CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
EXTRANUCLEAR DNA IN POLYTOMELLA PARVA
A thesis submitted in partial satisfaction of the
requirements for the degree Master of Science in
Biology
by
Karen B. Darnall
January,
1986
The thesis of Karen B. Darnall is approved:
Richard L. Potter
California State University, Northridge
ii
ACKNOWLEDGMENTS
I
wish
support
and
I
also
would
Brunk,
and
technical
and
to thank the members of my committee for
guidance
like
Dr.
the
preparation
to thank Dr.
Michael
advice.
Carolyn
in
I
Shuldig
am
for-
this
thesis.
Dr.
Cliford
Marvin Cantor,
Mann
also
of
for
their
grateful
their
in the laboratory.
iii
many
their
suggestions
to
Gayle
hours
of
and
Pomraning
assistance
TABLE OF CONTENTS
INTRODUCTION
1
.4
Isolation Techniques
Characterization of
the DNA
7
MATERIALS AND METHODS
.10
Cell Culture
.10
DNA Isolations
.10
S o u t her n B1 o t
Hy b r i d i z a t i o n -_ •
Fragment
Isolations
Labeling
and
12
12
Purification
of
the
Probe
• 13
Southern Transfer
Prehybridization
14
and
Hybridization
.14
DNA Characterization
DNase Treatment
17
Exonuclease Treatment
17
Mitochondrial
I.D.
.17
of
DNA
Pieparations
in
the Mitochondrial Fraction
Mitochondrial
Succinic
• 19
Isolation
Dehydrogenase
on
Sucrose
21
Gradient
Assay
.21
DNA Extraction
Molecular
Weight
21
• 22
Estimation
of
the
RESULTS
3. 7 kb
Band
.24
.25
Southern Blot Hybridization
DNase Dilution Series
25
• 26
DNA Characterization with Enzymes
Mitochondrial DNA Preparations
iv
28
.29
Succinic
Dehydrogenase
Activity
DISCUSSION
•
.30
31
38
REFERENCES
v
ABSTRACT
EXTRANUCLEAR DNA IN POLYTOMELLA PARVA
by
Karen Brower Darnall
Master of Science in Biology
When
parva,
DNA
and
was
subjected
molecular weight
to
3.7
kb.
chondrial
extracted
to
membrane
shown
to
fragments
plasmids,
the
common
a
no
to
exonuclease
telomeres
present.
performed with an
hybridized with
of
the
gel,
digestion
a
most
to
many
likely
plants
suggesting
exists
and
as
fungi.
and its susceptibil-
indicates
blot
that
there
are
hybridization
was
enzymatic labeling system.
itself,
low
be associated with mito-
and
A Southern
prominent
P.
position corresponding
The DNA molecule appears to be linear,
ity
phytoflagellate
electrophoresis,
band appeared at
The DNA was
mitochondrial
from
The fragment
but not with DNA in other portions
that
the
sequence
is not present in the larger DNA fragments.
vi
of
the
element
INTRODUCTION
Polytomella
parva
is
a
fresh-water
alga
that
has
been studied intensively at California State Northridge.
The
in
organism
a
simple
was
multiplies
broth medium at
obtained
experiment
The
purity
sample
and
A s har p ,
and
it
from
the
requiring
first
rapidly
large
DNA
intactness
d i s t inc t ,
migrated
mo l e c u l a r
the
cultured
A culture
laboratory
amount
agarose
past
easily
temperature.
of
was . extracted
by
l ow
well
room
is
protozoology
a
of
and
an
DNA.
examined
for
electrophoresis.
we i g h t
main
eukaryotic
and
gel
for
ban d wa s
v i s i-b 1 e
chromosomal
smear.
weight markers indicated a size of approximately
M~lecular
4 kilobases.
The
source
first
of
objective
the
small
'membrane-bound
or
of
DNA
free
this
project was
fragment
in
the
in
P.
to
find
parva.
cytoplasm?
Was
the
Was
the
it
DNA
in an organelle,
and if so, which one?
Several possibili-
ties
a
the
existed
includes
a
few
DNA
of
single
plastids
have
also
possible
nucleus,
and
been
microorganisms.
shown
and
a
as
to
contain
another
1984).
Viral
one
or
found
One
an
particles
in
of
Cytoplasmic
other
species
DNA:
P.
parva
more mitochondria,
proplastids.
various
harbors
source
of
protozoa
and
sources
of
in
the
form
Paramecium
has
been
types of Gram-negative bacteria,
endosymbiotic
are
also
karyotes.
1
alga
(Goodenough,
commonplace in many
eu-
After
DNA,
a
considering
decision
preparation.
was
The
made
were
not
of
to
of
appeared
to
4 kb
fragment
as
possible
with
this
a
sources
of
mitochondrial
technique
was
not
Attempts to isolate intact mitochon-
successful,
that
the
start
difficulty
anticipated, however.
dria
all
be
free
but
of
mitochondrial
chromosomal
DNA
fragments
yielded
the
well as two other bands at approximately
20 kb.
A second
It
was
not
objective
certain,
biotinylated
dUTPs
consisted
DNA
of
been
extracted
was
not
easily
were
performed
rest
of
a
the
variety
esed.
to
ment
to',·Characterize
the
fragment
nick-translation,
not
from
RNA.
low
obtained
without
Since
melting
in
~
Total
digestive
the
parva
enzyme
distinguish
much
the
changes
interference
from
the
the
band
4
DNA,
that
used
to
detect
kb
was
high
homologous
from
the
subjected
and
to
electophor-
it was possible
electrophoretic
the
agarose,
experiments
fragment
DNA
with
that
patterns
molecular
A Southern blot hybridization with the
was
labeled
other
treatments
in
fragment.
temperature
quantity,
separating
the
was
Since the fragment was so prominent,
without
DNA.
by
DNA.
of
until
and
had
was
weight
label~d
sequences
frag-
that
might
be repeated within the larger DNA fragments.
A survey of the literature revealed no previous publications
concerning
microscopic
morphological
DNA
in
examinations
details
of
P.
parva.
had
revealed
the
organism,
Extensive
many
and
of
electron
the
other
fine
papers
3
described physiological traits.
