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Mating types in cellular slime molds
BIOCHEMICAL
Vol. 52, No. 2, 1973
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATING TYPES IN CELLULAR SLIME MOLDS
Mary
Anne Clark*, D. Francis' and R. Eisenberg' *Department of Biology University of Texas, Arlington, Texas 76010 and A Department of Biological Sciences University of Delaware, Newark, Delaware 19711
Received
April
6,
1973 SUMMARY
We have discovered mating types in three species of cellular slime mold. One of these species is Dictyostelium discoideum, hitherto the subject of extensive biochemical investigations, and now amenable to genetic studies.
The cellular investigate
slime
the biochemistry progress
nately,
in this
sence of a practical however,
molds have been used by many workers
phase in the
life
direction
system
have recently cycle
development
of the macrocyst,
Very
meiosis
fact
led
us to
strains
of basically
results
of a search
slime
molds. Cellular
existence
before
from nature
mold,
heterothallic
might
organisms. types
Polysphondylium forms
in five
during struc-
of the macrocyst. slime
molds which
form macrocysts
these
be rare
homothallic
We report species
(3,4).
of
here the cellular
Methods slime
molds were isolated 672
from
(2),
sexual
thick-walled
germination
of cellular
ab-
Raper -et al.
shows a zygote
actually
suppose that
mating
slime
by the
of a true
a multicellular,
occurs
for
analysis.
evidence
few of the many strains
have been isolated This
the
cytological
Unfortu-
has been hindered
of a cellular
Their
and that
differentiation.
of genetic
reported
violaceum.
ture,
of cell
to
soils
of woodlands
BIOCHEMICAL
Vol. 52, No. 2, 1973
near
Newark,
(5),
except
strate
Delaware that
violaceum
growing
on Escherichia
it
the plate
bating
for
standard. way. all
with
Most
pairs
above;
latter
case,
were mixed
the
presence
in flask
in
0.25
macrocyst
__ coli
growing
culture
of macrocysts
flood-
and incuare
in this mixed
in
formation.
by inoculating
spores
on cerophyll slides
agar
amebae
by Gerisch's
method.
(8)
2~10~ amebae of each member of
ml of Bonner's
work equally
were then
in depression
approximately
(7)
when tested
for
by
then
These procedures
sterile
and by mixing
plate,
solution
(2,4).
on g.
well. after
Two or more independent
27OC.
salt
tested
Each isolate
to make macrocysts
of each species
together
10 Polysphondylium
on a cerophyll
self
and Raper
used as a sub-
were isolated.
were made in two ways:
two methods usually for
are
Cavender
11 Dictyostelium
ability
dark
and again
had been pregrown
pair
of
Banner's
isolates
two isolates
as described
the
coli
isolates
These mixtures
In the
its
3-4 days in the
possible
which
for
of
mucoroides,
clones
sterile
Self-sterile
of the
25 g.
and 9 2. pallidum
RESEARCH COMMUNICATIONS
was routinely
(6)
A total
8 Q. purpureum,
individually
technique
agar
of hay agar.
was tested
ing
the
Cerophyll
instead
discoideum,
using
AND BIOPHYSICAL
solution.
The mixtures
The
were scored
days in the
3-4
repeats
salt
dark
at
of each experiment
22-
were
performed. Results
Dictyostelium species
and Discussion
purpureum.
are shown in Table
1.
any of
ing
form no macrocysts
Since union,
these
the
formation
strains
of mixtures
Macrocysts
of DEP2 with isolates
isolates
Results
DEPI,
only
DEP6 or DEPT.
alone
of macrocysts
may be classified
673
form
with
this
in mixtures
The 4 remain-
or in any combination. is
the result
in two mating
of a sexual types:
Vol. 52, No. 2, 1973
Table
BIOCHEMICAL
1.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Formation of macrocysts in mixtures of D. - purpureum isolates
DEP2 DEP2
DEP6
DEPl
0
DEPl
DEP'I
@
t
t
0
0
0
0
0
DEP6 DEP7
0
0 - no macrocysts observed al - macrocysts sparse or irregular t - macrocysts numerous
Isolates DEP3, DEP4, DEP5, and DEP8 made no macrocysts in any combination.
mating
type
2.
