionine.
Colonies which
i
/proved to be revertant were then maintained on large slants
iof Vogel's minimal medium supplemented with 0.4 mg/ml
l
l
I L-methionine.
I
l Conidial Survival
'
--------------------
Hemacytometer counts were made on the 10- 2 , 10- 3 ,'
or 10-
4
dilutions of each culture.
These counts, which
reflect the number of conidia plated, were then compared
with the number of colonies which grew on the corresponding
dilution plates.
From these comparisons the percentage of
7
r-~---·~--~-·----J:-;~ffi~"i;n
tf.i i-t~;_t~g--;f- th~,~~~~i-cii-~i~·-~-~·~-p~:r;-~i;~~-
1
l
!leads to ambiguity in the hemacytometer counts due to the
!presence of conidial chains.
t
A chain of two or more co-
i
/nidia might yield a single colony when plated.
Therefore
a filtering technique which yielded single conidia was used
in later survival studies.
A ten milliliter disposable
glass pipette was packed with Pyrex glass wool to a density
which allowed the conidial suspension to filter easily.
The conidial suspension was aseptically filtered through
this column into a test tube and serial dilutions were made
as described previously.
!Ascospore Studies
'----~~------------
Strains
In order to obtain ascospores for study, the
auxotroph
~(JBM
cot-l(Cl02t).
4-13) was crossed to
~;
~(Y8743m),
fl(L);
The two cultures were obtained from silica
gel stocks supplied by Dr. Joyce B. Maxwell.
The cross was
carried out in petri plates containing Westergaard-!Jli tchell
'!
(1947) crossing medium supplemented with 0.3 mg/ml L-serine~
Some of the plates were coinoculated with both of the parental stocks, while in other cases the conidial parent
(male) was added one week after the protoperithecial (female) parent.
All matings were incubated in the dark at
room temperature.
Ten days after the initiation of the
crosses, petri plate coverlids were exchanged for clean
sterile ones so that the shot (dehisced) ascospores could
8
fbe collected on a surface free of conidial and mycelial
;
'growth.
After thirteen days the first shot ascospores were
noted on the plates which had ser(JBM 4-13) as the female
parent
parent.
and~;
~(Y8743m),
fl(L); cot-l(Cl02t) as the male
The perithecia were allowed to continue shooting
ascospores until the twentieth day after the initiation of
the cross.
The plates cQinoculated with both parental
types and the
plates which had -ser(JBM 4-13) as the male
' . .·.
parent showed poor ascus development and were therefore
discarded.
Maintenance of Spores
Ascospores were collected by swabbing the petri
plate covers with a cotton tipped applicator and rinsing
the surface of the crossing medium with a one percent solution of commercial laundry bleach consisting of six percent
sodium hypochlorite.
This solution was used in order to
!keep conidial spores and/or mycelial fragments from con!
!I taminating
-
the final ascospore suspension.
The ascospores
!were washed and centrifuged twice, and then resuspended in:
j
! sterile distilled water to a predetermined volume.
One
I milliliter
aliquots of the spore suspension were dispensed
into small (5 ml) ampules.
The ampules were divided into
six separate groups using a random numbers table (Dixon
and Massey, 1969).
Four groups of ampules were heat sealed
and separate groups were placed at one of the following
.
temperatures: -l9°C 1 4°C 1 25°C, and 32°C.
....
.. ......
, __________ ..-- ..
... .., ....
,.._.~ -~--~~-'--''"·~-----
.,_.._..~_,._.......,.....__
~-...-.,.
,._....._~-~--·
______
~
.....
--..
The remaining
--
.__~-------
,._,. _.,._.,_,,,.,......,
_________,......
.'
,,_,,._,___~~-~-"
9
!two groups of ampules were left unsealed to allow the
levaporation
of the water . contained inside.
One group vms
allowed to dry at room temperature while the other group
was placed in the 32°C incubator to dry.
Study of the
dried groups is referred to in this report as, "the drying
experiment."
Analysis of Reversion Frequency
Each of the six groups of ampules described above
was sampled at the start of the experiment and weekly
thereafter.
Every week one ampule at each temperature was
chosen for study.
In order to induce germination, the
ascospores were heat shocked in a 60°C water bath for forty
five minutes.
The ampules were opened and a 0.12 milli-
liter sample of spore suspension was plated onto each type
of medium.
Each sample was plated onto both an unsupple-
mented Vogel's minimal medium agar plate and a Vogel's
minimal medium agar plate supplemented with 0.1 mg/ml
L-serine.
Both types of media contained 2% (w/v) sucrose.
Ascospores were spread evenly across the surface of the
agar medium with an alcohol-flamed glass spreader.
plated ascospores were incubated at 32°C.
All
Twenty-four
>
hours later the ascospores were examined with a compound
dissection microscope at 20X magnification.
Ascospores
were scored on the basis of their phenotypic appearance.
Germinated ascospores which showed very dense growth were
scored as ser+, while germinated ascospores which showed
10
.sparce growth were scored
as~·
Random germinated asco-
spores of both phenotypic appearances were selected off of
the unsupplemented Vogel's minimal medium agar plates and
tested further for.requirements.
These germinated asco-
spores were cut from the petri plates on small agar blocks
and placed onto either small (12x75mm) slants of Vogel's
minimal medium or small slants of Vogel's minimal medium
supplemented with 0.3 mg/ml L-serine.
The slants were in-
cubated at 25°C for three days and growth observed.
Ascospores used in the drying experiment were
rehydrated with one milliliter of sterile distilled water
twenty-four hours prior to heat shocking and plating, as
suggested by the work of Davis and de Serres (1970).
i Freeze-Drying
The technique of freeze-drying was initially used
in this study for the prolonged maintenance of ascospore
samples.
The as cos pores were collected and wa.shed as de-
scribed previously.
One-tenth milliliter aliquots of
ascospore suspension were dispensed to small ampules.
These ampules were frozen in a solution of dry ice and
methyl acetate and quickly placed onto a freeze-drying apparatus.
After two hours the ampules were heat sealed with
an oxygen-flame torch and removed from the machine.
Re-
sults of ascospore viability tests conducted immediately
after freeze-drying proved negative, and thus the technique
of freeze-drying was not used in this study.
...
,~-..·-=
'
=<=~ ...... -~-·-""'-- ~,.....,,.,.. _ _ _...-=>-..,-~~-----------=---...--""-.,..
..
..,~..,,..,..,"""'~"'""""'<'~
......... ~,
;
!
'
'
J
RESULTS
Ser, Met Reversion Studies
Prior studies suggest that
~(JBM
4-13) cultures
will eventually undergo reversion from~ to ser+, and the
gene may never be stabilized.
I
One experiment carried out
by Bengston (1975) tested the revertibility of 102 ser isolates.
Eighty of the 102 cultures tested showed reversion
to wild-type while twenty-two appeared to be stable.
In
another experiment by Elaine Leboff and Dr. Joyce B.
Maxwell (unpublished) an apparently stable mutant isolate
of ser later reverted.
The question of reversion in ser
vegetative stock cultures was next investigated by Sievers
(1975).
Of the fifty-seven stock cultures studied, forty-
nine reverted to ser+ while eight appeared to remain stable
These results suggested that perhaps all
~(JBM
4-13) cul-
tures might revert if examined exhaustively, and formed the
basis for this experiment.
The cultures used in this study were obtained
from Marc Sievers as described in Materials and Methods.
The eight cultures used were double auxotrophs designated
~,
met which had not reverted in Sievers' previous study.
The met marker
was included in
the anlysis to eliminate
the
n·'·
.
.. .. . __._ '"
'
-
--•-'•»
,_._._,_,._•~• ·-~-~~---~-·-• ---~----"~' --~• ~------··~.....,.~ .._.,,__.,_""'"-'"'-~· .,..,,.~~----~-~---~--v=--~~
11
-..~
~.,
~-~- ·•·~_.,., •~--·'"' o>'-~·
12
revertants.
l
The eight cultures were subcultured weekly and
l
bne week old subcultures were analyzed for the presence of
I;revertant
nuclei each week until reversion was observed.
!'
~able I
lists the colony counts used in the calculation of
;reversion frequencies of the eight cultures during the
l
lcourse of the study. Reversion frequencies were calculated
l
comparing the number of colonies on methionine alone to
py
,the number appearing on serine and methionine, plated from
i
'serial dilutions and taking into account that 0.1 millilit.Iter of each dilution was plated •
1
;For example:
I
l
~
Methionine
Serine + Methionine
j
iDilution:
i
j
!Count:
24.2*
i
65
IThis particular culture had a reversion frequency of
!
124.2 X 10
!65
l
I
!
=
3.7 X 10- 5
X
*(in this culture the counts for the methionine
plates were an average of the number of colonies
appearing on ten duplicate plates.)
I
The results in Table I I indicate that
113)
t
l
4-
is mitotically unstable, and that it exhibits a vari-
!able reversion frequency in separate cultures.
l
~(JBM
Reversion
!
l; frequenc1es
.
