Volume 4 no. 1 January 1977
NUCleJC Adds Research
Heterogeneous base distribution in mitochandrial DNA of Neurospora crassa
Peter Terpatra, Marijke Holtrop and Ab Kroon
Laboratory of Physiological Chemistry, State University, Bloemsingel 10,
Groningen, Netherlands
Received 5 October 1976
ABSTRACT
The mitochondrial DNA of Neurospora crassa has a heterogeneous intramolecular
base distribution. A contiguous piece, representing at least 30% of the total
genome, has a G+C content that is 6% lower than the overall G+C content of
the DHA. The genes for both ribosomal RNAs are contained in the remaining,
relatively G+C rich, part of the genome.
INTRODUCTION
The most striking example of intramolecular base-composition heterogeneity
in mtDNA
is to be found in Saocharomyces cerevisiae, where half of the total
mtDNA has a G+C content lower than 5Z and is interpersed in small "spacer" regions between "gene" sequences of 32J G+C. This "genes and spacers" model also
applies to the mtDNA of Euglena gradlis,
which has a G+C content of 25Z (2),
but it is not clear whether it holds for other lower eukaryote mtDNAs as well,
especially those with a higher G+C content. Using buoyant density analysis
and denaturation-renaturation techniques we present evidence that the mtDNA
of Neurospora crassa has a heterogeneous base distribution, that is more in
agreement with the type of base—composition heterogeneity that can be seen in
phage X DNA, where the G+C bases are unequally distributed among long DNA sequences (3). This unequal distribution of G+C basepairs is exploited to establish an approximate localization of the mitochondrial rRNA
genes on the
mtDNA.
MATERIALS AND METHODS
Phage T 4 DNA and phage PM 2 DNA were a gift from F. Fase Fowler. Phage H} DNA
(4) was a gift from F. Arwert.
* Abbreviations: mtDNA « mitochondrial DNA; rRNA = ribosomal RNA; 1 x SSC =
0.15 M sodium chloride - 0.015 M sodiumcitrate; SDS ™ sodium dodecyl sulphate.
O Information Retrieval Limited 1 Falconberg Court London W1V5FG England
Nucleic Acids Research
Isolation of mitochondria: Growth and isolation of Neuroepora craesa strain
Em 5256 has been described (5). The buffers used are: Bl: 0.44 M sucrose, 10
mM Tris-HCl, 5 mM EDTA pH 7.6; B2: 0.44 M sucrose, 5 mM MgCl 2 , 10 mM TrisHC1 pH 7.6; B3: 0.44 M sucrose, 10 mM Tris-HCl, 50 mM EDTA pH 7.6; B4: 1.75
M sucrose, 10 mM Tris-HCl, 5 mM EDTA pH 7.6. The washed mycelium was
suspended in Bl using a Braun mixer for 30 sec. and homogenized in a grindmill (6). The homogenate was centrifuged 20 nin. at 2500 x g. The supernatant
was filtered through glasswool and centrifuged 20 min. at 20,000 x g. The
crude mitochondrial pellet was resuspended in Bl and layered on a cushion
of B4 and centrifuged 10 min. at 13,500 x g. The mitochondrial band at the
interphase was removed, suspended in Bl and centrifuged 10 min. at 20,000 x
3. The pellet was suspended in B2, DNase I (grade II, Boehringer) added to
50 yg/ml and incubated 1 h at 4°C, then diluted with 2 vol. of B3 and centrifuged 10 min. at 20,000 x g. This gives a purified DNaSe-treated mitochondrial pellet. All operations were performed at 4°C.
