Gene arrays at Pneumocystis carinii telomeres

Abstract

In the fungus Pneumocystis carinii, at least three gene families (PRT1, MSR, and MSG) have the potential to generate high-frequency antigenic variation, which is likely to be a strategy by which this parasitic fungus is able to prolong its survival in the rat lung. Members of these gene families are clustered at chromosome termini, a location that fosters recombination, which has been implicated in selective expression of MSG genes. To gain insight into the architecture, evolution, and regulation of these gene clusters, six telomeric segments of the genome were sequenced. Each of the segments began with one or more unique genes, after which were members of different gene families, arranged in a head-to-tail array. The three-gene repeat PRT1-MSR-MSG was common, suggesting that duplications of these repeats have contributed to expansion of all three families. However, members of a gene family in an array were no more similar to one another than to members in other arrays, indicating rapid divergence after duplication. The intergenic spacers were more conserved than the genes and contained sequence motifs also present in subtelomeres, which in other species have been implicated in gene expression and recombination. Long mononucleotide tracts were present in some MSR genes. These unstable sequences can be expected to suffer frequent frameshift mutations, providing P. carinii with another mechanism to generate antigen variation.

Figure 1.—

Maps of gene clusters. Arrows represent ORFs and point in the direction of transcription. Nonhatched arrows represent members of the PRT1 (solid arrows), MSR (open arrows), and MSG (shaded arrows) gene families. Rectangles with vertical lines represent subtelomeres. Solid circles represent telomeres. All features except the telomere are drawn to scale. M indicates the MSG gene that contained two point mutations. 5′F and 3′F indicate ORFs corresponding to the 5′ and 3′ ends, respectively, of either an MSG (shaded arrow) or an MSR (open arrow) gene. G12, G15, G18, and G20 indicate the MSR genes that have a poly(G) mononucleotide tract of the size denoted by the numeral. S and L indicate short and long MSR genes. The dashed-line boxes enclose regions that were at least 99% identical. The hatched arrows represent unique ORFs that had the following presumed functions, BLAST hits, and FASTA E-values: Aur1 (inositol phosphoryl-ceramide synthase, EMBL accession AF076692, 2.9 e-67), Prf (prefoldin-related, NCBI REFSEQ accession XM_331039.1, 3 e-21), Rbp (RNA binding, UniProt YAS9 SCHPO_Q10145, 7.6 e-8), 21H1.0001 (unknown function, no hits), Atp (P-type cation-pumping ATPase, NCBI accession NP_595246, 1 e-6), Nmp (nuclear migration, NCBI accession EAK84394, 2 e-61), Map (microtubule associated protein, NCBI accession XP_323888, 3 e-17), P55 (peptide similar to the p55 antigen of P. carinii, NCBI accession AAQ06671, 4 e-8), Chi (chitin synthesis, NCBI accession NP_013434, 0.084), U2S (U2 snRNP, NCBI accession NP_594538, 0), and 22C8.0001 (unknown function, NCBI accession Q09895, 6.2 e-49). All of the hatched ORFS (except Prf, Rbp, and 22C8.0001, which were not analyzed) hybridized to a single chromosome. Aur1 and Chi mapped to a 440-kb chromosome. Nmp, Map, and p55 mapped to a 290-kb chromosome. Genes 21H1.0001, Atp, and U2S mapped to chromosomes of 680, 620, and 550 kb, respectively.

Figure 2.—

Terminal sequence at the end of a cosmid array maps to a single PFGE band. (A) A Southern blot was made from a CHEF gel performed under standard conditions (Cushion et al. 1993). Hybridization probes were as follows: lane 1, CRJE; lane 2, ORF 21H1.0001; lane 3, 17D7 Atp; lane 4, 22C8 U2S; lane 5, 18A9 Aur; lane 6, 11H12 + 1B2 Chi. (B) Southern blot was made from a CHEF gel performed under conditions optimized to resolve the lower four chromosome bands (Cornillot et al. 2002). Hybridization probes were as follows: lane 7, CRJE; lane 8, 3G5 p55-like ORF (p55). Sizes of DNA markers in kilobase pairs are indicated to the left of each part.

Figure 3.—

Linked MSG genes were not more similar. The tree displays the synonymous p-distances (p_S) computed from alignments of synonymous nucleotide sites of the MSG genes in cosmid clones. When a gene was in more than one clone, such as the gene in the region completely shared by contigs 22C8 and 11H12 + 1B2, only one of the two copies of this gene was included in the analysis. Tree branches are labeled to indicate the cosmid and the MSG gene, reading Figure 1 left to right. For example, 11A11 MSG3 refers to the MSG gene that is most proximal to the telomere in cosmid 11A11. Trees produced from either all nucleotide sites or nonsynonymous sites had the same structure as the tree shown. Bar indicates synonymous p-distance (p_S).

Figure 4.—

MSR genes vary in structure. Depicted are gene, message, and peptide structures for the three classes (S, L, and G) of MSR genes. Open boxes, exon 1; solid boxes, intron; hatched boxes, regions common to all three classes; shaded boxes, region missing in class S genes; G_n, poly(G) tract.

Figure 5.—

Subtelomere structures. (a) Dotter plot made by comparing the last 7 kb at the end of the insert in cosmid 3G5 to itself. The DNA compared starts at the nucleotide immediately downstream of the stop codon of the last MSG gene (see Figure 1). The boxes enclose 10 blocks that either contained multiple copies of one or more short sequence motifs or contained copies of sequences found in other blocks. Cases of the second form of repetition are indicated by arrows. For example, sequences in blocks I and J were also present in blocks G and H, respectively. Below the Dotter plot is a diagram that depicts the structure of the 3G5 subtelomere. (b) Comparison of subtelomere structures in different cosmid inserts. Rectangles represent blocks as in a. In cases where a single diagram represents the subtelomeres from multiple cosmids, such as 17D7, 18A9, and 21H1, the width of a rectangle represents the average length of the block it represents.

Figure 6.—

Occurrence of subtelomere short repeats. Normalized occurrences [average occurrence/kilobase pair (+ standard deviation)] of various short subtelomeric repeated sequence motifs (“Srpt”) are shown in various chromosomal regions. The “o” indicates motifs that were present at least once in all members of the region considered. Results for genic regions at the ste3 locus were similar to the intergenic spacers ones (data not shown). Srpt1, T_3–4A_5–8; Srpt2, T₃A₅W; Srpt3, GA_1–2(GA)₂; Srpt5, GT₃AT; Srpt6, T4MT₂A₄; Srpt8, TRAT₄KYATYR₂; and InvSrpt7, BTGYBA₂MWA.