Vol 13, Issue 3, 399-406, March 2003
LETTER
Schizosaccharomyces pombe Essential Genes: A Pilot Study
Anabelle Decottignies1,3,4,
Isabel Sanchez-Perez2 and
Paul Nurse1,4
1Cell Cycle Laboratory, Cancer Research UK, London, WC2A
3PX, UK; 2Instituto de Investigaciones Biomedicas CSIC, c/
Arturo Duperier, 4, 28029 Madrid, Spain
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ABSTRACT
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After completion of the Schizosaccharomyces pombe genome
sequence, we have carried out a pilot gene deletion project to assess
the feasibility of a genome-wide deletion project and to estimate the
percentage of essential genes. Using a PCR-based gene deletion
procedure, we investigated 100 genes within a 253-kb region of
chromosome II. Eight of nine genes located within a region of 18 kb
could not be deleted, suggesting that systematic deletion of all
fission yeast genes may be difficult to achieve using this PCR
approach. The percentage of essential genes was found to be 17.5%.
Further deletion of selected S. pombe genes revealed that
whether a gene is essential or not is correlated with the timing of its
appearance on the tree of life and its conservation within all branches
of the tree. None of the investigated ancient genes in fission yeast
that have been lost in the Saccharomyces cerevisiae lineage
are essential. In agreement with S. cerevisiae and
Caenorhabditis elegans genome analyses, our data suggest that
natural selection has preferentially kept the genes required for vital
functions. We propose that many of the essential eukaryotic genes
appeared with the first eukaryotic cell and have remained conserved in
all species.
The fission yeast Schizosaccharomyces
pombe was the sixth eukaryote to be sequenced (Wood et al. 2002 ),
following the budding yeast (Goffeau et al. 1996) and four
multicellular organisms (C. elegans [The C. elegans
Sequencing Consortium 1998], Drosophila. melanogaster [Adams
et al. 2000], Arabidopsis thaliana [The Arabidopsis Genome
Initiative 2000], and Homo sapiens [International Human
Genome Sequencing Consortium 2001; Venter et al. 2001]). S.
pombe is predicted to have a maximum of 4940 protein-coding genes,
the smallest number of open reading frames (ORFs) in a eukaryote to
date (Wood et al. 2002). In comparison, there are 5300 to 5400 ORFs
predicted for the budding yeast Saccharomyces cerevisiae
(Mackiewicz et al. 2002). The genomes of multicellular organisms
contain more ORFs, with  15,000 for worm and fly and at least twice
as many for human and Arabidopsis. Up to 100 genomes of both
eubacteria and archaebacteria are now also publicly available at
http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html.
The plethora of ORF sequences from organisms located in various
branches of the tree of life has allowed the development of new
organism classification systems and the elaboration of novel genomic
trees (Tekaia et al. 1999; Korbel et al. 2002). Satisfyingly, these
trees are similar to the more traditional analyses based on molecular
phylogeny (Tekaia et al. 1999; Korbel et al. 2002). Paleontological
work suggests that the first prokaryotic cell may have arisen 3800
million years ago, whereas the acquisition of a closed nucleus may have
occurred 2000 million years ago (Feng et al. 1997). It has been
estimated that fungi separated from metazoa and plants 1000–1200
million years ago, and that fission yeast diverged from budding yeast
400 million years ago (Sipiczki 2000), although older time estimates
have been proposed for divergence of these yeasts by Heckman et al.
(2001).
The availability of a variety of genome sequences provides a powerful
tool to follow the history of a protein or protein family. Comparisons
of the S. pombe, S. cerevisiae, and C.
elegans gene sets led Wood et al. (2002) to conclude that 14% of
the S. pombe ORFs are found exclusively in that yeast and
therefore, are absent from S.cerevisiae, whereas 3% of the
S. pombe ORFs have homologs in C. elegans, which
appear to have disappeared from the S. cerevisiae lineage.
