Download The heterocaryon is inoculated into

The
heterocaryon
is inoculated
nutritional
element
by the slime
parent
sorbose
to
plats
the
which
and
the
standard
ore
when
colonies
ore
Fox,
the
to
filter
D. J.
is plated
and
of high
in part
media
growth
medium
requirement
time
some
may
spheroplosts
be expected
N.S.
Biology,
Rapid
F.
California
Institute
of
light
from
a Black
the
other
We
have
the
original
con
measured
be
make
found
it
This
principally
when
and
the
particular
Using
Laboratories,
this
table,
corrections
The University,
Laycock,
M.
D.
Boulter.
crosso
H. G.
The
separation
ore
acids,
it
with
numbers
large
from
this
after
50%
hove
of
and
gel
which
by
in
-
136,
to
work
may
incubation
out
gels
according
by
be expensive
time
several
Thurman
vividly,
This
resulting
will
to
the
Distillation
and
due
into
method
of
Ornstein
(Fox,
o Thunberg
Industries,
Thurman
and
Eastman
and
the
com-
o pole
blue
spectrophotometer.
Once
interference
the
localized
from
extinction
at
Z.
to construct
310,
280
384,
1941).
o Nomogram
observed
E280
and
absorption
the
from
E2@
any errors
mg.
protein
Boulter,
peaks.--The
to the
arising
by the
is in fact
due
conversion
electrophoresis
technique
tube.
Davis
Kodak
and
Co.,
Biochem.
four
ond
1962)
J.,X,
Science
The
gel
of
iroenzymes.
results
starch
adenine
d) ease
Polyacrylamide
(Omstein
(Tsoo,
using
advantages
electrophoresis
min.
os opposed
to E-20
hr.
volume
of incubation
medium
nicotinamide
bonds,
least
similar
over
Botanical
homogenates
electrophoresis.
time
is 40
only
a small
for
at
reports
gel
latter
defined
Trao
that
contain
communication
the
Hartley
demonstrated
crosso
requirement
clearly
fit
is
This
it has been
under
ultra-violet
Biochem.
when
elution
columns
when
cases
to the
thus eliminating
conversion
to
gel
1962)
electrophoresis
and require
to the
in sharp,
readily
Products
Boulter
are a)
ore smaller
the
usually
to meowre
some
starch
42,
Neurospora
electro-
studies
with
dehydrogenoses
b) polyacrylomide
gels
this
since
Fox,
mode
Using
Neurospora
California.
laborious
Christian,
of o
Grant,
establishing
In such
examination
converting
polyacrylamide
c) shorter
for
fractions,
of
Inasmuch
purification.
of
Preprinted
o table
been
of malic
polyocrylamide
for enzyme
using
starch,
carried
of
stand
chromatographic
in a spectrophotometer.
Owing
and
o pure
o U.S.P.H.S.
with
on S. P. 500
in pyrex
test tubes.
would
hove
given,
inherent
in direct
determined
by
very
fluoresce
is customary
(Worburg
in
components.
Pasadena,
curve.
visual
spectrophotometer.
phoresis.
tally
proteins
is measurable
ore contained
correction
and
of over
Liverpool.
Kblmark
isoenzymes
by
nucleic
figures
that
the
component
alone
acid
composition
proteins
V.,
dehydrogenase
The
in
dealing
Christian
protein
amino
port
become
elution
by rapid
sphero-
wool
supple-
osmotic
concentration,
the slime
component
procedure
con
on the
fractions
dark.
accurately
profitable,
in
equation.
The only
assumption
here
is that the extraneous
their
components.
time
be converted
to mg.
protein
E280 may ot ony future
or
corrected
for
in the
o Worburg-Christian
Warburg
Warburg-Christian
nucleic
acids
lamp
separated
any concentration
even
when
the fractions
compounds,
rnp and
value
of E280 that the
the variation
of aromatic
factor
U. V.
detect
absorbing
260
Glass
the eye con
is satisfactory
absorption
and
and ore widely
the protein-containing
both
Thigh
in only
position
of proteins
from
to measure
the E280 mp
procedure
color,
and
This method
results
os in
Technology,
customary
and
glass
Persistent
slime
growth
of the sphero-
further
frequently
ond
that
required
IO per cent
hyphae
component.
carried
GT6174,
The
heterocaryotic
to permit
oil
in the
with
by adding
by filtration
through
osmotic
concentration,
in liquid
media
if os is present
Grant,
locali-
of
slime
as this
hove
lacking
supplemented
obtained
is removed
of standard
ploting,
chromatogmphic
ore few
in number
possible
to localize
concentration,
consists
of the
tested
eluotes.
ponents
found
osmotic
advantageous
before
so far
by on
of
D.
liquid
Hyphol
on agor
It is sometimes
produce
described
thot
Boulter,
in
on
growth
plates.
