Cell, Vol. 40, 37-43, January 1985, Copyright 0 1985 by MIT 0092-8674/85/010037-07 $02.00/O The engrailed Locus of Drosophila: Structural Analysis of an Embryonic Transcript Stephen J. Poole, Lawrence M. Kauvar, types suggest controlling roles in Drosophila develop- Barry Drees, and Thomas Kornberg ment, two gene clusters, the Antennapedia complex and Department of Biochemistry and Biophysics the Bithorax complex, stand out. Mutations in these University of California, San Francisco “homeotic” genes produce normal structures in abnormal San Francisco, California 94143 locations. Flies with an Anfennapedia mutation have distal leg structures in place of normal distal antenna1 structures (Kaufman et al., 1980). Flies bearing simultaneous muta- Summary tions in the bx and pbx regions of the Bithorax complex have a wing in place of the normal haltere (Lewis, 1978). cDNA clones originating from the engrailed gene of Within these two gene clusters, which apparently perform Drosophila have been isolated from recombinant similar functions in different parts of the animal, several phage libraries that were made using poly(A)+ RNA ex- loci also share a common segment of DNA (McGinnis et tracted from early embryos. The DNA sequence of one al., 1984b). A strongly COIISeNed region of approximately of these clones includes a homeo box, a 180 bp se- 180 bp, present in several genes in both gene clusters, has quence present in several other Drosophila genes im- been designated the homeo box (McGinnis et al., 1984a). portant in formation of body pattern during develop- This sequence is strongly conserved among a number of ment. The homeo boxes found in the other Drosophila other segmented organisms including humans and Xeno- genes, as well as in cognate sequences from a wide pus (Levine et al., 1984; Carrasco et al., 1984). The homeo range of segmented animals, including higher verte- box contains an open reading frame and conservation brates, are highly conserved. By contrast, the homeo among the human, frog, and fly sequences is even box within the engrailed gene diverges substantially stronger at the amino acid level than at the nucleotide and, unlike the other homeo boxes, is interrupted by level. Thus, the homeo box appears to encode a peptide an intervening sequence. The engrailed homeo box is region of about 60 residues conserved over an immense located near the 3’ end of a 1700 bp open reading evolutionary span (Levine et al., 1984; Carrasco et al., frame. If translated, this sequence would produce a 1984). protein of unusual composition. We also show that a By using an extensive collection of engrailed mutations neighboring gene has a large region with strong ho- whose precise cytological aberrations had been mapped mology to engrailed, and that it also contains a homeo to the salivary gland polytene chromosomes (Kornberg, box. 1981a), DNA spanning the engrailed locus has been cloned by chromosomal walking (Kuner et al., unpub- Introduction lished data). Within the approximately 40 kb of DNA in which lethal engrailed mutations have been localized, a engrailed is one of a number of genes involved in specify- set of developmentally regulated poly(A)’ transcripts has ing the body pattern of Drosophila melanogaster. Em- been identified (Drees and Kornberg, unpublished). In this bryos homozygous for many alleles of engfailed display a report, we analyze several cDNA clones representing the severely disrupted segmentation pattern and die prior to most abundant of these transcripts. The engrailed gene hatching (Kornberg, 1981a). In these embryos pairs or contains an identifiable homeo box sequence that is diver- large groups of segments fuse because the segmental gent when compared to all of the other known homeo borders are not maintained. The requirement for wild-type boxes. Furthermore, the engrailed homeo box is set apart engrailed function extends beyond the embryonic period by being interrupted by an intervening sequence. A to later developmental stages as well. Adult flies that ei- nearby transcribed gene also contains a similarly diver- ther bear engrailedmutations that are not lethal (e.g., en’), gent and split homeo box. or lack engrailedfunction only in mosaic patches of tissue homozygous for embryonic lethal alleles, are unable to Results develop normally in many areas of the body. Specific defects have been observed in each of the adult segments Isolation of engrailed cDNA Clones examined and a remarkable position dependence has Isolation of the engrailed locus has made possible a been noted. In every segment the abnormalities occur molecular analysis of its structure and function. In the within the posterior portion only (Lawrence and Morata, map of the engrailed region (Figure lA), arrows mark the 1976; Kornberg, 1981a, 1981b; Lawrence and Struhl, breakpoints