INVESTIGATION
Heritable Loss of Replication Control
of a Minichromosome Derived from
the B Chromosome of Maize
Rick E. Masonbrink,* Shulan Fu,† Fangpu Han,† and James A. Birchler*,1
*Division of Biological Sciences, University of Missouri, Columbia, Missouri 65211, and †State Key Laboratory of Plant Cell and
Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese National Academy of Sciences, 100101
Beijing, China
ABSTRACT During an accumulation regime of a small telomere-truncated B chromosome, a derivative with large variations in size and
multiple punctate centromere loci exhibiting amplified copy numbers was discovered. Multiple centromere satellite loci or transgene
signals were documented in amplified chromosomes, suggesting over-replication. Immunolocalization studies revealed multiple foci of
biochemical markers characteristic of active centromeres such as CENP-C and phosphorylation of histones H3S10 and H2AThr133. The
amplified chromosomes exhibit an absence of chromosome disjunction in meiosis I and an infrequent chromosome disjunction in
meiosis II. Despite their unusual structure and behavior these chromosomes were observed in the lineage for seven generations during
the course of this study. While severely truncated relative to a normal B chromosome, the progenitor minichromosome is estimated to
be at least several megabases in size. Given that the centromere and transgene signals at opposite ends of the chromosome generally
match in copy number, the replication control is apparently lost over several megabases.
E
NDOREDUPLICATION is essentially the replication of
a genome without cell division and is typically found
in terminally differentiated cells in specialized tissues. This
process is thought to increase the availability of DNA templates to amplify gene expression because the transcriptional
and translational activities of the cell increase proportionally
with each genome doubling (D’Amato 1984; Larkins et al.
2001). Studies in plants and animals show that endoreduplication is correlated with the lowering of M-phase cyclins,
responsible for the G2-to-M transition, and with a higher
abundance of S-phase cyclins, responsible for the G1-to-S
transition (Evans et al. 1983). The oscillation of cyclins and
a regulatory protein degraded by the anaphase-promoting
complex (Dbf4) regulate the activity of cyclin-dependent
kinases (CDK) and Dbf4-dependent kinases (DDK), respectively. They phosphorylate the minichromosome maintenance
proteins 2–7 (MCM), which form the prereplication complex
Copyright © 2013 by the Genetics Society of America
doi: 10.1534/genetics.112.146126
Manuscript received September 22, 2012; accepted for publication October 29, 2012
Supporting information is available online at http://www.genetics.org/lookup/suppl/
doi:10.1534/genetics.112.146126/-/DC1.
1
Corresponding author: 311 Tucker Hall, University of Missouri, Columbia, MO 65211.
E-mail: [email protected]
necessary for replication licensing (Tuteja et al. 2011).
While all previous descriptions of endoreduplication involve
whole-genome duplication (e.g., Bauer and Birchler 2006),
we describe a single, supernumerary chromosome that routinely amplifies copy number without separation while the
remainder of the genome replicates normally.
The amplified chromosome originated from the B chromosome (B) of maize, a supernumerary chromosome that
persists in the genome with a selfish inheritance resulting in
higher-than-expected transmission (Longley 1927). The B
can accumulate in the genome by surviving meiosis as a univalent, by nondisjunction at the second pollen mitosis and
by preferential fertilization of the egg rather than the central
cell by the B-containing sperm (Roman 1948; Carlson 1969;
Carlson and Roseman 1992). Preferential fertilization is associated with a single gene in the normal karyotype (A chromosomes), but nondisjunction and univalent survival are
conditioned by the B chromosome (Carlson 1969; Carlson
and Roseman 1992; Chiavarino et al. 2001). Three regions
of the B chromosome are essential for nondisjunction at the
second pollen mitosis: one cis-acting site in the proximal
heterochromatin and two trans-acting sites in the proximal
and distal euchromatin (Roman 1949; Rhoades et al. 1967;
Rhoades and Dempsey 1972; Carlson 1973, 1978; Ward
Genetics, Vol. 193, 77–84
January 2013
77
1973; Lin 1978). Nondisjunction of the B chromosome has
also been observed in the tapetum, endosperm, and roots,
although these properties do not appear to contribute to its
selfish inheritance (Alfenito and Birchler 1990; Chiavarino
et al. 2000; Masonbrink and Birchler 2010).
