Volume 219, number ;I, ~]0;l FEBS 094~0 February I~91 '~ I~/I F¢detation of Europealt I|i,d~h¢ntkal ~odeti~ 001,~t'/U,IA)l/$~! $0 A D O N I S 0014 $'/9.1~11001,1 ~t W Cloning and sequencing of a leech homolog to the Drosophila engrailed gene Cathy J. Wcdeen, David J. Price* and David A. Weisblat Depttrltnent oj" bloleeuhtr and Cell BialoIl)'. University of Coil[amid, Berkeley. Cd ~4720, USA Received 26 November 1990 We have cloned and ~querleed tt tomolog (ht.en) to the Dra~phila cngritiled (on) gone from Iho glo~siphoniid leech, t/¢/ohdello tri~rrlali,*, Amino acid comparisons ol' the he.on homeodomain and C.tcrminal residues with tile corresponding residues encoded by emclass genes of other species reveal 75=79'~,~quenee identity. In addition the hi.on sequence appear=t to have a serin¢.rieh region 16 r~idues C,terminal from the homeodomain, which by analog)' to Dro~,phila may b¢a earlier site For pltofphorylation. The lee¢il gone eneodet mine amino acid substitutions for residttcs that are highl), conserved in other species, The~e arc found within the second and third orthe three putative helice=t or tile homeodomaifl, and in both of the intervening turn rellion~, Engrailed; Homeobox gent; Helobd¢lla trixerfatit 1. INTRODUCTION The homeobox gent, engrailed (en), encodes a DNA- binding protein that is necessary to establish the 'identi- ty' of the posterior compartment within each segment in Drosophila [1-3], The en gene encodes a serine-rich protein that has been shown to be the target of serine phosphorylation [4]; it has been proposed that other segmentation genes (e.g. fused) may regulate en func- tion by phosphorylation [5]. A closely related gone in DrosophUa is invected (inv), for which no function has yet been determined. Both en and inv are transcribed concurrently in the same tissues during embryogenesis [6]. In addition, en is expressed later in development in certain neurons of the central and peripheral nervous systems [7-10]. En-elass genes of divergent species are defined as a subfamily of homeobox-containin8 genes having an especially distinct and highly conserved homeobox region. This high degree of conservation has led to the identification and cloning of homologs from divergent species. In the fruit fly, honeybee, mouse, chicken, zebrafish, and human, two copies of en-class genes have been identified; in other species (grasshopper and sea urchin) only one on-class gone has been found [10-17]. Thus, it may be that a single en gone was pres- ent in a common ancestor to the arthropods, echinoderms and chordates and that this gone was Correspondence address: C,J. Wedeen, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA *Present address: Department of Physiology, University Medical School, Teviot Place, Edinburgh, EH8 9AG, UK duplicated independently in two, and maybe more, separate lines (i+e. the chordates and the insects). We have previously reported an en-class gone in the leech, He/obde/la triserialis [18]. We have now cloned and sequenced the t~omeobox and 3' nucleotides of this gone (hi-on) and we compare this sequence with those of other on-class genes. 2, MATERIALS AND METHODS 2.1. Libmo, ~z'reeai~/l The pl~ag¢, XHt.er, l, was one of 10 recombinants obtained by screening 6.8 x 104 plaque forming units from :t He/obdella triseriatts generate library [19) using tile low stringency hybridization conditions described by MeOinnls etal. [20]. 'File probe used was a 250 bp Pvull. Sail en eDNA fragment containing the Iton'Jeobox and upstream region from Ihe clone en-HB I [3}. 2.2. DNA sequencitz/~ Both strands of the 500 bp Pvull fragment (Fig. l) were sequenced. Most of the sequence reported in Fig. 2 (i.e. the 3' 147 bp of the homeobox and the downstream region preceding the first termination codon) ts a subset of these data, Homeobox sequence S' to the Pvull site was obtain,'d from a subclone of the 3.5 kb Hpal fragment, using oligonucleotide primers designed to anneal to already sequcttced por- tions of the clone. All sequencing was done using the dideoxy chain termination method. 3. RESULTS AND DISCUSSION 3.1. The ht-en sequence is highly conserved A recombinant clone homologous to Drosophila en was obtained by low stringency hybridization to a Helobdella triserialis library (Fig. 1, and section 2). The nucleotide and deduced amino acid sequence of the ht- en homeobox and C-terminal flanking region are given in Fig. 2, Given the probe used to clone XHt-eal con- tained the Drosophila en homeobox and 5' sequences, 300 Published by Elsevier Science Publishers B. V,