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FEMS Immunology and Medical Microbiology 22 (1998) 15^26
II. The genome of Pneumocystis carinii
James R. Stringer a; *, Melanie T. Cushion
a
b;c
Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Bethesda Ave.,
Cincinnati, OH 45267-0560, USA
b
Department of Internal Medicine, Division of Infectious Disease, University of Cincinnati College of Medicine, 231 Bethesda Ave.,
Cincinnati, OH 45267-0560, USA
c
Cincinnati Veterans Administration Medical Center, Cincinnati, OH, USA
Abstract
The best understood special form of P. carinii, P. carinii formae specialis (f.sp.) carinii, appears to be haploid and contains
about 8 million base pairs of DNA (8.5 fg) per nucleus. The genome of P. carinii f.sp. carinii is divided into 13^15 linear
chromosomes that range from 300 to 700 kb in size. Eight different P. carinii f.sp. carinii karyotypes have been observed. The
karyotypes of P. carinii f.sp. carinii differ due to slight variations in the lengths of chromosomes, but the 8 karyotype-forms of
P. carinii f.sp. carinii exhibit very little variation in DNA sequence. By contrast, the genome of P. carinii f.sp. carinii differs
markedly in sequence from the genomes of P. carinii from other hosts, such as mouse, ferret and human. In addition,
chromosomes and DNA sequences from P. carinii from mouse, ferret, and human also differ greatly from each other. The
genome of a ferret P. carinii appears to be up to 1.7 times larger than those of P. carinii from other hosts. Nearly two dozen
P. carinii genes have been cloned and sequenced. The typical P. carinii gene sequence is 60^65% A+T. P. carinii genes usually
contain introns, which are typically less than 50 bp in length, but can be as numerous as 9 per gene. A system for naming
P. carinii genes is proposed in which each gene would be designated by an italic three-letter lower case symbol. The first allele
(i.e. sequence) that is found would have a superscript 1, such as xyz11 . Any subsequent alleles would be designated as xyz12 ,
etc. A protein would have the same symbol as the gene that produced it, but written in roman print with the first letter an
uppercase, such as Msg1. Some of the P. carinii genome is comprised of DNA sequences that are present dozens of times. Three
families of such repeated DNA sequences have been described. Two of these families (MSG and PRT) encode proteins. The
third family is the telomere repeat, which is found at the ends of each chromosome, and sometimes at internal chromosomal
sites, in which case it has been called the alpha repeat. Determination of the complete sequence of the P. carinii genome is both
practicable and of primary importance to the understanding of this organism. The small size of the P. carinii genome and its
packaging into chromosomes that are resolvable by PFGE will facilitate sequence analysis. z 1998 Federation of European
Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Pneumocystis carinii; Genome; DNA; Gene; Chromosome
1. Introduction
Any discussion of the characteristics of Pneumo* Corresponding author. Tel.: +1 (513) 558-0069;
Fax: +1 (513) 558-8474; E-mail: [email protected]
cystis carinii must begin by recognizing that there are
many di¡erent organisms that go by this genus-species name. These di¡erent types of P. carinii are
called special forms, or formae specialis (f.sp.) [1].
Most of information about the genome has been
obtained from P. carinii f.sp. carinii, the organism
0928-8244 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 8 - 8 2 4 4 ( 9 8 ) 0 0 0 5 2 - 2
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found in most laboratory rats. Some data are available for other special forms, such as those from human, mouse and ferret. Current data are su¤cient to
conclude that the genomes of di¡erent special forms
of P. carinii resemble each other in certain respects,
but are by no means identical. Each special form has
its own particular set of genome characteristics.
Therefore, the general features of the genomes of
new special forms can be anticipated from those of
the special forms that have been analyzed, but the
speci¢c genomic features of new special forms are
likely to be distinctive.
2. Size of the P. carinii f.sp. carinii genome
Early studies attempted to determine the size of
the P. carinii genome by biochemical methods [2,3].
In these e¡orts, P. carinii were prepared from infected animals and puri¢ed as much as possible.
The organisms were observed in a microscope to
verify the absence of host cells, and to the count
number of P. carinii. DNA from a given number
of P. carinii was puri¢ed and quanti¢ed by standard
techniques. The genome size per organism was calculated from the amount of DNA divided by the
number of organisms. These studies produced genome size estimates on the order of 300 megabasepairs (Mb). These values were later found to be at
least 30-fold too high. The inaccuracy of the biochemical approach to measuring the genome size
was probably caused by the presence of host DNA
in the organism preparation.
The development of pulsed ¢eld gel electrophoresis
(PFGE) methods presented an opportunity to measure the size of the P. carinii genome with more accuracy. The ¢rst PFGE study suggested that the
P. carinii f.sp. carinii genome contains at least 13
chromosomes, ranging in size from 0.20 to 2.0 Mb
[4]. A second study resolved 16^20 bands, ranging
from 0.32 to 1.50 Mb in size [5]. Subsequent investigations revealed that the bands larger than 0.70 Mb
were artifacts caused by DNA from the rat host, and
that the true size range for P. carinii f.sp. carinii
bands is between 0.30 and 0.68 Mb [6^9]. The number of PFGE bands varies from 13 to 15, depending
on the rat colony from which the organisms were
obtained [6^9]. Summation of PFGE bands produces
a genome size of 7 Mb. However, the genome probably contains between 7.5 and 8 Mb, because two or
three bands (depending on the sample) appear to
contain more than one chromosome. A genome comprised of 8 Mb would contain about 8.5 fg of DNA.
To put the genome size of P. carinii in perspective,
the genomes of Escherichia coli and Saccharomyces
cerevisiae are 4.2 and 12 Mb, respectively [10,11].
This comparison of genome sizes is interesting to
consider in reference to speculations about the possible relationship between the gene content and the
life style of P. carinii. A stark feature of P. carinii is
its failure to multiply in vitro, which implies that the
microbe lacks certain biosynthetic capabilities, and is
therefore dependent on a mammalian host for survival [12]. Could P. carinii organisms lack biosynthetic capabilities because they lack su¤cient genes?
Possibly. The genome of P. carinii is only 70% as
large as that of S. cerevisiae. However, the genome
of P. carinii is twice the size of E. coli, which can
grow on a simple medium containing not much more
than sugar and ammonia. Although the P. carinii
genome contains twice as many basepairs as the genome of E. coli, it probably does not encodes twice
the number of proteins as this bacterium because a
substantial part of the P. carinii genome does not
encode protein. DNA that does not encode protein
in P. carinii includes the following: (1) spaces between genes; (2) spaces within genes (introns); (3)
sequences that encode structural RNAs; (4) sequences that comprise chromosomal structures such as
telomeres and centromeres. The spacing between
P. carinii genes has not been studied except for genes
encoding the major surface glycoprotein (see below),
but it seems unlikely that more than 10% of the
genome is occupied by intergenic spacers. Introns
are common in P. carinii genes, but are usually less
than 50 bp in length and appear to occupy no more
than 10% of the P. carinii genome (see below). Less
than 1% of the P. carinii genome encodes ribosomal
RNA [13]. It seems that no more than 10% of the
genome is occupied by telomeres [14] (see below).
