Mol Gen Genet (1984) 194:343-345
© Springer-Verlag 1984
Chromatographic resolution of nucleic acids extracted
from Penicillium chrysogenum
Gunter Saunders, Mark E. Rogers, Maxwell W. Adlard, and Geoffrey Holt
The Polytechnic of Central London, School of Engineering and Science, 115 New Cavendish Street,
London W1M 8JS, United Kingdom
Summary. High molecular weight D N A extracted from
Penicillium chrysogenum has been fractionated using RPC-5
Analog, into three distinct types designated 1, 2 and 3.
Types I and 2 have the same buoyant density of 1.710 g/cm 3
and together appear to comprise the nuclear DNA. Type
1 is enriched for repeated sequences which are normally
observed in restriction digests of P. chrysogenum total
DNA. Conversely, type 2 appears to be composed entirely
of non-repetitive sequences. Type 3 has been identified as
mitochondrial DNA, having a buoyant density of 1.695 g/
cm 3 and an estimated molecular weight of 31.6 x 106 Dal-
tons.
Introduction
The preparation and analysis of D N A from lower eukar-
yotes has been largely dependent on the use of differential
centrifugation employing a variety of gradient forming ma-
terials (see for example Maleszka 1978; Stahl et al. 1980).
Such techniques have proved successful, especially in the
separation of mitochondrial D N A from nuclear DNA. Ce-
sium chloride density gradient centrifugation, combined
with the use of 4"-6-diamidine-2-phenylindole ( D A P I ) h a s
led to the preparation of mitochondrial D N A from, for
example, yeast (Williamson and Fennel 1975) and more
recently Podospora anserina (Cummings et al. 1979). Al-
though an alternative method exists for the preparation
ofmitochondrial DNA, namely the isolation of intact mito-
chondria (Agsteribbe et al. 1972; Stepien et al. 1978), this
form of centrifugation appears to be the only method by
which ribosomal D N A has been purified from bulk nuclear
D N A for the study of ribosomal D N A organisation (Petes
et al. 1978).
It was the aim of this study to fractionate high molecular
weight D N A from Penicillium chrysogenum into its compo-
nents, most particularly in order to obtain preparations
of mitochondrial DNA.
Materials and methods
Organisms and culture conditions. Penicillium chrysogenum
(NRRL 1951) was used throughout these studies. The or-
ganism was maintained on slants of Aspergillus Complete
Medium (ACM), (see Ditchburn et al. 1974).
Offprint requests to: G. Saunders
Slants were incubated for 5 days at 26°C to provide
stock cultures. Large quantities of mycelium for D N A ex-
traction etc: were obtained by growth for 40 h at 26°C
in liquid ACM shaken at 200 revs min-1 on an orbital
incubator (Gallenkamp).
Extraction of high molecular weight DNA. Cultures of P.
chrysogenum (1.5 1) were grown as described above. Myce-
lium was harvested by filtration through Whatman filter
papers (porosity 1, diameter 22 cm) and washed with sterile
distilled water. After blotting dry, mycelium was distributed
into sterile plastic petri dishes and frozen at - 2 0 ° C for
4 h. Frozen mycelium was then placed in a lyophilisation
apparatus (Chemlab) for 48 h.
Lyophilised material was then ground in a cold mortar
with glass helices (Gallenkamp). Approximately 1 g of glass
was used per gram of lyophilised mycelium. After grinding,
100-150 ml of extraction buffer was added and the whole
mix placed in ice for 1-1.5 h. Extraction buffer consisted
of 25 mM Tris.HC1, 25 mM EDTA, 50 mM NaC1, 1%
SDS, pH 8.0.
After the specified time 5 M NaC1 was added to a final
concentration of 1 M and incubation in ice continued for
1 h. After this time the extraction was centrifuged at
10,000 x g for 30 min in an MSE 18 centrifuge cooled to
4 ° C.
The supernatant from this spin was removed and dia-
lysed overnight at 4 ° C against a dialysis buffer containing
50 mM Tris.HC1, 50 mM EDTA, 100 m M NaC1 p H 8.0.
Nucleic acids were then concentrated by ethanol precipi-
tation and treated with pancreatic ribonuclease (final con-
centration 100 lag m1-1) for 6-8 h at 37 ° C. Nucleic acids
were reprecipitated and dissolved in approximately 10 ml
of 10 mM Tris with 1 mM E D T A p H 8 (TE). This nucleic
acid solution was then ready for application to a sepharose
4B column.
Chromatography on sepharose 4B. D N A solutions obtained
as described above were applied to a sepharose 4B column
(60 cm x 2.5 cm) through a peristaltic pump at a flow rate
of 0.5 ml min-1. Once the sample was loaded, the flow rate
was reduced to 0.15 ml min -a and elution continued with
TE. Four and a half ml fractions were collected in a Uniscil
U F C 120 fraction collector. Fractions obtained were subse-
quently analysed by gel electrophoresis (see below).
