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Infection and Immunity, August 1999, p. 3960-3969, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Random Survey of the Cryptosporidium
parvum Genome
Chang
Liu,
Vladimir
Vigdorovich,
Vivek
Kapur, and
Mitchell S.
Abrahamsen*
Department of Veterinary PathoBiology,
University of Minnesota, St. Paul, Minnesota
Received 29 January 1999/Returned for modification 31 March
1999/Accepted 25 May 1999
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ABSTRACT |
Cryptosporidium parvum is an obligate intracellular
pathogen responsible for widespread infections in humans and animals. The inability to obtain purified samples of this organism's various developmental stages has limited the understanding of the biochemical mechanisms important for C. parvum development or
host-parasite interaction. To identify C. parvum genes
independent of their developmental expression, a random
sequence analysis of the 10.4-megabase genome of C. parvum was undertaken. Total genomic DNA was sheared by
nebulization, and fragments between 800 and 1,500 bp were gel purified
and cloned into a plasmid vector. A total of 442 clones were
randomly selected and subjected to automated sequencing by using one or
two primers flanking the cloning site. In this way, 654 genomic survey sequences (GSSs) were generated, corresponding to
>320 kb of genomic sequence. These sequences were assembled into 408 contigs containing >250 kb of unique sequence, representing ~2.5% of the C. parvum genome. Comparison of the GSSs
with sequences in the public DNA and protein databases revealed that
107 contigs (26%) displayed similarity to previously identified
proteins and rRNA and tRNA genes. These included putative genes
involved in the glycolytic pathway, DNA, RNA, and protein metabolism,
and signal transduction pathways. The repetitive sequence elements identified included a telomere-like sequence containing hexamer repeats, 57 microsatellite-like elements composed of dinucleotide or
trinucleotide repeats, and a direct repeat sequence. This study demonstrates that large-scale genomic sequencing is an
efficient approach to analyze the organizational characteristics and
information content of the C. parvum genome.
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INTRODUCTION |
Cryptosporidium parvum
has emerged as a well-recognized cause of acute gastrointestinal
disease in humans and animals throughout the world and is associated
with a substantial degree of morbidity in patients with AIDS
(15). C. parvum belongs to the phylum Apicomplexa
and is one of several genera that are referred to as coccidia. The
parasite primarily infects the microvillous border of the intestinal
epithelium and to a lesser extent the extraintestinal epithelium
(10). The life cycle of C. parvum resembles that of other coccidia and includes multiple asexual and sexual
developmental stages.
Despite the medical and veterinary importance of C. parvum,
studies of this organism at the genetic level have only begun in recent
years and are still in their infancy. Although a relatively small
number of basic metabolic and structural genes as well as several genes
encoding immunogenic antigens have been identified (10),
little is known about the basic cellular and molecular biology of this
pathogen in terms of virulence factors, genome structure, or
developmental biology. This is largely due to the inability to obtain
purified samples of the various developmental stages of the parasite
for biochemical studies. The relatively small size and simple
organization of the 10.4-megabase (Mb) C. parvum genome,
which is composed of eight chromosomes ranging from 1.04 to 1.5 Mb,
however, balance these disadvantages (3). Since the genomic
DNA sequence encodes all of the heritable information responsible for
parasite development, disease pathogenesis, virulence, species
permissiveness, and immune resistance, a comprehensive knowledge of the
C. parvum genome will provide the necessary information required for targeted research into disease prevention and treatment.
Over the past few years, large-scale sequencing of randomly selected
cDNA or fragments of genomic DNA has proven to be an efficient approach
for expanding the understanding of the biology of an organism,
including many pathogenic protozoa (6, 8, 21, 32, 36).
Recently, a large-scale expressed sequence tag (EST) sequencing project
was undertaken for C. parvum sporozoites (5). Due
to the inability to obtain purified samples of other developmental
stages, in particular, the intracellular stages, the ongoing C. parvum EST approach is limited to the discovery of genes that are
expressed in sporozoites. Considering the absolute dependence of
C. parvum development on the mammalian host cell, many
unique biochemical pathways and molecular mechanisms involved in
host-parasite interaction and pathogenesis are not likely to be
identified by the ongoing sporozoite EST project.