Polytomella
established
it
after
the
gellae.
and
single
a
enclosed
It
swims
similar
by
a
very
sometimes
cell,
initially
described
by
Arago,
organism as a new genus in 1910.
Polytoma,
spherical,
tion
was
alga.
thin membrane,
rapidly,
spinning
varying
Polytomella
in
in
The
He named
is
nearly
and has four
fla-
frequently changing direc-
size
a mean diameter of 10]Jm.
who
place.
by
a
It
exists
factor
of
.mitochondrial
as
two,
area
a
with
is
at
the anterior end where the flagellae arise from four separate
basal
bodies.
The
plastids,
proplastids,
cles
be
can
found
cells
accumulate
which
obscure
nucl.eus
Golgi
the
centrally
bodies,
scattered
numerous
is
throughout
organelles
and
and other dense vesithe
membrane-bound
other
located,
cell.
starch
when
The
granules
viewed
with
a
light microscope.
There
dria
per
sional
is
some
Burton
cell.
scale
question
models
as
to
and Moore,
of
the number
of mitochon-
1974,
three-dimen-
mitochondria
made
from
three
different
individual cells on the basis of electron micrographs.
One
individual
was
and
others
4
numbers
had
may
to
have
found
to
have
10.
It
is
been
due
to
a
single mitochondrion,
possible
that
artifactual
the
greater
breakage.
The
single mitochondrion was basket shaped and occupied approximately
drion
19%
may
of
also
the
lend
(Burton and Moore,
cell's
some
1974).
total
volume.
structural
The
support
to
mitochonthe
cell
4
Isolation Techniques
isolation
mitochondrial
Two
homogenizing
and
sonifying
the
involving
techniques
cells
proved
too
harsh,
rendering small membrane fragments instead of intact organelles.
A
for
the
isolation
The
amastigotes
membran~s
gentler
and
suspending
procedure
of
are
trypanosoma!
similar
cells
in
to
a
was
originally
amastigotes
P.
parva
shape~.
spherical
the
which
with
Lysis
hypotonic
was
was
used
found.
their
thin
achieved
solution.
The
by
proce-
dure was modified
by experimenting with buffers of various
ionic
pH,
made
strengths,
periodically
specific
order
for
to
the
and
with
temperatures.
Janus
oxidizing
monitor
the
green
stain
enzymes
lytic
Wet
of·
process.
mounts
(an
we.re
indicator
mitochondria)
Large
in
amounts
of
blue debris gave evidence that the mitochondria were burstDNA
ing.
appeared
isolations
to
include
were
dense
made
from
particles
preparations
and
a
small
that
amount
of debris.
Since
this
mitochondrial
organism,
technique
it
was
mitochondrial
was
pellet
Rather
a
had
necessary
are
not
to
usually
correlation
was
reported
to
d~ne
sought
from
that
our
of
the
demonstrate
the
micrographs
used
This was not
been
demonstrate
Electron
successful.
characteristic cristae.
gation.
DNA
in this investibetween
the
4
kb band and succinic dehydrogenase activity of the material
from which it was extracted.
5
Before mtDNA is isolated, the mitochondria are normally
treated
clinging
it
with
to
the
verifies
been
by
untreated
sizes
of
to
voluted
large
DNA
mtDNA
of
that
amounts
to
at
least
fragment
of
in
a
defrom
distinct
20 kb was
closely
1978).
related
This similar-
demonstrate
membranes.
of
Presumably,
would
isolations
some of
surface
contamination
repeatedly
that
the
in origin.
membrane
DNA.
was
have
incubation.
two
found
attempts
the
must
produced
(Ryan et al.
mitochondrial
inner
larger
mtDNA
~itochondrial
assumed
parva
DNA
importantly,
recovered
Mitochondrial
The
the
further
the
P.
nuclear
more
during
consistently
Chlamydomonas
was
but
membranes
to
digests
subsequently
process.
size
4 kb band was
cling
DNA
however.
prompted
It
membranes,
preparations
in
organism,
ity
this
enzyme
intact
the
DNA,
similar
the
by
Unfortunately
The
outer
that
protected
stroyed
DNase.
be
would
the mtDNA would
In
fact,
seem
any
con-
to
trap
likely
amount
negligible,
the
in
of
nuclear
comparison
to
the mtDNA extracted from the mitochondrial pellets.
Additional
purification
accomplished
was
The
central
that
was
high
sucrose
by
portion
in
of
of
the
succinic
mitochondrial
centrifugation.
gradient
gradient
fragments
contained
dehydrogenase
material
activity.
portion also produced the 20 kb and 4 kb fragments,
ting
a
of
that
the
DNA
mitochondrial
the
gradient,
was
enzyme.
which
extracted
from
Furthermore,
normally
indica-
material
the
collects
lowest
This
rich
in
fraction
unbroken
nuclei
6
(Cantor
and
Sheeler,
1970),
produced
some
weight DNA and none of the 4 kb fragment.
high
molecular
7
Characterization of the DNA
A
few
simple
experiments
much as possible about
was
a
size
more
was
careful
were
the fragment.
measurement
estimated
at
devised
3. 7
kb
The first
of
by
to
the
learn
experiment
fragment,
agarose
gel
as
and
the
electrophor-
esis.
Another
ability
the
test
of
was
made
enzymatic
DNA
biotinylated
tively
labeled
probes
DNA,
possible
detEction
are
they
by
not
as
the
recent
systems.
sensitive
hybridize
just
as
avail-
Although
as
radioac-
specifically,
and they can be obtained without a special license (Murasugi. and
a
1 ow
Wallace,
me 1 tin g
hybridize
The
1984).
3.7
point
agar o s e
total
P.
with
fragment
The
bound
kb
ge1 ,
parva
only
band
to
was
1 abe 1 e.d ,
DNA
on
itself,
a
removed
and
used
Southern
indicating
from
to
blot.
that
the
sequence is unique to the 3.7 kb band.
There
of
DNA
are
various
fragments.
The
electron
micrograph
plicated
strategy
conformation
of
of
was
the
electrophoretic
When
DNA
acrylamide
portional
on
of
the
is
size
most
a
the
and
used
to
of
on
rates
an
that
molecules.
of
an
com-
into
the
differences
forms
agarose
are
forms
A less
the
coefficients,
the
the
insight
disparate
through
migrational
frictional
some
based
of
is
specimen.
gain
molecule,
shapes
determining
informative
mobilities
the
of
purified
electrophoresed
gel,
to
means
of
or
DNA.
poly-
i.nversely
which
pro-
depend
Circular
DNA
8
elements can exist in three different conformations: loose,
open
compact,
circles;
molecules.
