Department of Biological Chemistry,
University of California, Irvine, Irvine, California
92697-1700,1 and Department of
Anatomy and Cell Biology, University of Florida, Gainesville, Florida
326102
Received 14 July 1999/Returned for modification 30 August
1999/Accepted 7 September 1999
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INTRODUCTION |
All eukaryotic cells have three
distinct RNA polymerases that normally transcribe different sets of
nuclear genes. RNA polymerase I (Pol I) is unique in that in most
eukaryotic organisms, its sole function is the transcription of genes
for large rRNAs (rDNA). Dedication of a separate RNA polymerase to rDNA
transcription is a feature unique to eukaryotes and must have had
strong selective advantages for eukaryotic organisms in evolution.
However, we have recently discovered that mutants of the yeast
Saccharomyces cerevisiae which are defective in
transcription factor UAF (upstream activation factor) give rise to
variants which now grow by transcribing endogenous rDNA by RNA
polymerase II (Pol II) (39). (In this paper, we use the term
transcription of rDNA to imply transcription of the gene encoding the
35S precursor rRNA, although the 5S RNA gene transcribed by RNA
polymerase III is a part of the rDNA repeat unit in S. cerevisiae.) Thus, yeast cells have an inherent ability to use Pol
II for rDNA transcription, but this transcription activity is
apparently silenced in normal cells. Studies of the processes which
enable yeast cells to grow without using the Pol I machinery may be
important for understanding the normal Pol I machinery and for gaining
insight into the significance of its evolution. In this paper, we
describe our finding that the switch to growth using the Pol II system
consists of two steps; the first step is a mutational alteration in
UAF, and the second step is an expansion of chromosomal rDNA repeats.
The first step, a UAF mutation, is sufficient to allow Pol II
transcription of rDNA, but the overall efficiency of rRNA synthesis is
apparently not sufficient for sustained cell growth. The second step,
rDNA repeat expansion, represents an adaptation process, which probably
involves a selection for faster-growing cells and leads eventually to a
semistable state of rDNA with an increase in repeat numbers and an
altered nucleolar location and morphology.
Like other eukaryotic rDNA promoters, the promoter for the gene
encoding 35S precursor rRNA in S. cerevisiae consists of two elements, the upstream element and the core element. Basal
transcription of yeast rDNA requires the core element and two
transcription factors, Rrn3p and CF (core factor), in addition to Pol
I. CF consists of three proteins encoded by RRN6,
RRN7, and RRN11. For high levels of
transcription, two additional factors, UAF and TBP (TATA binding
protein), as well as the upstream element are required in addition to
the components required for basal transcription (18, 19,
37). UAF contains three Pol I-specific protein subunits encoded
by RRN5, RRN9, and RRN10, histones H3
and H4, and the uncharacterized protein P30 (17). In
apparent agreement with the in vitro function of UAF, genes
RRN5, RRN9, and RRN10 are not
absolutely required for cell growth and yeast strains with mutations in
these genes can grow, albeit very slowly (19).
Slowly growing UAF mutants give rise to faster-growing variants which
do not require intact Pol I and synthesize rRNA using Pol II
(39). The slowly growing mutants defective in Pol I-specific UAF components are unstable because of the appearance of faster-growing variants, but they can be maintained stably by introducing a helper plasmid, e.g., pNOY103, which carries the 35S rRNA coding region fused
to the galactose-inducible GAL7 promoter. These cells can grow fairly well on galactose but extremely poorly on glucose due to
repression of the fusion gene. The faster-growing variants can grow,
with or without a helper plasmid, both on galactose and on glucose and
were previously shown to synthesize rRNA by transcribing endogenous
rDNA by Pol II (39). These cells were called PSW (polymerase
switched for growth) cells, thus defining the PSW state. The original
UAF mutant cells carrying the helper plasmid, which were unable to grow
on glucose, were called non-PSW (referred to as N-PSW in this paper),
thus defining the N-PSW state (39). The PSW state, once
established, is fairly stable and can be inherited through mitosis and
meiosis, but spontaneous reversion to the original N-PSW state can be
easily demonstrated (39). We have now studied the reversible
changes between the N-PSW and PSW states and discovered that they
represent expansion and contraction of rDNA repeats.
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MATERIALS AND METHODS |
Strains, plasmids, and media.
Yeast strains and plasmids
used are listed in Table 1. Disruption of
RPA135 was done as previously described (40). For disruption of SIR2, a DNA fragment covering the
SIR2 chromosome region, containing a 1.8-kb deletion
removing most of the SIR2 coding region except for the first
18 amino acids and replaced by a 1.6-kb fragment containing
LEU2, was used. For disruption of SIR3, a DNA
fragment covering the SIR3 chromosome region, containing a
2.5-kb BglII-XhoI deletion within the
SIR3 coding region replaced by the 2.5-kb
BglII-SalI fragment containing LEU2,
was used. For disruption of SIR4, a DNA fragment covering
the SIR4 chromosome region, containing a 330-bp
BglII-BamHI deletion inactivating SIR4
function (15) and replaced by the 3.1-kb
BglII-BglII fragment containing LEU2,
was used. Disruption of SIR2, SIR3, and
SIR4 genes was confirmed by the nonmating phenotype of the
strains constructed. Disruption of FOB1 was carried out by
using the EcoRI fragment obtained from
pUC-fob1::LEU2 as previously described (20). Disruption of RRN5 was done by using the
3.4-kb fragment carrying rrn5
::LEU2 described
previously (19).
YEP-galactose, YEP-glucose (YEPD), synthetic galactose, and glucose
media were described previously (26). The following supplements were added to the synthetic media as appropriate to satisfy
nutritional requirements: Casamino Acids (5 mg/ml), tryptophan (20 µg/ml), adenine (20 µg/ml), and uracil (20 µg/ml).
Spot test for PSW and N-PSW phenotype.
Individual colonies
formed on YEP-galactose media were picked and suspended in 100 µl of
H2O, and 5-µl aliquots of 10-fold serial dilutions were
spotted on YEP-galactose and YEPD plates. They were usually incubated
at 30°C for 7 days.
Analysis of chromosome XII by contour-clamped homogeneous
electric field (CHEF) electrophoresis.