Extraotion of mtDNA method A: The mitochondrial pellet was suspended in
0.25 M EDTA (pH 8.0) at 0°C, Sarkosyl (Geigy) was added till a final concentration of 2Z. After 5 min. at 30 C the lysate was gently extracted
with 1.5 vol. freshly distilled phenol-cresol (7) saturated with 10 mM TrisHCl (pH 7.5), the waterphase was extracted with 1 vol. chloroform/isoamylalcohol (24:1). The waterphase was dialyzed overnight against 0.1 x SSC*at
4°C treated with RNase A (Boehringer; heated 10 min. 80°C) 20 ug/ml for
30 min. at 30°C; NaCl was added to 1 M, Sarkosyl to 0.2Z and the DNA was
pelleted in a Ti60 rotor at 60,000 rpm, 6.5 h at 0°C. The pellet was
dissolved in 10 mM Tris-HCl, 0.25 mM EDTA, 0.05Z Sarkosyl pH 7.4, layered
on a isokinetic sucrose gradient (15Z - 30.9Z), in 0.5 M NaCl, 10 mM TrisHCl, 0.5 mM EDTA, 0.05Z Sarkosyl pH 7.4 in a SW27.1 rotor and centrifuged
19 h, 27,000 rpm, 4°C. Closed and open circular PM_ DNA were used as
sedimentation markers in a parallel tube. The gradients were fractionated
by upward displacement through a Uvicord flowcell. Fractions were dialyzed
against 1 x SSC.
Extraction of mtDNA method Bh The mitochondrial pellet was lysed in 50 mM
EDTA pH 8.0 with a cocktail of detergents (8) and extracted with phenol and
chloroform as described above. The waterphase was precipitated with alcohol;
after one night at -20 C, the precipitate was collected by centrifugation (15
min at 25,000 x g) and dissolved in IxSSC, treated with RNase as described
above, treated with Pronase (Calbiochem, nuclease free) with 0.1Z Sarkosyl
at 200 ug/ml for 2 hours at 30°C, extracted with chloroform after which
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CsCl (Merck, Suprapur) was added to a density of 1.70 gr/ml. Centrifugation
was in a Ti50 rotor for 64-72 hours at 34,000 rpm, 20°C. The tubes were
punctured at the bottom, fractions were taken by dripping and measured at
260 nm. Fractions were either dialyzed again
lxSSC or centrifuged again to
equilibrium in CsCl.
E. coli DNA was isolated from E. coli Q 13 following extraction method B and
purified on a hydroxyapatite column instead of a CsCl gradient.
Neurospora crassa nuclear DNA was extracted as described for E. coli DNA from
a crude nuclear pellet obtained after the first centrifugation step in the
preparation of mitochondria.
[321
L
1
P mitochondrial rRNA was extracted (5) from purified mitochondrial 80S
monosomes (9).
Analytical Ultraoentrifugation for CsCl buoyant density determination has
been performed as described (10) calculating the buoyant density according to
the method of Schildkraut et al. (11) using phage Hj DNA as a reference (4).
Alkaline band sedimentation was performed according to Studier (12).
Denaturation-Renaturation. DNA samples were sonicated at 0 C under nitrogen
with a Branson sonifier equipped with a micro-tip at maximal output in 6
bursts of 5 sec. Sonicated samples were dialyzed extensively against the
same solution of lxSSC. The Gilford spectrofotometer 2400 S was equipped with
4 water-jacketed 1 ml cuvettes with teflon stoppers, one cuvette containing
the Gilford thermosensor. Two waterbaths, one Haake F 423 with a Haake temperature programming device PG11, and one Colora Ultrathermostat at renaturation temperature were connected in parallel with two threeway stopcocks.
The degassed DNA samples (15-25 iig/ml in lxSSC) were denatured at a rate of
0.4 C/min. The hyperchromicity ranged from 30 - 36Z. The derivative melting
curve was obtained by plotting R •» (At£ - Ati)/(A98 - A2s)/(t2 - tj) against
temperature at 260 nm. After 10 min at 98 C the waterbath at renaturation
temperature (59 C) was switched on; after temperature stabilization (1 min)
the renaturation was followed to completion. The melting curves of the renatured samples were almost identical to the native curves and fell within 2 C
of the original curve. The kinetic complexity was calculated according to
Wetmur (13) taking into account a nonperfect hyperchromicity if necessary.
