In this report, we have determined that theE3ubiquitinligaseactivityofTrip12is indispensable for mouse development. Previous reports indicated that Trip12 promotes the ubiquitination and degradation of ARF . The down-regulation ofTrip12 expression stabilizes ARF and enhances ARF-dependent, p53-mediated cell cycle arrest in normal human fibroblasts . However, in our experiments, the p53 pathway was not activated in either Trip12 mt/mt mice or Trip12 mt/mt ES cells. Although Trip12 mt/mt mice had increased ARF expression levels, the p21 (which is a well known p53 target gene) expression levels were unchanged in these mice. These findings suggest that the growth arrest phenotype ofTrip12 mt/mt mice was caused by p16 expression rather than ARF induction. Previous reports also showed that Trip12-mediated ARF ubiquitination is inhibited by c-Myc . It is known that c-Myc expression levels are higher in mouse embryos and ES cells compared to differentiated cells [17,28,29,30]. Considering these results, we speculate that Trip12 minimally contributes to ARF ubiquitination during mice embryogenesis because of elevated c-Myc expression. On one hand, our experiments showed that BAF57 protein stability was regulated by Trip12-mediated ubiquitination and that the expression profiles of several genes were changed between wild-type and Trip12 mt/mt ES cells. This finding suggests that the ubiquitination activityofTrip12is tightly regulated by individual target proteins. Further experiments using conditional knockout approaches and theTrip12 protein complex that is purified from different cell types may help elucidate the exact in vivo functions oftheTrip12 protein.
While mutations in CUL4B cause mental retardation, short stature, impaired speech, and other abnormalities, the patients usually survive to adulthood. Thus, the Cul4b mutant mice are more severely affected. The mechanism underlying this species- specific difference in phenotypic severity caused by CUL4B deficiency remains to be elucidated. It is possible that certain mutations in humans may retain residual function of CUL4B and thus have less deleterious effect than the Cul4b null mutation in themouse. However, several mutations in human CUL4B resemble null mutations. The p.R388X mutation, for example, would have truncated the whole CULLIN domain and the rest ofthe C- terminus [16,17]. Moreover, this mutation caused nonsense mediated decay of CUL4B mRNA . Two reported deletions in CUL4B causing XLMR would have been devoid of most ofthe CULLIN domain, which isessential for CRL activity [19,20]. It should be noted that the more severe phenotype in mouse mutants Figure 6. Characterization of X chromosome inactivation by Cul4b expression in heterozygous mice. (A–C) Percentages of cells positive for Cul4b of Cul4b heterozygous mice and littermate wild-type female controls at 4 months (A), 3 weeks (B) and newborn (C). More than 2,000 cells of each tissue were scored. Hi, hippocampus; Ki, kidney; Li, liver; Lu, lung. Data were presented as mean6SD. *: p,0.05; **: p,0.01; ***: p,0.001. (D–E) Representative images of liver (D) and hippocampus (E) at 3 weeks stained with an antibody against Cul4b. Sections were counterstained with haematoxylin. Lower panels are the higher magnification ofthe upper panels. (F) Immunohistochemistry of paraffin sections of Cul4b heterozygous embryos at 7.5 dpc with an anti-Cul4b antibody. Embryos at 7.5 dpc were paraffin embedded and cross sectioned together with their surrounding deciduas. Middle and right panels are the higher magnification ofthe left panel.