Chromosomal DNA from yeast
strains was isolated as previously described (35) and
electrophoresed in 0.8% agarose (SeaKem LE; FMC BioProducts, Rockland,
Maine)-0.5× Tris-borate-EDTA buffer by using a CHEF Mapper (Bio-Rad,
Richmond, Calif.), programmed for a switch time of 300 to 900 s
for 68 h at 14°C and with an included angle of 120°
(21). After electrophoresis, the gel was stained with 0.5 mg
of ethidium bromide per ml for 30 min at room temperature, destained in
water for 1 to 2 h, and then photographed. The gel was transferred
to a nylon membrane (Zeta-Probe GT; Bio-Rad) and then analyzed by
Southern hybridization with 32P-labeled rDNA and
SIR3 probes (24). The rDNA probe used was the
613-bp SmaI-EcoRV fragment spanning positions
210 to +403 (+1 is the Pol I transcription start site). The
SIR3 probe used was the 754-bp
PstI-HindIII fragment within the
SIR3 coding region.
EM and FISH.
Yeast strains were grown at 25°C, and
electron microscopy (EM) analysis was carried out as previously
described, using a JEOL 100CX electron microscope (28).
Fluorescence in situ hybridization (FISH) analysis was also carried out
as described previously (28) except that the rDNA probe used
was a 6.9-kb DNA carrying the 35S rRNA coding region (+1 to +6922) with
extra 15 nucleotides derived from a multicloning site of a plasmid vector.
Other methods.
Isolation of DNA and Southern hybridization
analysis were carried out as described by Maniatis et al.
(24). Analysis of 5' ends of precursor rRNA by primer
extension was carried out as described previously (18, 39),
using the primer 5'-ACACGCTGTATAGAGACTAGGC-3', which
hybridizes to 35S precursor rRNA 130 nucleotides downstream of the Pol
I start site. Quantification in both analyses was done with a
PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).
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RESULTS |
Transition from the N-PSW state to the fully established PSW state
is an adaptation process.
Yeast strains carrying a deletion of
RRN9 were analyzed for growth by spot testing serial
dilutions on galactose and glucose plates. N-PSW strain NOY703 is able
to grow well on galactose due to the presence of a helper plasmid
carrying GAL7-35S rDNA (Fig.
1A Gal, rows a and b). Conversely, NOY703
grows poorly on glucose with colonies appearing after a long incubation
(7 to 10 days at 30°C) at a frequency of approximately
10
3 to 104 (Fig. 1A Glu, rows a and b).
When colonies were picked from the glucose plate, resuspended in water,
and spot tested again on glucose and galactose plates, growth on
galactose was still better than that on glucose as indicated by colony
size, and only a small fraction (ca. 1% in the example in Fig. 1A) of
the cells retained the ability to grow on glucose (Fig. 1A, rows c and
d). However, after repeated streaking on glucose plates, larger
colonies were easily obtained, and these clones showed better growth on
glucose than on galactose. Spot test analysis of one such established PSW strain, NOY878, which was derived from NOY703 and was used in the
present and previous work (39), is shown in Fig. 1B (rows c
and d). These observations demonstrate that between the N-PSW and PSW
states are intermediates which are able to grow weakly on glucose and
moreover are unstable such that they tend to lose the ability to form
colonies on glucose. Thus, switching from N-PSW to PSW states is an
adaptation process that occurs through selection for better-growing
variants on glucose plates.
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FIG. 1.
Spot test of rrn9 N-PSW strain NOY703 and
PSW variants derived from it. (A) Two independent colonies of NOY703
formed on a galactose plate were analyzed by spotting aliquots of
10-fold serial dilutions of suspension of colonies on YEP-galactose
(Gal) and YEPD (Glu) (rows a and b). Several discrete colonies, which
had just formed on YEPD by plating large numbers of NOY703 cells on
glucose (similar to colonies shown in rows a and b), were combined and
similarly analyzed (rows c and d). (B) Two independent colonies of
NOY703 (rows a and b), NOY878 (rows c and d), and an N-PSW revertant
derived from NOY878 (rows e and f) formed on galactose plates were
analyzed by the spot test as for panel A. Plates were incubated at
30°C for 10 days.
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Correlation of expansion of rDNA repeats with the switch from N-PSW
to PSW states.
In the course of analysis of rDNA in PSW strains,
we discovered that the amount of rDNA in these strains is much higher
than that in N-PSW strains. The results of a typical Southern analysis of rDNA in the wild-type (WT; NOY556), rrn9 N-PSW
(NOY703), and rrn9 PSW (NOY878) strains are shown in Fig.
2 (lanes 1 to 3). SIR3, a
single-copy gene on chromosome XII, the same chromosome that carries
the rDNA repeats, was used for normalization. It was found that the
rrn9 N-PSW strain showed about a 2-fold reduction and the
rrn9 PSW strain showed about a 2.6-fold increase in rDNA copy number relative to the WT strain. The reduction in the copy number
of rDNA repeats in the rrn9 N-PSW strain was not
unexpected, since a twofold reduction in rDNA copy number was
previously observed for a strain (NOY408-1a) carrying a deletion in an
essential Pol I subunit gene (RPA135) and growing on
galactose using the GAL7-35S rDNA fusion gene on a helper
plasmid (21). This strain was also analyzed, together with
its control RPA135 strain (NOY408-1b), and the previous
finding, a twofold reduction in rDNA copy number, was confirmed (Fig.
2, lanes 4 and 5). The absolute copy number of rDNA in this control
strain was previously estimated to be approximately 150 per genome.
Using this number for the current control strain (NOY556), one can
calculate that the rrn9 N-PSW strain has approximately 80 copies and the rrn9 PSW strain has approximately 400 copies. Thus, the switch from the N-PSW to the PSW state is accompanied
by an approximately fivefold increase in rDNA copy number.
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FIG. 2.
Comparison of chromosomal rDNA copy numbers of NOY556
(WT), NOY703 (9,N-PSW), NOY878 (9,PSW), NOY408-1a (A135), and
NOY408-1b (WT). DNA from these five strains were digested with
HindIII and PstI, and the digests were
subjected to agarose gel electrophoresis, followed by transfer to a
nylon membrane and hybridization with a mixture of
32P-labeled rDNA probe and SIR3 probe. An
autoradiogram is shown with an inserted gap between lanes 3 and 4. Radioactivity in each DNA band was quantified with a PhosphorImager.
