Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra (Barcelona) Spain
Correspondence: E-mail: alfredo.ruiz{at}uab.es.
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Abstract |
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Key Words: labial Hox complex Drosophila buzzatii chromosomal rearrangement genome evolution
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Introduction |
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A single Homeotic gene cluster (HOM-C) is postulated to have existed in the ancestor of the arthropods (Hughes and Kaufman 2002), although most sequences are partial and, usually, there are no data about the chromosomal localization of the genes (Finnerty and Martindale 1998; deRosa et al. 1999; Cook et al. 2001). The 10 Hox genes included in this ancestral complex are (in anteroposterior order): labial (lab), proboscipedia (pb), Hox3, Deformed (Dfd), Sex comb reduced (Scr), fushi tarazu (ftz), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B). This was probably also the organization in the ancestor of the Drosophila genus, although in insects Hox3 and ftz have lost their Homeotic function (Hughes and Kaufman 2002). Two rearrangements of this ancestral Hox complex are known to have occurred during the evolution of the Drosophila genus (fig. 1). In the lineage leading to D. melanogaster a split between Antp and Ubx separated the genes in two complexes: the Antennapedia complex (ANT-C) (Kaufman, Seeger, and Olsen 1990) and the Bithorax complex (BX-C) (Lewis 1978; Duncan 1987). These two complexes are located on the right arm of D. melanogaster chromosome 3, separated by approximately 9.6 Mb of euchromatic sequences. The ANT-C spans 400 kb (Adams et al. 2000) and comprises five Hox genes (lab, pb, Dfd, Scr, and Antp), which are expressed in those segments that will become the head and the anterior thorax. The BX-C spans
350 kb (Martin et al. 1995; Adams et al. 2000) and contains three Hox genes (Ubx, abd-A, and Abd-B), which are expressed in the posterior thorax and abdomen. A different arrangement of the Hox genes was found in D. virilis, a species of the Drosophila subgenus (vonAllmen et al. 1996). In this species Ubx mapped by in situ hybridization with Antp instead of with abd-A, indicating that in the lineage leading to this species a disruption of the Hox complex occurred between the genes Ubx and abd-A (fig. 1).
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Materials and Methods |
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In Situ Hybridization to Salivary Gland Chromosomes
Clones used as probes to map by in situ hybridization the Antp, Ubx, abd-A, and Abd-B genes have been reported elsewhere (Ranz, Segarra, and Ruiz 1997; Ranz, Casals, and Ruiz 2001). To localize the lab gene in D. buzzatii, D. hydei, D. mercatorum, D. repleta, and D. virilis, homologous probes were generated in each species by polymerase chain reaction (PCR) amplification of a 7251,172-bp segment encompassing exons 23 of the gene and including the homeobox (table 1), followed by cloning and sequencing of the amplified products. The clone of D. virilis was used to map lab in D. immigrans and D. canalinea.
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Southern Hybridization and Library Screening
Genomic DNA extraction was performed as described in Piñol et al. (1988). DNA was digested with different restriction enzymes and transferred from the agarose gels to nylon membranes by capillarity transfer under neutral conditions (Sambrook, Fritsch, and Maniatis 1989). Probes were labeled with digoxygenin-11-dUTP by random primer with the DIG DNA Labeling and Detection Kit (Roche). Hybridization was performed at 42°C for 16 h using standard hybridization buffer with formamide (50%). Filters were washed twice for 15 min in 2x SSC 0.1% sodium dodecyl sulfate (SDS) at room temperature and twice for 20 min in 0.1x SSC 0.1% SDS at 68°C.
A lambda genomic library derived from D. buzzatii line j-19 (Cáceres, Puig, and Ruiz 2001) was amplified following Sambrook, Fritsch, and Maniatis (1989) and used for cloning of lab. Screening was done by plaque hybridization under the same conditions as for the Southern hybridization. Two probes were used to clone lab in D. buzzatii, one corresponding to exons 23 and the other to exon 1, separated in D. melanogaster by a large intron of nearly 14 kb. The probe containing exons 2 and 3 was the same 1,122 bp fragment used in the in situ hybridization (see above). The exon 1 probe consisted in a 1,285-bp fragment amplified by PCR using specific primers (table 1). Sequencing of the PCR product confirmed its homology with exon 1 of D. melanogaster lab.