Glis3-mediated transcriptional activation is likely regulated at multiple levels, including tran- scriptional, translational and post-translational mechanisms that control Glis3 protein activity and/or its expression level. We previously reported that the central region of Glis3 containing the ZFD and the C-terminal transactivation domain are essential for Glis3-mediated transcrip- tional activation [1,33]. In this study, we discover a new function for the N-terminus of Glis3 in the regulation of Glis3 stability and its transcriptional activity. Using the N-terminus of Glis3 as bait, we identified by GeLC-MS and Y2H analyses a number of novel Glis3 interacting partners, including several members ofthe Nedd4/Rsp5 family of HECT E3ubiquitin ligases. We demonstrate that the HECT E3ubiquitinligase, Itch, promotes the polyubiquitination of Glis3, thereby targeting Glis3 for increased proteolytic degradation by the proteasome. Conse- quently, this leads to a substantially reduced Glis3 transcriptional activity as indicated by the decrease in Glis3-mediated activation ofthe mIns2 promoter as well as the transactivation of a reporter driven by 3xGlisBS. Itch and other E3 ligases have been reported to regulate the activ- ity of various transcription factors and biological processes through different mechanisms . For example, increased ubiquitination of p73 by Itch has been demonstrated to promote its degradation and consequently its transcriptional activity and function , while the hedgehog transcription factor Gli1 is targeted by Numb for Itch-dependent ubiquitination thereby inhib- iting growth and promoting cell differentiation . Itch has also been reported to interact with and target the pluripotency-associated transcription factor Oct4 for ubiquitination thereby affecting embryonic stem cell self renewal .
ES cells, derived from the epiblast of early blastocysts, can self- renew and maintain a pluripotent, undifferentiated state in vitro by extrinsic factors, such as LIF and Bmp, and intrinsic factors, including stem cell regulators Oct4 and Nanog [42–45]. Upon induction of lineage-specific genes, these stem cell regulators are downregulated through a negative-feedback-loop by not yet understood mechanisms [42,45,46]. In line with this, the transcript levels of stem cell specific genes Sox2, Dppa3/Stella and Zfp42/Rex-1 were strongly downregulated in Ring1b-deficient ES cells [43,47] (Figure 4D, Table S1). However, QPCR analysis showed that Nanog transcript levels had not altered and Oct4 transcript levels had reduced only to about 55% (Figure 4D). Importantly, Oct4 was not identified as an outlier on the microarray and Western blot analysis showed no significant changes in Oct4 protein levels (Figure 3A, Table S1). To examine Oct4 expression in more detail, we also quantified the immunofluorescence levels of Oct4 and Ring1b stainings in individual cells. We compared this to guided neural differentiation induced by retinoic acid (RA) treatment, which was demonstrated to result in significant downregulation of Oct4 . As expected, we found that three days after RA induced neural differentiation Oct4 was completely downregulated, in about 65% ofthe cells (Figure 5A, 5B). However, whereas four days following Ring1b deletion, 81% of all cells were negative for Ring1b, only 5% had lost Oct4 expression (Figure 5A, 5B, 5C). To assess the extent of neural differentiation, we next examined the expression of neuronal- lineage marker Nestin . We found that three days after RA treatment approximately 44% ofthe cells showed filamentous Nestin staining, compared to 32% ofthe cells after deletion of Ring1b (Figure 5D, 5E). This is remarkable considering the number of cells expressing Oct4. This suggests that expression of lineage markers co- exists with stem cell regulators in absence of Ring1b in ES cells. Finally, we examined theactivityof alkaline phosphatase, which is highly expressed in undifferentiated, pluripotent ES cells . We found that after deleting Ring1b a substantial number of cells still showed high activityof alkaline phosphatase compared to RA treated ES cells, which is indicative for a pluripotent, undifferenti- ated state of ES cells (Figure 5F). Combined, our findings suggest that Ring1b-deficient ES cells show features of differentiated cells, while retaining important ES cell characteristics.
Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub) activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitinligaseE3 catalyzes the transfer ofthe Ub peptide to an acceptor protein. TheE3 component is responsible for the specific recognition ofthe target, making the unveiling ofE3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7) gene encodes for a protein with characteristics from a C3HC4-type E3ubiquitinligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activityis dependent ofthe presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation isthe circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV–B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.
by the NetPhos score, figure 3a) and were subsequently mutated to phenylalanine in order to assess their role in regulating TRIM21 function. Y343 and Y388 are conserved between human and murine TRIM21 and are also predicted to be phosphorylated. Interestingly, Y393 in human TRIM21 corresponds to S396 in murine TRIM21 and this serine residue is also predicted to be phosphorylated in murine TRIM21 (data not shown). As TRIM21 has previously been demonstrated to be a potent inhibitor of TLR- driven IFN-b production , we employed an IFN-b promoter reporter gene assay driven by an upstream activator ofthe pathway, the adaptor protein TRIF, and assessed the effects of each ofthe tyrosine mutants in this system. As expected from previous reports, wild type full length TRIM21 dose dependently inhibited IFN-b promoter activity, in keeping with its role as a negative regulator of this response (figure 3b). Similarly transfec- tion of cells with increasing amounts of Y343F resulted in a similar inhibitory response, indicating that Y343 is not essential for TRIM21 activity (figure 3c). In contrast we observed a lack of inhibitory activity when Y388F and to a greater extent Y393F mutants were assessed (figure 3d and 3e, respectively). Further analysis to assess the effects of double (Y343,388F) and triple (Y343,388,393F) mutants of TRIM21 was also conducted, with both the double and triple mutants inhibiting the response in a similar manner observed in the Y393F mutant as seen in figure 3f and 3g respectively. To ensure all plasmids were expressing to a similar level, expression analysis ofthe mutants was carried out. Figure 3h illustrates that Y393F expresses to a higher level than the other TRIM21 plasmids. However the increased expression of Y393F does not lead to an increase in inhibition of IFN-b promoter activity, emphasising that the lack of inhibition in this
A study using a bioinformatic approach and validation with various translocation assays has identified multiple translocated effectors in EHEC including the NleL (ECs1560) locus . A sequence comparison and structural studies have identified NleL as a bacterial HECT-like E3ubiquitinligase [28,30,45]. The substrate and biological function of NleL remains unknown. Auto- ubiquitination is often used in the absence of a physiological substrate to measure theubiquitinE3ligaseactivity, we first tested if GST-NleL has theE3ligaseactivity in an in vitro auto- ubiquitination assay using E1, E2 (UbcH5a), ATP, and ubiquitin in the presence of purified recombinant GST-NleL 59–782 . Consistent with our previous work , poly-ubiquitinated GST-NleL was observed by Western blot when E1, E2 or ubiquitin were added to the reaction (Fig. 1A). No ubiquitination of GST-NleL was detected in the absence of E1, E2 or ubiquitin, indicating that each of these components were essential for GST- NleL ubiquitination. Furthermore, the mutant GST-NleL C753S (pZP1658) or the GST-NleL C753A (pZP2129) failed to form the poly-ubiquitination pattern seen with the wild-type GST-NleL in the in vitro ubiquitination assay (Fig. 1B). To assess the specificity ofthe cysteine mutation, a substitution mutant targeting C688 (C688S, pZP1657) of NleL was used in the same ubiquitination assay. Western blot analysis shows presence of mono- and poly- ubiquitinated GST-NleL C688S species similar to those observed when the wild-type GST-NleL was used (Fig. 1B).