The values for chromosomal rDNA (and plasmid rDNA) were first
normalized to the values for reference SIR3 DNA. These
values obtained for NOY703 (lane 2) and NOY878 (lane 3) were then
divided by corresponding values for control strain NOY556 (lane 1), and
the ratios calculated are shown below the pertinent bands. Similarly,
the normalized values obtained for NOY408-1a (lane 4) were divided by
corresponding values for control strain NOY408-1b (lane 5), and the
ratios calculated are shown below the pertinent bands. It should be
noted that growth of both NOY703 (lane 2) and NOY408-1a (lane 4) is
achieved by transcription of GAL7-35S rDNA on helper
plasmids (NOY103 and NOY102, respectively), and copy numbers of the
helper plasmids were higher than those carried by the control strains
due almost certainly to selection.
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To examine whether the increase in rDNA copy number is due to the
formation of extrachromosomal rDNA circles or an increase in rDNA
copies associated with chromosome XII (i.e., repeat expansion), we
separated chromosomes of these strains by CHEF electrophoresis and
analyzed chromosome XII by Southern hybridization using a rDNA probe
and a control probe for a single-copy gene on the same chromosome,
SIR3 (Fig. 3A). The Pol I
deletion strain previously studied was again analyzed in parallel. As
was found previously (21), chromosome XII of the wild-type
strain showed a broad band indicating a heterogeneous population with
an average estimated size of 2.8 Mb (Fig. 3A, lane 1). This size
roughly corresponds to the sum of the calculated size of non-rDNA
sequence, 1.1 Mb, plus 150 copies of the 9.1-kb rDNA unit. The Pol I
deletion strain showed a heterogeneous size distribution ranging from
1.4 to 1.9 Mb, with an average estimated size of 1.8 Mb (Fig. 3, lane
5), which corresponds to the sum of 1.1-Mb non-rDNA plus 80 copies of
the 9.1-kb rDNA unit. The 9 N-PSW strain showed one or sometimes two
heterogeneous chromosome XII bands (Fig. 3A, lane 2; Fig. 3B, lanes 2 and 9) which showed a mobility similar to bands seen for the Pol I
deletion strain (Fig. 3A, lane 5).
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FIG. 3.
Correlation between the sizes of chromosome XII as
analyzed by CHEF electrophoresis and PSW/N-PSW phenotypes. (A)
Chromosomal DNA was isolated from strains NOY505 (lane 1) and NOY703
(lane 2), PSW strain NOY878 (lane 3), an N-PSW strain derived
spontaneously from NOY878 (lane 4), and strain NOY408-1a (lane 5). The
size of chromosome XII was then analyzed by CHEF electrophoresis. Size
markers (lane M) are Hansenula wingei chromosomes, and their
sizes are indicated in megabase pairs. Left, chromosome patterns
revealed by staining with ethidium bromide; middle and right,
autoradiograms obtained after hybridization with a SIR3
probe and an rDNA probe, respectively. (B) Chromosomal DNA was isolated
from the following strains and analyzed by CHEF electrophoresis as for
panel A: NOY505 (lane 1), NOY703 (lane 2), NOY877 (lane 3) (NOY703 and
NOY877 are parents [P] of diploids [D] shown in lanes 5 to 7), a
diploid obtained after the cross of N-PSW strain NOY876 and N-PSW
strain NOY769 (lane 4), three independent diploid clones obtained after
the cross of N-PSW strain NOY703 and PSW strain NOY877 (lanes 5 to 7),
a diploid strain obtained after the cross of two PSW strains NOY877 and
NOY878 (lane 8), and two haploid segregants (D H) from the cross of
N-PSW strain NOY703 and PSW strain NOY877, one showing the N-PSW
phenotype (lane 9) and the other showing the PSW phenotype (lane 10).
Autoradiograms obtained after hybridization with a SIR3
probe and an rDNA probe, respectively, are shown. It should be noted
that in panel B, a portion of the gel containing the initial sample
plugs corresponding to lanes 1 to 5 was inadvertently lost before the
gel was subjected to autoradiography. Therefore, radioactive signals in
sample wells representing chromosome XII, which was present in
incompletely digested cells or spheroplasts and failed to enter the
gel, are not seen in these lanes.
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The PSW strain contained a very large chromosome XII which entered into
the gel but showed a very slow mobility, as judged by hybridization
with rDNA and SIR3 probes as well as by ethidium bromide
staining of gels (Fig. 3A, lane 3; Fig. 3B, lanes 3 and 10). Repeating
many CHEF analyses of chromosomes as well as standard agarose gel
electrophoretic analysis of DNA, we found that most rDNA in
rrn9 PSW strains is associated with chromosome XII and that the fraction present as extrachromosomal circles is small and is
not different from that in the control wild-type strain (data not
shown). It should be noted that intensities of the signals seen with
the rDNA probe relative to the signals seen with the SIR3
probe are clearly different among these strains and support the
conclusion that rDNA repeat numbers decrease in the rrn9 N-PSW strain and increase in the rrn9 PSW strain.
The increase in rDNA repeat numbers seen for PSW strains is a
reversible change correlated with their ability to grow without Pol I. A N-PSW clone obtained from the rrn9 PSW strain (as
described above and shown in Fig. 1B, rows e and f) was analyzed for
the size of chromosome XII. As shown in Fig. 3A, the size(s) of
chromosome XII was decreased and the mobility of the heterogeneous band
was similar to that seen before switch to the PSW state (and comparable to the A135 strain) (Fig. 3A; compare lane 4 with lanes 2 and 5).
Two rDNA states with different rDNA repeat numbers are inherited
through meiosis.
