Vol 13, Issue 2, 182-194, February 2003
LETTER
Sequence Analysis of a Functional Drosophila Centromere
Xiaoping Sun,
Hiep D. Le,
Janice M. Wahlstrom and
Gary H. Karpen1
Molecular and Cell Biology Laboratory, The Salk Institute,
La Jolla, CA 92037, USA
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ABSTRACT
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Centromeres are the site for kinetochore formation and spindle
attachment and are embedded in heterochromatin in most eukaryotes. The
repeat-rich nature of heterochromatin has hindered obtaining a detailed
understanding of the composition and organization of heterochromatic
and centromeric DNA sequences. Here, we report the results of extensive
sequence analysis of a fully functional centromere present in the
Drosophila Dp1187 minichromosome. Approximately 8.4%
(31 kb) of the highly repeated satellite DNA (AATAT and TTCTC) was
sequenced, representing the largest data set of Drosophila
satellite DNA sequence to date. Sequence analysis revealed that the
orientation of the arrays is uniform and that individual repeats within
the arrays mostly differ by rare, single-base polymorphisms. The entire
complex DNA component of this centromere (69.7 kb) was sequenced and
assembled. The 39-kb "complex island" Maupiti contains
long stretches of a complex A+T rich repeat interspersed with
transposon fragments, and most of these elements are organized as
direct repeats. Surprisingly, five single, intact transposons are
directly inserted at different locations in the AATAT satellite arrays.
We find no evidence for centromere-specific sequences within this
centromere, providing further evidence for sequence-independent,
epigenetic determination of centromere identity and function in higher
eukaryotes. Our results also demonstrate that the sequence composition
and organization of large regions of centric heterochromatin can be
determined, despite the presence of repeated
DNA.
[Supplemental material is available online at
www.genome.org. The sequence data from this study have been submitted
to GenBank under accession nos.:
Beagle = AY183918, F = AY183919,
412 = AY183920, Bel = AY183921,
You = AY183922, Maupiti = AY183926,
AATAT = AY183925, AY183931–AY184007, and
TTCTC = AY183923–AY183924, AY183927–AY183930,
AY184008–AY184069].
The last few years have witnessed the publication
of complete or nearly complete euchromatic physical maps and sequence
assemblies for Caenorhabditis elegans, Arabidopsis
thaliana, Drosophila melanogaster, and most recently,
Homo sapiens (The C. elegans Sequencing Consortium
1998 ; Adams et al. 2000; The Arabidopsis Genome Initiative 2000; Lander
et al. 2001; Venter et al. 2001). The heterochromatin comprises  30%
of both the fly and human genomes, yet it has been virtually ignored by
these large-scale genome projects. This enigmatic part of the genome
has unusual cytological, molecular, and genetic properties, including
differential control of replication, condensation throughout the cell
cycle, and the ability to silence gene expression (John 1988; Weiler
and Wakimoto 1996; Elgin and Workman 2002). Heterochromatin is
concentrated in large (megabase-sized) blocks, predominantly in the
centric and subtelomeric regions of all chromosomes, and contains
tandemly repeated short sequences (satellite DNAs), middle repetitive
elements (e.g., transposons), and some single-copy DNA.
Heterochromatin has been unflatteringly referred to as "junk DNA,"
because it is more difficult to assign functions to repeated sequences
than to protein-encoding sequences. However, heterochromatin is not
inert and has been demonstrated to be essential for cell and organismal
viability in multicellular eukaryotes. Essential genes (e.g., lethal
mutable genes, ribosomal RNA genes) and fertility genes (e.g., Y-linked
male fertility factors) reside in heterochromatin (Gatti and Pimpinelli
1992). Essential cis-acting chromosome inheritance functions
are also mediated by heterochromatin, including centromeres (Sullivan
et al. 2001), meiotic pairing (McKee and Karpen 1990; Dernburg et al.
1996; Karpen et al. 1996), and sister chromatid cohesion (Moore and
Orr-Weaver 1998; Bernard et al. 2001). Thus, a complete understanding
of eukaryotic genomes requires detailed structural and functional
analysis of heterochromatin.
One essential heterochromatic element is the centromere, which is
associated with the kinetochore. The centromere/kinetochore complex is
necessary for spindle attachment, prophase and anaphase chromosome
movements, and the activity of the spindle assembly checkpoint (SAC)
(for review, see Dobie et al. 1999; Sullivan et al. 2001). One key
question currently under debate is what determines centromere identity.
How is only one site (in most eukaryotes) chosen for centromere
function and kinetochore formation? Cytological and biochemical studies
have demonstrated a physical association between tandemly repeated
satellite DNAs and centromere regions and proteins in higher eukaryotes
(Willard 1990; Vafa and Sullivan 1997; Sullivan 2001; Sullivan et al.
2001). However, normal centromeric DNA is neither necessary nor
sufficient for centromere function (Karpen and Allshire 1997; Choo
2000; Sullivan et al. 2001). Different heterochromatic properties and
functions have been demonstrated to be regulated in a
sequence-independent manner by epigenetic mechanisms (Allshire et al.
1994; Hendrich and Willard 1995; Wakimoto 1998; Jenuwein and Allis
2001). In comparison to models that emphasize the importance of primary
DNA sequence to centromere identity and propagation, epigenetic models
parsimoniously account for both the stability and plasticity of
centromeres (Karpen and Allshire 1997; Choo 2000; Sullivan et al.
2001).
The involvement of epigenetic regulation does not preclude a role for
DNA sequence in centromere identity or function, or other functions
encoded by heterochromatin. For example, secondary sequence
characteristics, such as A+T richness, or the ability to form
higher-order structures through bending, may facilitate the conversion
of an epigenetic mark into the formation of a functional kinetochore
(Vig 1994; Murphy and Karpen 1998; Koch 2000; Sullivan et al. 2001). In
addition, specific sequences may serve as boundary elements that
constrain the spreading of centromeric chromatin (Maggert and Karpen
2001; Blower et al. 2002). Thus, understanding heterochromatic and
centromeric sequences would reveal insights into their functional
regulation, in addition to providing a more complete understanding of
genome structure and sequence.