Therefore, it appears that both acquisition and loss of genes have
occurred since the divergence of S. pombe and S.
cerevisiae from their common ancestor (Aravind et al. 2000). In
this paper, we carry out a pilot gene deletion project in S.
pombe using a PCR-based procedure (Bähler et al. 1998), to assess
the feasibility of a genome-wide project and to determine the
percentage of essential genes in S. pombe. Included in this
analysis are genes that have been gained recently in the S.
pombe and S. cerevisiae lineages, ancient genes that have
been lost in the budding yeast lineage, and genes that have remained
conserved throughout evolution.
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RESULTS
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The availability of the S. pombe genome sequence and
methods for PCR-mediated deletion of ORFs (Bähler et al. 1998) has
allowed us to carry out a pilot gene deletion project to assess the
number of essential S. pombe genes and to address several
evolutionary questions. The first step of the work was the
classification of fission yeast ORFs based on phylogenetic criteria
(Fig. 1). We performed BlastP analysis
(Altschul and Lipman 1990) on 450 S. pombe proteins
out of the 4929 predicted by PombePD (see Wood et al. 2002). These
proteins were encoded by three sets of 150 successive protein-encoding
genes located on each of the three chromosomes. We searched for the
presence of homologs in prokaryotes, S. cerevisiae, metazoa,
and plants using a threshold value for E of 10–5,
and classified the fission yeast proteins into eight different classes
according to the distribution of homologs within these organisms (Ia,
Ib, Ic, Id, II, III, IV, and V) (classes defined in legend of Fig. 1).
A similar analysis was performed on 450 predicted proteins of
S. cerevisiae on chromosomes I, IV, and VIII (Fig. 1).
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Figure 1. Comparison of S. pombe and S. cerevisiae proteome.
(A) A consensus phylogeny of fission yeast and budding yeast
adapted from Sipiczki (2000). (B) 450 proteins from S.
pombe and S. cerevisiae (YPD and PombePD) were compared to
proteins from prokaryotes, metazoa, plants, S. pombe, and
S. cerevisiae using BLASTP (Altschul and Lipman 1990) with a
cutoff E value of 10–5. The proteins from both
yeasts were classified into eight different classes, according to the
distribution of homologous proteins in other species. Class I:
homologous proteins are found in both S. pombe and S.
cerevisiae and in either metazoa + plants + prokaryotes (Ia),
or metazoa + plants with no homolog in prokaryotes (Ib), or
metazoa + prokaryotes (Ic), or only metazoa (Id). Class II:
homologous proteins are found in both yeasts and in plants or
prokaryotes, but there is no homolog in metazoa. Class III: homologous
proteins are present in both S. cerevisiae and S.
pombe, but there is no homolog outside the fungal branch. Class IV:
homologous proteins of S. pombe are not found in S.
cerevisae and S. cerevisiae proteins do not have an
homolog in S. pombe, but homologous proteins of both yeasts
are found at least in the metazoa branch. Class V: there is no
homologous protein of one yeast in the other yeast, and no homolog in
other branches. Numbers of genes in each category are shown for both
yeasts. Percentages are given in the table. The third column gives the
gene distribution in the 253-kb region that we selected for systematic
gene deletion in this study.
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The distribution of proteins in the eight classes is very similar in
the two yeasts. In both organisms, 28% of the total proteins have
homologs in prokaroytes and eukaryotes (S. cerevisiae, plants,
and metazoa) (class Ia, Fig. 1), whereas 21.5%–24 % are specific to
eukaryotes with no homologous protein being detected in prokaryotes
(class Ib, Fig. 1). About 10% of the proteins do not have an homolog
within the metazoa but do have an homolog in other eukaryotes and
prokaryotes (classes II and III, Fig. 1). The class IV proteins share
homology with proteins from other species (with at least an homolog in
the metazoa branch) but do not have homologs in the other yeast; this
class accounts for 4.4% of the S. pombe proteins and 3.5% of
the S. cerevisiae proteins. The only significant difference
between the two yeasts is found in class V, which consists of proteins
specific to either S. pombe or S. cerevisiae. This
group comprises 19% of total fission yeast proteins and up to 28% of
total budding yeast proteins (class V, Fig. 1). The budding yeast class
V proteins may be too high by 10% because of overestimation of the
total number of proteins in S. cerevisiae, especially those
that are not conserved (Mackiewicz et al. 1999, 2002; Blandin et al.