)--Division
of proteins
the
a second
(Supported
GM-0965.
in such
heterocaryons
t-es
from
heterocoryon.
zation
from
Caution:
os pure os strains
behovioaifferent
medium
composition.
to sotisfy
and
slime.
N. 1. H.
in
of spheroplosts
picked
a liquid
Growth
slime
necessary,
suspension,
persistent
into
by the hyphol
parent
of the heterocoryon
but
one.
High
osmotic
concentration
is ordinarily
medium.
largely
suspension
mented,
plost
required
if it has
of
gel
dinucleotide,
incubation
electrophoresis
onaerobiwas
Davis,
Disc Electrophoresis,
with
the modification
introduced
29,
1963).
from
to
Neurospora
25”.
crassa
About
squeezed
I g.
out
(74A)
fresh
through
tion
medium
in
The
were
incubation
Three
blue
strong
but
bands
above
below
one
pH
weak
with
four
the
or
presence
showed
that
the
The
following
may
Neurospora
Department
by E.
Gowda,
of
Barry,
J.
buted.
Methods
other
original
laboratories,
source
that
Biological
Sciences,
these
have
P. St.
Singleton,
been
or
extracts
from
it
may
geared
to using
Neurosporo,
documented.
This note
has
N.
Murray
may
be described
I) Standard
and
standards
No.
for
532).
Two
for
highly
inbreeding
They
fluffy
(flp:
-2)
Biotin
supplied
were
ore
(linkage
and
for
from
group
scoring
is that
No.
Crosser:
concentration
as K2HPO4
The
no
and
synthetic
has been
and
KH2P04
are
min.
odenine
which
stained
present
complete
the
The
dinucleo-
of solution
with
at
the
and
methylene
front.
incubation
malic
formazan
but
bonds
further
No
medium
on
work
dehydrogenase
It is designed
incubated
is necessary
isoenzymes.--The
to be
only.
methods
listed
notebook
in
This
published
below
the
are
to others,
this
laboratory
has
D.
Ishitani,
from
are
Lawrence
F.
( see
been
shared
sources,
many
laboratories
of summary
is worthwhile,
and
(FGSC
NN
NO.
-2:25),
are
at various
of them
not
homozygous
feel
already
or incompletely
prepared
by
Brockman
and
in
the
we
additional
H. E.
986)
times
Bolecontri-
credit
properly
for
to the notebook,
in
de Serres
a work-
Perkins,
Newmeyer,
B.N.
all of whom
have
though
undocumented
involved;
it was
J.
from
D.
even
people
74-OR23-IA
74A
of
unpublished
especially
sort
extracted
laboratory
Strickland,
and C.
and
methods
ore
used
74-ORB-la
for
as
(FGSC
heterokoryon
favorable.
that
and
originated
spontaneously
in
according
to defective
spores
develop
so that
perithecia
rapidly
contamination
No.
297).
cross medium
(SC) is basically
increased
IOO-fold
(it probably
rather
the
60
blue
incuba-
(v/v)acetic
100 ml.
also
difficult
to tmce
and assign
as we ourselves
refer
constantly
types
produced,
(P605:
pure
ing
is that
aberrations
fla
are
them
available,
with
most of the
wild
parents
conidia
295),
II)
7%
up to
band
gels,
The
useful
cytologically
(IS protoperitheciol
FGSC
be
These
St.
I g./ml.,
for
B. in
nicotinamide
was
bands;
stained
purposes
various
consensus
Ridge
black
Murray,
W. N.
H. R. Cameron,
making
checked
reference.
derived
and
strains
sex-tests
fertile
If the
and
factors,
convenience
flA
Perkins.
with,
acid.
protein
University.
N.