We have previously shown that the PSW state can
be inherited not only through mitosis but also through meiosis
(39). In crosses between PSW and N-PSW strains, both PSW and
N-PSW haploid segregant clones were obtained after growth as diploids,
followed by sporulation and tetrad dissection. Although an exact 2:2
segregation pattern was not always observed because of an apparently
increased frequency of switching in the PSW/N-PSW diploid state and
perhaps during meiosis, a clear segregation of the phenotypes indicated their association with a chromosome, presumably chromosome XII (39). We have now examined the state of chromosome XII in
such diploids as well as haploid segregants in such a cross. As shown in Fig. 3B, independent diploid clones obtained from the cross showed a
copy of chromosome XII with expanded rDNA repeats and a copy with
decreased rDNA repeats (lanes 5 to 7). Such diploid clones, analyzed
after the cross without extensive colony purification, almost always
showed the PSW phenotype (39). Thus, the copy of chromosome
XII with the expanded rDNA repeats derived from the PSW parent strain
apparently functions as a dominant allele. The two chromosome XII
species with different rDNA repeats can coexist within the same diploid
nucleus and can be segregated into individual haploid spores through
meiosis, as confirmed by CHEF analysis of clones of haploid segregants;
segregant clones with the PSW phenotype showed a chromosome with
expanded rDNA repeats and those with the N-PSW phenotype showed a
chromosome with reduced rDNA repeats (Fig. 3B, lanes 9 and 10). Thus,
the expanded state of rDNA on chromosome XII is stable enough to be maintained through meiosis. Figure 3B also includes the analysis of two
control diploids, one obtained by a cross between two N-PSW strains and
the other obtained by a cross between two PSW strains. The former
showed the chromosome XII with decreased rDNA repeats (lane 4), and the
latter showed the chromosome with expanded rDNA repeats (lane 8). These
results give further support to the correlation between the expanded
rDNA state on chromosome XII and the PSW phenotype. Interestingly, the
shorter chromosome XII derived from the rrn9 N-PSW parent
in the PSW/N-PSW diploids showed a band with a size distribution more
homogeneous than that of the N-PSW haploid parent or the N-PSW diploid
(Fig. 3B; compare lanes 5 to 7 with lanes 2 and 4) or the N-PSW haploid
segregants (lane 9), and their sizes varied depending on the diploid
clone examined (lanes 5 to 7). A possible reason for this observation
is discussed below.
Mutation of FOB1 decreases the frequency of switching
from the N-PSW state to the PSW state.
The observed correlation of
expansion of rDNA repeats with the PSW phenotype suggests that rDNA
repeat expansion is necessary to attain the PSW state. Alternatively,
the expansion of rDNA could simply be a consequence of PSW. To
distinguish between these two alternatives, we examined the effects of
deletion of the FOB1 gene on the switching from N-PSW to PSW
states. The FOB1 gene was previously demonstrated to be
required for expansion/contraction of rDNA repeats (21). It
was found that strain NOY408-1a, which carries the rpa135
mutation (and helper plasmid pNOY102), showed a reduction in the number
of rDNA repeats to about 80 repeats on average (Fig. 3A, lane 5) and
that the introduction of the missing RPA135 gene induced a
gradual increase in repeat number back to the normal level, about 150. Derivatives of the rpa135 strain carrying a
fob1 deletion were constructed; these showed a reduced rDNA
repeat number with more homogeneous repeat number distribution. These
fob1 rpa135 strains did not show a significant increase in repeat numbers upon reintroduction of the RPA135
gene, demonstrating a requirement of FOB1 for rDNA expansion
(21). It should be noted that DNA replication fork blocking
aided by Fob1 protein at the site distal to the 35S rRNA coding region (20) appears to stimulate recombination, leading to a
stimulation of rDNA expansion/contraction. Thus, rDNA expansion and
contraction take place almost certainly during DNA replication and may
require many generations of cell growth to attain large changes in
repeat numbers.
We constructed fob1 deletion derivatives of
rrn9 N-PSW strain NOY703 and compared the frequency of
switching of these strains to the PSW state with that of the control
N-PSW strain NOY703. Many independent colonies which formed after
streaking these strains on galactose plates were analyzed by a spot
test. Examples of the results are shown in Fig.
4B. It was found that the control N-PSW
FOB1 strain formed PSW variants with a frequency ranging from 104 to 103. In contrast, the N-PSW
fob1 strain showed a frequency less than
105 in all the colonies analyzed, i.e., a large
reduction, by a factor of 10 to 100 or higher, in the frequency of the
switch. Thus, the fob1 mutation inhibits switching from
the N-PSW to the PSW state. We conclude that the expansion of rDNA
repeats is the cause of the switch to the PSW state and not a
consequence of the switch.
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FIG. 4.
Effect of a fob1 mutation on the frequency of
switch from the N-PSW to PSW states. (A) Chromosomal DNA was isolated
from the following strains and analyzed by CHEF electrophoresis: NOY505
(lane 1), NOY703 (lane 2), four independent fob1 deletion
isolates obtained from NOY703 by disruption of FOB1 (lanes 3 to 6), and a fob1 deletion isolate obtained from the control
strain NOY505 (lane 7). The left and right panels show autoradiograms
obtained after hybridization with a SIR3 probe and a rDNA
probe, respectively. (B) Three independent colonies of NOY703 (9,
N-PSW) and those of a fob1 mutant derived from NOY703
were analyzed by spot test on YEP-galactose (Gal) and YEPD (Glu).
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It should be noted that the rrn9 fob1 N-PSW strains
used in this experiment showed a more homogeneous chromosome XII band than the band for the parent FOB1 strain and that mobility
differed depending on the fob1 clones obtained after the
transformation used for their construction (Fig. 4A, lanes 3 to 6 compared with lane 2 or with lanes 2 of Fig. 3A and B). The rDNA
repeats appear to continue to expand and contract in rrn9
strains as well as in rpa135 and WT strains, showing a
heterogeneity in the size of chromosome XII, but such expansion and
contraction are greatly inhibited by deletion of FOB1
(21). The rrn9 fob1 N-PSW strain was
constructed from the rrn9 FOB1 N-PSW strain by a standard gene disruption method. Individual fob1 transformants may
have carried different rDNA repeat numbers at the time of
FOB1 deletion; upon depletion of the Fob1 protein during
growth on selective plates, rDNA expansion and contraction ceased, and
individual clones may have been left carrying different, yet relatively
homogeneous, repeat numbers.
We also note that the homogeneous and variously sized shorter
chromosome XII bands seen in PSW/N-PSW diploid clones (Fig. 3B, lanes 5 to 7) could be explained in a similar way. Fob1p was shown to be
present in the nucleolus (9). Perhaps it may be bound mostly
to rDNA repeats on chromosome XII. Fob1p that had allowed
expansion/contraction of the chromosome XII in the N-PSW nuclei may
have been sequestered by competition to the expanded chromosome XII
from the PSW strain after nuclear fusion in the cross, leading to
cessation of expansion and contraction of the N-PSW chromosome XIIs.
