Parkin-Co-Regulated Gene (PACRG) Product Interacts with Tubulin and Microtubules

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FEBS Letters 582 (2008) 1413–1418

Parkin-co-regulated gene (PACRG) product interacts with tubulin and microtubules

Takashi Ikeda*,1 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received 7 December 2007; revised 18 February 2008; accepted 25 February 2008

Available online 1 April 2008

Edited by Judit Ova´di

tility in mouse [5] and in the stable assembly of axonemal outer Abstract Parkin-co-regulated gene (PACRG) is a gene that shares a bidirectional promoter with ParkinsonÕs disease-related doublet microtubules in Trypanosoma [6]. Our study of Chla- Parkin/Park2 gene. Recently, the PACRG gene product was mydomonas PACRG showed that it is densely localized along implicated in the function of flagella. However, its exact function the flagellar outer doublet microtubules, possibly buried in the remains unknown. Here, I assessed the interaction between microtubule wall [7]. Despite these findings, the biochemical PACRG and tubulin. Co-sedimentation experiments revealed nature and physiological function of PACRG remain to be that PACRG directly binds to microtubules and a/b-tubulin het- studied. Here, I report that PACRG associates with both erodimers with high affinity. Microscopic studies showed that microtubules and tubulin dimers in vitro. PACRG bundles microtubules and forms branched aggregates with unpolymerized tubulin dimers. The amino acid sequence of the microtubule-binding region of PACRG is highly conserved among various organisms, suggesting that tubulin binding is a 2. Materials and methods basic property of PACRG. Structured summary: 2.1. Alignment By BLAST search using the amino acid sequence of C. reinhardtii MINT-6439731, MINT-6439753, MINT-6439768: PACRG (GenBank Accession No. BAG12189) [7] as the query, PACRG (genbank_protein_gi:169219363), tubulin alpha (uni- homologous sequences were obtained from T. brucei (XP_843825, protkb:P02550), tubulin beta (uniprotkb:Q2XVP4) bind XP_827292), Paramecium tetraurelia (CAK71037, XP_001443020), (MI:0407) by cosedimentation (MI:0027) Caenorhabditis elegans (NP_495496), Tetrahymena thermophila MINT-6439796: (XP_001023392, XP_001022215, XP_001022189), Strongylocentrotus purpuratus (XP_791188, XP_784874), Drosophila melanogaster PACRG (genbank_protein_gi:169219363), tubulin alpha (uni- (NP_609957, XP_843825), Apis mellifera (XP_625146), Danio rerio protkb:P02550), tubulin beta (uniprotkb:Q2XVP4) bind (NP_001007399), Mus musculus (NP_081308), and Homo sapiens (MI:0407) by pull-down (MI:0096) (AAH30642). Alignment was created using CLUSTALW ver. 1.83 [8].

