Genetics, Vol. 165, 1823-1830, December 2003, Copyright © 2003

Characterization of a Male-Predominant Antisense Transcript Underexpressed in Hybrids of Drosophila pseudoobscura and D. persimilis

Mohamed A. F. Noora, Pawel Michalaka, and David Donzea
a Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana

Corresponding author: Mohamed A. F. Noor, 206 Life Sciences Bldg., Louisiana State University, Baton Rouge, LA 70803., mnoor{at}lsu.edu (E-mail)

Communicating editor: S. SCHAEFFER


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

Characterizing genes that are misregulated in hybrids may elucidate the genetic basis of hybrid sterility or other hybrid dysfunctions that contribute to speciation. Previously, a small segment of a male-predominant transcript that is underexpressed in adult male hybrids of Drosophila pseudoobscura and D. persimilis relative to pure species was identified in a differential display screen. Here, we obtained the full sequence of this 1330-bp transcript and determined that it is an antisense message with high sequence similarity to the D. melanogaster TRAP100 gene, part of the Mediator protein complex that regulates transcriptional initiation during development. Both the sense and the antisense messages are transcribed in D. pseudoobscura, but only the sense message (TRAP100) is transcribed in D. melanogaster complex species. Unlike the antisense message, the sense message is transcribed similarly in D. pseudoobscura males and females and in hybrids of D. pseudoobscura and D. persimilis. The high sequence similarity between distantly related species suggests that the sense message is functionally constrained within the genus. We speculate that the antisense transcript may have evolved a role in male-specific post-transcriptional regulation of TRAP100 in the D. pseudoobscura lineage and that its underexpression in sterile hybrid males may cause an overproduction of TRAP100 protein, possibly yielding deleterious effects.

OUR understanding of the genetic basis of hybrid dysfunctions has grown dramatically over the past 15 years, largely as a result of many high-resolution genetic mapping studies where one starts with the phenotype and attempts to identify contributing genomic regions (see reviews in COYNE and ORR 1998 Down; WU and HOLLOCHER 1998 Down). However, despite this progress, only a handful of candidate genes or genetic pathways have been identified that contribute to hybrid problems (see review in ORR and PRESGRAVES 2000 Down). While estimating the numbers of genes that contribute to hybrid sterility and knowing the relative abundances of those affecting males vs. females greatly helps our understanding of speciation, more genes or genetic pathways must be directly identified and characterized to determine the nature of the genetic interactions that cause speciation. New molecular approaches can help to complement these results from mapping studies. One avenue could be a "reverse genetics" approach, where one begins with a genetic disruption and attempts to characterize it and determine its phenotypic consequences.

A growing body of evidence suggests that at least some hybrid dysfunctions result from transcriptional deregulation in hybrids. For example, overexpression of Xmrk-2 (Xiphophorus melanoma receptor kinase) causes tumor formation and lethality in hybrids between swordtails (Xiphophorus helleri) and platyfish (X. maculatus; SCHARTL 1995 Down; SCHARTL et al. 1999 Down). The Odysseus gene, a putative factor contributing to hybrid sterility in D. simulans and D. mauritiana (TING et al. 1998 Down), also appears to be regulatory. In these species, genes with male-specific patterns of expression are disproportionately underexpressed in hybrids (MICHALAK and NOOR 2003 Down), possibly contributing to sterility in F1 male hybrids. Finally, theoretical studies suggest that binding strength of regulator proteins to promoter regions can provide biologically plausible and powerful postmating isolation, such as hybrid sterility (JOHNSON and PORTER 2000 Down; PORTER and JOHNSON 2002 Down).