Effects of mutations in SIR genes and genes for Pol I
and CF subunits.
We have previously shown that in addition to UAF,
both Pol I and transcription factor CF are important in the maintenance of normal yeast nucleolar structures (27-29). In addition,
there are some proteins known to be involved in the maintenance of rDNA chromatin structure. For example, Sir2 protein is present in the yeast
nucleolus in addition to being present at telomere regions (11). Sir2p is known to be required for silencing of some
Pol II reporter genes inserted into rDNA (1, 10, 36) and for decreasing the rate of recombination within the rDNA repeats
(12). Therefore, we examined mutations in some of these
genes with respect to switching to the PSW state.
PSW strains can grow in the absence of Pol I (39) or CF (see
below). Yet analysis of individual colonies of NOY408-1a (which is
rpa135 and carries a helper plasmid) by the
galactose-glucose spot test did not reveal the appearance of cells able
to form colonies on glucose, i.e., no switching, while a control
rrn9 strain showed switching to PSW (Fig.
5A). By spreading more cells on glucose
plates, we failed to detect appearance of any PSW colonies from
NOY408-1a cultures grown in galactose medium (less than 1 in
107 cells [data not shown]). However, strains carrying
both the UAF mutation and the Pol I mutation and growing on galactose
with a helper plasmid (e.g., NOY896, which is rrn9
rpa135 pNOY103) can switch to the PSW state (Fig. 5A). Analysis
of many independent colonies by the spot test indicated that the
rrn9 rpa135 strain showed a switching frequency
similar to that for the control rrn9 strain. Thus, the
rpa135 mutation does not inhibit or significantly stimulate the switch from the N-PSW to PSW states and is unable to
cause the switch by itself.
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FIG. 5.
Efficiency of switching from the N-PSW to PSW states.
(A) Two independent colonies of strains carrying rrn9
(NOY703; N-PSW), rpa135 (NOY408-1a), and rrn9
rap135 (NOY896) were analyzed by spot test on YEP-galactose and
YEPD. (B) Two independent colonies of strains carrying
rrn6 (NOY566), rrn6 sir2 (NOY918), and
rrn6 rrn5 (NOY919) were analyzed by spot test on
YEP-galactose and YEPD. (C) N-PSW strains carrying rrn9
(NOY703), rrn9 sir2 (NOY901), rrn9
sir3 (NOY911), and rrn9 sir4 (NOY912) were
grown on a YEP-galactose plate. Three single colonies from each strain
were analyzed by spot test.
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Strain NOY566 carrying a deletion in one of the CF subunit genes
(rrn6::LEU2) and growing on galactose with the
helper plasmid pNOY103 also failed to produce cells able to grow on
glucose (Fig. 5B, upper samples). When this mutation was combined with
a UAF mutation (rrn5), switching to the PSW state was
observed (Fig. 5B, lower samples), and its efficiency was similar to
that observed for the control rrn5 N-PSW strain carrying
a helper plasmid (data not shown). Thus, the presence of intact UAF,
but not Pol I or CF, appears to be important for preventing switching
to the PSW state.
SIR3 and SIR4 are required for silencing at the
telomeres and silent mating loci as is SIR2 but are not
required for silencing of a Pol II reporter gene inserted into rDNA
(23, 36). We examined the effects of individual deletion of
SIR2, SIR3, and SIR4 genes on the
switch from N-PSW to PSW in the rrn9 background. N-PSW
rrn9 strain NOY703 and its sir2,
sir3, and sir4 deletion derivatives were grown on
YEP-galactose plates. All had similar growth rates. Twelve single
colonies from each strain were analyzed for the frequency of PSW
variants by the spot test. The frequency for the rrn9
sir2 colonies in this and other similar experiments ranged from
~104 to 101, whereas that for the control
NOY703 strain ranged from ~104 to 103
(Fig. 5C and other data not shown). The median value for the former was
clearly much (at least 10-fold) higher than for the latter. In
contrast, sir3 and sir4 did not show such
an increase (Fig. 5C; apparent negative effects observed with these
mutations were not studied further).
Although the sir2 mutation increased the frequency of
switching to the PSW state when combined with a UAF mutation, the
sir2 mutation itself was unable to allow switching to the
PSW state. This conclusion can be drawn from the fact that the
introduction of sir2 into NOY566 (rrn6
pNOY103) did not allow the strain to form PSW variants, whereas
introduction of a UAF mutation (rrn5) was able to do so
(Fig. 5B, middle compared to lower samples). Thus, disruption of
SIR2 stimulates switching to the PSW state, presumably by
stimulating the rate of expansion and contraction of rDNA repeats, but
does not by itself cause the switching.
Altered localization of the nucleolus in polymerase switched
strains.
We examined the structure of the nucleolus in PSW strains
by using EM and immunofluorescence microscopy (IFM). Since the presence of helper plasmids such as pNOY103, which allows transcription of the
GAL7-35S rDNA fusion gene by Pol II, may lead to formation of several mininucleoli (27, 28), thus complicating the
analysis, we examined PSW strains without such helper plasmids. Figure
6 shows electron micrographs of thin
sections of cells of a rrn9 rpa135 PSW strain (NOY794)
and cells of a wild-type strain (NOY505). The control cells showed
electron-dense nucleolar materials at or near the nuclear periphery,
forming the normal crescent-shaped nucleolus (Fig. 6A). In contrast,
the nucleolus of the rrn9 rpa135 PSW strain (without
any helper plasmid) showed a round nucleolus, which is distant from the
nuclear periphery. Quite often, the nucleolus in the PSW strain
revealed two different parts, one with higher and the other with lower
electron density. Although we have not studied the basis of the
presence of two subnucleolar regions, a similar feature as well as the
interior localization of the nucleolus was observed for strains with
chromosomal rDNA deletions complemented by the GAL7-35S rDNA
fusion gene on a multicopy plasmid (28). IFM using
antibodies against nucleolar protein Ssb1p (3) also
supported the interior localization of the nucleolus in PSW strains,
which was clearly different from the crescent structure along the
nuclear periphery seen in the control strains (data not shown).