These estimates suggest that about 6 Mb of the
P. carinii genome encodes proteins. If an average
P. carinii protein is 50 kDa, 6 Mb would encode
4000 such proteins. This estimate agrees with an estimate derived by comparison with the S. cerevisiae
genome, which contains 6000 open reading frames
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17
Fig. 1. Electrophoretic karyotypes of four special forms of P. carinii. Lanes marked rPc, hPc, fPc and mPc contained : P. carinii f.sp. carinii (sample Sprague^Dawley rat number 1288), P. carinii f.sp. hominis (human), P. carinii f.sp. mustelae (from ferret number 2245, provided by F. Gigliotti, University of Rochester), P. carinii f.sp. muris (one from a SCID mouse, provided by C. Sidman, University of Cincinnati, one from a C3H mouse provided by A. Harmsen, Trudeau Institute, and one from a S4-9 mouse at the University of
Cincinnati). Chromosomes were separated by clamped homogeneous electric ¢eld (CHEF) electrophoresis through 1% agarose in
0.5UTBE bu¡er at 14³C. The gel in (A) was run at 135 V with a 80^100 second ramp for 144 h. The gel in (B) was run at 135 V with a
70^90 s ramp for 144 h, then for an additional 24 h with a ramp of 100^110 s. The gel in (C) was run at 135 V with a 50^100 s ramp
for 104 h. Chromosomes were stained with ethidium bromide and photographed under illumination with ultraviolet light. The largest
band in the hPc lane is host DNA. Numbers at the left of each panel indicate positions of standard chromosomes that had been run in
an adjacent lane (bands not shown).
[11]. P. carinii has 70% as much DNA as S. cerevisiae, so might be expected to have 4200 open reading
frames. This number is similar to the number of
open reading frames in the genome of E. coli [10].
Determination of the number and identities of all
proteins encoded in the P. carinii genome will require
its sequencing.
3. Chromosomes
Because P. carinii chromosomes can be resolved
and visualized by electrophoresis, the collection of
DNA bands produced by this technique has come
to be called an electrophoretic karyotype. Electrophoretic karyotyping has shown that the P. carinii
f.sp. carinii genome is divided into at least 13 linear
chromosomes (see Fig. 1) [8]. The exact number of
chromosomes is di¤cult to specify for two reasons.
First, some bands may contain more than one chromosome. Second, P. carinii from di¡erent rat colonies can produce electrophoretic karyotypes with different numbers of bands, ranging from 13 to 15. It
seems reasonable to construe these data to mean that
the genome of P. carinii f.sp. carinii has 15 chromosomes, but further work is needed to con¢rm this.
The variation in electrophoretic karyotype seen
among di¡erent populations of P. carinii f.sp. carinii
indicates that this special form encompasses a variety
of strains, called karyotype forms (see Fig. 2) [8].
The report describing this phenomenon examined
67 populations of P. carinii from 10 di¡erent com-
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mercial rat colonies, and found four distinct karyotype forms. Further analysis raised the number of
distinct karyotype forms in rats to eight [15]. Karyotype forms of P. carinii f.sp. carinii have been shown
to be stable for at least a year [8]. A particular karyotype form can occur in multiple colonies and rat
strains. This suggests that the various karyotype
forms can infect any type of rat. It seems most likely
that the presence of a karyotype form in a particular
colony stems from the history of the colony. A strain
associated with a rat colony may have been in the
rats that were used to found the colony.
The di¡erent karyotype forms of P. carinii f.sp.
carinii tend to not vary at the DNA sequence level
(see article by Cushion in this issue). One interpretation of this phenomenon is that an individual organism is more likely to undergo a change in chromosome length than to acquire a point mutation. A
non-mutually exclusive alternative is that chromosome length variation is less deleterious than point
mutation. At any rate, the data from PFGE analysis
of populations of P. carinii f.sp. carinii suggest that
the most sensitive index of genetic diversity in
P. carinii is chromosome-length polymorphism. Electrophoretic karyotypes also di¡er among di¡erent
P. carinii special forms. Karyotype di¡erences led
to the discovery that rats can be infected by either
of two special forms, originally called prototype and
variant, and now called P. carinii f.sp. carinii, and
P. carinii f.sp. ratti [6,8,16]. Unlike the karyotype
forms of P. carinii f.sp. carinii, which exhibit very
little sequence divergence, P. carinii f.sp. carinii and
P. carinii f.sp. ratti appear to di¡er at all loci
[6,8,16,19^21] This has been shown by sequence
analysis of genes, and by hybridization experiments.
This sequence variation was ¢rst detected in a DNA
sequence from the locus encoding ribosomal RNA
[17]. These investigators called the organisms with
di¡erent ribosomal RNA sequences Pc1 and Pc2,
which correspond to P. carinii f.sp. carinii and
P. carinii f.sp. ratti, respectively. In addition to ratderived organisms, electrophoretic karyotypes of
P. carinii from humans (P. carinii f.sp. hominis),
mice (P. carinii f.sp. muris), and ferrets (P. carinii
f.sp. mustelae) have been visualized [6,9,18]. Fig. 1
shows these electrophoretic karyotypes compared to
each other and to that from rat P. carinii. The pulsed
¢eld gel procedure resolved 13 bands in P. carinii
f.sp. hominis, ranging in size from 0. 37 to 0.81 Mb
(see lane labeled hPc in Fig. 1A), and 15 bands in
Fig. 2. Karyotypic variation and detection of putative mitochondrial DNA in P. carinii f.sp. carinii. Five populations of P. carinii f.sp. carinii were subjected to ¢eld inversion gel electrophoresis (FIGE) through a 1% agarose gel (20U25 cm, total volume
of 200 ml) cast in 0.5UTBE. The electrophoresis bu¡er was
0.5UTBE plus 0.1 M glycine and was maintained at 6^8³C. For
the ¢rst 48 h, the gel was run at 132 V (with polarity alternated
between 50 s forward and 25 s backward). The bu¡er was then
changed and electrophoresis was continued at 100 V (50 s forward, 25 s backward) for an addition 96 h. (A) The DNA bands
visualized by staining with ethidium bromide and illumination
with ultraviolet light. Samples in lanes 3^5 are all karyotype
form 1 [8]. Samples in lanes 1 and 2 di¡er from form 1 in the
spacing of bands migrating between 485 and 700 kb. Non-alignment of bands in lanes 1 and 2 were an artifact of FIGE. (B) A
radiograph produced by hybridizing the bands in the gel to a radioactive 346 bp segment of the mitochondrial gene encoding the
large subunit rRNA (mt LSUrRNA) [57]. Two discreet bands
are present near the bottom of (B). The sizes of these two bands
were determined by CHEF (not shown) to be approximately
50 kb.