Inhibitor of apoptosis proteins (IAPs) are phylogenetically conserved proteins characterized by the presence of at least one Baculovirus IAP Repeat (BIR) motif, a zinc-binding structure of approximately 70 amino acid residues that mediates protein- protein interactions. As indicated by their name, the function of these proteins was first believed to be restricted to inhibition of cell death, mostly by direct interference with the proteolytic activities of caspases. However, several studies later proved that IAPs have a much wider spectrum of action, and that XIAP is probably the only member capable of direct caspase inhibition. In addition to their BIR domains, an interesting feature of XIAP, cIAP1 and cIAP2 isthe presence of a RING finger domain conferring E3ubiquitinligaseactivity. Cellular IAP proteins can promote Lys48- and Lys63-linked polyubiquitination of RIP1 and RIP2[29,30,31,32]. The addition of Lys63-linked ubiquitin chains to these proteins was reported to create a platform for the recruitment and activation ofthe TAB-TAK1 complex and ofthe IkB Kinase (IKK) complex IKKa-IKKb-NEMO, which drives NF-kB activation and thereby induces gene transcription [22,23]. However, recent studies have shown that Lys63-linked ubiquitin chains might not be essential for NF-kB activation, suggesting existence of other chain types for the recruitment and activation ofthe these kinase complexes[35,36]. Interestingly, cIAP1 has been shown to induce Lys6-, Lys11-, Lys48-, and Lys63-auto-ubiqui- tination, and a recent study demonstrated the ability of cIAP1 to conjugate RIP1 with Lys11-linked ubiquitin chains[33,37]. In addition, cIAP1/2-mediated ubiquitination of RIP1 was also reported to prevent RIP1 from integrating and activating death complexes[31,38].
It is well established that RIG-I is an essential cytosolic sensor for the innate immune response to certain viruses. However, the regulatory mechanism of RIG-I-mediated signaling has not been adequately characterized. To identify potential proteins that interact with RIG-I and regulate its signaling, we performed yeast two-hybrid screens of a human fetal kidney cDNA library using full-length RIG-I as bait. From 5610 6 independent screened clones, we identified 17 b-galactosidase-positive clones, among which 5 clones encoded the C-terminal regions of RNF135 (Aa 125–432, GenBank accession number NM_032322.3, and also identified as Riplet in recent publication ). This gene is located in a chromosomal region known to be frequently deleted in patients with neurofibromatosis [17–19]. On the basis of its functions described below, we designated this protein as REUL (for RIG-I E3ubiquitinligase). Since the REUL clone obtained from the yeast two-hybrid screening was not full-length, we amplified full-length REUL cDNA from the human fetal kidney cDNA library using PCR. Human REUL contains 432 amino acid residues and shares 57% sequence identity at the amino acid level with its mouse ortholog (Fig. 1A). Structural analysis with several programs indicated that REUL contains an N terminus RING- finger domain, and a C terminus PRY and SPRY domain (Fig. 1A). The middle region of REUL has no detectable similarity to any other proteins.
Our results support a model in which Hoxa2 acts as a stabilizer for p53 protein level, via its negative effect on RCHY1. This could not be expected from the known roles of Hoxa2. Indeed, p53 is known to be mainly involved in cell cycle arrest, senescence and apoptosis . Thus, a p53 stabilization would, at a first glance, induce cell cycle arrest, senescence and/or increase in apoptosis, which is not in line with Hoxa2 functional studies that led to postulate anti- differentiation and pro-proliferative roles for Hoxa2 [43,46,68]. Nevertheless, besides its role in cell cycle and apoptosis, p53 has also been involved in DNA repair. The p53 response to DNA damage varies according to its subcellular localization, the cell cycle status and the extent of DNA damage, from apoptosis induction to DNA repair . Efficient DNA repair is a crucial requirement during embryonic development . It allows cells sustaining a high division rate, implying shorter cell cycles, but preserving quality divisions essential for developmental processes. In that context, analyses with the KEGG PathwayFinder module from the R2 microarray analysis and visualization platform (http://r2.amc.nl) allowed highlighting a wide range of transcripts positively correlated to HOXA2 expression which correspond to genes involved in DNA repair (data not shown). In support of this hypothesis, RCHY1 has been shown to induce the degradation of PolH, a member ofthe Y family translesion DNA polymerase involved in double- strand break repair via homologous recombination [71,72]. In addition, the study ofthe knock-out mouse for Rchy1 has recently been published and confirmed its role in DNA repair .
The regulation of fundamental aspects of neurobiological function has been linked to theubiquitin signaling system (USS), which regulates the degradation and activityof proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3ubiquitinligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect ofthe APC ubiquitinligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) ofthe gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes ofthe APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets ofthe APC in specific sensory neurons. Because ofthe similarity between human and C. elegans nervous systems, the role ofthe APC in sensory neurons may also advance our understanding of human sensory function and disease.