2008 Federation of European Biochemical Societies. Pub- 2.2. Preparation of PC-tubulin lished by Elsevier B.V. All rights reserved. Purified a/b-tubulin heterodimer (PC-tubulin) was obtained from porcine brain by cycles of temperature-dependent polymerization Keywords: Axoneme; Outer doublet microtubule; ParkinsonÕs and phosphocellulose chromatography [9]. disease; Park2; Microtubule bundle; Chlamydomonas 2.3. In vitro synthesis and stabilization of microtubules PC-tubulin was polymerized by incubation at 37 C in a tubulin assembly buffer (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, 1 mM GTP, pH 6.8) containing 20% (v/v) dimethyl sulfoxide. Microtubules 1. Introduction were stabilized by addition of 20 lM taxol and collected by centrifugation at 30 C. The precipitated microtubules were washed with the Parkin-co-regulated gene (PACRG) was originally identified tubulin assembly buffer containing 20 lM taxol and resuspended in in human genome as a gene whose transcription is co-regulated an appropriate volume of the buffer. with a ParkinsonÕs disease-related gene Parkin/Park2 [1] by a bidirectional promoter [2]. PACRG has been suggested to 2.4. Expression and purification of recombinant PACRG proteins Recombinant PACRG proteins were expressed using the Bac-to-Bac interact with Parkin/Park2 and form a large chaperonin com- Baculovirus Expression Systems (Invitrogen, Carlsbad, CA). Total plex with chaperones [3]. Taylor et al. [4] showed that PACRG RNA was prepared from wild-type C. reinhardtii cells using TRIzol is regulated by a ubiquitin-proteasomal system that involves Reagent (Invitrogen) and first strand cDNA was synthesized by Super- Parkin/Park2. These observations have thus suggested the Script II reverse transcriptase (Invitrogen) using an oligo(dT)12–18 primer. Using it as the template, the cDNAs encoding the full-length and involvement of PACRG in parkinsonism. truncated coding sequences of PACRG were amplified by PCR using Recently, several studies have unexpectedly reported that the primer sets, PACRG_BamHI_F [50-CGG GAT CCA TGA ACG PACRG is also present in the flagella of various eukaryotes. GAG ACG TGG C-30] and PACRG + 6His_EcoRI_R [50-CGG This protein is implicated in the spermatogenesis and male fer- AAT TCA GTG ATG GTG ATG GTG ATG TGC GTA GTT CAC GCT GG-30] (full length), PACRG301 + ATG_BamHI_F [50- CGG GAT CCA TGA AGC CGT CCC CAC CTC AAG-30] and *Fax: +81 42 739 8453. PACRG + 6His_EcoRI_R (DN), PACRG_BamHI_F and PAC- E-mail address: [email protected] RG852 + 6His_EcoRI_R [50-CGG AAT TCA GTG ATG GTG ATG GTG ATG CTT TTG CTC GAA CAG CG-30](DC1), PAC- 1Present address: Honeybee Science Research Center, Tamagawa RG_BamHI_F and PACRG693 + 6His_EcoRI_R [50-CGG AAT University Research Institute, Machida, Tokyo 194-8610, Japan. TCA GTG ATG GTG ATG GTG ATG CAC CAG GTC CGC