Repetitive DNA poses significant challenges to cloning, sequencing, and
assembly. Nevertheless, substantial progress has been made in the
analysis of the nonsatellite component of Drosophila,
Arabidopsis, and human heterochromatin (Copenhaver et al.
1999; Kotani et al. 1999; Adams et al. 2000; The Arabidopsis Genome
Initiative 2000; Horvath et al. 2000a,b; Kumekawa et al. 2000; Haupt et
al. 2001). These regions predominantly contain transposons and
transposon fragments, as well as genes and gene fragments. Less
progress has been made in genomic analysis of satellite sequences. The
nucleotide composition of individual repeats has been obtained from
sequencing small clones of satellite DNA (Lohe and Brutlag 1986; Jabs
and Persico 1987; Warburton et al. 1996; Losada et al. 1997; Haaf and
Willard 1998). In situ hybridization to mitotic chromosomes, along with
classical cytogenetic banding methods, have revealed the gross
distribution of some repeated DNAs within heterochromatin in organisms
such as Drosophila (Lohe et al. 1993; Pimpinelli et al. 1995)
and humans (Dunham et al. 1992; Grady et al. 1992; Mullenbach et al.
1996). However, cytological methods do not provide high-resolution
information about the size and complexity of simple sequence arrays.
Although pulsed field gel electrophoresis (PFGE) provides one method
for spanning the gap between short-satellite sequences and cytological
mapping (Sun et al. 1997), the dispersion of repetitive DNAs throughout
the genome, and tandem repetition of satellites, has impeded extensive
restriction mapping of specific heterochromatic regions.
Chromosome-specific satellite DNAs and PFGE have been used to
successfully map satellite domains in mammals; however, these maps are
limited by the lack of direct molecular access deep within individual
satellite blocks (Willard et al. 1986; Jabs and Persico 1987; Jabs et
al. 1989; Arn et al. 1991; Schueler et al. 2001).
Recent studies have revealed significant, unprecedented information
about centromere region sequences. Detailed sequence analyses of human
centromeres have been published recently, revealing the substructure
and composition of the satellite arrays (Lee et al. 2000; Schueler et
al. 2001). However, the sequence assemblies of these regions of
satellite DNA are not complete, leaving open the possibility that other
types of sequences are present in the arrays. In Arabidopsis,
the pericentromeric regions on all five chromosomes have been cloned
and sequenced (Copenhaver et al. 1999; Kotani et al. 1999; The
Arabidopsis Genome Initiative 2000; Kumekawa et al. 2000; Haupt et al.
2001). Detailed studies of some of these pericentromeric regions
demonstrate that they contain intact and fragmented transposons as well
as single-copy genes. However, the satellite regions present in
Arabidopsis centromere regions have not been completely
sequenced. Although we have a better understanding of the organization
and composition of some higher eukaryotic centromere regions, we lack
an understanding of the exact primary sequence of any functional,
higher-eukaryotic centromere. Similarly, a number of important
questions about the global and detailed organization of heterochromatic
DNA, and the relationship between heterochromatin sequences and
functions, remain unanswered.
We have studied the molecular genetics of chromosome structure and
inheritance using the Drosophila minichromosome
Dp1187. This minichromosome contains the components and
inheritance functions associated with normal higher eukaryotic
chromosomes, yet is amenable to molecular analysis and manipulation. It
is relatively small (1.3 Mb, or 1/30th the size of the normal
X chromosome), contains easily scorable genetic markers, and
is not essential for the viability of the cell or organism. Studies
utilizing this minichromosome system have generated information about
chromosome structure, and the cis and trans
regulators of chromosome inheritance, nuclear organization, and gene
expression (e.g., Karpen and Spradling 1990; Murphy and Karpen 1995a;
Karpen et al. 1996; Dobie et al. 2001; Hari et al. 2001; Maggert and
Karpen 2001; Donaldson et al. 2002). Analysis of the transmission
behavior of molecularly defined Dp1187 deletion derivatives
localized the sequences necessary for chromosome inheritance to within
a specific 420-kb portion of the centric heterochromatin (Murphy and
Karpen 1995b). Pulsed-field Southern analysis revealed the gross
composition and organization of this functional centromere, as well as
the rest of the minichromosome centric heterochromatin (Sun et al.
1997). These studies revealed the presence of two large blocks of
simple, highly repeated satellite sequences in the functional
centromere, as well as complex DNA in the form of intact and fragmented
transposons.
We wanted to gain a more precise understanding of the sequence
composition and organization of centric heterochromatin and
specifically of a centromere demonstrated to function in vivo.
Therefore, we have cloned, sequenced, and assembled all the complex DNA
within the Dp1187 centromere, and 8.4% of the satellite
arrays. Here, we describe the results of these studies, which
demonstrate that centric heterochromatin sequencing and assembly can be
accomplished. Furthermore, these studies confirm the uniformity of the
satellite arrays, reveal that the transposons have very high identity
to euchromatic copies, and show that the structure of one end of the
centromere is complex, and includes higher-order repeat structures. We
discuss the relevance of these findings to the evolution of
heterochromatin and to current models for the determination of
centromere identity, and discuss the possibility that genome projects
can ultimately be truly completed through inclusion of assembled
heterochromatic sequences.
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RESULTS
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Generation of a Centromere-Enriched Library From Gel-Purified Minichromosome DNA
The presence of the same repeats in different parts of the genome is
the major impediment to mapping and sequencing a specific region of
heterochromatin. To circumvent these problems, we have used gel
purification of a minichromosome derivative, allowing us to focus
cloning and sequencing efforts on one specific region of centric
heterochromatin, namely a genetically defined functional centromere.
The only centric heterochromatin in the 620-kb Dp1187
derivative  1230 corresponds to the 420-kb, fully functional
centromere (Fig. 1). Previously, PFGE of
gel-purified 1230 DNA was used to restriction map the
centromere, which revealed its gross organization and composition (Fig.
