Proc. Natl. Acad. Sci. USA Vol. 85, pp. 5582-5586, August 1988 Developmental Biology Structure and expression of Hox-2.2, a murine homeobox-containing gene (development/in situ hybridization/Hox 2.1 gene/amino terminus/conserved hexapeptide) KLAUS SCHUGHART*, MANUEL F. UTSETt, ALEXANDER AWGULEWITSCH*, AND FRANK H. RUDDLE*t Departments of *Biology and tHuman Genetics, Yale University, New Haven, CT 06511 Contributed by Frank H. Ruddle, April 1, 1988 ABSTRACT The Hox-2.2 gene is one of a cluster of terns of the Hox-2.2 gene with that of the Hox-2.1 gene in the homeobox-containing genes on mouse chromosome 11. A developing central nervous system. cDNA clone containing theHox-2.2 homeobox has been isolated from an adult spinal cord library. Our analysis of the Hox-2.2 MATERIAL AND METHODS cDNA and genomic clones indicates that there are at least two exons and one intron. The largest open reading frame includes Isolation and Sequencing of cDNA Clones. Approximately 4 the homeobox and codes for a 224 amino acid protein of x 105 AgtlO recombinant phage clones representing a cDNA molecular weight 25,312. Comparisons of the predicted Hox- library from polyadenylylated RNA of adult murine spinal 2.2 protein with other homeodomain-containing proteins re- cord (A.A., unpublished work) were screened with a 353-bp vealed four regions of sequence similiarity: an N-terminal genomic Hae III-fragment of the Hox-2.2 gene (ref. 12 and octapeptide, a hexapeptide upstream of the homeodomain, the Fig. 2; bp 556-908) containing the Hox-2.2 homeobox. homeodomain, and a glutamic acid-rich region at the C Overlapping fragments suitable for DNA sequencing were terminus. Possible functions ofthese regions are discussed. The prepared from the isolated cDNA by treatment with BAL-31 Hox-2.2 gene is expressed in 13.5-day embryos in the devel- exonuclease and subcloned as described (14) into vectors oping hindbrain and spinal cord. The expression patterns of pGEM-4 and pGEM-3 (Promega Biotec, Madison, WI) or a Hox-2.2 and Hox-2.1 in 13.5-day embryos are compared. pGEM derivative (a gift of L.D. Bogarad, Yale University). The nucleotide sequences from both strands ofthe cDNA and In Drosophila melanogaster a highly conserved DNA region from some genomic DNA fragments (Fig. 1) were determined been in by the dideoxy method (15, 16). Sequence analyses and of 183 base pairs (bp), the homeobox, has identified comparisons were performed with programs provided by several genes affecting embryonic development of the fruit DNAStar (Madison, WI) and the University of Wisconsin fly, including maternal-effect genes, segmentation genes, and Genetics Computer Group (17). homeotic genes (reviewed in refs. 1 and 2). Maternal-effect In Situ Hybridization. In situ hybridization to cryostat genes specify the spatial coordinates ofthe egg, segmentation sections of 13.5-day-postcoitus embryos was performed with genes determine the number and polarity of body segments, 35S-labeled single-stranded RNA probes (18). Two restriction and each of the homeotic genes is required for the proper fragments were cloned into pGEM plasmids to prepare morphogenesis of a distinct region along the anterior-poste- probes specific for Hox-2.2 transcripts and negative control rior axis. To a first approximation, the region-specific func- probes of the opposite orientation: the 353-bp genomic Hae tions of Drosophila homeobox genes can be superimposed III fragment containing the Hox-2.2 homeobox and the onto their region-specific patterns of expression (1, 2). 511-bp upstream EcoRI-fragment (Figs. 1 and 2) from the Homeobox genes have also been detected in many other cDNA. The 511-bp cDNA probe does not contain homeobox species, including mouse and human (3). The homeobox sequences. The Hox-2.1 probe, a 265-bp genomic Hae III sequence codes for a protein domain that exhibits structural fragment containing the homeobox, was described previ- similiarity to certain prokaryotyic and eukaryotic DNA- ously (18). binding proteins (4, 5). Experiments that show nuclear localization of homeodomain proteins (refs. 6 and 7 and references therein) and the specific DNA-binding properties RESULTS AND DISCUSSION of homeodomain-containing fusion proteins (8, 9) suggest a Structure of the Hox-2.2 mRNA and Protein. RNA blot role for homeodomain proteins in controlling gene expres- hybridizations detected a Hox-2.2-specific transcript of 1.5 sion. Most of the 15 homeobox loci described in the mouse kilobases (kb) in 13.5-day mouse embryos (13) and spinal genome are organized in two gene clusters on chromosomes cord of adult mice (data not shown). Therefore, a cDNA 6 and 11 (reviewed in ref. 10). The Hox-2 cluster on library prepared from spinal cord poly(A)+ RNA of adult chromosome 11 contains at least 