A transcriptional role for conserved footprinting sequences within the larval promoter of a Drosophila alcohol dehydrogenase gene

• Published in: Journal of molecular biology Vol. 249; no. 2; pp. 259 - 269
• Main Authors: Hu, Jie, Qazzaz, Hassan, Brennan, Mark D.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 1995
• Subjects: adh gene; adh promoter; Adh proximal promoter; alcohol dehydrogenase; Alcohol Dehydrogenase - biosynthesis; Alcohol Dehydrogenase - genetics; Animals; Base Sequence; binding sites; Conserved Sequence; DNA footprinting; DNA Primers; DNA-binding proteins; DNase I footprinting; Drosophila; Drosophila - enzymology; Drosophila - genetics; Drosophila affinidisjuncta; Drosophila melanogaster; Drosophila melanogaster - enzymology; Drosophila melanogaster - genetics; embryo (animal); fat body; gene expression; Genes, Insect; genetic regulation; in vitro transcription; Larva; larvae; Molecular Sequence Data; Mutagenesis, Site-Directed; nucleotide sequences; Point Mutation; promoter regions; Promoter Regions, Genetic; proximal promoters; Restriction Mapping; Sequence Homology, Nucleic Acid; somatic transformation; structural genes; transcription (genetics); transcription factors; Transcription Factors - metabolism; Transcription, Genetic; transcriptional regulation; Adh proximal promoter; Adh; somatic transformation; ABF; transcriptional regulation; in vitro transcription; DNase; SNF; DNase I footprinting; β-gal
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1995.0295

All
Drosophila alcohol dehydrogenase (
Adh) genes that are expressed in larvae display strong transcription in the larval fat body. To identify and characterize elements needed for
Adh promoter function, footprinting analysis of the
Drosophila affinidisjuncta Adh gene was performed with stage-specific nuclear proteins from embryos and larvae. Multiple sites upstream of the larval promoter were protected from deoxyribonuclease digestion by both embryonic and larval extracts. Comparison with foot-printing results for
Adh genes from other
Drosophila species revealed only one nuclease-protected region that is conserved in both sequence and position. Clustered point mutations in this sequence were analyzed by footprinting analysis, transient transformation and
in vitro transcription. Two separate sequences in this footprinting region exerted positive effects on transcription from the
Adh proximal promoter in the larval fat body. The effects of these sequences on gene expression were synergistic. One of these sequences, TGATAA, bound
in vitro to
Drosophila melanogaster box A binding factor protein, as shown by gel mobility shift assays. This is the first direct demonstration of specific protein-DNA interactions influencing transcription of a
Drosophila Adh gene in the larval fat body.