Polymerase Chain Reaction Amplification
Primers (table 1) were designed with Primer Designer (1.011990 Scientific and Educational Software). Primers used to amplify the lab segment comprising exons 23 (homeobox) in all species were designed according to the D. melanogaster lab sequence. Exon 1 of D. buzzatii and D. virilis were amplified using the regions conserved between Labial proteins of D. melanogaster (AAD19811.1) and Tribolium castaneum (AAF64147.1). To amplify the cDNA of D. buzzatii and D. virilis, forward primers were designed in each species exon 1 sequence. Primers located in the transcription initiator (Inr) sequence of D. buzzatii and the beginning of the coding region of D. virilis were used to amplify the 5' UTR in the latter species.
Polymerase chain reaction was carried out in a volume of 50 µl, including 100200 ng of genomic DNA or cDNA or 1 µl of phage lysate, 20 pM of each primer, 200 µM dNTPs, 1.5 mM MgCl2, and 1 unit Taq DNA polymerase. Temperature cycling conditions were 30 rounds of 30 s at 94°C, 30 s at the annealing temperature, and 60 s at 72°C, with annealing temperature varying between 55° and 61°C depending on the primer pair used.
RNA Isolation and Reverse Transcriptase-Polymerase Chain Reaction
Total RNA from 012-h-old embryos from D. buzzatii (line j-19) and D. virilis was isolated with TRIZOL (GIBCO) following the manufacturer's instructions. Glassware was washed with DEPC-water to eliminate RNases. cDNA was obtained by random primer with the First Strand cDNA Synthesis Kit for reverse transcriptase polymerase chain reaction (RT-PCR; AMV-Roche). Amplification conditions for RT-PCR were as described for PCR.
DNA Sequencing and Sequence Analysis
Polymerase chain reaction products and restriction fragments from lambda phages were cloned into pGEM-T (Promega) and Bluescript II SK (Stratagene) and sequenced with universal primers. In some cases internal specific primers were designed for primer walking. Sequencing was performed in an ALF express DNA automated sequencer (Pharmacia Biotech) and an ABI 373 A (PerkinElmer) automated sequencer. Nucleotide sequences were analyzed with the Wisconsin Package (Genetics Computer Group). Similarity searches were done with algorithms Fasta, BlastN, and BlastX. ClustalW (Thompson, Higgins, and Gibson 1994) was used to perform multiple alignments. AVID was used to align long sequences (>3 kb) and mVISTA to visualize the alignments (Mayor et al. 2000; Dubchak et al. 2000). Untranslated region (UTR) functional elements were searched with UTRscan (Pesole and Liuni 1999).
Maximum likelihood estimates of the number of synonymous and nonsynonymous substitutions per site (dS and dN, respectively) were obtained with the program codeml of the PAML package (Yang 1997). For hypothesis testing, the base model assumed a rooted tree with constant nucleotide substitution rate (global clock) and a single = dN/dS ratio. Codon equilibrium frequencies
i were estimated from the nucleotide frequencies of the three codon sites (F3X4 option of codeml; Yang 1997). The base model included 13 parameters: the transition/transversion rate ratio (
), the nonsynonymous/synonymous substitution ratio (
), two relative branch lengths (rs), and nine nucleotide frequencies.
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Results and Discussion |
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In D. virilis three genomic fragments of the lab gene (Dv lab) where amplified by PCR and sequenced (table 2): a 1,172-bp fragment corresponding to exons 2 and 3 and intron 2, a 1,268-bp fragments corresponding to exon 1, and a 1,818-bp fragment containing the 5' UTR. In addition, a 548-bp cDNA fragment comprising the area from the end of exon 1 to the beginning of exon 3 was also cloned and sequenced. Overall, 3,184 bp were sequenced, with only intron 1, part of exon 3, and the 3' UTR remaining.