6 homeobox genes (10, 11). mice was screened with a DNA fragment containing the Previous reports described the homeobox sequences of the Hox-2.2 homeobox. Offour cDNA clones isolated, clone c5, Hox-2.2, Hox-2.3, and Hox-2.4 genes (12) and demonstrated which contained the largest insert and hybridized to the their expression in mouse embryos (13). genomic Hox-2.2 DNA (3.2-kb and 2.4-kb EcoRI-Hind III In order to understand their functions during murine fragments; Fig. 1) was selected for further analysis. Sequence development, a detailed analysis of homeobox gene tran- analyses of clone c5 and of appropriate genomic DNA scripts, gene products, and specific patterns of expression is fragments have enabled us to describe the entire coding required. In this paper we present the complete amino acid region of a Hox-2.2 transcript, to identify two exons and one sequence of a Hox-2.2 protein,t as predicted from a cDNA intron, and to predict a putative polyadenylylation signal clone, and we compare the region-specific expression pat- (Figs. 1 and 2) These data can account for a Hox-2.2 transcript The publication costs of this article were defrayed in part by page charge tThe sequence reported in this paper is being deposited in the payment. This article must therefore be hereby marked "advertisement" EMBL/GenBank data base (IntelliGenetics, Mountain View, CA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. and Eur. Mol. Biol. Lab., Heidelberg) (accession no. J03782). 5582 Downloaded by guest on September 26, 2021 Developmental Biology: Schughart et al. Proc. Natl. Acad. Sci. USA 85 (1988) 5583 4.- The 8 N-terminal amino acid residues of the Hox-2.2 -40. .0- protein are highly conserved in the N-terminal regions of II several other homeodomain proteins (Fig. 3A), including H P P Pv E Pv P H B mouse Hox-1.3 (7, 21), mouse Hox-2.1 (20), mouse Hox-3.1 (ref. 22 and A.A., unpublished data), human c13 (23), and 0.5kb I-H Drosophila Antp (24). The N termini of other homeodomain Rox-2.2 proteins, such as Xenopus Xhox-1A (25), Drosophila Ubx c:5 (1 ..4kb) Pv E p (26), and Drosophila Dfd (27), are less conserved. The N termini ofthe mouse Hox-1.1 (6). mouse Hox-2.3 (28), human cl (29), Xenopus XIHbox 2 (30), and Xenopus Xhox-36 (31) 51 3' proteins, representing related homeodomain proteins (30), FIG. 1. Restriction map of the Hox-2.2 genomic region and can be grouped into a second class of N termini. The schematic representation of the isolated cDNA. The open reading conservation of N-terminal regions among different homeo- frame is represented by open boxes. Stippled boxes indicate the box genes of widely diverged species suggests an important homeobox region. Abbreviations for restriction enzymes: E, EcoRI; functional role for the N terminus, perhaps in intracellular B, BamHl; P, Pst I; Pv, Pvu II. Arrows above the genomic map localization. The N-terminal region of the yeast MATa2 indicate which genomic regions have been sequenced. Open arrow homeodomain protein, for example, is necessary for its indicates the direction of transcription. transport to the nucleus (35). The predicted Hox-2.2 amino acid sequence also contains of 1.4 kb with an open reading frame of 672 bp, preceded by a highly conserved hexapeptide 14 amino acid residues a 5' untranslated region of 109 bp and followed by a 3' upstream ofthe homeodomain (residues 127-132, Fig. 2). The untranslated region of about 650 bp. The structure and sequence and position of these hexapeptides, originally organization of mouse Hox-2.2 shows several similiarities to described by Mavilio et al. (23) are conserved in a number of other homeobox genes within the coding region and the homeodomain proteins (Fig. 3B). As shown at the bottom of untranslated regions. Fig. 3B, the Hox-2.2 hexapeptide is almost identical to a The predicted Hox-2.2 protein. Based on our sequence conserved ,-type globin sequence (30, 36) that is involved in analyses of cDNA clone c5, the entire amino acid sequence protein-protein interactions between al and ,82 subunits (34). of a putative Hox-2.2 protein can be predicted (Fig. 2). The This suggests that a possible function of this hexapeptide in largest open reading frame in clone c5 is in-frame with the homeodomain proteins is to influence protein-protein inter- homeobox and codes for a protein of 224 amino acids with a actions and thereby modify the DNA-binding properties of calculated molecular weight of 25,312. In addition to the possible multimeric homeodomain proteins. Interestingly, Antennapedia-class homeodomain (12), the 224 amino acid alternatively spliced transcripts of the Drosophila Ubx gene Hox-2.2 protein contains three regions conserved with other that differ in the spacing between the hexapeptide and the homeodomain proteins.