Previously, maize embryos with B chromosomes were
bombarded with telomere repeats, resulting in chromosomal
truncations of A and B chromosomes (Yu et al. 2007). One of
these truncations (86-B23) had most of the B chromosome
long arm removed, resulting in a chromosome slightly larger
than a B chromosome centromere. This chromosome accumulated to multiple numbers when a normal B chromosome
was in the genome. During this program of accumulation,
we fortuitously discovered a lineage in which this chromosome replicated multiple times, but remained a single entity.
Materials and Methods
Mitotic in situ hybridization
Root-tip FISH was performed as described with a slight
modification (Masonbrink and Birchler 2010). To make cellwall digestion times more predictable, distilled water was
substituted in washes for ethanol. Fluorescently labeled oligonucleotide probes consisted of a telomere repeat that
strongly cross-hybridizes to the B repeat and CentC, which
hybridizes to the centromere of all chromosomes and in the B
chromosome long arm (Alfenito and Birchler 1993; Ananiev
et al. 1998; Lamb et al. 2005). The telomere oligonucleotide
will label the B chromosome centromere because of the partial homology of the B centromere-specific repeat (Alfenito
and Birchler 1993) and produces such a strong signal that
the exposure time for the A chromosome telomeres is seldom
long enough for visualization. Nick-translated PCR products
consisted of the Stark repeat (Lamb et al. 2007), the centromeric retrotransposon of maize (CRM) (Nagaki et al. 2003),
the 180-bp knob repeat (Peacock et al. 1981), and the cassette from the telomere-truncation experiments without the
telomere array (WY96) (Yu et al. 2006).
Meiotic fluorescence in situ hybridization
Meiotic FISH was performed as described previously
(Masonbrink et al. 2012). FISH probes consisted of fluorescein-labeled telomere oligonucleotide (green) and a Texas
Red-labeled CentC oligonucleotide.
Immunocytochemistry
Immunolocalization for mitosis and meiosis were performed as
described (Han et al. 2009). The polyclonal antibody against
maize CENP-C was raised in rabbit (GL Biochem, Shanghai). A
monoclonal rabbit antibody (04-817) raised against histone
H3 phosphorylated at Ser-10 was obtained from Upstate.
The phosphorylated H2a antibody was previously described
(Dong and Han 2012). The images were taken as a confocal
z-stack (Zeiss LSM 710 NLO), and a flat projection of the
three-dimensional image was created with the ZEN 2009 Light
Edition (Zeiss) and processed with Photoshop CS 3.0.
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R. E. Masonbrink et al.
Results
A small B chromosome derived-minichromosome (86-B23) (Yu
et al. 2007) (Figure 1A) was accumulated to multiple copies
using the maize B chromosome accumulation mechanism. In
two generations, this minichromosome was accumulated to
five copies before finding an unusually large chromosome with
multiple distinct centromere loci. In the next generation, we
observed many of these minichromosomes changing in size and
quantity between cells of the same root. Smaller-sized chromosomes typical of the progenitor were usually found in every cell,
while the larger-sized chromosomes were restricted to fewer
cells as if they were present only in certain developmental
lineages. These chromosomes had multiple distinct CentC signals per minichromosome (Figure 1B). In addition, we probed
the chromosome with CRM, the centromeric retrotransposon of
maize (Figure 1C); Stark repeat (Lamb et al. 2007), which is
found in the centromere and the distal heterochromatin of the
B chromosome (Figure 1D); WY96, the truncation transgene
cassette without the telomere array (Figure 1E); and the 180bp knob repeat, a constituent of maize heterochromatin (Figure
1F) (Peacock et al. 1981). Each probe hybridized to multiple
sites on the endoreduplicated chromosomes, and the quantity
of these signals was correlated with the size of the chromosome. For the medium-sized chromosome in Figure 1E, the
transgene, which exhibits the most discrete signals, hybridized
to between 16 and 20 sites. In a single cell of one individual, the
chromosome deviated from the typical spherical appearance
and was observed to form a large circle (Supporting Information, Figure S1). While we attempted to increase the copy
number of the amplifying chromosome, we were not able to
observe a cell with more than eight. As a sample of the frequency of their occurrence, the numbers were recorded from
three different root tips. Of 48 cells, 29 (60%) showed a large
chromosome, whereas the normal-appearing minichromosome
was observed in the remaining cells.