2000; Wood et al. 2001).
Fission Yeast Genes Essential for Vegetative Growth
To estimate the percentage of S. pombe genes required for
vegetative growth, we screened a contiguous region of chromosome II
containing 100 ORFs. The 253-kb region, selected at random, is likely
to be appropriate to estimate the percentage of essential genes. First,
according to phylogenetic criteria, the gene distribution within this
region containing 100 genes is similar to our estimated distribution
for the S. pombe genome (Fig. 1B). Second, assuming that
fission yeast genes play a similar role than their budding yeast
homologs, the genes appear to be organized randomly relative to their
function, with the exception of four genes encoding putative ribosomal
proteins (for an estimated total of 55 on the entire genome).
Interestingly, cytoplasmic ribosomal subunit-encoding genes also appear
to cluster in S. cerevisiae (CYGD,
http://mips.gsf.de/proj/yeast/CYGD/db/index.html).
We designed 85 pairs of primers for targeted PCR-based gene deletion
(Bähler et al. 1998) because deletion of the other 15 genes had
already been published (Table
1). After transformation of
a fission yeast diploid strain with the PCR-amplified deletion
cassette, geneticin-resistant clones were selected and the gene
deletion was checked by colony PCR (see Methods). Diploids were
sporulated and four-spored asci dissected on rich medium. When tetrads
did not contain spores that could form geneticin-resistant colonies,
the gene deletion was classified as giving a lethal phenotype (Table
1). We deleted 65 of the 85 genes by this procedure. Of the 20
remaining genes, the deletion cassette could not be PCR amplified for
3, and correctly deleted geneticin-resistant colonies could not be
obtained for the other 17, despite repeating the deletion procedure up
to six times. Eight of these undeletable 17 genes were located within a
string of 9 contiguous genes that lie between SPBC106.10 and
SPBC106.20 ORFs (Table 1), suggesting that this whole
chromosomal segment of 18 kb was refractory to gene deletion. We
measured the recombination frequency between two markers (cut
4 and cdc13) flanking this segment to check whether the
region was a cold spot for recombination. Analysis of random spores
generated by a cross between cut4-533 and cdc13-117
indicated that the genetic distance between the two markers was 5 cM,
compatible with their physical separation of 30 kb, suggesting that
this region is not a recombination cold spot. The efficiency with which
genes were correctly deleted varied from 5%–100% with an average of
51% based on 650 geneticin-resistant clones analyzed. Including the
genes for which the deletion phenotype had already been published, 14
of the 80 genes analyzed were essential for fission yeast vegetative
growth (Table 1). This suggests that the percentage of essential genes
in S. pombe is 17.5%, compared with 17.8% in budding yeast
(YPD; see Garrels 2002), with an interval of confidence (P90)
for S. pombe essential genes of 9.5%–25.5%.
Nine of the 14 S. pombe essential genes of Table 1 have been
previously described and can be classified into the functional
categories of genes described by MIPS (CYGD,
http://mips.gsf.de/proj/yeast/CYGD/db/index.html). For the other five
fission yeast essential genes, a putative function can be assigned by
homology with S. cerevisae. Classification into functional
categories reveals that among the 14 S. pombe essential genes
of Table 1, 6 belong to the so-called Protein Fate functional category
that includes genes involved in protein folding, modification, and
targeting. They include stt3, SPBC106.06, cut4, SPBC582.07c
SPBC1685.03, and sec61, with cut4, and possibly
SPBC106.06 and SPBC582.07c, being also required for
completion of mitosis. Another four genes (cdc13, mob1, alp6,
cut12) are essential for mitosis. SPBC582.11c is likely to
encode the fission yeast homolog of Nup84p nucleoporin; cdt1
is required for DNA replication initiation (Nishitani et al.
2000); rhb1 encodes a Rheb-related GTPase that putatively
regulates alternative responses to limiting nutrients (Mach et al.
2000); and SPBC1271.13 probably encodes a ribosomal protein.