Hsu,
be sufficiently
in future
issuer.
strains:
Two Oak
compatibility
used
D.
at
was
2,000
x g. for IO min.
on top of each
gel
at 34’
IO
mode
or with
in publication.
it would
Inasmuch
to warrant
not been
A thick
bands
cited
from
cases
mg.;
block
omido
hr.
Liverpool.
Stanford
collected
and in most
for modifications.
50
90
water
so that
the bromophenol
and some placed
in an
black
IO mg.;
separated.
information
Lawrence,
K.S.
acid,
for
surplus
at
layered
in an oven
with
on amide
be
for
placed
components
Methods.
B. Moling,
Mayo,
J. R.
malic
twenty
matching
not
then
the
homogenized
o ( w / v ) omido
oxoloacetic
University,
used
Stanford
of
about
was
sucrose)
and
about
40 min.
the apparatus
chloride,
of the
bands
for
from
mg.;
stained
one
in the
protein
mycelium
0.70/
were
not
of any
Laboratories,
The
bands
was
black
of the
600
w/v
water
slurry
was centrifuged
of the sample
was
and
by
neotetrazolium
HCI.
and
conclusion
Batonicol
evacuated
protein
(FriesT%
deionized
The
2’ at 4 mA/gel
were
removed
formazan
amido
gauze.
of tris,
absence
7.0
with
to substantiate
Hartley
for
in the
stained
matched
were
no formazan
formed
50°,
Gels
gels
and
contained
were
which
stained
consisted
with
and pestle,
the
sample.
0. I ml.
out at
The gels
blue,
IO mg.;
with
concentrated
medium
washed
of cotton
and
medium
tide,
IO mg.;
methylene
the pH adjusted
to 8.4
liquid
was
tubes
fixed
aerated
in a mortar
used as the
carried
3 cm.
Thunberg
gels
in
mycelium
layers
solution
fluid
was
and electrophoresis
dye moved
about
remaining
acid.
grown
of
several
large
pore acrylamide
and the supernatont
column
tracking
was
weight
risks
St.
Lawrence
projected.
background
These
and
simultaneously.
are
minimized.
The
are
strains
are
Their
main
strains
FGSC
than
as the
monobasic
that of Westergaard
makes
no difference
salt
alone.
pH
and Mitchell
(1947).
(and phosphate
is
is 6.5
without
adjustment.
are:
For
I liter
of SC:
KN03
I.09
K2HP04
0.7
g
KH2P04
0.5
g
hS04.7H20
1.0
g
N&l
0.1
g
CaCl2
biotin
0.1
0.5
g
mg
trace
Trace
SC
element
element
Cu
0. I mg,
ond
Totum
1945,
All
tration,
but
crosses
without
agor
Genet.
the
shaker
or with
I%
Neurosporo
lists
coding
ploting
or
Francisco).
tubing
This
medium.
The
solutions
inert,
stable
5)
Agar
it should
because
crude
gel.
of
to use
amount
the
0. I ml TE
some concen-
for
SC
and
in
unautocloved
Petri
dishes.
because
25”
stock
agor
(2%)
ore
after
autocloving.
added
Rondom
osco-
germination
to 30’
or
6O’C
water
bath)
conidio
or hyphae.
for
o few
doys,
beginning
special
circumstances
two
stock
(e.g.,
cornmeal
solution
I2 below],
the
by
following
sequentially.
H.
(Bll4),
Vogel
added
Make
accomplished
crosses
glucose
os described
with
(conveniently
when
ogorsTw?th
precautions:
certain
that
in o large
flask
everyond
o
source.
N.
to school
teachers
Media
N.
available
water
nontoxic.
for
plus
orstudents
who
grocery
stores
propylene
ond
isolation:
4% ogor
is routinely
used
of solubility
for relatively
much
higher
difficulties
concentrations
mentioned
these
about
orange,
In our
here
For
by
the
yellow,
benefit
of them
Prokosh
os o
have
suggested
coded
prior
Baltimore
and
0.05
ml dye per
green
and
these
ore
blue,
to
Son
100
and
ml
come
biologically
St. L.).
here
for ascospore
pure
ogors
such
by
Inc.,
os well
projects.
be readily
experience
P.
but
Neurosporo
moy
and Co.,
opproximotely
at
in red,
OSl7-01),
ourselves,
doing
supplements
glycol.