The
because
offers
it
and
it
cannot
the
linear
open
circular
more
"snake"
strand
supercoiled
circles;
DNA
migrates
resistance
its
way
can.
than
through
The
linear
most
slowly
the
the
relative
or
supercoil;
gel matrix as
mobilities
of
the
linear and supercoiled molecules depend on electrophoretic
conditions,
but
they
generally migrate at different rates
(Rodriguez and Tait, 1983).
Total
different
DNA
extracts
degrees
of
from
parva
P.
digestion
were
DNase
by
subjected
If
1.
the
to
3.7
kb band had consisted of supercoiled molecules, low concentrations
of
the
enzyme
would
have
nicked
the
strands,
causing the supercoil to relax and assume an open circular
form.
Higher
linearized
concentrations
the
would
have
would have digested away all the material.
The experiment
any
several
of
change
in
the
DNase
very
enzyme
concentrations
no
and
the
high
showed
molecule,
of
mobility
of
the
concentrations.
3. 7 kb
This
band
suggests
at
that
the molecule is linear.
The mechanism by which the ends of linear DNA replicate
is
a
organisms.
strands
in
the
leave
problem
DNA
starting
5'
gaps.
to
3'
The
that
is
polymerase
from
an
direction.
gaps
can
solved
differently
synthesizes
RNA
primer
Excision
be
avoided
by
various
complementary
and
of
proceeds
the RNA
by
the
DNA
only
primers
formation
of circular intermediates which are subsequently cleaved.
9
Another
solution
to
the
problem
strands
together
at
the
ends
formed.
These
found
at
the
structures
ends
of
is
so
are
to
that
called
eukaryotic
link
a
the
two
hairpin
telomeres
chromosomes
DNA
loop
and
is
are
as
well
as
the
ends
of
the termini of linear yeast plasmids.
A
a
common
method
of
determining
whether
linear molecule are blunt-ended or not,
to replace the terminal phosphates.with
system
utilizes
this
method
made
use
requires
was
of
a
free
nucle9~ides,
labeled
not
feasible.
digestive
3'0H
enzyme,
groups
for
An
32
is by attempting
P~
not
The enzymatic
phosphates,
so
alternative procedure
exonuclease
activity.
III,
It
which
catalzyes
the release of 5' mononucleotides from the 3' end of duplex
DNA,
but
sites
to
are
is
also
able
(New England
digest
the
present,
band,
but
to
cleave
apurinic
Biolabs
1983-84).
The
which
indicates
that
does
gaps within the strands.
not
rule
out
the
or
apyrimidinic
enzyme
the
was
3'0H
able
groups
possibility
of
MATERIALS AND METHODS
Cell Culture
P.
parva
containing
was
seven
tryptone,
(Moore
et
sterile
filter
and
temperature,
of
the
cells
room
liters
of
a
it
yeast
al.
grown
in
four
100 ml
the
time
of
Cells
Scientific)
counted
were
and
harvested.
was
passed
th!"ough
the
medium.
of
five
days
phase.
the
stained
in a
for
At
the
was
with
Kava
bub-
to
the
bacterial
stain
chamber
generally
to
consisted
added
to· prevent
a
room
cells
medium without
hemocytometer
counts
sodium
through
Inocula
same
inoculation
Cell
0.1%(w/v)
Air
to
were
broth:
carboys
0.2%(w/v)
Chloramphenicol (SO wg/ml)
at
in
and
growth
contamination.
they
extract,
bubbled
took
temperature
complex
1970).
stationary
bling air.
carboy
at
0.2%(w/v)
acetate
reach
grown
(ICL
before
ranged
from
6
2 to 3 x 10 /ml.
DNA Isolations
When
growth
the
phase,
the
bottom.
the
cells
cells were deprived of air at the stationary
they
When
formed
tended
the
a
to
carboy
pellet
in
agglutinate
was
tip_ped
at
approximately
and
a
one
settle
45
0
to
angle,
hour,. and
the broth was siphoned off the top.
The
pellet
was
washed
10
once
with
distilled
water,
11
then
immediately
(0.5
NaCl,
syl.
The
suspended
0.05 M EDTA,
high
in
100
ml
0
of
60 C NET
buffer
0.05 M Tris-8.5) with 0.1% Sarko-
concentration
of
salt
and
the
heat
were
used to inactivate DNases I and II normally found in eukaryotic cells (Brunk,
The
frequent
mixture
personal communication).
was
incubated
agitation.
Protein
at
60°
and
for
starch
hour
one
were
with
extracted
three times with a 24:1 chloroform/n-amyl alcohol solution.
The
aqueous
salt
layer
was
concentration
and
phenol/chloroform.
ether.
DNA
volume
3
was
(20
The
then
M sodium
isopropanol.
~g/ml
dialyze_d
The
then
residue
precipitated
acetate
pellet
and
was
two
by
to
once
was
again
the
with
removed
adding
volumes
redissolved
reduce
with
one-tenth
of
ice-cold
in. RNase
buffer
RNase, 0.5 mM NaOAc).
isolations;
were
extracted
nonpolar
The same procedure was
DNA
overnight
however,
taken
from
the
isopropanol
were
needed
followed
when
sucrose
the
for the mitochondrial
mitochondrial
gradient,
instead
of
two
five
fractions
volumes
because
of
of
the
residual sucrose in solution (Maniatis et al. 1983).
The DNA was checked to make certain that the molecular
weight
was
phoresis
loaded
on
with
high
a
and
0.5%
Hind
that
the RNA was digested by electro-
agarose
gel.
III-digested
An
lambda
provided a molecular weight standard.
additional
DNA
well
fragments
was
which
Tris-icetate buffer,
pH 8.0, was used for all gels and electrophoresis.
12
Southern Blot Hybridization
I~olation
Fragment
The band of homogeneous DNA was isolated by extraction
from
a
made
with
0.5%
applied
to
was
melted
trough
would
.
agarose
low
the
be
slice.
melting
comb
formed
in
in
The
in
the
at
a
60
one
the -_.gel.