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|
FIG. 6.
EM analysis of the nucleolus in control strain NOY505
(A) and PSW strain NOY794 (B). The strains were grown in YEPD at 25°C
to an A600 of about 0.5, and samples were
prepared for EM analysis. The nuclear envelope is marked with arrows to
serve as a point of reference, and the nucleolus (electron-dense areas
within the nucleus) is indicated as N. The vacuole is indicated as V. Bars, 1 µm.
|
|
Localization of expanded rDNA repeats in a rrn9 PSW
strain was then examined by FISH, and the results were consistent with the interior localization of the nucleolus in the PSW strain revealed by EM and IFM described above. As shown in Fig.
7, 4',6-diamidino-2-phenylindole (DAPI)
staining of PSW cell nuclei carried out under the conditions of FISH
revealed a "hole" with much reduced DNA staining, and rDNA was seen
surrounding this hole. For the control cells, this rDNA arrangement was
not observed and rDNA was seen as either a cap, a bar, or a collection
of dots mostly located at the nuclear periphery as reported previously
(13, 28). Although the conditions for sample preparation are
different for EM, IFM, and FISH, the holes surrounded by rDNA seen in
PSW cells by FISH may correspond to round nucleoli seen by EM and IFM.
We conclude that the sites of rDNA transcription as well as ribosome
assembly in PSW cells are different from those in normal yeast cells
and are at more interior locations compared with the normal site at the
nuclear periphery.
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FIG. 7.
FISH analysis of rDNA in PSW strain NOY852 and control
strain W303-1a. Yeast strains were analyzed for rDNA and DNA as
described in Materials and Methods. Images of rDNA and DNA were
pseudocolored green and red, respectively, giving overlapped regions
yellow in overlay. Individual images are shown in black and white. Note
that in the PSW strain, many of the DAPI-stained nuclei have a hole
with decreased DAPI staining and that rDNA appears to surround these
holes. In the control strain, such a hole surrounded by rDNA was rarely
seen.
|
|
Transcription of rDNA by Pol II in N-PSW rrn9
strains.
Cells which can grow reasonably well on glucose (i.e.,
are able to form colonies on glucose) are defined to be in the PSW state, whereas cells that cannot grow or can grow only extremely slowly
on glucose (no visible colonies after 7 to 10 days of incubation) are
defined to be in the N-PSW state (39). By defining the N-PSW state in this way, we originally assumed that transcription of rDNA by
Pol II takes place only in the PSW state and not in the N-PSW state.
Weak residual transcription observed in the UAF mutants under
conditions of repression of the GAL7 promoter was
interpreted to be due to the basal transcription of rDNA by Pol I, as
was observed in in vitro experiments (19, 39). We have now
examined this previous assumption and found that it is incorrect;
transcription of rDNA by Pol II actually takes place in
rrn9 strains even in the N-PSW state, though very weakly.
Using a primer extension analysis, we have previously shown that
transcription of rDNA by Pol II in established PSW strains starts at
several positions upstream from the start site (+1) seen for
transcription by Pol I, ranging from 9 to 95 and with a major site
at 29 (39). We used the same method and examined the
question of whether Pol II transcription of rDNA takes place in N-PSW
strains. In experiments shown in Fig. 8A,
fob1 derivatives of rrn9 N-PSW and
rrn9 PSW strains (NOY921 and NOY920, respectively) were
used to minimize the occurrence of switching to PSW in N-PSW cultures.
Control strains W303-1a and NOY408-1a were also analyzed. Cells were
grown in galactose medium and divided into two parts; one part was
shifted to glucose medium, and the other was kept in galactose medium.
One hour after the shift, cells were harvested and RNA was prepared.
Primer extension was then carried out to examine 5' ends of rRNA
precursors from these strains.
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FIG. 8.
Primer extension analysis of primary transcripts for
detection of rDNA transcription by Pol II. RNA was prepared from the
following strains growing on synthetic galactose medium supplemented
with Casamino Acids, tryptophan, and adenosine (lanes G) and 1 h
after shift from galactose to glucose synthetic medium with the same
supplements (lanes D): W303-1a (lanes 1, 2, 9, and 10), NOY408-1a
(lanes 3 and 4), NOY920 (lanes 5 and 6), NOY921 (lanes 7 and 8), NOY566
(lanes 11 and 12), NOY918 (lanes 13 and 14), and NOY919 (lanes 15 and
16). Primer extension reactions were done in parallel, but gel
electrophoresis and autoradiography were done in two separate groups
(shown in A and B) with the same WT samples included. Autoradiograms
(exposure times, 12 h [A] and 18 h [B]) are shown. Lane
8' is the same as lane 8 after a longer exposure, which was equivalent
to ~30 h. Positions indicated as +1, G, and P correspond to the start
site for the Pol I rDNA promoter, that for the GAL7
promoter, and a major site (29) among the 5' ends identified for rDNA
transcripts in PSW strains, respectively. Three independent experiments
were carried out, and the amounts of Pol II-specific precursor rRNAs
found in NOY921 in glucose (lane 8 or 8') were 15.4% ± 2.0% of those
in control PSW strain NOY920 (lane 6).
|
|
We found, contrary to the original assumption, that N-PSW strains
showed the presence of transcripts, which corresponded to the
transcripts made by Pol II in the control PSW strain under the
condition of repression of GAL7-35S rDNA on the helper
plasmid (Fig. 8, lanes 8' and 6).The amounts of those Pol II-specific precursor rRNAs were simply much lower than that found in the control
PSW strain (approximately 15% of the control PSW strain level; see the
legend to Fig. 8). No transcript with the Pol I start site (+1) was
detected. It should be noted that Pol I deletion (rpa135)
and CF deletion (rrn6) strains did not show any
transcripts unique to the PSW strains; only faint transcripts which
corresponded to the one with the GAL7 promoter start site as
the 5' end were observed (Fig. 8; compare lanes 4 and 12 with lanes 3 and 11, respectively). Figure 8 also includes the results obtained for the rrn6 sir2 strain and the rrn6
rrn5 N-PSW strain used in the experiments shown in Fig. 5. The
former did not show any PSW-specific transcripts (lane 14), as was the
case with the rrn6 strain, whereas the latter showed
PSW-specific transcripts (lane 16), as did the rrn9 N-PSW
strain. The difference between the two strains is correlated with the
difference in their abilities to switch to the PSW state shown in Fig.
