Sequence of the Tribolium castaneum Homeotic Complex: The Region Corresponding to the Drosophila melanogaster Antennapedia Complex

Corresponding author: Susan J. Brown, Ackert Hall, Kansas State University, Manhattan, KS 66506., sjbrown{at}ksu.edu (E-mail)

Communicating editor: T. C. KAUFMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The homeotic selector genes of the red flour beetle, Tribolium castaneum, are located in a single cluster. We have sequenced the region containing the homeotic selector genes required for proper development of the head and anterior thorax, which is the counterpart of the ANTC in Drosophila. This 280-kb interval contains eight homeodomain-encoding genes, including single orthologs of the Drosophila genes labial, proboscipedia, Deformed, Sex combs reduced, fushi tarazu, and Antennapedia, as well as two orthologs of zerknüllt. These genes are all oriented in the same direction, as are the Hox genes of amphioxus, mice, and humans. Although each transcription unit is similar to its Drosophila counterpart in size, the Tribolium genes contain fewer introns (with the exception of the two zerknüllt genes), produce shorter mRNAs, and encode smaller proteins. Unlike the ANTC, this region of the Tribolium HOMC contains no additional genes.

HOX genes encode transcription factors that regulate developmental fate along the anterior-posterior (A-P) body axis of virtually all bilaterians (FINNERTY and MARTINDALE 1998 ). Hox genes, located in clusters, are spatially colinear in that their chromosomal organization reflects their functional domains along the A-P axis. Multiple Hox clusters have been described for several vertebrates including mice (GRAHAM et al. 1989 ), humans (ACAMPORA et al. 1989 ), and fish (KIM et al. 2000 ). In contrast, single Hox clusters have been identified in a number of invertebrates including amphioxus (GARCIA-FERNANDEZ and HOLLAND 1994 ), sea urchins (POPODI et al. 1996 ), and several insects such as mosquitoes (DEVENPORT et al. 2000 ; POWERS et al. 2000 ), beetles (BEEMAN 1987 ), and locusts (FERRIER and AKAM 1996 ). In Drosophila, the single complement of Hox genes is divided into two clusters, the Antennapedia complex (ANTC) and the bithorax complex (BXC), separated by 7.5 Mb on the right arm of LG 3 (ADAMS et al. 2000 ). The ANTC contains genes required for proper development of the gnathal and thoracic segments [labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), and Antennapedia (Antp); KAUFMAN et al. 1990 ], while the BXC genes [Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)] direct developmental fate in the posterior thorax and abdomen (BENDER et al. 1983 ). The ANTC contains additional genes: zerknüllt (zen), a Hox gene that lost its function along the A-P axis and gained a function in the extraembryonic region of the insect egg (FALCIANI et al. 1996 ); zerknüllt-related (zen2), a recent duplication of zen; bicoid (bcd), another recent derivative of zen (STAUBER et al. 2000 ) that encodes the anterior morphogen of the Drosophila embryo (STRUHL et al. 1989 ); and fushi tarazu (ftz), a pair-rule gene related to the Antp-class homeobox genes (SCOTT and WEINER 1984 ). Eight cuticle genes, five lysine tRNA genes, and amalgam (ama), which encodes a member of the immunoglobulin superfamily, are also located in the Drosophila ANTC (ADAMS et al. 2000 ).

Sequence analysis of the relatively compact Hox clusters (80–120 kb) of several deuterostomes has revealed that the order and orientation of Hox genes is well conserved. However, among protostomes, only the considerably larger ANTC and BXC (400 and 350 kb, respectively) of Drosophila melanogaster (MARTIN et al. 1995 ; ADAMS et al. 2000 ) and the highly degenerate Hox cluster of Caenorhabditis elegans (C. ELEGANS SEQUENCING CONSORTIUM 1998) have been sequenced. Analysis of homeobox sequences from several arthropod classes (for a recent synthesis see COOK et al. 2001 ) and an onychophoran (GRENIER et al. 1997 ) indicates that precursors of eight Hox genes, as well as zen and ftz, existed in the protoarthropod. Although these gene fragments are often aligned with their Drosophila counterparts for comparison, the number, order, and orientation of these genes, or whether they are actually clustered, is not fully understood for any other arthropod.