Identifying and characterizing genes that are differentially expressed in pure species vs. hybrids may lead to a better understanding of the genetic disruptions that occur in hybrids. In a previous study, REILAND and NOOR 2002 Down used differential display to identify transcripts underexpressed in sterile male hybrids of D. pseudoobscura and D. persimilis relative to pure species. They detected fairly limited differences in expression between hybrids and pure species, although they did confirm one transcript that was underexpressed in hybrids (GenBank accession no. AF510848). This transcript was male specific and polyadenylated, but the 100 bp of sequence that was obtained, presumably at the 3'-end of this transcript, bore no significant sequence homology to any known gene as determined by BLAST (ALTSCHUL et al. 1990 Down). Here, we isolate and characterize the complete transcript in D. pseudoobscura and other Drosophila species and demonstrate it to be an antisense transcript to the TRAP100 gene.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Fly strains:
Drosophila pseudoobscura strains used in this study were Mather, California 17; Flagstaff, Arizona 1993; Zapotitlan, Puebla, Mexico; Mount St. Helena, California 10; and American Fork, Utah 12. The other lines used were D. melanogaster OregonR, D. simulans Florida City; D. yakuba Tai 18; D. miranda Mount St. Helena, California 22; D. subobscura Mount St. Helena, California 1998; and D. mojavensis A993. All fly strains (or DNAs) are available upon request.

Inverse PCR:
Circularized D. pseudoobscura Mather 10 genomic DNA was obtained from Jody Hey (Rutgers University). Circularized DNA samples had been ligated after digestion by one of the following enzymes: HindIII, EcoRI, and TaqI. We designed primers from the published sequence of the transcript misexpressed in hybrids (GenBank accession no. AF510848) that elongated from the middle toward the outside of the fragment. PCR was performed in a 20-µl volume with 1.5 mM MgCl2, 0.2 mM dNTPs, 1 µM of each primer, and 1 unit of Taq polymerase. These concentrations were used for all PCRs in this study unless otherwise specified. Samples were cycled 32 times at 94° for 1 min, 55° for 1 min, and 72° for 3 min. An 800-bp amplification product was obtained from the TaqI-digested DNA and reamplified a second time in a 50-µl reaction volume. This product was purified using the QIAGEN (Valencia, CA) gel extraction kit, and sequenced with Applied Biosystems (Foster City, CA) BigDye version 3, following the manufacturers' instructions. Sequences were visualized at the Pennington Biomedical Research Center.

Northern blot:
Total RNA from 25–30 adult male D. pseudoobscura (Mather 17 line) was isolated using the QIAGEN RNeasy kit, following the manufacturer's directions. A DNase step was included in the purification. Duplicate 5-µg samples of the total RNA were resolved on a 1% agarose gel and blotted to Zeta-probe membrane (Bio-Rad, Richmond, CA). The duplicate membrane strips were separately probed with riboprobes complementary to the sense and the antisense sequence. Probe templates were generated by PCR primers that attached the T7 RNA polymerase promoter to the appropriate end, and riboprobes were synthesized using the Maxiscript T7 kit (Ambion, Austin, TX). Blotting was performed using a NorthernMax kit (Ambion).

Southern blot:
Genomic DNA from 30 adult male D. pseudoobscura (Mather 17 line) was isolated using the Gentra Systems (Research Triangle Park, NC) PureGene DNA isolation kit, following the manufacturer's directions. This DNA was further purified by phenol-chloroform extraction. DNA (25 µg) was digested with the indicated restriction enzymes, resolved on a 1% agarose gel, and blotted to Zeta-probe membrane. Hybridization and washing (at 65°) was carried out according to the Zeta-probe standard hybridization protocol, using as a probe a 0.75-kb PCR fragment of TRAP100 extending upstream from the HindIII site in exon 7. The probe was labeled using the NEBlot random prime labeling kit (New England Biolabs, Beverly, MA).

Quantitative PCRs:
For D. melanogaster and D. simulans samples, total RNA was isolated from 30 adult male flies as above. Reverse transcription was performed in a 20-µl reaction volume with 0.5 µmol total RNA, 5 mM MgCl2, 1 mM dNTPs, 2.5 µM primer, 20 units Invitrogen SuperScript II reverse transcriptase, and 20 units RNasin. Reactions were incubated at room temperature for 10 min, 42° for 15 min, and 99° for 5 min. Two microliters of this reverse transcriptase (RT) reaction was then used in a 10-µl PCR (conditions as described above) consisting of 30 cycles of 94° for 30 sec, 56.5° for 30 sec, and 72° for 30 sec. Products of this PCR were visualized on an EtBr-stained 2% agarose gel.

