Voiume3 no.io Octoberi976
Nucleic Acids Research
Interspersion of different repeated sequences in the wheat genome revealed by
interspecies DNA/DNA hybridisation.
Derek B. Smith, Jiirgen Rimpau and Richard B. Flavell
Department of Cytogenetics, Plant Breeding Institute, Trumpington,
Cambridge, CB2 2LQ, UK
Received 20 August 1976
ABSTRACT
The repeated sequences in oats DNA have been used to study chromosomal
repeated sequence organisation in wheat. Approximately 75% of the wheat
genome consists of repeated sequences but only approximately 20% will form+
heteroduplexes with repeated sequences from oats DNA at 60°C in 0.18 M Na .
The proportion of wheat DNA that forms heteroduplexes with oats DNA is
shown to be independent of the wheat DNA fragment length. However, the
proportion of wheat DNA that is retained with the heteroduplexes when
fractionated on hydroxyapatite is very dependent upon the wheat fragment
length up to 3500 nucleotides. This is because more non-renatured wheat
DNA is attached to the heteroduplexes with longer fragments. The results
indicate that the repeated sequences in the wheat genome homologous to
repeated sequences in oats are not clustered in the chromosomes but
distributed amongst other repeated and possibly non-repeated sequences.
INTRODUCTION
The organisation of different kinds of nucleotide sequences has been
investigated in many genomes in recent years (1, 2, 3, k). In many
organisms most of the intermediate repeated sequences are comparatively
short (e.g. 300 base pairs) and separated by non-repeated sequences 800 to
several thousand base pairs long (A). However this general pattern is not
universal; Drosophila for example has a somewhat different genome
organisation (2).
Many higher plant species contain a high proportion of repeated
sequence DNA. Our own detailed studies on the wheat and rye genomes (3, 5)
have shown that about 75% of the DNA of these genomes consists of repeated
sequences and most of the single or few copy sequences are less than 5000
base pairs long. With such a high proportion of repeated sequences, there
must be relatively long lengths of repeated sequence DNA in these genomes.
It is the organisation of this repeated sequence DNA with which we are
primarily concerned in this paper.
Some of the repeated sequences in wheat are sufficiently closely
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© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
Nucleic Acids Research
related to repeated sequences in the related cereal species oats to form
heteroduplexes in vitro (6, 7). Other repeated sequences are not. We
have therefore investigated whether the sequences common to both genomes
are clustered in the wheat genome or interspersed with other repeated
sequences as a way of finding out more about sequence organisation within
the repeated sequences.
MATERIALS AND METHODS
Isolation of 3 H labelled and uniabeiled DNAs
Unlabelled wheat (var. Chinese Spring) and oats (var. Maris Titan)
DNAs were isolated from leaves as described by Smith and Flavell (6).
Tritium labelled wheat DNA was purified from 3 day old seedlings germinated
in the presence of 3 H thymidine (CH3 labelled, 41 Ci/m mol) as previously
described (6). The specific activity was 28,400 cpm/pg. Calf thymus DNA
was purchased from Sigma Chemical Co. 98 + 1% of the native DNAs were
retained on hydroxyapatite at 60°C in 0.12 M PB*.
DNA fragmentation and estimation of average fragment sizes
Unlabelled DNA was sheared by sonication as previously described (6, 8).
Weight average single strand fragment lengths were determined by boundary
velocity sedimentation in 0.9 M NaCl 0.1 M NaOH, as described by Studier
(9) using an MSE centriscan ultracentrifuge. Distribution curves of fragment sizes in similarly treated samples have been presented previously (8).
Tritium labelled wheat DNAs of different average fragment sizes were
obtained by fractionating native and sheared DNA in 5 to 11% w/w linear
sucrose gradients made in 0.1 N NaOH after sedimentation for 19 and 40 hr
at 24,000 rpm at 20°C. Weight average single stranded fragment lengths in
each gradient fraction were determined as described by Burgi and Hershey
(10) after sedimentation for 16 to 40 hr in identical gradients.
Approximately 1 yg of 3 H labelled DNA was seditnented in a 14 ml gradient
together with 150 yg unlabelled marker DNA whose molecular weight was
determined separately in the analytical ultracentrifuge.