The molecular structure of Db lab and Dv lab is similar to that of Dm lab, comprising three exons and two introns (table 2 and fig. 3). Therefore, the structure of the lab transcription unit has not been altered by the lab-pb split in D. buzzatii and the exact breakpoint must be located at least 8 kb upstream from the lab transcription start point. In contrast, Db lab is 32% larger than Dm lab, mostly because of changes in noncoding regions. The total length of the CDS is about 4% larger (1,968 bp versus 1,890 bp), whereas intron 1 is 5 kb longer and intron 2 and the 5' UTR are more than twice as large in D. buzzatii (table 2). In general, the D. virilis gene is more like that of D. buzzatii than that of D. melanogaster (table 2), as expected from their phylogenetic relationships (fig. 1). Interestingly, the lab structure differs widely between Drosophila and Tribolium castaneum (table 2). In the latter species, the CDS is 1,061 bp long with two exons only, and intron 1, the 5' UTR, and the 3' UTR are also significantly shorter (Nie et al. 2001).
Expression of labial and Identification of Regulatory Sequences
To verify the predicted exon structure of Db lab, total RNA from 012-h-old embryos was amplified by RT-PCR. Primers were designed on exon 1 and exon 3 (table 1 and fig. 3), allowing us to examine both exon junctions at the same time. The amplified cDNA fragment had the expected size of 570 bp, and its sequence confirmed precisely the predicted exon-intron junctions. In certain D. melanogaster lines, an alternative splicing of lab intron 1 that produces two mRNAs with 18 nucleotides of difference has been described (Mlodzik, Fjose, and Gehring 1988). This alternative splicing is caused by a small duplication at the end of intron 1 that provides a new functional splice acceptor site and gives rise to the longer form of the mRNA (FlyBase 2002). The analysis of the genomic and cDNA sequences of Db lab indicates that in D. buzzatii there is no alternative splicing and only the short form of mRNA is present. In D. virilis, the sequenced cDNA fragment also corresponds to the short mRNA. To identify the end of the 3' UTR, polyadenylation signals were searched along the sequence 3' from the stop codon. Four possible polyA signals were identified between 623 and 825 bp from the stop codon of Db lab. No conserved sequences were found between Dm lab and Db lab on the 3' UTR.
The open reading frame and translation start site for Db lab were predicted by identification of consensus sequences and homology with Dm lab. To find the promoter and transcription start sites, 1,308 bp of genomic DNA sequence from the 5' region of Db lab were aligned with 630 bp from the 5' region of Dm lab (X13104 X12834) including the 5' UTR and 400 additional nucleotides upstream (fig. 4). The most conserved region between the two sequences corresponds to the transcription start site described for Dm lab (Mlodzik, Fjose, and Gehring 1988; Diederich et al. 1989). In Db lab, we find in this conserved region the sequence TCAGTC that fits well the proposed consensus TCA+1G/TTT/C of the initiator (Inr) of Drosophila genes, where A+1 is the transcription start site (Arkhipova 1995). No other sequence similar to the Inr consensus was found in the 5' upstream region of Db lab. An additional alignment including the 738-bp upstream sequence of Dv lab and using the ClustalW algorithm (Thompson, Higgins, and Gibson 1994) with a gap extension penalty of 0.05 indicated also a high identity around both the transcription (Inr) and the translation (Met) start sites between the three Drosophila species (fig. 4). There is a high homology between the Inr and position +32 in the three species (fig. 4), with the sequence CACG located between positions +29 and +32 being fully conserved. This agrees with the characterization of this sequence as the Downstream Promoter Element (DPE) in Dm lab (Kutach and Kadonaga 2000). Because no TATA box was found upstream of the transcription start site, we conclude that Db lab and Dv lab present a TATA-less promoter with Downstream Promoter Element (DPE), as does Dm lab and all other homeotic genes described in Drosophila (Nie et al. 2001). Analysis with the UTRscan software (Pesole and Liuni 1999) of the 5' UTRs of the three Drosophila species identified internal ribosome entry site (IRES) elements in all of them. These IRES elements promote cap-independent translation in different genes (Hellen and Sarnow 2001). In the Hox genes Ubx and Antp, they regulate translation during development (Ye et al. 1997).