.
var1ed
from 1. 0 6 x 1 0 - 6 to 7 • 77 x 10-l
Colony
13
___
-------~--.-------·---~~--·----
t
i
Table I
j
Colony Counts
I
I
l
:culture Medium Dilution
l
I
!I
.....,._,;"""""""""'~-,.,....-""""~"-...,....,>u?.
Weeks
1
MS 2B
M
100
2
3
0
425
4
5
6
S/M
10- 3
362
289
S/M
10- 4
36
35
0
0
0
0
0
!
MS 6B
MS
58
MS 88
M
10°
0.6*
S/M
10- 3
72
123
162
193
213
327
S/M
lo- 4
2
12
16
20
21
45
10°
0
0
0
M
24.2*
S/M
lo-3
+
551
+
672
S/M
10- 4
58
57
66
65
0
252
M
10°
S/M
lo- 3
91
+
S/M
lo- 4
11
94
M methionine
S/M serine/methionine
+ plates overcrowded, colonies too
nt~erous
to count
plates no longer made, reversion has occurred
* numbers represent an average of ten duplicate plates .
l
I
!
I
14
. ...
.
.....
r---~- ,.--~--·---=-""'------·---·----~ ---~_d -,,.~-
'!
.
'
-"""',..,..,_~,;
''
Table I cont.
j
Colony Counts
l.
!
l
I culture Medium Dilution
Weeks
1
MS 95
MS 130
MS 138
3
0
S/M
353
133
151
S/M
33
18
18
M
0
0
S/M
8
267
286
S/M
1
26
18
M
5
6
0.3*
554*
· S/M
553
S/M
74
14.3*
0
0
S/M
87
168
311
S/M
5
18
59
M
4
0.9*
0
M
MS 108
2
M methionine
S/M serine/methionine
+ plates overcrowded, colonies too numerous to count
- plates no longer made, reversion has occurred
*
numbers represent an average of ten duplicate plates
!
!
i
L~-~~~,.....~~,.~=...-~,.,.·----~........,~--=·--~·-. .....,,..,.,.....~~--_._,_-,_,~..-
------
'"""""""'""--··----------····------ --------"-·-··----- --·------- ---
------- --------------·---------·--- -·------- ----·--- ·--
Table II
I'
Reversion Frequencies
Culture
2
*
1.47xlo- 3
MS 6B
*
*
*
MS 58
*
*
*
MS 88
*
MS 95
*
*
5.96xlo- 6
MS 108
*
*
l.OSxlO
MS 130
7.77xlo-l
MS 138
*
MS 2B
*
!
Weeks
1
2.74xl0
*
3
-1
5
*
3.7xl0
-
*
1. 33xl0
-5
-
I
6
-
-6
I
I
I
II
I
I
-6
4.6xl0-5
no revertant colonies seen
- not studied further
4
-
----,
I
l
!
I'
·--~ ..,i
1--'
VI
16
lsize varied on both methionine plates and methionine/serine
·plates.
All presumptive revertants were tested further for
requirements in order to distinguish revertant from nonrevertant, leaky (limited growth on methionine plates) col-.
onies.
Colonies which proved to be revertant were then
designated as such and kept for further study.
For example
revertant colonies which originated from culture M.S. 2B
were labelled M.S. 2B R.
Each time a culture was studied, the percentage
of conidia which survived to produce colonies was calculated.
Hemacytometer counts which represent the number of
plated conidia were compared with the number of colonies
which grew on the agar plates forty-eight hours after
plating.
i For example :
i
II Source
J
l
Volume plated
or counted
Dilution
agar plate
362
0.1 ml
i hemacytometer.
I
0.4 mm
Counts
3
103
I
!
I
l Because these counts were made for different dilutions,
I
! values
th~
were adjusted to represent the number of conidia per
'
milliliter of concentrated (10°) conidial suspension, as
follows:
Plates:
362 x 10 3 x 10
Hemacytometer:
'
~.>'<"~--~
. . . . _ , ___
,.,_.._L..,."~><>---~._.--.-----
103 X 10
0.4 mm3
=
3.62 x 10 6 conidia/m1
2
X
=
2.58 x 10 7 conidia/m1'
17
iThis particular culture had a viability (percentage of co,nidia which survive to produce colonies) of
I
i3.62 X 106
)
X 100 = 14.0%
j2.58 X 10 7
i
The filtering technique used in this study re!sulted in ambiguity in the hemacytometer counts.
The fil-
'
Lter was effective in removing mycelial fragments and pieces
/
Jof agar which had become dislodged in the preparation of
i
!the conidial suspension, but i t allowed the passage of
I
i
·;chains of conidia consisting of two or more connected or
I
!immature, nonseparated conidia. For the purposes of this
i
i study, conidial chains were counted as one unit regardless
I
I
!of their length.
Thus each single conidirun or conidial
I
l chain was regarded as having the potential to form one co!
I
I
j lony when plated.
Hemacytometer counts reported in Table
i
l
i III represent the total number of individual conidia plus
l
Il the
nrunber of conidial chains.
I tered
Plate I shows a non-fil-
conidial suspension consisting of conidia, mycelial
Il! fragments,
I conidial
and other debris.
Plate II shows a filtered
suspension consisting of individual conidia as
1
well as chains of conidfa.
Table IV gives the conidial
viability for the eight cultures.
The results of the conidial viability studies
suggest that there may be an increased conidial viability
in cultures which revert to ser+, met.
For example, cul-
ture MS 138 at 10- 2 dilution demonstrated a conidial via-
18
..
..
..,
~----·~>··~ -··--~~---~~--·-~--·---·-·--------·----·~-~--·-~--·~··~~--- -~.,
!
Table III
i
Hemacytometer Counts
Culture
Dilution
2
1
MS 2B
MS 6B
10- 2
103
47
10- 3
9
3
10- 2
52
32
43
29
*
2
11
4
9
4
*
54
*
93
*
15
67
11
*
34
*
*
10- 2
130
29
14
10- 3
13
2
1
10- 2
158
71
23
10- 3
13
4
3
10- 2
39
10- 3
8
10- 2
38
36
33
·10- 3
5
*
10- 2
10- 2
10- 3
MS 95
MS 108
IviS 130
MS 138
6
33
10- 3
MS 88
5
*
10- 3
MS 58
Weeks
3
4
3
Counts reflect the total number of individual conidia
plus chains for a volume of 0.4 rnm3 of spore suspension.
*
hemacytometer count not made
not studied further, reversion had occurred
19
Plate I
Nonfiltered Conidial Suspension
20
Plate II
Filtered Conidial Suspension
21
"··· ,_
'~
... ,_, ....
'-"
~,~.,,...,,,,
_
,__...~,,...- __._~,•-~• .·~.-o
-<o.o-.'..... ,..,,...,....,-~...,.,._....,.~.,..=~•~--·'-'">.'•
•...-,.•...__.,_=·
·"""'~--...-
~---
..-•.-...
-.~.....-. =--,.~=-
....
~-"~~~~~
...-...
~-···"-·~,......,.__..."'-- ----~ ~-
.. ---
...,_,_.. ...
"~""-"'"'•"
..
~
..........
-~~----...---~·.
, ..
-·
Table IV
Percent Conidia Surviving To Produce Colonies
Culture Dilution
Weeks
1
MS 2B
MS 6B
10- 2
14.0
24.6
10- 3
16.1
38.5
10- 2
10- 3
MS 58
10- 2
10- 3
MS 88
10- 2
10- 3
MS 95
10- 2
10- 3
108
MS
MS 130
MS 138
*
2
3
4
5
*
14.9
12.5
24.1
19.8
43.9
*
24.6
5.9
19.3
9.5
31.8
*
40.8
*
2 8. 9
*
14.7
3.9
24.4
*
32.9
*
10.9
*
*
18.3
43.2
26.6
60.4
10- 2
0.20
15.0
49.7
10- 3
0.25
26.7
38.1
10- 2
13.3
10- 3
7.9
10- 2
9.2
18.7
37.7
10- 3
7.0
*
41.5
percent surviving conidia not calculated due to lack
of hemacytometer counts
not studied further, reversion had occurred
I
6
\
1
I
i
;,. ·-~~--''>''"'-'--~"""~ '<-""-""'"· ..... .-.,~~•<=.,_..,X"''~~~"'"'"""'''~-_,._,.,...~,_-,~-~----~~ _.._., ,_.. ,__ ___..,.~_,,__.~,_.."""""'-.,..... H.,.,.,__.,...,.-""""""''""=".........,...~4'>~-··~"'~'-'~->0~~..-.•'"'-"_._' __,~,~-"'-~»-"••-=>_, ,,._.,.__,..,__,,..__.~~-~,_,.~,,..,.......,,...,..,,~--""""¥'''"
22
.
---~-,,~..,.._....,.,.,.._...,~-'~ .-~-.,~nv,..,_.,,....,....-...,
..
.
-
..,._.,.,....~,.._~..._._...,~~tw.....-~"-~""',.._"*"'-"''~'••""'"~~.,.,.....,...,.,~,_~---=.,_,..._.,,..c>:,<-~
r•• ,., .. , .