Reassociation of homologous and heterologous DNAs
Unlabelled oats DNA in 0.12 M PB at 500 yg/ml was sheared to an
average single strand fragment size of about 400 nucleotides (see figure
legends for actual sizes in different experiments). Aliquots were taken
and to each a small volume of H labelled wheat DNA from one of the sized
stock samples (see above) was added. The final ratio of unlabelled to
labelled DNA was usually in excess of 8000:1. The samples were denatured
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at 100°C for 5 minutes and incubated at 60°C for 18-20 hours (Cot = 90-100).
After incubation the samples were diluted to 2 mis with 0.12 M PB at 58°C
and applied to hydroxyapatite columns (BioRad HTP; 2cc bed volume)
previously equilibrated to 0.12 M PB at 58°C. The columns were washed with
0.12 M PB at 58°C to elute unrenatured DNA before the DNA in the duplex
fraction was removed, either by (a) elution with 0.12 M PB at 95°C, (b)
a phosphate gradient or (c) a thermal gradient.
S^ nuclease digestion
Si nuclease was prepared from a-amylase (Sigma; Type IVA) using the
method of Sutton (11). After renaturing DNA in 0.12 M PB at 60°C, four
volumes of 0.5 M NaCl and five volumes of assay buffer (0.05M acetate
buffer pH 4.3 containing 0.2 mM ZnS04 and 5.5 mM mercaptoethanol (12)) were
added, these solutions having been previously equilibrated to 24°C. Si
nuclease was then added and the mixture was incubated at 24°C for 90
minutes. Preliminary experiments were conducted to ensure the enzyme
concentration was not limiting (see Results). The reaction was terminated
either by the direct addition of trichloroacetic acid (final concentration
5%) or by adding 0.2 volumes of 0.5 M PB and gently shaking the mixture
with chloroform containing 1% octanol. The aqueous phase was dialysed to
0.05 M PB before fractionation of the S-| nuclease resistant duplexes on
hydroxyapatite.
"Zero time binding" DNA'
Hydroxyapatite chromatography of denatured wheat DNA that has been
momentarily returned to renaturing conditions reveals a fraction that
renatures extremely rapidly (3, 8). This has been called the zero time
binding fraction (1) and shown to contain reverse repeat sequences which
renature by a first order reaction (1, 13). Thus it is necessary either to
remove these sequences or to correct for their effect upon the proportion
of DNA in the duplex fraction. Davidson et al (1) have suggested a
correction procedure which we have applied to our results.
Estimation of DNA
Unlabelled DNA was estimated by OD26O measurements after mixing and
centrifuging at low speeds to sediment any hydroxyapatite. Labelled DNA
was measured by precipitation with 5% trichloroacetic acid in the presence
of 100-150 yg of bovine serum albumin at 0-4°C, collection on Whatman GF/B
filters and counting in a toluene based scintillation mixture (6).
Percentages of reannealed DNA were corrected for the zero time binding
fraction.
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RESULTS AND DISCUSSION
The hybridisation of different sized wheat DNA fragments to oats DNA
Tritium labelled wheat DNA fragments of different known average lengths,
were mixed with an 8000 fold or greater excesses of either unlabelled
wheat or unlabelled oats DNA, denatured and incubated at 60°C in 0.12 M PB
to a C o t of between 90 and 100. At this C Q t value in the wheat + wheat
DNA experiment essentially all fragments carrying repeated sequences are
incorporated into duplexes and therefore retained on hydroxyapatite in
0.12 M PB at 58°C (6, 8). Fragments consisting of only non-repeated
sequence DNA do not commence renaturation until a C t value greater than
100 (8) and consequently are not adsorbed on hydroxyapatite at 58°C in
0.12 M PB. The proportion of labelled DNA of each fragment size in the
hydroxyapatite duplex fraction was determined and the results are shown in
figure 1.
too
c
o
Fragment
size - n u c l e o t i d e s x 10
Figure 1.