In the red flour beetle, Tribolium castaneum, we have previously identified and characterized the cDNA sequences, expression patterns, and, for some genes, the mutant phenotypes of the orthologs of pb (SHIPPY et al. 2000A , SHIPPY et al. 2000B ), zen (FALCIANI et al. 1996 ), Dfd (BROWN et al. 1999 , BROWN et al. 2000 ), ftz (BROWN et al. 1994 ), Scr (CURTIS et al. 2001 ), Ubx (BENNETT et al. 1999 ), and abd-A (STUART et al. 1993 ; SHIPPY et al. 1998 ). We recently described a bacterial artificial chromosome (BAC) clone containing the Tclab transcription unit and the 3' end of the Tribolium homeotic complex (HOMC; NIE et al. 2001 ). We found no genes in the interval between the 5' end of Tclabial (Tclab) and the 3' end of maxillopedia (mxp, the ortholog of pb), indicating that the cuticle genes found in the corresponding location in the Drosophila ANTC are likely to be a recent addition. We also noted that different genes are located downstream of lab in Tribolium and Drosophila, indicating a lack of synteny outside the HOMC between these two insects. Further, although even-skipped (eve) homologs are linked to homeotic gene clusters in mammals (D'ESPOSITO et al. 1991 ; FAIELLA et al. 1991 ), we previously determined that, similar to eve in Drosophila, Tceve is not linked to the HOMC (BROWN et al. 1997 ). We have sequenced the rest of the BAC clone containing Tclab, as well as two additional BAC clones containing the interval delimited by the 5' half of mxp and prothoraxless (ptl, the ortholog of Antp). In a brief description of the second BAC clone (BROWN et al. 2001 ) we noted that it contains mxp, TcDeformed (TcDfd), Cephalothorax (Cx, the ortholog of Scr), and Tcfushi tarazu (Tcftz), as well as two Tczerknüllt (Tczen) genes (more closely related to each other than to the Drosophila zen genes), but no other genes. The third BAC clone contains ptl and overlaps the second BAC clone in the region containing Tcftz. Here we describe the organization of the region of the Tribolium HOMC corresponding to the Drosophila ANTC, including gene order and orientation, as well as intergenic distances.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BAC library screens:
One BAC library was constructed in our laboratory by partially digesting GA-1 (wild-type) genomic DNA with HindIII and ligating it into pBeloBac11 (ZHU et al. 1997 ). This library contains inserts averaging 40–60 kb in length. The identification of Bmxp1 from this library was previously described (SHIPPY et al. 2000A ). A second BAC library (kind gift of Exelixis Pharmaceutical, San Francisco), constructed from GA-2 genomic DNA that had been partially digested with EcoRI and ligated into pBACe3.6, contains inserts averaging 125 kb. GA-2 (a kind gift of Scott Thomson, University of Wisconsin, Parkside), was derived from GA-1 by 20 generations of single brother/sister matings and is also being used in the construction of a high-density molecular map of the Tribolium genome. Twenty-two BAC clones were identified by screening the second BAC library with radiolabeled Tcftz cDNA and a fragment from the 3' end of an Ultrathorax (Utx) cDNA (Utx, the ortholog of Ubx). EcoRI fingerprint blots of each clone were hybridized under stringent conditions with radiolabeled fragments from Tclab, mxp, TcDfd, Cx, Tcftz, ptl, and Utx. A preliminary contig was assembled based on the hybridization patterns. Finally, the extent of overlap between individual clones was determined by radiolabeling selected BAC clone inserts and hybridizing them to the fingerprint blots. Clones with minimal overlap were chosen for sequencing.