For real-time PCR analyses in D. pseudoobscura and hybrids, 250 ng of total RNA prepared as described above was reverse transcribed in a reaction containing 5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, 1 mM dNTPs, 20 units RNasin, 20 units reverse transcriptase (SuperScript II), 2.5 µM of the target primer, and 50 nM of 18S rRNA reverse primer (ABI Applied Biosystems). PCR primers and the fluorescent probe were designed using Primer Express 2.0 software (ABI) and their sequences were: upper, 5'-CGTGAAGAACATTTTGGACAACA-3'; lower, 5'-GGTTGCGCAGACGGAGAA-3'; and probe, 5'-AGCGACGTCTCTTCA-3'. The target probe was FAM labeled (Biosearch Technologies) and the normalizer probe (18S rRNA) was VIC labeled (ABI). Predeveloped ABI TaqMan assay reagents and ABI standard protocols were used to prepare the RT-PCR reaction mixture. The probes contained a reporter dye at the 5'-end of the probe and a quencher dye (BHQ-1) at the 3'-end of the probe. During the reaction, the reporter dye and quencher dye are separated, resulting in increased fluorescence of the reporter. All PCRs consisted of an initial 2 min at 50° and 10 min at 95° and then 40 cycles of 95° for 15 sec and 60° for 1 min. ABI Prism 7000 SDS software was used for visualization and quantification of the amplification products. The mean difference in threshold cycle number (CT) was tested with a post hoc Tukey test following a two-way repeated measures ANOVA analysis, in which the genotype (D. pseudoobscura males, D. pseudoobscura females, and D. pseudoobscura x D. persimilis F1 hybrid males) effect was significant (P < 0.05). We also compared mean difference in CT between D. pseudoobscura females, D. persimilis females, and F1 hybrid females. For all analyses, each sample was replicated in three independent RT-PCR reactions. All results were repeatable when different cycle thresholds were tested and largely independent of whether or not normalization for 18S rRNA was applied.

Rapid amplification of cDNA ends reactions:
To obtain the full transcript sequences and to identify spliced introns, we used the Ambion FirstChoice RNA ligase mediated-rapid amplification of cDNA ends (RLM-RACE) kit, following the manufacturer's directions. RNA from adult male D. pseudoobscura Mather 17 was used for all RACE reactions. Briefly, for the 5'-RACE, 1 µg of total RNA was treated with both calf intestinal phosphatase and tobacco acid pyrophosphatase, each for 1 hr. An adapter was ligated to the 5'-end of the RNA, and reverse transcription was performed using random decamers. The specific transcript was then amplified in two reactions using a primer that bound to the adapter and another from the known sequence. This amplification product was cloned into competent Escherichia coli cells using the Invitrogen TOPO-TA cloning kit, following the manufacturer's instructions, and colonies bearing the insert were amplified and sequenced as above.

For the 3'-RACE, a reverse transcription reaction was performed using a primer that attached an adapter past the poly(A) tail of transcripts. The specific transcript was then amplified in two reactions using a primer that bound to the adapter and another from the known sequence. This amplification was cloned into chemically competent E. coli using the Invitrogen TOPO-TA cloning kit, following the manufacturer's instructions, and colonies bearing the insert were amplified and sequenced as above.

Sequence analyses:
A primer was designed from the coding sequence of the D. melanogaster CG7375 gene, which maps immediately adjacent to the gene being studied. This primer was then used to amplify an ~2-kb segment for sequence comparisons within and between species. We were unable to optimize this reaction or any of several others attempted to obtain much sequence from D. subobscura or D. mojavensis, so only sequence from one end is reported from these two species. PCR products were gel purified prior to direct sequencing, and no cloning was involved. All sequencing used BigDye version 3 as above. Sequencing reactions were performed in both directions, and base calls were manually confirmed.

DNA and inferred amino acid sequences for all species were aligned using BioEdit (HALL 1994 Down). Introns were frequently unalignable between taxa belonging to different species groups, so only coding sequences were analyzed for between-group comparisons. Analyses of sequences were performed by the computer programs SITES (HEY and WAKELEY 1997 Down), DnaSP (ROZAS and ROZAS 1999 Down), and PAML (YANG 1997 Down) or were performed manually.