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P. carinii f.sp. mustelae, ranging in size from 0.50 to
0.87 Mb (see lane labeled fPc in Fig. 1B). Fig. 1C
shows a gel that resolved the genomes of three isolates of P. carinii f.sp. muris into 15 bands, ranging
in size from 0.35 to 0.61 Mb. The bands in mouse,
rat, human, and ferret P. carinii sum to 6.5, 7.0, 7.7,
and 11 Mb, respectively. Thus, the genomes of P.
carinii from di¡erent hosts appear to di¡er in size
by as much as 1.7-fold. Such large di¡erences in
genome size seem di¤cult to reconcile with the
view that this genus contains a single species, but
more work will be necessary to clarify this issue.
DNA hybridization has been used to identify the
chromosomes that carry speci¢c P. carinii f.sp. carinii genes [9,15]. In one of these studies, 15 genespeci¢c DNA probes were hybridized to the bands
produced by PFGE of the eight karyotype forms of
P. carinii f.sp. carinii. This procedure showed that
each probe hybridized to a single band in all electrophoretic karyotypes tested. None of these probes
hybridized to the same band. Consequently, there
is currently no information regarding gene linkage
in P. carinii. While no data on gene linkage are
available, hybridization to PFGE bands has suggested that di¡erent special forms may have di¡erent
linkage groups. In these experiments, seven genes
were mapped to a PFGE band in each of the two
special forms of P. carinii from rats (P. carinii f.sp.
carinii and P. carinii f.sp. ratti). Five of the 7 genes
fell on similarly sized chromosomes in the two special forms [6,19^22]. However, the IMP dehydrogenase gene mapped to chromosomes of 430 and 630 kb
in P. carinii f.sp. carinii and P. carinii f.sp. ratti,
respectively [23]. Similarly, the p55 gene mapped to
chromosomes that di¡ered by more than 100 kb in
length [24]. These large di¡erences in the size of the
chromosome carrying a speci¢c gene suggest that
translocations may have occurred during the divergence of these special forms. If this were the case, it
would tend to reduce the ability of homologous
chromosomes to pair during meiosis, thus increasing
the chance of reproductive isolation, and speciation.
To clarify the basis of major shifts in the sizes of
PFGE bands carrying a speci¢c gene, linkage groups
must be identi¢ed. This information can be acquired
by hybridizing a large number of DNA probes to
band resolved by PFGE. However, this tedious
task will not be necessary if a physical map of the
19
genome is constructed. Such a map will also facilitate
the determination of the complete sequence of the P.
carinii genome (see below).
4. Ploidy
The number of copies of each chromosome that
are contained within the nucleus of a single P. carinii
organism is not clear, but haploidy seems most
likely. Haploidy is suggested by the results of experiments that mapped genes to speci¢c bands in electrophoretic karyotypes. DNA hybridization has been
used to identify the chromosomes that carry 15
cloned P. carinii f.sp. carinii sequences [15]. In these
studies, the 15 DNA probes were hybridized to the
bands produced by PFGE of eight karyotype forms
of P. carinii f.sp. carinii. This procedure showed that
each probe hybridized to a single band in all electrophoretic karyotypes tested, indicating that if P. carinii are diploid, their homologous chromosomes do
not exhibit length polymorphism. The possibility
that chromosome length polymorphism does not occur in P. carinii seems unlikely because the DNA
hybridization experiments described above showed
that the chromosome carrying a given gene varied
in size from one karyotype form to another. Therefore, the absence of doublet bands in a electrophoretic karyotypes cannot be ascribed to lack of chromosome-length polymorphism in P. carinii. It is not
surprising that P. carinii exhibit chromosome-length
polymorphism because it is common in fungi [25].
Haploidy is also suggested by studies that have
measured the amount of DNA in an individual nucleus. One way to assess the DNA content per nucleus would be to measure the amount of DNA obtained from a known number of organisms.
Unfortunately, such measurements are di¤cult to
make with accuracy due to the high probability
that P. carinii preparations are contaminated with
rat DNA. In addition, populations of P. carinii contain two major di¡erent morphological forms (cysts
and trophic forms), which may not have the same
ploidy [26]. Typically, trophic forms far outnumber
cysts, but it is not uncommon for di¡erent populations of P. carinii to vary with respect to the cyst/
trophic form ratio. Therefore, the best way to measure the DNA content per nucleus is to use a dye that
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J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26
binds to DNA and stains the nucleus of individual P.
carinii organisms. The amount of DNA per nucleus
can be estimated by relating staining intensity to
DNA mass. Such studies have suggested that a P.
carinii nucleus contains 9 fg of DNA, which is very
close to the 8.5 fg predicted from the cumulative
molecular masses of bands in electrophoretic karyotypes [27]. Thus, these staining data suggest that
both the cyst and trophic forms of P. carinii are
haploid.
5. Gene structure and nomenclature
Nearly two dozen P. carinii genes have been
cloned and sequenced. Analysis of cloned genes
[19^22,28^46] has shown that the genome of P. carinii tends to be rich in adenine and thymidine residues. The typical P. carinii gene sequence is 60^65%
A+T. P. carinii genes usually contain introns, which
are typically less than 50 bp in length, but can be as
numerous as 9 per gene [20]. P. carinii introns are
often more AT-rich than exons, and are bordered by
canonical splice donor and acceptor sequences.
A standard system for naming P. carinii genes has
not been devised. Now is the opportune time to
adopt a standard nomenclature for naming P. carinii
genes because the entire sequence of the P. carinii
genome will be deciphered over the next few years.
Since P. carinii is a fungus, it would seem appropriate to adopt a nomenclature system similar to one
used for other fungi, such as the well studied ¢ssion
yeast, Schizosaccharomyces pombe, which is one of
the closest known relatives of P. carinii [34^36,47^
51]. In S. pombe, a gene is represented by three lower
case italic letters such as his. Genes that function in
same pathway are usually given the same three letter
symbol followed by an italic number to designate the
particular gene, such as his3 and his7 for two genes
that are needed for histidine synthesis. Wild-type alleles are represented by a superscript +, so the two
wild-type histidine genes would be written his3‡ and
his7‡ . Mutant alleles are written without the +, and
each di¡erent mutant allele is designated by a hyphen followed by a letter, number, or both, such as
his7-1.
While the S. pombe system has merit, it is not the
best for P. carinii, because most P. carinii genes will
be de¢ned by their sequence, not by their functionality. In the absence of a functional basis for de¢ning
a wild-type allele, there will be little need for including a + in the gene symbol. One could argue that the
most prevalent allele should be designated as wildtype, but most P. carinii genes are described in a
single isolate so the prevalence of the speci¢c sequence ¢rst obtained is usually not known. This limitation will apply to the vast majority of genes because these will be discovered when the P. carinii
f.sp. carinii genome is sequenced (see below). Therefore, we propose the the following set of rules for
naming P. carinii genes, alleles of the same gene,
gene families, and proteins. (1) Genes: genes will
each be symbolized by three italic letters. In cases
where function is known, or is suggested by DNA
or peptide sequence homology, the three letter italic
symbol will be derived from the function. Genes that
function in a related way will have the same three
letter symbol, followed by an italic numeral. (2) Alleles of a gene: the ¢rst allele (i.e. sequence) that is
found will be designated with a superscript 1, such as
xyz11 , and any subsequent alleles will be designated
as xyz12 , etc. (3) Gene families: genes that are members of gene families will all be given the same three
letter italic name, followed by an italic numeral.