Shotgun subcloning and sequencing:
BAC clone DNA was isolated using the Qiagen large construct kit (QIAGEN, Valencia, CA), sheared with a nebulizer and subcloned into pCR4Blunt-TOPO using the TOPO Shotgun subcloning kit (Invitrogen, Carlsbad, CA). White colonies were restreaked to verify color selection. Colonies containing inserts were picked and transferred to 96-well microtiter plates. Plasmid DNA was purified and sequenced by automated protocols at the Sequencing and Genotyping Facility in the Department of Plant Pathology at Kansas State University. Files containing Escherichia coli DNA sequence were removed from the data set and BAC contigs were constructed using PHRED, PHRAP, and CONSED (EWING et al. 1998 ; GORDON et al. 1998 ). The finished sequence averages greater than sixfold coverage. Gaps in the BAC insert sequences were filled by sequencing available subclones on both strands with primers designed from the ends of contigs.

Sequence analysis:
The previously identified cDNAs were mapped on the BAC sequences by comparing cDNA and genomic sequence. The following GenBank accession numbers correspond to the cDNA sequences used: Tclab, AF231104 and AF230312; mxp, AF187068 and AF187069; Tczen1, X97819; Tczen2, AF452568; TcDfd, U81038; Cx, AF227628; ftz, U14734; and ptl, AF228509 and AF452569. The BAC contig sequences were also analyzed using BLASTX [National Center for Biotechnology Information (NCBI)], FGENESH (BCM Gene Finder), GENIE (REESE et al. 1997 ), and GENESCAN (BURGE and KARLIN 1997 ) to identify putative genes. MatInspector (QUANDT et al. 1995 ) was used to search Tribolium sequences for conserved regulatory sites. Annotated sequences are available in GenBank under the following accession numbers: Bmxp1, AY043293; 35E10, AF321227; and 35F4, AY043292. These entries provide corrected and updated sequences for previous entries of Tczen1, Tcftz, and ptl with corresponding changes in the deduced amino acid sequences. The Tribolium sequence was analyzed for repeats and regions of simple sequence using RepeatMasker (University of Washington Genome Center).

RT-PCR:
We used total RNA from 0- to 16-hr eggs as template for reverse transcription using the Thermoscript RT-PCR system (GIBCO BRL, Gaithersburg, MD). Primers designed to amplify the second Tribolium zen gene were located 56 bp downstream of the first methionine codon in the newly identified open reading frame (ORF) and 86 bp upstream of the translation stop codon in frame with the homeobox. The single RT-PCR product was cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced at the Sequencing and Genotyping Facility in the Department of Plant Pathology at Kansas State University (GenBank accession no. AF452568).

Protein alignment:
Protein sequences were aligned using Clustal W with the Blosum62mt score matrix (Vector NTI, Informax, North Bethesda, MD). The dendogram shown in Fig 2 was produced using the neighbor-joining algorithm (SAITOU and NEI 1987 ). The GenBank accession numbers of the Zen protein sequences used in this analysis are the following: Megaselia abdita, CAB40893; Schistocerca gregaria, CAB61208; D. melanogaster, AAF54087 and AAF54088; and T. castaneum, AAK16425 and AAK16424.

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. Comparison of Drosophila ANTC and the corresponding region of the Tribolium HOMC. The Tribolium BAC clones are shown above the Tribolium map. The transcription units are shown with boxes denoting exons and vertical or diagonal lines denoting introns. Differential splicing is indicated by introns above and below the exons. Homeobox genes are shown below the chromosome and nonhomeobox genes above, with the exception of Tcchp. Arrows indicate the direction of gene transcription. The Tribolium and Drosophila orthologs are color coded. The Drosophila map was constructed using the annotations from GenBank entries AE001572, AE001573, AE001574.

