Genetica 83: 9-16, 1990.
0 1990 Kluwer Academic
Publishers.
Printed
9
in the Netherlands.
Evolution of a telomere associatedsatellite DNA sequencein the genome of
Drosop?datristi and related species
L. Bachmann, M. Raab & D. Sperlich
LehrstuhlPopulationsgenetik,
Fakultiitfir
FRG
Received
18.4.1990
Accepted
in revised form
Biologie, Universitiit Tiibingen, Auf der Morgenstelle 28, D-7400 Tiibingen,
4.9.1990
Abstract
A highly repetitive satellite DNA sequence from the genome of Drosophila tristis with a length of 18 1 bp has been
cloned in the pUC plasmid. The sequence hybridizes to the telomeres of all chromosomes but the Y ofD. tristis and
produces a ladderlike hybridization pattern with filterbound genomic DNA of D. tristis digested with Eco RI or Pst
I with the hybridization bands at fragment lengths in multiples of 181 bp. A similar pattern is found when the
genomic DNA comes from D. ambigua or, though less clear, from D. microlabis. Additional bands appear in the
zones of high fragment lengths, too. In D. obscura and D. kitumensis, however, the 181 bp sequence is found in
fragments with a length of a few kb only. The 18 1 bp sequence is tandemly arranged in the genome of D. tristis and
has a copy number of about 82,000 per haploid genome (i.e. 10 per cent of the total DNA). A sequence comparison
among four independently cloned copies of the family from D. tristis and another homologous sequence from D.
obscura, found by chance, shows a one to six per cent variation in basepair composition. However, low divergence
(only one per cent) between two copies of D. tristis and between the one of D. obscura and one of D. tristis was
observed, and high divergence (six per cent) between these two pairs. This is discussed and explained as the
evolutionary consequence of an existing homogenization process by unequal crossing over.
Introduction
Satellite DNA is known to consist usually ofa number
of highly repetitive DNA sequences which are typically arranged in large tandem arrays in the genome.
With specific staining techniques and by chromosomal in situ hybridizations with satellite specific
probes it could be shown that the bulk of satellite
DNA corresponds with those regions of the chromosomes that correspond cytologically with heterochromatin which is mainly located in the centromeric
but sometimes also in the telomeric regions of the
chromosomes.
Although it is still an open question, whether
satellite DNA has any function for the organisms, a
common organization pattern and some other common properties are visible when results from so
different taxonomic groups as Primates (Musich et al.,
1980) Rodents (Dod etal., 1989), Drosophila (Lohe &
Brutlag, 1987 a, b; Bachmann et al., 1989), corn (Jones
& Flavell, 1983), and other organisms are compared.
It is specifically obvious that highly repetitive DNA
can diverge drastically between closely related species
with respect to sequence composition, number of
copies and length of repetition units but is rather
uniform in all respects intraspecifically. Amplification
and homogenization events are regarded as the main
forces determining the evolution of satellite DNA
leading to the phenomenon of the so-called concerted
evolution (Dover, 1982).
Most of the investigations on satellite DNA evolution accomplished until today deal with sequences
from the centromeric regions of the chromosomes.
Only little is known about telomeric satellites. Jones
and Flavell (1983) have described a number of
different repetitive units of telomere associated sat-
10
ellites in rye and related species having a length of 120,
480 and 610 bp respectively. A 340 bp long sequence
was found in the telomere regions of Chironomus
chromosomes by Saiga and Edstrom (1985). More
complex repetitive DNAs have been reported for some
Drosophila species. A 3kb fragment was isolated by
Rubin (1978) from the telomere regions of the chromosomes of D. melanogaster. Later, Staller-Young et
al. (1983) sellected a 12 kb DNA fragment that
hybridizes to the telomeres but also to the pheterochromatin of the chromosomes of D. melanogaster
and related species. This fragment shows also partial
homology with the 3 kb DNA fragment isolated by
Rubin (1978). A similar behaviour, i.e. hybridization
to telomeres and to the chromocenter, was observed
for another sequence by Renkawitz-Pohl and Biolan
(1984). All these sequences exist in the genome in
moderately repetitive copy numbers and it was proposed by Staller-Young et al. (1983) that some of them
contain different repetitive motives in a clustered and
scrambled organization. This hypothesis concerning
the general organization pattern has been also discussed by Felger and Sperlich (1989) who analysed the
distribution of hybridization sites of various middle
repetitive sequences ofD. subobscura. About thirty per
cent of the different probes gave signals in the
telomeric and centromeric regions of the chromosomes but were also detectable in the entire euchromatic parts of the chromosomes in a dispersed
arrangement. Steinemann (1984) and Steinemann and
Nauber (1986) observed that one of their cloned,
moderately repetitive sequences hybridized exclusively to the telomeres of all chromosomes and additionally to a single euchromatic site of the neo-Y chromosome of D. miranda. They explain this with the
assumption that the neo-Y of this species arose by an
end to end fusion of two acrocentric chromosomes
homologous to the Y-chromosome and to the III
chromosome of D. pseudoobscura.