~
.,_.,,.~,- ~''""'"-'-"~'
tant colonies were first observed for MS 138 at the third
iweek, the conidial viability increased to 37.7%.
This in-
1
jcrease in conidial viability at the time of reversion was
lobserved in five of the eight cultures. Two of the remain_;
I
ling three cultures reverted early so that only one value of
I
/conidial viability could be made.
-~did
Only one culture, MS 58,
not show this increase in viability but rather demon-
lstrated variable viability.
I
1
A new study was therefore undertaken to determine
ll if mutant cultures differed from revertant cultures in the ·
lI
!percentage of conidial viability.
Revertant cultures which
I
!had been isolated during the reversion study described pre...:
:
Ilviously
were compared to the mutant cultures from which
i
'
I
!
t h ey
!
! R.
•
•
or~g~nate
d•
i
For example, MS 2B was compared to MS 2B
Table V shows the viability for the mutant and revert-
!
1 ant conidia.
I this
'I
An improved filtering technique was used in
part of the study in order to reduce the number of
conidial chains in the final conidial suspension.
This
filter, descibed in Materials and Methods, resulted in a
conidial suspension consisting of single conidia only, as
seen in Plate III.
The results in Table V indicate no pre-
ferential survival of conidia for either mutant or revert- •
ant.
In some cases, revertant cultures demonstrated a
higher percentage of surviving conidia, as in MS 130 R.
In other cases the mutant cultures demonstrated the higher
survival percentage, as in MS 108.
These results do not
I
23
______
r·---·-···----------~-_,
~
---·---~-·--~-~-·-~---1
Table V
i
'
Percent Conidia Surviving To Produce Colonies
Culture
Mutant
Revertant
MS 2B
54.9
49.6
MS 58
30.0
41.0
MS 88
59.2
18.2
MS 6B
!
!
i
;
!
!
MS 95
56.9
58.3
MS 108
72.2
35.4
MS 130
46.9
68.7
MS 138
29.1
71.7
- culture was lost
I
l
24
Plate,III
Filtered Conidial Suspension (New Technique)
25
r;i a];ii ityth-;~- ~~t~t
nu~lei.
-·~-·-·~
I
!Ascospore Studies
I
-~~~·stored
Ascospores of Neurospora crassa which had been
at 7°C for eight months, showed a marked decline in
I
!the ratio of ~(JBM 4-13) to wild-type, compared with the
lratio obtained from the same mating when young (Bengston,
1975).
During that study the genotypic ratio changed from
,one ser: one ser+ prior to storage to one ser: two ser+
I
-
-
I
!after storage without change in ascospore viability.
The
)following experiment was done to determine whether ser(JBM.
I
.
!
4-13> reverts to wild-type in stored ascospores and whether
1
!
I
1 reversion frequency is affected by storage temperature.
I
I
1 13)
l
Ascospores collected from a mating of
and~;
~(Y8743m),
fl(L);
cot~l(Cl02t)
~(JBM
4-
were dispensed
to ampules and freeze-dried as described in Materials and
!Methods.
The technique of freeze-drying is often used fori
j the storage of cultures (Rhoades, 1970).
Ithis portion of the study
It was used in
in an effort to retain the asco1
spores in as "dormant" a state as possible, thereby reduc- •
' ing the effects of uncontrolled environmental factors.
I
Following freeze-drying, all tested ascospores proved to
I be
inviable.
Various treatments were given in an effort
Il
soaking of ascospores in distilled water, a method which
1
was shown to increase germination in old spores by Davis
i to revive the ascospores.
!
These treatments included the
land de Serres (1970); addition of Vogel's salt solution at
IL----------...-..-.. . . . . . . . . . .~-~-----~
----·-·---·--~~,_, .._,~. . J
26
.
r,·"·---------~---~---------------~---~-~ ------~---~-~,
1SOX, 2X, and lX concentrations; and addition of a 2% (w/v)
lsucrose solution.
In some cases the treatments were given
lprior to heat shocking, while in other cases treatment fol!
'
!lowed
heat shocking.
All attempts to revive the ascospores
\
!were unsuccessful.
Due to the resultant ascospore invia-
1
!bility, the method of freeze-drying was abandoned.
I
Another mating of
~(JBM
4-13)
and~;
pe(Y8743rn)
.fl(L); cot-1 (Cl02t). was made to obtain a new group of asco-:
l-
jspores for study.
J
As an alternative to freeze-drying,
I
!these ascospores were stored in sterile distilled water.
I
1
lOne vial £rom each group was tested prior to placing the
lfour groups of ampules at the selected incubation ternperajtures.
Results from these unstored spores are designated
las Week 0 in the data.
Ampules were then assigned to the
Ipredetermined incubation temperatures
I and
32°C.
of -l9°C, 4°C, 25°C,
Every week thereafter, one ampule from each
!perature was chosen and tested.
'
!
tern~
These results are desig-
'
!nated as Week 1, Week 2, etc., and represent data gathered·
I
!
after one week of storage at a designated temperature, two
l
)Weeks of storage, etc.
I
! from minimal
Table VI lists the ascospore counts
medium plates for the four temperatures.
1
Ascospores were scored on the basis of their phenotypic
l!
appearance twenty-four hours after heat shocking.
I
I
I
As co-
spores which showed dense growth were scored as wild-type
(ser+) whereas ascospores showing sparce growth were scored
~s
serine (ser-).
Plate IV shows an ascospore scored as
.'!!.-!!§~:_-t:::YP~-~cg~pC!::r.~.~--!'-~:!=:-~__c.!f.l_~_f:;_~-2~E~~~-.§_C::.~~§_<?:__~~----~-~E.!.~~--5??_______
~~·
____
............-...-.......,
''
.,_··~-·-------·--~-~
-------------------·~...---~--------·-··--··---- .
Table VI cont.
II
!
I
II
l
Storage
Temp.
-19°C
Type
11
12
13
14
15
Week
16
17
15
26
13
11
6
2
9
5
145
88
145
Wild
60
59
Serine
17
Non-germ.
17
18
19
20
17
4
9
7
6
3
4
3
4
2
2
105
161
146
141
111
103
136
70
103
70
80
68
78
89
64
16
18
29
19
15
16
14
28
17
12
20
12
14
25
21
19
27
16
18
Wild
80
81
90
60
95
109
72
85
31
56
Serine
23
18
19
8
11
16
21
19
8
17
9
12
19
5
15
24
27
23
7
11
32
56
70
55
102
95
67
66
72
71
Serine
3
8
15
14
20
13
15
11
11
11
Non-germ.
6
12
12
11
22
28
14
29
10
22
Wild
Serine
Non-germ.
4°C
25°C
Non-germ.
I
i
!
l
l
i
I
......
------1
Ascospore Counts from Minimal Plates
l
II
----------··- ..
32°C
Wild
----. --~~-..,·-··~--..........._,.-.._.-. ~·
'"-'-'"'"""~~~~----·------
. . .--"---------··•••o.----···--·••""". . . . . __
N_"
-~---· ·-····'-'--••••·---~· ··~---:-~--------·''·-·"-•""'------··~·-·--·--~
-·
•
~.,,,, .............
'"'
•• ' "
1\.)
CX)
29
Plate IV
Comparison of Germinated Ascospores on Minimal Medium
30
;-;i~,i~~l-m;di-~ aft~ ty- ~~~rs ·g~owth :----·---··--~----,
I
Germination frequencies were determined by com-
lparing the number of germinated ascospores to the total
I
,
I
jnumber of ascospores observed on the plate. In addition,
I
jthe percentages of ascospores which were scored as wildjtype and serine were calculated.
!
!Fo:r;- example:
i
I
Ascospore Count
I
l
Wild
40
Serine
12
Non-germ.
28
!
I
80 total ascospores observed
I
!This particular sample had a germination frequency of
l
! 40 +
j
12
X 100
80
=
65.0%
Of the 80 spores observed, wild-type comprised
l
I
iQ. X 100
80
= 50.0%
'
i and serine comprised
l
l ~~
!
X 100 = 15.0%
!
l Figure I summarizes the germination frequencies for the
!
I four
I
I
!
I
II
J
I
incubation temperatures during the twenty week study.
IAt Week
1
I
O, the four unstored samples had germination fre-
quencies which are generally the same. This result is ex-
1 ::::::
::a:::::: ::::s:::::c::: ::i:~:a:::f:::tt::o:::
con~~-'!:~_9n~.~---J?~E-~!?-.9. __!_~~~--~~~E.~~-~-~--_!:!l_e___J:_~~-~-~Y.--~~e~--~~':1-':IX. _____ ._,,
31
1-
z
UJ
(.)
a:
UJ
a.
---
-
0
2
4
6
8
10
12
14
16
32°C
25°C
4°C
·19°C
18
TIME, WEEKS
FIGURE
GERMINATION
FREQUENCY
ON
MINIMAL
----
20
32
(a~-~ospor~-5,·-a.t--;a:~h te'mper.ature-·exilTbiteCf-ffio"derateiy ~-v·a:ri.::·~-·,
(able germination frequencies.