In order to identify C. parvum genes, independent of their
developmental expression, we conducted large-scale sequencing of random
C. parvum genomic segments. In this report, we described the
identification of 654 genomic survey sequences (GSSs) obtained by the
random sequencing of clones from a small-insert C. parvum total genomic DNA library. The relatively high number of GSSs with
similarity to previously characterized genes from other organisms implies that genomic sequencing is an efficient method for gene discovery in C. parvum. Furthermore, the identification of
putative C. parvum genes and repetitive elements laid the
foundation for studies directed toward understanding the biology of
C. parvum and the development of strategies for subspecies
differentiation and epidemiological surveillance of the parasite.
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MATERIALS AND METHODS |
DNA preparation.
C. parvum oocysts (Iowa isolate;
originally obtained from C. Sterling, University of Arizona, Tucson)
were sterilized by incubation in Clorox (3 × 107
oocysts/ml; sodium hypochlorite, 5.25%; dilution rate, 1:3) for 7 min
on ice. The oocysts were washed five times in phosphate-buffered saline
(PBS) by centrifugation at 3,500 × g for 10 min at
4°C. The oocysts were resuspended in PBS at a concentration of
108 oocysts/ml. An equal volume of 2× excystation medium
(0.05 g of trypsin and 0.15 g of sodium taurocholate in 5 ml of
Hanks' buffered salt solution [pH 7.2 to 7.4]) was added, and the
oocysts were incubated at 37°C for 1 h. The unexcysted oocysts
and sporozoites were washed three times in PBS by centrifugation. The
pelleted oocysts and sporozoites were suspended in 400 µl of DNA
lysis solution (120 mM NaCl, 0.1 M EDTA, 25 mM Tris base, 1%
Sarkosyl), and the suspension was subjected to three freeze-thaw cycles
with liquid nitrogen and a 70°C water bath. The lysate was incubated with protease K (1 mg/ml) for 2 h at 37°C followed by
phenol-chloroform extraction and ethanol precipitation by using
standard methods (29). The DNA precipitate was resuspended
in 0.5 ml of TE (10 mM Tris base, 1 mM EDTA [pH 8.0]) and treated
with RNase A (1 mg/ml) for 1 h at 37°C. The DNA sample was
extracted with phenol-chloroform, precipitated, and resuspended in TE
as described above.
Library construction.
Total genomic DNA (100 µg) was
randomly sheared by using a gas-driven nebulizer as previously
described (28), blunted with Escherichia coli DNA
polymerase, and phosphorylated with T4 polynucleotide kinase. The DNA
fragments were fractionated by electrophoresis, and fragments between
800 to 1,500 bp were excised from the agarose gel and purified with
QIAEX II kits (Qiagen, Chatsworth, Calif.). The purified DNA fragments
were cloned into the SmaI site of pBluescript II SK (+)
vector (Stratagene, La Jolla, Calif.).
Sequencing and analysis.
Randomly selected clones from the
unamplified library were grown overnight, and plasmid DNA was purified
with SNAP kits (Invitrogen, Carlsbad, Calif.) or Qiagen plasmid
minikits (Qiagen). DNA sequencing was performed at the Advanced
Genetics Analysis Center (College of Veterinary Medicine, University of
Minnesota) by using dye termination cycle sequencing technology with
AmpliTaq DNA polymerase (Perkin-Elmer, Foster City, Calif.) and was
analyzed on an ABI fluorescence automated sequencer (PE Applied
Biosystems, Foster City, Calif.). Sequence data were edited with
EditSeq (DNASTAR, Inc., Madison, Wis.), to remove the vector sequence
and/or to delete sequences of low reliability. Contig assembly and
statistical analysis were performed by using SeqMan (DNASTAR). Public
databases, including GenBank (release 105.0), EMBL (release 53.0), PIR
(release 55.0), SWISS-PROT (release 35.0), PROSITE (release 14.0), and Profile Library, were searched for similarity to known sequences or
motifs by using NETBLAST, MOTIFS, and PROFILESCAN (GCG Wisconsin Package, version 9.1; Genetics Computer Group [GCG], Madison, Wis.).
Previously identified C. parvum sequences in GenBank were searched and retrieved with STRINGSEARCH and FETCH (GCG). The sequences
were further compiled into a local database by using GCGTOBLAST
(GCG) and were searched for similarities to our C. parvum GSSs by using BLAST (GCG). The mono-, di-, and
trinucleotide compositions were calculated with COMPOSITION (GCG).