View larger version (10K): In this window In a new window Download PPT slide   Figure 2. The phylogenetic relationship of insect Zen proteins. We compared the Zen proteins from Tribolium (TcZen1 and TcZen2) with those from S. americana (SgZen), D. melanogaster (DmZen and DmZ2), and M. abdita (MaZen). The Tribolium and Drosophila Zen proteins form separate groups, suggesting that the zen gene pairs in Tribolium and Drosophila arose via independent duplication events in each lineage.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Two Tribolium BAC libraries were screened for clones containing homeotic genes. We sequenced three clones with minimal overlap (Bmxp1, 35E10, and 35F4), which span a region corresponding to the Drosophila ANTC. The resulting 279,643-bp Tribolium genomic contig is delimited at one end by the 3' end of chaoptic (chp), the gene flanking Tclab. The other end of the contig extends 25 kb upstream of ptl (Fig 1). This portion of the Tribolium HOMC is two-thirds the length of the Drosophila ANTC (as defined by the distance between the agt and Soh-1 genes, which flank lab and Antp, respectively). The G/C content of the Tribolium contig is 33.5%. Fifteen simple sequence repeats comprise 0.3% of the total sequence, only two of which contain any G/C. A total of 203 regions of low complexity, which are dispersed randomly throughout the contig, are predominantly A, T, or A/T rich and comprise 3.25% of the total sequence.

Polymorphisms in Tribolium DNA:
The sequences of Bmxp1 and 35E10 match perfectly in the 4.2 kb by which they overlap. In contrast, we found several single nucleotide polymorphisms (SNPs) within the 8-kb overlap of BAC clones 35E10 and 35F4, indicating that the inbred strain used as the source of DNA for this BAC library is not completely isogenic and still contains heterogeneous chromosomal regions (if not entire chromosomes). SNPs were detected at a rate of 1/85 bp, which is similar to the rate of SNP detection in Drosophila (MORIYAMA and POWELL 1996 ).

Genes within the Tribolium HOMC:
The transcription units of the Tribolium homeotic genes Tclab, mxp, TcDfd, Cx, and ptl, as well as those of Tczen and Tcftz, were located within the contig by comparing genomic and previously identified cDNA sequences (Fig 1). In the following descriptions of Tribolium homeobox genes, the total length of the transcription units and the length of the large intron upstream of the homeobox (when present) were determined from the sequence described here (Table 1). Exon sizes reported here confirm previous analyses and are included for completeness. Tclab is located at the 3' end of the HOMC. The 13-kb transcription unit is composed of two exons (658 and 816 bp) separated by an 11.6-kb intron located 125 bp upstream of the homeobox. The mxp gene, located 13.8 kb upstream of Tclab, contains three exons (561, 142, and 1430 bp). The first two are separated by a large intron (18.5 kb) located 11 bp upstream of the homeobox. The second intron (only 51 bp) is located within the homeobox. TcDfd is located 57.7 kb upstream of mxp. This 9.78-kb transcription unit contains a single intron (7.7 kb) located 29 bp upstream of the homeobox. The Tribolium Cx gene is located 24.6 kb farther upstream. It contains three exons (753, 328, and 633 bp). The first two are separated by a 20.1-kb intron located 20 bp upstream of the homeobox. The second exon (160 bp) is located in the 3' untranslated region (UTR) 24 bp downstream of the translation stop codon. The 922-bp Tcftz transcription unit is located 9.7 kb upstream of Cx. It contains a single intron (49 bp) located 17 bp upstream of the homeobox.