~1
200
of
mini-gel
agarose.
was
Tape
well
and
one
long
Buffer
and
gel
were
parva
DNA
was
P.
trough and lambda Hind III-digest was loaded
marker
band
with
that
0
the
hours
temperature
so
chilled to 4 C before use.
loaded
A preparative
well.
volts
was
and
DNA
was
then
stained with ethidium bromide.
visualized
razor
blade.
by
The
electrophoresed
UV
illumination
slice
was
for
and
turned
on
three
cut
its
out
edge
and excess agarose was removed.
The
Two
gel
volumes
was
of
melted
65°C
in
buffer
an
C
eppendorf
(50 mM Tris-8.0,
10 mM EDTA) were added,
mixed well,
tional
equal
used
the
half
hour.
to
extract
tube
was
upper
phase
being
taken
moved
by
the
agarose.
shaken
was
not
two
An
to
remove
chloroform
was removed with ether.
at
-20°C
(Perbal
after
1984).
the
at
65°C.
lOmM NaCl,
and incubated an addi-
volume
After
vigorously
removed
tube
of
the
for
65°C
phenol
phenol was added,
three
minutes.
centrifugation,
interface.
extractions,
was
and
with
Phenol
the
was
The
care
re-
chloroform
The DNA was precipitated overnight
A large
portion
of
the
isolated
13
DNA
was
made
smaller
directly
portion
was
into
a
biotinylated
restricted
and
probe,
cloned
and
into
a
pUC-19
for later use.
Labeling and Purification of the Probe
The
the
BRL
DNA
nick-translation
with
biotin
product insert.
according
~g
3
of
system
to
was
the
plasmid
used
to
instructions
DNA
was
label
in
the
labeled,
and
200-300 ng of DNA from the gel was labeled with biotin-11dUTP (BRL).
Biotinylated
by· filtration
plastic
glass
DNA
over
pipette.
wool,
and
was
a
The
column
and
equilibrated
was
a
Sephadex
The
~ip
two
inch
with
was
loaded
from
G-50
was
length
5
ml
siliconized
from
the
top.
1 X sodium chloride-sodium citrate,
on
the
~1
The 50
top
of
nick-translation
the
was
tube,
rinse
a
and one half mls of resin
~1
to
cut
nucleotides
using
with
was
it was absorbed, an additional 200
used
free
column,
packed
packed with four
0.1% SDS eluting solution.
mixture
separated
the
reaction
column,
of
and
eluting
and
was
after
solution
added
to
the column.
Fractions
were
collected
in
20
tubes.
tube contained 1 ml of eluting solution, and 150
collected in each of the remaining tubes.
were
taken
paper.
The
from
each
tube
and
spotted
2
on
~1
The
~1
first
was
aliquots
nitrocellulose
paper was developed with the BRL Biotin Detec-
,, .
14
tion
System
found
that
according
the
eliminated
for
to
filter
the
the
inst_ruc tions,
blocking
purpose
of
step
with
isolating
but
BSA
the
was
it
could
probe.
be
DNA
was generally detected in 3 or 4 of the tubes in the middle
of the series.
Southern Transfer
A "Baby
phorese
and
a
Gel"
sample
apparatus
of
Tetrahymena
P.
(BRL
parva
thermophila
H6)
DNA,
was
used
to
lambda-Hind
DNA.
The
electo-
III marker,
1.1%
agarose
gel
was run at 60 V for one and one half hours.
Nitrocellulose
pads
for
in
were
the
3
I1
contained
transfer.
HCl
denatured
tions
and
and
and
in
gel
rinsed
baby
was
with
the
sheets,
blot
kit.
nitrocellulose
and
kit
depurinated
distilled
neutralized
with
the
wicking
the
The
then
supplied
night,
paper,
The
gel
was
and
were
for
5
water;
according
to
was
baked
blotting
minutes
the
the
DNA
was
instruc-
blotted
for
used
two
overhours
in an 80°C vacuum oven.
Prehybridization and Hybridization
Prehybridization
tions
(Leary et al.
cellulose
paper
amide ,
X
5
SSC,
was
5
treatment
and
hybridization
1983) were recommended by BRL.
prehybridized
X
Den hard t ' s
in
10
ml
so 1 uti on ,
of
50%
25
mM
condiNitroformsodium
15
~g
phosphate pH 6.5, and 250
of
salmon
sperm
DNA.
The
salmon sperm DNA was sheared by forcing a 4 mg/ml solution
through
The
a
25
sheared
needle
gauge
DNA
was
several
boiled
for
times
10
(Perbal
minutes
and
1984).
chilled
in an ice-ethanol bath before it was added to the hybridization mixture.
The nitrocellulose paper and prehybridization solution
were
sealed
in
a
Dazey
Seal-a-Meal
bag
and
incubated
for
4 hours at 65°C.
The
except
hybridization
for
the
reduction
of
band
the
and
solution
addition
formamide
of
had
10%
to
III
same
dextran
45%.
lambda-Hind
the
sulfate
Probes
marker
ingredients
for
were
and
the
the
3.7
kb
denatured
in
the same manner as the sperm DNA and added to the prewarmed
(42°C)
hybridization
solution.
Both
filter
and
solution
were put into a new bag and bubbles were carefully eliminated
before sealing.
Hybridization was allowed to continue
for 24 hours at 42°C.
Post-hybridization
by
BRL
in
washed
two
the
seven
detection
times
incubations
at
steps
rinsing
kit
with
50°C.
were
instructions.
SSC,
The
SDS
paper
recommended
The
solutions,
was
not
paper
was
including
dried
before
developing.