from flies collected in Vienna by W. Pinsker. A line of
D. microlabis and of D. kitumensis were provided by
M. L. Cariou. D. guanche and the stock H 271 of D.
subobscura were given to us by A. Prevosti. The D.
melanogaster strain used was the Canton S strain. All
lines have been kept at least for some time in the
laboratory before they were used for the experiments
described here.
Isolation and cloning of highly repetitive DNA
Total genomic DNA of adult Drosophila flies was
extracted following the protocol of Preiss et al. (1988).
Highly repetitive DNA was isolated from restriction
satellite DNA bands appearing after polyacrylamide
gelelectrophoresis of Eco RI or Pst I digested genomic
DNA. Restriction fragments were recovered by overnight incubation of small pieces of the gel containing
the satellite bands in 500 mM NaAc; 1mM EDTA and
ligated into the plasmid pUC 19 according to King and
Blakesley (1986). Cells of E. coli JM 103 were
transformed with the recombinant plasmids and
selected using the blue-white colour system of the
/3-galactosidase reaction (Davis et al., 1986).
Hybridization
of labelled probes to filterbound DNA
Labelling of probe DNA, hybridization to lilterbound
DNA and detection of the hybridization signals were
performed using the ‘DIG DNA Labelling and Detection Kit nonradioactive’ (Boehringer: No. 109 36 57),
as described in the manual, or according to standard
protocols (Davis et al., 1986) when 32P-labelled probes
were used. DNA was transferred to nitrocellulose or
Hybond N (Amersham) membranes by the procedure
of Southern (1975).
In situ hybridization
Material and methods
Drosophila strains
The strains of D. tristis and D. obscura were derived
from flies collected in Tubingen, that of D. ambigua
Preparation of mitotic chromosomes and in situ
hybridization were carried out as described by Bachmann et al. (1989).
11
DNA sequencing
CsCZ-density gradients
Plasmid DNA was prepared according to the manual
of ‘Diagen Plasmid Kit Hi-purity’ (Diagen: No 41014).
Sequencing was carried out as described in the
instruction of ‘T7 Sequencing Kit’ (Pharmacia: No.
27-1682-01).
50 to 100 kg of genomic DNA was centrifuged in a
CsCl solution, adjusted to a refraction index of 1.398
for 48h at 42,000 rpm using a TI 50 rotor (Beckman).
The gradient wascollected in 3.5fractions by an ISCOfractionizer and the DNA concentration recorded
photometrically at h = 253 nm. The gradients containing 0.85 pg Hoechst 33258/pg DNA (Manuelidis,
1977) were prepared to achieve a better resolution.
They were treated otherwise in the same way as
described above.
Estimation of copy number of satellite DNA
Defined concentrations of genomic DNA and purified
cloned satellite DNA were blotted on a Hybond N
(Amersham) membrane by means of a Schleicher and
Schuell Minifold II apparatus. 32Plabelled (Feinberg
& Vogelstein, 1983) satellite DNA, was hybridized to
these filters and autoradiographed. It was assumed
that spots of the same intensity contain also the same
amount of homologous satellite DNA. The validity of
this method was confirmed by measuring and comparing the bound radioactivity quantitatively in the
scintillation counter. With both methods it is only
possible to determine the relative proportion of a
sequence in the genome. For the calculation of
absolute copy numbers it is necessary to know the size
of the total genome. For D. t&is a genome size in the
magnitude of that of D. melanogaster was assumed, i.e.