A recent report suggest that mammalian KLHL10 and Cullin-3 can interact in vitro and that Cullin-3 is highly expressed during late murine spermatogenesis . In addition, KLHL10 was shown to be exclusively expressed in the cytoplasm of developmentally advanced murine sperma- tids, and mice carrying a null klhl10 allele are infertile due to defects during late spermatid maturation . These data suggest that a similar E3 complex may function in late mammalian spermatogenesis and that the defects in klhl10 mutant mice may be due to lack of caspase-3 activity. Despite apparent anatomical differences between insect and mamma- lian spermiogenesis, there are similarities in the removal of bulk spermatid cytoplasm. Like in insects, intracellular bridges between spermatids and the bulk ofthe cytoplasm are eliminated during mammalian spermatogenesis. In addition, residual bodies, which contain the extruded cytoplasm ofthe mammalian spermatids show high levels of active caspase-3 expression and may be homologous to the insect waste bag [99,100]. Furthermore, targeted deletion ofthemouse Sept4 locus, which encodes the pro-apoptotic protein ARTS, causes defects in the elimination of residual cytoplasm during sperm maturation . Finally, a recent study reported a high frequency of mutations in klhl10 from infertile oligozoospermic men . These intriguing ana- tomical and molecular similarities between spermatid indi- vidualization processes in Drosophila and mammals suggest that further studies on the link between theubiquitin- proteasome system and apoptotic proteins during sperm differentiation in Drosophila may provide new insights into the etiology of some forms of human infertilities.
Despite the organizational complexity of this organelle, it is increasingly appreciated that the Golgi is quite plastic and dynamic. For example, its morphology changes during cell mitosis , cell migration, in which the whole organelle orients towards the direction of cell move- ment , and apoptosis, during which it fragments into dispersed stacks . Given its role in many dynamic cell processes, it is not surprising that the Golgi is subject to considerable reg- ulation in response to environmental as well as cell-intrinsic cues. Indeed, recent work has uncovered a number of regulatory mechanisms controlling the organization and function ofthe Golgi [13–15]. For example, ERK signaling controls the reorientation ofthe Golgi towards the leading edge during cell migration  and heterotrimeric GTPases and SRC signaling have been proposed to allow the Golgi to adjust to changes in secretory cargo load [9, 17].
. Moreover, the mutant used here, rsp5-1, was described earlier as showing no defect in the ubiquitination of carboxypep- tidase S precursor (pCPS) . Also the defect in Sna3-GFP fusion protein sorting into the vacuole caused by the rsp5-1 mutation is not enhanced by ret1-1. Another possibility is that the observed genetic interaction is a result of an additive defect in anterograde trafficking caused by ret1-1 and rsp5-1. The ret1-1 mutation inhibits the transport of Gas1p, a glycosylphosphatidylinositol (GPI)- Figure 6. Overexpression ofubiquitin suppresses ret1-1 rsp5-1 mutant defects. (A) Growth defect of ret1-1 rsp5-1 mutant is suppressed by overexpression ofubiquitin or its variants. ret1-1 rsp5-1 mutant was transformed with empty vector [-] or with plasmids encoding wild type ubiquitin [UBI], ubiquitin with only single lysine 48 [K48] or 63 [K63] present and all other lysines replaced with arginine. Serial 1:10 dilutions of transformants were spotted on YPD medium and incubated for 2 days at indicated temperatures. (B) Localization of GFP-Rer1 to vacuole in ret1-1 rsp5-1 mutant is suppressed by overexpression ofubiquitin. Transformants from Figure 6A were additionally transformed with plasmid encoding GFP-Rer1 and were grown on SC -ura -leu at 28uC. Expression ofubiquitin variants was induced by addition of 100 mM CuSO 4 for 2 hours before observations. GFP-Rer1