Biotinylated
alkaline
and
DNA
phosphatase
washes
were
was
detected
system
made
performed
by
according
by
the
BRL.
to
streptavidin,
The
the
reactions
instructions
provided by BRL, and the filter blocking step was
included~
16
The
strate,
the
last
was
nylon
reaction,
processed
component
in
precipitation of
in
the colored sub-
a
Sears
sealing
most
other
bags
bag,
reacts
because
with
substrate and causes a high background (BRL Focus 1985).
the
17
DNA Characterization
DNase Treatment
1
of
mg
of
buffer
0. 5
(0.2
mg/ml
tested
DNase
(Sigma)
M NaCl,
nuclease
in
I
order
BSA).
dilution
the
wit~
series
of
plasmid
DNase
was
the
buffer
as
A 15 ul cocktail of 0.06 Ug
plasmid
(BRL)
tube.
were
added
were
incubated
ture
was
to
at
each
37°C for
loaded
on
an
The
same
experiment
DNA
The
was
gel
through
dilution
original
and
was
stock.
core
reaction
45 minutes.
agarose
at 60 V for one hour (Mann,
DNA
carried
made in 5 ul
same
ml
gradual nicking by DNase.
Each
the
1
change in the electro-
eight tubes starting with 0.5 ug/ml.
of
in
10 mM sodium acetate,
Purified
demonstrate
phoretic pattern of the DNA
A 1:2
reconstituted
5 mM MgC1 ,
2
free
to
was
buffer
mixtures
Half of each mixand
electrophoresed
personal communication).
was
repeated
with
~
parva
DNA.
The first tube of the dilution series contained 0.1 ug/ml
DNase,
Another
and
one
lane
dilution
was
series
used
was
to
made
show
with
undigested
Eco
Rl
in
DNA.
water.
The initial tube contained 0.4 units of enzyme.
Exonuclease Treatment
P.
to
test
parva
for
DNA
the
was
treated
presence
of
with
exposed
exonuclease
3 'OH
III
groups.
(BRL)
A low
18
salt
buffer
mM NaCl,
at
used
5 mM DTT),
37°C.
that
was
(66
of
the
Tris-7.5,
66
mM
MgC1 2 ,
and the DNA was digested for
Electrophoresis
all
mM
DNA,
6.5
one hour
1.1% agarose gel indicated
on a
including
the 3. 7 kb
band,
had
been
degraded, because a hazy pool of DNA appeared at the lowest
portion
have
of
been
gel.
internal
procedure,
ligation
the
ligase
On
nicks
was
reaction
incubated
III
was
the
buffer
the
digestion was carried
to
(ligase
to
mixtures,
were
at
one
buffer
reaction mixtures and
try
with
37°C
of
assumption
introduced
used
ATP
added
the
the
is
for
by
to
phoresed on a 1.1% agarose gel.
there
may
extraction
them.
Two
appropriate
buffer
and
one
Exonuclease
hour.
very
without
changing
low in salt),
out at 37°C for
untreated P.
the
repair
mixtures,
also
that
one hour.
and
Both
parva DNA were electro-
19
Mitochondrial Preparations
P.
and
parva cells were harvested as described previously
pelleted
tion
for
Cells
5
minutes
were
again.
in 250 ml polyethylene bottles by centrifugain
a
resuspended
Sorval
in
GSA
rotor
distilled
at
water
3,000
and
rpm.
pelleted
This treatment did not seem to reduce their motil-
ity.
Cells
solution
within
were
lysed
one
to
one
periodically
tion
was
under
decreased
to
of
mls
of
Samples were exam-
the microscope.
When
or
two
intact
tonic~ty
of
the
solution
up
0.25 M sucrose
by
adding
hypotonic
the cells were lysed
one
field,
Janus
the
Most
750
and one half hours.
power
erose,
32 C· in
(0.02 M Tris-8.0).
ined
to
0
at
the
cells
populaper
was
low
brought
750 mls of cold 0.5 M su-
thereby stabilizing the unlysed cellular components.
green,
a
vital
by
mitochondria,
of
the
lysis
indicated
stain which
was
used
procedure.
that
many
lysate
was
of
to
turns blue when oxidized
.the
A large
the
check
to
amount
mitochondria
effectiveness
of
were
blue
debris
being
lysed
also.
The
GSA
rotor
was
at
2,000 rpm for
caref~lly
cells and free
sion,
were
centrifuged
poured
off,
nuclei.
pelleted
by
10 minutes (Cantor 1978).
in
bottles
250 ml
five minutes.
leaving
the
centrifugation
the
The supernatant
pellet
The mitochondria,
in
of
whole
still in suspen-
at
6,000
rpm
for
20
The pellets were suspended in 40 ml H 0.
2
bound
and
DNA
salts
digested
(0.2
M NaCl,
DNase),
3 l..lg/ml
enzyme
(final
was
was
and
TE
ting
the
resulted
5 mM MgC1 ,
2
inactivated
after
50 mM).
buffer
(10
DNA
(Brunk
in
DNase
incubating
concentration
with
with
mM
complete
the
two
The
Tris-8.0,
1967).
adding
the
solution
hours
by
1 mM EDTA)
This
enzyme
10 mM sodium acetate,
at
0
4 C.
The
adding
EDTA
pellet was washed
disappeaqtnce
that in the mitochondria.
by
Non-membrane
before
treatment
of
all
twice
isola-
consistantly
DNA,
including
21
Identification of DNA in the Mitochondrial Fraction
Mitochondrial Isolation on a Sucrose Gradient
The
the
initial
method
treated
mitochondrial
described
with
DNase.
pellet
was
previously,
except
The
was
pellet
prepared
that
it
suspended
by
was
in
not
5
ml
of NET buffer.
A sucrose
tube.
the
of
one
to
bottom
and
cushion
and
55%
make
sion
space
was
made
in
a
35
ml
polya1lomer
One and one half mls of saturated CsCl was deposited
on
a
gradient
to
one
catch
half
for
the
mls
the
of
whole
solutions
a
gradient.
was
ml
layered
left
at
on
the
67%
were
pellet,
sucrose was
cells
sucrose
21
starch
and
and
added
unbroken
mixed
in
a
a
layer
to make
nuclei.
gradient
17%
maker
The 5 ml mitochondrial suspengradient
the top of the tube.
and
there
was
a
3
ml
The gradient was cen-
trifuged
for two hours at 27,000 rpm in the AH 627 Sorvall
swinging
bucket
rotor.
A yellow
band
of
material
could
be seen in the middle portion of the tube.
Fractions were collected from the bottom of the tube.
A peristaltic
pump
delivered
30
drops
(one
and
one
half
mls) to each of 18 tubes in a fraction collector.
