Residence at the expression site is necessary and sufficient for the transcription of surface antigen genes of Pneumocystis carinii.

Susan M. Sunkin and James R. Stringer*Department of Molecular Genetics, Biochemistry andMicrobiology, College of Medicine, University ofCincinnati, Cincinnati, OH 45267-0524, USA.SummaryThe major surface glycoprotein (MSG) of P. carinii f.sp. cariniiis a family of proteins encoded by a familyof heterogeneous genes. Messenger RNAs encodingdifferent MSG isoforms start with the same sequence,called the upstream conserved sequence (UCS), whichis encoded by a single locus. The mechanism by whichthe UCS becomes part of different MSG mRNAs is notobvious becauseatleast15loci, which aredistributedthroughout the genome, encode MSGs. One possibi-lity is that attachment to the UCS locus is requiredfor the transcription of an MSG gene. The alternativeto this expression site model is that mRNAs acquirethe UCS by RNA splicing. To distinguish betweenthese two models, UCS/MSG junctions in the genomewere compared with UCS/MSG junctions in mRNA.The UCS/MSG junctions in the mRNA matched thosein the genome, as would be expected if splicing didnot contribute to the attachment of the UCS to the 58ends of MSG mRNAs. Given that few if any MSGmRNAs lack the UCS, the correspondence betweenthe UCS/MSG junctions in transcripts and those inthe genome indicates that attachment to the UCS isboth necessary and sufﬁcient for transcription of anMSG gene.IntroductionPneumocystis carinii is a fungus that can cause pneu-monia in immunocompromised humans (Walzer et al.,1974; Mills, 1986). Other mammals can also be infectedby P. carinii, but each host species harbours a differentgenetic variety of the microbe (Sinclair et al., 1991; Edlindet al., 1992; Stringer, 1993; J.R. Stringer et al., 1993; Dei-Cas et al., 1994; De Luca et al., 1994; Keely et al., 1994;Peters et al., 1994a,b; Weinberg et al., 1994). The geneticdivergence among these varieties is substantial, but deﬁ-nitive evidence of multiple species of Pneumocystis islacking. Accordingly, the term formae specialis(meaningspecial form) is used to designate the varieties of P.carinii(Pneumocystis Workshop, 1994). None of the spe-cialformsofP.cariniigrowswellenoughinculturetoallowpropagation of clonally derived stocks, but large numbersof organisms can be obtained routinely from laboratoryanimals. The organisms used in this study were obtainedfrom immunosuppressed rats that were infected with P.carinii formae specialis carinii(P. carinii f. sp. carinii)byexposure to airborne organisms produced by other immu-nosuppressed infected rats (Frenkel et al., 1966; Arm-strong and Cushion, 1993; Cushion et al., 1993).All studied special forms of P. cariniiare coated with amajor surface glycoprotein (MSG) (Walzer and Linke,1987; Kovacs et al., 1988; Pesanti and Shanley, 1988;Linke and Walzer, 1989; Radding et al., 1989; Tanabe etal., 1989; Lundgren et al., 1991), also known as gpA(Gigliotti et al., 1988; Gigliotti, 1992). In P. cariniispecialforms recovered from rats, ferrets, mice and humans, ithas been shown that MSG is actually a family of proteinsencoded by a family of heterogeneous genes (S.L. Strin-ger et al., 1991; 1993; Haidaris et al., 1992; Kovacs etal., 1993; Garbe and Stringer, 1994; Wright et al., 1994).The genome of P. carinii f. sp. carinii contains approxi-mately 100 different MSG genes (Stringer et al., 1991),which are organized in clusters that are located at theends of each of the 15 chromosomes (Wada et al., 1993;Sunkin et al., 1994; Sunkin and Stringer, 1996; Underwoodet al., 1996; Wada and Nakamura, 1996). Populations ofP. carinii f. sp. carinii contain mRNAs encoding multipleMSG isoforms (Linke et al., 1994), but expression of thisgene family within an individual organism is thought to belimited, based on both phenotypic and molecular geneticdata. Phenotypic data indicating limited expression includeindirect immunoﬂuorescence studies that showed thatnot all organisms within a population could be labelledwith an antibody directed against a subset of MSG iso-forms (Angus et al., 1996; Sunkin and Stringer, 1996). Inaddition,thefractionof organismslabelledbysuchananti-body varied among populations(Angus et al., 1996). Addi-tional phenotypic evidence of limited MSG expression hasbeenprovidedbyWesternblotting, whichshowedthat someP. carinii f. sp. carinii populations contained a particular