Recombinational mapping:
We sought to confirm the genomic location of the identified transcript. Female D. pseudoobscura Mather 17 were crossed to male D. persimilis, and the F1 female progeny were backcrossed to D. pseudoobscura. The DPSX010 microsatellite was then amplified from 50 of the male offspring of this backcross and visualized on a LiCor DNA analyzer. This microsatellite was selected because it is on the XR chromosome, which is homologous to the chromosome arm found to bear the identified sequence in D. melanogaster.

We identified a difference between these two species in the sequence of the misexpressed transcript at a HaeIII restriction site (GGCC), where an additional 20 bp was cleaved off digests of the D. persimilis fragment but not the D. pseudoobscura fragment. We amplified a 600-bp fragment from this gene in the same 50 backcross progeny in a 20-µl reaction volume, digested 5 µl of this amplification in a 10-µl reaction volume with five units of HaeIII for 1 hr, and visualized the products on a 2% TBE agarose gel. Recombinants between the microsatellite alleles and the alleles at this gene were noted.


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

Identification of transcript:
The 100-bp fragment initially obtained from the 3'-end of the polyadenylated transcript putatively misexpressed in hybrids of D. pseudoobscura and D. persimilis did not match any published D. melanogaster sequence using BLAST (ALTSCHUL et al. 1990 Down). We obtained circularized D. pseudoobscura DNAs used in a previous study (MACHADO et al. 2002 Down), designed pairs of primers going toward the outside of the fragment, and attempted inverse polymerase chain reactions (OCHMAN et al. 1988 Down; OFFRINGA and VANDERLEE 1995 Down) from them to extend the sequence. The reaction from TaqI-digested DNA yielded an 800-bp amplification product that contained the original sequence.

BLAST analysis of this longer amplification product suggested that the sequence of the D. pseudoobscura transcript was homologous to the D. melanogaster TRAP100 gene (CG7999). However, the D. pseudoobscura transcript was transcribed in the opposite direction, with its 3'-end falling within the 57-bp penultimate intron of the D. melanogaster gene. TRAP100 maps to chromosome 3L in D. melanogaster, which is homologous to the XR chromosome in D. pseudoobscura. The location of TRAP100 was confirmed in D. pseudoobscura via recombinational mapping. Among backcross F2 males between D. pseudoobscura and D. persimilis, we found 0/50 to have X chromosomes recombinant between TRAP100 and DPSX010, the latter of which is known to be on the XR (NOOR and SMITH 2000 Down; MACHADO et al. 2002 Down).

Features of the transcripts:
Northern blot analysis confirmed the presence of the antisense TRAP100 transcript (see Fig 1A). Probing a blot of D. pseudoobscura total RNA with a sequence complementary to the D. melanogaster transcript gave a band at approximately 3.5 kb, a size that corresponds to that of D. melanogaster. This transcript is hereafter called the "sense" transcript. A probe complementary to the antisense sequence hybridized to an RNA of ~1.7 kb. This analysis confirms that two transcripts with high homology to the D. melanogaster TRAP100 gene are present in D. pseudoobscura but that one is transcribed in the opposite direction. Southern blot analysis of D. pseudoobscura DNA indicated the presence of a single copy of the TRAP100 gene (see Fig 1B), suggesting that the antisense transcript indeed originates from the downstream end as proposed.



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  		Figure 1. (A) Northern blot showing two transcripts from the TRAP100 sequence in D. pseudoobscura. Lane 1 was from D. pseudoobscura RNA hybridized with a probe complementary to the sense transcript. Lane 2 was from D. pseudoobscura RNA hybridized with a probe complementary to the antisense transcript. (B) Southern blot analysis indicates the presence of a single TRAP100 locus. Digests were HindIII for lane 1, EcoRI for lane 2, HindIII + EcoRI for lane 3, and DraI for lane 4. The blot was probed with a 0.75-kb fragment extending upstream from the HindIII site in exon 7 of TRAP100.