150 X lo6 bp.
Results
As can be seen from Figure 1, showing the results of
buoyant density centrifugation of genomic DNA of D.
tristis in a standard CsCl-density gradient (Fig. la)
and in a Hoechst 33258 CsCl-density gradient (Fig.
lb), no satellite DNA can be separated from the
mainband DNA under either condition. If there is any
satellite DNA in the genome of D. tristis it must be
cryptic. Yet, a prominent restriction satellite can be
detected if genomic DNA of D. tristis is digested with
the restriction endonucleases Eco RI, Pst I, Hae III or
Sau 3A respectively and separated electrophoretically
in agarose gels or, giving even more conspicous
b
c
&
5
t
ZQ
0
A
buoyant density
Fig. 1. Distribution
of fragments
addition of Hoechst 33258 (b).
of different
buoyant
buoyant density
densities
of genomic
DNA
of D. fristis in a C&I-density
gradient
without
(a) or with
12
fragment bands, in polyacrylamide gels. To find out,
whether the basic band with approximately 180 bp
fragments is really formed by a class of satellite DNA
units, this fraction wascloned into the pUC 19 plasmid
(see Material and methods). Four of the clones
obtained (pTET 181/l to 4) were choosen for further
analysis. All the four recombinant plasmids carried a
18 1 bp Eco RI insert and were derived independently
from Eco RI digested DNA of D. t&is.
The plasmids pTET 181/l-4 were first used for
hybridization to filterbound genomic DNA of D.
tristis digested with the restriction enzymes Eco RI or
Pst I. The pattern achieved is depicted in Figure 2. The
typical satellite ‘ladder’ with signals of decreasing
intensity at 18 1,362,543,724 bp, and so on fragment
lengths is a proof that the satellite is composed of a 181
-724
bp
-543
bp
-362
bp
-181
bp
Fig. 2. Hybridization
of Digoxigenin
labelled DNA from the clone
pTET 181/l to filterbound
genomic DNA of D. tristis digested
with Eco RI. Moderate
stringency conditions
(65°C 0.2 X SSPE).
Fig. 3. In situ hybridization
of the cloned sequence pTET
mitotic chromosomes
of D. tristis.
18 I to the
bp long basic unit that is tandemly arranged in the
genome and that it posessesa single restriction site for
Eco RI or Pst I at most. Variability with respect to
presence or absence of the proper restriction site (or
uncomplete digestion) will result in the production of
monomeric, dimeric, trimeric fragments (and so on)
giving a ladderlike hybridization pattern. The fact that
each of the four clones hybridizes in the same way is in
accordance with the assumption that all four clones
are members of the same satellite family.
The cytological location of the 18 1 bp satellite was
determined by means of in situ-hybridization of the
cloned DNA to the mitotic chromosomes ofD. t&is.
As can be seen from Figure 3 strong signals appear in
the telomeric regions of all chromosomes but the Y,
which have been already previously known to show a
large heterochromatic block at telomeres that can be
made visible by the staining technique of C-banding
(Raab, unpubl.).
Hybridization of labelled pTET 181/l DNA to
filterbound Eco RI and Pst I digested genomic DNAs
of the related Drosophila species D. ambigua. D.
obscura, D. microlabis, D. kitumensis, D. subobscura,
D. guanche, and D. melanogaster, the latter not
belonging to the D. obscura group, gives almost no
cross-hybridization at high stringency conditions with
exception of D. ambigua, for which a ladder of the
same shape appears as in D. tristis but with considerably lower intensity. At very low stringency conditions
the ladder becomes a smear in D. ambigua and a
13
12345676
-
5.4
kb
_
-
3.35
3.1
kb
kb
Fig. 4. Hybridization
of Digoxigenin
labelled DNA from the clone
pTET 181/l tofilterboundgenomic
DNA ofD. kirumensis(lanes
1
& 5), D. micro(nbis (lanes 2 & 6), D. obscure (lanes 3 & 7) and D.
ambigua (lanes 4 & 8). Low stringency conditions
(60°C, 2 X
SSPE).
similar but less pronounced signal can be detected in
D. microlabis (see Fig. 4). No comparable hybridiza-
tion is visible with the DNA of any of the other species
listed above (only D. kitumensisand D. obscura are
shown in Fig. 4). Yet, another somewhat unexpected
and additional group of bands emerges in the DNA of
the species of Figure 4. A group of two bands at 3.35
kb and 3.1 kb respectively is present in all the four
lanes of Figure 3 and a clear band at 5,4 kb in D.