Hydroxyapatite binding of homoduplexes and heteroduplexes as a
function of labelled DNA fragment size. 3H wheat DNA of various average
fragment sizes was mixed with unlabelled wheat, oats or calf thymus DNA of
average single stranded fragment size 300 to 400 nucleotides. The concentration of unlabelled DNA was 500 yg/tnl in 0.12 M phosphate buffer and the
ratio of labelled to unlabelled DNA exceeded 1:8000. After denaturation
and incubation to Cot 90-100 at 60°C the samples were applied to
hydroxyapatite columns and the single stranded DNA eluted with 0.12 M
phosphate buffer. DNA in the duplex fraction was recovered by elution with
0.12 M phosphate buffer at 95°C.
Unlabelled DNAs were
• Wheat
• Oats
A Calf thymus
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The proportion of labelled wheat DNA in the duplex fraction after
incubation with oats DNA increased with fragment length up to a fragment
length of 3500 nucleotides. With an average fragment length of 250
nucleotides only 30% of the wheat DNA was in the duplex fraction while at
4000 nucleotides, nearly 70% was in the duplex fraction. In the
corresponding intraspecies hybridisation curve, nearly 80% of the
labelled DNA was in the duplex fraction at an average fragment size of 250
nucleotides and this proportion increased to 95% with fragments around 4000
nucleotides long.
The shapes of these curves are very similar to those described and
analysed in detail by Davidson et al (1) and Graham et al (1*») after
renaturation studies with different length Xenopus and-sea urchin DNA
fragments. From these intraspecies hybridisation studies, they concluded
that the proportion of the DNA which renatures is represented by the
ordinate intercept and the additional DNA in the duplex fraction with
increasing fragment size is non-renatured segments of the partially
renatured fragments. We have previously used these conclusions to
interpret the curve gained when labelled wheat fragments were hybridised to
unlabelled wheat DNA. The intercept value of 75% (figure 1) implies that
75% of the wheat DNA renatures after incubation to Cot 100 at 60°C in 0.12
M PB and is therefore the proportion of the genome that consists of
repeated sequences. The additional 20% of the labelled DNA in the duplex
fraction with fragment sizes of 4000 nucleotides consists of non-repeated
single stranded DNA on fragments which also contain a repeated sequence (3).
Utilisation of these same arguments to interpret the curve when
labelled wheat fragments were incubated with oats DNA did not seem
justified without further investigation since a number of different
circumstances prevail in this situation. Firstly, less than a third of the
wheat repeated sequences hybridise to oats DNA, thus a much higher
concentration of denatured labelled fragments carrying repeated sequences
are present throughout the incubation. These fragments could possibly
hybridise with each other at longer fragment lengths where renaturation is
more rapid (15), and then be included in the duplex fraction on hydroxyapatite. To test this, labelled wheat DNA fragments were incubated at the
same concentration as in the oats DNA experiments with unrelated calf
thymus DNA. Some labelled wheat DNA was included in the duplex fraction
after incubation to Cot 100 but this did not increase substantially with
fragment size over the range studied here (see figure i).
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Secondly, wheat repeated sequences form very mismatched duplexes when
hybridising with oats repeated sequences. A relatively high proportion of
the duplexes are only just stable at 60°C in 0.12 M PB (see figure 2, and
reference 6). In this situation short labelled DNA fragments may form
such poor duplexes that they are unstable and will not bind to hydroxyapatite at 0.18 M Na + , 58°C while longer fragments are able to form
sufficient duplex structure to be retained in the duplex fraction on
hydroxyapatite. Alternatively, longer labelled wheat DNA fragments may
bind more than one short unlabelled oats DNA fragment and by this means
form sufficient duplex structure to be retained on hydroxyapatite at 0.18 M
Na + , 58°C.
100
80
90
100
Centigrade
Figure 2.
