Susan M. Sunkin

Genes Encoding Antigenic Surface Glycoproteins in Pneumocystis from Humans

Pneumocystis is a eukaryotic microbe that causes pneumocystosis, an AIDS‐associated pneumonia. Pneumocystosis also occurs in many other mammalian species, and animal‐derived organisms have been extensively utilized in Pneumocystis research. Pneumocystis from diverse hosts contain a large glycoprotein (gpA/MSG) on the surface. Antibodies elicited against gpA/MSG of Pneumocystis from humans sometimes cross‐react with epitopes on proteins of similar size from Pneumocystis from other host species. Here we report the isolation and partial sequence of two presumptive gpA/MSG genes from human‐derived Pneumocystis. The cloned human‐derived Pneumocystis gpA/MSG genes and predicted peptides were different from those previously isolated from Pneumocystis from rats and ferrets. The genome of human‐derived Pneumocystis contained multiple copies of sequences related to the two cloned gpA/MSG genes.

A Tandem Repeat of Rat‐derived Pneumocystis carinii Genes Encoding the Major Surface Glycoprotein

ABSTRACT. A fragment from the genome of rat‐derived Pneumocystis carinii was found to contain two MSG genes arranged as a direct repeat. The sequences from one gene (MSG B), the region between the two genes, and part of the second gene (MSG A) were determined. The two MSG genes were not identical in sequence. The open reading frames of MSG A and MSG B encode non‐identical proteins, both of which are similar to that encoded by a previously published cDNA. The MSG B gene sequence showed no evidence of introns. The 5’and 3’untranslated regions of the MSG gene pair were highly conserved, but the regions immediately upstream of the open reading frames of MSG A and B were different from the region upstream of a previously characterized MSG cDNA. Primers designed to extend upstream of the 5’end of MSG and downstream of the 3’end of MSG were used in a polymerase chain reaction with total genomic P. carinii DNA as template. Presumptive intergenic amplification products from this reaction were cloned and sequenced. The sequences of these regions were similar but distinct, indicating that tandem arrangement of MSG genes is a common organizational motif.

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

The major surface glycoprotein (MSG) of P. carinii f. sp. carinii is a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSG isoforms start with the same sequence, called the upstream conserved sequence (UCS), which is encoded by a single locus. The mechanism by which the UCS becomes part of different MSG mRNAs is not obvious because at least 15 loci, which are distributed throughout the genome, encode MSGs. One possibility is that attachment to the UCS locus is required for the transcription of an MSG gene. The alternative to this expression site model is that mRNAs acquire the UCS by RNA splicing. To distinguish between these two models, UCS/MSG junctions in the genome were compared with UCS/MSG junctions in mRNA. The UCS/MSG junctions in the mRNA matched those in the genome, as would be expected if splicing did not contribute to the attachment of the UCS to the 5′ ends of MSG mRNAs. Given that few if any MSG mRNAs lack the UCS, the correspondence between the UCS/MSG junctions in transcripts and those in the genome indicates that attachment to the UCS is both necessary and sufficient for transcription of an MSG gene.

Transcription factor genes from rat Pneumocystis carinii.

Genes encoding the TFIID TATA‐box binding protein (TBP) from two probable species of rat Pneumocystis carinii (prototype and variant) were sequenced. The two P. carinii TBP gene sequences were 91% identical to each other, and 65‐77% identical to TBP genes from other species. A cDNA from one of the two P. carinii TBP genes was sequenced, which showed that four small introns resided in identical positions within the TBP genes from the prototype and variant rat P. carinii. Conservation of the 180 amino acids that constitute the conserved core of TBP was 97% between the P. carinii TBP, which were 95% and 97% identical to conserved core sequences of TBP from Saccharomyces cerevisiae and Schizosaccharomyces pombe respectively.

A new family of Pneumocystis carinii genes related to those encoding the major surface glycoprotein

Abstract A new family of Pneumocystis carinii genes (called MSR for MSG-related) that encodes peptides related to the major surface glycoprotein (MSG) is described. Members of the MSR sequence family are linked to MSG genes and are located near the ends of at least 13 chromosomes. Transcripts encoding different MSR isoforms were present in a single population of P. carinii f. sp. carinii, showing that multiple MSR genes were expressed. Two size classes of MSR mRNA, 2.4 and 3.5 kb, were detected. Both sizes of MSR mRNA lacked the upstream conserved sequence (UCS), which is found on the 5′ end of MSG mRNAs because MSG genes must be linked to the UCS to be transcribed. The absence of the UCS from MSR mRNAs suggests that expression of MSR genes does not require linkage to the UCS locus.