1)(Sun et al. 1997). This analysis identified and positioned two large
blocks of AATAT and AAGAG satellites, five islands of complex DNA
(Motus 1–5, Tahitian for "small island")
embedded in the AATAT array, and a large complex island at the right
end of the centromere (Maupiti). Hybridization analysis and
restriction mapping suggested that Motus 1–4
contained known transposable elements: three Long Terminal Repeat
(LTR)-containing retrotransposons (HMS Beagle, 412,
and Belshazar/3S18), plus one non-LTR (LINE-like)
retroposon (F).
To gain a more detailed understanding of the sequence composition and
organization of the Dp1187 centromere, we used PFGE-purified
1230 as an enriched source for cloning and sequencing.
Heterochromatin contains a nonrandom distribution of restriction sites,
as a result of the predominance of simple repeats. Therefore, purified
1230 DNA was randomly sheared then cloned into the 
ZAPII vector (see Methods for details). The 1230 library
includes sequences from the centric heterochromatin, the subtelomeric
heterochromatin (100 kb), and the euchromatin (100 kb). We
screened the library with clones corresponding to the five major
components previously demonstrated to be present in the centromere: the
transposons, the AAGAG and AATAT satellite regions, the
transposon-satellite junctions, and the complex DNA "island"
Maupiti (Fig. 1; Sun et al. 1997). Previously unidentified
types of DNA were isolated by randomly picking clones, and by probing
the library with whole, gel-purified 1230 and a specific
restriction fragment from the centromeric region (see Methods).
Plasmids were then generated by superinfection/excision of the
clones. All clones were initially sequenced using the flanking T3 and
T7 primers present in the pBluescript polylinker, and then sequences
were extended with internal primers (see Methods). The positions of the
clones within 1230 were confirmed by hybridization to
digested 1230, using PFGE Southern analysis (data not
shown).
We isolated, converted to plasmid, and sequenced 459 clones, and a
total of 947.9 kb of sequence was generated from 1230
(Table 1). This includes 495.6 kb of
centromere sequence, which was assembled into 13 contigs (79.9 kb) that
cover 19% of the 420 kb centromere. All complex DNA within the
centromere (69.7 kb) has been sequenced using this library, and the
average coverage for these regions was five- to sevenfold. In addition,
the 1230 library sequences include 10.2 kb (3%) of the
satellite DNA, predominantly from the regions flanking the transposons
(fivefold–sevenfold coverage) (see below). However, additional
satellite sequence has been obtained by polymerase chain reaction (PCR)
methods (see below). Most of the clones and sequence corresponded to
subtelomeric, euchromatic, or unassigned regions of 1230
(Table 1), and were not analyzed further.
Single Transposons Are Intact and Nearly Identical to Previously Sequenced Elements, and Are Inserted Directly Into the AATAT Array
The 1230 library clones have been used to generate
complete sequences of the Motus, and have allowed us to
characterize the junctions with the AATAT satellite (Table 1, Fig. 1).
The Beagle, F, 412 and Bel
transposons are intact and complete; all four elements have >99%
identity with the reference transposon sequences deposited in GenBank.
Motu 5 is >99% identical to the recently described
transposon You, a non-LTR (LINE-like) retroposon (FlyBase
2002). All five transposons are inserted directly into the AATAT
arrays, not into complex DNA, and are oriented as shown in Figure
2, confirming the orientations predicted
from previous restriction mapping (Sun et al. 1997). The two
long terminal repeats (LTRs) present in all three retrotransposons
(Bel, Beagle, 412) are 99.7, 99.2, and
99.8% identical (respectively). Thus, these elements are likely to be
recent insertion events (SanMiguel et al. 1998), or the sequences are
under severe functional constraints. The completeness of the
transposons and their recent origin raise questions about the evolution
and function of these centromeric elements (see Discussion).
"Tagged" PCR Produces Sequence From Pure Satellite Arrays
The analysis of sequences from the 1230 library also
demonstrated that the AATAT satellite is oriented as AATAT (from left
to right in Fig. 1) at the euchromatic/centromere breakpoint and in the
transposon flanks, and that the AAGAG satellite is oriented as TTCTC
(designated as such hereafter). Analysis of the 1230
library clones produced a total 7027 bp of AATAT contigs from the
transposon flanks and euchromatin-heterochromatin junction, and 807 bp
of contiguous TTCTC sequence from the AATAT-TTCTC and
TTCTC-Maupiti junctions (Table 1). However, only six clones
that contained pure satellite arrays were recovered from the library
(Table 1). We have determined that the absence of pure satellite clones
occurs during the initial cloning step into ZAP, and not in the
subsequent excision step that generates the plasmid (data not shown).
Because clones from the transposon-satellite junctions only contained
up to 970 bp of satellite DNA, we suspect that clones containing
satellite arrays larger than 1 kb are unstable, and that the lack of
pure satellite clones reflects the 2-kb minimal size cutoff used to
generate the library (see Methods).
To further investigate the organization and composition of the
satellite arrays, we developed PCR methods that would randomly sample
satellite sequences. First, we generated random satellite clones and
sequence using PCR amplification of gel-purified 1230, with
hybrid primers that contained satellite sequence plus a unique tag
(Fig. 3). This approach was used to
generate clones and sequences from the transposon-satellite junctions
and from pure satellite arrays. One challenge in attempting to amplify
junctions or pure satellite arrays is template shrinkage (Fig. 3A). We
successfully ameliorated this problem by using hybrid satellite-tagged
primers; transposon-satellite junctions required only one tagged primer
plus one standard primer corresponding to the complex (transposon) DNA
(Fig. 3B; supplemental material). Amplification and cloning of pure
satellite arrays poses a problem in addition to template shrinkage—two
primers that contain complementary satellite strands frequently form
primer dimers, even when hybrid primers are used. Thus, to amplify pure
satellite arrays, we used two different tagged primers and a primer
corresponding to one of the tags (Fig. 3C). Second, we also generated
pure satellite sequences using bacterial transposon insertion into
gel-purified minichromosome DNA in vitro, followed by amplification
with one primer from the bacterial transposon and a hybrid
satellite-tagged primer (Fig. 3B).