Immunocytochemistry was used to characterize the
chromatin of the amplified chromosomes and to determine
if multiple centromere loci on a single chromosome could
condition multiple active centromeres. CENPC is an inner
kinetochore protein that is a biochemical marker of centromere activity. An antibody against CENPC was found to
localize to multiple loci on the large amplified chromosomes
(Figure 2). The larger of such chromosomes were found to
exhibit up to three or more detectable foci. In contrast, the
smaller versions and the A chromosomes never had more
than one binding site for each chromosome. Sister-chromatid cohesion is indicated by phosphorylation of serine 10 of
histone H3 (Kaszas and Cande 2000). The amplified chromosomes had multiple sites of sister-chromatid cohesion indicated by bound phosphorylated H3S10 antibody (Figure 3).
An antibody to phosphorylated H2AThr133, a histone modification associated with centromere function (Dong and Han
2012), also had multiple foci (Figure 4).
Throughout meiosis the amplified chromosome continued
to vary in size and frequency between cells of the same tassel
Figure 1 Centromeric sequences in the
amplified chromosome. Telomere
probes, which cross-hybridize to the B
repeat and were used for detection of
the B repeat, are green. The inset pictures are the red channel of an amplified
chromosome. Red arrows denote amplified chromosomes, and white arrows
denote unmodified full-sized maize B
chromosomes. (A) CentC (red), a centromeric satellite repeat, localizes to a single
locus on the progenitor chromosome of
the amplified chromosome. (B) CentC
(red) had multiple distinct signals on
the amplified chromosome. (C) Multiple
CRM (red) signals are on the larger amplified chromosome, while the smaller
versions have only two. (D) Stark repeat
(red), which is found in the distal heterochromatin of the long arm and in
the centromere of the B chromosome,
hybridized throughout the amplified
chromosome. (E) WY96 (red), the singlecopy truncation cassette probe, hybridized to multiple distinct loci on the
amplified chromosome. (F) Knob (red) is
found in the proximal heterochromatin of
the B chromosome and also in the amplified chromosome. Bars, 10 mm.
and anther. In one anther, nearly all the stages of meiosis
were observed, excluding pachynema, from the same plant
that had multiple amplified chromosomes. In pachynema,
these chromosomes appeared small and compact, a deviation
from the typical string-like morphology of pachytene chromosomes (Figure 5A). In diplonema, two spherical copies of
the chromosome had “sticky” behavior, a characteristic of B
chromosomes (Figure 5B) (McClintock 1933). In diakinesis,
we observed three smaller-sized chromosomes of various
sizes, and a notable division of the B repeat to opposite ends
of a chromosome (Figure 5C). At metaphase I, the amplified
chromosomes appeared to nondisjoin as demonstrated by
being closer to one pole than all other chromosomes (Figure
5D). This was also seen in somatic cells of the anther (Figure
S2). All amplified chromosomes that did not nondisjoin
lagged at the metaphase plate. They apparently eventually
moved to one pole as evidenced by the lack of micronuclei in
subsequent stages. Chromosome disjunction was never observed with this chromosome at meiosis I (Figure 5E). Unequal numbers of amplified chromosomes were observed in
prometaphase II, probably the result of precocious monopolar
movement at metaphase I and nondisjunction at anaphase I
(Figure 5F). At anaphase II, some sister separation was apparent, but many times the chromosome nondisjoined to one
Minichromosome Amplification
79
Figure 2 Multiple sites of bound
CENPC on larger amplified chromosomes. The yellow arrows denote amplified chromosomes with multiple sites of
bound CENPC, which is a marker for
centromere activity. The red arrows denote small amplified chromosomes with
one site of CENPC binding. The top inset
is the red CENPC antibody channel
showing multiple signals on each large
amplified chromosome. The box in the
top inset shows multiple CENPC signals
on the amplified chromosome. The middle inset is the B repeat, which is found
on the B chromosome and throughout
the amplified chromosomes. The bottom inset is the DAPI channel. The white
arrows denote three B chromosomes.