With every cycle, Ti, DNA was used as a control.
Hybridization: Filters (Sartorius, 0.1 ti) were loaded (14) with 0.3 yg DNA
each and hybridized with the ^
^P rRNA (spec. act. 191.000 cpm/ygj in 1 ml
3xSSC - 0.2Z SDS* at 65°C for 19 h. Each vial contained also a blank and a E.
coli DNA filter. The filters were washed 10 min in 2xSSC at 25°C (3 times) and
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at 60°C (once), treated with RNase A (20 ug/ml in 2xSSC at 25°C, 1 h) and
washed again in 2xSSC at 25 C (3 times). Filters were counted in a toluene
based scintillation cocktail. Blank and E. coli filters contained less than
0.011 of the input counts.
RESULTS
On preparative scale we are unable to isolate the 40 x 106 dalton circular
mtDNA of Neuroepora Craaea (15) in intact form. A method to iuolate full
length linear molecules has been described (16), but we could not reproduce
this. DNA preparations with a mean fragment length of about 5 x 106 dalton
were obtained with a preparative CsCl equilibrium gradient, an example of
which is shown is Fig. 1A. The DNA is clearly heterogeneous in density: a
main peak banding at 1.699 g/ml and a minor peak at 1.693 g/ml, which contains about 30Z of the total (see also Fig. IB, trace a ) . Contamination with
nuclear DNA as an explanation for this heterogeneiLy is excluded, because
its density is 13rag/foilhigher (Fig. IB, trace b). To investigate the nature
of the minor light peak, we purified this fraction and compared its properties
with total mtDNA and a fraction from the heavy side of the peak in Fig. 1A.
The indicated fractions (Fig. IA) were pooled and after two successive CsCl centrifugations a heavy (fraction H) and a light fraction (fraction L) were obtained with a buoyant density profile as shown in Fig. IB (trace c and d ) .
These fractions, together with total mtDNA were sonicated and subjected to
denaturation-renaturation in ixSSC. As can be seen from the differentiated
melting curves in Fig. 2 there is considerable heterogeneity in melting behaviour, although not as extreme as for example in Eugtena (2) and Sacchapomycee
(17) mtDNA. T 4 DNA melts sharply, but in total mtDNA (Fig. 2A) five transitions can be observed: the main one at 83.2 C (Table I ) . In fraction H (Fig.
2B) the main transition is shifted to 85 C and in fraction L (Fig. 2C) to
81.4 C. There is no evidence for a large proportion of very A+T rich stretches
in the mtDNA as the early melting regions still have a T m of 80.2°C (Fig. 2C),
which corresponds to a G+C content of 27Z (Table I). The G+C content, calculated from the buoyant density for total mtDNA (Table I) is in close agreement with the chemically determined G+C content of 40Z (19), but is higher than
the corresponding G+C content calculated from the Tm's (Table I). This may be
caused by the base distribution heterogeneity itself (17,20) and the small
fragment length employed in the denaturation analysis (21) (Table II).
To asses in an independent way the relative amount of the A+T rich fraction
in a total genome, we performed quantitative renaturation with total mtDNA,
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density(g/ml)
1.693 11.701 1712
1.699
1.741
Fig. 1. A: Preparative CsCl density gradient from mtDNA prepared by method B.
The indicated fractions (L and H) have been pooled and recentrifuged twice in
CsCl. B: Analytical CsCl centrifugation of a) total mtDNA; b) nuclear DNA; c)
purified fraction H; d) purified fraction L. Marker DNA is from phage HI
SO
90
80
90
80
90
trmperatuTt'C
Fig. 2. Differential melting curves of A: 0 — 0 — 0 T 4 DNA and #-•-• total
mtDNA. jJ: fraction H mtDNA and £: fraction L mtDNA (see Fig. IB; trace c and
d). The ordinate R is defined in Methods. The arrows in panel C indicate the
six melting transitions listed in Table I.
