Three regulatory regions of the Aedes aegypti glutamine synthetase gene differentially regulate expression: identification of a crucial regulator in the first exon.
Aedes aegyptiglutamine synthetase (GS) is expressed constitutively at various developmental stages and its relative mRNA abundance increases in the midgut following blood feeding in support of the biosynthesis of chitin, a component of the peritrophic matrix. To understand the regulation of GS expression better, GS-luciferase reporter fusion genes were constructed and analysed in transiently transfected C6/36 cells. These studies have identified three GS regions: GS-A, -B and -C (C1, C2) that are required for efficient transcription. The crucial regulatory DNA sequence is located within 140 nucleotides of the GS-C region in the first exon. GS-B region between -209 and +4 contains a negative modulator that represses transcription of the GS-C promoter, but the 5'-GS-A region, between -476 and -282, can negate the transcription inhibition of GS-B and promote GS transcription of the GS-C promoter. Electrophoretic mobility shift assays showed that nuclear proteins for GS-A, GS-B and GS-C1 are present in the C6/36 cells, and therefore that GS-A, GS-B and GS-C1 indeed possess regulatory function. By contrast, nuclear proteins isolated from both cultured cells and midgut tissues bound to GS-C2, suggesting that GS-C2 plays an important role in GS transcription and that GS-C2 is regulated by several different and redundant transcription factors to achieve constitutive expression in a wide variety of tissues.
Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.
L L
LL
Niu
L M
LM
Kiley
R
R
Dasgupta
P
P
Kohler
B M
BM
Christensen
D000330:Aedes / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D008156:Luciferases; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection
D017931:DNA Primers; D014157:Transcription Factors; D008156:Luciferases; D005974:Glutamate-Ammonia Ligase