The Tribolium zen genes:
The previously described Tczen gene is located 31.5 kb upstream of mxp (Fig 1). Two short introns (53 and 57 bp) located upstream of the homeobox separate three exons (122, 96, and 613 bp). A homeobox encoding a zen-related homeodomain, identified by BLASTX (NCBI), was found between Tczen and mxp (BROWN et al. 2001 ). The ORF including this homeobox does not contain an initiation methionine. Therefore, we identified an additional upstream ORF, including a candidate initiation methionine, and designed primers to amplify a cDNA derived from this putative gene. The sequence of the resulting 730-bp fragment (GenBank accession no. AF452568) was compared with genomic DNA to identify introns. This newly identified zen gene contains three exons (180, 84, and 610 bp) separated by two small introns (55 and 47 bp) that are located upstream of the homeobox. We have changed the name of the previously identified zen gene to Tczen1 and named the newly identified gene Tczen2. There are only 216 bp between the 3' end of Tczen1 [as determined by the sequence of a cDNA clone (FALCIANI et al. 1996 )] and the first candidate initiation methionine within the Tczen2 ORF. The proteins encoded by the Tribolium zen genes share regions of similar amino acids, one of which extends downstream of the homeodomain for several residues. The two Tribolium Zen proteins are more similar to each other than they are to either of the Drosophila Zen proteins, which do not contain sequence similarity to one another outside the homeodomain. We compared the Zen proteins from Tribolium with those from the Orthopteran S. americana and two Dipterans, D. melanogaster and M. abdita. In the dendogram shown in Fig 2, the Drosophila Zen proteins are most closely related to one another within the group of dipteran Zen proteins, while the two Tribolium Zen proteins form a separate group. These results support the hypothesis that the zen gene pairs in Tribolium and Drosophila arose via independent duplication events in each lineage.

The Tribolium ortholog of Antp:
Comparison of cDNA and genomic sequences provides evidence that at least four different messages are transcribed from the Tribolium ptl locus encoding the ortholog of Antp. These messages differ at the 3' end, indicating the use of three different polyadenylation sites. The ptl locus contains three exons (512, 862, and 1613 bp) separated by two introns (14.4 and 7.2 kb). In addition to the single cDNA containing all three exons, we sequenced several different cDNAs that lack the first exon and instead contain sequence from the 3' end of the first intron contiguous with the second exon, indicating the presence of a second promoter downstream of the first (Fig 1). Messages initiated from this promoter contain two exons (1539 and 1613 bp) and the 7.2-kb intron immediately upstream of the homeobox. All identified mRNAs from the Tribolium ptl gene encode the same protein, which contains the conserved motifs previously described for Antp orthologs in other hexapods. A detailed analysis of ptl will be required to assess differential expression from the two promoters and the use of the multiple polyadenylation sites during development.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Comparison of Tribolium Zen proteins:
The amino and carboxy terminal sequences, as well as the homeodomain sequences of the eight Tribolium Hox proteins, are aligned in Fig 3. The putative Zen proteins show similarity to the central class proteins (group C in Fig 3) within the N-terminal octapeptide (ZHAO et al. 1993 ). Zen1 is particularly interesting in that it contains the same inserted Q residue as the Scr/Cx orthologs. Another shared region includes the carboxy terminus. Three of the last four residues (LTxL) are shared not only with each other but also with TcDfd (Fig 3) and several of its orthologs (data not shown). The Zen/Hox 3 proteins of grasshopper, S. gregaria (GenBank entry CAB61208), and a polychaete worm, Chaetopterus variopedatus (GenBank entry AAD55934), also end in LTxL. Short motifs, such as the WRPY motif located at the carboxy terminus of Hairy and Runt proteins (WAINWRIGHT and ISH-HOROWICZ 1992 ), can be functionally important. Although the function of the motifs in the Tribolium proteins is not known, such sequence conservation outside the homeodomain supports the hypothesis that the Hox 3 gene family arose via duplication of the central class precursor (ZHANG and NEI 1996 ) rather than the anterior class precursor (SCHUBERT et al. 1993 ).

View larger version (26K): In this window In a new window Download PPT slide   Figure 3. Comparison of Tribolium Hox protein sequences. Sequences from the amino terminus, the homedomain, and the carboxy terminus of each protein are shown. (A) Proteins encoded by anterior-class genes. (C) Proteins transcribed by central-class genes. Residues outside the homeodomain that are shared by the Zen proteins (B) and proteins in C are in boldface type.