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Nucleotide Sequence Comparisons
The coding sequences of Db lab and Dv lab were aligned and compared to that of Dm lab (NM_057265). Table 3 shows the number of synonymous and nonsynonymous substitutions per site (dS and dN) for pairwise comparisons between the three Drosophila species. These numbers have been estimated using maximum likelihood methods (Yang and Bielawski 2000; Yang and Swanson 2002) for the entire coding sequence (CDS) as well as for three separate (non-overlapping) regions: exon 1, exon 2 + 3 (excluding the homeobox) and the homeobox. The ratio between the number of nonsynonymous and synonymous substitutions ( = dN/dS) provides information on the type of selection acting on amino acid sequences. The low average
values for the entire gene (0.06960.0910) suggest a relatively high degree of functional constraint, but there is wide variation among regions (see below).
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Outside those conserved domains there is a very low conservation, which is surprising when compared with other proteins. The Labial proteins from D. buzzatii and D. melanogaster have an overall identity of only 62.8%, much lower than the 95% identity found by Hooper et al. (1992) between the Antp proteins from D. virilis and D. melanogaster (with the same divergence time). There are very few direct comparisons of homologous genes between D. melanogaster and D. buzzatii, but D. melanogaster and D. virilis homologous proteins usually have an identity between 80% and 90%, with values ranging from 36% to 97% (O'Neil and Belote 1992). Interestingly, all regions conserved between Db lab and Dm lab are also more or less conserved in all other sequences studied, including the human gene HoxA1. The domain conservation suggests that the protein functionality relies basically on a few domains, leaving the rest of the sequence with more freedom to change. This fact could explain why minigenes carrying the chicken Hoxb1 gene are able to rescue null mutants for the lab gene in D. melanogaster (Lutz et al. 1996). Thus, Labial proteins seem to be built on highly conserved blocks joined by rapidly evolving sequences. For example, the spacer or "gene specific region" (Peterson et al. 1999) localized between the YKWM motif and the homeodomain is much more variable, both in sequence and in length, in the lab gene than in other Hox genes even between Drosophila species.
Concluding Remarks
Although Hox gene colinearity and cluster conservation represents a paradigm in Evo-Devo, in the genus Drosophila the Hox cluster has been split at least three times. The lab-pb split described here is the most recent example. Even after moving to a new location, labial appears to evolve consistently, showing no alteration of its molecular evolutionary patterns. A paracentric inversion as well as a gene transposition event might, in principle, account for the lab-pb disruption and the relocation of lab near the genes abd-A and Abd-B observed in the repleta group of Drosophila. However, the former possibility seems more likely given the extensive reorganization of the Drosophila genome produced by chromosomal inversions (Powell 1997; Ranz, Casals, and Ruiz 2001). The molecular characterization of the chromosomal regions 5' of lab and 3' of pb in D. buzzatii should help to determine which was the molecular mechanism that caused such a split and the precise location of the breakpoint between lab and pb. This study would also elucidate the exact physical relationship, if any, between lab and the genes abd-A and Abd-B. Furthermore, although the rearrangement has not altered the lab coding region, and although some of its regulatory sequences seem to be in place, whether the relocation of lab had any consequences upon the level or spatiotemporal pattern of expression of this gene remains an open question, one that we are already investigating.
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Acknowledgements |
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Footnotes |
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2 Present address: Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia.
William R. Jeffery, Associate Editor
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, M. D., S. E. Celniker, and R. A. Holt, et al. (192 co-authors). 2000. The genome sequence of Drosophila melanogaster. Science 287:2185-2195.
Arkhipova, I. R. 1995. Promoter elements in Drosophila melanogaster revealed by sequence analysis. Genetics 139:1359-1369.
Cáceres, M., M. Puig, and A. Ruiz. 2001. Molecular characterization of two natural hotspots in the Drosophila buzzatii genome induced by transposon insertions. Genome Res. 11:1353-1364.