The tagged PCR experiments produced significant coverage of both pure
satellite arrays and junctions with complex sequences (Table
2). The PCR sequences increased the
coverage for the AATAT that flanks the single transposons to >10-fold,
from the fivefold–sevenfold coverage produced by the 1230
library. Genome Priming System (GPS) insertion into gel-purified
1230, using one tagged satellite primer and one primer
homologous to the bacterial transposon (Fig. 3B), produced 11 kb of
pure AATAT sequence. This total does not include 3 kb of shorter AATAT
sequences (<200 bp) that were identical to larger PCR product
sequences. We conservatively chose to exclude these sequences from
totals presented here and in the tables, as they could represent
shrinkage products from the same sites as the larger products. PCR with
two tagged TTCTC primers (Fig. 3C) generated 6.4 kb of pure satellite
sequence, and GPS insertion produced 3 kb of TTCTC sequence. However,
we recovered no clones using two tagged AATAT primers (Fig. 3C). This
result was likely caused by primer-dimer formation that occurs at the
low annealing temperature that had to be used with the AATAT primer.
Regardless, these results demonstrate the utility of the tagged PCR and
transposon insertion approaches to analyzing satellite DNA, methods
that can be used to analyze similar regions in the Drosophila
and human genomes.
To estimate the error rate in the 1230 sequences, we
compared the sequences derived from the 1230 library
clones to the homologous PCR-generated sequences. The transposon and
satellite components of the junction fragments were 100% identical in
the and PCR-derived consensus sequences. The depth of coverage
allowed us to eliminate errors generated by sequencing, PCR, or
cloning. Differences of only 0.1% were observed when comparing
individual reads from the GPS transposon sequences in the clones that
were generated after insertion into isolated 1230, and with
the published transposon sequence. Similarly, comparison of the
flanking satellite DNA sequence with the consensus sequence derived
from either the PCR and sequences only showed a 0.1% difference.
Importantly, in almost all cases where there was a base pair ambiguity,
only one sequenced strand was different; the remaining sequences were
in complete agreement. This gives us a valid prediction for the error
rate present in the pure satellite sequences. The observed differences
of 0.1% are roughly fourfold higher than what we would expect from
previously determined TAQ polymerase error rates (Cline et al.
1996). This error in some of the PCR clones could be from
the result of the insertion process (gene conversion), two rounds of
PCR (isolation and screening), sequencing error, or different PCR
conditions. It is likely that most of the observed differences among
individual reads were generated during PCR or cloning, rather than
sequencing. Thus, the quality of the sequences presented here is high,
and in most cases the depth of coverage allowed us to generate
consensus sequences with high confidence.
The AATAT and TTCTC Satellite Arrays Are Highly Conserved and Uniform
Tables 2 and 3, and Figure 2 summarize
the satellite sequence totals and characteristics obtained from
analysis of the library and the amplified products. In total, 31 kb of
satellite DNA sequence was obtained from analysis of the
1230 library and the PCR approaches, corresponding to
8.7% and 7.5% of the AATAT and TTCTC satellites present in the
centromere, respectively. The small size of the clones allowed us to
generate complete sequences using vector primers (see below). The
validity of the assembled sequences was supported by the fact that
consensus sequence information obtained from the clones and the PCR
approaches for the transposons and satellite flanks are identical
(differences for individual reads = 0.1%, see above).
These approaches have produced the largest data set of
Drosophila satellite sequences to date, providing an
opportunity to perform a detailed analysis of the sequence and
organization of the satellite arrays. First, tandem repeats within
satellite arrays are oriented in the same direction ("head to
tail"), AATAT and TTCTC (left to right, Fig. 1). Second, an
AATAT/TTCTC junction is located 950 bp from the right end of the
You element. Thus, the two satellites appear to be directly
juxtaposed, with no intervening complex DNA, or other type of repeat.
Third, the arrays are uniform in sequence; only 2.2% and 0.3% of the
AATAT and TTCTC sequences contained base changes, respectively. The
vast majority of alterations (92%) were one base changes to the simple
sequences, and there were no significant insertions of complex DNA
(besides the transposons in the Motus). The
remaining changes were small insertions and deletions (<5 bases),
which could also represent multiple single-base changes. Only a small
proportion of the observed AATAT variation results from mutation during
PCR amplification or cloning or sequencing errors (expect 0.1%
variation, see above). The absence of significant variability across
the bulk of the sequenced satellites is consistent with the high
resolution restriction mapping (Sun et al. 1997), and demonstrates that
the satellite arrays are uniform and lack significant insertions of
complex DNA (besides the transposons in AATAT).
Important information about the composition of the satellite arrays was
revealed by analyzing the frequencies of different types of nucleotide
changes (Table 3). First, TTCTC displayed significantly fewer sequence
changes than AATAT (0.3% versus 2.2%,  2 = 124,
P < .0001). Second, the AATAT arrays at the junctions
displayed nearly identical frequencies of different types of changes as
the "pure" arrays (Table 3). The only exception was a moderate but
statistically insignificant increase in insertions/deletions near the
junctions, from 9% to 13% (2 = 0.52,
P < .50). Thus, transposon insertions did not alter the
frequencies of changes in the flanking satellite or the expansion of
altered repeats. Third, the overall frequency of transversions (68%)
was twofold greater than transitions in both pure AATAT arrays and
junctions (32%; 2 = 19, P < .0001; see
Discussion). This ratio was reversed for the TTCTC satellite; however,
the significance of this result is unclear, as only 27 single base
alterations were identified in these arrays. Finally, we observed
clustering of polymorphisms and regular spacing (5 and 10 bp) of units
with the same base changes (data not shown), perhaps reflecting
expansion of individual altered repeats during array evolution. In
summary, we conclude that the sequence and organization of the AATAT
and TTCTC arrays are highly conserved, and that the two types of
satellites display different frequencies and types of base changes.
Assembly and Analysis of the Maupiti Region
One of the greatest challenges we faced was assembling a complete
contig of the complex island Maupiti. The high-resolution
restriction map suggested that this island was 35 kb in size, and
contained multiple, partial G-like transposons, an incomplete
Doc transposon, and at least two large blocks of a novel
A+T-rich sequence (Sun et al. 1997). The large size and repetitive
nature of this region created significant difficulties for sequence
assembly. Existing alignment/assembly programs are not able to
automatically assemble repeated sequences, even if the repeats are
variable; sequences are frequently misplaced within the contig. We were
able to overcome these difficulties by isolating multiple clones from
the library for each region, which confirmed the presence and
validity of polymorphisms that were necessary to correct assembly.
