Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
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Abstract |
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Introduction |
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The key role that cell cycle regulation plays in developing organisms might imply that its participating components will be highly conserved among diverse eukaryotic taxa. Indeed, cyclins and CDKs are well conserved and easily recognizable from yeast to humans (Pines 1994
; Jeffrey et al. 1995
; Liu and Kipreos 2000
). Recent analysis of the Drosophila genome sequence supports previous suggestions of strong parallels between many fly and vertebrate cell cycle regulators (Rubin et al. 2000)
. In addition, there are numerous reports demonstrating proper function of cell cycle regulators in heterologous systems (e.g., Lehner and O'Farrell 1990
; Lew, Dulic, and Reed 1991
; Geng et al. 1999
). Significantly, Xenopus cyclin A (CycA) can bind to and activate the yeast Cdc28 kinase in vivo (Funakoshi et al. 1997
). Conversely, several genes that have been previously shown to contribute directly to different stages of cell cycle regulation in flies do not as yet have homologs in other genomes analyzed (e.g., thr [D'Andrea et al. 1993
], rux [Thomas et al. 1994
], pim [Stratmann and Lehner 1996
], rca1 [Dong et al. 1997
]). This observation challenges the concept of the conservation of the cell cycle machinery and raises questions about the evolutionary origin of these genes. One gene that has no apparent homolog in other eukaryotes is the CycA inhibitor rux.
Rux is an essential cell cycle regulator in Drosophila, and viability in loss-of-function rux alleles is reduced to roughly 10% of wild type (Gonczy, Thomas, and DiNardo 1994
; Thomas et al. 1994
). It has been demonstrated that rux acts to down-regulate CycA-dependent activity during the G1 phase and is responsible for a temporary G1 arrest during several stages of Drosophila development (Sprenger, Yakubovich, and O'Farrell 1997
; Thomas et al. 1997
). In addition, Rux regulates the second meiotic division during Drosophila spermatogenesis (Gonczy, Thomas, and DiNardo 1994
; Thomas et al. 1994
) and functions during embryogenesis in the exit from mitosis (Foley and Sprenger 2001)
. Recent studies (Foley, O'Farrell, and Sprenger 1999
; Avedisov et al. 2000
) revealed different levels of regulation of CycA by Rux and suggested a mechanism by which Rux mediates cell cycle arrest. Specifically, we defined two small but functionally important structural motifs within the Rux protein (Avedisov et al. 2000)
. An N-terminal Leu-31 has been found to be critical for the association between Rux and CycA and for the inhibition by Rux of CycA-dependent kinase activity. This residue is a part of a canonical RXL sequence which mediates, in part, the binding of human CKIs p21, p27, and p57 to CycA-Cdk2 and CycE-Cdk2 (Adams et al. 1996
; Chen et al. 1996
). The second motif is a C-terminal bipartite nuclear localization signal (NLS) which is responsible for targeting Rux to the nucleus. Overexpression of wild-type Rux protein in the developing eye disc also results in translocation of CycA to the nucleus, where CycA protein is destroyed, and this activity of Rux is also dependent on the C-terminal NLS. Thus, Rux may influence the intracellular distribution of CycA and promote its destruction (Avedisov et al. 2000)
.
The rux gene is present in a single copy at cytological position 5C on the X chromosome of Drosophila melanogaster. The gene encodes a 335-amino-acid protein (fig. 1 ) with no homologs detectable in current protein databases. In this study, we performed a phylogenetic analysis of Rux in eight species of the D. melanogaster subgroup and showed that the protein evolves unexpectedly rapidly. This report represents one of the first studies of a hypervariable gene with a known essential function in closely related Drosophila species.
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Materials and Methods |
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DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted from 50 adult flies from each strain as described (Chia et al. 1985
). Different combinations of degenerate primers were used for PCR amplification of regions of the rux gene from eight Drosophila species. Standard PCR amplification conditions were 30 cycles of denaturation at 95°C for 30 s, primer annealing at 50°C or 55°C for 30 s, and primer extension at 70°C for 3 min. Oligonucleotide sequence information and details of PCR strategy are available on request.
The amplified fragments were cloned into the pCR2.1 vector (Invitrogen) and sequenced. Sequences were verified by sequencing directly from the amplified genomic DNA. Sequencing was done on an automated sequencer (Applied Biosystems) using the ABI PRISM dye-terminator cycle-sequencing kit (Perkin-Elmer). The D. simulans, D. mauritiana, D. sechellia, D. teissieri, D. yakuba, D. erecta, and D. orena rux sequences have been deposited in the GenBank database under accession numbers AF327884AF327890.
Data Analysis
The nonredundant protein sequence database at the National Center for Biotechnology Information (NCBI, National Institutes of Health, Bethesda, Md.) was searched using the gapped BLAST program (Altschul et al. 1997
). Protein sequences were compared with domain-specific sequence profiles using the SMART system (Schultz et al. 1998
) and the Conserved Domain (CD) search at the NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Multiple protein and nucleotide sequence alignments were constructed using the CLUSTAL W (version 1.8) program (Baylor College of Medicine software package) (Thompson, Higgins, and Gibson 1994
) and then adjusted by hand. Protein secondary-structure prediction was performed using the PHD program with a multiple-protein-sequence alignment supplied as the query (Rost and Sander 1994
). Sequence-structure threading was performed using the hybrid fold prediction method (Fischer 2000)
.