Chouinard, S., and T. C. Kaufman. 1991. Control of expression of the homeotic labial (lab) locus of Drosophila melanogaster: evidence for positive and negative autogenous regulation. Development 113:1267-1280.[Abstract]
Cook, C. E., M. L. Smith, M. J. Telford, A. Bastianello, and M. Akam. 2001. Hox genes and the phylogeny of arthropods. Curr. Biol. 11:759-763.[CrossRef][ISI][Medline]
DeRosa, R., J. K. Grenier, T. Andreeva, C. E. Cook, A. Adoutte, M. Akam, S. B. Carroll, and G. Balavoine. 1999. Hox genes in brachiopods and priapulids and protostome evolution. Nature 399:772-776.[CrossRef][ISI][Medline]
Devenport, M. P., C. Blass, and P. Eggleston. 2000. Characterization of the Hox gene cluster i the malaria vector mosquito, Anopheles gambiae. Evol. Dev. 2:326-339.[CrossRef][ISI][Medline]
Diederich, R. J., V. K. L. Merrill, M. A. Pultz, and T. C. Kaufman. 1989. Isolation, structure, and expresion of labial a homeotic gene of the Antennapedia Complex involved in Drosophila head development. Genes Dev. 3:399-414.[Abstract]
Dubchak, I., M. Brudno, G. G. Loots, L. S. Pachter, C. Mayor, E. M. Rubin, and K. A. Frazer. 2000. Active conservation of noncoding sequences revealed by three-way species comparisons. Genome Res. 10:1304-1306.
Duboule, D., and G. Morata. 1994. Colinearity and functional hierarchy among genes of the homeotic complexes. Trends Genet. 10:358-364.[CrossRef][ISI][Medline]
Duncan, I. 1987. The Bithorax complex. Annu. Rev. Genet. 21:285-319.[CrossRef][ISI][Medline]
Finnerty, J. R., and M. Q. Martindale. 1998. The evolution of the Hox cluster: insights from outgroups. Curr. Opin. Genet. Dev. 8:681-687.[CrossRef][ISI][Medline]
FlyBase. 2002. The FlyBase database of the Drosophila genome projects and community literature. Nucleic Acids Res. 30:106-108 (http//flybase.bio.indiana.edu/).
Gehring, W. J., M. Affolter, and T. Bürglin. 1993. Homeodomain proteins. Annu. Rev. Biochem. 63:487-526.[CrossRef][ISI]
Gehring, W. J., and Y. Hiromi. 1986. Homeotic genes and the homeobox. Annu. Rev. Genet. 20:147-173.[CrossRef][ISI][Medline]
Gubenko, I. S., and M. B. Evgen'ev. 1984. Cytological and linkage maps of Drosophila virilis chromosomes. Genetics 65:127-139.
Hartl, D. L., and E. R. Lozovskaya. 1994. Genome evolution: between the nucleosome and the chromosome. Pp 579592 in B. Schierwater, B. Streit, G. P. Wagner, and R. DeSalle, eds. Molecular ecology and evolution: approaches and applications, Birkhauser Verlag, Basel.
Hazelrigg, T., and T. C. Kaufman. 1982. Revertants of dominant mutations associated with the Antennapedia gene complex of Drosophila melanogaster: cytology and genetics. Genetics 105:581-600.[ISI]
Hellen, C. U. T., and P. Sarnow. 2001. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15:1593-1612.
Hong, Y. S., S. Y. Kim, A. Bhattacharya, D. R. Pratt, W. K. Hong, and M. A. Tainsky. 1995. Structure and function of the Hox A1 human homeobox gene cDNA. Gene 159:209-214.[CrossRef][ISI][Medline]
Hooper, J. E., M. Pérez-Alonso, J. R. Bermingham, M. Prout, B. A. Rocklein, M. Wagenbach, J. E. Edstrom, R. de Frutos, and M. P. Scott. 1992. Comparative studies of Drosophila Antennapedia genes. Genetics 132:453-569.
Hughes, C., and T. C. Kaufman. 2002. Hox genes and the evolution of the arthropod body plan. Evol. Dev. 4:459-499.[CrossRef][ISI][Medline]
Johnson, F. B., E. Parker, and M. A. Krasnow. 1995. Extradenticle protein is a selective cofactor for the Drosophila hometics: role of the homeodomain and YPWM amino acid motif in the interaction. Proc. Natl. Acad. Sci. USA 92:739-743.[Abstract]
Kaufman, T. C., M. A. Seeger, and G. Olsen. 1990. Molecular and genetic organization of the Antennapedia gene complex of Drosophila melanogaster. Adv. Gen. 27:309-362.
Kutach A. K., and J. T. Kadonaga. 2000. The downstream promoter element DPE appears to be as widely used as the TATA Box in Drosophila core promoters. Mol. Cell Biol. 20:4754-4764.