Comparison of Tribolium and Drosophila genes:
In Table 1 the transcription unit length, number of exons per gene, and size of the proteins encoded by the Tribolium genes are compared with their Drosophila orthologs. The length and gene content of the intergenic regions are compared in Table 2. Tclab is 4 kb shorter than lab. The location of the first intron, between the YKWM motif-encoding region and the homeobox, is conserved. However, the lab homeobox contains an intron not found in Tclab. Sequence information from additional species will be required to determine whether lab gained this intron or Tclab lost it. In the 29.6-kb region between lab and pb, there are eight cuticle genes (GenBank accession no. AE003674). However, no genes are in the corresponding interval in Tribolium, which is only 13.8 kb (Table 2 and NIE et al. 2001 ). pb is 13 kb larger than mxp and contains five more exons. The location of the two introns in mxp, one in the homeobox and one 11 bp upstream of the homeobox, are conserved in pb. Again, in the absence of additional data it is impossible to determine whether the Drosophila pb gene gained introns or the Tribolium mxp gene lost them.

Both insects contain two zen genes; however, these genes appear to have arisen via independent duplication events in each lineage and show species-specific features. For example, the two Drosophila zen genes contain two exons while the two Tribolium zen genes contain three. In Tribolium there is very little distance between the zen genes (217 bp), and they are located 31 kb upstream of mxp (Table 2). In contrast, zen2 is within 1.2 kb of pb in Drosophila, and >8 kb separates zen1 and zen2. Three genes (bcd, ama, and CG1162) are located in the 37-kb region between zen1 and Dfd in Drosophila. The corresponding region is somewhat smaller in Tribolium (26 kb) and does not appear to contain any genes. TcDfd contains fewer exons than Dfd (two vs. five, Table 1; BROWN et al. 1999 ). However, the transcription units are similar in size. This is due in large part to the presence of a long intron (>9 kb in each insect) immediately upstream of the homeobox. TcDfd is transcribed in the same orientation as the other genes. In contrast, Dfd is transcribed in the opposite orientation. Six lysine tRNA genes are in the 20.7-kb region between Dfd and Scr. Although slightly larger (24.6 kb), the corresponding region in Tribolium does not contain any genes. Scr and Cx contain four and two exons, respectively. Scr transcripts initiate at two promoters (ANDREW 1995 ; Fig 1). However, we have found evidence only for a single promoter in Cx (CURTIS et al. 2001 ; Fig 1). Tcftz and ftz are similar in that they are relatively short transcription units (1.4 and 1.9 kb) located upstream of the Cx/Scr orthologs (9.7 and 14.3 kb, respectively) and contain a single small intron upstream of the homeobox. However, Tcftz is transcribed in the same direction as the other genes, whereas ftz is transcribed in the opposite direction. The distance between Tcftz and ptl is more than twice that between ftz and Antp (66 vs. 30 kb, Table 2). Antp contains two promoters and eight exons that are differentially spliced to produce different mRNAs. ptl is similar to Antp in that it contains two promoters and produces multiple mRNAs. However, it contains only three exons.

Although the Tribolium genes encode smaller proteins (Table 1) and, with the exception of the zen genes, contain fewer introns, they are only slightly shorter than their Drosophila counterparts (Table 1). The Antp/ptl orthologs are an exception to this observation in that the longest Antp transcription unit is >100 kb in length, while the longest ptl transcription unit is only 24 kb. Most of this difference is due to the distance between promoters. Several of the Drosophila Antp gain-of-function (GOF) mutations result from translocation breakpoints located between the first and second promoter (SCHNEUWLY et al. 1986 ). Assuming that corresponding breaks would produce similar mutant phenotypes in Tribolium, it is tempting to speculate that the lack of GOF mutations mapping to the ptl locus reflects the considerably smaller target region between the two promoters.