5B. It appears that inactivation of UAF by the rrn9
deletion (or the rrn5 deletion) is sufficient to allow
Pol II to transcribe chromosomal rDNA, even though this transcription
is apparently not enough to allow cells to grow without helper plasmid,
and the expansion of rDNA repeats is required for improved cell growth.
| |
DISCUSSION |
Model for RNA polymerase switch.
The results presented in this
report demonstrate that switching to the PSW state, i.e., the state
that allows growth using the Pol II system, involves two steps: a
mutation in UAF and an expansion of rDNA repeats. Table
2 summarizes information obtained on the
WT, N-PSW, and PSW states. We found that some mutational alterations of
UAF are sufficient to allow Pol II transcription of rDNA, although
transcription is weak (~15% of the control PSW strain) and is
apparently not sufficient to allow cellular growth. After repression of
the GAL7 promoter by glucose, N-PSW UAF mutants can continue
to grow and divide at least for several generations by using
preexistent ribosomes, and possibly also some new ribosomes, that would
continue to be synthesized by the weak rDNA transcription by Pol II.
This residual growth may be sufficient to allow UAF mutants to continue
FOB1-dependent expansion and contraction of rDNA repeats at
least for a while so that a small fraction of cells (103
to 104) can form colonies, enabling some of them to
establish a fully competent PSW state.
UAF is unique in playing an essential role in silencing Pol II
transcription of rDNA. Mutations in other genes, those for subunits of
CF or Pol I or Sir2p, a known component of rDNA chromatin, do not allow
Pol II transcription. The simplest possible mechanism for silencing is
that UAF binds to the upstream element of the rDNA promoter
(19) and by itself or in combination with other interacting
proteins forms a structure that inhibits Pol II transcription. According to this model, UAF may be a crucial component of
rDNA-specific chromatin and is probably bound to the promoter region
regardless of the state of Pol I activity. The second step, a
FOB1-dependent expansion of rDNA repeats, is slow and
involves intermediate states, presumably reflecting states of
chromosome XII with somewhat increased, but not yet fully expanded,
rDNA repeats. Cells in such intermediate states may be able to grow
somewhat more efficiently than the original N-PSW cells, leading to
formation of tiny colonies, but may lose the increased rDNA repeats due
to an instability of the expanded states, returning frequently back to
the N-PSW state, as the experiments shown in Fig. 1 indicate. We
suggest that only fully or nearly fully expanded rDNA repeats (about
400) can establish a relatively stable structure(s), i.e., a Pol
II-specific nucleolar structure, thus preventing further increase in
rDNA repeats or frequent loss of the increased repeats.
It should be noted that switch from the rrn9 N-PSW to
rrn9 PSW states is clearly an adaptation process which
takes place on glucose plates. Fully established PSW cells do not exist
in N-PSW cultures grown in galactose by transcribing the artificial fusion gene on a helper plasmid.
Deletion of SIR2 was shown to increase the efficiency of
switching, though it does not cause switching to the PSW state by itself; i.e., it stimulates the second step without causing any change
equivalent to the first step. Since sir2 mutations are known
to increase the frequency of recombination within rDNA repeats (12), stimulation of FOB1-dependent repeat
expansion can be explained on this basis; sir2 mutations are
expected to increase the rate of both expansion and contraction and
consequently increase the frequency of rDNA repeats with sufficiently
high numbers (about 400) to form a reasonably stable Pol II-specific
nucleolar structure and establish the PSW state.
Significance of the requirement of rDNA repeat expansion for
establishment of the PSW state.
There are two different models to
explain the requirement of rDNA repeat expansion for establishing the
PSW state. The first model assumes that in rrn9 N-PSW
strains, all of the rDNA repeats are accessible to Pol II and
transcribed in a productive way leading to ribosome formation and that
a higher gene dosage is required simply to increase the overall rate of
rDNA transcription to meet the need for growth. In this case, one has
to explain the formation of a distinct nucleolar structure in PSW
cells, which is localized at an interior site rather than at the
original site at the nuclear periphery. Perhaps UAF is a key element
for retaining rDNA at the nuclear periphery and rrn9
cells in the N-PSW state have rDNA (and the Pol II-specific nucleolus)
at an interior site. Expansion of rDNA repeats may simply increase the
rRNA synthesis rate without changing the location of the nucleolus.
Unfortunately, it has been difficult, for technical reasons, to study
the nucleolus in rrn9 N-PSW cells which do not carry a
helper plasmid, and thus, we have no information on this question
(Table 2).
The second model assumes that only a fraction of the rDNA repeats is
accessible to the Pol II machinery or is transcribed productively to
lead to ribosome formation. Perhaps the Pol II machinery is not freely
diffusible in the yeast nucleus, as suggested by previous studies for
higher eukaryotic cells (4, 14). In addition, mobility of
rDNA repeats on chromosome XII in interphase may also be limited within
certain "chromosome territories" (25), thus making
formation of the nucleolus at an interior site(s) difficult. According
to this model, DNA repeat expansion is required to form a nucleolar
structure at a suitable site in addition to (or rather than) a simple
increase of repeat numbers to increase transcription rate by a high
gene dosage. Such a Pol II-specific nucleolar structure may be required
for more efficient rDNA transcription by Pol II and/or subsequent
steps, rRNA processing, rRNA modification, and ribosome assembly. In
connection with the second model, it should be noted that transcription
of some Pol II reporter genes integrated into rDNA repeats is known to
take place. However, it has not been demonstrated that these reporter
genes integrated into any of the repeats can be transcribed by Pol II.
Clearly, further experiments are required to settle these issues and
distinguish between the two (and other possible) models.
The presence of a round nucleolus localized at an interior site(s) in
PSW strains is consistent with the results of our previous studies
using yeast mutants with chromosomal rDNA completely deleted (28). Such a mutant, when complemented by a plasmid carrying a single rDNA repeat transcribed by Pol I, contained many mininucleoli preferentially localized at the nuclear periphery. In contrast, the
same mutant, when complemented by a plasmid carrying the
GAL7-35S rDNA fusion gene transcribed by Pol II and growing
on galactose, contained a rounded nucleolus that lacked extensive
contact with the nuclear envelope and resembled that observed in the
PSW strains studied in this work. These observations indicate the
presence of separate nuclear subregions, one favorable for rDNA
transcription by Pol I followed by ribosome assembly and the other
favorable for rRNA synthesis by Pol II (using the fusion gene) followed by ribosome assembly.