Chromatography on RPC-5 Analog. Fractions obtained
from the sepharose 4B column, containing high molecular
344
weight DNA, were pooled and concentrated by ethanol pre-
cipitation and redissolved in 4-5 ml of 10 mM Tris. HC1,
10 mM EDTA and 0.5 M NaC1, (TES buffer) pH 7.2. This
sample of D N A was then loaded through a peristaltic pump
(Pharmacia) onto an RPC-5 Analog column (10 cm x 1 cm)
previously equilibrated with the same but at a flow rate
of 0.15 ml min 1. A Bio-rad 'Econo column' was used.
Once the loading of the sample was completed, the flow
rate was maintained at 0.15 ml min-1 and the salt concen-
tration of the flow through the column changed in either
a stepwise fashion or by application of a salt gradient using
an LKB "Ultrograd" gradient maker. (For details of salt
solutions used and gradients applied see results section.)
Three to four ml fractions were collected and analysed by
agarose gel electrophoresis. The elution of D N A from the
column was monitored using an LKB, 8300 Uvicord II
detector, connected to a Hewlett-Packard 339OA reporting
integrator.
Isolation of nuclei and mitochondria. Intact nuclei were pre-
pared using the procedure described by Gealt et al. (1976).
For the isolation of mitochondria 35 g wet weight of myce-
lium was harvested from liquid ACM flasks and washed
with PBS. After resuspension in approximately 100 ml of
PBS and distribution into a sterile 1 1 conical flask a lytic
mix was added. The lytic mix consisted of 0.5 ml Helix
juice (Sigma), 4.5 ml of PBS, plus 25 mg of cellulase (Novo
Industries). The flask was then incubated overnight at 26 ° C
with gentle agitation. PBS consisted of 0.1 M phosphate
buffer, 0.67 M KC1 pH 6.4. After overnight digestion proto-
plasts and osmotically fragile mycelia were harvested by
centrifugation at 4,000 x g, for 10 min at 4 ° C. The pellet
obtained was then resuspended in approximately 75 ml of
homogenisation buffer (Gealt et. al. 1976) and the suspen-
sion placed at - 7 0 ° C for 20 min. The semi-frozen mixture
was then homogenised at low speed for 3 min in a Silverson
laboratory emulsifier. After addition of a further 125 ml
of homogenisation buffer and filtration through 4 layers
of sterile gauze the filtrate obtained was centrifuged at
9,000 x g for 15 min, at 4 ° C. The supernatant obtained was
then re-centrifuged at 34,000 x g for 30 rain at 4 ° C to ob-
tain a crude mitochondrial pellet. The crude mitochondrial
pellet, resuspended in a small volume of homogenisation
buffer was treated with pancreatic deoxyribonuclease
(Sigma) for i h at 26 ° C, final concentration 50 lag ml- 1.
Mitochondria were then repelleted, resuspended in approxi-
mately 1 ml of TE and nucleic acids extracted in a manner
similar to that described for the preparation of nuclear
DNA.
Determination of buoyant densities. Buoyant densities were
determined in essentially the manner described by Shild-
kraut et al. (1962).
Restriction enzyme digests. Restriction enzymes were pur-
chased from Bethesda Research Laboratories and reactions
were carried out in the buffers recommended by the manu-
facturers. Approximately 0.5-1 lag of D N A were mixed with
4 units of EcoRI restriction enzyme in a final reaction vol-
ume of 40 gl. Tubes were incubated for 4-6 h at 37 ° C.
Reactions were terminated by heating at 65 ° C for 10 min.
HindIII digests of 2 D N A (BRL) were used routinely as
molecular weight standards.
D N A concentrations in stock solutions were assayed
using mithramycin as described by Hill and Whatley (1975).
Agarose gel electrophoresis. Fractions collected from se-
pharose 4B and RPC-5 columns were analysed on 0.7%
agarose gels. Running buffer was 0.089 M Tris-base,
0.089 M boric acid, 0.002 M EDTA pH 8.3. All chemicals
for eleetrophoresis buffer were purchased from Sigma. Gels
were electrophoresed for 2 h at 150 V.
Restriction enzyme digests were analysed on 1% agar-
ose gels in tris-borate buffer. Electrophoresis was for 16 h
at 40 V. Gels were stained for I h in electrophoresis buffer
containing 1 lag/ml- 1 ethidium bromide. Stained gels were
visualised with a Mineralight transilluminator (UV light
Products Ltd.) and photographed through a Wratten No. 9
filter, using a Polaroid MP-4 land camera and Polaroid
type 665 film.
Results
Under the conditions described in materials and methods
high molecular weight D N A is obtained, free from contami-
nating low molecular weight R N A by passage through se-
pharose 4B. This is confirmed both by the UV absorbance
profile of the column eluent and gel electrophoresis of frac-
tions obtained (data not shown). Unfortunately, intact viral
R N A co-elutes with D N A off the sepharose 4B column
and therefore a prior treatment with pancreatic ribonucle-
ase is necessary before R N A / D N A separation. Passage
through sepharose 4B column also effectively removes con-
taminating proteins present inl the initial extract. Before
sepharose 4B treatment, the extract contained 230 lag/ml- 1
protein whilst after treatment less than 10 gg/ml- 1 was de-
tected.