Kwiatowski, J., and F. J. Ayala. 1999. Phylogeny of Drosophila and related genera: conflict between molecular and anatomical analyses. Mol. Phylogenet. Evol. 13:319-328.[CrossRef][ISI][Medline]
LeCalvez.. 1953. Carte cytologique des chromosomes geants des glandes salivaires de Drosophila immigrans Sturt. Chromosoma 6:170-174.[ISI][Medline]
Lewis, E. B. 1978. A gene complex controlling segmentation in Drosophila. Nature 276:565-570.[ISI][Medline]
Lutz, B., H. C. Lu, G. Miller, and T. C. Kaufman. 1996. Rescue of the Drosophila labial null mutant by chicken ortholog Hoxb-1 demonstrates that the function of Hox genes is phylogenetically conserved. Genes Dev. 10:176-184.[Abstract]
Maier, D., A. Preiss, and J. R. Powell. 1990. Regulation of the segmentation gene fushi tarazu has been functionally conserved in Drosophila. EMBO J. 9:3957-3966.[Abstract]
Maier, D., D. Sperlich, and J. R. Powell. 1993. Conservation and change of the developmentally crucial fushi tarazu gene in Drosophila. J. Mol. Evol. 36:315-326.[ISI][Medline]
Martin, C. H., C. A. Mayeda, C. A. Davis, C. L. Ericsson, J. D. Knafels, D. R. Mathog, S. E. Celniker, E. B. Lewis, and M. J. Palazzolo. 1995. Complete sequence of the bithorax complex of Drosophila. Proc. Natl. Acad. Sci. USA 92:8398-8402.[Abstract]
Marty, T., M. A. Vigano, C. Ribeiro, U. Nussbaumer, N. C. Grieder, and M. Affolter. 2001. A HOX complex, a repressor element and a 50 bp sequence confer regional specificity to a DPP-responsive enhancer. Development 128:2833-2845.[ISI][Medline]
Mayor, C., M. Brudno, J. R. Schwartz, A. Poliakov, E. M. Rubin, K. A. Frazer, L. S. Pachter, and I. Dubchak. 2000. VISTA: visualizing global DNA sequence alignments of arbitrary length. Bioinformatics 16:1046-1047.[Abstract]
McGinnis, W., and R. Krumlauf. 1992. Homeobox genes and axial patterning. Cell 68:283-302.[ISI][Medline]
Mlodzik, M., A. Fjose, and W. J. Gehring. 1988. Molecular structure and spatial expression of a homeobox gene from the labial region of the Antennapedia-complex. EMBO J. 7:2569-2578.[Abstract]
Nie, W., B. Stronach, G. Panganiban, T. Shippy, S. Brown, and R. Denell. 2001. Molecular characterization of Tclabial and the 3' end of the Tribolium homeotic complex. Dev. Genes Evol. 211:244-251.[CrossRef][ISI][Medline]
O'Neil, M. T., and J. M. Belote. 1992. Interspecific comparison of the transformer gene of Drosophila reveals an unusually high degree of evolutionary divergence. Genetics 131:113-128.
Pesole, G., and S. Liuni. 1999. Internet resources for the functional analysis of 5' and 3' untranslated regions of eukaryotic mRNAs. Trends Genet. 15:378.[CrossRef][ISI][Medline]
Peterson, M. D., B. T. Rogers, A. Popadi, and T. C. Kaufman. 1999. The embryonic expression pattern of labial, posterior homeotic complex genes and the teashirt homologue in an apterygot insect. Dev. Genes Evol. 209:77-90.[CrossRef][ISI][Medline]
Piñol, J., A. Francino, A. Fontdevila, and O. Cabré. 1988. Rapid isolation of Drosophila high molecular weight DNA to obtain genomic libraries. Nucleic Acids Res. 16:2736-2737.[ISI][Medline]
Powell, J. R. 1997. Progress and prospects in evolutionary biology: the Drosophila model. Oxford University Press, New York.