Large clones (e.g., 4–7 kb) that spanned multiple polymorphisms and
components were especially helpful in validating the assembly of
smaller contigs. In vitro insertion of a modified yeast transposon
(AT-2 [Devine and Boeke 1994], see Methods) was used to complete the
sequences of plasmids that were difficult to assemble because of the
presence of repetitive DNA. Finally, Sequencher 3.0 software
(GeneCodes) was helpful, because it allowed us to manually align the
most difficult sequences, and to simultaneously view the sequence
traces to confirm polymorphisms. Other regions of heterochromatin
(e.g., pure satellite arrays with few polymorphisms) will likely be
even more challenging, but assembly of the Maupiti contig has
served as an important stepping stone and learning experience that will
help us design strategies to assemble even more challenging
heterochromatic sequences.
The assembled Maupiti contig (Fig. 2) was based on an average
of fivefold–sevenfold coverage, and revealed information about this
region of heterochromatin that was not visible in the high-resolution
map. The size of Maupiti is 38.924 kb (Table 1, Fig. 2),
10% larger than the 35 kb estimated from the restriction map (Sun
et al. 1997). Maupiti contains only four types of sequences:
16.2 kb of A+T-rich sequence (Sun et al. 1997), 15.1 kb of
G-like transposon sequence, 4.3 kb of
F/Doc-like transposon sequence, and 4.2 kb of a
Doc transposon. This analysis also identified the junction
between the TTCTC array and the left end of Maupiti, which
demonstrated that the orientation of this satellite is the same at the
left and right ends of the block (Fig. 1).
The assembled Maupiti contig revealed the exact size,
distribution, and sequence of the A+T-rich DNA, previously identified
in a small clone in the Sun et al. study (1997). The A+T-rich sequence
is present in six blocks of 1550, 5900, 6500, 550, 1450, and 270 bp,
from left to right (brown cylinders, Fig. 2). These blocks share
between 88% and 100% identity, predominantly in the 95–98% range.
All blocks except block number five are oriented in the same direction.
The dot plot (Fig. 4A) and sequence
comparisons (Fig. 4B; see Methods) demonstrate that the A+T-rich blocks
contain related, small tandem repeats (small colored arrows, Fig. 4B).
The largest blocks (repeats 2 and 3) are nearly identical; each
contains a 4.65-kb direct repeat, plus different combinations of
subrepeats in the left ends. The smaller blocks contain different
combinations of these subrepeats. Blocks 1 and 5 are identical, but are
in opposite orientation (see below).
View larger version (28K):
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Figure 4. Sequence composition and organization of Maupiti. (A)
Dot-plot analysis of Maupiti reveals the presence and
organization of internally repeated DNAs. Arrows above each bar
indicate relative orientations of each component. Solid arrows below
the bars indicate the presence of inverted repeats at the ends, and
large internal direct repeats. Note that most of the internal elements
are oriented in the same direction. (B) The substructures of
the A+T-rich, G-like, and Doc elements are shown
relative to each other, and to previously sequenced elements (bottom).
Blocks are numbered from left to right (relative to A). The
homologies (colors) and relative orientations (arrowheads) of
subrepeats within the A+T-rich elements are shown. The inverted repeats
at the ends are composed of G-like block 1/A+T-rich block 1
and A+T-rich block 5/G-like block 6.
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The assembled Maupiti sequence also revealed the positions and
orientations of the transposons (Fig. 4). All the transposons in
Maupiti are incomplete or fragmented, unlike the complete
transposons in the AATAT Motus (Fig. 2). The Doc
element is 98% identical to sequenced euchromatic Doc
elements, and is nearly complete (green, Fig. 4). The
F/Doc elements (red with green cross-hatch, Fig. 4)
represent fragments that are 80%–87% identical to sequenced
euchromatic elements (parts of F and Doc elements
share significant homology). These elements appear to be a subfamily
that shares more sequence homology with each other than with previously
sequenced F and Doc elements reported in GenBank. A
similar conclusion can be drawn from analysis of the G-like
elements. The six blocks (blue cylinders, Fig. 4B) are 91%–100%
homologous to each other, and share 80%–89% identity with parts of
previously sequenced G elements. None of these G-likeelements are complete, and all copies are oriented in the same
direction, except block 1 (Fig. 4B). Thus, these elements appear to be
a subfamily of G-like repeats that are more closely related to
each other than to previously sequenced G elements. These
observations suggest that local repeat expansion played an important
role in generating the structure of Maupiti (see Discussion).
Finally, identical 3-kb blocks composed of G-like and A+T-rich
sequences are present at both ends, in inverted orientation (Fig. 4A).
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DISCUSSION
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Successful Methods for Sequence Analysis of Centric Heterochromatin
Current genome projects have not produced truly complete genome
sequences; the human and Drosophila assemblies lack the
30% of these genomes considered to be heterochromatic.
Heterochromatin sequence and organization has been notoriously
difficult to analyze, as a result of the presence of repeated DNA.
Heterochromatin mapping, cloning, and sequencing requires approaches
that are not necessary in the analysis of euchromatic, predominantly
single or low copy number DNA (Schueler et al. 2001). One approach is
to use methods that isolate specific regions of heterochromatin from
the rest of the genome. Sequences that are repeated many times in the
genome are often present in only one or a few copies in a particular
region, which reduces the complexity of mapping, sequencing, and
assembly. Single-copy entry points, in the form of marked transposon
insertions (e.g., P elements in Drosophila), or
rearrangements that juxtapose euchromatin with heterochromatin, have
been used successfully for heterochromatin genome analysis
(Karpen and Spradling 1990; Karpen and Spradling 1992; Howe
et al. 1995; Le et al. 1995; Sun et al. 1997; Yan et al. 2002).