Essential Genes: Comparison Between S. pombe and S. cerevisiae
We then tested whether deletion of homologous genes in the two
yeasts showed the same deletion phenotype (whether essential or not),
choosing the closest S. cerevisiae homolog of each of the
S. pombe genes from Table 1 and Figure
2B (see below). Among the 81 S.
pombe genes with an homolog in budding yeast, 88% (71 genes) show
the same deletion phenotype in both yeasts, 6% (5 genes) are essential
for S. cerevisiae but not for S. pombe, and 6% (5
genes) are essential for S. pombe but not S.
cerevisiae (Table 2). This means that
of the 15 fission yeast essential genes included in our study, only 10
(67%) are also essential for budding yeast growth. This number is
similar to that calculated from previously published data (PombePD),
which reveals that 135 of the 198 (68%) S. pombe essential
genes with S. cerevisiae homologs are also essential for
S. cerevisiae. These data indicate that, although the absolute
percentage of essential genes is similar between S. cerevisiae
and S. pombe, surprisingly only two-thirds of the essential
genes in one yeast have essential homologous genes in the other yeast.
One hypothesis would be that, in budding yeast, the genes have been
duplicated to compensate for essential gene loss. However, analysis of
Table 1 genes reveals that it is unlikely in fission yeast. The
SPBC1271.13 and SPBC582.11c ORFs, which are required
for S. pombe growth but do not have essential homologous genes
in budding yeast, have not been duplicated in S. cerevisiae.
Similarly, the YGR147c, EXO70, and YMR093w
ORFs of S. cerevisiae, which do not have essential homologs in
fission yeast, do not belong to gene families in S. pombe. On
the other hand, CCT4, which is required for growth of S.
cerevisiae and S. pombe, belongs to gene families in both
yeasts. Moreover, most of the paralogous genes of budding yeast
CCL4 are also essential, suggesting the existence of essential
gene families. Other gene families, like the MFS superfamily of
permeases, which includes the nonessential SPBC1271.10c ORF
(Table 1), are very unlikely to comprise a high percentage of essential
genes, as revealed by S. cerevisiae studies (for review, see
Sa-Correia and Tenreiro 2002).
Age of S. pombe Genes and Whether They Are Essential
We then tested whether a gene was essential and correlated it with
the time of appearance of a gene on the life tree. Sipiczki (2000) has
proposed a consensus tree for eukaryotes based on molecular phylogeny
of both 18s rRNA and HMG-CoA reductase sequences. If we refer to this
tree shown in Figure 2A, we can postulate that our gene classes
appeared in the following order: Ib > Id > III > V. To
estimate the percentage of essential genes in each of these classes, we
deleted another 24 genes from both class Id and class III (Fig. 2B).
Together with the data from Table 1, we estimate that lethality in the
classes is as follows: Ib, 25% (6/24); Id, 18% (3/17); III, 7%
(1/15), and V, 5% (1/19). From these data, if we consider only the
fission yeast genes that do not have homologs within the prokaryotic
branch, the more ancient the gene is, the more likely it is to be
essential (Fig. 2C). Data from the S. cerevisiae genome (YPD,
see Garrels 2002) give a similar profile (Fig. 2C). Focusing on the
genes with homologs in both eukaryotic and prokaryotic cells (Table 1,
class Ia), we find that 22.5% (4/18) are essential for S.
pombe. Data in S. cerevisiae are similar as we estimate
that 26% of class Ia genes are required for budding yeast growth.
Comparing this data with the average of 17.5% of essential genes for
the whole genome, we conclude that ancient genes maintained in all
eukaryotic species or in both eukaryotic and prokaryotic species, are
more likely to be essential. In contrast, yeast-specific genes (class
V), which have appeared recently, are less likely to be essential.
We then focused on S. pombe class IV genes, which have an
homolog in the metazoa branch but do not have an homolog in the S.
cerevisae lineage, deleting another 36 genes within this class
(Table 3). Of these 40 genes, only 1
(cdt1) was found to be essential (Hofmann and Beach 1994).