The B repeat is green and CENPC is
red. Note that A chromosomes have
one distinct signal per sister chromatid.
Bar, 10 mm.
pole (Figure 5G). In telophase II, the large spherical-shaped
chromosomes were still present, as well as a higher quantity
of small versions (Figure 5H).
While screening the amplifying chromosome line, a small
version was found that resembled the progenitor of the amplifying chromosome with two CentC FISH signals and
a characteristic small size. It retained these characteristics
and remained at one copy in every cell examined in the root.
Because this minichromosome was stable, we self-pollinated
and outcrossed the plant to see if amplification would reinitiate. In screening the next generation, we observed
enlarged chromosomes in root tips from 10 of 18 seedlings,
demonstrating that the amplifying phenotype could reinitiate. We also screened 40 seeds by FISH from the original
transformant seed stocks to determine if amplification was
a property of this chromosome but were unable to find an
amplifying minichromosome. While the earliest seed stocks
did not contain an amplified chromosome, we did find one
plant in which the minichromosome frequently nondisjoined
between cells of the same root, resulting in quantity shifts from
zero to four minis and increased chromosome size (Figure S3),
but the amplifying phenotype was not recapitulated.
Discussion
Here we describe a chromosome that changes size and
structure among cells of the same plant. Its behavior is similar
to a univalent chromosome during meiosis and mitosis,
frequently nondisjoining even in the absence of a normal B
chromosome. The fact that the normal karyotype replicates
80
R. E. Masonbrink et al.
and separates normally indicates the presence of a cis-acting
modification in this amplifying chromosome.
A potential explanation for this behavior is that there is an
enhancement of nondisjunction without the subsequent
sister separation. The sister chromatids of the normal B
chromosome eventually separate, but the replicated sisters
may remain adhered through successive replication cycles,
thus doubling the size of the chromosome. For this hypothesis to explain the observed structure, the chromosome’s sister chromatids would have to remain adhered through
multiple mitoses and thus would drastically lower its presence among monitored cells. This possibility would halve the
number of cells in the lineage receiving such a chromosome
for each instance of nondisjunction. Considering that we
found an instance of eight such chromosomes in one cell
with 10–16 centromere sites (Figure 1B), the odds of finding this would be extremely low. In this scenario, nondisjunction could explain the number of centromere loci present,
but the observed frequency of amplified chromosomes would
be much lower than that observed. Moreover, nondisjunction
of the B chromosome has never been observed to produce
such a conglomerate (Masonbrink and Birchler 2010).