We obtained the remainder of the D. pseudoobscura antisense transcript using 5'-RLM-RACE (LIU and GOROVSKY 1993 Down). Excluding the poly(A) tail, we determined the length of this transcript to be 1330 bp. To confirm that this was the entire transcript, we performed a large-volume RT reaction using a primer whose 5'-end matched the RNA sequence 175 bp from the putative 5'-end of the antisense transcript. We then attempted a series of PCRs from this RT reaction as well as for genomic DNA controls using primers both within and <100 bp outside of the transcript. Reactions using primers from within the putative 5'-end generated amplification products from cDNA and genomic DNA. Reactions using primers outside the putative 5'-end of the transcript amplified only products from genomic DNA.

Subsequent PCRs and sequencing from genomic DNA demonstrated that the antisense transcript bore no spliced introns. Within the antisense sequence, a potential open reading frame (ORF) was detected that could encode for a 155-amino-acid product, beginning ~690 bp from the 5'-end of the antisense sequence. This putative translated product does not bear significant similarity to any other known genes.

We used 3'-RLM-RACE beginning in the sixth exon to confirm that the sense transcript in D. pseudoobscura had the same intron/exon boundaries and 3'-end as the D. melanogaster transcript. The RACE was successful, indicating that the sense transcript is also polyadenylated. The same intron-exon boundaries and 3'-end were identified in D. pseudoobscura as documented in D. melanogaster, although the sixth intron was substantially shorter in D. pseudoobscura (68 bp) than in D. melanogaster (143 bp). No frameshift mutations were identified between the species, and the D. pseudoobscura sequence bears a 90-bp 3'-untranslated region (3'-UTR).

The sense and antisense transcripts overlap broadly in D. pseudoobscura (see Fig 2). The 5'-end of the antisense transcript begins 227 bp before the 3'-end of the translated region of the sense transcript, in the middle of exon 7. The antisense transcript terminates in the intron separating exons 5 and 6 of the sense transcript. Two polyadenylation signals (AAUAAA) were noted in the antisense transcript immediately before its termination.



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  		Figure 2. Diagram of TRAP100 transcripts in D. pseudoobscura. For the sense transcript, open boxes indicate coding regions of exons 4–7 of sense transcript, hatched box indicates 3'-untranslated region, and lines indicate introns. The antisense transcript is shaded.

Expression of transcripts:
Northern blot analyses described above demonstrated that both the sense and antisense transcripts are present in D. pseudoobscura adult males. REILAND and NOOR 2002 Down noted previously that the antisense transcript was both male specific and expressed at a lower level in hybrids between D. pseudoobscura and D. persimilis than in pure parental species. We confirmed the results of REILAND and NOOR 2002 Down using fluorescent real-time RT-PCR: expression of the antisense transcript was unambiguously higher in D. pseudoobscura males than in females (P = 0.0018) and also higher in pure-species males than in hybrid males (P = 0.032). No difference in expression of the antisense transcript was noted between pure-species females and hybrid females. In contrast, the sense transcript was not differentially expressed in D. pseudoobscura males compared to hybrid males or in males compared to females.

We also attempted RT-PCRs of both transcripts in adult male D. melanogaster and D. simulans. While we obtained clear amplification of the sense transcript in both species, we could not amplify a product corresponding to the antisense transcript in these two species. Positive control amplifications from genomic DNA were successful, suggesting that the RT-PCR was unsuccessful because of the absence of antisense RNA transcripts in these species rather than PCR conditions.

DNA sequence analyses:
Using primers designed from coding regions in D. melanogaster, we amplified and sequenced a 1895-bp region of TRAP100 including all of the antisense transcript in five strains of D. pseudoobscura, one strain of D. persimilis, and one strain of D. miranda; the corresponding 1998-bp sequence in D. yakuba and in D. simulans; and a 621-bp sequence from the 3'-end of the antisense transcript in D. mojavensis. These sequences have been submitted to GenBank under accession nos. AY208899, AY208900, AY208901, AY208902, AY208903, AY208904, AY208905, AY208906, AY208907, AY208908 and AY221127. Approximate phylogenetic relationships of these species are presented by POWELL 1997 Down.