I
species
g.
purpureum.
of
6 others
The pattern
indicates One isolate,
(Del,
than
structure
those (Fig.
Shortly erroneous)
a third
sent
two mating
De5,
among 11 isolates
types
exist
forms macrocysts De8).
make macrocysts.
DEP6, DEP7).
here,
only
Three
with
other
These macrocysts
but
are of
similar
as in each isolates
are much
internal
1).
after results
us his
mating
DEPl,
of interaction
of g. purpureum,
-D. discoideum
kindly
that
(isolates
De3, De6, De7, Dell,
De4, Deg) never
larger
with
DEP2) and II
discoideum.
of this
(De2,
(isolate
type
we had communicated to
Professor
K. B. Raper,
were begun in his findings,
our early
which
may be present
674
(and in fact,
similar
laboratory.
extend in g.
ours
experiments He has very
in indicating
discoideum
(9).
that
Vol. 52, No. 2, 1973
Fig.
1.
BIOCHEMICAL
Macrocysts. After g After -* Scale
g. Gcoroides. far
failed
to
abundant
demonstrate
tensive
in
inhibit
isolates,
(Table
2).
induced
formation exists
Details
have
make
and slowly
when
or more ex-
2).
One strain self-sterile
when mixed
self-fertile
respect
with
to this
interaction
so
form
the more nearly
with
of this
species
Nineteen
to quicker (Table
(DEPlxDEP2). (DelxDe5).
of 25 isolates
sparsely,
Further,
macrocyst
this
Five
in any combination
and a hierarchy
ability
types.
irregularly,
made macrocysts.
isolates
with
when grown alone.
and are not
development
has never
studies
mating
3 days
more or less
grown in isolation
RESEARCH COMMUNICATIONS
four days in JJ. purpureum four days in D. discoideum = 30 u.
Exhaustive
macrocysts
macrocysts
AND BIOPHYSICAL
will
inhibitory be published
elsewhere. Polysphondylium types
were
found
cross-reactions
violaceum.
In this
in each of two groups occur
between
pattern
of reactions
indicates
species
2.
consists
violaceum
species of
isolates
members of different that
the
single
of two syngens,
675
a pair
of mating
(Table groups.
3).
NO
This
morphological or breeding
species.
Vol. 52, No. 2, 1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
2
Formation of macrocysts of D. mucoroides
Group Isolate
No.
12
in mixtures isolates
a
b
C
d
e
f
g
h
i
12
4
3
g
2
25
21
22
20
t
t
t
t
t
t
t
t
t
$
t
t
t
t
t
t
0
t
t
t
t
t
0
0
t
t
t
0
0
0
t
0
fB
0
0
0
0
0
0
0
0
0
0
0
4 3 9 2 25 21 22
0
20
group
a. - isolates
1, 10,
group
b. - isolates
4,
group
c. - isolates
3, 7, 8
group
d. - isolate
9
group
e. - isolate
2
group
f.
group
g. - isolates
15, 16,
group
h. - isolates
17,
22
group
i.
13,
20
- isolates
Mating
may occur
in the
case of
gated
24
25 21
each syngen,
Paramecium
23,
(10).
This
but
not
situation
between
syngens,
is being
as
investi-
more fully. 2.
tion
within
14,
5, 6, 19
18,
- isolates
12,
pallidum.
of isolates
No macrocysts in
this
species.
have yet
been seen in any combina-
Vol. 52, No. 2, 1973
BIOCHEMICAL
AND BIOPHYSICAL
Table Formation
of macrocysts
RESEARCH COMMUNICATIONS
3
in mixtures
of p.
violaceum
DFl
DF2
DF4
DF5
D5’7
0
t
@
t
t
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
t
t
0
0
DFl DF2 DF4 DF5 DF7
i
DF6
isolates
DF6
MAC1
MAC1
MAC2
MAC2
0
Symbols as in Table 1. Isolates DF3 and MAC3 make no macrocysts
Currently nature.
under
That
by the
fact
isolated
study
breeding
that
DF2 of -P. violaceum
these
ranges
- multiple
discoideum, control large
g.
from single allele
does occur
of forest
type
is
floor
II.
