Journal of Heredity Advance Access published online on February 28, 2008
Journal of Heredity, doi:10.1093/jhered/esn006
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Brief Communications |
An Intron Loss of Dfak Gene in Species of the Drosophila melanogaster Subgroup and Phylogenetic Analysis
From the College of Life Science, Hubei University, Wuhan 430062, People's Republic of China
Address correspondence to Q. T. Zeng at the address above, or e-mail: zengqit{at}hubu.edu.cn
Drosophila focal adhesion kinase (Dfak) gene is a single-copy nuclear gene. Previous study revealed that Drosophila melanogaster and Drosophila simulans had lost an intron precisely within the tyrosine kinase (TyK) domain of this gene. However, this did not happen in several other Drosophila species, including Drosophila elegans, Drosophila ficusphila, Drosophila biarmipes, Drosophila jambulina, Drosophila prostipennis, Drosophila takahashii, and Drosophila pseudoobscura. In the current study, homologous sequences of Drosophila sechellia, Drosophila mauritiana, Drosophila yakuba, Drosophila teissieri, Drosophila santomea, and Drosophila erecta were amplified by polymerase chain reaction, and further sequencing analysis indicated that these species were missing a TyK domain intron, indicating they were closely related. The relationship of the D. melanogaster species group was reconstructed using TyK domain nucleotide sequences. The resulting phylogenetic tree revealed that these 8 species were the most related species in the melanogaster group. These results strongly support previously proposed classifications based on morphological and molecular data.
The intron was discovered in 1977 (Berget et al. 1977; Chow et al. 1977), and the first intron loss was found in the rat insulin gene in 1980 (Perler et al. 1980). Since that time, precise in-frame spliceosomal intron deletions have been repeatedly reported in other species, including Drosophila (Kwiatowski et al. 1992; Anderson et al. 1993; Luque et al. 1994; Fitzgibbon et al. 1995; Dalage et al. 1996; Wilanowski and Gibson 1998; Jin et al. 2005; Hu and Leung 2006). These deletions can provide valuable clues in the study of genome evolution.
Drosophila focal adhesion kinase (Dfak) gene, cloned and characterized in Drosophila melanogaster, is a single-copy nuclear gene and contains 13 short introns, ranging from 54 to 69 bp in size, and 1 long intron (Fujimoto et al. 1999). This gene encodes focal adhesion kinase, a nonreceptor cytoplasmic tyrosine kinase (TyK), activated by cell matrix adhesion. TyK plays important roles in several cell functions, including cell migration, cell cycle progression, cell adhesion, and cell survival (Natarajan et al. 2003). Previous study revealed that both D. melanogaster and Drosophila simulans, 2 members of the D. melanogaster species subgroup, have lost a short intron (about 60 bp) within the TyK domain of the Dfak gene. The loss of this intron, named the Y intron, did not occur in 7 other close relatives, including Drosophila elegans, Drosophila ficusphila, Drosophila biarmipes, Drosophila prostipennis, Drosophila takahashii, Drosophila jambulina, and Drosophila pseudoobscura (Jin et al. 2005). In this study, we examined whether other species of the melanogaster subgroup had lost the Y intron. The homologous sequences of the melanogaster subgroup species Drosophila sechellia, Drosophila mauritiana, Drosophila yakuba, Drosophila teissieri, Drosophila santomea, and Drosophila erecta were obtained by polymerase chain reaction (PCR). All 6 sequences had similar intron structure to D. melanogaster and had lost the Y intron during the evolutionary process.
The loss of the Y intron of the Dfak gene in these 8 species supports morphological and molecular evidence that they are the most closely related species in the melanogaster group. In order to explore this hypothesis, the phylogenetic relationships of the melanogaster group were reconstructed on the basis of the nucleotide sequences of the Dfak TyK domain. Here, we report the results.