Another potential consideration might be that the minichromosome formed a ring structure. Then, with sisterchromatid exchange within the ring, multiple centromeres
would be present. This scenario also seems unlikely because
the sister centromeres of rings typically separate at anaphase
and rupture the chromosome. With this hypothesis an atypical
behavior of rings would need to occur to form larger and
larger structures due to repeated failure of sister-chromatid
Figure 3 Active sites of sister-chromatid cohesion (H3S10) in the amplified chromosome. The yellow arrow denotes an amplified chromosome
with multiple sites of bound H3S10 antibody, which is a marker for sisterchromatid cohesion. The red arrows denote smaller amplified chromosomes. The white arrows denote B chromosomes. Note that all A
chromosomes have one distinct signal per sister chromatid. The red channel is the H3S10 antibody. Bar, 10 mm.
separation. Also, this scenario would not predict the finding
of rare cells with many different-sized chromosomes spanning 10–20 copies (Figure 1), nor is it consistent with the
observation that, when they are not present, the normal minichromosome was observed. Finally, McClintock studied
small-ring chromosomes in maize (McClintock 1938), and
their structure and behavior are not concordant with the
chromosomes described here.
A more likely scenario might be that an initial modification
occurred that circumvents replication licensing, thus initiating
multiple rounds of replication per cell cycle in select cells
(Nishitani and Lygerou 2002). The minichromosome could
then increase to various sizes between cells and permit the
accumulation of multiple copies. This scenario would also
group the larger amplified chromosomes in selected cells, while
the smaller versions would be present in most cells. A combination of over-replication and nondisjunction could also allow
multiple chromosome copies of varying sizes to accumulate,
while also limiting the number of cells having large variants.
The latter mechanism would double the number of
centromere signals at each replication cycle, which would
create chromosomes with 2, 4, 8, 16, 32, etc., centromere
signals. The number of centromere signals is directly proportional to the number of single copy transgenes, which
have distinct condensed FISH signals (Figure 1E, inset). In
counting the number of transgene signals, we noted that the
number deviates from the strict doubling predicted. This
deviation might result from the splitting of some of the
larger conglomerates in some mitoses, not all copies of the
chromosome continuing to replicate in synchrony, or from
limitations on the detection of all copies.
The number of centromeric FISH signals (Figure 1) and
antibody-binding sites associated with sister-chromatid cohesion and centromeric activity (Figures 2–4) were often at
unequal frequencies. Inactivated centromeres, i.e., centromeres that do not recruit a kinetochore (Han et al. 2006),
within the amplified structure may account for this signal
discrepancy, possibly explaining the relative stability of these
chromosomes. The precocious monopolar movement and
Figure 4 Sites of functional centromeres (phosphorylated H2A) in the amplified chromosome. The yellow arrow
denotes an amplified chromosome with
multiple sites of bound phosphorylated
H2AThr133 antibody, which is a marker
for centromere function. The red arrow
denotes a small amplified chromosome.
The top inset is the red H2A antibody
channel showing multiple signals per
large amplified chromosome. The middle inset is the B repeat channel, which
is found on both B chromosome arms at
distal positions and throughout the amplified chromosome. The bottom inset
is the DAPI channel. The white arrow
denotes a B chromosome. B-repeat
probes are green. Note that A chromosomes have one distinct H2A signal per
sister chromatid. Bar, 10 mm.
Minichromosome Amplification
81
Figure 5 The meiotic behavior of an
amplified chromosome. All meiocytes
were obtained from a single anther, except the pachytene cell. Yellow arrows
denote amplified chromosomes, and
white arrows denote B chromosomes.
CentC probes are red, and telomere
probes, which strongly cross-hybridized
with the B repeat, are green. (A) The
amplified chromosome appears small
at pachynema with diffuse B-repeat
chromatin. (B) Two amplified chromosomes in diplonema. (C) Three amplified
chromosomes in diakinesis. (D) Two amplified chromosomes, both with early
monopolar movement typical of univalent B chromosomes at metaphase I. (E)
Two amplified chromosomes, the larger
with persistent early monopolar movement and the smaller with late monopolar movement at anaphase I. A
univalent B chromosome lagging and
separating sister chromatids is present
(white arrow). (F) Prometaphase II with
the top cell having two amplified chromosomes, while the bottom cell has one
larger amplified chromosome and a univalent B chromosome. (G) Anaphase II
with the left cell segregating sister chromatids of three amplified chromosomes
and one large amplified chromosome
nondisjoining to one pole without sister-chromatid separation. The right cell
has one amplified chromosome lagging
at the metaphase plate while a larger
amplified chromosome has nondisjoined
to one pole. (H) Telophase II with seven
amplified chromosomes. The larger amplified chromosomes are in distinct
spherical shapes. Bars, 10 mm.