manipulation
0460-15,
used
inquire
food
color
(McCormick
before
outocloving
introduced
the
not
various
use was
that
(0324-15,
W e h eve
(Th e!r
be mentioned
that
4% is near the limit
ogors
suggests
This may explain
from
minimal
l:l3).
containing
by the use of Schilling
is odded
to the medium
substrate
mg,
Beadle
propose
The
or
from
lists
components
Neurosporo
see
in distilled
and
section
Add
(2x).
tubes,
crosses
Difco
component
(0321-15;
ore
(see
ore he&hocked
(30 min.
is therefore
necessary
to kill
up as o 50x
carbon
varieties
of media:
dyes
we
fertilization
under
that
see
Bo 0.01
medium
perithecia.
crosses
heat.
next
15 cm
moving
the
is mode
not
os standard
convenience
Color
os 4%
N
13:42-43)
Do
solution
after
Ascospores
hypochlorite
be noted
8114.
future,
Sucrose
(2%)
from
precipitation
in
I8 doys
for
used
amounts
final
acid.
25’C,
be
in the
liter
Medium
N, in the
metals
in approximately
stock
by
metals
mg per
stirrer).
three
complete
for
4)
ot
from
Bull.
adding
is used
now
the
citric
be shot
Medium
o mechanical
for
provide
no
It should
hove
used
stock:
before
sucrose
Difco
them
50x
2.0
In the
Vogel
be speeded
to
moy
medium:
Microb.
is dissolved
with
stiff
may
started
using
Zn
32:678-686).
out
isolation;
(Difco)
been
and
os preservative.
some cloudiness
carried
after
unsuccessful
on SC).
without
(8286).
We
thing
hove
mg,
Botany
below)
os o concentrated
routinely
have
In preparing
J.
We
in containing
of oscospores
3)Minimal
0.02
not isolated
until
ot least
not good
before
this time.
ascospores
[ (1956,
SC:
MO
2 ml/l
chloroform
There
is normally
aging
Cornmeal
ore
and
for
prepared
ore
further
ripening
(see
solution
given7
SC.
This will
it differs
with
ore usually
ratios
ore
mg,
Am.
element
per liter
is conveniently
spores
allele
The
solution
Fe 0.2
the trace
solution
is stored
at 5’C,
before
autoclaving.
after
solution
ore
(NN
of workers
in
manipulation
os Difco.
required
3:ll).
other
countries,
ond isolation
Our
small
experience
to produce
on
equally
6)
Stock
solutions
of
supplements:
Amount
Supplement
p-aminobenzoic
choline
per
stock
acid
0.4
chloride
ml
Stock
solution
solution
100
“lg.
used
ml
medium
0.5
ml.
2.0
1’
1.5
‘1
inositol
5.0
I’
1.0
”
nicotinclmide
50
‘1
0.2
I’
Ca-pontothenote
1.0
‘I
1.0
”
1.0
I’
1.0
I’
1.0
‘)
1.0
‘I
40.0
I’
I.25
I’
25.0
”
2.0
‘I
10.0
I’
2.0
8’
indole
1.0
‘I
2.0
”
L-leucine
5.0
I’
4.0
‘I
L-lysine
20.0
‘(
2.5
C’
L-methionine
10.0
8’
5.0
‘I
L-phenylolonine
10.0
‘I
2.0
I’
L-pro1
10.0
”
5.0
4’
rulfanilamide
3.4
‘I
I.0
I’
L-threonine
5.0
”
2.0
‘I
2.5
”
3.0
‘I
pyridorine
HCI
thiamine
L-arginine
L-histidine
HCI
DL-homoserine
ine
L-arginine
+ L-lysine
20.0
I’
40.0
I’
3.0
4’
7.0
I’
L-isoleucine
+ L-valine
Individual
adenine
supplements
sulphate,
concentrated
0.4
mg/ml.
stock
As a rough
vitamins,
and
general
0.2-0.5
greater
than
higher
optimal
mide--several
time
os protoperithecial
7 days
in
at
and
the
amount
25’
tests:
the
before
center
tests
as necessary
-nit-
Pairs
with
are
for
made.
fluffy
mg/ml,
tester
recovery
Petri
plates,
strains.
23
chloroform.
ot 1.0
about
0.2-0.5
concentrations
in
(P.
each
Plates
e.g.,
St.