We show that Roc2 and Cul5 only bind to each other, and that knocking out one protein greatly reduces the level ofthe other. The Roc2-Cul5 complex is conserved in other species including C. elegans and humans [39,40]. What isthe function ofthe Roc2-Cul5 complex in vivo? A previous Drosophila study used viable P element insertions in the 59UTR of Cul5 for over-expression experiments that suggested Cul5 is involved in cell fate specification and bouton formation in the larval CNS . This study indicated that the insertion alleles were very weakly hypomorphic, and consistent with this we were not able to detect a difference in the Cul5 mRNA levels of these alleles by RT-PCR (data not shown). Here we report the identification of Roc2 and Cul5 transposon insertion alleles in which we cannot detect protein by immunoblot analysis of mutant embryos. These mutants develop into morphologically normal adults. Thus, Roc2-Cul5 is not required for development. It is possible that Roc2-Cul5 is redundant with other CDL. A recent paper showed that Roc1-Cul2 and Roc2-Cul5 complexes may act redundantly during meiotic cell cycle progression in C. elegans . RNAi knockdown of Roc2 or Cul5 did not reveal an obvious phenotype, consistent with our results. However, RNAi knockdown of either Roc2 or Cul5 mRNA in a cul-2 mutant background caused complete sterility, whereas cul-2 mutants only display partial sterility. We occasionally observed a small, but inconsistent reduction in female fecundity in both the Drosophila Roc2 and Cul5 mutants, perhaps reflecting such redundancy. Cul2- and Cul5-based E3 ligases use similar substrate adapter machin- ery, consisting of ElonginB, ElonginC, and a variable BC box protein [40,42,43], suggesting that Cul2 and Cul5 complexes could have overlapping substrates in some organisms. Drosophila Cul2 forms a complex with Rbx1, Elongins B and C, and VHL that supports polyubiquitin chain formation in vitro, and that is capable of ubiquitylating the HIF-1a transcription factor as occurs in mammals [44,45,46]. Our data indicate that any potential redundancy between Cul2 and Cul5 in Drosophila must occur by utilizing different Roc proteins, as Roc1a is not part of a Cul5 complex, and Roc2 is not part ofthe Cul2 complex. Whether redundancy exists or not, that the Cul5-Roc2 complex has been evolutionarily conserved suggests that it plays an important role in many organisms.
In order to investigate the effects of reduced expression of lamin A/C on HP1 stability, we employed a vector-based shRNA system to knock down endogenous lamin A/C levels as described in Materials and methods. In initial transient transfection experi- ments, HeLa cells expressing lamin A/C shRNAs and co- expressing GFP were observed to be depleted in HP1a and b but not HP1c, whereas cells transfected with control shRNA vector did not show any effects on HP1 levels (Fig. 1A). Subsequent experiments were carried out with HeLa cells stably expressing shRNA against lamin A/C transcripts. In one clone that has been described earlier (cl27) , there was ,90% reduction in lamin A/C protein by western blot analysis and nearly complete loss of HP1a and b proteins with no detectable effects on the levels of HP1c (shown in Fig. 1B). Although the inner nuclear membrane protein emerin is redistributed in cells expressing laminopathic mutants that disrupt the lamina , it was not significantly mislocalized to the cytoplasm upon lamin A/ C knock-down, suggesting that the small amount of lamin A/C that remains at the nuclear rim in these cells is sufficient for the proper localization of emerin. A previous study has also reported the normal distribution of emerin in lamin A/C depleted cells .
Closer to this paper isthe work of Gallego, Montero, and Salas . These authors analyze two policies, in Mexico City and Santiago (Chile), aimed at reducing congestion and pollution. They find the policies that impose driving restrictions may lead to a higher number of cars on the city. Our results point on the same direction. Batarce and Ivaldi  estimate the demand for transportation mode taking into account traffic congestion in an equilibrium setup. In their work, traffic congestion isthe equilibrium of a game with a continuum of drivers. De Borger and Proost  theoretically analyze the political economy aspects of congestion pricing. Their results corroborate the emprical observation that road pricing is politically difficult to implement. We analyze the same problem empirically.