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Materials and Methods |
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Drosophila Species
Six species were used: D. mauritiana, D. sechellia, D. yakuba, D. teissieri, D. santomea, and D. erecta, obtained from the Laboratory of Life Science, Hubei University. Nucleotide sequences of D. melanogaster (AY886895 [GenBank] ), D. simulans (AY882925 [GenBank] ), D. elegans (AY882926 [GenBank] ), D. ficusphila (AY886896 [GenBank] ), D. biarmipes (AY882924 [GenBank] ), D. jambulina (AY886894 [GenBank] ), D. prostipennis (AY886893 [GenBank] ), D. takahashii (AY882927 [GenBank] ), and D. pseudoobscura (AY886897 [GenBank] ) were recovered from the National Center for Biotechnology Information (NCBI) GenBank database.
DNA Preparation and Amplification
Total DNA was extracted from 3–5 fresh adult flies and stored at –20 °C for later PCR amplification. Two primers, Fak01 (5'-AAG TCA GGG AAA ACC GAT GCT-3') and Fak02 (5'-TTG CTC CCG CTT CAG TGT CT-3') were reported elsewhere (Jin et al. 2005), and locations are shown in Figure 1. PCR (50 µl) was performed using conditions as follows: 50 ng template DNA, 20 pM of each primer, 2.5 mM MgCl2, and 2 U Taq DNA polymerase. Amplification was implemented with denaturing at 95 °C for 3 min, 30 cycles of denaturing at 94 °C for 1 min, annealing at 58 °C for 50 s, and extension at 72 °C for 1 min and 10 s, followed by extension at 72 °C for 10 min.
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DNA Cloning and Sequencing
The amplified samples were run on 1% agarose gel and the bands excised using Gel Extraction Kit (Axygen, Hangzhou, China) and cloned into PMD-18T cloning vector (Takara, Dalian, China). The cloned PCR products were sequenced using universal M13-47 forward (5'-CGC CAG GGT TTT CCC AGT CAC GAC-3') and M13-48 reverse (5'-AGC GGA TAA CAA TTT CAC ACA GGA-3') primers in an ABI3730 sequencer. Two independent PCR syntheses of the same species were sequenced separately for assuring accuracy. The nucleotide sequences reported in this study have been deposited in NCBI GenBank nucleotide sequence database under accession numbers EU301822 [GenBank] –EU301827 [GenBank] .
Sequence Alignment and Phylogenetic Analysis
Sequences were aligned using Clustal W program (Thompson et al. 1994). The phylogenetic tree was constructed using PAUP* (Swofford 1998) by neighbor-joining (NJ) method. The confidence levels of the clusters were evaluated with the bootstrap test with 1000 replications. Drosophila pseudoobscura, a member of the obscura species group, was used as the outgroup.
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Results |
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All 6 Drosophila Species Lost Y Introns within Dfak TyK Domains
As shown in the schematic Dfak gene structure of Figure 1, genomic DNA fragments of about 1.3 kb covering TyK domain were amplified from 6 Drosophila species (Figure 2). The sequencing results showed that DNA fragments from D. sechellia, D. mauritiana, D. yakuba, D. teissieri, D. santomea, and D. erecta were 1262, 1264, 1262, 1270, 1269, and 1276 bp in size, respectively. In addition, 2 published homologous sequences from D. melanogaster and D. simulans were used for the sequence alignments (Jin et al. 2005). The result of alignments showed that the 8 species had the same genomic structure within Dfak TyK domains, which consisted of 6 exons and 5 introns. Previous study revealed that there was a Y intron loss within the Dfak TyK domain of D. melanogaster and D. simulans (Figure 3) (Jin et al. 2005). So, it could be concluded that the 6 species also lost the Y introns.
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Phylogenetic Analysis
The phylogenetic tree produced by NJ analysis is shown in Figure 4. As supported by bootstrap values, D. melanogaster, D. simulans, D. sechellia, D. mauritiana, D. yakuba, D. teissieri, D. santomea, and D. erecta are more closely related to each other than other species which also belong to the melanogaster group.