Powers T. P., J. Hogan, Z. Ke, K. Dymbrowski, X. Wang, F. H. Collins, and T. C. Kaufman. 2000. Characterization of the Hox cluster from the mosquito Anopheles gambiae (Diptera: Culicidae). Evol. Dev. 2:311-325.[CrossRef][ISI][Medline]
Randazzo, F. M., M. A. Seeger, C. A. Huss, M. A. Sweeney, J. K. Cecil, and T. C. Kaufman. 1994. Structural changes in the Antennapedia complex of Drosophila pseudoobscura. Genetics 133:319-330.
Ranz, J. M., F. Casals, and A. Ruiz. 2001. How maleable is the eukaryotic genome? Extreme rate of cromosomal rearrangement in the genus Drosophila. Genome Res. 11:230-239.
Ranz, J. M., J. González, F. Casals, and A. Ruiz. 2003. Low occurrence of gene transposition events during the evolution of the genus Drosophila. Evolution 57:1325-1335.[ISI][Medline]
Ranz, J. M., C. Segarra, and A. Ruiz. 1997. Chromosomal homology and molecular organization of Muller's elements D and E in the Drosophila repleta species group. Genetics 145:281-295.
Rauskolb C., and E. Wieschaus. 1994. Coordinate regulation of downstream genes by extradenticle and the honeotic selector proteins. EMBO J. 13:3561-3569.[Abstract]
Russo, C. A. M., N. Takezaki, and M. Nei. 1995. Molecular phylogeny and divergence times of Drosophilid species. Mol. Biol. Evol. 12:391-404.[Abstract]
Ruvkun, G., and O. Hobert. 1998. The taxonomy of developmental control in Caenorhabditis elegans. Science 282:2033-2041.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Shippy, T. D., S. J. Brown, and R. E. Denell. 1998. Molecular characterization of the Tribolium abdominal-A ortholog and implications for the products of the Drosophila gene. Dev. Genes Evol. 207:446-452.[CrossRef][ISI][Medline]
Spicer, G. S. 1988. Molecular evolution among some Drosophila species groups as indicated by two-dimensional electrophoresis. J. Mol. Evol. 27:250-260.[ISI][Medline]
Stone, W. S. 1962. The dominance of natural selection and the reality of superspecies (species groups) in the evolution of Drosophila. Univ. Texas Publ. 6205:507-537.
Struhl, G. 1984. Splitting the bithorax complex of Drosophila. Nature 308:454-457.[ISI]
Terol, J., M. Perez-Alonso, and R. Frutos. 1991. Molecular characterization of the zerknüllt region of the Antennapedia complex of D. subobscura. Hereditas 114:131-139.[ISI][Medline]
Tiong, S. Y., J. R. Whittle, and M. C. Gribbin. 1987. Chromosomal continuity in the abdominal region of the bithorax complex of Drosophila is not essential for its contribution to metameric identity. Development 101:135-142.[Abstract]
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract]
Von Allmen, G., I. Hogga, A. Spierer, F. Karch, W. Bender, H. Gyurkovics, and E. Lewis. 1996. Splits in fruitfly Hox gene complexes. Nature 380:116.[CrossRef][ISI][Medline]
Wasserman, M. 1992. Cytological evolution of the Drosophila repleta species group. Pp. 455552 in C. B. Krimbas and J.R. Powell, eds. Drosophila inversion polymorphism. CRC Press, Boca Raton, Fla.
Wharton, L. T. 1942. Analysis of the repleta group of Drosophila. Univ. Texas Publ. 4228:23-59.
Wilder, J., and H. Hollocher. 2001. Mobile elements and the genesis of microsatellites in Dipterans. Mol. Biol. Evol. 18:384-392.
Yang, Z. 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. Comp. Appl. Biosci. 13:555-556.[ISI][Medline]
Yang, Z., and J. P. Bielawski. 2000. Statistical methods for detecting molecular adaptation. Trends Evol. 15:496-503.[CrossRef][ISI]
Yang, Z., and W. J. Swanson. 2002. Codon substitution models to detect adaptive evolution that account for heterogeneous selective pressures among site classes. Mol. Biol. Evol. 19:49-57.
Ye, X., P. Fong, N. Iizuka, D. Choate, and D. R. Cavener. 1997. Ultrabithorax and Antennapedia 5' untranslated regions promote developmentally regulated internal translation initiation. Mol. Cell Biol. 17:1714-1721.[Abstract]