Each gene in the family will have a di¡erent numeral
appended, such as msg1, msg2, etc. The entire gene
family will be symbolized by the same three letters
written in capital roman font, such as MSG. (4)
Proteins: when possible, a protein will be symbolized
by the same three letters as the gene that produced
that protein, but the three letters will be written in
roman print with the ¢rst letter an uppercase, such
as Xyz1, Msg1, etc.
6. Sequence heterogeneity among special forms of
P. carinii
Genetic divergence has been demonstrated and
quanti¢ed by gene sequencing. The broadest P. carinii gene sequence database is for a 300-bp segment
of the mitochondrial gene encoding the large subunit
rRNA (mt LSUrRNA). A sequence from this locus
has been determined for special forms of P. carinii
from nine host species (rat, mouse, shrew, rabbit,
ferret, pig, horse, monkey, and human) [16,34,52^
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J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26
56]. Sequence variation at this locus ranges between
4 and 27%. Several of these nine special forms of
P. carinii have been analyzed at other loci, which
showed that DNA sequence di¡erences are not peculiar to the mt LSUrRNA locus [16,19,28,30,33,34,43,
44,54,55,57^71].
Eight di¡erent loci have been compared in P. carinii from humans and rats [34,43,54,60,61,70,72,73].
All of these loci di¡ered between the two organisms,
with variation approaching 50% at the internal transcribed spacer (ITS) region of the nuclear gene encoding ribosomal RNAs [70].
The divergence seen in the gene sequences of different P. carinii special forms appears to be genomewide. Genomic DNA from P. carinii f.sp. carinii hybridized very poorly to genomic DNA from P. carinii f.sp. ratti [16] or to human P. carinii [60,61].
Similarly, three genes from P. carinii f.sp. carinii
did not hybridize to chromosomes from ferret or
mouse P. carinii [9].
7. Repeated DNA
Some of the P. carinii genome is comprised of
DNA sequences that are present dozens of times.
Three families of repeated DNA sequences have
been described. Two of these families (MSG and
PRT) encode proteins. The third family is the telomere repeat, which is found at the ends of each
chromosome, and sometimes at internal chromosomal sites, in which case it has been called the alpha
repeat [18]. It is interesting to note here that genes
encoding the 18S, 26S and 5.8S ribosomal RNAs
(rRNA) are not repeated (more than twice) in
P. carinii f.sp. carinii [13] (no data are available for
other special forms). This low copy number of the
genes encoding rRNA is rather unusual.
7.1. The protease gene family
Lugli et al. cloned a gene they call PRT1, which
encodes a subtilisin-like serine protease [40]. DNA
hybridization showed that sequences related to
PRT1 are distributed on all but one of the 11
P. carinii f.sp. carinii chromosomes that were resolved by the experiment. The sequences of additional cloned PRT family members indicated that the
21
genome encodes multiple subtilisin-like serine proteases, which di¡er in sequence. The presence of a
gene family encoding multiple forms of this kind of
protease is unusual and intriguing. There may be a
connection between the PRT protease family and the
expression of the major surface glycoprotein (Msg),
which is also encoded by a multi-gene family. PRT
genes tend to be linked to MSG genes, and production of Msg may involve a proteolytic cleavage of a
pre-Msg by a subtilisin-like serine protease [40,74]
(see below).
7.2. The major surface glycoprotein gene family
P. carinii are coated with a major surface glycoprotein (Msg) [75^85], also known as gpA [86,87]. In
P. carinii, special forms recovered from rats, ferrets,
mice, and humans, it has been shown that Msg is
actually a family of proteins encoded by a family
of heterogeneous genes [58,59,61,66^68,74,88,89].
The genome of P. carinii f.sp. carinii contains approximately 100 di¡erent MSG genes [6,88], which
are organized in clusters that are located at the ends
of each of the chromosomes [6,58,59,61,62,67]. Expression of the MSG gene family within an individual organism is thought to be limited. Phenotypic
data indicating limited expression include indirect
immuno£uorescence studies that showed that not
all organisms within a population could be labeled
with an antibody directed against a subset of Msg
isoforms [90]. In addition, the fraction of organisms
labeled by such an antibody varied among populations [90]. Additional phenotypic evidence of limited
expression of the MSG family has been provided by
Western blotting, which showed that some P. carinii
f.sp. carinii populations contained a particular Msg
epitope in abundance, and other populations did not
[91].
Antigenic variation appears to be achieved by allowing only one MSG gene to be transcribed in a
given organism. The transcribed MSG gene resides
at a speci¢c locus, called the expression site, which
can be occupied by di¡erent MSG genes in di¡erent
organisms [92,93]. It is believed that the expressed
MSG can be changed by changing the gene that is
attached to the expression site [92^96]. Switching is
presumably accomplished by some form of DNA
recombination, but the frequency and mechanism
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of these recombination events have not yet been determined. One possibility is that a site-speci¢c recombinase is involved [92]. Such a recombinase would
provide the organism with a means to switch surface
antigens at high frequency. Alternatively, switching
may occur via recombination between homologous
DNA sequences in donor and expressed MSG genes.
It is possible that MSG gene switching contributes to
the high frequency of chromosome length polymorphism among populations of P. carinii f.sp. carinii.
The expression site contains the upstream conserved
sequence (UCS), a 300-bp sequence that is at the 5Pend of every MSG mRNA [92^94,96]. It is probable
that translation of MSG mRNA begins in the UCS,
because a large protein containing the UCS is
present in P. carinii [97]. However, the UCS is not
present on the mature Msg found on the cell surface,
suggesting that the UCS is removed from the Msg
precursor by a protease [97]. The UCS peptide contains a site that would be cut by a protease like Prt1
[40]. The linkage of MSG genes and PRT genes presenting the intriguing possibility that the organism
coordinates expression of the two gene families.
7.3. Telomere repeats
The ends of P. carinii f.sp. carinii chromosomes
are made of tandem repeats of the sequence
TTAGGG [14]. Such repeated structures are typical
of the telomeres of eucaryotic chromosomes. The 6bp repeat found at P. carinii telomeres appears to be
located at some non-telomeric loci as well, such as
upstream of the K-tubulin gene [18]. Such internal
telomere-like sequences are thought to be generated
by the action of telomerase, which is the enzyme
responsible for adding new telomere repeats to chromosome ends. Internal repeats may have been added
after a double-strand break. If the modi¢ed break
were then joined, an internal telomere-like sequence
would be left. Another possibility is that internal
telomere-like sequences were formed when two telomeres fused.
8. Mitochondrial genome
As far as is known, the mitochondrial genome of
P. carinii f.sp. carinii resembles that of other fungi. A
6.8-kb segment of the mitochondrial genome has
been cloned. Sequence analysis and DNA hybridization studies have shown that this cloned segment
contained seven genes (ribosomal RNA, NADH subunits 1, 2, 3, and 6, cytochrome oxidase II and apocytochrome b) found in fungal mitochondria [31].