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T A B L E
DNA
T*
I
G+CZ**
pCsCl
G+CZ***
80.2
27
fraction L
81.4
29.5
1.693
33.7
total mtDNA
83.2
34
1.699
39.8
1 .701
41.8
fraction H
85
38
87.7
45
89
48
* Melting transition temperatures (T) indicated in Fig. 2.
** calculated from T (18).
*** calculated from buoyant density (11).
T A B L E
II
k 2 (L/mol/sec)(13)
DNA
kinetic
complexity
106 x 10 6 *
10.9
12*
total mtDNA
11.4
23.1
58 x 10 6
fraction H
8.3
32.1
28 x 10 6
fraction L
9.2
31.6
32 x 10 6
* k2 for Ti, is taken to correspond to 106 x 10 6 dalton (23). Data are not
corrected for differences in G+C content (24).
fraction L and fraction H (Fig. 3 ) . The mitochondrial fractions followed
second-order kinetics till about 50Z renaturation, whereafter renaturation
slowed down as can be seen for fraction L in Fig. 3. The kinetic complexity
relative to T 4 DNA has been calculated from the first 50Z renaturation part
of the lines (Table II). The outcome for total mtDNA is in reasonable agreement
with the complexity of 66 x 10 6 as determined by Wood et al. (22) with the
same technique. Both values however are too high as compared with other, and
to our opinion, better values of 40 - 44 x 10 6 for the mtDNA molecular weight,
based on electron microscope measurement (15,19) and the added molecular
weight of restriction enzyme fragments (25,26). A similar situation holds
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Fig. 3. Renaturation second order
rate plot for •-•-# T4 DNA, 0-0-0
total mtDNA, x-x-x fraction H mtDNA
and Q-O-O fraction L mtDNA (see
Fig. IB; trace c and d ) . The upper
arrow corresponds to 50Z renaturation for total mtDNA, the lower
arrow to 50Z renaturation for both
fraction H and L.
-=
10-,
1
2
time(secx10"3)
for yeast mtDNA under standard renaturation conditions (8,10), which are considered to be not optimal for that DNA (10). For Neurospora crasea mtDNA this
remains a point for further investigation. Nevertheless it may be concluded
that both fraction L and H have a complexity of about half of the total genome.
Whether the A+T rich sequences are dispersed in the genome or located in one
stretch cannot be decided from this experiment. If they are dispersed in
short stretches, the CsCl density heterogeneity should disappear when one
looks at longer fragments. This is not the case as can be seen from Fig. A,
where at a fragment length longer than 302 of the intact genome the A+T
rich shoulder is still present in an amount of 30Z of the total DNA, from
which we conclude that there is one long segment of at least 30Z genome
length present in the mtDNA, that contains 6Z less G+C than the average for
the whole genome (Table I).
This kind of distribution gives us the possibility to localize in a first
approximation the ribosomal RNA genes on the DNA. The rRNA from mitochondrial
ribosomes was hybridized to total mtDNA, fraction H and fraction L with
increasing amounts of RNA (Fig. 5 ) . The plateau value with total mtDNA (3.3Z)
is comparable to previously established data (27,28), from which it was
estimated that there is no more than one gene for each rRNA on the mtDNA.
Fraction H is clearly enriched in rRNA genes, whereas fraction L is poor in
these sequences. Taking into account that fraction H and L have much lower
complexities than the total molecule (Table II), it can be concluded that
the ribosomal genes are localized on the relatively G+C rich region of the
genome.
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T
1
T
1599 1.701
fig. 4. Relation between mtDNA fragment length and CsCl density. A: Isokinetic
sucrose gradient of mtDNA prepared by method A. Fractions a,b and c have been
pooled and subjected to analytical CsCl centrifugation in panel 13: Trace a, b
and c resp. The mean fragment length of a,b and c was 3, 6 and 12 x 10 6 as calculated from the PM2 DNA sedimentation marker (closed (I) and open (II) circles: see arrows in panel A ) .
density <g/ml)
Fig. 55.