The sequences of all the D. pseudoobscura lines were very highly conserved: among the 1443 bp of coding sequence in exons 6 and 7 and 214 bp of noncoding sequence (introns 5 and 6 and the 3'-UTR), only six polymorphisms were documented. The nonsynonymous polymorphism was a conservative amino acid change of leucine to valine in one line. Codon usage bias was moderate (effective number of codons, 49.6; codon bias index, 0.41; WRIGHT 1990 Down; MORTON 1993 Down). Two of the D. pseudoobscura sequences were identical (Mather, California, and Puebla, Mexico), and this sequence is used for comparisons to the other species. This sequence differs from D. persimilis by two nonsynonymous, three synonymous, and three noncoding base differences and from D. miranda by three nonsynonymous, seven synonymous, five noncoding base differences, and one noncoding one-base indel.

Only one polymorphic site (one of the synonymous sites) bore variants shared among multiple strains within D. pseudoobscura. Coupled with the low overall level of polymorphism, this caused TAJIMA's (1989) D-statistic for this sequence to be -0.6682. While this value was not significantly different from zero, it was consistent with other genes on the XR in this species (X009 has D = -0.6977; MACHADO et al. 2002 Down) in exhibiting a general trend for negative Tajima's D values. The weighted average value of WATTERSON's (1975) estimator, {theta}, of the population mutation rate parameter 3Nµ (where N is the effective population size and µ is the neutral mutation rate) was 0.00152 for sense TRAP100 in D. pseudoobscura, which is much lower than that for all other loci surveyed in the species (0.0099 on average; MACHADO et al. 2002 Down).

The coding sequences of TRAP100 from D. melanogaster group species readily aligned to those of the D. pseudoobscura group species: sequence similarity was ~80% for exon 6 and 70% for exon 7 (see Table 1). Estimated ratios of nonsynonymous to synonymous substitutions (dN/dS) were calculated using NEI and GOJOBORI's (1986) method and found to be similar to the maximum likelihood estimates derived by the approach of YANG et al. 2000 Down as implemented in the PAML (YANG 1997 Down) computer software package (data not shown). We eliminated D. simulans and D. persimilis from these comparisons because of high sequence similarity to D. melanogaster and D. pseudoobscura, respectively. All ratios were well below 0.2 (Table 2), suggesting strong functional constraint on the gene resulting from purifying selection against amino acid changes and consistent with the lack of polymorphism within D. pseudoobscura.


 
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  		Table 1. Sequence differences between species in two exons of TRAP100


 
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  		Table 2. Ratio of nonsynonymous substitutions to synonymous substitutions (dN/dS)

The 3'-end of the antisense transcript (intron 5, between exons 5 and 6) was also surveyed in two additional species, D. subobscura and D. mojavensis. This intron was very A-T rich and mostly unalignable between the more distantly related species. However, one of the two possible polyadenylation signals for the antisense transcript in D. pseudoobscura was present in all species except D. melanogaster (off by one base) and D. mojavensis (completely missing; see Fig 3).



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  		Figure 3. Sequences of TRAP100 intron 5. This intron is at the 3'-end of the antisense transcript. Possible polyadenylation signals in D. pseudoobscura and D. persimilis are underlined, and the poly(A) cleavage site is noted in boldface type.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

We have determined that a 1330-base antisense transcript to the TRAP100 gene is underexpressed in male F1 hybrids of D. pseudoobscura and D. persimilis relative to pure species. TRAP100 is part of the mediator (MED) transcription factor complex (BOUBE et al. 2000 Down; PARK et al. 2001 Down), which is conserved across kingdoms (see review in BOUBE et al. 2002 Down). The antisense transcript that we identified is polyadenylated, expressed at a higher level in males than in females, and not transcribed in species of the melanogaster group. The sense TRAP100 transcript is highly conserved in DNA sequence among all Drosophila species studied, is expressed normally in male F1 hybrids of D. pseudoobscura and D. persimilis, and does not exhibit sex-specific patterns of gene expression.

While a putative 155-amino acid ORF was detected within the antisense message, we consider it more likely that this message serves in regulation rather than in encoding a protein. The resultant amino acid sequence from the ORF does not bear detectable similarity to any known sequences. Further, virtually the entire antisense message is contained within amino acid coding exons of the sense message, so it seems unlikely that a usable protein product could have evolved from the long reverse TRAP100 complement.

