* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Standard PDF - Wiley Online Library
Copy-number variation wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Whole genome sequencing wikipedia , lookup
X-inactivation wikipedia , lookup
DNA vaccination wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Epigenomics wikipedia , lookup
Molecular cloning wikipedia , lookup
Gene desert wikipedia , lookup
Primary transcript wikipedia , lookup
Oncogenomics wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Ridge (biology) wikipedia , lookup
Mitochondrial DNA wikipedia , lookup
Public health genomics wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Point mutation wikipedia , lookup
Genomic imprinting wikipedia , lookup
Metagenomics wikipedia , lookup
Transposable element wikipedia , lookup
Gene expression programming wikipedia , lookup
Genetic engineering wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Pathogenomics wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Human Genome Project wikipedia , lookup
Gene expression profiling wikipedia , lookup
Genome (book) wikipedia , lookup
Human genome wikipedia , lookup
Non-coding DNA wikipedia , lookup
Minimal genome wikipedia , lookup
Genomic library wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Designer baby wikipedia , lookup
History of genetic engineering wikipedia , lookup
Microevolution wikipedia , lookup
Genome editing wikipedia , lookup
Helitron (biology) wikipedia , lookup
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 FEMSIM 890 6-10-98 16 J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26 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 FEMSIM 890 6-10-98 J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26 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- FEMSIM 890 6-10-98 18 J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26 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. FEMSIM 890 6-10-98 J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26 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 FEMSIM 890 6-10-98 20 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^ FEMSIM 890 6-10-98 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 FEMSIM 890 6-10-98 22 J.R. Stringer, M.T. Cushion / FEMS Immunology and Medical Microbiology 22 (1998) 15^26 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. FEMSIM 890 6-10-98