Direct repeats and simple sequence repeats were identified with
FINDPATTERNS (GCG).
Nucleotide sequence accession numbers.
Nucleotide sequences
reported in this paper are available in the GenBank database under
accession no. AQ023473 to AQ024123.
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RESULTS AND DISCUSSION |
Characteristics of sequencing data.
To generate a uniformly
distributed, representative sequencing template library,
high-molecular-weight C. parvum genomic DNA was mechanically
sheared by nebulization as previously described (28). The
sheared DNA was separated by gel electrophoresis, and fragments with a
size distribution from 800 to 1,500 bp were purified and used to
construct the genomic library in the vector pBluescript II SK (+).
Automated DNA sequencing was performed on a total of 432 random clones.
Among them, 212 clones were sequenced with primers flanking each side
of the cloning site (T3 and T7 primers). The remaining 230 clones were
sequenced with only one flanking primer (T3). A total of 324,076 bp of
genomic sequence was generated. In order to identify overlapping
sequences, all sequences were subjected to contig assembly. This
analysis generated 408 contigs containing 256,935 bp of unique genomic
sequence. This represented ~2.5% of the estimated 10.4-Mb C. parvum genome. The majority of nonunique sequence was the result
of overlapping sequences generated from individual clones with both
flanking primers. A total of 94% (408 individual contigs generated
from 432 random clones) of the random clones contained unique
sequences. To assess the quality of our sequence data, GSSs matching
previous C. parvum database entries were aligned with their
corresponding database entries. The accuracy of our sequences,
indicated by the percentage of the identical nucleotides between
the aligned sequences, was found to be greater than 99% (data not
shown). Other than vector sequences used to construct the genomic
library, no contaminating bacterial or bovine sequences were found
among the generated GSSs. This is likely due to the harsh chemical
treatments and extensive washing of the oocysts prior to DNA isolation,
which greatly reduced the chance of contamination of the
C. parvum genomic DNA library with host or other
microbial DNA fragments.
Identification of putative genes.
Database searching with the
GSSs was performed by using the program NETBLAST against
the nonredundant GenBank, PDB, SWISS-PROT, and PIR (1)
databases. This analysis revealed that 134 GSSs, corresponding to 107 individual contigs (26%), displayed significant similarity (smallest
probability [P 
10
5]) to sequences
present in the databases. Among them, 129 GSSs displayed similarity to
known protein sequences, one displayed a significant similarity to
telomeric sequences of several eukaryotes (CpGR254), and two (CpGR12A
and CpGR12B) contained sequences representing C. parvum rRNA
genes (GenBank accession no. AF040725). Seven of the GSSs represented
previously characterized C. parvum sequences (precursor of
oocyst wall [GenBank Z22537], tubulin beta chain [PIR A25342],
C. parvum DNA segment B [GenBank M59420], C. parvum open reading frame [ORF] 2 gene [GenBank U18112], thrombospondin-related adhesive protein (TRAP) [GenBank
AF017267], elongation factor [GenBank U71180], and protein
disulfide isomerase [GenBank U48261]). In addition, searching
the GSSs with the program tRNAscan-SE (20) identified
one GSS (CpGR309B), which was highly homologous to the isoleucine tRNA
gene of Thiobacillus ferrooxidans (GenBank U18089).
The GSSs which displayed significant similarities to database entries
were grouped based on the biological roles of their matches (Table
1)
by using the classification system developed by Riley (27).
The distribution of putative genes in different functional groups is
shown in Fig. 1. It is evident that genes involved in macromolecular and small-molecular biosynthesis are well
represented in the C. parvum genome, as well as genes
potentially involved in cellular signaling, energy production, and the
regulation of mRNA and protein expression. Of special interest are
those genes potentially involved in parasite survival, pathogenesis, and host-parasite interaction. Below, we described several groups of
proteins that fall into these categories, which provide new insights
into C. parvum biology.
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FIG. 1.
Functional classification of C. parvum GSSs,
showing the proportions of predicted genes according to their putative
biological functions. GSSs having a P value of
105 were classified into 12 functional categories.