Succinic Dehydrogenase Assay
The
presence
of
enzymatic
activity
was
used
as
a
22
marker
The
to
identify
of
rate
(DCPIP)
was
recording
adjusted
the
fractions
reduction
measured
of
at
2,6-dichlorophenolindophenol
660
nm
spectrophotometer.
to
give
readings
containing mitochondria.
with
The
between
a
Beckman
amount
0.9
and
Model
24
of
DCPIP
was
0.4
absorbance
units over a three minute interval.
0.1
ml
samples
from
each
fraction
were
added
to
1.5
ml of 0.03 M, pH 7.4 phosphate buffer, 0.5 ml 0.1 M succinate, and 0.1 ml 1 mM KCN.
last.
The
u~
40
solution was mixed
immediately.
The
change
of
calculated for
each fraction
of 2.5 mM DCPIP was added
in
the cuvette and measured
absorbance
per
minute
was
from the chart paper (Rendina
1971).
DNA Extraction
The
make
the
had
18
six
fractions
sequential
from
the
portions.
gradient
The
three
were
fractions
greatest activity were put into one pool.
to
be
removed
by
dialysis
were
fitted
in
order
pooled
to
to
with
The sucrose
isolate
and
precipitate the DNA.
Dialysis
bags
to
pipette
tips
(made
for
the Pipetteman 5000) with half an inch of the tube removed.
The
bags
were
additional
5
filled
ml
with
space
in
the
the
a two-fold increase in volume.
for two days against TE buffer.
sucrose_ solutions,
pipette
tips
and
the
allowed
for
The solutions were dialyzed
23
The
DNA was
extracted as
described
earlier,
and
pre-
cipitated in 50 ml polypropylene tubes in a dry ice-ethanol
bath.
The
tubes
20,000
rpm
for
was
marked
were
30
as
it
centrifuged
minutes.
was
The
removed
in
an
outer
from
SW
side
the
34
of
rotor,
rotor
each
at
tube
because all
but one of the DNA pellets were too small to be seen.
the
0. 5
ml
DNA
pellets.
The
portion
with
from
the
completely
of
by
amount
buffer
dissolve
contaminating
DNA
TE
protein.
RNA in
the
large
high
because
electrophoresis
of
was
A
used
pellet
~o
of
each
wash
which
~~zymatic
there
was
preliminary
showed
that
sample also.
was treated with RNase (40 ~g/ml)
A portion
to
sample was
a
each
of
precipitated
activity
did
large
amount
inspection
of
not
of
the
there was a very large
That particular sample
then
reprecipitated.
electrophoresed
the results of the enzyme assays.
out
and
compared
24
Molecular Weight Estimation of the 3.7 kb Band
A log.:..molecular
Hind
III
fragments
weight/migration
was
the
results
section,
the
sucrose
gradient.
edge of
made
from
representing
a
plot
the
photograph,
the
Measurements
of
DNA
were
lambdafound
isolated
made
from
in
from
the
the well to each of the molecular weight standards
and used to make a standard curve on semi-log graph paper.
A similar
measurement
the
in kilobases,
size,
and Tait 1983).
of
the
band
in
lane
5 was
made
and
was read from the graph (Rodriguez
RESULTS
Southern Blot Hybridization
1
2
Total
3
cell
P. parva and
kb
was
kb
ly
to
paper,
probe,
from
nitro-
and
taken
an
from
thermophila
transferred
cellulose
3 .7
~
DNA
the
direct-
agarose
gel ,
was used for hybridization.
23 . 1
Most
of
the
gel
from
was
taken,
DNA
which
on
the
the
made
a
blot
smear
4. 4
in
lanes
1
and
ranging
2,
from 22 to 25 kb.
There
appears
strong
hybridization
2. 3
2. 0
be
with
P.
the
3 .7
kb
band
parva
DNA,
and
a
amount
A
very
of
2.6
kb.
not
seem
to
ization
l ane 2: Polytomella
mophila
signal
25
small
shows
There
be
with
DNA
any
the
does
h y bridT.
\vhich
used as a control .
lane 3: lambda-Hind III marker
of
background.
faint
at
lane 1 : Tetrahymena thermophila
~
to
therwas
26
DNase Dilution Series
A
at
portion
varying
change
of
plasmid
stock
concentrations
in
was
order
in electrophoretic mobility
ing amounts of digestion.
series
shown
below
digested
to
with
DNase
demonstrate
the
patterns with diminish-
The photograph of a 1:2 dilution
starts
with
the
highest
concentration
on the left.
DNA
Lanes
in
3,
4,
nicked,
II).
lar,
the
first
and
relaxing
Plasmids
and
in
lanes
5
two
show
that
them
to
lane
6
7 and
8
lanes
some
the
were
was
comple.tely
of
the
digested.
plasmids
were
open
circular
form
almost
entirely
open circu-
show some of
the
supercoiled
(form
forms
(form I).
This
lish
a
experiment
suitable
method
the 3.7 kb DNA in P.
1
2
3
4
was
5
carried
for
out
as
a
model
characterizing
the
to
estab-
form
of
parva.
6
7
8
+
form II (supercoiled)
+
form I
+
form
(open circular)
III
(linear)
27
'
P.
plasmid
parva
eighth
DNA
in
of
lane
change
gel.
This
DNA.
was
in
the
stock
the
to
DNA
sample
treated
complete,
mobilities
pattern
The faint
previous
contained
one was
in
in
would
any
be
no
same
manner
experiment,
the
the
typical
band
did
other
of
a
as
linear
the
of
not
lanes
the
that
except
Digestion
DNase.
and
of
the
appear
of
the
fraction
bands that appear to run slightly ahead
of the 3.7 kb fractions are degraded fragments which appear
after the DNA has been stored for a long period.
In
lanes
seven
and
eight,
there
are
distinct
high
molecular weight bands appearing just below the main chromosomal
fraction.
This
DNA
might
also
belong
to
the
mito-
chondrion.
1
2
3
4
5
6
7
8
+
mt DNA?
+
3.7 kb
.
28
DNA Characterization with Enzymes
Exonuclease III
When
P.
the
entire
the
DNA
parva
3. 7
with
kb
DNA
was
band
ligase
treated
with
disappeared.
did
not
exonuclease
Prior
effectively
III
treatment
prevent
any
of
of
the exonuclease activity.