L.).
containing
are
wrapped
acids,
are in
in
Even
40
when
in
of nit
pg/ml
SC
SC agar,
towels
The
order
mutants,
nicotina-
type
when
I5 ml
paper
in
wild
is better
about
assays.
case
of
ot
IO micrograms
many
cases
media
the
use
tyrosine
in flask
Gpprmches
segregonts
the
and
germination
medium
of - nit-
mg/ml,
growth
and
segregants,
growth
to allow
mg amino
provided
maximum
crossing
Adenorine,
soluble
uracil
in crossing
optimal
to fertilize,
over
sufficiently
to provide
necessary
supplement
of standard
the
ot 5°C
not
of auxotrophic
when
necessary
and
0.5
medium
The
of cares
germination
only
stored
are
at
use per ml final
and pyrimidines.
literature
attained
parent,
supplemented.
7) Mating-type
inoculated
are
and
tyrosine
is used
in a number
ripening
ratios
and
Menosine
in the
are
in water
uracil,
rule,
we
mg purines
indicated
allele
dissolved
acid,
solutions.
concentrations
to obtain
good
are
odenylic
per
and
is used
is
are
incubated
Plates
tested
ore
incubated
ore
marked
is spotted
to
NN
at
25’
for
visibly
turbid
marked
the
points
is used.
few
&nod.
J.
numbers
ore
be scored
may
ore
con
be
read
8:591
etched
on
undiluted
nitric
Control
tubes
with
of
Gommexane
pointed
on
incubators,
given
in the
In 750
ml.
water,
” ore
5 l/2
and
Solution
(see
distilled
Citric
ZnS04,
Vogel,
Bulletin
dissolve
concentrated
did
at
microspotulo
preincubation
“, Chicago
they
mixed
spotted
for
sulfuric
1962,
stock
Tool
and
(Some
so ten
acid
o
Mfg.
Co.).
domestic
years
ago.
(tech)
)
plus
For
100
distilled
(and
etc.
prevented)
by
in 95%
alcohol;
where
cultures
using
o concentrated
this
ore
solution
is
to be stored
we
include
here
13:42-43,
1956.
successively
with
his
directions
stirring
ot
room
Add
with
for
preparing
Medium
(or
temperature
N,
(see
os
(Molinckrodt
groma
The
2.5
1.00
liter.
is stored
at
resulting
with
Chloroform
(2 ml.)
room
temperature.
single-strength
o suitable
ml.
medium
carbon
source
is added
For use,
dissolve
successively
I H20
7 H20
6 H20
with
stirring
5.00
5.00
grams
grams
I .oo
gram
0.25
gram
MnS04,
I H20
0.05
gram
H3B03,
anhydrour
0.05
gram
0.05
gram
2 H20
24
os a preservative,
and the
this medium
is diluted
50-
is designated
such
at
N;
os sucrose
medium
is sterilized
by autoclaving.
solution
(containing
citric
acid
as o solubilizing
water,
stirring
5 ml.
below)
agent)
room
it
(20
has a pH
grams
is mode
temperature:
per
of about
liter),
up os follows:
acid
ml
150 gram*
250 grams
is about
obtained
FdNHd~(so~)~,
CuSO4,
5 H20
NqMo04,
ore
A flamed
strains,
in
thoroughly
solids)
in closed
containers.
nontoxic
detergents.
least
desk-tops,
below)
water.
acid,
ond
mode
A
solution).
is supplemented
supplemented
element
ml.
patterns
Smith,
ore
in detail
by Perkins,
tubes
ore convenient;
(“Honditool
at 5’
IO grams
5 grams
Solution
In 95
some
controlled
100
Element
thus
trace
readily
7 H20
N
With
Hexochlorocyclohexane)
onhydrous
Biotin
the
The
ore
2 H20
Medium
tool
of 2 liters
H20
Trace
with
pencil).