ambigua and somewhat longer in D. obscura independent from the restriction enzyme used (Eco RI
or Pst I). This indicates the existence of rather long
fragments that contain the 18 1 bp sequences or at least
a part of them. It is probable from the pattern that
these fragments are not comprised from large tandem
repeats of the 181 bp subunits that have lost their Eco
RI as well as the Pst I restriction site but that they are
more complex clusters in which only a part of the
sequence, having no Eco RI and Pst I restriction sites,
is present. In conclusion it can be assumed that the 18 1
bp sequence of the pTET 181 clones exists in the
genome of D. tristis in a very high copy number but in
that of D. ambigua in a comperatively lower and in
that of D. microlabis still lower amplification stage. It
can be seen from Figure 5 that, using the same
hybridization solution, the signal intensity of about 2
ng pTET 18 l/l corresponds to the signal of somewhat
less than 25 ng of genomic DNA of D. tristis. That
means that roughly 10 per cent of genomic DNA is
made up by copies of the pTET 18 1 bp sequence. If the
entire haploid genome contains 150 X lo6 bp then
there must exist about 82,000 copies of this sequence in
the haploid genome of D. tristis.
To learn more about the character and especially
about the intraspecific variability of the 181 bp
subunits the clones pTET/l, pTET/2, pTET/3, and
pTETM were sequenced (Fig. 6). When the sequences
were available it became obvious that another Pst I
clone existed in our collection which has not only
exactly the same size of 18 1 bp but also an extremely
high degree of homology with the pTET 181 clones.
This clone comes from D. obscuraand its sequence has
been aligned to the pTET 181 sequences and is also
shown in the Figure 6. As can be seen the sequences
are neither significantly AT- (58.3%) nor GC-rich
(41.7%) and the satellite composed by this sequence
must be included in the main band DNA as a cryptic
satellite in the CsCldensity gradient (see Fig. 1).
Further, the independent copies are not identical
among each other indicating that some variability
among copies exists. The degree of divergence between
the copies can be expressed by per cent base pair
differences. The values are given in Table 1. The
smallest divergence is found between pTET 18 l/l and
pTET 181/4 and between pTET 18 l/2 and the
obscura clone pOPT 18 1, while pTET 18l/3 is rather
distant to either similar pairs. This internal existence
of clusters or subfamilies of similar copies might be
interpreted as the result of evolution by inhomologous
crossing over.
B
A
13405
3229
6730
2273
3720
1089
1478
681
730
346
402
124
a
Fig, 5. Hybridization
experiment
for the determination
of the copy number of the pTET 18 1 fragment in the genome of D. trisris. Genomic
DNA was blotted in amounts of 250,125,62.5,25,12.5,
and 6.25 ng (A)andcloned
satellite DNA in amounts of 10,5,2,1,0.5,
andO. 1 rig(B)
on a Hybond N membrane.
The filter was hybridized
with “P-labelled
pTET 181/I DNA (a). The radioactivity
of the various hybridization
bands was measured additionally
in the scintillation
counter. The corresponding
counts per minute (cpm) are listed in b.
10
20
40
30
50
60
70
pTET
181/l
GAATTCCAAT
TCGCATTTTG
ATTGTGGTGT
TGCGGATATG
GATTGCAGAT
TATTGTGCCA
TATATACATT
pTET
181/Z
------A---
C---m-----
---A----A-
--e----T/,-
A---------
____------
_____-----
pTET
181/3
------____
-------m-T
--------A-
-------T/C,-
A---------
______-_--
______----
pTET
181/4
-----___--
------e--T
______----
_____-----
_____-----
_______---
_______---
pOPT
181
------A---
C--------s
---A----A-
-------TA-
A---------
__________
__________
100
110
60
50
80
TTGCAGCAGG
80
70
100
90
----------
----------
----------
----------
----------
----------
----------
pTET
181/3
__----____
-_________
-------m-G
__________
-_________
__________
-_________
pTET
181/4
----------
----------
-------m-G
__-_______
_----_____
__-----___
____------
pOPT
181
-----_____
--T-----v-
-------e-G
__________
__________
-_________
---_______
150
160
170
180
160
170
181/l
AGGGCATATT
AAACTGCAAT
CTTGATCCCA
AATTTCAATC
G
pTET
181/2
_____-----
___-------
-___------
-----G---m
_
pTET
181/3
----------
----------
----------
------____
_
pTET
181/4
---------_
----------
-----cm---
--------__
_
pOPT
181
_____-----
_________10
-_________
30
CTATGCCTGC
180
pTET
20
ATATATGTCG
140
181/2
150
TGTCGCCAGA
130
pTET
140
CCAGGAAATG
120
181/l
130
CTAGGGCCTT
110
pTET
120
AGATAGGTGG
90
-----G---m
_
40
Fis. 6. Base pair sequences of the four cloned copies of the 18 1 bp satellite sequence pTET
of D. obscura.