Eco R1
Restriction
sistent
banding
of
P.
parva
pattern
with
cellular
Eco
DNA
Rl.
It
revealed
is
not
a
con-
possible
from this gel to determine whether the bands are the result
of digestion of the 3.7 kb fragment,
or whether they result
from digestion of chromosomal sequences.
1
2
3
4
lane 1 : >. <Px174-Hae III digest
lane 2 : Eco R1
lane
3:
Eco
R1
(high
lane 4: undigested
kb
1353+
1078+
872+
602+
cone.)
29
Mitochondrial DNA Preparations
DNA
that
was
fragments,
without
appears
three
as
isolated
prior
distinct
from
sucrose
bands.
mitochondrial
gradient
It
is
membrane
purification,
not known whether
the two high molecular weight bands are different conformations
of
the
same
molecule
or
whether
the
bands
represent
two different species of DNA.
1
2
lane 1: mitochondrial DNA preparation
lane 2:
cellular DNA preparation
Seventeen fractions were taken from the sucrose gradient
and
analyzed
Consecutive
tions
tions.
in
for
fractions
order
to
succinic
were
simplify
dehydrogenase
consolidated
the
dialysis
to
activity.
form
and
DNA
six
por-
extrac-
30
Succinic DH Activity
Fractions four and
4
five,
taken from the sucrose
gradient, show evidence
that the 3 .7 kb band was
isolated from solutions
with enzymatic activity.
Fraction four originally
A/min
contained the more visible
pellet of nucleic acid ,
I
but there was a large amount
'
\.
of RNA and protein contamin-
2
1
ation.
\. 5
I
3
',
\
.
clean up the sample, much
.-
i~_;: ~-~·,E~ ~~"? ---,
of the DNA may have been
lost.
(enzyme fractions)
(DNA portions)
1
2
In an attempt to
3
4
5
6
2 3. 1
9.4
6.6
4.4
2.3
2.0
DISCUSSION
This
fragment
alga,
to
was
found
P.
to
exist
The
the
characterize
of
investigation
great
the
kb)
bands
made
Although
traditional
were
feasible,
were
was·
a
3. 7
in
and
the
green
study
to
band
as
recovered
from
a
well
by
as
mitochon-
centrifugation.
identifying . the
shown
was
partially
kb
of
DNA
small
this
by , d:Lfferential
methods
it
of
fragment,
The
of
abundance
objective
molecule.
preparation
not
in
main
source
the
(20
larger
drial
preliminary
parva.
identify
two
a
enzyme
mtDNA
assays
that
the DNA in question associated with mitochondrial membrane
fragments.
There
not
RNA,
would
is
and
be
mtDNA
that
that
the size
is
that
to
also
ends,
and
kb
its
our
is
these
mitochondria
to
seems
results
and
3.7 kb.
It
as
a
linear with
be
present
need
to
for
the
however.
be
The
source
characterize
be
to
DNA
support
mitochondrial,
to
is
mol~cule
approximately
ability
appears
the
additional
intact
sequence
but
find
DNA
isolate
hindered
DNA,
to
the
The molecule
ment.
3. 7
evidence
desirable
supposition
inability
firm
the
of
frag-
unprotected
only
in
confirmed
the
by
additional experiments.
The
problems
preparations
can
be
dures
made
that
have
on
had
the
of
to
acquiring
be
resolved
project.
potential
adequate
before
There were
and
31
were
not
mitochondrial
more
two
tried
progress
other
proce-
because
of
32
time constraints and lack of equipment.
Simpson has devel-
oped an excellent method for isolating trypanosomal kinetosomes
a
by
small
forcing
hypotonically
orifice
nitrogen
gas
under
a
(personal
swollen
constant
organisms
pressure
communication,
through
controlled
A
1985).
by
French
pressure cell also might have been used to break the cells
open
a
be
without
ripping
sufficiently
obtained,
the
pure
then
mitochondrial
mitochondrial
.the
mtDNA
could
membranes.
preparation
be
If
were
to
and
ex-
circumvent
the
identified
tracted in the usual manner.
Another
approach
mitochondrial
First,
by
the
"in
been
isolation
location
situ"
used
mounted
would
show
beled
DNA
of
be
the
to
thin
the
mtDNA
technical
to
by
using
could
be
as
on
micrographs
well
representative,
have
sequences
Electron
membranes
steps.
probes
homologous
sections.
two
demonstrated
Biotinylated
identify
mitocondrial
(BRL
used
difficulties
hybridization.
recently
paraffin
could
as
the
personal
la-
commun-
ication 1985).
The
of
3. 7
P.
the
kb
parva
second
problem,
fragment,
DNA
in
DNA.
a
could
that
be
CsCl-EtBr
of
obtaining
eliminated
gradient
by
a
pure
sample
isolating
preparation
of
the
total
A clean supply of DNA would be more easily
characterized.
Ideally,
the
shape
of
the
mined by electron microscopy.
domonas
reinhardi,
was
molecule
should
be
deter-
A similar organism, Chlamy-
originally
found
to
have
linear
33
mtDNA.
When
microscopy,
circular.
a
linear
fragment
or
restriction
but
was
(<1%)
conformation
should
restriction
DNA,
portion
inspected
replication mechanism
drial
a
small
were
by
were
electron
found
to
be
information also contributed to the under-
its
apparent
by
molecules
very
This
standing of
The
the
be
site
map
not
et
al.
of
P.
parva's
confirmed
by
a
mapping.
was
(Ryan
made
An
by
successful
digesting
due
to
the
mitochon-
similar
attempt
1978).
to
method
construct
total
cellular
presence
of
many
would
have
rather
than
incompletely digested fragments.
Because
been
of
preferable
the
enzymatic
the
3.7
to
its
kb
to
use
system,
sequence
Murasugi
difference
greater
and
in
a
to
sensitivity,
radioactive
try
within
it
probe
to
detect
the
the
larger
DNA.
Wallace
(1984),
sensitivity
between
there
the
is
presence
of
According
a
1,000-fold
two methods.
There
was enough material to make a satisfactory probe by isolating
but
the
purification
tedious.
tion
fragments
I
was
solution
months.
