Assembly and promoter analysis of variant-specific surface protein (vsp) genes of Giardia lamblia

by Nigam, Anuranjini.

Giardia lamblia undergoes antigenic variation of variant-specific surface proteins (VSPs) that are encoded by a family of ~150 vsp genes only one of which is expressed at a time. The vsp gene promoters have not been previously studied. A comparison of the upstream non-coding region of vsp genes shows that they lack the AT-rich regions found in other Giardia gene promoters. We have determined that the core promoters of vsp A6 and vsp C5 genes extend from -57 to + 6 and -50 to + 6 respectively. Through linker scanning analysis, we have also identified regions within the vsp A6 core promoter important for promoter activity that span -7-3, -12-8, -17-13 and -42-38. There is no sequence similarity in the upstream regions of the previously characterized vsp genes that were analyzed, with the exception of a seven nucleotide region that encompasses the translation initiation site: Py A A T G T T. We have demonstrated that the four nucleotides flanking the start codon are essential for promoter activity. This result suggests that it may be an Inr element, which by definition determines the site of transcription initiation. In addition, this element loosely resembles the metazoan Inr consensus: Py Py A A/T Py Py. Using 5? RACE I have determined that for two vsp genes, the translation and transcription start sites are synonymous and reside within this conserved element. However, we were unable to identify protein factors that bind this region using electrophoretic mobility shift assays. A search for characteristic VSP motifs, such as CRGKA, amongst identified ORFs in the Giardia genome assembly in turn identified 180 ORFs which may be VSPs. 16 Eighty one of these are found within contigs while 99 of these are found at contig and 80 ORFs have the Inr element identified in this study. This study supports the hypothesis that longer upstream non-coding regions of vsp genes play a role in regulating the expression of these genes and hence antigenic variation in G. lamblia. 17