Yellow fever mosquito sterol carrier protein-2 gene structure and transcriptional regulation

Abstract

AeSCP-2, a sterol carrier protein, is involved in sterol trafficking in mosquitoes. The activity of the AeSCP-2 gene is important for mosquito development. An earlier study demonstrated that the transcription of this gene was upregulated by 20-hydroxyecdysone (20E) in cultured gut tissues. To investigate 20E-regulated transcription of the AeSCP-2 gene we truncated the upstream flanking region of AeSCP-2 gene and linked it to a reporter gene. The mosquito Aag-2 cell line was transfected with these promoter/reporter constructs and treated with 20E at various concentrations. Expression vectors of different transcription factors such as HR3 and beta FTZ-F1 were also co-transfected with the AeSCP-2 promoter/reporter constructs. The observed results demonstrated that varied combinations of transcription factors produce different promoter activities of the AeSCP-2 gene. This observation leads us to the conclusion that the partnership of transcription factors is crucial in regulating the transcriptional activity of the AeSCP-2 gene.

Figure 1

Genomic DNA organization of the AeSCP-2 gene. “*”- indicates a start codon, “x” – indicates a stop codon. Exons are shown as boxes, coding sequences are shown in gray, arrow indicate the transcription direction

Figure 2

The map of putative responsive elements of transcription factors identified by computational search in upstream, flanking sequence of the AeSCP-2 gene and constructs of truncated AeSCP-2 promoter.

Figure 3

The induction of AeSCP-2 expression by 20E in tissue cultures. A. Dosage-response of AeSCP-2 gene transcription in larval gut tissue cultures after 12 hours incubation. B. Time course-response of AeSCP-2 gene transcription in larval gut tissue cultures. C. Time course-response of AeSCP-2 gene transcription in larval carcasses tissue cultures. Bar = mean values ± S.D. (N= 3–6). Relative mRNA level = normalized to the level detected in newly molted 4^th instars which is arbitrarily set at 1.

Figure 4

Transient transfection assay of AeSCP-2 promoter activity as determined by the increase in CAT protein after co-transfection with pIE1^hr-HR3 and treated with 5 µM of 20E for 12 hours. Bar = mean values ± S.D. (N= 6–12). Relative CAT levels = normalized to the level detected in untreated cells which is arbitrarily set at 1.

Figure 5

Transient transfection assay of AeSCP-2 promoter activity as determined by the increase in CAT protein after co-transfection with pIE1^hr-βFTZ-F1 and treated with 5 µM of 20E for 12 hours. Bar = mean values ± S.D. (N= 6–12). Relative CAT levels = normalized to the level detected in untreated cells which is arbitrarily set at 1.

Figure 6

EMSAs for protein-binding of βFTZ-F1-1A. ³²P-labaled double-stranded βFTZ-F1-1A oligonucleotide probes (20,000 cmp) were incubated with 5 µg of Aag-2 cell extract in the presence or absence of unlabeled probe-specific competitors. A. Cell extracts with no co-transfection, B. Cell extracts cotransfected with pIE1^hr-βFTZ-F1, and C. Cell extracts cotransfected with pIE1^hr-HR3

Figure 7

EMSAs for protein-binding of βFtz-F1-1B. ³²P-labaled double-stranded βFTZF1-1B oligonucleotide probes (20,000 cmp/lane) were incubated with 5 µg of Aag-2 cell extract in the presence or absence of unlabeled probe-specific competitors. A. Cell extracts with no co-transfection, B. Cell extracts co-transfected with pIE1^hr-βFTZ-F1 and C. Cell extracts co-transfected with pIE1^hr-HR3