It has been suggested that the large transcription units in Drosophila prevent them from being expressed prematurely during rapid, long-germ development (counted in hours; SHERMOEN and O'FARRELL 1991 ). Similar correlation in Tribolium seems unlikely, since the embryos require 3–4 days to develop at 30° and the early divisions alone are counted in hours (HANDEL et al. 2000 and our unpublished observations).

Search for a bicoid ortholog:
Comparison of Bicoid and Zen homeodomain sequences from Megaselia, a basal cyclorrhaphan fly, and Drosophila suggests that bicoid may have resulted from a recent duplication of the zen gene (STAUBER et al. 2000 ). Further, bicoid is located upstream of zen in both Megaselia and Drosophila (BROWN et al. 2001 ), suggesting this might be its ancestral location. However, we find no evidence for any additional genes, including bicoid, in the Tribolium contig described here. Thus, if Tribolium does contain an ortholog of bicoid it must be located outside this portion of the HOMC.

Large intergenic distances:
The distance between homeobox genes in Tribolium and Drosophila is compared in Table 2. Additional genes found in the Drosophila ANTC are noted in the appropriate intervals. As noted above, the Tribolium zen genes are quite distant from neighboring genes. In addition, the distance between ftz and Antp/ptl orthologs is twice as far in Tribolium (60 kb) as it is in Drosophila (30 kb). Such differences in intergenic spacing may contribute to the evolution of regulatory differences between the Drosophila and Tribolium orthologs.

The intergenic distances in Drosophila and Tribolium are considerably larger than those in any deuterostome Hox cluster. It has been suggested that the differences between Drosophila and vertebrate complexes (larger intergenic distances, longer transcription units, additional nonhomeotic genes, inversions, and breaks in the Drosophila complexes) reflect a loosening of structural and regulatory constraints in Drosophila that, in other organisms, might be required to maintain temporal, if not spatial, colinearity (FERRIER and AKAM 1996 ). These large intervals are known to contain widely spaced regulatory elements in Drosophila (GINDHART et al. 1995 ) and may be important to regulation in Tribolium as well. In the mosquito Anopheles gambiae, the interval containing the orthologs of lab and Antp is >350 kb. Long distance Southern mapping indicates similarly distant spacing of Hox genes in the grasshopper, S. gregaria (FERRIER and AKAM 1996 ). Thus, it is likely that large size and dispersed regulatory regions are common to hexapod Hox clusters.

Putative regulatory regions upstream of identified genes were searched for known binding sites. Several consensus homeodomain binding sites were identified by MatInspector in the 5-kb regions immediately upstream of mxp, TcDfd, and Cx. To understand the significance, if any, of these sites, it will be necessary to analyze these regions for their ability to direct gene-specific expression patterns of reporter genes. Such studies are now feasible in Tribolium using a recently described transformation system (BERGHAMMER et al. 1999 )

The lack of inversions, breaks, and nonhomeobox genes in the region of the Tribolium HOMC corresponding to the Drosophila ANTC suggests that the structure of the Tribolium HOMC is more constrained than that of the Drosophila clusters. To determine whether such structural constraints have regulatory consequences, it will be necessary to determine whether Tribolium Hox genes can function outside the chromosomal context of the complex. This can be accomplished by examining homeotic mutants that might carry translocations or other chromosomal rearrangements or by attempting to rescue the effects of a homeotic mutation by reintroducing a copy of the wild-type gene into the Tribolium genome.

	ACKNOWLEDGMENTS

We thank Barb Van Slyke for expert technical assistance. We thank Scott Thomson for the inbred Tribolium strain and Exelixis for the kind gift of the Tribolium BAC library. This work was supported by grants from the National Institutes of Health and the National Science Foundation to R. Denell and S. Brown. E. Richardson was supported by a Howard Hughes Medical Institute undergraduate research fellowship. Contribution no. 02-7-J is from the Kansas Agricultural Experiment Station.

Manuscript received July 30, 2001; Accepted for publication December 13, 2001.

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