Initial fractionation of nucleic acid from P. chrysogenum
on RPC-5 Analog involved stepping up the salt concentra-
tion through the column and monitoring the eluent ob-
tained by measuring UV absorbance and analysis on agar-
ose gels. It was found that the majority of D N A (greater
than 85%) could be washed from the column using TES
buffer at a salt concentration of 0.75 M. Further D N A
eluted from the column when the salt concentration was
stepped up to 1 M. The D N A contained within the 0.75 M
wash was re-concentrated by precipitation and bound again
to RPC-5 Analog and a gradient (illustrated schematically
in Fig. 1) to fractionate this D N A was applied. In addition
Fig. 1 shows the elution profile, measured as absorbance
at 254 rim, of D N A over the whole of the gradient applied.
As can be seen two peaks were obtained. The elution pat-
0'65
1'21
1.0,
~ 0,8 0.60
~ 0.6 m
~0"4 3o~ zo
0,55
0,2
0,0 [0.50
4 6 12 16 20 24
Time (h)
Fig. 1. UV absorbance profile of eluent obtained from an RPC-5
Analog column after application of the gradient shown (right hand
scale)

Fig. 2. Mitochondrial DNA of P.
chrysogenum treated with EcoRI
restriction enzyme. Track (1)
Mitochondrial DNA, (2) 2 DNA
digested with HindlII
tern as visualised by agarose gel electrophoresis of samples
of the fractions collected, showed clearly that each p e a k
present in Fig. 1 corresponded to the presence of high m o -
lecular weight D N A . D N A from the two peaks were desig-
nated types 1 and 2, in order o f elution. D N A obtained
in the 1 M buffer using the original step gradient was desig-
nated type 3. F r o m the areas under the peaks obtained
on the U V absorbance profile it was determined that P.
chrysogenum total D N A was comprised o f 16% type 1
D N A , 76% 2 and 8% 3.
All 3 D N A types were subsequently analysed in terms
of their respective b u o y a n t densities and response to restric-
tion enzyme treatments. In addition the b u o y a n t density
of D N A obtained from isolated nuclei and m i t o c h o n d r i a
was determined. Total D N A extracted as described gives
two peaks, one m a j o r p e a k at a b u o y a n t density o f 1.710 g/
cm 3 and another, m i n o r peak, which is seen as a shoulder
on the side of the main peak, at a b u o y a n t density o f
1.695 g/cm 3. D N A isolated from purified nuclei gives only
the 1.710 g/cm 3 p e a k whilst the mitochondrial D N A extrac-
tion yielded only the 1.695 g/cm 3 type. D N A of types 1
and 2 b o t h had the same b u o y a n t density o f 1.710 g/cm 3
and can therefore be assumed to constitute the nuclear
D N A . W h e n D N A o f these two types were recombined
and analysed on a cesium chloride gradient only one peak
was observed. Type 3 D N A had a b u o y a n t density o f
1.695 g/cm 3 and m a y therefore be assumed to represent
the mitochondrial D N A .
Restriction enzyme analysis of D N A types 1, 2 and 3
gave 3 distinct restriction profiles. Type 1, when digested
with EcoRI yields two distinct bands of molecular weight
4.2 x ]06 D and 1.1 x ]06 D respectively, (these bands are
just visible in restriction digests of total D N A ) whilst type
2 gave only a fluorescent, striated smear. Type 3 shows
eight distinct bands o f combined molecular weight
31.6 x ]06 D, (see Fig. 2).
Discussion
The fractionation o f high molecular weight D N A from P.
chrysogenum into three distinct types, using RPC-5 Analog,
is almost certainly a consequence of differences in base com-
position o f one sort or another. Thus, in the case o f the
separation of types 1 and 2 from 3 it is most likely that
the high A + T content (in this case 64%), characteristic
345
of mitochondrial D N A , provides the basis for fractionation.
It was found by Wells et al. (1980), that retention of D N A
on RPC-5, an ion exchange resin from which RPC-5 Ana-
log is derived, could be correlated directly with A + T con-
tent.
Conversely, the separation o f 1 and 2 cannot be occur-
ring on the basis of differences in base composition. It is
possible in this case that although the overall base composi-
tion of type 1 is similar to that o f type 2, localised differ-
ences within type 1 molecules m a k e them slightly less able
to bind to RPC-5 A n a l o g a n d / o r remain b o u n d (Wells et al.
1980). It is well k n o w n that eukaryotic D N A contains a
number of repeated genes, the most c o m m o n of which are
the ribosomal and transfer R N A genes (Long and D a w i d
1980). F r o m the restriction profile o f type 1 D N A it could
clearly by seen to contain repeated sequences and m a y
therefore be enriched for either one or b o t h of the gene
types mentioned above.
Acknowledgments, This work was supported by SERC research
grant no. GR/C 20864. M.E.R. is in receipt of an SERC student-
ship no. BI/312975.
We would like to thank T.E. Price for his technical assistance.
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C o m m u n i c a t e d by F. Kaudewitz
Received October 10 / November 28, 1983