Also,
mating
type
in
suggested
I of g. discoideum
manner of mating
control
was
as strains
strains
Del,
DFl and
were isolated
and p.
that
types
- one allele
from
violaceum locus
inheritance in
fungi
systems
Our results
(10,ll).
at a single isolates
type
of mating
locus
systems
purpureum,
of
type
exchange
sample.
Genetic
by 2 alleles proportion
meter
of mating
know the
organisms.
protozoa loci
yet
of genetic
conditions
and of opposite
10 gram soil
We do not
frequency
De5 of mating
same square
De6, De7, De9 and Dell
a single
the
in natural
strain
from the
is
in any combination.
with
each syngen.
do not make macrocysts
677
and
to multiple
are consistent
within
in
2. with The in any
Vol. 52, No. 2, 1973
combination however.
suggests
that
simple
Until only
slime
molds,
tool
by means of
investigation
great
of
interpretation
cell
on this
is
i;Ici,n!>.!,::<;j,
point. D. disccideum, -
biochemical
analysis
of develop-
in this
organism
has been pos-
analysis
a most cumbersome parasexual
of a complete be of
RESEARCH COMMUNICATIONS
and particularly
in the
now, genetic
The finding molds will
this
investigations
have become a major
sible
AND BIOPHYSICAL
We continue
The cellular
ment.
BIOCHEMICAL
sexual
practical
system value
system
in the cellular in permitting
(1).
slime genetic
differentiation. Acknowledgments
This ration
work was supported
to M. A. Clark,
and by a UNIDEL grant the
University
critically
by NIH grant to the
of Delaware.
reading
by a grant
from the
Research
No. GM 19003-02
Department We thank
of Biological Dr.
Walter
Corpo-
to D. Francis, Sciences
Vincent
of
for
the manuscript. References
2.
11.
Katz, E.R., and Sussman, M., Proc. Nat. Acad. Sci. @, 495 (1972). Erdos, G.W., Nickerson, A.W., and Raper, K.B., Cytobiologie 6, 351 (1973). Blaskovics, J.C., and Raper, K.B., Biol. Bull. 113, 58 (1957). Filosa, M.F., and Chan, M., J. Gen. Microbial. 71, 413 (1972). Cavender, J.C., and Raper, K.B., Amer. J. Bot. 52, 294 (1965). Jones, W.R., III, and Francis, D., Biol. Bull. E, 461 (1972). Bonner, J.T., J. Exp. Zool. 106, 1 (1947). Gerisch, G., Naturwiss. 3, 6% (1959). Raper, K.B., et al., manuscript in preparation. -7 Sonneborn, T.M. In Mayr, E., ed., The Species Problem, pp. 155-324. AAAS, Washington(195r Esser, K. and Kuenen, R. Genetics -of Fungi. Springer-Verlag, New York (1967).
678
Vol. 52, No. 2, 1973
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATING TYPES IN CELLULAR SLIME MOLDS
Mary
Anne Clark*, D. Francis' and R. Eisenberg' *Department of Biology University of Texas, Arlington, Texas 76010 and A Department of Biological Sciences University of Delaware, Newark, Delaware 19711
Received
April
6,
1973 SUMMARY
We have discovered mating types in three species of cellular slime mold. One of these species is Dictyostelium discoideum, hitherto the subject of extensive biochemical investigations, and now amenable to genetic studies.
The cellular investigate
slime
the biochemistry progress
nately,
in this
sence of a practical however,
molds have been used by many workers
phase in the
life
direction
system
have recently cycle
development
of the macrocyst,
Very
meiosis
fact
led
us to
strains
of basically
results
of a search
slime
molds. Cellular
existence
before
from nature
mold,
heterothallic
might
organisms. types
Polysphondylium forms
in five
during struc-
of the macrocyst. slime
molds which
form macrocysts
these
be rare
homothallic
We report species
(3,4).
of
here the cellular
Methods slime
molds were isolated 672
from
(2),
sexual
thick-walled
germination
of cellular
ab-
Raper -et al.
shows a zygote
actually
suppose that
mating
slime
by the
of a true
a multicellular,
occurs
for
analysis.
evidence
few of the many strains
have been isolated This
the
cytological
Unfortu-
has been hindered
of a cellular
Their
and that
differentiation.
of genetic
reported
violaceum.
ture,
of cell
to
soils
of woodlands
BIOCHEMICAL
Vol. 52, No. 2, 1973
near
Newark,
(5),
except
strate
Delaware that
violaceum
growing
on Escherichia
it
the plate
bating
for
standard. way. all
with
Most
pairs
above;
latter
case,
were mixed
the
presence
in flask
in
0.25
macrocyst
__ coli
growing
culture
of macrocysts
flood-
and incuare
in this mixed
in
formation.
by inoculating
spores
on cerophyll slides
agar
amebae
by Gerisch's
method.