CGA CAG-30](DC2), and PACRG301 + ATG_BamHI_F and PAC- fugation, the solution was mixed with Ni-NTA Agarose (QIAGEN). RG693 + 6His_EcoRI_R (DNC2). The PCR products were ligated After incubation, the beads were washed with the buffer and the bound into pFastBac1 vector (Invitrogen). Baculoviruses expressing 6· His- proteins were eluted with an elution buffer (80 mM PIPES, 1 mM tagged PACRG proteins were produced according to manufacturerÕs MgCl2, 1 mM EGTA, 0.1 mM GTP, 0.1% Triton X-100, 5 mM colchi- recommendations. Sf9 insect cells (Invitrogen) infected with the viruses cine, 300 mM imidazole, pH 6.8). All procedures were carried out at were lysed in a lysis buffer (0.6 M NaCl, 50 mM NaH2PO4,10mM 4 C. The eluates were analyzed by SDS–PAGE followed by CBB imidazole, 0.1% TritonX-100) and the PACRG recombinants fused staining. to 6· His-tag were primarily purified using Ni-NTA Agarose (QIA- GEN, Valencia, CA). Subsequently, these solutions were fractionated 2.8. Dark-field and electron microscopic observations by gel filtration using Superdex 200 PC 3.2/30 column (GE Healthcare Microtubules were mixed with PACRG in the assembly buffer con- UK Ltd.) in SMART System (GE Healthcare UK Ltd.), and the frac- taining 0.1% Triton X-100 and 20 lM taxol, and incubated at 37 C. tions containing pure PACRG proteins were collected. This fraction- As a negative control, microtubules were mixed with solutions that ation step was not carried out for the DC2 and the DNC2 had been obtained from baculovirus/Sf9 cells not expressing recombi- recombinants because these recombinants were obtained at very low nant PACRG. These mixtures were diluted to appropriate concentra- levels in a soluble state. These solutions were dialyzed against the tubu- tions with the same buffer and observed using a dark-field microscope lin assembly buffer to be subjected to the following experiments. For BX50 (OLYMPUS, Tokyo, Japan). For negative-staining electron negative control for microscopic studies (see below), solutions from microscopy, the mixtures were negatively stained with 1% uranyl ace- baculovirus/Sf9 that do not express recombinant PACRG were also tate. For thin-section observations, the PACRG-microtubules/tubulin prepared in the same way as those from the recombinant PACRG- complexes were collected by centrifugation and fixed with 2% glutaral- expressing cells. dehyde and 1% OsO4. Samples were dehydrated, embedded in EPON812 resin, thin-sectioned and stained with uranyl acetate and 2.5. Microtubule co-sedimentation assay lead citrate. These specimens were observed using JEM1010 electron The recombinant PACRG proteins were mixed with stabilized microscope (JEOL Ltd., Tokyo, Japan). For observation of tubulin microtubules in the tubulin assembly buffer containing 0.1% Triton in the presence of PACRG, tubulin was mixed with PACRG below X-100 and 20 lM taxol. After incubation at 37 C, microtubules were the critical concentration for polymerization in the presence of 5 mM collected by centrifugation and washed with the buffer. Both the pre- colchicine at room temperature. The solutions were observed using cipitates and the supernatants were analyzed by SDS–PAGE. The the dark-field microscope. presence of the recombinant PACRG proteins were detected by staining with Coomassie brilliant blue (CBB). 3. Results 2.6. Quantitative analysis of PACRG-microtubule interactions For quantification of microtubule-PACRG binding, varying amounts of the full-length and truncated PACRG recombinants were 3.1. PACRG directly binds to taxol-stabilized microtubules and mixed with microtubules at a constant concentration of tubulin hetero- unpolymerized tubulin heterodimers dimer. PACRG proteins that were bound and not bound to microtu- We have found that PACRG is associated with the outer bules were separated by centrifugation and analyzed by SDS–PAGE. doublet microtubules in the Chlamydomonas axoneme [7].To Quantities of both bound and unbound PACRG in the CBB-stained examine whether PACRG directly binds to microtubules or SDS–PAGE gel were estimated by densitometry using a CS Analyzer (ATTO, Tokyo, Japan). Data for a given set of conditions were those not, I conducted a co-sedimentation assay using microtubules averaged from 4 to 5 experiments. polymerized from brain PC-tubulin and recombinant PACRG. Recombinant PACRG had been produced by baculovirus/in- 2.7. His-tag pull-down assay sect cells and extensively purified using affinity chromatogra- PC-tubulin was diluted below the critical concentration for polymer- phy on a nickel column and gel filtration. SDS–PAGE ization with a pull-down buffer (80 mM PIPES, 1 mM MgCl2,1mM indicated that it was highly purified (Fig. 1A). When the puri- EGTA, 0.1 mM GTP, 0.1% Triton X-100, 5 mM colchicine, pH 6.8). Subsequently, PACRG was mixed with this solution containing 1% fied PACRG was incubated with taxol-stabilized microtubules BSA, and incubated. After insoluble proteins were removed by centri- and then centrifuged, a large portion of the PACRG was pre-

Fig. 1. PACRG directly binds to microtubules. Microtubule co-sedimentation assay shows a direct association between PACRG and microtubules. (A) Purification of recombinant PACRG using sequential purification steps. Chlamydomonas PACRG produced in a baculovirus/insect cells system was purified by affinity chromatography on a nickel column and gel filtration. The purified protein was subjected to SDS–PAGE followed by staining with CBB. (B) Co-precipitation of recombinant PACRG with taxol-stabilized microtubules. PACRG was mixed with microtubules and centrifuged. They were co-precipitated. In contrast, PACRG without microtubules was not precipitated. (C) Direct association of PACRG with unpolymerized a/ b-tubulin heterodimers. His-tag pull-down assay was carried out under conditions where tubulin does not polymerize. SDS–PAGE analysis indicates that PACRG binds to unpolymerized tubulin. T. Ikeda / FEBS Letters 582 (2008) 1413–1418 1415