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The deduced amino acid sequence of CpGR24B displayed significant
similarity (
P = 2.8e
53) to members of the prohibitin
gene
family (Fig.
2). In mammals, the
prohibitin gene product has been
shown to negatively regulate cell
proliferation (
22). In addition
to gene structure, the
function of this protein is conserved across
many lower and higher
eukaryotes. For example, the
Pneumocystis carinii prohibitin
gene expressed in human fibroblasts has been
shown to arrest the cell
cycle in the G
1 phase (
23). The similarity
between CpGR24B and members of the prohibitin family suggests
that this
GSS represents a portion of a
C. parvum gene which may
function in controlling
C. parvum proliferation and
development.
It is interesting to note that in yeast, prohibitin has
been found
to be localized within the inner mitochondrial membrane and
appears
to play a role in mitochondrial inheritance and regulation of
mitochondrial morphology (
2). However, there is no evidence
for the existence of mitochondria in
C. parvum, suggesting
that
not all prohibitin functions are conserved.
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FIG. 2.
Multiple sequence alignment of the deduced amino acid
sequences of CpGR24B and members of the prohibitin family. The origins
and accession numbers of the prohibitin sequences used in this
alignment are as follows: Arabidopsis thaliana (At), U69155;
Nicotiana tabacum (Nt), U69154; C. parvum (Cp),
AQ023505; Homo sapiens (Hs), S85655; Rattus
norvegicus (Rn), M61219; Toxocara canis (Tc), U97204;
S. cerevisiae (Sc), U16737; Trypanosoma brucei
(Tb), AF049901. The amino acid numbers for each sequence are indicated
on the right. In the sequence alignment, identical residues are shown
with a black background, and similar residues are shown with a gray
background.
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A total of 21 GSSs displayed limited similarity to proteins involved in
the cell signaling pathway, including protein ligands,
cell surface
receptors and their associated proteins, and protein
kinases and
phosphatases. For example, CpGR231B displays limited
homology to the
shk1 kinase-binding protein (
P = 4.0e
10). This
kinase
is an essential component of the Ras- and Cdc42-dependent
signaling
cascade, which has been demonstrated to be required
for cell viability,
normal morphology, and mitogen-activated protein
kinase-mediated signal
response in the fission yeast (
11). Although
these GSSs are
not conserved to the extent that CpGR24B is within
the prohibitin gene
family, the similarity of these GSSs to proteins
involved in
intracellular signaling provides evidence that signal
transduction
pathways in
C. parvum are similar to those used by
other
eukaryotic organisms. These
C. parvum proteins are likely
involved in the coordination of complex host-parasite
interactions,
signaling with other parasites, and the regulation
of growth and
differentiation of parasites in response to
external
signals.
In addition to the GSSs that displayed similarity to known genes
involved in responding to extracellular signals, several
GSSs displayed
limited similarity to genes involved in cell adhesion
and/or
recognition. CpGR176A and CpGR260A displayed similarity
(
P = 4.3e
6 and
P = 5.1e
14) to the F-spondin
precursor gene of
amphibians and mammals which plays a role in cell
signaling and
adhesion (
18). In addition, CpGR176A and
CpGR260A are also similar
but not identical to the previously
identified
C. parvum family
of TRAPs (GenBank accession no.
AF017267,
AF073838,
AF033828,
X77587, and
U42213) and to the
phylogenetically related
sporozoan proteins including the
Eimeria
maxima EM100 antigen
(
M99058), the
Eimeria
tenella Etp100 protein (
AF032905),
the
Toxoplasma
gondii MIC2 microneme protein (
U62660), and
the
Plasmodium
falciparum circumsporozoite protein-TRAP-related
protein
(
U34363). Members of the TRAP family of proteins have
been shown to be
localized in the apical end of
C. parvum sporozoites
and are
structurally related to the micronemal proteins of
Eimeria and
Toxoplasma, which are involved in
host-cell attachment and/or
invasion (
33). Another GSS,
CpGR17B, displayed similarity (
P = 1.3e
14) to human
tyrosyl-tRNA synthetase and to endothelial
monocyte-activating
protein II (EMAP II) (
P = 7.6e
11). Recently,
an EMAP
II-like domain has been found at the carboxyl-terminal
end of
human tyrosyl-tRNA synthetase. The human tyrosyl-tRNA synthetase
is
secreted as cells undergo programmed cell death (apoptosis)
and is
cleaved into two cytokines including the EMAP II-like molecule
(
37). EMAP II is a multifunctional tumor-derived cytokine
that
has been shown to activate endothelial cells, resulting in the
elevation of cytosolic free calcium concentration, release of
von
Willebrand factor, induction of tissue factor, and expression
of
adhesion molecules such as E-selectin and P-selectin (
17).