Probe
from
of
the
a
low
point
biotinylated
not
able
to
after
it
had
stability
melting
agarose
probes
was
gel,
rather
reuse the leftover hybridizabeen
was
stored
supposed
at
to
4°C
have
for
two
been
the
major advantage to the system.
A more
thorough
characterization
of
the
ends
of
the
3.7 kb DNA molecules would include an attempt at end-labeling with
with
terminal
biotinylated
transferase.
dUTPs
as
This
well.
could
Like
have
been done
exonuclease
III~
34
the
enzyme
have
been
requires
tested
free
by
to
be
from
3 2
maize
sensitive
ase,
but
that
proteins
It
was
yATPs.
was
to
were
(Sederoff
1984).
the
This
and
It
of
linear
mtDNA
transfer-
later
the
of
the
polynu-
terminal
DNA-protein
aspect
with
appeared
to
repl-ication
ends. could
fragments
was
linked
the
5'
similar
the
kinase.
that
priming
a
III
covalently
The
fragments
When
tested,
to
hypothesized
in
the
exonuclease
insensitive
involved
groups.
incubating
cleotide kinase and
plasmid
3' OH
determined
5
1
termini.
complexes
the
P.
were
linear
parva
DNAs
molecule
sould be studied further.
Chlamydomonas
mtDNA
reinhardi
publications
to
its
singular
mtDNA
molecule
16
(Most
plant
kb.
times
a 11
that
1 in ear
was
1978).
The
larger
than
major
C.
is
that
at
alga
organism
are
has
small,
at
with
a
about
least
ten
the molecules are nearly
molecule
reinhardi 1 s
green
The
mtDNAs
u n expected
mtDNA
only
surprisingly
algal
fact
an o·t her
the
credit.
that
and
The
size.)
is
discovery
of
20
P.
(Ryan
parva
It
kb.
is
would
a1•
et
not
much
be
very
interesting to find out if it is linear also.
Small
in
mitochondrial
yeast,
fungi,
trypanosomes
circular
Some
and
contain
molecules
plant
mtDNAs
DNA
elements
higher
plants.
huge
network
a
called
also
The
of
minicircl~s
consist
of
have
a
been
found
kinetoplast
large
and
and
of
small
maxicircles.
heterogeneous
mix
of sizes.
Bean
mtDNA
isolated
from
the
supercoil
regions
of
35
CsCl-EtBr
30
gradients
bands.
complex,
The
Corn
29
to
was
mtDNA
having
mtDNA
two
from
kb.
these
as
complex
dominant
tobacco
among
organisms
as
an
species
10-12
had
the
1.5
of
size
that
were
ranging
less
kb.
10
from
that
classes
the
of
1.8
and
indicated
different
mtDNA
series
patterns
bands
studies
suggesting
their
oligomeric
electrophoretic
Hybridization
homology
of
displayed
there
in
genomes
each
were
not
electrophoretic
patterns
had
anserina
harvested
first indicated (Dale 1982).
Cultures
of
different
phases
sequences
were
cence.
with
N.
during
found
Unlike P.
the
but they
loops,
and
its
to
life
were
span.
increase
in
Amplified
quantity
with
at
mtDNA
senes-
parva, the sequence was able to hybridize
large
intermedia
kb,
Podospera
juvenile
also
mtDNA.
have
small
Neurospora
circular
crassa
mtDNAs,
and
about
4
do not hybridize with the large mitochondrial
there
is
no
significant
homology
between
the
small circles in the two different species (Sederoff 1984).
It
appears
are
amplified
mtDNA.
it
that
but
not
duplications
all,
of
small
portions
mtDNA
of
fragments
the
primary
Before assuming this is not the case for P.
would
with
some,
be
wise
radioactive
to
repeat
probes
to
the
prove
parva,
hybridization
experiment
that
kb
the
3. 7
segment
was not present in the major mtDNA band.
The
unknown.
frames,
purpose
Some
of
of
these
the
extra
mtDNA
sequences
elements
contain
open
is
still
reading
such as the linear maize plasmids (Sederoff 1984),
36
but
many
based
that
of
on
them
mtDNA
mutations
the
do
and
are
to
fixed
than
Unicellular
the
extremes
the
most
of
mtDNA
primitive
to
have a
as
have
well
as
algae
tures
and
C.
in
recent
10
the
times
have
more
do
quickly
(Wilson
not
only
in
seminar
some
know
one
been
indicates
demonstrated
We
had
have
evidence
nucleus
design.
organisms
trees
size
of
whether
category
It is fairly obvious now that animals
small
mtDNA _loop,
and
plants most
often
loop that is ten-fold larger than the animal mtDNA,
green
or
one
5
organisms
of mtDNA or several.
tend
Evolutionary
differences,
mitochondrion
1985).
not.
the
a
mixture
have
of
both
cannot
be
other.
smaller
molecules.
"plant-like" and
unequivocally
Now
we
have
of
"animal-like"
assigned
two
Many
to
organisms
the
fea-
one kingdom
to
compare.
reinhardi and P. parva both appear to have small, "anim-
al-like" primary mtDNA strands.
chonrial
plasmids,. P.
parva
With the additional mito-
is
more
similar
to
plants
than C. reinhardi.
Both algae
vocales
and
the
have
been
classified
class Chlorophyceae
in
(W.
Protozoologists put them in the class:
The
is
most
that
contains
obvious
C.
distinction
reinhardi
cellulose,
between
the order of VolD.
P.
parva
1974).
Phytomastigophorea.
the
has chlorophyll and a
whereas
Stewart
has
two
organisms
cell wall that
no
chlorophyll
ans lacks a cell wall.
There
are
many
"colorless"
algae
that
are
distin-
guished from the photosynthetic forms solely by the absence
37
of
chlorophyll.
of
the
cells,
terpart
which
i s
photosynthetic
the
to
accident
Polytomella
It
not
can
to
would
was
ent i r e1y
apparaturs
be
Chlamydomonas
corresponding
1974).
It
is
very
The
Polytoma
named),
is
the
1os s
for
some
colorless
coun-
occurs,
survived.
Polytomella
be
c 1 ear
and
to
organism
colored
Dunaliella
interesting
but
(the
the
h Q_w
(W.
D.
for
species
Stewart
see if Dunaliella
contains the same 3.7 kb fragment as Polytomella.
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