In this study, PFGE isolation of the 1230 minichromosome
derivative provided a purified template for cloning and PCR, which
allowed us to clone, sequence, and assemble a significant proportion of
the repeat-rich, 420-kb functional centromere. The entire complex DNA
component of the minichromosome centromere (69.7 kb) was sequenced and
assembled, including Maupiti, and the five transposons
inserted in the AATAT array. Analysis of 1230 library
clones produced 10.5 kb of satellite sequence, predominantly from the
AATAT arrays that flank the transposons. Tagged PCR and GPS transposon
insertion using purified 1230 were very successful at
producing satellite DNA sequence from both pure arrays and junctions.
From all methods, 19.1 kb of AATAT sequence and 11.9 kb of TTCTC
sequence were generated, corresponding to >8% of the amount of each
satellite in the minichromosome centromere (Table 2). The sequence
coverage averaged fivefold–sevenfold for the transposons and
Maupiti, and >10-fold for the AATAT flanking the single
transposons. Comparison of sequences derived from cloning to the
PCR-generated sequences demonstrated that they differed by only 0.1%,
which suggests that the quality of the sequence is high. We conclude
that heterochromatin can be successfully analyzed using these
modifications of standard genomic approaches. One useful application of
these approaches will be in the analysis of other heterochromatic
regions that are isolated from "contaminating" genomic background
sequences, for example the heterochromatic Bacterial Artificial
Chromosome (BAC) clones generated by the Drosophila, human,
and Arabidopsis genome projects.
Sequence Composition and Organization of the Dp1187 Centromere
Although the satellite arrays have not been completely sequenced,
this study has produced the largest collection of Drosophila
satellite sequences to date (31 kb), providing an opportunity to gain a
greater understanding of the composition and organization of
Drosophila satellites. We found that the AATAT and TTCTC
components of the Dp1187 centromere are organized as tandem
repeats in a uniform "head-to-tail" orientation, with few examples
of "head-to-head" or "tail-to-tail" orientations. The arrays
are oriented as AATAT and TTCTC, from left to right (Figs. 1 and 2), at
all sites where orientation could be determined unambiguously (i.e., in
the transposon flanks, the AATAT/TTCTC junction, and the
Maupiti junction). We also determined that the AATAT and TTCTC
arrays are directly juxtaposed 950 bp from the right end of the
You element, with no intervening sequences.
The level of sequence variation among repeats was low; only 2.2% of
the bases in the AATAT array were altered, consisting of predominantly
single base insertions or deletions. This is similar to the level of
variation observed in comparisons of different human X chromosome 
satellite arrays (1%) (Durfy and Willard 1989), and in interspecies
comparisons of the PRAT satellite family in Coleoptera (1.3%)
(Mravinac et al. 2002). In contrast, only 0.3% of the TTCTC bases were
altered. The TTCTC array could be newer than the AATAT array, and thus
may not have had as much time to accumulate base alterations.
Alternatively, molecular-drive mechanisms (Dover 1982), for example
array expansion and contraction resulting from unequal exchange, and
repeat homogenization through gene conversion, may act differently on
the two sequences. It is also possible that TTCTC contains fewer
variations from functional constraints, such as sequence requirements
that facilitate centromere function (see below). Unfortunately, there
are no published, large-scale compilations of naturally occurring
sequence variations for Drosophila heterochromatin or
euchromatin. Thus, the generality of these findings, and their
relevance to the evolution and function of these satellite arrays in
Drosophila require further detailed analysis of
naturally occurring variations in different satellites, and in
similar satellite sequences located in different sites in the genome.
Such studies may also help distinguish between the molecular mechanisms
that are responsible for satellite evolution in Drosophila.
The pattern of base alterations in the satellite arrays was unusual.
Approximately two-thirds of the AATAT changes were transversions, and
only one third were transitions. This result is surprising, as
transitions are thought to be significantly more frequent than
transversions. The predominance of transversions could be from the
molecular characteristics of the mechanisms responsible for mutation
and homogenization, and how they act on this particular sequence, or
could result from functional selection. Again, additional large-scale
studies of natural variation in Drosophila and other species
are necessary to determine if the high frequency of transversions
occurs in other satellite arrays, and if heterochromatin and
euchromatin display similar frequencies of transversions and
transitions.
We generated complete sequences for the five transposons inserted into
the AATAT array. Heterochromatic transposons previously identified in
Drosophila heterochromatin are often organized as scrambled
clusters of different, incomplete elements (Devlin et al. 1990; Trapitz
et al. 1992; Hochstenbach et al. 1994). Surprisingly, all five elements
in the Dp1187 centromere are intact and complete, in
comparison to sequenced euchromatic elements. The two LTRs in the
Bel, Beagle, and 412 elements displayed very
high levels of cis homology (99.2–99.8% identity).
Interestingly, these transposons are inserted directly into the AATAT
arrays, with no insertion of other sequences at the junctions. In fact,
the satellites present at the junctions display similar levels of
variation in comparison to the "pure" satellite arrays obtained by
random sampling (Table 3). The completeness of the transposons, the LTR
homologies, and the lack of AATAT divergence at the junctions suggest
that these elements were recent additions to the array (SanMiguel et
al. 1998). Alternatively, the transposon sequences may be constrained
by functional requirements, such as centromere activity. Recent studies
have demonstrated that centromeric silencing in
Schizosaccharomyces pombe and DNA elimination in
Tetrahymena thermophila require RNA interference (RNAi)
mechanisms, which act on repeats that apparently evolved from
transposons (for review, see Dernburg and Karpen 2002). Finally, no
transposons were found in the TTCTC array; this is another piece of
evidence in favor of a more recent origin of this array, relative to
AATAT (see above), but could also reflect insertion sequence bias of
the transposons. It will be interesting to determine if other TTCTC
arrays lack transposons, and whether other AATAT arrays contain single,
intact, presumably recent insertions.
The overall organization and sequence composition of the 39 kb
Maupiti contig provides some clues concerning its molecular
evolution. The ends, consisting of A+T-rich and G-like
sequences, are in inverted orientation (Fig. 4A). However, the elements
present in the rest of the contig are organized as direct repeats, even
though they are separated by other components. This observation,
combined with the high homology of the different blocks, suggest that
Maupiti may have evolved by successive duplications, unequal
exchanges, and partial deletions. The basic building block may have
been an A+T-rich unit, which served as a target for insertion of a
G-like element, followed by rounds of expansion and deletion.