To investigate the tissue-specific expression pattern of AtR- DUF1, a fusion gene comprising the native AtRDUF1 promoter, a 1.3-kb fragment upstream ofthe start codon of AtRDUF1 CDS, and the b-glucuronidase (GUS) gene  coding sequence as the reporter gene were constructed and transformed into wild-type Arabidopsis. Histochemical staining showed that AtRDUF1 ex- pressed abundantly in seeds, but was also locally detectable in flowers, hypocotyls, leaves and roots (Figure 2). The staining was strong in immature seeds (Figure 2A), whereas in intact desiccated seeds, the GUS expression was only detectable at the funiculus attachment region (Figure 2B). In broken seeds, GUS staining was uniformly presented throughout the seed (Figure 2C), indicating that the limitation of GUS staining in intact seeds was due to blocking by the seed coat. To exclude possible false observations caused by the diffusion ofthe soluble intermediate of GUS substrates [27,28], seeds were dissected and stained separately. The GUS staining signal could be observed uniformly throughout the embryo, but only in the funiculus attachment region ofthe seed coat (Figure 2D). During germination, a reduction in GUS staining was detectable early on (Figure 2E–H), which is consistent with the decrease of AtRDUF1 expression detected by real-time qRT-PCR (Figure S2 in File S1). In 4-d-old seedlings, the GUS expression was mainly detected in the junction ofthe root and hypocotyl, in leaf tips, and around the meristem (Figure 2H). In 2- week old seedlings, the GUS staining was only detectable in leaf tips and root tips (Figure 2I and 2J). In reproductive tissue,
multiple synchronous and/or metachronous cancers ofthe oesophagus, lungs, and head and neck region (i.e. oral cavity, oropharynx, hypopharynx, or larynx). 90% ofthe tumours in head and neck are squamous cell carcinomas, and at least 75% of them are attributable to the combination of tobacco and alcohol consumption. The odds ratio of OSCC may be as high as 50.1 for those who are both heavy smokers and heavy drinkers in comparison to people who neither drink nor smoke. 13 It has been estimated
In general, c-tubulins are highly conserved proteins in all eukaryotes. At the amino acid sequence level, human c-tubulin 1 and c-tubulin 2, respectively, show 98.9% and 97.6% identity with the corresponding mouse isoforms (Table S1) . To study the subcellular localization and function of human and mouse c- tubulin 2, we have chosen human osteosarcoma cells U2OS. Because of their flat shape, they are excellent for immunofluores- cence analysis and are easily transfectable. Moreover, thethe selection of U2OS made it possible to answer the question whether or not themouse c-tubulin 2 is capable of replacing human c- tubulin 1. We have used exogenously expressed FLAG-tagged mouse and human c-tubulins to evaluate the subcellular localization of c-tubulin 2 proteins and their interactions with GCPs. It was reported previously that exogenous mouse c-tubulin 2 located to interphase and mitotic centrosomes in mouse Eph4 epithelial cells . Our data corroborate this finding by showing that both human and mouse c-tubulin 2 are recruited to interphase and mitotic centrosomes in human U2OS cells. By immunoprecipitation experiments we found that c-tubulin 2 interacted with GCP2, an integral component of cTuSCs. Reciprocal coimmunoprecipitations of c-tubulin 2 and GCP4 (T. Sulimenko, unpublished data) indicated that c-tubulin 2 normally also incorporated in cTuRCs. We found no differences between c-tubulin 1 and c-tubulin 2 with regard to their localization and interactions. Intriguingly, antibody to GCP2 coimmunoprecipitated more endogenous than exogenous c- tubulins (Fig. 1B, Fig. S2). A similar result was obtained with Figure 6. Quantitative evaluation of microtubule formation in