Apparent discontinuous transcription of Trypanosoma brucei variant surface antigen genes

David A. Campbell, Deborah A. Thornton, John C. Boothroyd
1984 Nature  
The repeated mini-exon sequence that encodes the first 35 base pairs of all variant surface antigen mRNAs of Trypanosoma brucei directs the synthesis of a discrete I37-nucleotide transcript. It thus seems that variant surface antigen mRNAs are transcribed discontinuously, and we present two alternative models for how this might occur. THE ability of African trypanosomes to establish chronic infections in their mammalian hosts depends on a highly developed system of antigenic variation, whereby
more » ... ndividual members of the parasite population change the composition of their surface coae-3 . This antigenic variation is controlled at the level of gene expression 4 ,5, each trypanosome possessing a large repertoire of genes (estimated at over 100; refs 6, 7) coding for the antigenically distinct variant surface glycoproteins (VSGs) which comprise the coat. At anyone time and on anyone trypanosome, only one species ofVSG can be detected 8 , suggesting that mutual exclusion operates between the VSG genes. Activation of VSG genes occurs by a two-step process requiring first the duplication and transposition of a silent basic copy (BC) of the gene into one of a few telomeric expression sites by a process equivalent to gene conversion, then selection of the expression site over others for transcription9-ls. The extra copy of the gene thus produced is called the expression-linked copy (ELC). The socalled 'non-duplication-activated' VSG genes described by others probably represent genes which had already undergone the first step in activation (that is, gene conversion into an expression site) before the variants were isolated for study. Although the structure of expression sites and the sequences involved in the gene conversion event have been identified for some variants l 6--19, little is known about the second step, particularly how activation occurs and how the mutual exclusion operates between the different expression sites. Recently, a major clue to this problem has come from the finding that the 5' 35 nucleotides of VSG mRNAs are not contiguously encoded with the protein-coding portion of the gene i3 , and that this spliced leader segment is identical for different VSG mRNAs regardless of which telomeric expression site they occupyl4. The genomic location of the 35-base pair (bp) mini-exon coding for the spliced leader has recently been reported to be a 1.35-kilobase pair (kb) segment tandemly repeated 100-200 times (as one or more clusters) at an unidentified locus in the ~enome but at least 30 kb upstream of the active VSG gene 20 ,2 . Two other important observations concerning the spliced leader are that it is detected on many RNA molecules of varying size in blot analyses using RNA from different life stage forms of Trypanosoma brucei (ref. 21 and M. Parsons et al., personal communication) and that homologous sequences have been detected in the genomes of related species and genera 22 . Several models have been proposed to explain how a contiguous mRNA is produced from such an unusual arrangement of exons 2 0-23 and how the activity of different expression sites is regulated. These have included chromosome end exchange with splicing of very long transcripts, and discontinuous transcription whereby the mini-exon and ELC are flanked by their own initiation and termination sites. To determine how VSG mRNAs are produced and processed, we have studied the 1.35kb repeats and identified their transcriptional products. Molecular cloning of a mini-exon repeat To enable nucleotide sequence analysis of the mini-exon repeat, recombinant plasmids containing individual repeats were generated. This was initially done using a PvuII digest of the genomic DNA of T. brucei as this enzyme has been reported to release the mini-ex on-containing portion of the repeat as a discrete band at -720 bp21. Such a band was excised from an agarose gel, the DNA purified by electroelution 24 and ligated into the PvuII site of the plasmid vector pBR322 (ref. 25). A sample of this same excised material was radiolabelled by nick-translation 26 and used as a probe in colony hybridization 27 , thereby identifying recombinants containing repetitive DNA. Four plasmids thus identified were further characterized and three were found to contain indistinguishable inserts with at least one RsaI site. As the mini-exon should include an RsaI site, nucleotide sequence analysis in the vicinity of this site was performed on one of the plasmids (pMEP.1). The strategy and sequence obtained are shown in Figs I and 2, respectively. This demonstrated unambiguously that pMEP.I contains a portion of the mini-exon repeat. To obtain a recombinant plasmid containing an insert representative of the complete 1.35-kb repeat, a ligation was performed using the 1.35-kb fraction of trypanosome genomic DNA digested with Sau3A and the plasmid vector pAT153 (ref. 28) digested with BamHI. Recombinants were screened with the
doi:10.1038/311350a0 pmid:6090933 fatcat:dwzaxcrtarecpkiowbs2q76fe4