Thermal elution profiles from hydroxyapatite of duplexes
formed by the repeated sequences of oats DNA ( • ) and heteroduplexes
formed between the repeated sequences of oats DNA and labelled wheat DNA
( • ) . Labelled wheat DNA was mixed with unlabelled oats DNA (500 yg/ml
in 0.12 M phosphate buffer) such that the ratio of labelled to unlabelled
DNA exceeded 1:8000. The mixture was sheared to an average single strand
fragment size of 400 nucleotides. After denaturation and incubation at
60°C to Cot 90 the sample was applied to hydroxyapatite at 60°C and the
denatured DNA eluted with 0.12 M phosphate buffer. The duplexes were
removed by elution with the same buffer at the temperatureandintervals
labelled DNA
indicated and the concentration of unlabelled DNA (OD?6o)
was determined for each fraction that eluted above 60oc.
Both of these possibilities would result in an increase in the
proportion of the labelled wheat DNA in relatively unstable duplex
configuration with oats DNA, when long wheat fragments were incubated with
oats DNA. This would therefore invalidate the assumption that all the
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additional DNA in the hydroxyapatite duplex fraction with longer fragment
lengths was single stranded and unrelated to oats DNA under the renaturation criteria. We therefore investigated the proportion of labelled wheat
DNA which was in a duplex configuration and the stability of these
duplexes after incubation of different length fragments with unlabelled
oats DNA. The duplex DNA was separated from denatured DNA, including the
denatured portions of the partly base paired fragments, by degrading the
denatured DNA with S] nuclease. The quality of the duplex structures was
assayed by their ability to bind to hydroxyapatite at different phosphate
concentrations and different temperatures.
Properties of Si nuclease resistant labelled wheat DNA hybridised to oats
DNA
Although S-j nuclease digests single stranded DNA, the degree to which
it attacks small single stranded regions within regions of duplex DNA is
dependent upon the temperature and salt concentration (12, 16). After
testing a number of incubation temperatures and salt concentrations we
found that S] nuclease under standardised, convenient DNA and enzyme
concentrations would completely digest denatured DNA in 1.5 hr at 24°C in
0.2 M NaCl, except for the few percent of renatured DNA in denatured
eucaryotic DNAs formed from intrastrand reassociation (see figure 3).
These conditions are considerably less stringent than those used by others
(e.g. 12, 16) to preserve mismatched duplexes. By adopting these very
relaxed conditions we hoped to preserve as much as possible of the very
mismatched heteroduplexes whilst digesting all the unrenatured regions.
The results shown in figure 3 show that denatured DNA is rapidly degraded
under these conditions.
Mixtures of unlabelled oats DNA and labelled wheat DNA fragments 300,
2500 or 5500 nucleotides long, incubated to Cot 90 were treated with S]
nuclease for 1.5 hr using the conditions detailed in figure 3. The
results in table 1 show that the proportion of TCA precipitable S]
nuclease resistant labelled wheat DNA was independent of the labelled
fragment size incubated with oats DNA. In all experiments the
proportions of TCA precipitable S] resistant labelled wheat DNA were very
similar to each other and to the proportion of duplex DNA indicated by the
extrapolation of the curve in figure 1 to the ordinate. These results
imply that most if not all the additional labelled DNA in the hydroxyapatite duplex fraction with long fragments in figure 1 is single stranded.
"
"
"
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•
D
i
O
:
X
\
E 10
\
TCA
insoluble
Q.
V ^ .—
1
2
s
—
20
HOURS
Figure 3.
DNA digestion by S-\ nuclease at, 24°C.
H wheat DNA was added to calf thymus DNA (500 vg/ml) in 0.12 M phosphate
buffer such that the ratio of labelled to unlabelled DNA exceeded 1:8000.
DNAs were either native (open symbols) or melted for 5 minutes at 100°C
(closed symbols). Assay buffer (see Materials and Methods) and 0.5 M NaCl
were added at 24°C and samples taken immediately. S-j nuclease was added to
the remaining DNAs which were then incubated at 24°C. Samples were removed
at various intervals, neutralised with phosphate buffer, diluted to give a
final phosphate concentration of 0.05 M and applied to hydroxyapatite
columns. The columns were washed with 0.12 M phosphate buffer at room
temperature and then at 58°C. Si resistant duplexes were eluted with 0.12
M phosphate buffer at 95°C.
o • TCA precipi table radioactive wheat DNA in the
room temperature and 58°C fractions combined.
a • TCA precipi table radioactive wheat DNA in the
95°C fraction.