The most detrimental storage
!'
jtemperature in terms of germination frequency was -l9°C.
i
IAscospores at -l9°C showed a steady decline in germination
i
i frequency
;
from 64.6% a·t Week 1 to 5. 6% after twenty weeks
!
lof storage. Ascospores at the other three storage temper!
.
! atu~es exhibited a common range of germination frequencies ••
~Germination
frequencies ranged from 65.6% to 90.4% for as-!
I
!
!cospores stored at 4°C, from 65.3% to 95.6% for ascospores
i
!stored at 25°C, and from 64.0% to 94.1% for ascospores
I
)stored at 32°C.
iI
1
Figures II, III, IV, and V show the per-
centages of ascospore types for the -l9°C, 4°C, 25°C, and
!
l 32°C storage temperatures respectively.
Ii
i
!
These figures show
1
that wild-type ascospores comprise a substantial portion of
J
! the germinated ascospores.
I
These results also indicate a
deviation from the expected 1:1 ratio of wild-type (ser+)
I versus serine (ser) which had been observed by Bengston
I
- - .
I (19 75) •
l
Greater variation in germination frequency is ap-
i
1
parent for wild-type ascospores compared to serine ascospores.at all four temperatures.
This suggests that the
,,l
l variations in total germination frequency could be due in
I mostpart
to changing germination frequency among wild-type;
j ascospores.
II Control
I
j
Experiment
Since the
~
types were infrequent compared to
~
1 ser+, it seemed possible that ser were selected against by·
'"-··--6··-·
' . .....- .... .....,,_.,..__...._,_ _
_..~··"'-~·-""
.....,..-.,.. _ _ _ _ _ _ _ _ _ _ .._... _
33
80
70
60
1-
z
w
0
a:
w
a.
50
- - total germination
.......- · wild type
serine
40
10
0
2
4
6
8
10
12
TIME, WEEKS
FIGURE
II
ASCOSPORE
TYPES
14
16
18
20
34
100
90
80
60
1-
z
UJ
0
a:
UJ
0.
-
40
-~
30
total germination
wild-type
serine
20
10
2
4
6
8
10
12
14
16
18
20
TIME, WEEKS
FIGURE
ASCOSPORE
Ill
TYPES
4°C
.._.,..,_,,,.,,,,,_"",._-=---A>-A.,<=;>""'-~-=~--~-~~~-~---~·~·~.--=,_..~-,.·<"~=""''''''''."'-_.,_..,,.,..A,-<",'"='"""-=~~~-·~--,._---"--~--.,........_.._,."_ __
35
100
90
80
70
60
1-
z
UJ
(.)
a:
50
UJ
a.
--
40
total germination
,.___..
wild~type
se rt ne
30
20
10
QL-~_.~~~-L~--~~~--~~-L~--~~-L~~~~~~
0
2
4
6
8
10
12
14
16
18
TIME, WEEKS
FIGURE
ASCOSPORE
IV
TYPES
25°C
---~·~-~.._..,._.,.,,~~•~·~~~,,,.,.._._~~-~--·~--~-------~-=~-___,,_~,.-.,.-=r•<"'*"~-=--=-<--'•---.--·~~'"""~'~__,,.,.,.,._.,_...~.-
------
20
I
36
80
70
60
..,_
z
UJ
(.)
a:
50
UJ
c..
total germination
-----. wild-type
---.. serine
0
2
4
6
8
12
10
TIME, WEEKS
FIGURE
ASCOSPORE
V
TYPES
14
16
18
37
'
lfrequency was dependent on the nutritional requirements of
ljthe
ascospores used, eaCh sample was also plated onto mediI
lum supplemented with serine as described in Materials and
!
!
l
!Methods.
Table VII lists the ascospore counts from the
lserine plates for each of the four temperatures.
Because
i!
!the serine plates contained all the necessary growth re!quirements for both the wild-type and serine ascospores
1
I
jall germinated ascospores appeared phenotypically similar.
l
1
I
i
jTherefore ascospore counts indicate only the number of ger-,
1
.
!minated or nongerminated ascospores.
;
Germination frequen-
j.
lcies were calculated as before and are shown in Figure VI.
I
.
INo substantial change in germination frequency was observe~
lfor ascospores germinated on medium supplemented with ser- j
1 ine
as compared to germination frequencies for ascospores
'germinated on minimal medium (Figure I).
II
Reversion of ser(JBM 4-13) in ascospores stored
.
Ij at
-
four different temperatures was studied.
Ratios of
jwild-type versus serine differed from the expected 1:1
i
I ratio,
but the proportion of serine ascoSpores did not de-
l
. .
j cline during the course of the study as one might expect
! if
!
I
,1_
serine were reverting to wild-type.
I
The Drying Experiment
This study has shown that the technique of freeze
I; drying
l
i
causes inviability of ascospores of Neurospora
~
crassa.
Freeze-drying requires that organisms undergo the
;
_, .... ~-~.,..~ ......i
r--'"'"'"•"~...__._.,,. _ _ _ _ _ _ _ _ _ _ ,.,.._,._.._, _
,
.
__,_~~-..---M-~-·-..,---~-----------·---~----··-~·----- ~---~----~--···----·•••• ·----~---·---·-"'-·---·--------·-·~--·--·---- ....-~'"' ~......._.,_..,.__.,_. ,ow,.....,_.,_._._1
!
l
!.
'
I
!:
Ij
!!
Ascospore Counts from Serine Plates
Storage
Temp. Type
.Q.
-19°C
I
4°C
!.
~
2.
!
~
.§..
l
~
1
10
-
46
78
63
46
24
20
10
9
15
22
Non-germ.
-
24
39
51
57
87
70
72
42
98
139
55
58
70
92
100
82
71
87
96
91
7
20
17
10
17
11
22
17
25
20
62
72
85
84
88
81
. 66
99
80
111
5
13
12
8
15
4
6
10
9
13
69
88
87
74
85
143
82
62
76
125
9
16
_7
9
11
7
10
43
19
5
Germ.
!I
25°C
Germ.
Non-germ.
I
32°C
Germ.
Non-germ.
I
1
,
!
I
I
- data not available
I
I
I
1
Germ.
Non-germ.
!
I
Week
I
I
!
!
~
Table VII
!
L. _____..... ~----------~
............................. ____ ..,,_.~---
.
. . . _. __,. ______ _. ........ -~------. ~-~.-... -...- ......,___.. _.. . _ ____,_ _ _
. _________ __.. __.... ___________,__"...,.'··----- ____ _..A..,--···· ••
w
co
,. . .,
..
·~-~·-··~~--,..,. ····-·----~·------- -~----------
...
------~-·«---
. . . .-.. . .
~---~-··--·------~
. .-.. .-··---··•><
~
. . .··----~---~"'---···· --..~----··-·---........................~~'""'------·-·-----
"-··---~---
...........--··------..
--·-··-·-·--··-·---··-··-·1
Table VII cont.
Ascospore Counts from Serine Plates
Storage
Temp.
~
11
12
13
14
15
Week
16
17
I!
18
19
20
II
I
-l9°C
27
11
17
7
15
23
17
19
. 4
3
121
84
122
103
153
123
144
130
105
109
71
102
92
93
87
126
82
92
78
80
Non-germ.
16
13
27
21
32
10
35
22
19
21
Germ.
89
100
102
90
129
106
94
111
72
122
3
9
23
4
14
11
20
20
9
13
28
93
82
77
103
140
127
89
88
116
4
5
8
26
25
18
19
16
9
17
Germ.
Non-germ.
4°C
Germ.
'
25°C
Non-germ.
32°C
Germ.
Non-germ.
I'
I
I
'!
!
- - -. · · · -·· I
w
1.0
40
.....
z
UJ
u
a:
UJ
a.
32°C
25°C
4°C
-19°C
0
2
4
6
8
TIME,
10
16
WEf~'S
FIGURE
GERMINATION
14
12
VI
FREQUENCY
ON
SERINE
18
20
41
~~-
...--..,..,..._...,....
""·"~"""..-""~--
.............___
~..,.,,.-,,.._
_________
~---
.........
....- - - · - - _ _ . . , - _ . . . _........
~
~
...
-·...._..,..~"""-""""-"=""'""'"'""···
!stresses of freezing followed by desiccation and subsequent
l
\maintenance in vacuo.
The results descibed previously for
!the -l9°C storage temperature indicate that to some extent
l
'
lascospores have the ability to withstand the stresses of
!freezing temperature.
I
This part of the study tested the
jability of Neurospora ascospores to withstand the stresses
J
of desiccation.
Ampules containing ascospore suspension to be
l
!used in the drying experiment were left unsealed to allow
l
/evaporation of the water contained inside.
One group of
I!
i ampules was allowed to dry at room temperature, while the
l
j other was placed in a 32°C incubator to dry.
At the start
l1
j of the experiment, designated Week 0, dehydration was not
J
I
yet complete; therefore, ascospores were not sampled.