(8)
2~10~ amebae of each member of
ml of Bonner's
work equally
were then
in depression
approximately
(7)
when tested
for
by
then
These procedures
sterile
and by mixing
plate,
solution
(2,4).
on g.
well. after
Two or more independent
27OC.
salt
tested
Each isolate
to make macrocysts
of each species
together
10 Polysphondylium
on a cerophyll
self
and Raper
used as a sub-
were isolated.
were made in two ways:
two methods usually for
are
Cavender
11 Dictyostelium
ability
dark
and again
had been pregrown
pair
of
Banner's
isolates
two isolates
as described
the
coli
isolates
These mixtures
In the
its
3-4 days in the
possible
which
for
of
mucoroides,
clones
sterile
Self-sterile
of the
25 g.
and 9 2. pallidum
RESEARCH COMMUNICATIONS
was routinely
(6)
A total
8 Q. purpureum,
individually
technique
agar
of hay agar.
was tested
ing
the
Cerophyll
instead
discoideum,
using
AND BIOPHYSICAL
solution.
The mixtures
The
were scored
days in the
3-4
repeats
salt
dark
at
of each experiment
22-
were
performed. Results
Dictyostelium species
and Discussion
purpureum.
are shown in Table
1.
any of
ing
form no macrocysts
Since union,
these
the
formation
strains
of mixtures
Macrocysts
of DEP2 with isolates
isolates
Results
DEPI,
only
DEP6 or DEPT.
alone
of macrocysts
may be classified
673
form
with
this
in mixtures
The 4 remain-
or in any combination. is
the result
in two mating
of a sexual types:
Vol. 52, No. 2, 1973
Table
BIOCHEMICAL
1.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Formation of macrocysts in mixtures of D. - purpureum isolates
DEP2 DEP2
DEP6
DEPl
0
DEPl
DEP'I
@
t
t
0
0
0
0
0
DEP6 DEP7
0
0 - no macrocysts observed al - macrocysts sparse or irregular t - macrocysts numerous
Isolates DEP3, DEP4, DEP5, and DEP8 made no macrocysts in any combination.
mating
type
2.
I
species
g.
purpureum.
of
6 others
The pattern
indicates One isolate,
(Del,
than
structure
those (Fig.
Shortly erroneous)
a third
sent
two mating
De5,
among 11 isolates
types
exist
forms macrocysts De8).
make macrocysts.
DEP6, DEP7).
here,
only
Three
with
other
These macrocysts
but
are of
similar
as in each isolates
are much
internal
1).
after results
us his
mating
DEPl,
of interaction
of g. purpureum,
-D. discoideum
kindly
that
(isolates
De3, De6, De7, Dell,
De4, Deg) never
larger
with
DEP2) and II
discoideum.
of this
(De2,
(isolate
type
we had communicated to
Professor
K. B. Raper,
were begun in his findings,
our early
which
may be present
674
(and in fact,
similar
laboratory.
extend in g.
ours
experiments He has very
in indicating
discoideum
(9).
that
Vol. 52, No. 2, 1973
Fig.
1.
BIOCHEMICAL
Macrocysts. After g After -* Scale
g. Gcoroides. far
failed
to
abundant
demonstrate
tensive
in
inhibit
isolates,
(Table
2).
induced
formation exists
Details
have
make
and slowly
when
or more ex-
2).
One strain self-sterile
when mixed
self-fertile
respect
with
to this
interaction
so
form
the more nearly
with
of this
species
Nineteen
to quicker (Table
(DEPlxDEP2). (DelxDe5).
of 25 isolates
sparsely,
Further,
macrocyst
this
Five
in any combination
and a hierarchy
ability
types.
irregularly,
made macrocysts.
isolates
with
when grown alone.
and are not
development
has never
studies
mating
3 days
more or less
grown in isolation
RESEARCH COMMUNICATIONS
four days in JJ. purpureum four days in D. discoideum = 30 u.
