Transcript initiation, polyadenylation, and functional promoter mapping for the dihydrofolate reductase-thymidylate synthase gene of Toxoplasma gondii

Molecular and Biochemical Parasitology
Mariana MatrajtDavid S Roos

Abstract

The fused dihydrofolate reductase/thymidylate synthase gene of Toxoplasma gondii contains ten exons spanning approximately 8 kb of genomic DNA. We have examined the ends of DHFR-TS transcripts within this gene, and find a complex pattern including two discrete 5' termini and multiple polyadenylation sites. No TATAA box or other classical promoter motif is evident in 1.4 kb of genomic DNA upstream of the coding region, but transcript mapping by RNase protection and primer extension reveals two prominent 5' ends at positions -369 and -341 nt relative to the ATG initiation codon. Upstream genomic sequences include GC-rich regions and the (opposite strand) WGAGACG motif previously identified in other T. gondii promoters. Mutagenesis of recombinant reporter plasmids demonstrates that this region is essential for efficient transgene expression. Sequencing the 3' ends from multiple independent mRNA clones demonstrates numerous polyadenylation sites, distributed over >650 nt of genomic sequence beginning approximately 250 nt downstream of the stop codon. Within this region, certain sites seem to be preferred: 14 different positions were found among the 32 polyadenylated transcripts examined, but approximately 40% of the transcripts map...Continue Reading

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Citations

Jun 28, 2005·International Journal for Parasitology·Corinne MercierMarie-France-Cesbron Delauw
Jun 5, 2010·DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes·Junya YamagishiXuenan Xuan
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