Relation to rDNA silencing of Pol II reporter genes.
Silencing
of certain Pol II reporter genes inserted into rDNA has been reported
(1, 10, 36). One feature of this silencing system is the
requirement of SIR2 but not SIR3 and
SIR4. Similarly, SIR2, but not SIR3 or
SIR4, was shown to play a role in decreasing the rate of
recombination within rDNA repeats (12). In the present system, the second step in switching to the PSW state is stimulated by
a sir2 mutation but not by a sir3 or
sir4 mutation. Thus, a rDNA chromatin structure containing
Sir2p postulated to be important for rDNA silencing of reporter genes
or inhibition of recombination appears to play a role in decreasing
switching to the PSW state by decreasing the rate of expansion and
contraction of rDNA.
It is clear that the roles of SIR2 and UAF in silencing of
Pol II transcription of rDNA are fundamentally different. In contrast to UAF mutations, sir2 deletion itself does not allow rDNA
expansion to the PSW state (or rDNA transcription by Pol II without
expansion). It appears that UAF, presumably together with other
unidentified components, prevents Pol II transcription of rDNA
directly, and Sir2p is not involved in this repressive chromatin
structure, which is almost certainly located at rDNA promoter regions.
In this connection, it should be noted that in in vitro experiments both Pol I and CF, together with TBP and Rrn3p, join the UAF-promoter complex to form a preinitiation complex (18, 19). In
addition, both Pol I and CF are known to be important, like UAF, for
the maintenance of intact nucleolar structures (28), yet
neither Pol I subunit deletion nor CF deletion allows rDNA expansion to the PSW state. Perhaps the weak rDNA transcription by Pol II, which was
observed in UAF deletion but not Pol I subunit deletion or CF subunit
deletion mutants, is important, allowing residual growth and selective
pressure to continue, leading to an eventual establishment of the PSW state.
Significance of large tandem repeat numbers of rDNA genes.
In
most eukaryotes, rDNA genes are tandemly repeated at one or a few
chromosomal loci, the nucleolar organizers. The repeat number at a
locus is generally large but is highly variable among organisms,
ranging from less than 100 to over 10,000 per haploid genome. The
repeat numbers often vary significantly not only among related species
but also among different strains of the same species (reviewed in
reference 22; for variations within S. cerevisiae, see references 2 and
30). It was often assumed that the presence of large
numbers of rDNA genes reflects a demand for high rates of rRNA
synthesis to meet cellular growth needs. However, the large variations
of gene number among closely related organisms are difficult to explain
on this basis. In addition, a given organism does not appear to require
the presence of all rDNA gene copies for normal growth rates. We have
previously described the construction of a yeast strain which carries
only 25% (i.e., ca. 40 repeats) of the normal rDNA repeats
(21). This strain was constructed by first deleting
RPA135 in the presence of a helper plasmid, which caused
reduction of the rDNA repeat numbers followed by deletion of
FOB1, preventing expansion and contraction of the repeats,
and finally by reintroducing the missing RPA135 gene. This
yeast strain and a control yeast strain with ~150 rDNA repeats showed
identical growth rates (21) and rRNA synthesis rates (16). Thus, it is clear that normal yeast cells use only a
fraction of the ~150 rDNA repeats to synthesize rRNA by Pol I, even
under conditions of near maximum growth rates. Previous analyses of rDNA chromatin structure using psoralen cross-linking also showed that
only a fraction of the rDNA copies is transcribed in actively growing
cells in a variety of systems including yeast (5, 8). As we
discussed above in connection with the requirement of rDNA repeat
expansion for the formation of a new Pol II-specific nucleolar structure, extra rDNA repeats might be present simply to form suitable
nucleolar structures rather than to function as template for rRNA
synthesis. Perhaps the number of rDNA repeats unique to each organism
reflects the presence of particular nucleolar structures unique to
these organisms (and environmental or developmental conditions).
We consider rDNA expansion and the alteration of nucleolar structures
in PSW strains as an extreme example of a general plasticity of the
nucleolar structure. The FOB1-dependent expansion and
contraction of rDNA repeats might be used for changing nucleolar
structure in response to environmental changes, in addition to the
well-discussed role in the maintenance of sequence homogeneity
throughout many rDNA repeats. In this regard, we note that there have
been studies reporting heritable changes in rDNA copy numbers in flux
induced by specific environmental changes (6, 7). For
S. cerevisiae, it was reported that cells grown at the
optimal temperature of 30°C led to an increase in rDNA repeat numbers
relative to cells grown at 22°C (32). The plasticity of
rDNA repeat numbers as well as nucleolar structures may be advantageous
to organisms. However, nucleolar structures are complex. In addition,
the nucleolus may play functional roles other than synthesizing
ribosomes (31, 33, 34, 38). Thus, clear understanding of
this subject must await further studies of nucleolar structure and functions.
There are three features of rDNA transcription in most eukaryotes that
distinguish it from rRNA synthesis in eubacteria or archaea: (i) the
use of a distinct RNA polymerase, Pol I, (ii) the presence of tandemly
repeated rRNA genes (with exceptions of some lower eukaryotes which
carry many copies of rDNA plasmids or minichromosomes), and (iii) the
presence of the nucleolus as the site of transcription, rRNA
processing, and ribosome assembly. Separation of the site of rDNA
transcription, using a unique polymerase presumably for the purpose of
efficiency and regulation, may have had selective advantages for
eukaryotic organisms, and the evolution of the three features of rDNA
transcription might have been interrelated. The RNA polymerase switch
induced by mutations in UAF affects all of these three features. Thus,
further studies on this system may provide some insight into not only
the functional significance of these features in general cell biology
but also the question of their evolution in eukaryotes.
M.O. and I.S. contributed equally to this work.
This work was supported by Public Health Service grants GM35949 (M. Nomura) and GM48586 (John Aris).
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Abstract
Introduction