In addition, mononuclear phagocytes exposed to EMAP II
demonstrated
the induction of tumor necrosis factor alpha (TNF-
) and
tissue
factor.
The above-mentioned database search could identify only
C. parvum genes which were similar to sequences
currently present
in the public databases. Consequently, GSSs
representing unique
C. parvum genes or genes that have not
been characterized in other
organisms would not be identified. In order
to estimate the actual
coding capacity of
C. parvum genome,
all sequences were subjected
to analysis for the presence of ORFs by
using the program ORF
Finder (
25). Since the expected
frequency of the three stop
codons is 3/64, the longer an ORF is, the
more likely it represents
a coding sequence. This analysis revealed
that 615 of the 654
GSSs (94%) had the potential to encode proteins,
under the condition
that an ORF longer than 100 amino acids was
considered to be a
coding sequence (
25). The high percentage
of potential coding
sequences in our GSSs suggests that the
C. parvum genome has a
high gene density with little intergenic
spacing.
In order to further characterize potential
C. parvum genes,
GSSs which contained ORFs that did not display a high degree of
similarity to those in the databases were further analyzed by
using the
programs MOTIFS and PROFILESCAN (GCG) to determine the
presence of
functional protein motifs. In our analysis, only motifs
with a low
false-positive rate and within ORFs of >100 amino acids
were
characterized. This search resulted in the identification
of 11 functional protein motifs or profiles (Table
2). These
included ATP or GTP binding
domains, signatures for transport
proteins, and surface receptors. This
analysis suggests that these
GSSs may represent additional
C. parvum genes.
Identification of repetitive sequences.
Microsatellite DNA
sequences, also called simple sequence repeats or simple tandem
repeats, are ubiquitous elements of eukaryotic genomes. The function of
these repeats is not well understood, despite a number of hypotheses
that have been proposed, including modulation of gene regulation, sites
of frequent recombination, and formation of left-handed DNA
conformation (or Z-DNA) (13). These tandem repeats of 1- to
5-bp motifs have been found to be distributed throughout eukaryotic
genomes and have been demonstrated to be useful markers for the rapid
and sensitive genetic fingerprinting of an organism (34).
In order to identify potential genetic markers for strain typing and
tracking of
C. parvum, we analyzed the nature and
frequency
of microsatellite DNA sequences including all possible
dinucleotide
and trinucleotide repeats in the
C. parvum
GSSs. The GSSs containing
a di- or trinucleotide repeat and the
number of repeats they contained
are listed in Table
3 and Table
4. Among the 57 GSSs found to
contain
microsatellite-like elements, the most abundant dinucleotide
repeats included (TT)
n, (AA)
n,
(TA)
n, and (AT)
n. Similarly,
(AAT)
n,
(TAA)
n, (TAT)
n,
(ATA)
n, (TTA)
n, and (ATT)
n
constitute the most
abundant trinucleotide repeats. A
potential role for several of
these microsatellite DNA sequences as
genetic markers is currently
being investigated.
To further characterize structural features of the
C. parvum
genome, we examined the GSSs for the presence of additional
repetitive
sequences. This analysis identified two GSSs, CpGR265A
and CpGR254,
that contained complex repetitive sequences. CpGR265A
contained
multiple direct repeats of 14 bp with a consensus sequence 5'
TCTCTTTCAATYCT 3'. Twenty-five copies of the direct repeat were
present within 512 bp of sequence. Database searching revealed
no
significant identity with any other sequences. Similarly, CpGR254
contained 48 copies of an imperfect direct repeat sequence
T
(2-12)AG
(3-5).
This basic repeat unit was
similar in base composition and structure
to telomeric sequences
characterized from other lower and higher
eukaryotes (
14).
Further characterization demonstrated that
this repetitive sequence
represents a portion of a
C. parvum telomeric
DNA sequence
(
19).