The size of the mitochondrion genome is not known,
but recent PFGE studies (shown in Fig. 2) have detected a band that hybridized to a probe from P.
carinii f.sp. carinii mitochondrial DNA. This band
migrated between linear DNA markers 50 and 100
kb in size. These data suggest that the mitochondrial
genome of P. carinii may be similar in size to that of
S. cerevisiae, which is 78 kb [98].
9. The P. carinii genome project
Determination of the complete sequence of the P.
carinii genome is both practicable and of primary
importance to the understanding of this organism.
The small size of the P. carinii genome and its packaging into chromosomes that are resolvable by
PFGE will facilitate sequence analysis. The DNA
sequence will move the ¢eld forward in a variety of
ways. It will identify all of the proteins encoded by
P. carinii, which will provide an incomparably valuable view of this fastidious organism's metabolic
capabilities and de¢ciencies. These data may lead
to improvements in our ability to culture P. carinii,
and could suggest new targets for anti-Pneumocystis
drugs. The sequence will also reveal how genes are
organized into chromosomes, and de¢ne the distribution and identity of repeated genes and other repeated DNA sequences. This information will be
necessary for understanding how MSG genes are
used to achieve surface variation, and whether other
surface proteins, like those of the PRT family, can
vary as well. Understanding such variation and the
mechanism underlying it may also present new avenues to therapy.
The genome sequencing project will be accomplished by ¢rst constructing a physical map of each
chromosome by creating and characterizing libraries
of cloned DNA fragments, as has been done for
other fungi [99]. Libraries of the P. carinii genome
have been made previously, including one in which
relatively large DNA segments were inserted into the
FEMSIM 890 6-10-98
J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26
genome of phage P1 [100]. For the sequencing project, segments carrying 50 kb of P. carinii DNA will
be inserted into cosmid vectors. Cosmids carrying
DNA from a particular chromosome will be identi¢ed and the inserts in these cosmids mapped along
the length of that chromosome [99]. Once a collection of clones that cover the chromosome is obtained, then each cosmid clone in this collection
will be divided into smaller pieces, which are sequenced. This approach allows the sequencing e¡ort
to be divided into biologically relevant segments
(chromosomes), each of which can be analyzed independently by di¡erent laboratories. The physical
maps and collections of ordered clones that will be
produced during the ¢rst phase of the genome project will also provide useful DNA reagents for
P. carinii research.
[7]
[8]
[9]
[10]
[11]
[12]
10. Conclusions
Analysis of the genome of P. carinii has already
revealed much of what is known about diversity
among these organisms, and has provided novel insights into their biochemical and structural properties. Ultimately, genome research will give us the
capacity to identify every protein encoded by these
mysterious organisms.
References
[13]
[14]
[15]
[16]
[1] Anonymous (1994) Revised nomenclature for Pneumocystis
carinii. The Pneumocystis Workshop. J. Eukaryotic Microbiol. 41, 121S^122S.
[2] Pifer, L.L., Pifer, D.D., Woods, D.R., Joyner, R.E. and Edwards, C.C. (1986) Preliminary studies on the development of
a vaccine for Pneumocystis carinii. I. Immunological and biochemical characterization. Vaccine 4, 257^265.
[3] Gradus, M.S., Gilmore, M. and Lerner, M. (1988) An isolation method of DNA from Pneumocystis carinii : a quantitative comparison to known parasitic protozoan DNA. Comp.
Biochem. Physiol. B 89, 75^77.
[4] Fishman, J.A., Ullu, E., Armstrong, M.Y. and Richards, F.F.
(1989) Organization of DNA and RNA from rat Pneumocystis
carinii. J. Protozool. 36, 4S^5S.
[5] Yoganathan, T., Lin, H. and Buck, G.A. (1989) An electrophoretic karyotype and assignment of ribosomal genes to resolved chromosomes of Pneumocystis carinii. Mol. Microbiol.
3, 1473^1480.
[6] Hong, S.T., Steele, P.E., Cushion, M.T., Walzer, P.D., String-
[17]
[18]
[19]
[20]
[21]
23
er, S.L. and Stringer, J.R. (1990) Pneumocystis carinii karyotypes. J. Clin. Microbiol. 28, 1785^1795.
Lundgren, B., Cotton, R., Lundgren, J.D., Edman, J.C. and
Kovacs, J.A. (1990) Identi¢cation of Pneumocystis carinii
chromosomes and mapping of ¢ve genes. Infect. Immun. 58,
1705^1710.
Cushion, M.T., Kaselis, M., Stringer, S.L. and Stringer, J.R.
(1993) Genetic stability and diversity of Pneumocystis carinii
infecting rat colonies. Infect. Immun. 61, 4801^4813.
Weinberg, G.A. and Durant, P.J. (1994) Genetic diversity
of Pneumocystis carinii derived from infected rats, mice, ferrets, and cell cultures. J. Eukaryotic Microbiol. 41, 223^
228.
Blattner, F.R. and Plunkett, G., III, Bloch, C.A., Perna, N.T.,
Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D.,
Rode, C.K., Mayhew, G.F. et al. (1997) The complete genome
sequence of Escherichia coli K-12. Science 277, 1453^1474.
Go¡eau, A., Barrell, B.G., Bussey, H., Davis, R.W., Dujon,
B., Feldmann, H., Galibert, F., Hoheisel, J.D., Jacq, C., Johnston, M. et al. (1996) Life with 6000 genes. Science 274, 546,
563^567.
Sloand, E., Laughon, B., Armstrong, M., Bartlett, M.S., Blumenfeld, W., Cushion, M., Kalica, A., Kovacs, J.A., Martin,
W., Pitt, E. et al. (1993) The challenge of Pneumocystis carinii
culture. J. Eukaryotic Microbiol. 40, 188^195.
Giuntoli, D., Stringer, S.L. and Stringer, J.R. (1994) Extraordinarily low number of ribosomal RNA genes in P. carinii.
J. Eukaryotic Microbiol. 41, 88S.
Underwood, A.P., Louis, E.J., Borts, R.H., Stringer, J.R. and
Wake¢eld, A.E. (1996) Pneumocystis carinii telomere repeats
are composed of TTAGGG and the subtelomeric sequence
contains a gene encoding the major surface glycoprotein.
Mol. Microbiol. 19, 273^281.
Cushion, M.T., Balows, A. and Sussman, M., Eds. (1997)
Topley and Wilson's Microbiology and Microbial Infections,
Pneumocystis carinii, 9th edn., Edward Arnold, London.
Cushion, M.T., Zhang, J., Kaselis, M., Giuntoli, D., Stringer,
S.L. and Stringer, J.R. (1993) Evidence for two genetic variants of Pneumocystis carinii coinfecting laboratory rats.
J. Clin. Microbiol. 31, 1217^1223.
Liu, Y., Rocourt, M., Pan, S., Liu, C. and Leibowitz, M.J.
(1992) Sequence and variability of the 5.8S and 26S rRNA
genes of Pneumocystis carinii. Nucleic Acids Res. 20, 3763^
3772.