The arrow indicates the incubation time chosen for the heteroduplex studies.
3
We further characterised the S-| nuclease resistant wheat-oats heteroduplexes and the unlabelled oats homoduplexes by adsorbing them to
hydroxyapatite at room temperature in 0.05 M PB and eluting them with
either a phosphate gradient at 58°C or a temperature gradient in 0.12 M PB.
The elution profiles are shown in figures 4 and 5 respectively.
Only a small proportion of oats homoduplex DNA eluted at 0.10 to 0.15 M
PB (figure 4) or between 20 and 65° (figure 5); most of it eluted in the
0.18 to 0.30 M PB (figure 4) and 72 to 93° (figure 5) fractions. This is
the expected behaviour for duplex DNA formed between single strands having
some mismatched bases (6, 17). In contrast to this, a considerable
proportion of the S-| nuclease resistant TCA precipitable labelled heteroduplex DNA eluted in the 0.10 and 0.12 M phosphate fractions (figure 4) and
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Table 1.
Effect of wheat DNA fragment length on Si nuclease resistant
heteroduplex DNA formation.
Percent of wheat DNA in S] nuclease
resistant heteroduplex
3
Experiment Number
H wheat fragment length
300 nucleotides
2500
5500
•I
1
18
20
18
2
3
20
16
18
13
10
17
Unlabelled oats DNA (average single stranded fragment size 430 nucleotides)
at 500 ug/ml in 0.12 M phosphate buffer was mixed with labelled wheat DNA
of average single stranded fragment sizes 300, 2500 and 5500 nucleotides.
The ratio of labelled to unlabelled DNA exceeded 1:15000. After denaturing
and incubation to Cot 105, the samples were digested with Si nuclease as
described in Materials and Methods and immediately precipitated with TCA.
The percentages shown were derived from the TCA precipitable radioactivity
in duplicate samples with and without S] nuclease treatment. They have
been corrected for the "zero time binding DNA" as described by Davidson
et al (1).
at 20 and 58°C (figure 5). Such DNA would normally be considered as
single stranded, S-j nuclease digestible. However, in this case these Si
nuclease resistant DNAs probably consist of very imperfect duplexes which
are too short, as a result of Si nuclease cleavage from other duplex
regions, to bind to hydroxyapatite in 0.12 M phosphate at 20 or 58°C.
This conclusion seems justified because the Si nuclease satisfactorily
degraded the 3200 fold excess of unlabelled single stranded DNA.
Wilson and Thomas (18) have shown duplex DNA lengths of around 50 base
pairs are required for quantitative binding to hydroxyapatite at 60°C in
0.12 M phosphate buffer. Thus we consider it reasonable that Si nuclease
digestion of long oats-wheat very mismatched heteroduplexes should release
some duplexes that are too short to bind to hydroxyapatite yet are TCA
precipitable.
Irrespective of the different elution behaviour of the Si nuclease
resistant labelled wheat DNA versus the unlabelled oats DNA, the important
conclusion for this paper is that the elution profiles of the duplexes
formed with 300, 2500 and 5500 nucleotide long wheat fragments were not
only quantitatively but also qualitatively similar. The longer wheat
fragments did apparently form some more Si nuclease resistant unstable
duplexes than shorter wheat fragments but these differences involved only
about 2% of the wheat genome (10% of 20% Si nuclease resistant DNA). The
results do not suggest therefore that the longer labelled fragments formed
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10
10
12
15
18
-20
-25
30
30
Phosphate concentration (M)
Figure 4.
Phosphate gradient elution profile of Si resistant H wheat
DNA/oats DNA heteroduplexes and oats unlabelled DNA homoduplexes. Oats DNA
at 500 vg/ml in 0.12 M phosphate buffer (average single strand fragment
size 410 nucleotides) was mixed with aliquots of labelled wheat DNA such
that the ratio of labelled to unlabelled DNA exceeded 1:8000. After
denaturation and incubation to Cot 105 at 60°C the samples were treated
with Si nuclease as described in Materials and Methods. The Si nuclease
resistant DNA was applied to hydroxyapatite columns in 0.05 M phosphate
buffer at room temperature and the DNA eluted with increasing concentrations of phosphate buffer at the temperatures indicated.
