Molecular cloning of engrailed: a gene involved in the development of pattern in Drosophila melanogaster

Abstract

The engrailed gene acts early in Drosophila embryogenesis and plays an essential role in the processes that establish and maintain the repeating segmental pattern. To begin molecular analysis of the role of the engrailed gene in embryonic pattern formation, we used a chromosomal walk to clone genomic sequences that encompass the locus, and have physically mapped the positions of 15 engrailed mutations. The positions of engrailed rearrangement mutations indicate that the engrailed complementation unit includes a minimum of 70 kb. The locus can be divided into two regions. Rearrangement mutations interrupting the centromere proximal 50 kb of the locus result in embryonic lethality while mutants altered in the distal 20 kb of the locus survive to show morphological abnormalities in several adult segments. It appears that long-range cis interactions play a role in the function of the engrailed gene.

Figure 1. A Recombinant DNA Clone from Df(2R)en^SF31 Spans the Polytene Region of 48A

In situ hybridization (Pardue and Gall, 1975) has grains at 48A and 48B. The chromosomes are from a wild-type strain and the probe was from the clone E31, a clone containing the en^SF31 breakpoint with sequences from 48A1 and 48B5.

Figure 2. Molecular Map of Polytene Region 48AB

Coordinates are in kb, based on a zero point at the insertion site of the en¹ transposition, and the map is orientated with the centromere to the left. Individual phage (E1-20) and cosmid (cos 189B, CH1A, and 190) clones are shown above the coordinate scale and below it are shown restriction maps that were determined for the individual phage. The arrowheads indicate the end points of the en^SF31 deletion.

Figure 3. Demonstration of the Positions Altered by Chromosomal Rearrangements

In each panel, DNA extracted from wild-type or parental flies (designated +) is compared to DNA extracted from an engrailed mutant (right lane). DNA was digested with a restriction endonuclease, transferred to nitrocellulose, and hybridized with a nick translated Eco RI fragment of DNA from the walk. Arrows indicate the novel restriction fragments created by the rearrangement. In some digests both the proximal and distal rearrangement fragments are seen, whereas in others only one of the new fragments is detected because of either limited sensitivity or resolution. Because of the presence of a wild-type allele of engrailed on the balancer chromosome, generally the DNA fragment broken by a DNA rearrangement mutation is still present in the mutant heterozygotes. However, in a few cases there is a polymorphism between the parental chromosome and the balancer; in these, the mutation causes a band to disappear (e.g., en^SF50). Digestions of genome DNA and positions (see Figure 2) of the Eco RI fragments used for probes were: en¹, Xho I (−0.2, +2.7); en^c2, Bam HI (−0.2, +2.7); en^LA3, Hind III (+25.3, +34.2); en^SF24, Xho I (+2.7, +12.0); en³⁰, Eco RI (+13, +20.5); en³², Xho I (−1.0, −4.7); en^SF37, Xho I (−28.0, −33.9); en^SF37, Bgl II (+2.7, +12.0); en^SF42, Xho I (−10.6, −15.2); en^SF49, Bam HI (−10.6, −15.2); en^SF50, Xho I (−1.0, −4.7); en^SF52, Xho I (−28.0, −33.9); en^SF61, Eco RI (+13, +20.5); en^SF82, Eco RI (−10.6, −15.2); en^SF83, Bam HI (−5.4, −10.6); and en^Es, Bgl II (+25.3, +34.2).

Figure 4. In Situ Hybridization with Breakpoint Fragment Probes

Wild-type polytene chromosomes from larval salivary glands were hybridized with nick translated probes from a subclone containing the en¹ insertion element (A), the Bam HI breakpoint restriction fragment of the proximal en^SF37 chromosome rearrangement (B), the Eco RI breakpoint restriction fragment of the distal en^SF37 chromosome rearrangement (C) and the breakpoint restriction fragment of en^SF24 (D). Note multiple sites of hybridization in (A) and (C), sites of hybridization at 48A and 46C in (B), and sites of hybridization at 48A and 65A in (D).

Figure 5. Physical Location of engrailed Breakpoint Mutations

The locations of the engrailed breakpoint mutations on the restriction map of the region (see Figure 3) are given. The distances are measured in kb and the accuracy of localization of the breakpoints is indicated by brackets. Note the physical separation of the lethal and nonlethal rearrangement alleles. The two distinct breaks mapped for en^SF37 are shown.