The Pamilo-Bianchi-Li (PBL) method (Li 1993
; Pamilo and Bianchi 1993
) and the K-Estimator (Comeron 1999
), MEGA2 (Kumar, Tamura, and Nei 1994
), and YN00 (Yang and Nielsen 2000)
programs were used to estimate the numbers of synonymous (Ks) and nonsynonymous (Ka) substitutions per site. Phylogenetic trees based on multiple alignments of nucleotide sequences were constructed using the neighbor-joining (Saitou and Nei 1987
) and maximum-parsimony (Fitch 1971
) methods as implemented in the PAUP* program (Swofford 1998
) with default parameters.
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Results and Discussion |
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As a next step in identifying Rux homologs, we tested the eight known species from the melanogaster subgroup. According to the most recent estimates for individual species from this subgroup, the D. melanogaster lineage split from D. yakuba approximately 5.1 ± 0.8 MYA, from D. mauritiana 2.7 ± 0.4 MYA, and from D. simulans 2.3 ± 0.3 MYA (Li, Satta, and Takahata 1999
). Positive results were obtained in cross-hybridization experiments, and the corresponding genomic sequences were recovered by PCR. The alignment of the inferred polypeptides is shown in figure 2
. The most surprising result is the unusually low level of similarity between Rux proteins from these closely related species, in which orthologous proteins typically show negligible amino acid sequence difference (table 1
). Thus, Rux seems to be one of the most diverged Drosophila proteins described to date (see table 1
and below).
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Interspecific Comparison of rux Within the melanogaster Subgroup |
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By aligning the sequences from all eight species, evolutionarily conserved regions and variations in selective constraints along the Rux protein sequence were identified. The predicted size of the Rux protein differed for each species (fig. 2 ). These differences in length were largely due to different copy numbers of a six-amino-acid repeat unit in the C-terminal region of the protein. Interestingly, expansion of this sequence has occurred only in the species of the melanogaster complex, while both the yakuba and the erecta/orena lineages retain a single unit. While the function of these repeats is unclear, the observed length divergence among the species of the melanogaster complex may reflect the evolution of an as yet unknown regulatory pattern.
The distribution of replacements along the Rux protein sequence is nonuniform. To reveal patterns that may be specific to previously described functional domains of this protein (Avedisov et al. 2000)
, the coding region was subdivided into two partially overlapping regions and further characterized (table 2
). The region that is important for interaction with CycA resides within the N-terminal two thirds of the protein (amino acids 1217), whereas the C-terminal portion (amino acids 188335) has been shown to be responsible for the proper intracellular localization of wild-type Rux and contains the NLS (Avedisov et al. 2000)
. Most of the amino acid substitutions have occurred in the C-terminal third of the protein. This region also harbors seven of the 10 insertions/deletions detected in the alignment. This suggests that the C-terminal part of the protein is under noticeably reduced selective constraint, although most of the residues that compose the bipartite NLS consensus are conserved between the eight species. In some of the species, the first arginine residue in the distal basic cluster of the NLS consensus is replaced by proline (fig. 2
). To examine the possibility that this point substitution changes the subcellular localization of Rux protein in these species, we expressed in Drosophila cultured cells a chimeric protein in which the N-terminal 39 amino acids were from D. melanogaster Rux and the remaining 301 amino acids were from D. erecta Rux. The C-terminal portion derived from the D. erecta protein contained the entire region required for nuclear localization (Avedisov et al. 2000)
, including an NLS of the structure RKR-(X)10-PKR. Similar to the D. melanogaster Rux, this chimeric protein localized predominantly to the nucleus (data not shown), indicating that the subcellular localization of Rux is conserved in different fly species despite the deviation of the NLS sequence from the consensus in some of those species.
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Synonymous and Nonsynonymous Substitutions in the rux Gene
We calculated the numbers of synonymous (Ks) and nonsynonymous (Ka) substitutions per site between rux gene sequences from different species (table 3
). The Ka/Ks ratio is normally used to estimate the mode and the strength of selection acting on a coding sequence (for a recent review, see Kreitman and Comeron 1999
). For Rux, each pairwise comparison produces Ks values exceeding Ka; i.e., there is no clear evidence of positive selection. However, the Ka/Ks ratios for this protein are among the highest reported for Drosophila species (Schmid and Tautz 1997
; Schmid et al. 1999
), which suggests relaxed purifying selection. Table 3 shows the data produced by the PBL (Li 1993
; Pamilo and Bianchi 1993
) and maximum-likelihood (Yang and Nielsen 2000)
methods; several other methods for Ks and Ka calculation implemented in the MEGA and K-Estimator packages yielded very similar results (data not shown). These observations confirm that there are low overall constraints on nonsynonymous substitutions in the rux gene.
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Conclusions |
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Acknowledgements |
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Footnotes |
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1 Present address: Engelhardt Institute of Molecular Biology, Moscow, Russia.
2 Present address: National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland.
1 Keywords: roughex
cell cycle inhibitor
cyclin A
evolution
Drosophila
2 Address for correspondence and reprints: Sergei N. Avedisov, Engelhardt Institute of Molecular Biology, 32 Vavilov Street, Moscow 117984, Russia. ave{at}genome.eimb.relarn.ru
.
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