I week
One
later, ascospores appeared to be dehydrated and test-
,
1
I:::lb::::~ :::::sv::: :::t:r:::ga::::::::n:~un::r::::t:::~
, frequencies were calculated as described previously and
I are shown in Figure VII. Ascospores stored by drying showl ed a greater variation in germination frequencies than as' 'lll
cospores stored in water.
Germination frequencies ranged
from 44.5% to 87.8% for room dried ascospores and from
33.1%. to 87.4% for ascospores dried at 32°C.
This increas-
ed variation in germination frequencies could be due to
the changes occurring in the relative humidity in the lab- .
oratory, but this possibility was not investigated.
The
I
~
}
( percentages of ascospo:res
scored as wild-type or serine
"
...
;
•~-~.
• ' "-"•'·"·•·"
~ •••
-._.,,_ •• ~.•-~ ' "
-- - - • -'"•.=.«o<
--..~-u-.o•A~-·-••-•·~-~~-.
·~··A••"~--··-~-·~---·~·~·~·-~-~~-,.~....,~"""-'•_,..--.......,•-~- ·--~-· _._,._,,_,,._,.,,,_..~...,__..,~
~.v ~-'-••~---•
·-·--••-«·-'P."•f
r·--------··
·--··~~---.,.------·-------·-----------------.........------------~----·
....
~~----•-....-....w
~~------~--~·-·---·------·-··--~'"----
--·------;-••·•·--"•
''""-~·--·•"l
I
Table VIII
It
j
I
I
I
I
I
I
Ascospore Counts from Minimal Plates
Drying
Temp.
1
2
3
4
---;--
12
113
84
Serine
4
34
Non-germ.
6
Wild
-
6
-
7
-
8
-
9
10
48
34
22
24
231
92
110
14
10
9
8
6
62
29
20
130
64
51
8
6
12
72
27
77
1
96
121
43
29
74' 146
40
58
Serine
2
28
36
9
6
17
46
11
10
Non-germ.
6
73
74
13
20
23
111
28
46
0
Room
32°C
Wild
II
I
Week
Type
-
I
l
I
!
- data not available
I
I
l~~ --~- - , _.,. ., ,. . .,_.,_., . . .,_, . _. ,.___.,_ ~-·- - ·w" " - " - •
i
!
·--
-
··--·-·--
,,.,... _
_,,,..,~~
... -•ow _ _ , . . . _ , _ , , , _ _ _ _ _ , _ _ _ _ _ _ _ ,.,,..,,... _
.. . - . , , ........ _ . . _ , . _ , _ _ _ • • · - • ,_..., __ ,
••-··"~
-''<•~~·
!
••' 0.•"
~
1\J
..
~~--~·-···-
. . ,,,,_____,._"" ___,_ _,_..,._,____ ,___
"'_..._.~----··--~·-···--'"-------~
..
--·-··-----·-~-,.,~~-~---------··--·~-·
------ ----···
·-~-~-----•-·»-·----"-""------
...
~~~~·--·~· ···--------~--~·-·-------
t
Drying
Temp.
Room
32°C
~---~---~
i
Table VIII cont.
~
Ascospore Counts from Minimal Plates
1
Type
I
I
11
12
13
14
15
Week
16
Wild
61
95
42
40
54
37
83
34
40
12
Serine
20
24
11
1
11
8
15
9
14
3
Non-germ.
84
120
66
15
9
22
55
45
18
11
Wild
23
56
122
21
29
25
107
42
25
71
Serine
9
7
31
9
8
6
17
12
3
12
Non-germ.
7
14
64
7
20
16
48
32
10
12·
t. . .,~---"""~---·-""'""'_"_. . ,. . . .,._" "__..............,....__,~-··""'~'"'___,. _., ________._.
........... "..
17
18
19
20
.---·----.. ------~-----'"----'""'___. ____,__ _._. . ____________ .. ,. ~-~····------·--. -----······<>--- ·---------~-. . . .
1
I
~
l
. "'·-·r ..._. .. t
.:::..
w
44
1-
z
u
UJ
0:
UJ
a.
.__......, room temperature dried
.....___.. 32° C dried
0
2
4
6
8
TIME,
16
WEEKS
FIGURE
GERMINATION
14
12
10
VII
FREQUENCY
ON
MINIMAL
18
20
45
j'~..,.__..,..,.__...._.......,....._~·
..
_ _ _ _ ,...,.._...,.~,...,.,~,..r.--·-~-·- · - - - - - - · - - - - - · - ·--=-,...,...~""'"'-
"<P-N"""""-~""-,
!for the room dried and the 32°C dried ascospores are shown
I
.
lin Figures VIII and IX, respectively.
These figures show
I
!that wild-type ascospores comprise a substantial portion of
I
!the germinated ascospores, and that the variation in the
frequency is a reflection of the varia!itotal germination
.
!
ltion occurring in the germination of the wild-type ascoi
!Spores.
Table IX lists the ascospore counts from the ser-
l
line medium plates for the drying experiment.
1
Germination
frequencies were calculated and are shown in Figure X.
No
!
!substantial changes in germination frequencies were noted
'
I
ifor ascospores germinated on serine medium compared to
Ijthose
I
lito the
I .
! water.
germinated on minimal medium.
The results of the drying experiment correspond
results obtained for ascospores stored in distilled
Desiccation, therefore, has little effect on the
j survival of Neurospora crassa ascospores.
I
I
'
46
100
90
70
60
1-
z
UJ
u
a:
50
UJ
a.
40
30
0
2
4
6
8
10
12
-
total germination
wild-type
-
serine
14
16
18
20
TIME, WEEKS
FIGURE
ASCOSPORE
TYPES
ROOM
VIII
TEMPERATURE
DRIED
47
100
90
80
70
.~'v
;
1-
z
UJ
u 50
a:
UJ
I
a..
40
-
total germination
<>---<>
--
30
0
2
4
6
8
10
12
14
wild~type
serine
16
TIME, WEEKS
FIGURE
ASCOSCOSPORE
IX
TYPES - 32°C DRIED
18
20
. . . . . . _. ____________. . __________. ______________
__. ______ . _________._______________________________________:. . . · ----- . . . 1
i~t;i;--ix
I
1
l;
Ascospore Counts from Serine Plates
I
!
Drying
Temp.
Room
Type
Germ.
Non-germ~
32°C
Germ.
Non-germ.
0
1
2
3
4
-
14
141
110
65
4
137
59
83
-
3
2
117
53
120
51
Week
5
6
7
8
9
10
30
25
23
317
102
94
12
8
13
87
37
62
46
43
18
12
I'
-
I
!
i
!!
I!
I'
56
14
208
130
63
30
83
I;
!
'
''I
36
Table IX cont.
Drying
Temp.
Room
32°C
I
I
I
I
11
12
13
14
15
Week
16
17
18
19
20
116
85
56
34
66
52
88
51
43
39
Non-germ.
99
94
65
13
11
11
52
72
30
36
I
Germ.
52
64
176
39
48
72
43
53
23
91
I
Non-germ.
14
21
96
12
30
30
40
33
13
8
!
~
Germ.
!
I
i
II
j
t
II
I
~
- data not available
"
~
\
L. -· -· ... ,..~---·~. .,·--~---,...,-.."--·------~. . .~,··-· -~----~-~·--------------
•·•--···-"-'•'•··-~""''•·-~··"'-' ••· ~~·--•-·• '' ,,.,,. -'"'•'"'"·-"'"'"''"'"""'--'-""'"'''...,._......,.,.~---...\•••·• ~ ···-~··•• .. ~"---~---'"''"''""__.__ .. _w••~•"_,'._.._......_..._~-••••~-.r-"..,_.,_,._,
l
_ _:... ........... ,,_,......._,..,,, • •''''''''' • "••'•• ' ,....,.
" •
.>
'
.s:::.
(X)
49
100
80
I
1-
z
w
() 50
ex:
w
a.
30
temperature
dried
dried
18
20
20
10
0
2
4
6
8
TIME,
10
14
16
WEEKS
FIGURE
GERMINATION
12
FREQUENCY
X
ON
SERINE
~-~~"'"'"'"""''''"'""'__..___,..,,.,_,..
i
_ _ _= ..~--------·~~----~-,._,.,._,,.,"'""'""'_ _ _ __...,...._.~--·---.......""""'...,..~''"""~""==-"'"""''"'··-""''"·
.
'
f
I
DISCUSSION
i
lser, Met Reversion Studies
i1==~~===----------------------
The phenomenon of reverse mutation of
l
~(JBM
4-
1
)13) has been investigated by Bengston (1975) and Sievers
I
(1975) who observed characteristic high frequencies of
1
jspontaneous reversion of the mutant and variation in rever-
!
jsion frequencies among individual spore isolates.
!jObserved
Bengston
reversion frequencies ranging from 1.0 x 10- 5 to
!
)1.0, and Sievers observed a range of 8.0 x 10- 7 to 3.7 x
\
l1o- 2 •
j
I
In general, studies concerning reverse mutation
J
at other genetic loci have not seen reversion frequencies
I
!' as
high as those obtained for
i
1 exceptions
lt reported
I
~(JBM
4-13).
have been noted for Neurospora.
reversion frequencies as high as 10
However, two
Giles (1951)
.