Fig. 2. Microtubule-binding property resides in the conserved region of PACRG. (A) Alignment of PACRG amino acid sequences of various eukaryotes. A large portion of PACRG is well conserved among organisms while its N-terminal region is divergent. Underlined sequences indicate the regions that are missing in the recombinant proteins used in B and C (green), D and E (red), and F (green and blue). Highly conserved residues are highlighted in dark gray, and residues conserved in the majority of sequences are highlighted in light gray. (B) High purity of the truncated recombinant protein that lacks the N-terminal region (DN) as assessed by SDS–PAGE. (C) The recombinant PACRG protein that lacks the N-terminal region binds to microtubules although its affinity is attenuated. The truncated PACRG recombinant was mixed with microtubules and separated into precipitated and supernatant fractions by centrifugation. The amount of the truncated PACRG used was the same as that of the full-length protein used in Fig. 1C. (D) High purity of the truncated recombinant protein that lacks the C-terminal 23 residues (DC1, underlined in red in A) as assessed by SDS–PAGE. (E) The recombinant PACRG that lacks the C-terminal 23 residues binds to microtubules although its affinity is attenuated. The amount of the protein was the same as that of the full-length protein used in Fig. 1C. (F) The recombinant protein that lacks the C-terminal 76 residues (underlined in blue in A), and the recombinant protein that lacks both of the 76-residue-C-terminus (underlined in blue in A) and the N-terminus (underlined in green in A), bind to microtubules. The amounts of the recombinants used were smaller than that of the full-length protein used in Fig. 1C. 1416 T. Ikeda / FEBS Letters 582 (2008) 1413–1418 cipitated. In contrast, almost no PACRG was precipitated beads were eluted with an imidazole-containing buffer. SDS– when centrifuged without incubation with microtubules PAGE indicated that PACRG is directly associated with unpo- (Fig. 1B). Essentially the same result was obtained when the lymerized tubulin dimers (Fig. 1C). buffer contained 1% BSA (data not shown). These results indicate direct binding between microtubules and the recombinant 3.2. Microtubule-binding property is present in the conserved PACRG. region of PACRG Next, to determine whether PACRG directly binds to unpo- Blast search showed that the sequences homologous to lymerized tubulin dimers also, I carried out a His-tag pull- PACRG are conserved within a wide range of flagellate down assay using Ni-agarose beads. Tubulin and Ni-agarose eukaryotes and C. elegans, suggesting that PACRG plays an beads were mixed with 6· His-tagged PACRG at 4 C in the important role in the cilia/flagella as suggested by the previous presence of 5 mM colchicine at a tubulin concentration lower studies [5–7] (Fig. 2A). Some organisms such as Trypanosoma than the critical concentration for polymerization. They were [6] were found to have multiple PACRG-homologous se- centrifuged after incubation, and the proteins bound to the quences.

Fig. 3. PACRG-induced formation of microtubule bundles and branched aggregates of unpolymerized tubulin. (A) Dark-field micrographs of microtubules in the absence (upper, left panel) and presence (upper, right panel) of PACRG, and electron micrographs of negatively stained (middle panels) and thin-sectioned (lower panels) microtubules in the absence (left panels) and presence (right panels) of PACRG. (B) Dark-field micrographs of unpolymerized tubulin in the absence (left panel) and presence (right panel) of PACRG. Bars: 10 lm (dark-field micrographs), 200 nm (negatively stained samples), or 100 nm (thin-sectioned samples). T. Ikeda / FEBS Letters 582 (2008) 1413–1418 1417