Exhaustive
macrocysts
macrocysts
AND BIOPHYSICAL
will
inhibitory be published
elsewhere. Polysphondylium types
were
found
cross-reactions
violaceum.
In this
in each of two groups occur
between
pattern
of reactions
indicates
species
2.
consists
violaceum
species of
isolates
members of different that
the
single
of two syngens,
675
a pair
of mating
(Table groups.
3).
NO
This
morphological or breeding
species.
Vol. 52, No. 2, 1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
2
Formation of macrocysts of D. mucoroides
Group Isolate
No.
12
in mixtures isolates
a
b
C
d
e
f
g
h
i
12
4
3
g
2
25
21
22
20
t
t
t
t
t
t
t
t
t
$
t
t
t
t
t
t
0
t
t
t
t
t
0
0
t
t
t
0
0
0
t
0
fB
0
0
0
0
0
0
0
0
0
0
0
4 3 9 2 25 21 22
0
20
group
a. - isolates
1, 10,
group
b. - isolates
4,
group
c. - isolates
3, 7, 8
group
d. - isolate
9
group
e. - isolate
2
group
f.
group
g. - isolates
15, 16,
group
h. - isolates
17,
22
group
i.
13,
20
- isolates
Mating
may occur
in the
case of
gated
24
25 21
each syngen,
Paramecium
23,
(10).
This
but
not
situation
between
syngens,
is being
as
investi-
more fully. 2.
tion
within
14,
5, 6, 19
18,
- isolates
12,
pallidum.
of isolates
No macrocysts in
this
species.
have yet
been seen in any combina-
Vol. 52, No. 2, 1973
BIOCHEMICAL
AND BIOPHYSICAL
Table Formation
of macrocysts
RESEARCH COMMUNICATIONS
3
in mixtures
of p.
violaceum
DFl
DF2
DF4
DF5
D5’7
0
t
@
t
t
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
t
t
0
0
DFl DF2 DF4 DF5 DF7
i
DF6
isolates
DF6
MAC1
MAC1
MAC2
MAC2
0
Symbols as in Table 1. Isolates DF3 and MAC3 make no macrocysts
Currently nature.
under
That
by the
fact
isolated
study
breeding
that
DF2 of -P. violaceum
these
ranges
- multiple
discoideum, control large
g.
from single allele
does occur
of forest
type
is
floor
II.
Also,
mating
type
in
suggested
I of g. discoideum
manner of mating
control
was
as strains
strains
Del,
DFl and
were isolated
and p.
that
types
- one allele
from
violaceum locus
inheritance in
fungi
systems
Our results
(10,ll).
at a single isolates
type
of mating
locus
systems
purpureum,
of
type
exchange
sample.
Genetic
by 2 alleles proportion
meter
of mating
know the
organisms.
protozoa loci
yet
of genetic
conditions
and of opposite
10 gram soil
We do not
frequency
De5 of mating
same square
De6, De7, De9 and Dell
a single
the
in natural
strain
from the
is
in any combination.
with
each syngen.
do not make macrocysts
677
and
to multiple
are consistent
within
in
2. with The in any
Vol. 52, No. 2, 1973
combination however.
suggests
that
simple
Until only
slime
molds,
tool
by means of
investigation
great
of
interpretation
cell
on this
is
i;Ici,n!>.!,::<;j,
point. D. disccideum, -
biochemical
analysis
of develop-
in this
organism
has been pos-
analysis
a most cumbersome parasexual
of a complete be of
RESEARCH COMMUNICATIONS
and particularly
in the
now, genetic
The finding molds will
this
investigations
have become a major
sible
AND BIOPHYSICAL
We continue
The cellular
ment.
BIOCHEMICAL
sexual
practical
system value
system
in the cellular in permitting
(1).
slime genetic
differentiation. Acknowledgments
This ration
work was supported
to M. A. Clark,
and by a UNIDEL grant the
University
critically
by NIH grant to the
of Delaware.
reading
by a grant
from the
Research
No. GM 19003-02
Department We thank
of Biological Dr.
Walter
Corpo-
to D. Francis, Sciences
Vincent
of
for
the manuscript. References
2.
11.
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