-4
~
for separate
isolates of an inositol-dependent mutant of Neurospora, JH5202, and a variability in reversion frequency of fifteen
j to 726 revertants per 10
I
I
6
microconidia tested.
Barnett and
I
1 de Serres
1
(1963) reported the unstable ad-3B mutant 137 to
revert at a frequency of 4.29 x 10
-6
variability in reversion frequency.
50
, and up to a 50-fold
51
Various models have been proposed to account for
:gene instability and associated reversion phenomenon.
~odels
I
!the
I
Some
attribute instability to the intrinsic properties of
gene itself,. and primarily focus on base-pairing errors:
\in the DNA.
Freese (1959) proposed a model of mutation
!
based on transitional changes, in which a purine is substi-
1
ltuted for a purine, and a pyrimidine substituted for a pyr!imidine.
Another base-pairing error is caused by trans ver-
I
\sional changes in which a purine is replaced by a pyrimi;dine and vice versa.
Freese suggested ·that transversional
}
!changes
may be increased
by an unbalanced nucleotide pool
I
.
i
!caused by starvation.
This hypothesis was also discussed
I
I
iby Ryan et al. (1961) in a paper about the independence of
'
l
!spontaneous reversion and DNA replication. Barnett and de
--
I
iserres
(1963)
explained the spontaneous gene instability at .
J
i
jthe ad-3B locus in Neurospora on the basis of a mutational
l
!model involving complementary transitional changes
I
(TA~CG)
.:
Gene instability may also be due to extrinsic
i
I
!factors, such as mutator genes or episomes.
l
Mutator genes
j are factors which are located at specific sites in the gen-:
I
jome.
For example, Siegel and Kamel (1974) working with E.
!coli found that the mutant rout Tl gene induces only the
i
!transversion of adenine-thymine to cytosine-guanine.
Epi-
1
!somes are thought to be responsible for induced localized
)genetic instability.
Hill (1963) and Schwartz (1965) pro-
/posed that episomes modifying
the expression of a suppres.
!
i
!sor were responsible for the...... unstable revertants found
in
,.--u-·-· , ,___ , •
~ ••
•
~
•
• <
; , ""'""".,. .• . , ••
·~·•'' •-·'""-'·F•'~
q
"- • "'· •'·-
• -. · · - · "-•-""""
·-·~ v"•· ··~-~~-~'--•-•• •~~ «~·-~-~--~....,_.
~.~~·•--..,-
«<-,..,.• _.~.._.._._..,_ _._~·-·-----~~~...- •.,,._, ------~~-~~~"'""--~"'=..,....~-"''
"-"·-"·''"""-""·"·~'-'•;
52
e ,
!
iity of a mutant without correcting the original mutation.
Il
l.~~n
The working hypothesis regarding reverse mutation
~(JBM
4-13)
is that high frequency of spontaneous re-
'
)version is due to the inherent instability of the gene.
I
,iThis study was undertaken to answer a question which neii
!ther of the previous studies of ~(JBM 4-13) had examined,
I
!namely, whether reversion to wild-type is a property of all
I
!ser(JBM 4-13) progeny.
If the ~(JBM 4-13) site were in-
\
iherently unstable, one would predict that all cultures
I(Would
revert to wild-type.
!
!causing instability at the
If an extrinsic factor were
~(JBM
4-13) site, one would
lexpect to find some stable isolates in which the extrinsic
I
!factor had separated from the ser(JBM
i
l (1975)
4~13~
site.
Bengston·
i
I
j
tested the revertibility of .102 serine' isolates.
J.
!Eighty of the 102 cultures showed reversion to wild-type
I
!while twenty-two appeared to be stable. Sievers (1975)
I
!studied fifty-seven serine containing cultures and observed
I
!reversion in forty-nine, while eight appeared to remain
I
!stable.
I
j
For this study, the eight previously tested and
I
!apparently stable serine cultures of Sievers were used.
j
!under exhaustive testing of repeated serial subcultures
I
lover a six week test period, all eight cultures reverted to:
i
!wild-type.
I
.
This study, unlike those of Bengston and
!Sievers, utilized the testing of repeated.serial subcul-
53
number of nuclei, and thus increased the chances that revertant nuclei would be observed if present. Reversion
l
.
-6
-1
ifrequencies ranged from 1.1 x 10
to 7. 7 x ·10
, and are
l
;in agreement with those observed by Bengston and Sievers.
I
'\
.The results of this study indicate that reversion to wild-
1
'
;type is a property of all ser(JBM 4-13) containing cultures
,I
!suggesting that instability is due to the peculiar nature
I
jof this site and not due to the influence of an extrinsic
imutator.
!Ascospore Studies
Bengston (1975) suggested that reversion of ser
j
i
/(JBM 4-13) occurred in stored ascospores.
During that in-
1
!vestigation, ascospores which had been stored at 7°C for
i
!eight months showed a marked decline in the ratio of serine
l
!
ito wild-type ascospores compared to the ratio obtained from
!
lascospores isolated from the same mating prior to storage
l
(one ~: two ser+ after eight months storage compared to
I
lone ser: one ser+ before storage).
l
-
Additional observations
--
1
imade during that study suggested that reversion in ~(JBM
!
14-13)
vegetative
cultures is time dependent and can occur
I
..
!in the absence of growth. During a three week test period,·
l
jthe number of cultures showing revertant microconidia among
I
lplated cells increased from twenty-four cultures the first
lweek to £orty-two and thirty-nine cultures the second and
I
!third weeks respectively.
l increased
This suggests that revertants
in frequency as a function of time.
In addition,.
54
r·-~----·-·~------·--·---~--,------------·--·---------~-~---.-··-······-~""·'"·~·.
'a portion of a vegetative culture that had a reversion frei
~uency of 8.5 x 10- 4 was placed in a desiccator without
'·
putrients
so that no growth could occur.
!
After one week,
i
lthe reversion frequency had increased to 2. 8 x 10- 3 •
Thus
;reversion seemed to take· place in the absence of growth.
i
l
~other
portion of the same culture that had remained on
l
!the nutrient medium over the same week, showed a reversion·
.
-3
/frequency ·of 2.1 x 10
• No greater amount of reversion
I
:occurred with growth than without growth.
Whereas most models for gene mutation are based
on replication errors during DNA synthesis, in rare in-
!stances investigators have concluded that a small amount of
!
I
•
;mutat~on
can occur without gene replication during periods
l
lof storage.
In both cases the mutational process has a
!
!temperature coefficient characteristic of chemical reac-
l
ltions.
Auerbach (1959) working with Neurospora conidia
I1stored
dry at 4°C and 30°C, observed that lethal conidia
1
I
!
!accumulate in strict linear proportion with time,. and at
l
J4°C the accumulation was much slower than at 30°C.
Conidia
I
!transferred from 4°C to 30°C during the course of the study
I
1
jshowed a faster increase in the accumulation of lethal colnidia than the spores stored continuously at 30°C.
Auer-
l
;
i
)bach suggested that the mutational process involved several
I
steps with different temperature coefficients. Drake (1966) -.
!
I
jobserved a slow accumulation of mutations in bacteriophage
T4 which lacked cellular hosts and thus were metabolically
t
Line :r:--t -~Ci:rlq __ !}() t. --~§.P 1! g9:t i:qg ___tll.§. i._:r:__)_)~ h~-----~.t?Y~:r:..:!::c:t:rl."t§ . .':fl'? r e.. ob-
55
..
.. ··----...~------~------~---~--~--~-----------..·-·-~,..-~--------··-"·---·~-·---~-~----·"·-·---·-··~--·~·
r~ ~-~~
:served to accumulate linearly with time, about twenty times
:faster at 20°C. than at.0°C.
Ryan (1955) observed mutation
1
)from histidineless to prototrophy in E. coli that had
I
- - -
!
Ryan observed ~ew clones of his+ bac-
!ceased multiplying.
I
.
!teria appearing during the stationary phase of parental his
;bacteria in both liquid and agar medium.
During that time
i
i
/there was no increase in the number of parents.
The origin
I
lof the new his+ clones occurred at the rate of about one1
ffortieth the rate observed during growth.
This reduced
·i
:rate of reverse mutation is characteristic of changes obi
I
;served in stored material.
I
In addition, other investiga-
ltions have tested whether DNA replication is necessary for
I
.
;mutat1on_ to occur.
Ryan et al.
(1961) working with
i
.!.·
coli'
lfound that mutations gradually increased over a period of
l
I
i
/time when DNA synthesis was prevented.
These investigators.
I
!suggest that mutations arise from minor and selective baseI
I
•
:pa1r
s ub s t•1tut•1ons taking place in the DNA during starva-
!
I
1
tion, or because a minute amount of DNA replicates and
1
l
1
base-pairing errors occur with an unusually high frequency.!
1j
The purpose of this study was to see if reversion
Jof ~(JBM 4-13) occurred in stored ascospores in the ab!sence of cellular division, and whether reversion is af1 fected by the temperature at which the spores were stored.