The substructure of the A+T-rich subrepeats also suggests duplication
of a basic subunit and evolution via similar molecular drive mechanisms
(Dover 1982). Finally, the nearly complete Doc element is
present in only one copy and may have inserted into the repeated array
subsequent to the repeat duplications.
What Is the Role of DNA Sequence in Centromere Identity?
Analysis of the sequence characteristics of centromeres in different
organisms, and the fact that centromeric satellites are neither
necessary nor sufficient for centromere function, suggest that primary
DNA sequence does not determine centromere identity and propagation
(see Introduction, and Choo 2000; Sullivan et al. 2001).
Sampling of 8.4% of the satellite arrays in the Dp1187
centromere demonstrated that they are uniform, and do not contain any
blocks of complex DNAs. Furthermore, the transposons inserted in the
AATAT array are nearly identical to elements present at many sites
within the euchromatin and the heterochromatin, and Maupiti
does not contain unique, centromere-specific sequences. These results
confirm our previous assertion that the Dp1187 centromere does
not contain sequences that are centromere-specific, or are located at
all Drosophila centromeres (Murphy and Karpen 1995b; Sun et
al. 1997). There is significant additional evidence in favor of
epigenetic models for the propagation of centromere identity
mechanisms, which can parsimoniously account for both centromere
stability and plasticity (see Introduction and Karpen and Allshire
1997; Sullivan et al. 2001).
If epigenetic mechanisms determine centromere identity and function, do
secondary sequence characteristics play a role, such as repetition and
sequence composition, or the ability to form bent structures (Vig 1994;
Murphy and Karpen 1998; Koch 2000; Sullivan et al. 2001)? All sequenced
endogenous eukaryotic centromeres are A+T-rich, and contain repeated
DNA, usually in the form of simple or complex satellite DNA. This study
demonstrates that the minichromosome centromere is also A+T-rich and
replete in highly repeated satellite DNA. However, neocentromeres in
humans are established on normal euchromatic DNA, which does not share
these properties, or any obvious conserved motifs (Barry et al. 2000;
Lo et al. 2001a,b; Satinover et al. 2001).
Higher-order organization of centromeric DNA, such as the inverted
repeat structure at the ends of Maupiti, may be important for
function. All three S. pombe centromeres contain
transposon-like, middle-repetitive elements that flank a single copy
central core in an inverted orientation (Takahashi et al. 1992; Clarke
et al. 1993). Interestingly, the ends of Maupiti are also
organized as inverted repeats. It has been hypothesized that inverted
repeats may cause centromeric chromatin to assemble a stem-loop
structure in S. pombe, and similar types of structures have
been proposed for flies and mammals (Sullivan et al. 2001; Blower et
al. 2002). Centromeric chromatin in fly and mammalian metaphase
chromosomes exhibit unique, cylindrical structures, which may rely on
the presence of repeated DNA, inverted repeats, or some other
characteristic of the sequence, such as A+T composition (Blower et al.
2002). This higher-order structure may be necessary to "present"
the centromeric chromatin to the outside of the condensed chromosome,
ensuring its contacts with components responsible for kinetochore
formation and interactions with microtubules. Further analysis is
necessary to determine if any property of centromeric sequences affects
formation of this structure or other aspects of centromere structure
and function.
How Can We Generate Truly Complete Genome Sequence Assemblies?
We and others (e.g. Schueler et al. 2001) have successfully
demonstrated that the difficulties associated with analyzing the
sequence composition of heterochromatin, including satellite DNA, can
be overcome using modifications of extant genomic methods. Can the
methods described here be used to produce truly complete genome
sequences? Many of these methods, such as gel-purification and cloning
of heterochromatic DNA and tagged PCR, can be applied to studies of
other regions of heterochromatin in flies and other organisms. However,
these approaches are not efficient or robust enough to be scaled up to
analyze the 100 Mb of heterochromatin in the Drosophila
genome (Adams et al. 2000). Complementary methods for focusing on
specific regions of heterochromatin involve generating new, single-copy
entry points. Chromosome rearrangements that juxtapose single-copy
euchromatic regions with heterochromatin have provided a method for
long-range restriction mapping and gene localization, allowing the
single-copy region to be used as a probe to tag one end of the
restriction fragments (Karpen and Spradling 1990; Howe et al. 1995; Le
et al. 1995). Single P-transposable elements provide another method for
marking and analyzing specific heterochromatic regions (Karpen and
Spradling 1992; Thompson-Stewart et al. 1994; Howe et al. 1995; Roseman
et al. 1995; Wallrath and Elgin 1995). We have generated over 600
single P-element insertions into Drosophila centric
heterochromatin, which can now be used to map, clone, sequence, and
assemble many different regions (A. Konev et al., in prep; Yan et al.
2002).
Most importantly, even if significant amounts of heterochromatic
sequence are generated, the manual approach used to assemble
Maupiti is too inefficient. We need to develop automated
assembly software that can appropriately join regions rich in repeated
sequence. Regardless, our study demonstrates that one key to assembling
repeated sequence is to focus on small regions of heterochromatin.
Sequences repeated throughout the heterochromatin are often present
"locally" in only one or a few copies. Focus on specific
heterochromatic regions can thus lead to accurate contig assemblies, as
we have shown for Maupiti. The constrained assembler used to
assemble Drosophila and human whole-genome shotgun (WGS)
sequences could be very useful for heterochromatin sequence assemblies,
because retention of "mated-pair" information can overcome some of
the misalignment problems caused by repeats (Myers et al. 2000). The
manual Maupiti assembly we have generated can provide a good
test case for the abilities of new assemblers to work successfully with
repeated DNA.