nondisjunction observed in metaphase I and II and anaphase
I and II were more frequent with larger versions of the
amplified chromosome. In fact, the only sister-chromatid
separation observed in meiosis occurred with small versions
82
R. E. Masonbrink et al.
of the chromosome in anaphase II. Early monopolar movement is frequent with univalent B chromosomes (Carlson
and Roseman 1992), but is at much lower levels than what
was seen with the amplified chromosome.
This unusual chromosome was found in a lineage derived
from a truncated B chromosome. This amplifying behavior
acts in cis; namely only the minichromosome is affected,
thus suggesting some change, be that mutational or epigenetic, that was perpetuated over the seven generations that
the chromosome was under scrutiny. Nevertheless, a progenitor-sized chromosome was still observed in this lineage,
suggesting that single copies were possible and that the
amplification could repeatedly occur from a single copy.
Many small B chromosome derivatives have been studied
in our laboratory over many years (Han et al. 2007) without
any similar cases being observed.
This minichromosome contains the knob heterochromatin
that is adjacent to the B centromere, indicating that the knob
is proximal to the site of truncation and that the B centromere is intact. The core of the B centromere is 700 kb
and the B repeat cluster spans a minimum estimate of 3.7 Mb
(Jin et al. 2005). Thus, this example provides insight into the
size range of replication control. Re-replication of this chromosome is regularly unrestricted and apparently spans megabases, given that the transgene signals at the chromosome
terminus generally match the number of centromere signals.
The discovery of this chromosome will provide a model system for future analyses of replication control.
Acknowledgments
This work was supported by National Science Foundation
grants DBI0701297 and DBI0922703.
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Communicating editor: A. Houben
GENETICS
Supporting Information
http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.112.146126/-/DC1
Heritable Loss of Replication Control
of a Minichromosome Derived from
the B Chromosome of Maize
Rick E. Masonbrink, Shulan Fu, Fangpu Han, and James A. Birchler
Copyright © 2013 by the Genetics Society of America
DOI: 10.1534/genetics.112.146126
Figure S1 An amplified chromosome twisted into a figure eight (observed once in a single cell over the course of seven generations of study) when sister chromatids were separating in the remainder of the cell. The top inset is CentC in the red channel showing multiple distinct signals. The middle inset is B repeat in the green channel. The bottom inset is DAPI in the blue channel. The yellow arrows denote an amplified chromosome in a figure eight. Red arrows denote smaller amplified chromosomes. White arrows denote B chromosomes. Telomere probes were used to detect the B repeat. Scale bar is 10 microns. 2 SI R. E. Masonbrink et al. Figure S2 Somatic cells of the anther showing early monopolar movement occurring in mitosis. While screening for meiocytes, somatic cells of the anther were found in anaphase. The amplified chromosome nondisjoined with early monopolar movement (yellow arrow). The white arrow denotes a B chromosome. Telomere probes, which hybridize with the B repeat, are green. CentC probes are red. Scale bar is 10 microns. R. E. Masonbrink et al. 3 SI FIgure S3 A minichromosome from the first generation following telomere mediated chromosomal truncation that is increased in size, but still retains one centromere signal per sister chromatid. The insets show from top to bottom: the CentC (red) channel, the green telomere (B repeat) channel, and the DAPI (blue) channel. The yellow arrow denotes the minichromosome. The white arrow denotes a B chromosome. Telomere probes, which cross-­‐hybridize to the B repeat, are green. CentC probes are red. Scale bar is 10 microns. 4 SI R. E. Masonbrink et al.