18 l/l
to 4 and a homologous
sequence of pOPT
18 I
15
Fig. 1. Base pair divergence
between the cloned copies pTET
181/l-4 of D. rristis and pOPT 181 of D. ohcura in per cent.
pTET
pTET
pTET
pTET
181/l
181/2
181/3
181/4
pTET
181/l
pTET
181/2
pTET
181/3
pTET
181/4
pOPT
181
-
4.40
3.30
3.30
1.66
6.10
2.80
5.50
1.10
3.30
6.10
Discussion
The cloned satellite DNA sequences pTET/l-4 represent a family of a tandemly arranged telomere
associated 181 bp satellite-DNA of D. tristis. It could
be shown for this species that the copies of the family
are located at the telomeres of all chromosomes, but
the Y, and that they are species specifically amplified
to a very high copy number of roughly 82,000 per
haploid genome. This high degree of amplification can
be regarded as characteristic for D. tristis although
homologous sequences could be detected in other
species, too.
A length of 181 bp as the repetition unit of this
telomeric satellite DNA is identical in length with that
of the basic unit of a satellite DNA located in the
centromeres of D. tristis. It is, however, not homologous to the centromeric satellite DNA sequence (Bachmann, in prep.). This observation supports the hypothesis of John et al. (1986), that particular repeat
families are restricted to specific heterochromatic
regions, i.e. either to proximal or to distal chromosome regions, but not to both. Yet, the correspondence of the telomeric and centromeric repeat families
with respect to sequence length could be interpreted, if
not assumed as a random coincidence, as due to the
mechanism of amplification. It may be supposed that
the homogenization events on repetitive units is size
dependent in the same way in centromeric and
telomeric regions but otherwise independent of the
actual nucleotide sequence that is amplificated and
homogenized. If this hypothesis would be true this
mechanism should be assumed to be more precise at
the telomeres than at the centromeres, since the
variability between the cloned telomeric repeats pTET
181/l-4 is pretty low, on average 3,75% (see Table l),
compared to centromeric repeats. E.g., for a 290 bp
centromeric satellite DNA of D. guanche the average
variability proved 11.6% (Bachmann et al., 1989) and
it is of similar magnitude for the 181 bp centromeric
repeats of D. tristis (Bachmann, in prep.). Following
the idea that the homogenization mechanism is always
the same, the differences in the observed average
variability between copies indicate most probably a
difference in the frequency of recombination between
telomeric and centromeric repetitive DNA sequences.
Another explanation would be that strong selection is
effective specifically on the pTET 181 bp repeat
family. This assumption is supported by the high
degree of homology between pTET 18111-4 from D.
tristis and pOPT 181 from D. obscura.
Hybridization
of pTET to lilterbound genomic
DNAs of D. ambigua, D. obscura, D. microlabis, D.
kitumensis and also of D. tristis detected repetitive
DNAs homologous to pTET 181 repeats but of a
length of several kb. Perhaps, these moderately repetitive sequences could represent in anology to the long
telomeric repetitive DNAs described for D. melanogaster (Rubin, 1978) and D. miranda (Steinemann,
1984) an ancient type of telomeric repetitive DNA,
which was partially amplified in the species listed
above to the pTET 181 repeat family.
Acknowledgement
The work was supported by the grant Sp 14616 of
‘Deutsche Forschungsgemeinschaft’.
We are extremely grateful to Dr. Cariou for giving us the strains of D.
kitumensis and D. microlabis. Mrs. Elke Mtiller helped
us during the experiments with skilled technical
assistance and Ms. Charlotte Rehm typed the manuscript carefully.
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