§fe 3 H wheat, average single strand size 300 nucleotides
2500
5500
430
Unlabelled oats DNA
Ordinate shows the percentage of the TCA insoluble counts (Si resistant
wheat DNA) or the percentage of OD26O °f the unlabelled oats DNA.
duplex structures with different stability as might have been expected if
(a) longer regions of duplex were formed or (b) several unlabelled oats
fragments bound with each of the longer wheat fragments.
Although the heteroduplexes derived from different length wheat fragments were quantitatively and qualitatively similar, we also investigated
the phosphate elution profile of the heteroduplexes with their denatured
DNA "tails" undegraded.
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c
30
._ o
<
o
20
CM
!l O
"o
12 M PB
05 M
o
I
I
20
I LJ I
•
11!
20
'Centigrade
Figure 5.
This experiment was identical to that described in the legend
to figure 4 except that the S] resistant duplexes were eluted from
hydroxyapatite with 0.12 M phosphate buffer at the temperature intervals
indicated.
H wheat, average single strand size of 300 nucleotides
@
2500
5500
450
Oats unlabelled DNA
Phosphate gradient elution of wheat-oats heterodupiexes from hydroxyapatite
Labelled wheat DNA fragments with average lengths of 300, 2500 and
5500 nucleotides respectively were hybridised to Cot 90 at 60°C in 0.12 M
PB with a 21,000. fold excess of unlabelled oats DNA. The samples were
then diluted to 0.10 M phosphate and applied to hydroxyapatite at 58°C.
The columns were first washed with 0.10 and 0.12 M PB to remove the DNA
considered as completely unrenatured fragments. The DNA assumed to
contain some duplex structure was then eluted with steps of increasing
phosphate concentration at 58°C terminating with a 0.5 M phosphate wash at
95°C. The percentages of the total DNA eluted with phosphate
concentrations higher than 0.12 M were 33 and 30; 65 and 64; 69 and 74 for
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duplicate experiments using 300, 2500 and 5500 nucleotide long fragments
respectively. These values agree with those expected from figure 1. The
proportion of labelled wheat DNA containing duplex structure that eluted
at each phosphate concentration is shown in figure 6 together with the
corresponding elution profile for the unlabelled oats DNA.
25
-30
-50
-50
Phosphate concentration (M)
Figure 6.
Phosphate gradient elution of oats DNA and three samples of
^H labelled wheat DNA of different average fragment sizes hybridised to
oats DNA. Oats DNA was mixed with three labelled wheat DNAs samples as
described in the legend to figure 4. After denaturation and incubation at
60°C to Cot 90 the samples were diluted and applied to hydroxyapatite
columns at 58°C. After eluting unrenatured DNA with 0.10 M and 0.12 M
phosphate buffer, the renatured duplexes were eluted with increasing
concentrations of phosphate buffer at 58°C with complete recovery being
assured with a final wash at 95°C. A mean of two replications is shown
for each labelled sample and the mean of six unlabelled oats samples is
also shown.
$fa Wheat (average single strand size 300 nucleotides);
32% in duplex fraction
< ^ Wheat (average single strand size 2500 nucleotides);
65% in duplex fraction
illl; Wheat (average single strand size 5500 nucleotides);
72% in duplex fraction
«^>
Oats (average single strand size 410 nucleotides);
83% in duplex fraction
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Between 10 and 20% of the labelled wheat DNA in the duplex fraction
could be removed from hydroxyapatite only by melting the duplexes. This
DNA is in the form of complex aggregates (unpublished results) and nearly
40% of the unlabelled oat DNA eluted in this fraction.
Independent of labelled wheat fragment length, a considerably higher
proportion of wheat DNA than oats DNA eluted at the lower phosphate
concentrations indicating that the heteroduplexes between wheat and oats
fragments contained less faithful duplex structures than the oats DNA
homoduplexes. This is consistent with the much lower thermal stability of
the heteroduplexes (Smith and Flavell, 1974 and figure 2 ) .