Zhang, J., Cushion, M.T. and Stringer, J.R. (1993) Molecular
characterization of a novel repetitive element from Pneumocystis carinii from rats. J. Clin. Microbiol. 31, 244^248.
Meade, J.C. and Stringer, J.R. (1995) Cloning and characterization of an ATPase gene from Pneumocystis carinii which
closely resembles fungal H+ ATPases. J. Eukaryotic Microbiol. 42, 298^307.
Smulian, A.G., Ryan, M., Staben, C. and Cushion, M. (1996)
Signal transduction in Pneumocystis carinii: characterization
of the genes (pcg1) encoding the alpha subunit of the G protein (PCG1) of Pneumocystis carinii carinii and Pneumocystis
carinii ratti. Infect. Immun. 64, 691^701.
Sunkin, S.M. and Stringer, J.R. (1995) Transcription factor
FEMSIM 890 6-10-98
24
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26
genes from rat Pneumocystis carinii. J. Eukaryotic Microbiol.
42, 12^19.
Stedman, T.T. and Buck, G.A. (1996) Identi¢cation, characterization, and expression of the BiP endoplasmic reticulum
resident chaperonins in Pneumocystis carinii. Infect. Immun.
64, 4463^4471.
Weinberg, G.A., O'Gara, M.J. and Cushion, M.T. (1994) Coinfection of rats with genetically diverse forms of Pneumocystis carinii demonstrated by P. carinii inosine monophosphate
dehydrogenase gene polymorphism. J. Eukaryotic Microbiol.
41, 119S.
Smulian, A.G., Theus, S.A., Denko, N., Walzer, P.D. and
Stringer, J.R. (1993) A 55 kDa antigen of Pneumocystis carinii : analysis of the cellular immune response and characterization of the gene. Mol. Microbiol. 7, 745^753.
Zolan, M.E. (1995) Chromosome-length polymorphism in
fungi. Microbiol. Rev. 59, 686^698.
Ru¡olo, J.J. and Walzer, P.D., Eds. (1994) Pneumocystis carinii Pneumonia, Vol. 2, Pneumocystis carinii Cell Structure,
2nd edn., pp. 25^43. Marcel Dekker, New York.
Wyder, M.A., Rasch, E.M. and Kaneshiro, E.S. (1994) Assessment of Pneumocystis carinii DNA content. J. Eukaryotic
Microbiol. 41, 120S.
Edman, J.C., Kovacs, J.A., Masur, H., Santi, D.V., Elwood,
H.J. and Sogin, M.L. (1988) Ribosomal RNA sequence shows
Pneumocystis carinii to be a member of the fungi. Nature 334,
519^522.
Edman, J.C., Edman, U., Cao, M., Lundgren, B., Kovacs,
J.A. and Santi, D.V. (1989) Isolation and expression of the
Pneumocystis carinii dihydrofolate reductase gene. Proc. Natl.
Acad. Sci. USA 86, 8625^8629.
Edman, U., Edman, J.C., Lundgren, B. and Santi, D.V. (1989)
Isolation and expression of the Pneumocystis carinii thymidylate synthase gene. Proc. Natl. Acad. Sci. USA 86, 6503^6507.
Pixley, F.J., Wake¢eld, A.E., Banerji, S. and Hopkin, J.M.
(1991) Mitochondrial gene sequences show fungal homology
for Pneumocystis carinii. Mol. Microbiol. 5, 1347^1351.
Ypma Wong, M.F., Fonzi, W.A. and Sypherd, P.S. (1992)
Fungus-speci¢c translation elongation factor 3 gene present
in Pneumocystis carinii. Infect. Immun. 60, 4140^4145.
Banerji, S., Wake¢eld, A.E., Allen, A.G., Maskell, D.J., Peters, S.E. and Hopkin, J.M. (1993) The cloning and characterization of the arom gene of Pneumocystis carinii. J. Gen. Microbiol. 139, 2901^2914.
Keely, S., Pai, H.J., Baughman, R., Sidman, C., Sunkin, S.M.,
Stringer, J.R. and Stringer, S.L. (1994) Pneumocystis species
inferred from analysis of multiple genes. J. Eukaryotic Microbiol. 41, 94S.
Li, J. and Edlind, T. (1994) Phylogeny of Pneumocystis carinii
based on b-tubulin sequence. J. Eukaryotic Microbiol. 41,
97S.
Fletcher, L.D., McDowell, J.M., Tidwell, R.R., Meagher,
R.B., Dyskstra and C.C. (1994) Structure, expression and
phylogenetic analysis of the gene encoding actin I in Pneumocystis carinii. Genetics 137, 743^750.
Volpe, F., Ballantine, S.P., Delves, C.J. (1993) The multifunctional folic acid synthesis fas gene of Pneumocystis carinii
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase. Eur.
J. Biochem. 216, 449^458.
O'Gara, M.J., Lee, C.H., Weinberg, G.A., Nott, J.M. and
Queener, S.F. (1997) IMP dehydrogenase from Pneumocystis
carinii as a potential drug target. Antimicrob. Agents Chemother. 41, 40^48.
Narasimhan, S., Armstrong, M.Y., Rhee, K., Edman, J.C.,
Richards, F.F. and Spicer, E. (1994) Gene for an extracellular
matrix receptor protein from Pneumocystis carinii. Proc. Natl.
Acad. Sci. USA 91, 7440^7444.
Lugli, E.B., Allen, A.G. and Wake¢eld, A.E. (1997) A Pneumocystis carinii multi-gene family with homology to subtilisinlike serine proteases. Microbiology 143, 2223^2236.
Fletcher, L.D., Berger, L.C., Tidwell, R.R. and Dykstra, C.C.
(1993) Cloning and characterization of the calmodulin-encoding gene from Pneumocystis carinii. Gene 129, 307^308.
Fletcher, L.D., Berger, L.C., Peel, S.A., Baric, R.S., Tidwell,
R.R. and Dykstra, C.C. (1993) Isolation and identi¢cation of
six Pneumocystis carinii genes utilizing codon bias. Gene 129,
167^174.
Edlind, T.D., Bartlett, M.S., Weinberg, G.A., Prah, G.N. and
Smith, J.W. (1992) The beta-tubulin gene from rat and human
isolates of Pneumocystis carinii. Mol. Microbiol. 6, 3365^3373.
Dyer, M., Volpe, F., Delves, C.J., Somia, N., Burns, S. and
Scaife, J.G. (1992) Cloning and sequence of a beta-tubulin
cDNA from Pneumocystis carinii: possible implications for
drug therapy. Mol. Microbiol. 6, 991^1001.
Christopher, L.J., Fletcher, L.D. and Dykstra, C.C. (1995)
Cloning and identi¢cation of Arp1, an actin-related protein
from Pneumocystis carinii. J. Eukaryotic Microbiol. 42, 142^
149.
Banerji, S., Lugli, E.B., Miller, R.F. and Wake¢eld, A.E.
(1995) Analysis of genetic diversity at the arom locus in isolates of Pneumocystis carinii. J. Eukaryotic Microbiol. 42,
675^679.