1.693
Plateau hybridization of
32
L ]p rRNA with •-•-• total mtDNA,
0-0-0 fraction H mtDNA and x-x-x
fraction L mtDNA.
10
RNA/DNA
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DISCUSSION
The density heterogeneity displayed by fragmented mtDNA from Heuroapova aras8a
has also been observed for other Neuroapora species by Reich and Luck (29),
who suggested that it might be caused by an intermolecular heterogeneity.
This explanation becomes very unlikely in view of the renaturation data and the
more recently obtained restriction enzyme maps (25, 30, 31) which exclude intermolecular macroheterogeneity. From the foregoing experiments we propose the
following intramolecular base distribution pattern for the fleuroepora araaea
mtDNA: it is heterogeneous, although not very extreme, with a maximal difference in G+C content between the most abundant fragments of 10Z and with
two minor components of high G+C content (Table I ) . The most remarkable
feature is a stretch of base sequences with a length of at least 30Z of the
total molecule, containing 6Z less G+C than the average. This last observation
agrees very well with the recently published denaturation map of Bernard et
al. (19), where it was shown that one stretch consisting of one-third to onehalf of the circular mtDNA melts at a lower temperature than the remaining
part. Random mechanical and enzymatic breakdown (19) during the isolation of
the mtDNA can then explain the occurence of the heterogeneous CsCl density profiles in Fig. 1A and Fig. 4.
To explain the extensive base sequence heterogeneity in yeast mtDNA (1) and
Euglena mtDNA (2), a genes and spacers model has been proposed (1), with the
speculation that it could reflect a common coding structure for other mtDNAs
as well (1,2). This will be difficult to test, because the majority of mtDNAs
have a higher overall G+C content than yeast mtDNA (182 G+C (1)) and Euglena
mtDNA (25Z G+C (2)). Although sequence heterogeneity is still present in other
mtDNAs, there is no longer an easy distinction between potential spacer and gene
sequences because sequences with an extreme G+C content are not abundant. On the
basis of denaturation analysis this can be concluded for Acanthamoeba mtDNA (32)
with 31Z G+C, Newrvapora craaea mtDNA (Table I) with 40Z G+C, animal mtDNAs (33)
with 40 - 43Z G+C and for plant mtDNAs (34,35) with 47Z G+C. Base sequence heterogeneity as measured by denaturation analysis tends to decrease in these
mtDNAs with increasing G+C content and is absent in plant mtDNAs (34,35). A
similar trend can be noticed in chloroplast DNAs (36). Spacerregions may be present in these mtDNAs with approximately the same G+C content as the genes, but
their existence may be unrelated to sequence heterogeneity. At this moment the
only known spacerregions in Neuroapora cra8ea mtDNA are represented by the unconserved regions of the ribosomal RNA precursor (28). This precursor is 25Z
longer than the sum of both the mature rRNAs and has the same overall G+C
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content as the rRNAs (28). Such an organization of rRNA genes is a strongly
conserved feature in E. ooli DNA (37) and animal mtDNAs (38). It does not reflect a common coding structure with yeast mtDNA, where the two rRNA genes are
at a distance of nearly half a genome length (39).
The localization of the rRNA genes on the relatively G+C rich part of Neurospora mtDNA (fig. 5) is a first start in our trial to map these genes on the
DNA. Recent investigations, using restriction fragments of the mtDNA (for a
preliminary account see ref. 40) agree with the localization predicted by the
existence of a precursor rRNA (28).
ACKNOWLEDGEMENT
We thank Dr. G. Venema from the Department of Genetics, Haren, for offering us
the analytical ultracentrifugation facilities. These studies were supported in
part by a grant from the Netherlands Foundation for Chemical Research (SON)
with financial aid from the Netherlands Organization for the advancement of
Pure Research (ZWO) .
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