Analysis of nucleotide compositions.
To investigate the
correlation between the nucleotide composition of a sequence and its
coding potential in the C. parvum genome, the nucleotide
compositions of the GSSs were calculated with the program COMPOSITION
(GCG) and compared with those of known C. parvum coding
sequences. The coding sequences used in this study (accession nos.
AF001211, U24082, U90628, L31806, AF013984, U34390, U95995,
AF017267, U41365, U95996, S76665, U48261, U35027, S76666, U11761, U48717, U35028, U18120, U65981, U42213, U21667, U71181, L08612, U22892,
U83169, and M86241) were retrieved from GenBank with the FETCH program
(GCG). The overall AT contents were 62.4% for the coding sequences and
68.0% for the random sequences, suggesting that there is no bias
against AT content in the coding region as has been found for
other eukaryotes (24). However, an interesting discrepancy
was observed when the frequencies of individual nucleotides in the
coding and the random sequences were compared. The frequencies of
A (33.1%) and C (17.0%) in the coding sequences were nearly identical
to those in the random sequences (A, 33.7%; C, 16.1%). In contrast,
the occurrence of G in coding sequences (20.6%) is 32% greater
than that in the random sequences (15.9%). This was offset by the
corresponding decrease in the occurrence of T (29.3% in the coding
sequences and 34.3% in the random sequences). Previously, a bias of GC
content has been reported in C. parvum (10). Our
analysis demonstrated that this bias is due to the preference of G in
the coding sequences. The relevance, if any, of this finding is not clear.
To investigate the presence of dinucleotide bias in the
C. parvum genome, the dinucleotide preference
(DiP) of the random
genomic sequences was calculated by dividing the
observed frequency
of the dinucleotide by its expected frequency (Fig.
3). Dinucleotides
CG (0.54), AC (0.76),
GT (0.76), and TA (0.78) are significantly
disfavored in the
C. parvum genome. The low dinucleotide frequency
(DiF) of
dinucleotides CG and TA is consistent with the fact that
both these
dinucletides are underrepresented in the genes of
Drosophila and a wide range of bacteria, yeast, primates, and other apicomplexans
(
7). Previously, the low DiF of dinucleotide CG was
observed,
based on the study of four
C. parvum sequences
(
4).
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FIG. 3.
DiP in C. parvum GSSs. The observed DiF in
C. parvum GSSs was calculated with the COMPOSITION program
(GCG). The expected DiF was calculated by multiplying the observed
frequencies of the two mononucleotides that constitute the
dinucleotide. The DiP for a dinucleotide was calculated as the ratio of
its observed DiF to its expected DiF. The DiPs were plotted against
their corresponding dinucleotide sequences. The actual value for each
DiP is indicated on the right of each column.
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Comparison of different sequencing approaches.
The
efficiencies of C. parvum gene discovery with random genomic
sequencing and EST sequencing were compared to determine the usefulness
of each of these approaches. The GSSs generated in this study were
compared to 567 C. parvum sporozoite ESTs (5). The average sequence length of the GSSs (496 bp) is longer than that of
the ESTs (476 bp), which may be attributed to the length of the cDNA
insert, which is shorter than that of the genomic sequence insert. A
total of 384 unique ESTs were generated, at a redundancy rate of
32.3%, which is significantly higher than that of the GSS project
(6%; 408 individual contigs generated from 432 random clones).
However, as ESTs are derived from expressed sequences, all 384 unique
ESTs are assumed to represent expressed C. parvum genes
regardless of their matching with database entries. Among the unique
ESTs, 37% (142 of 384) displayed significant similarity with sequences
in the current databases. In contrast, 26% of the individual genomic
contigs (107 of 408) displayed significant similarity with sequences in
the current databases. This difference is not unexpected, as GSSs do
not necessarily represent coding sequences. In general, the
characteristics of the C. parvum GSS and EST projects are
comparable with those conducted on other organisms, in terms of total
sequence length, percentage of sequences with database match, and
redundancy rate (6, 8).