Van der Peer, Y., Hendriks, L., Goris, A., Neefs, N., Vancanneyt, M., Kersters, K., Berny, J., Hennebert, G.L. and De
Wachter, R. (1992) Evolution of basidiomyceteous yeasts as
deduced from small ribosomal subunit RNA sequences. System Appl. Microbiol. 15, 250^258.
Bruns, T.D., Vigalys, R., Barns, S.M., Gonzalez, D., Hibbett,
D.S., Lane, D.J., Simon, L., Stickel, S., Szaro, T.M., Weisberg, W.G. et al. (1992) Evolutionary relationships within the
fungi : analyses of nuclear small subunit rRNA sequences.
Mol. Phys. Evol. 1, 231^241.
Edman, J.C., Sogin M.L. and Walzer, P.D., Eds. (1994) Pnemocystis carinii Pneumonia, Vol. 5, Molecular Phylogeny of
Pneumocystis carinii, 2nd edn., pp. 91^105. Marcel Dekker,
New York.
Baldauf, S.L. and Palmer, J.D. (1993) Animals and fungi are
each other's closest relatives : congruent evidence from multiple proteins. Proc. Natl. Acad. Sci. USA 90, 11558^11562.
Eriksson, O.E. (1994) Pneumocystis carinii, a parasite in lungs
of mammals, referred to a new family and order (Pneumocystidaceae, Pneumocystidales, Ascomycota). Systema Ascomycetum 13, 165^180.
FEMSIM 890 6-10-98
J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26
[52] Sinclair, K., Wake¢eld, A.E., Banerji, S. and Hopkin, J.M.
(1991) Pneumocystis carinii organisms derived from rat and
human hosts are genetically distinct. Mol. Biochem. Parasitol.
45, 183^184.
[53] Furuta, T., Fujita, M., Mukai, R., Sakakibara, I., Sata, T.,
Miki, K., Hayami, M., Kojima, S. and Yoshikawa, Y. (1993)
Severe pulmonary pneumocystosis in simian acquired immunode¢ciency syndrome induced by simian immunode¢ciency
virus : its characterization by the polymerase-chain-reaction
method and failure of experimental transmission to immunode¢cient animals. Parasitol. Res. 79, 624^628.
[54] Peters, S.E., Wake¢eld, A.E., Whitwell, K.E. and Hopkin,
J.M. (1994) Pneumocystis carinii pneumonia in thoroughbred
foals : identi¢cation of a genetically distinct organism by DNA
ampli¢cation. J. Clin. Microbiol. 32, 213^216.
[55] Peters, S.E., English, K., Laakkonen, J. and Gurnell, J. (1994)
DNA analysis of Pneumocystis carinii infecting Finnish and
English shrews. J. Eukaryotic Microbiol. 41, 108S.
[56] Wake¢eld, A.E., Keely, S.P., Stringer, J.R., Christensen, C.B.,
Ahrens, P., Peters, S.E., Bille-Hansen, V., Henriksen, S.A.,
Jorsal, S.E. and Settnes, O.P. (1997) Identi¢cation of porcine
Pneumocystis carinii as a genetically distinct organism by
DNA ampli¢cation. APMIS 105, 317^321.
[57] Wake¢eld, A.E., Pixley, F.J., Banerji, S., Sinclair, K., Miller,
R.F., Moxon, E.R. and Hopkin, J.M. (1990) Ampli¢cation of
mitochondrial ribosomal RNA sequences from Pneumocystis
carinii DNA of rat and human origin. Mol. Biochem. Parasitol. 43, 69^76.
[58] Haidaris, P.J., Wright, T.W., Gigliotti, F. and Haidaris, C.G.
(1992) Expression and characterization of a cDNA clone encoding an immunodominant surface glycoprotein of Pneumocystis carinii. J. Infect. Dis. 166, 1113^1123.
[59] Kovacs, J.A., Powell, F., Edman, J.C., Lundgren, B., Martinez, A., Drew, B. and Angus, C.W. (1993) Multiple genes
encode the major surface glycoprotein of Pneumocystis carinii.
J. Biol. Chem. 268, 6034^6040.
[60] Stringer, J.R., Stringer, S.L., Zhang, J., Baughman, R., Smulian, A.G. and Cushion, M.T. (1993) Molecular genetic distinction of Pneumocystis carinii from rats and humans. J. Eukaryotic Microbiol. 40, 733^741.
[61] Stringer, S.L., Garbe, T., Sunkin, S.M. and Stringer, J.R.
(1993) Genes encoding antigenic surface glycoproteins in
Pneumocystis from humans. J. Eukaryotic Microbiol. 40,
821^826.
[62] Wada, M., Kitada, K., Saito, M., Egawa, K. and Nakamura,
Y. (1993) cDNA sequence diversity and genomic clusters of
major surface glycoprotein genes of Pneumocystis carinii. J.
Infect. Dis. 168, 979^985.
[63] Zhang, J. and Stringer, J.R. (1993) Cloning and characterization of an alpha-tubulin-encoding gene from rat-derived Pneumocystis carinii. Gene 123, 137^141.
[64] Banerji, S., Lugli, E.B. and Wake¢eld, A.E. (1994) Identi¢cation of two genetically distinct strains of Pneumocystis carinii
in infected ferret lungs. J. Eukaryotic Microbiol. 41, 73S.
[65] Dei-Cas, E., Mazars, E., Ferragut, C.O., Durand, I., Aliouat,
E.M., Dridba, M., Palluault, F., Cailliez, J.C., Seguy, N.,
Tibayrenc, M. et al. (1994) Ultrastructural, genomic, isoenzy-
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
25
matic and biological features make it possible to distinguish
rabbit Pneumocystis from other mammal Pneumocystis
strains. J. Eukaryotic Microbiol. 41, 84S.
Garbe, T.R. and Stringer, J.R. (1994) Molecular characterization of clustered variants of genes encoding major surface
antigens of human Pneumocystis carinii. Infect. Immun. 62,
3092^3101.
Sunkin, S.M., Stringer, S.L. and Stringer, J.R. (1994) A tandem repeat of rat-derived Pneumocystis carinii genes encoding
the major surface glycoprotein. J. Eukaryotic Microbiol. 41,
292^300.
Wright, T.W., Simpson Haidaris, P.J., Gigliotti, F., Harmsen,
A.G. and Haidaris, C.G. (1994) Conserved sequence homology of cysteine-rich regions in genes encoding glycoprotein A
in Pneumocystis carinii derived from di¡erent host species.
Infect. Immun. 62, 1513^1519.
Wake¢eld, A.E., Pixley, F.J., Banerji, S., Sinclair, K., Miller,
R.F., Moxon, E.R. and Hopkin, J.M. (1990) Detection of
Pneumocystis carinii with DNA ampli¢cation [see comments].
Lancet 336, 451^453.
Ortiz-Rivera, M., Liu, Y., Felder, R. and Leibowitz, M.J.
(1995) Comparison of coding and spacer region sequences of
chromosomal rRNA-coding genes of two sequevars of Pneumocystis carinii. J. Eukaryotic Microbiol. 42, 44^49.