Antisense RNAs and double-stranded RNAs (causing RNAi) have attracted much recent attention and have been identified from all kingdoms of life (e.g., EDDY 2001 Down; BRANTL 2002 Down; HANNON 2002 Down; WAGNER and FLARDH 2002 Down). Many of these RNAs appear to function to inhibit gene expression through forming RNA duplexes, sometimes by targeting RNA for cleavage and degradation, or to block RNA translation directly. Antisense RNA homologous to the downstream portion of a transcript, as we observed in D. pseudoobscura TRAP100, has been demonstrated to inhibit translation of the complementary sense message. The C. elegans lin-4 gene codes for a 22-nucleotide antisense RNA complementary to the 3'-untranslated region of the lin-14 mRNA, and lin-4 inhibits translation of the lin-14 message at a postinitiation step (OLSEN and AMBROS 1999 Down). This suggests one possible mechanistic role of the TRAP100 antisense RNA, as a post-transcriptional regulator of translation of the TRAP100 mRNA.

In mouse tissues, antisense transcripts exist that correspond to ~7% of transcribed genes (OKAZAKI et al. 2002 Down). Antisense RNAs have been detected in Drosophila previously, although most are <100 bp long (e.g., LAI 2002 Down). One notable exception is found within the micropia retrotransposon in Drosophila hydei: a 1.0-kb antisense RNA is encoded that is complementary to the reverse transcriptase and RNaseH coding regions of the element (LANKENAU et al. 1994 Down). This antisense transcript is also male specific, and LANKENAU et al. 1994 Down proposed that it may function in the control of germ-line expression of transposon-encoded proteins. Unlike the transcript we identified, however, the micropia antisense RNA does appear to be expressed in very distantly related Drosophila species.

The antisense message of TRAP100 may serve in the post-transcriptional regulation of the sense message. Given that the antisense message is more abundant in males than in females, this hypothesis would predict that the TRAP100 protein is more abundant in females than in males. If true, underexpression of the antisense message in hybrid males would yield overproduction of TRAP100 protein, and hybrid males would have a TRAP100 expression profile similar to pure-species females. Hypothetical overproduction of TRAP100 may be associated with sterility or other dysfunctions in these hybrid males. This idea is further supported by the broad utilization of the Drosophila MED complex by highly diverse transcription factors (PARK et al. 2001 Down), the known requirement of other TRAP genes for Drosophila cell viability (BOUBE et al. 2000 Down), and the recruitment of the TRAP-MED complex to reproductive-tissue-specific promoters in prostate cancer cells (WANG et al. 2002 Down).

From the data obtained thus far, we can only speculate as to the consequence of the underexpression of this transcript in F1 hybrids. We do not yet know that underexpression of antisense TRAP100 leads to overexpression of TRAP100 protein or has any particular fitness consequence. Nonetheless, this approach to studying the genetics of hybrid dysfunctions may complement genetic mapping studies in the future. The traditional approach has been to identify obvious hybrid dysfunction phenotypes and genetically map them to regions of the genome. While mapping studies have been fruitful, it is typically very difficult to isolate and/or identify single genes involved in the phenotype. In contrast, studying regulatory disruptions in hybrids via differential display (e.g., LIANG 2002 Down), microarray analysis, or serial analysis of gene expression (e.g., CHEN et al. 2002 Down) immediately identifies genes that are differentially expressed in hybrids vs. pure species. Follow-up experiments identifying the consequences of disruptions of such genes may immediately elucidate part of the genetic basis of hybrid dysfunctions and, thus, speciation in general.


*  FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY208899, AY208900, AY208901, AY208902, AY208903, AY208904, AY208905, AY208906, AY208907, AY208908 and AY221127. Back


*  ACKNOWLEDGMENTS

We thank Jeff Feder and Daniel Ortiz-Barrientos for constructive comments on the manuscript and G. Kilroy, S. Newman, and R. Staten for technical assistance. M.A.F.N. is funded by National Science Foundation grants 9980797, 0100816, and 0211007 and Louisiana Board of Regents Governor's Biotechnology Initiative grant 005. P.M. is funded by a fellowship from the Lalor Foundation. D.D. is funded by Human Frontier Science Program grant RGY0011 and National Institutes of Health-National Institute of Child Health and Human Development grant HD01339.

Manuscript received February 3, 2003; Accepted for publication May 6, 2003.


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*TOP
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