I
j I.n
the life cycle of Neurospora crassa the ascospore repre-
l sents
l
a period of dormancy in which no cellular division
; and presumably no DNA
r~plication
is occurring.
Thus the
I
!
1--~~~osp()re .:W.~l:3 ... ~-~;t:~-<?~~9.-~--~g::___~!::~~x. ~-~---'r..~~.!~~-~-~~!~-~~y-~~·--~~-~-'!:~e:.
56
••~-'r"'-'-.'"*-'<-~Y,_,....""'"~-""'"",......_._...-.,_.~_,..,......,_.~.,,,,,
__
,,.,._._...~,_,_.._c..,.__._
...-...,..,.,.,._,,__..,.,....-""...,..-..=_..,.,h-><-....•>r.O_,__ _ ,._............._._,_ _ ,_.,_.
........-,_...,......,__..._., '
--~
·"'"''~·-~•-"-""''""''""'""~~·
_._.,-.,
'amounts of cryptic DNA synthesis, freeze-drying of as cospores was to be utilized.
Rhoades (1970), in an investi-
f
;gation of culture viability of selected molds, yeasts, bact
lteria, and viruses after freeze-drying, reported that out
l
!
:of fifty-two separate species prepared, forty remained vial
\
.ble after twenty years of storage.
Cultures remaining via-
!ble included species of Salmonella, Streptococcus, and
!corynebacteria.
In the present investigation, free.ze-
ldrying of Neurospora ascospores proved to be lethal.
I
Irrune-
!
i
idiately
after the freeze-drying process, ampules containing
i
I
:ascospores were tested.
)
Ascospores which had demonstrated
I
!a
germination frequency of approximately: 80% prior to
I .
I
!freeze-drying, were found to be 100% inviable following
1
jfreeze-drying.
Mazur (1968) has proposed several factors that
l
!could be responsible for the lethality caused by freeze-
!
!drying, freezing, or thawing, which include:
l
1. Lethal freezing or thawing damages the plasma
I:membrane
and/or various internal membranes.
Mazur reports
1
l
jthat yeast cells killed by freezing and thawing are not
1
!physically disintegrated, but rather exhibit marked permei
!
.
.
[ab1l1ty changes.
Intracellular solutes of low molecular
!
!weight enter the surrounding medium, while the cells become'
j
!permeable to solutes that are normally nonpenetrating.
i
I
2. Decreased quantities of liquid water produce
i
!
l
i a progressive increase in the concentration of solutes,
(
iboth within the cell and outside of it.
~
"
'
•
,••
-•
·•·
'
-- •
•
~·-~•·•
.,
·•
,.,,_,,_" •
,~
_
_., .,,
•
~~
''
-
······~•---
·--• • ' ' '
··~·---·~·~ r•<.-~-
_,.,,,..,, •• ,..<,
""~-------....
__
Thus a progressive
__._.,,_,_~-----·-·•·~· -~~~---~·
' • · - - • - · .,,,
·•·•·•· -·•
'••
•
•'
• •
•
57
'decrease in the separation between macromolecules is pro:duced.
The concentration of ions could influence the sta-
1
~ility
of both proteins and DNA, and lead to irreversible
;
)damage.
I
l
3. Freezing and freeze-drying remove much of the
l
i
~ater from a solution.
In many microorganisms, 90% of the
i
Mater can be removed by freezing without much loss in viai
I
:bili ty. The removal of the residual 10% water, which in
I:yeast
i
and E. coli remains unfrozen at temperatures as low
-
'
:as -70°C, is presumably bound to proteins and nucleic acids
;
I
:as waters of hydration.
Experiments to determine which of these explana!
itions might account for the effects of freeze-drying on
l
lascospores will be described in the section entitled "The
j
:Drying Experiment" presented later in this report.
l
Since freeze-drying proved to be lethal to asco-
I
I
!Spores, a method of storage using sterile distilled water
I
iwas employed.
Itstored
·I
Smith (1973) reported that ascospores can be
in sterile distilled water for periods up to one
!
i-
'
i
lyear at room temperature and up to eighteen months at 4°C
I
!without much loss in viability.
Smith observed that mutant
i
!ascospores stored for eighteen months at 4°C showed a loss
\
lin
viability of from 2% to 12% with no detectable change in
I
!viability due to genotype.
It
In the present study ascospores were suspended in'
:sterile distilled water and placed at four different tern.. . peratures.
The results show that reversion of ser -(JBM
4-J3)t
- . . -- ... -- .. --"--------------------------------- --------------"·------------------------------ -·---~- --------~--------------- ---------~~-»-.........
. - ,I
58
'does not occur in stored ascospores.
Ratios of wild-type
j
jversus serine ascospores differed from the expected 1:1
l
!ratio as observed by Bengston {1975).
i
The frequency of
iserine ascospores did not decline during the course of the
I
lstudy for any of the four temperatures as one would expect
!
)if serine were reverting to wild-type.
!
Due to the absence
'
!of reversion in stored ascospores, the effects of temper;
I
jature on spontaneous reverse mutation could not be deteri
'
i
•
;ml.ned.
However, the -l9°C storage temperature was observed
ito be more deleterious to the ascospores in terms of viai'
/bility than the other three temperatures of 4°C, 25°C, and
These results are in marked contrast to those ob!tained by Bengston {1975).
Bengston observed an initial
l
!ratio of serine to wild-type ascospores of 1:1 and later a
li
jl:2 ratio after eight months.
'I
The present study observed
.
Ia serine to wild-type ratio of approximately 1:3 during the'
Itcourse
of the twenty week experiment.
This difference
!could
be resolved by considering the method of random asco!
1
!
!spore isolation.
Prakash {1963) reported that there is a
lrisk of selection due to "differential ripening" of asci
I
i connected
with "differential discharge" of ascospores.
(
jPrakash suggests that the genotypic make-up of an ascus,
!
i along with other developmental factors, may be responsible
for differential maturity.
Thus a perithecium that con-
tains asci at different stages of maturity, may stop dis-
59
!If this is the case, then any sample taken from the ejected
l
,spores may be biased in the genotypes represented.
There-
!
jfore ascospores isolated by Bengston may have been ejected
.!
from perithecia or asci which became mature earlier than
1
1
;others 1 and thus represented a biased sample.
The pro-
'
i
!longed storage period at 7°C could have allowed for the reI
I
lmaining perithecia or asci to become fully mature, and sub-:
I
.
I
lsequently eject a different genotypic proportion of ascol'
!Spores.
In the study reported in this thesis, ali. spores
!
;were collected at one time which eliminated this variable.
:The Drying Experiment
l
In an effort to determine the causes of lethality·
·!
)to Neurospora ascospores as the result 'of freeze-drying,
'
'
1
!the iaeas of Ma~ur (1968) were incorporated into the exper-
l
limental design.
if
.
i reez1.ng
For example, to determine the effects of
..
temperatures on ascospores, ampu 1 es conta1.n1.ng
i;ascospores
suspended in sterile distilled water were placed
!
at -l9°C.
The results show that inviability gradually in-
creases with time in contrast to the immediate inviability
that was observed in freeze-drying.
After one week of
storage at -l9°C ascospores showed a germination frequency
of approximately 65%..
Continued maintenance at this tern-
perature for a twenty week period reduced the germination
frequency to 5%.
Therefore, damage caused to plasma mem-
branes due to freezing and thawing as proposed by Mazur,
cannot be the cause of the immediate inviability seen in
60
..
----------.~~---~--~.~
1
The drying experiment was performed to test
j
I
whether decreased quantities of liquid water influenced the
(stability of DNA and proteins and the ultimate viability of.
!
!ascospores.
Ascospores allowed to dry at room temperature
1
;and at 32°C showed no immediate loss in viability as was
I
/observed following freeze-drying.
Germination frequencies
l
jafter nineteen weeks of dehydrated storage were approxi1
mately 75% for the room temperature dried ascospores, and
1
l
;approximately 73% for the ascospores dried at 32°C.
Thus
I
\decreasing quantities of liquid water in the surrounding
!
!environment seems to have little effect on ascospore via-
l
lbility. Mazur's last proposal for lethality caused by
i
!freeze-drying suggests that removal of residual water from
l
jcells by in vacuo treatment and maintenance is deleterious.
!
iin the present study, ascospores which had been dehydrated
I
/at 32°C for twenty weeks were placed in vacuo for a twelve
Iihour
period before testing. All ascospores tested follow~
I
jing this treatment were found to be inviable. Therefore,
jlethality in Neurospora crassa ascospores caused by freeze-:
i
jdrying is probably due to the removal of a critical volume
II
!of water by in vacuo treatment.
l
!The Status of Ser(JBM 4-13)
j
I.
1 t~ve
,.
The reversion of the mutant gene in all vegetacultures tested exhaustively provided evidence that
Jhigh frequency of spontaneous reversion is due to inherent
'
l
.
