Analysis of the Brugia malayi HSP70 promoter using a homologous transient transfection system.
Biolistic transient transfection of Brugia malayi embryos with constructs driving the expression of a luciferase reporter gene was used to identify regions of the upstream sequence of the heat shock protein 70 (HSP70) gene of B. malayi necessary for transgene expression. Analysis of 1160 nucleotides upstream of the start codon of the HSP70 gene identified several potentially important elements, including putative CAAT and TATA boxes, a core promoter domain, a polypurine stretch, and a spliced leader addition site. Nested deletion analysis of the HSP70 upstream domain mapped the promoter of the HSP70 gene to the region 396 to 31 nucleotides upstream of the start codon. This encompassed the putative CAAT and TATA boxes, and putative core promoter. Deletion of the putative CAAT box did not result in any diminution of reporter activity, while constructs in which the TATA box or core promoter were deleted retained roughly half of the activity of the undeleted construct. Unlike the native gene, transcripts derived from constructs containing the HSP70 upstream sequences were not trans-spliced. However, incorporation of the 495 nucleotides downstream of the start codon (encompassing exon 1, intron 1 and part of exon 2) resulted in the production of transcripts that were correctly cis- and trans-spliced. Similarly, a construct containing the 495 downstream nucleotides in which most of exon 1 was deleted, was correctly cis- and trans-spliced. This finding suggests that downstream intron sequences in addition to the splice leader addition site are necessary for trans-splicing in B. malayi.
Shu
Limin
L
Division of Geographic Medicine, University of Alabama at Birmingham, BBRB 203, 1530 3rd Avenue South, Birmingham, AL, USA.
Katholi
Charles R
CR
Higazi
Tarig
T
Unnasch
Thomas R
TR
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D017178:Brugia malayi / Q000196:embryology / Q000235:genetics* / Q000302:isolation & purification; D005091:Exons; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000235:genetics*; D015801:Helminth Proteins / Q000032:analysis / Q000235:genetics*; D006358:Hot Temperature; D007438:Introns; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation; D014162:Transfection / Q000379:methods*
D018840:HSP70 Heat-Shock Proteins; D015801:Helminth Proteins; D008156:Luciferases