However, in this case, a functionally equivalent gene to cdt1
has been reported in S. cerevisiae, which has very low
sequence similarity (Tanaka and Diffley 2002). This may be a highly
diverged gene derived from a common ancestor or may be an example of
nonorthologous gene replacement (Koonin et al. 1996), when a gene is
functionally replaced by another that is unrelated by descent. The two
genes may, however, share limited sequence similarity acquired by
convergent evolution. Assuming that class IV genes are more likely to
be the result of gene loss instead of lateral transfer from
plants/animals to S. pombe, we conclude that a fission
yeast gene for which the homolog has been lost in the budding yeast
lineage is very unlikely to be essential, although its origin may
be ancient.
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DISCUSSION
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This pilot work has shown that a systematic deletion of all S.
pombe ORFs using a PCR-based gene deletion procedure may be
difficult to achieve compared with the equivalent work in S.
cerevisiae because the efficiency of homologous recombination is
lower in S. pombe than in S. cerevisiae (Kaur et al.
1997). Bähler et al. (1998) have shown that the use of longer
flanking sequences (60–80 bp instead of the 40 bp used for budding
yeast) increases the efficiency of homologous integration in the
PCR-based procedure. However, increasing the length of flanking
sequences up to 80 bp as used in our study was not sufficient to delete
all target genes. We identified one region of 18 kb in length on
chromosome II containing 9 genes, within which we were unable to delete
8 of the genes. Meiotic recombination frequency was normal between two
markers flanking this region, indicating that it is not a cold spot
region for meiotic recombination. One possibility is that the chromatin
structure may be different and gene transcription low in this region.
Because S. pombe transcription factors have been shown to
alter local chromatin structure and to activate meiotic recombination
hotspots (for review, see Davis and Smith 2001), we speculate that some
regions of the genome may contain poorly transcribed genes with
"closed" chromatin structure, resulting in a low efficiency of
targeted gene deletion. Alternatively, integration of the deletion
cassette may have occurred but, because of the silent chromatin, the
kanamycin resistance gene was insufficiently expressed, reminiscent of
transcriptional silencing observed at mating-type, telomeric and
centromeric regions of S. pombe chromosomes (for review, see
Huang 2002).
From our data, we calculate that the percentage of essential genes in
fission yeast growing on a rich medium is 17.5%, similar to the 17.8%
of genes that are essential for S. cerevisiae growth on rich
medium. However, taking into account our failure to delete six genes
with essential homologs in budding yeast, the percentage of essential
genes in fission yeast may be higher (between 18% and 20%). The
probability of an unknown gene being essential for S. pombe is
dependent on whether homologs are found within other branches of the
life tree. We estimate that 27% of the proteins conserved in
prokaryotes, mammals, plants, and S. cerevisiae (our class
Ia), and 27% of the proteins conserved in mammals, plants, and S.
cerevisiae, but not found in prokaryotes (class Ib), are essential
in S. pombe. If we calculate the absolute number of essential
genes in each of these two classes of genes, we estimate that class Ia
and Ib genes account, respectively, for 43% and 38% of the total
number of essential S. pombe genes (Fig.
3). This means that 80% of the S.
pombe essential genes are found in highly conserved gene classes
(Ia and Ib), although they account for only 50% of the total number of
protein-encoding genes. A lower fraction of fungal-specific proteins
(III and V) is essential for S. pombe growth (Fig. 3); these
account for 10% of the essential genes, although they form 26% of the
total number of protein-encoding genes (Fig. 3).
Analysis of data contained in the YPD database (see Garrels 2002)
revealed similar conclusions for budding yeast. Classes Ia and Ib
contain 82% of S. cerevisiae essential genes, whereas
fungal-specific proteins only comprise 12% of the essential proteins
(Fig. 3). Using RNA-mediated interference in C. elegans,
Gönczy et al. (2000) have shown that the essential genes in this
nematode worm are mainly those conserved in other organisms. They found
that genes conserved in both eukaryotes and prokaryotes (our class Ia)
account for 40% of the total essential genes in C. elegans
embryos, whereas genes conserved in eukaryotes but not prokaryotes (our
class Ib) account for 45%. Nematode-specific genes form only 7.5% of
the essential genes, although they account for 40% of the total genome
(Gönczy et al. 2000). Therefore, a general consensus emerges
suggesting that genes essential for eukaryotic cells are mainly found
in gene classes that have homologs within all the eukaryotic branches.