.:
1...-~--~-_,~ ... -L'.-_..,..,.,....... ~......... ~ ,.._.......,__~-·----~-·----·~-""'=-~--------~--~-"'-~,..~~---~·---__,_..~-~~~-~·,_ _,.,_~.,·
61
instability in the gene.
Two results obtained by Bengston
'(1975) suggested that reversion could occur in the absence
!
~f cell division.
-The ascospore study reported here showed
j
jthat reversion does not occur in dormant, nondividing cells
i
!indicating
that cell growth is necessary for the reversion
I
.
!of
~(JBM 4-13).
l
An alternate hypothesis to explain the high fre-
quency of spontaneous reversion observed in ser(JBM 4-13)
l
would be that revertant nuclei have an increased chance for
l
i
l
:survival compared to mutant nuclei.
The results of conid-
1ial viability studies conducted on the eight original nonreverted cultures, suggested that there might be an increased conidial survival in cultures which reverted to
.ser+, met.
Culture MS 138 demonstrated
a conidial viabil-
ity of 9.2% and 18.7% prior to reversion.
When revertants
were first observed in this culture, the conidial viabilitY,
increased to 37.7%.
A similar increase was noted for five
of the eight cultures at the time of reversion.
A study was undertaken to determine if revertant
cultures showed increased conidial viability compared to
mutant cultures.
The results obtained indicate that re-
vertant nuclei do not have a higher viability when compared
to mutant nuclei.
In some cases the revertant culture dem-
onstrated a higher percentage of viable conidia, while in
other cases the mutant culture showed the greater
viabilit~
Thus selected survival of revertant nuclei compared to mu-
62
tv8rsTon.~·£i::eCi'uenci.es~~>c;t;5"erve·<r·Tn-se-x:-c:Ji3M-··4..:·i3f':~~····-···~-··-·~--"'-·-""~""·
l
---
!
Recent studies by Joyce B. Maxwell (unpublished)
l
!indicate that high frequency of spontaneous reversion obli
lserved in ~(JBM 4-13) cultures may not be due to gene in-·
I
:stability alone.
iI
Maxwell's experiment was designed to test
;nuclear competition between serineless and revertant nuclei
lat various concentrations of serine supplement; in partie-
!jular,
the possibility was tested that a positive selection
!
jfor growth of revertant nuclei might be operating at the
i
I
iconcentration of serine used routinely for culture mainte-
i
I
;nance and study.
In a similar experiment 1 Ryan and Leder-
'i
[berg (19 46) working with a leucineless strain of Neurospora
j
:crassa, found that in a heterocaryon between the leucine/
jless and wild-type or revertant strains, the leucineless
I
inuclei had an advantage in the presence of leucine.
The
1
i
[reversion frequency was higher in the presence of low conI
lcentrations of leucine than in the presence of high concen-
!
i t rat1ons.
.
1
In Maxwell 1 s experiment, a known number of mu-
I
ltant conidia were .added to a known number of revertant co/nidia, thus establishing a certain input nuclear ratio.
i
~The
culture initiated from the mixed conidia was allowed to
!grow for one week at 25°C.
Conidial suspensions were pre-
!
!pared from the one week old cultures were plated onto minI
limal medium plates and minimal medium plates supplemented
j
!
!with 0.4 mg/ml, 1.0 mg/ml, or 2.0 mg/ml of L-serine.
The
t
!results obtained indicated that during growth on low coni
i centrations of serine (0.4 mg/ml) the revertant to mutant
63
~~ti·~·--i;;~;;~·~~-~d:·"""a·o a·:F;id.-;-;h·ii;--~~t···-;h;·h'igh~;t~.t~;.;~-~~·t;~:·······
tion of serine used (2.0 mg/ml) the revertant to mutant
i
~atio increased less than 2-fold.
Based on these findings,
:it is apparent that all previous investigations of
~(JBM
.4-13) were carried out at serine concentrations which would
i
;favor the revertant nuclei over the mutant nuclei.
Thus
I
[the high reversion frequencies obtained may be explained by
l
i
!strong selection for rare revertant nuclei which arise in
I
.
I
dividing mutant cultures.
If selection accounts for the
!I
:unusual reversion frequencies observed in the studies of
'ser(JBM 4-13), the absence of reverse mutation in stored
'
'
;ascospores is explained.
I'
!l
BIBLIOGRAPHY
Auerbach, Charlotte, 1959 Spontaneous Mutations in Dry
Spores of Neurospora crassa. z. Vererbungslehre
90 : 335-346.
,Barnett, W.E., and F.J. de Serres, 1963 Fixed Genetic In.
stability in Neurospora crassa. Genetics. 48: 717-723
,Bengston, R., 1975 I. The Analysis of Spontaneous Reversion in a Serine Auxotroph of Neurospora Crassa. II.
Linkage Data on a New Serine-Requiring Mutant: Ser-5.
Masters Thesis, California State University, Northridge, California.
Davis, Rowland H., and F.J. de Serres, 1970 Edited by
Herbert and Celia vlliite Tabor. Genetic and Microbiological Research Techniques for Neurospora crassa.
~ethods in Enzymology.
Academic Press, Inc., New York
17 : 79-143.
Drake, John W., 1970 The Molecular Basis of Mutation,
Holden-Day, San Francisco. 273 pages.
Drake, John W., 1966 Mutations Occurring in Bacteriophage
T4 in the Complete Absence of DNA Replication. Proc •
. Nat. Acad. Sci. 22 : 738-743.
:Freese, Ernst, 1959 The Difference Between Spontaneous and
·
Base-Analogue Induced Mutations of Phage T4. Proc.
Nat. Acad. Sci. 45 : 622-633.
I
'Giles, N., Jr., 1951 Studies on the Mechanism of Reversion
:
in Biochemical Mutants of Neurospora crassa. Cold
Springs Harbor Symposia Quant. Biol. 16 : 283-313.
Hill, Ruth F., 1963 The Stability of Spontaneous and Ultra
violet Induced Reversions From Auxotrophy in Escheri~ coli.
J. Gen. Microbiol. 30 : 289-297.
64
65
Kline, Fereshteh, 1973 Biosynthesis of Serine in Neurosuora £Lassa. Masters Thesis, California State
University, Northridge, California.
[Massey, F. J., Jr., and W. J. Dixon, 1969 Introduction to
Statistical Analvsis, McGraw-Hill, New York, 3rd Ed.,
638 pages.
.Maxwell, Joyce B., unpublished·data; personal communication
'
·Mazur, Peter, 1968 Survival of Fungi After Freezing and
Desiccation, in the Fungi, An Advanced Treatise, Ed.
by G. c. Ainsworth, A. s. Sussman, Vol. III : 325-394.
Prakash, V., 1963 Random Ascospore Isolation, Neurospora
Newsletter 3 : 10-11.
:Rhoades, H. E., 1970 Effects of Twenty Years Storage on
Lyophilized Cultures of Bacteria, Molds, Viruses, and
Yeasts, Am. J. Vet. Res. 31, 10 : 1867-1870.
Ryan, F.J., and Joshua Lederberg, 1946 Reverse-Mutation
and Adaptation in Leucineless Neurospora, Proc. Nat.
Acad. Sci. 32, No. 6 : 163-173.
fRyan, Francis J., 1955 Spontaneous Mutation in Nondividing
'
Bacteria, Genetics 40 : 726-738.
:Ryan, F.J., D. Nakada, and M.J. Schneider, 1961 Is DNA Replication a Necessary Condition for Spontaneous Mutation.
z. Vererbungslehre 92 : 38-41.
Schwartz, N., 1965 Genetic Instability in Escherichia coli.
J. Bacteriology 89 : 712-717.
Siegel, E.C., and F. Kamel, 1974 Reversion of Frameshift
Mutations by Mutator Genes in Escherichia coli.
J. Bacteriology 117 : 994-1001.
•sievers, M., 1975 Genetics of An Unstable J:..iutant of Neurospora crassa. Masters Thesis, California State
University, ~orthridge, California.
,Smith, B.R., 1973 Storage of Ascospores in Water, Neurospora Newsletter 20 : 34.
'Sussman, A.S., 1966a Types of Dormancy as Represented by
Conidia and Ascospores of Neurospora, Proc. Symp. Col.
Res. Soc. 18 : 235-257.
66
Sussman, A.S., 1966b Dormancy and Spore Germination, in
the Fungi, An Advanced Treatise, Ed. by G.C. Ainsworth
and A.S. Sussman, Vol. II : 733-764.
jSussman, A.S., and H.O. Halverson, 1966 Spores, Their
'
Dormancy and Germination. Harper and Row, New York,
354 pages.
;vogel, H.J., 1956 A Convenient Growth Medium for Neurospora crassa (Medium N). Microbial. Genet. Bull.
13 : 42.
!Wellman, A.M. and D.B. Walden; 1971 Long Term Cryogenic
·
Storage of Neurospora crassa Spores. Cryobiology
8 : 566-569
'
;Westergaard, M., and H.K. Mitchell, 1947 Neurospora V.
A Synthetic Medium Favoring Sexual Reproduction.
Am. J. Bot. 34 : 573.
\