The amino acid sequence of PACRG is well conserved ex- tached between the A- and B-tubules and within and inside the cept for the N-terminal region (Fig. 2A). To determine whether microtubule walls [10,11]. PACRG may constitute such a the tubulin-binding nature resides in the conserved region or structure, and play an important role in the doublet microtu- not, I carried out a co-sedimentation assay using a recombi- bule assembly [6]. In this context, the observations that PAC- nant PACRG that lacks the Chlamydomonas-specific N-termi- RG can bind with both microtubules and tubulin dimers (Figs. nal region (Fig. 2A and B). As shown in Fig. 2C, this truncated 1 and 3) are interesting. Tubulin polymerization in the pres- PACRG became bound to microtubules although its affinity ence of PACRG resulted in the formation of a large amount was attenuated. This result indicates that the conserved region of aggregates in addition to normal microtubule bundles contains microtubule-binding domain(s) and suggests that the in vitro. The aggregates are most likely produced by non-stoi- microtubule-binding property may well be common to the chiometric association. However, it is conceivable that PAC- PACRG of other organisms. RG is integrated in the microtubule cytoskeleton in an While moderately conserved region extends over 180 resi- ordered manner in vivo [7]. The exact mode of PACRG-tubu- dues, there is an ultra-conserved region at the extreme C-termi- lin interaction in vivo, as well as in vitro, awaits further studies nus (Fig. 2A, red underline). To examine whether this region is that quantify the interaction and examine whether or not essential for the microtubule-binding of PACRG, I performed PACRG itself forms a homo-dimer or a homo-oligomer. a co-sedimentation assay using a truncated PACRG that lacks PACRG is expressed in various tissues of mammals [5,12]. this region (Fig. 2D). This truncated PACRG was found to Although I examined only Chlamydomonas PACRG in this bind with microtubules (Fig. 2E). However, the binding of this study, tubulin-binding properties may well be common to truncated protein saturated at about two thirds of the concen- the PACRG of other organisms (Fig. 2). In support of this tration at which the full-length PACRG saturates (Fig. S1). idea, previous studies have provided indirect evidence for Thus, it is possible that the extreme C-terminal region contains PACRG-tubulin interaction. For example, Imai et al. [3] de- a site that binds to tubulin at a site different from the site to tected a- and b-tubulin as PACRG-associated proteins by which other PACRG regions bind. I also examined the micro- immunoprecipitation from the lysates of human embryonic tubule-binding activity in PACRG recombinants that lack kidney cells that over-express PACRG. In addition, Taylor either the C-terminal 76 residues (Fig. 2A, blue underline) et al. [4] showed that the localization of PACRG in neurons (DC2) or both of the N-terminus (Fig. 2A, green underline) of the locus coeruleus in the progressive supranuclear neuron and the C-terminal 76 residues (Fig. 2A, blue underline) palsy brain is displaced by hyperphosphorylated tau, a micro- (DNC2), and found that these proteins also bind with microtu- tubule-associated protein [13–15]. bules (Fig. 2F). These results suggest that at least the middle Abnormalities in the promoter region of Parkin/Park2 and region contains microtubule-binding site(s). PACRG are implicated in various human diseases that include leprosy [12,16,17], typhoid and paratyphoid fever [18], and leu- 3.3. PACRG bundles microtubules and forms branched kemia [19], as well as in male sterility [5] and ParkinsonÕs dis- aggregates with unpolymerized tubulin dimers ease [3,4]. Considering such a wide range of abnormalities for To examine the effects of PACRG on the gross appearance which PACRG may be responsible, PACRG can be involved of microtubules, I observed taxol-stabilized microtubules by in some fundamental cellular function. It may well be related dark-field microscopy after addition of PACRG. As shown to its activity to bind with tubulin and microtubules. in Fig. 3A, microtubules formed thick bundles in the presence of PACRG. No such bundle was observed when microtubules Acknowledgements: The author would like to thank Professor Ritsu Kamiya (University of Tokyo) for encouragement and critically read- were mixed with solutions obtained from baculovirus/insect ing the manuscript. I also thank Dr. Toshiki Yagi (Kyoto University) cells not expressing recombinant PACRG (data not shown). for technical help for protein purification, Mr. Susumu Aoyama and I also observed tubulin heterodimers in the presence of col- Mr. Soh Nishio (University of Tokyo) for help for tubulin preparation, chicine and PACRG at tubulin concentrations below the crit- and Dr. Tetsuhiko Sasaki (Tamagawa University) for support to prep- ical concentration for polymerization, and found that many aration of the manuscript. This study was supported by the Research Fellowship of the Japan Society for the Promotion of Science (JSPS) branched aggregates were formed in the solution (Fig. 3B). for Young Scientists. Electron microscope observations indicated that the aggregates did not contain any microtubule-like structures (data not shown). Finally, tubulin was polymerized in the presence of PACRG. Appendix A. Supplementary data This resulted in production of a large amount of branched aggregates, in addition to microtubule bundles (data not Supplementary data associated with this article can be shown). found, in the online version, at doi:10.1016/j.febslet.2008. 02.081.

4. Discussion References

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