The more pertinent observation for this paper is that with the
exception of the aggregate fraction, the phosphate elution profiles of the
heteroduplexes were very similar whatever the lengths of the labelled wheat
fragments involved in the duplexes. The duplexes carrying longer wheat
fragments did not preferentially elute at lower phosphate concentrations, as
would be expected if the additional wheat DNA bound to hydroxyapatite was
associated with oats DNA in poor duplex structures.
CONCLUSIONS
The proportion of wheat DNA in the hydroxyapatite duplex fraction after
hybridisation to oats DNA to C o t 90-100 is very fragment size dependent
(figure 1). Several pieces of evidence indicate that most, if not all of
this variation is due to the single stranded DNA content of the hydroxyapatite duplex fraction. The phosphate elution profiles from hydroxyapatite
of the duplex fractions (figure 6 ) , the proportions of S] nuclease
resistant heteroduplex DNAs (table 1) and phosphate and thermal elution
profiles from hydroxyapatite of the S-| nuclease resistant heteroduplexes
(figures 4 and 5), provide no evidence that long wheat fragments form
significantly more or different heteroduplex structures with oats DNA than
short wheat fragments.
The quantitative agreement between the proportion of the wheat genome
that hybridises to oats repeated sequences at 60°C In 0.18 M Na + estimated
from (a) extrapolation of the curve in figure 1 to the ordinate and (b)
the amount of S-j nuclease resistant DNA (table 1) endorses the conclusion
that only approximately 16-22% of the wheat genome consists of repeated
sequences that are related, albeit somewhat distantly, to repeated
sequences in the oats genome while the rest of the wheat genome is unable
to form stable heterodup-lexes with oats DNA during incubation at 60°C in
0.18 M Na+ to Cot 90-100.
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Because some 67% of the wheat genome is included in the hydroxyapatite
duplex fraction when wheat fragments longer than 3500 nucleotides are
hybridised to oats DNA those common repeated sequences accounting for 1622% of the wheat genome are not clustered but must be distributed through
67% of the wheat genome at intervals of less than 3500 nucleotide pairs.
Seventy five percent of the wheat genome consists of repeated sequence DNA
and 20% of non-repeated sequences shorter than 5000 thousand base pairs (3).
Therefore, even if all these non-repeated sequences are interspersed in
that part of the wheat genome which contains repeated sequences homologous
to sequences in oats, repeated sequences homologous and not homologous to
oats, must also be interspersed with each other.
This arrangement rules out the possibility that much of the large
repeated sequence DNA content of the wheat genome consists of groups of
thousands of essentially identical copies of a few base pairs (e.g. 4 to 20)
tandemly arranged, as found for example in much of the repeated sequence
DNA complement of Drosophila and in other repeated sequence fractions
recognised as 'satellite' DNAs (19, 20, 21). However these results for
wheat do not eliminate the possibility that similar repeating units
several hundred or thousand base pairs long are tandemly arranged
throughout a substantial proportion of the wheat genome. In this case
only part of each repeating unit would be related to a repeated sequence
DNA in oats while the rest would have no homology with oats DNA.
The findings reported in this paper are consistent with our other
studies which have previously suggested that different repeated sequences
between 400 and 800 base pairs are interspersed with each other throughout
a substantial proportion of the wheat genome (3).
We plan to discuss in more detail elsewhere further implications of
the shape of the wheat-oats heteroduplex DNA curve in figure 1 together
with similar curves involving other cereal DNAs. In this paper we
particularly wished to investigate whether DNA of a distantly related
species could be used to probe repeated sequence organisation in complex
genomes.
ACKNOWLEDGEMENTS
We thank Michael 0'Dell for his technical assistance. J. Rimpau was on
leave from the Institut fllr Pflanzenzlichtung der UniversitSt Gottingen and
was supported by Deutsche Forschungsgemeinschaft.
+ Present address: Institut fur Pflanzenziichtung der Universitat Gottingen,
34 Gottingen, West Germany.
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* Abbreviations:
PB, an equimolar mixture of Na^PCty and Na2HP04, pH 6.8;
C o t, concentration of DNA (moles per litre) x time (seconds).
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