Mazars, E., Odberg-Ferragut, C., Dei-Cas, E., Fourmaux, M.,
Aliouat, E., Brun-Pascaud, M., Mougeot, G. and Camus, D.
(1995) Polymorphism of the thymidylate synthase gene of
Pneumocystis carinii from di¡erent host species. J. Eukaryotic
Microbiol. 42, 26^32.
Liu, Y. and Leibowitz, M.J. (1993) Variation and in vitro
splicing of group I introns in rRNA genes of Pneumocystis
carinii. Nucleic Acids Res. 21, 2415^2421.
Banerji, S., Lugli, E., Miller, R. and Wake¢eld, A. (1995)
Analysis of genetic diversity at the arom locus in isolates
of Pneumocystis carinii. J. Eukaryotic Microbiol. 42, 675^
678.
Wada, M. and Nakamura, Y. (1994) MSG gene cluster encoding major cell surface glycoproteins of rat Pnuemocystis carinii. DNA Res. 1, 163^168.
Walzer, P.D. and Linke, M.J. (1987) A comparison of the
antigenic characteristics of rat and human Pneumocystis carinii by immunoblotting. J. Immunol. 138, 2257^2265.
Kovacs, J.A., Halpern, J.L., Swan, J.C., Moss, J., Parrillo,
J.E. and Masur, H. (1988) Identi¢cation of antigens and antibodies speci¢c for Pneumocystis carinii. J. Immunol. 140,
2023^2031.
Pesanti, E.L. and Shanley, J.D. (1988) Glycoproteins of Pneumocystis carinii : characterization by electrophoresis and microscopy. J. Infect. Dis. 158, 1353^1359.
Linke, M.J. and Walzer, P.D. (1989) Analysis of a surface
antigen of Pneumocystis carinii. J. Protozool. 36, 60S^61S.
Radding, J.A., Armstrong, M.Y., Ullu, E. and Richards, F.F.
(1989) Identi¢cation and isolation of a major cell surface glycoprotein of Pneumocystis carinii. Infect. Immun. 57, 2149^
2157.
Tanabe, K., Takasaki, S., Watanabe, J., Kobata, A., Egawa,
K. and Nakamura, Y. (1989) Glycoproteins composed of ma-
FEMSIM 890 6-10-98
26
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26
jor surface immunodeterminants of Pneumocystis carinii. Infect. Immun. 57, 1363^1368.
Lundgren, B., Lipschik, G.Y. and Kovacs, J.A. (1991) Puri¢cation and characterization of a major human Pneumocystis
carinii surface antigen. J. Clin. Invest. 87, 163^170.
Lundgren, B., Koch, C., Mathiesen, L., Nielsen, J.O. and
Hansen, J.E. (1993) Glycosylation of the major human Pneumocystis carinii surface antigen. APMIS 101, 194^200.
Zimmerman, P.E., Voelker, D.R., McCormack, F.X., Paulsrud, J.R. and Martin, W.J. (1992) 120-kD surface glycoprotein of Pneumocystis carinii is a ligand for surfactant protein
A. J. Clin. Invest. 89, 143^149.
Linke, M.J. and Walzer, P.D. (1991) Identi¢cation and puri¢cation of a soluble species of gp120 released by zymolyase
treatment of Pneumocystis carinii. J. Protozool. 38, 176S^
178S.
McCormack, F.X., Festa, A.L., Andrews, R.P., Linke, M.
and Walzer, P.D. (1997) The carbohydrate recognition domain of surfactant protein A mediates binding to the major
surface glycoprotein of Pneumocystis carinii. Biochemistry 36,
8092^8099.
Gigliotti, F., Ballou, L.R., Hughes, W.T. and Mosley, B.D.
(1988) Puri¢cation and initial characterization of a ferret
Pneumocystis carinii surface antigen. J. Infect. Dis. 158, 848^
854.
Gigliotti, F. (1992) Host species-speci¢c antigenic variation of
a mannosylated surface glycoprotein of Pneumocystis carinii.
J. Infect. Dis. 165, 329^336.
Stringer, S.L., Hong, S.T., Giuntoli, D. and Stringer, J.R.
(1991) Repeated DNA in Pneumocystis carinii. J. Clin. Microbiol. 29, 1194^1201.
Linke, M.J., Smulian, A.G., Stringer, J.R. and Walzer, P.D.
(1994) Characterization of multiple unique cDNAs encoding
the major surface glycoprotein of rat-derived Pneumocystis
carinii. Parasitol. Res. 80, 478^486.
Angus, C.W., Tu, A., Vogel, P., Qin, M. and Kovacs, J.A.
(1996) Expression of variants of the major surface glycoprotein of Pneumocystis carinii. J. Exp. Med. 183, 1229^1234.
[91] Vasquez, J., Smulian, A.G., Linke, M.J. and Cushion, M.T.
(1996) Antigenic di¡erences associated with genetically distinct Pneumocystis carinii from rats. Infect. Immun. 64, 290^
297.
[92] Wada, M., Sunkin, S.M., Stringer, J.R. and Nakamura, Y.
(1995) Antigenic variation by positional control of major
surface glycoprotein gene expression in Pneumocystis carinii.
J. Infect. Dis. 171, 1563^1568.
[93] Sunkin, S.M. and Stringer, J.R. (1996) Translocation of surface antigen genes to a unique telomeric expression site in
Pneumocystis carinii. Mol. Microbiol. 19, 283^295.
[94] Edman, J.C., Hatton, T.W., Nam, M., Turner, R., Mei,
Q., Angus, C.W. and Kovacs, J.A. (1996) A single expression site with a conserved leader sequence regulates variation
of expression of the Pneumocystis carinii family of
major surface glycoprotein genes. DNA Cell Biol. 15, 989^
999.
[95] Wada, M. and Nakamura, Y. (1996) Unique telomeric expression site of major-surface-glycoprotein genes of Pneumocystis carinii. DNA Res. 3, 55^64.
[96] Sunkin, S.M. and Stringer, J.R. (1997) Residence at the expression site is necessary and su¤cient for the transcription
of surface antigen genes of Pneumocystis carinii. Mol. Microbiol. 25(1), 147^160.
[97] Sunkin, S.M., Linke, M., McCormack, F.X., Walzer, P.D.
and Stringer, J.R. (1998) Identi¢cation of a putative precursor to the major surface glycoprotein of Pneumocystis carinii.
Infect. Immun., in press.
[98] de Zamaroczy, M. and Bernardi, G. (1986) The primary
structure of the mitochondrial genome of Saccharomyces cerevisiae ^ a review. Gene 47, 155^177.
[99] Xiong, M., Chen, H.J., Prade, R.A., Wang, Y., Gri¤th, J.,
Timberlake, W.E. and Arnold, J. (1996) On the consistency
of a physical mapping method to reconstruct a chromosome
in vitro. Genetics 142, 267^284.
[100] Metcheva, I.S., Stedman, T.T. and Buck, G.A. (1996) An
arrayed bacteriophage P1 genomic library of Pneumocystis
carinii. J. Eukaryotic Microbiol. 43, 171^176.
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