100 mm
stored
effective,
If spore
B. R.
satisfactory):
The resulting
total
volume
50 times
strength
medium
fold
medium,
is described
and
ore
(See
(usually
wax
Neurosporo--at
onhydrous
NH4N03,
5.8.
to
containers,
H. J.
Genetics
citrate,
CaC12,
34”.
grinding
hours.
be
re-
tests.)
minimal
with
to
ore
in o 75 or 100 mm tube)
the tube
or by pipetting.)
variations).
plug,
Event
culture
plates
inverted.
substances
plate
ot
for
type
The
48-72
plates
ogar
test
procedure
l:l3,
1,2,3,4,5,6
the
of
distilled
WQ,
1.5%
incubation
consisting
rocks,
in
permission
KH2P04,
mating
of each
types.
after
ond
- 1.0 ml
by shaking
of the
toxic
infestations
shelves,
reagents
No3
is very
Mite
Microbial
‘bnolytical
base
a corborundum
(Lindane;
be dipped
the
for
0.5
inoculum
mating
acid.
mites:
solution
may
tape,
in molten,
the
o small
both
perithecia
has solidified,
NN
is used
of
spray
over
o cotton
Alconox
and
which
on
plastic
(e.g.,
diTtr&buted
hours
Ogata,
mixture
of
With
12-24
(see
a residue
leave
12)
on
after
detergents
containers
presence
mutant
medium
and
mode
with
the
indicated
with
porofilm
of glassware:
II)
the
is plated
the
sectors,
ore
spotting
may give
better
results.
of stocks
by silica
gel:
The
Microb.
an
using
When
Tubes
ore sealed
IO)
Cleaning
cleaning
concentrated
for
be secured
suspension
(which
Tests
suitable
Tests
Conidial
suspensions
conidia
should
be well
plate.
hours
before
9) Prerervotion
into
sector.
method
conidial
swirling
bottom
lids
o similar
sterile
by
may
the
Auxanogrophy:
water.
(The
the
Q marked
and
be checked,
1:14,
8)
on
onto
The resulting
trace
element
The
biotin
solution
solution
obtained
0.5%
I%
glycerol
“complete
Note
(Sheffield).
os carbon
that
by
in test
medium,”
N-Z-Case
purposes
the
5.0
stored
Medium
To prepare
source,
above
dissolving
tubes
and
supplement
and
moy
solidify
not
mg.
in
V.
of
biotin
Perithecial
1.5%
Since
it
slants
slant,
the
in about
ml.
distilled
source,
0.5%
“complete”slants,
It is designed
The
cotton
cotton
is generally
where
use
to be used
employment
of
has
found
below
of
just
normally
media.
of
blind
blind
end)
end
the
cotton
well
a large
above
University
that
where
cotton
better
was
crossing
medium
type
of conditions
right
wrapped
the
around
medium.
of perithecia
of Malaya,
CI 3 x 3/8
in the crossing
the mouth
of
ffiola
In
certain
been
Lumpur,
tend
conditions
useful.
the
water.
yeast
The
extract,
I%
sucrose
plus
for
information
Malaya.
in
I50
for
agor
cotton
(with
most
ml
the
in
found
depending
after
on the
6 to 8 days
regions
in the
submerged
cotton
was
flasks,
the
formation
the
crossing
of
other
Erlenmeyer
of
slants,
fruited
in the medium,
3/4”of
of
crawa.
profuse-regions
than
partially
in
described
in CI number
Neurosporo
exist
When
surface
technique
medium
of crossing.
in the
thick
uncovered
below
kept
used
perithecia.
luxuriantly
about
l/2”
tube
with
on extra
pad of the cotton
The tube acted
QS a float
and
crosses,
often
test-tube
fruiting
effective
to form
contained
conventional
of the
inch
the
mode
aerobic
on aerial
medium
the flask.
hove
crosses
agor)
and
been
perithecio
found
(without
few or no perithecia
with
to ensure
particll
submergence
WCS placed
tube
towards
amount
Neurosporo
and
non-obrortant
the
was
in
is thin
liquid
producing
In order
which
of the
cotton
medium
of the
provided
layer
observed
the
utilization
85 ml
Crosses
cottened
Botany,
in 50
and
ogor.
publication.
production-
non-absorbant
agor
the
as Q preservative,
(Merck)
“and
different
used,
) is added
in the frozen
state.
N with
CI carbon
“minimal
with
be cited
(I ml.
only.
Prokash,
use
is prepared
is dispensed
To prepare
and
Chl oroform
100 ml.
room
temperature.
total
volume
is about
solution
is stored
ot
and
a fair
and
--Department
at
the
with
the
amount
of
the
strains
of