In conclusion, a truly complete understanding of genome organization
and sequence requires much more extensive mapping and sequencing of
heterochromatin, in both model organisms and humans. This study
demonstrates that significant amounts of heterochromatin sequence can
be generated and assembled, and that important information about
heterochromatin structure and biology can be produced by such an
analysis. However, a large-scale genomics attack on heterochromatin
will require development of high-throughput approaches, especially in
the area of assembly software development.
|
METHODS
|
|---|
Drosophila Strains
The generation and gross structural analysis of 1230
are described in Figure 1 and in (Le et al. 1995; Murphy and Karpen
1995b; Sun et al. 1997). The genotype for the strain used in all
experiments was y; ry506;
Dp1230, y-
ry+. Standard culture conditions and media were
used.
Library Construction and Screening
PFGE purified 1230 DNA (Sun et al. 1997) was used to
generate small insert clones for sequence analysis. Briefly,
1230 DNA was randomly sheared to 2–7 kb, to avoid
restriction-site biases that would preclude inclusion of many
heterochromatic regions. The sheared DNA was blunted, EcoRI methylated,
ligated to EcoRI linkers, digested with EcoRI, and fragments <1 kb
were removed using a Sephacryl-drip column. The 2–7 kb fractions were
then ligated to the EcoRI digested and dephosphorylated ZAPII
vector. Characterization of clone numbers and insert size demonstrated
that 1230 was represented >80-fold. A high-quality,
random-sheared library from total Drosophila genomic DNA was
generated at the same time, with the same protocol; this library may be
useful for analyses of other Drosophila heterochromatin
regions.
The library was screened by hybridization to colonies. Probes were
generated using information from the 1230 restriction map
(Sun et al. 1997), including all the previously identified transposons,
the A+T-rich sequence, the AATAT and TTCTC satellites, gel purified,
intact 1230, and a 1230 centromeric restriction
fragment (235 kb SpeI). Approximately 100 colonies were also picked
randomly and analyzed. The locations of clones within 1230
(Sun et al. 1997) were then determined by PFGE Southern analysis.
Cloning Satellite DNA From Gel-Purified 1230 by "Tagged" PCR and Bacterial Transposon Insertion
1230 DNA Template Preparation
To eliminate background from other genomic regions with similar
repeats, 1230 was gel-isolated away from genomic DNA using
PFGE (Sun et al. 1997). Isolated 1230 DNA was cut with NotI
and SfiI restriction enzymes resulting in a 490-kb fragment (420-kb
heterochromatin/centromere + 70-kb euchromatin). The 490-kb fragment
was gel-isolated and treated with  -agarose to produce a suitable
template for PCR.
Satellite "Tagged" PCR for Junctions
To reduce product shrinkage resulting from primers annealing to
proximal repeat units, PCR primers composed of six units of AATAT or
five units of TTCTC were tagged at the 5' end with a high Tm, short 6
mer sequence (CCCGGG or GC GCGC). PCR amplification starting with
0.1 ng of DNA was carried out in 10 mM Tris-HCL (pH 8.8), 50 mM KCl,
1.5 mM MgCl2, 0.001% (w/v) gelatin, 200 µM of each dNTP,
and 200 ng of each primer, in a 50-µL volume. PCR was performed under
the following conditions: 94°C for 2 min, then 2–10 cycles of 94°C
for 15 sec, 36°C for 15 sec, 72°C for 1 min (slow ramping
0.6°C/sec); 30 cycles of 94°C for 15 sec, 56°C for 30 sec, 72°C
for 1 min; and a final extension time of 72°C for 8 min. Increasing
the annealing temperature takes advantage of the 5' primer tags that
were incorporated in the initial cycles. This results in an increase of
the product size from 50–75 bp of satellite flank to 500–600 bp of
satellite flank (see supplemental materials). PCR products were cloned
using PCR-Script Amp cloning kit (Stratagene) or TOPO TA kit
(Invitrogen). Colonies were screened with colony PCR using M13 Rev and
Fwd primers for pCR2.1 TOPO clones, and T3 and T7 primers for
pPCR-Script AMP clones. PCR products were visualized on 2% Metaphor
agarose gels and then clones containing inserts were purified using
either QIAquick PCR Purification Kit (QIAGEN) or Wizard PCR Preps DNA
Purification System (Promega). The same primers were used for both
amplification and DNA sequencing. Sequences were analyzed with
Sequencher (Genecodes).
PCR on Satellite Arrays
Amplification of satellite DNA from 1230 was done by
using a single satellite primer (five units of TTCTC) that was tagged
at the 5' end with an 18-mer sequence (5'-CATGACTGGAC GCTCCAC-3' or
5'-GAGTTGCATCTGGGATCG-3'). PCR was performed under the following
conditions: 94°C for 2 min; 30 cycles of 94°C for 15 sec, 36°C
for 15 sec, (slow ramping 0.4°C/sec) 72°C for 2 min, one final
extension of 72°C for 8 min. This produced single-stranded satellite
sequences that incorporated the 5' tagged 18-bp primer. After cleanup
of the PCR reaction, 1–2 µL of the reaction was used as a template
for PCR with the 18-mer and the junction primers. Amplification,
isolation, and sequencing were the same as for the junctions.
Transposon Tagging
The Genome Priming System (NEB) was used according to
manufacturer's instructions to introduce a modified bacterial
transposon into the purified and isolated 1230 DNA. The
flanking satellite DNA was amplified using a primer from the end of the
transposon and a satellite-tagged primer. The products were
cloned and analyzed in the same manner for the satellite-tagged PCR
method.
Sequence Determination and Analysis
All sequencing was performed in The Salk Institute DNA Sequencing
Facility, using big dye-termination reagents and ABI/PE 377 automated
sequencers. Sequencer 3.0 (Genecodes) was used for all sequence
assemblies. Accession numbers are included in supplemental materials.
|
Acknowledgements
|
|---|
We thank Michael Blower, Patrick Heun, and Beth Sullivan for
critical editorial comments. This research was supported by the
National Institutes of Health/National Human Genome Research Institute
(R01 HG00747).
The publication costs of this article were
defrayed in part by payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance with 18
USC section 1734 solely to indicate this fact.
|
Footnotes
|
|---|
1 Corresponding author. 
E-MAIL karpen{at}salk.edu; FAX (858) 622-0417.
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.681703.
|
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ABSTRACT
RESULTS
