Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients.
The entire functional even-skipped locus of Drosophila melanogaster is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3' of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is 'carved out' by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314-24), but at different concentrations. The 3' region also contains a single element for neurogenic expression in ganglion mother cells 4-2a and 1-1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were found for expression in EL neurons, U/CQ neurons and the mesoderm. The even-skipped 3' untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels rather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3' end of the locus, but was not found to contribute to efficient functional rescue.
Fujioka
M
M
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Emi-Sarker
Y
Y
Yusibova
G L
GL
Goto
T
T
Jaynes
J B
JB
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins; D001756:Blastoderm / Q000378:metabolism*; D019521:Body Patterning / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000196:embryology* / Q000235:genetics; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D018398:Homeodomain Proteins / Q000235:genetics*; D051741:Kruppel-Like Transcription Factors; D008648:Mesoderm / Q000378:metabolism; D009420:Nervous System / Q000196:embryology; D010641:Phenotype; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D019076:Transgenes / Q000235:genetics*
D020413:3' Untranslated Regions; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; C497244:Kr protein, Drosophila; D051741:Kruppel-Like Transcription Factors; D012097:Repressor Proteins; D014157:Transcription Factors; C052837:eve protein, Drosophila; C118475:gt protein, Drosophila; C074089:kni protein, Drosophila