One interpretation of this data is that essential genes evolve more
slowly as proposed by the adaptive theory of mutation rates, which
argues that essential protein-encoding genes evolve at lower rates than
nonessential ones. However, Hurst and Smith (1999) have shown that, in
rat and mouse, mutation rate is not correlated with the severity of the
knockout phenotype, suggesting that, for these organisms, essential and
nonessential genes evolve at the same rate. Moreover, Jordan et al.
(2001) have shown that relative rates of amino acid sequence evolution
are very similar in all three domains of life (eubacteria,
archaebacteria, and eukaryotes). Therefore, we interprete the data
gathered from the S. pombe, S. cerevisiae, and C.
elegans genomes as indicating that natural selection has
preferentially kept genes that are required for essential functions.
This interpretation gains further support from the observation that
S. pombe conserved genes, which have been lost in the S.
cerevisiae lineage (class IV), are unlikely to be essential. We
conclude that genes that have been maintained throughout evolution are
more likely to be essential.
However, what about genes that appeared late in the tree of life? We
have established that the more ancient a gene is, the more likely it is
to be essential. Therefore, organism-specific genes (class III and V)
that have arisen more recently appear to be less likely to be required
for vital functions of the cell. It has been suggested that these
proteins may be required for more specialized functions (Chervitz et
al. 1998; Rubin et al. 2000). In C. elegans, for example,
processes that are unique to the metazoa and have arisen more recently
are carried out by proteins that do not have homologs in yeast, whereas
core biological functions use orthologous proteins (Chervitz et al.
1998). We propose that generally in eukaryotes, many of the essential
genes are those that appeared with the origin of eukaryotic life and
have remained conserved within all branches of the tree of life.
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METHODS
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Protein Sequence Comparison
S. pombe proteins from PombePD (see Wood et al. 2002) were
compared to nr database using BLASTP (Altschul and Lipman 1990) with a
cutoff E value of 10–5.
Gene Deletion
PCR-based gene deletion, using the kanR marker,
was performed according to Bähler et al. (1998) with flanking
sequences of 80 bp. An h+/h– ura4-D18/ura4-D18
leu1-32/leu1-32 ade6-M210/ade6-M216 S.
pombe diploid strain was transformed with the PCR product, and
geneticin-resistant colonies were selected on YES medium containing 100
µg/mL G418 (Life Technologies). Gene deletion was checked by colony
PCR. Diploid strains were sporulated and tetrads were dissected as
described in http://www.bio.uva.nl/pombe/. The deletion phenotype was
assessed on YES medium.
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WEB SITE REFERENCES
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http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html;
microbial genomes.
http://mips.gsf.de/proj/yeast/CYGD/db/index.html; MIPS
Comprehensive Yeast Genome Database with annotation for all yeast
ORFs.
http://www.bio.uva.nl/pombe; S. pombe site with an online
fission yeast handbook.
http://www.uni-frankfurt.de/fb15/mikro/euroscarf/; collection of
S. cerevisiae deletion strains.
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Acknowledgements
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We are grateful to Adam Bermange and Damien Hermand for their help.
We thank Pierre Tilquin for help with statistical analysis of the data.
We thank all members of the Cancer Research UK Cell Cycle Laboratory
for their support. We thank Jacky Hayles, John Sgouros, André
Goffeau, and Françoise Foury for critical reading of the manuscript.
This work was supported by the European Commission, Biotechnology
Programme (A.D.), Comunidad Autonoma de Madrid (I. S.-P.), and Cancer
Research UK.
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.
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Footnotes
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3 Present adress: Catholic University of Louvain, Institute
of Cellular Pathology, 1200 Brussels, Belgium. 
4 Corresponding authors.
E-MAIL anabelle.decottignies{at}gece.ucl.ac.be; FAX
32-2-762-9405.
E-MAIL Paul.Nurse{at}cancer.org.uk; FAX 44-20-72693610.
Article and publication are at
http://www.genome.org/cgi/doi/10.1101/gr.636103.
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Received July 17, 2002;
accepted in revised format December 10, 2002.
13:399-406 © by 2003 Cold Spring Harbor Laboratory Press ISSN 1088-9051/03 $5.00
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