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Discussion |
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Despite study for 30 years and exciting recent advances, considerable important questions about the origin and evolution of the intron remain unanswered or uncertain (Roy and Gilbert 2006). However, there is no doubt that there have been gains and losses of introns during the evolutionary process. The Y intron loss in 8 species of the melanogaster subgroup displays a new pattern of intron loss, featuring a precise loss of a single intron in a multiple-intron gene, with neighboring introns retained. This occurrence is hard to explain by the theory that homologous recombination (HR) between a full-length or a prematurely terminated cDNA and its genomic counterpart should be responsible for intron loss (Fink 1987; Derr and Strathern 1993). However, a new model of intron loss, defined as intron exclusion, can explain this case well (Hu 2006). Compared with the previous cDNA-mediated homologous recombination (cDMHR) theory, the new model suggested that a DNA double-strand break repair (DSBR) machinery is involved in the cDMHR process as a single internal intron was removed precisely. According to the cDMHR/DSBR theory, a double-strand break within a specific intron could stimulate cDMHR for DSBR and cause loss of the specific intron. In fact, there are some examples of intron exclusion which have been reported. It was found that there was an allelic intron presence–absence polymorphism for the 4f-rnp gene in D. robusta in which intron 7 was lost precisely with the surrounding introns intact (Feiber et al. 2002). Large-scale comparison of intron positions in mammalian genes showed 5 in-frame losses of a single internal intron in 5 genes of mouse and 1 exact loss of an internal intron in rat (Roy et al. 2003). Alignment of introns for some selected plant homologous genes revealed that the Adh gene of Oryza sativa lost a single intron with the upstream and downstream introns retained (Hu 2006). The current study provides additional evidence of the new intron loss model.
The melanogaster group includes more than 160 species, most of which have been classified into 12 subgroups (Lemeunier et al. 1986). The melanogaster subgroup includes 9 species that appear to be of Afrotropical origin. Drosophila melanogaster and D. simulans are cosmopolitan in their distributions. Drosophila sechellia and D. mauritiana are endemic island species which are close relatives of D. simulans (Caccone et al. 1996). Drosophila teissieri and D. yakuba have similar geographic distribution ranging from northwest to southeast Africa. Drosophila teissieri, mainly a forest species, is more western in distribution, whereas D. yakuba, an open field species, is found widely in eastern regions. Drosophila erecta and Drosophila orena are restricted to west central Africa (Lachaise et al. 1988). Finally, D. santomea, closely related to D. yakuba, was recently discovered on São Tomé island in the Gulf of Guinea in west-equatorial Africa (Lachaise et al. 2000). In this study, the analysis of gene structural features (deletion or insertion of an intron or other nucleotides) and the phylogeny of the Dfak TyK domains were congruent and strongly supported previously proposed classifications which were based on morphological and molecular data.
In this study, it is a pity that we could not obtain D. orena. Many studies examining phylogenetic relationships of the melanogaster subgroup indicate that D. erecta and D. orena are the most closely related (Solignac et al. 1986; Lachaise et al. 1988; Arhontaki et al. 2002). It is likely that D. orena has also lost the Y intron within the Dfak TyK domain. If all members of the melanogaster subgroup have lost their Y introns, it suggests the Y intron was lost in their common ancestor. Generally, the melanogaster subgroup carries divergence time from 0.2 million to 15.0 million years ago (Lachaise et al. 1988, 2000). This suggests that the Y intron was lost in the common ancestor of the melanogaster subgroup species 15.0 million years ago.
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Funding |
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National Natural Science Foundation of China (NO.39901010 and NO.30470970).
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Acknowledgments |
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We thank Dr Wang Wen from Kun-ming Institution of Zoology for kindly providing some Drosophila species. We are grateful to our colleagues Y. F. Zhang, L. Tian, S. Wan, Z. X. Li, Y. Song, and T. T. Yi for numerous insightful discussions during the course of this study and for their critical reading of the manuscript.
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Footnotes |
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Corresponding Editor: James Thompson
Received October 25, 2007
Accepted December 15, 2007
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