of alleles with cytologically visible chromo- 1982) an area that coincides with the posterior develop- some rearrangements. Because most engrailed mutations mental compartment (Garcia-Bellido et al., 1973). No are embryonic lethals (Kornberg, 1981a), suggesting em- engrailed-related defect has ever been observed in the bryonic expression, DNA fragments (l-4 kb) from this en- cells of the anterior compartments: cells in the anterior tire region were used as probes for detection of homolo- compartments develop normally in the absence of en- gous embryonic RNA. Several such probes hybridize on grailed function. Northern blots to poly(A)’ embryonic RNAs of 3.6,2.7, and Among the many other genes whose mutant pheno- 1.4 kb. Cell 38 engrailed probes tested (see lane A in Figure 6 below). A) 4SA REGION engroiled engroiled Eleven cDNA clones hybridizing with the 0.9 kb probe related gene cDNA H H were isolated. They range in size from ml.1 to 2.6 kb. All clones have been examined in detail by digestion with re- H lttt to tt I striction endonucleases and by heteroduplex mapping. 40 kb 20 kb 0 Nine are apparently derived from the same transcript; I LETHAL ENGRAllED REGION I their size and relative abundance suggests that they are copies of the major 2.7 kb RNA, and one of these (c-2.4) has been analyzed in detail. 6) ENGRAILED REGION +-AA Structure of the engrailed cDNA Clones RI RI Both strands of cDNA clone c-2.4, as well as much of the I I corresponding genomic DNA and an independently iso- lated cDNA clone, have been sequenced. With the excep- tion of a few differences attributed to single-base polymor- - cDNA phisms, the homologous sequences are identical. The . ccentromere H-box cDNA clone c-2.4 contains 2449 bp (Figure 2) and has one 1 adenylate-rich end. Comparison with the genomic se- C) ENGRAILED-RELATED REGION quences indicates that two intervening sequences, 1.1 -c-B+ and 0.28 kb in length, had been removed from the RNA RI Bgl II H3 RI template for c-2.4. In the genomic sequences, both the do- If I I nor splice junction (AGGT) and the acceptor splice junc- tions (TTXCAG) are similar to other eukaryotic splice junc- homologous with tions (Mount, 1982; see Figure 5 for the splice junctions engrailed region of the second intron). The splice points of c-2.4 are at posi- -centromere ;I-bo: tions 1489 and 1587. The location of the introns is di- 1 agrammed in Figure 1B. The sequences at positions 238-336 consist largely of Figure 1. The engrailed Chromosomal Region repeating trinucleotides of the form CAX, where X is usu- (A) Overview of the 48A region of the second chromosome. The centro- mere is to the left of the map, and the zero point is the insertion site ally A or G. This CAX repeat is homologous to a repeated of the en’ transposition (Kuner et al., unpublished). The vertical arrows element that has been found in numerous Drosophila mark the locations of the mapped engrailed chromosomal breakpoint RNAs, including the Notch (S. Artavanis, personal com- lethal mutations. These mutations are (left to right): ensF52, ensF=, munication) and Antennapedia (R. Garber, personal com- enSF42, enSFa, enSFM, enSF32, enz, enSF=, enSF24, erPJ, enSF61, and munication) transcripts and may in some instances con- e&m (Kornberg, 1981a; Kornberg and Ali, unpublished data). Shown above on the right is the region of homology with the engrailed cDNA sist of 100 tandem CAA or CAG repeats (S. Artavanis, clone. Above and to the left is the location of a related gene that shares personal communication). A restriction fragment that con- some homology with the engrailed cDNA clone. tains this region of the engrai/edcDNA hybridizes to many (6) Structure of the engrailed cDNA clone c-2.4. The polarity on the genomic restriction fragments (data not shown); other chromosome is the same as in (A). The direction of transcription (5’to 3’) is from right to left. The horizontal lines show the inferred engrailed repeating trinucleotides in this region (e.g., GCX, posi- exons that are spliced in the cDNA. Also indicated is the location of the tions 379-420) may also contribute to the multiple bands homeo box sequences within the cDNA. The 0.9 kb Eco RI fragment of hybridization. designated A is used as a probe in Figure 6. The orientation of the poly(A) stretch of c-2.4 and the (C) Structure of the engrailed-relatedregion. The centromere lies to the orientation of c-2.4 with respect to the genomic DNA are left and the engrailedgene is 17 kb to the right. The regions within the 3.6 kb genomic Eco RI fragment that are homologous to the engrailed consistent with the known direction of transcription of the cDNA clone c-2.4 are shown and include the split homeo box. Direction engfailed region (Drees and Kornberg, in preparation). In of transcription of this gene is from left to right.