In order to examine the redundancy of sequence data generated between
the ESTs and GSSs, the ESTs were compiled into a local
database and
searched with our GSSs. Forty-eight of the 654 GSSs
(7.3%) matched
sequences present in 33 of the
C. parvum ESTs (33
of 568 [5.8%]). Eighteen of these 33 EST sequences matched database
entries. Among the 18 sequences, five sequences encoded rRNA and
proteins, eight sequences encoded proteins with known functions,
and
five sequences encoded hypothetical
proteins.
During the course of our study, another
C. parvum GSS
project (
5) and a
C. parvum sequence tagged site
project (
26) were
initiated. To determine the redundancy of
sequences generated
from different sources with the same genomic DNA
sequencing approach,
sequences generated from these projects were
retrieved from GenBank,
compiled into separate local databases, and
searched with our
GSSs. One hundred twenty-nine of our
C. parvum GSSs (19%) matched
134 GSSs (8.9%) retrieved from the
public databases. Eight (1.2%)
of our GSSs matched seven (7 of 149 [4.7%]) retrieved
C. parvum sequence tagged sites. The
above analysis indicates that currently
there are relatively few
redundancies among
C. parvum sequences
generated by
different approaches or from different sources using
the same genomic
DNA sequencing approach. This will change as
more sequences become
available.
Concluding comments.
In this study, we employed a random
genomic sequencing approach to conduct a general survey of the
organizational characteristics and informational content of the 10.4-Mb
C. parvum genome. Of the 408 assembled contigs, 107 displayed significant similarity with gene sequences currently in the
public databases. These 107 putative C. parvum genes were
identified from a total of 256,935 bp of unique genomic sequence. This
predicts a minimum gene density of approximately 1 gene/2,500 bp of
genomic sequence. In related work, we have obtained more than 15 kb of
contiguous DNA sequence from the smallest C. parvum
chromosome. Within this locus, eight expressed ORFs were identified
(unpublished data). The gene density identified at this locus (8 genes/15 kb) is approximately 1 gene/2,000 bp, consistent with that
predicted from the GSS data. This predicted gene density suggests that
the 10.4-Mb C. parvum genome may contain ~4,000 to 5,000 genes, comparable to the coding capacity of Saccharomyces cerevisiae, which has a genome size of 13.5 Mb and contains 5,800 genes (12). Other data (16, 30, 31) and analysis
of the transcript sizes of the eight ORFs on the smallest C. parvum chromosome (unpublished data) suggest that the average size
of the transcript (untranslated and coding sequences) of a C. parvum gene is 1,000 to 2,000 bases (unpublished data). Together
these data predict that approximately 50 to 75% (the number of genes
times the average length of a gene) of the C. parvum
genome is transcribed into RNA sequences. As the GSS analysis could not
identify those genes without database matches, the above-estimated
coding capacity of the C. parvum genome may be less than the
actual capacity. Indeed, the ORF analysis of nonmatching GSSs indicates
that many of these sequences likely represent additional C. parvum genes.
Repetitive sequences are known to be present in eukaryote genomes at
significantly different frequencies (
9). Of the 408
contigs generated in this study, only two contained direct repeat
sequences, one of which represented a telomeric sequence
(
19).
This suggests that repetitive sequences may
comprise < 0.5% of
the
C. parvum genome. This
percentage is significantly lower than
that reported in similar
studies for other organisms. In addition
to direct repeat sequences,
diverse microsatellite sequences have
been identified in this
study, constituting less than 1% of the
C. parvum genome
that was characterized (2,308 of 250,000 bp).
The paucity of repetitive
sequences is consistent with the notion
that a large percentage of the
C. parvum genome contains coding
sequences.
| |
ACKNOWLEDGMENTS |
We thank Bruce A. Roe (University of Oklahoma) for help in
initiating this project. We are also grateful to Alison A. Schroeder and Cheryl A. Lancto for technical assistance, Yuan Wang for batch submission of GSSs to GenBank, and Elizabeth Shoop from Computational Biology Center (University of Minnesota) for the analysis and web
publication of GSSs.
This work was supported in part by grants from the NIH (AI-35479) and
the Minnesota Agricultural Experiment Station to M.S.A.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Veterinary PathoBiology, University of Minnesota, 1988 Fitch
Ave., St. Paul, MN 55108. Phone: (612) 624-1244. Fax: (612)
625-0204. E-mail: abrah025{at}tc.umn.edu.
Editor:
J. M. Mansfield
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Abstract
Introduction
