Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Protein (nutrient) wikipedia , lookup
Protein phosphorylation wikipedia , lookup
Signal transduction wikipedia , lookup
Magnesium transporter wikipedia , lookup
Protein moonlighting wikipedia , lookup
List of types of proteins wikipedia , lookup
Gene expression wikipedia , lookup
ã Oncogene (1999) 18, 7026 ± 7033 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc A novel exon within the mdm2 gene modulates translation initiation in vitro and disrupts the p53-binding domain of mdm2 protein Nik Veldhoen1, Su Metcalfe2 and Jo Milner*,1 1 YCR P53 Research Group, Department of Biology, University of York, York, YO10 5DD, UK; 2Department of Surgery, Addenbrooke's Hospital, University of Cambridge, Cambridge, CB2 2QQ, UK The mdm2 protein interacts with a number of proteins involved in cell growth control. Such interactions favour cell proliferation and may explain the oncogenic potential of mdm2 when over-expressed in cells. Interaction with the tumour suppressor p53 involves the N-terminus of mdm2 and targets p53 for rapid degradation by the ubiquitin pathway. We now describe a novel, highly conserved exon of mdm2 (exon a) which includes an in-frame UGA stop codon. Expression of exon a disrupts in vitro translation of the p53 binding domain of mdm2. We propose that exon a induces translation re-initiation at an internal AUG codon within the mdm2a mRNA isoform. The putative mdm2a protein lacks the N-terminus of mdm2 and shows little, if any, binding capacity for p53. Mdm2a mRNA is expressed in a tissue-speci®c manner and is observed predominantly in testis and peripheral blood lymphocytes. We propose that mdm2a expression may provide a mechanism for uncoupling mdm2-p53 interaction in certain cell types and/or under speci®c conditions of cell growth. Keywords: mdm2; p53; regulatory exon; alternative splicing Introduction In normal cells the mdm2 protein can bind proteins involved in cell growth suppression and in cell proliferation. The eects of such interactions favours cell proliferation and this may account for the oncogenic nature of mdm2 when over-expressed (Haines, 1997; Momand and Zambetti, 1997; Piette et al., 1997). Two major tumour suppressors, p53 and pRB, are bound by mdm2 with subsequent inhibition of their cell growth suppressor properties (Momand et al., 1992; Oliner et al., 1993; Xiao et al., 1995). Conversely, transcriptional activation by E2F-1/DP1 is stimulated when complexed with mdm2 protein (Martin et al., 1995). The E2F-1/DP1 heterodimer facilitates the progression of cells through S phase and mdm2 is predicted to enhance this function and promote cell division. Indeed, targetted over-expression of an mdm2 minigene in the mammary gland results in uncontrolled entry into S phase, polyploidy *Correspondence: J Milner Received 17 March 1999; revised 9 August 1999; accepted 23 August 1999 and tumour formation in transgenic mice (Lundgren et al., 1997). The eect is independent of p53 since both wild type and p53 null mice were similarly aected. The mdm2 protein can be divided into sub-domains (reviewed by Piette et al., 1997). Human mdm2 is composed of 491 residues (see Figure 1) and interactions with p53 and E2F-1/DP1 occur within the N-terminal half of the protein (Chen et al., 1993; Leng et al., 1995; Martin et al., 1995; Freedman et al., 1997). Other interactions occur at distinct sites within mdm2, including L5 protein binding within the central acidic domain (residues 237 ± 260) and RNA binding at the C-terminal RING ®nger domain (residues 438 ± 477) (MareÂchal et al., 1994; Elenbaas et al., 1996). Interaction between mdm2 and Numb, a cell fate regulator, has recently been reported and raises the possibility that this pluripotential protein may also in¯uence cell dierentiation and survival (JuvenGershon et al., 1998). The interaction between p53 and mdm2 has attracted a lot of attention and the molecular structure of the binding domain of p53 in complex with the Nterminus of mdm2 has now been resolved (Kussie et al., 1996). The p53 binding domain of mdm2 (residues 15 ± 125 for the human form) contains a deep hydrophobic cleft into which p53 residues TFSDLWKLL (amino acids 18 ± 26) bind as an amphipathic alpha helix. Binding eectively blocks the transactivation domain of p53 and targets p53 for rapid degradation by the ubiquitin-mediated proteolytic pathway (Haupt et al., 1997; reviewed by Kubbutat and Vousden, 1998). Indeed, there is evidence that mdm2 directly functions as a ubiquitin ligase for p53 (Honda et al., 1997; Honda and Yasuda, 1999). Nuclear-cytoplasmic shuttling by mdm2 may also be important in accelerating the degradation of p53 (Roth et al., 1998). These various properties of mdm2 serve to down-regulate p53 activity and this is essential for normal embryonic growth and development (Montes de Oca Luna et al., 1995). The oncogenic potential of mdm2 was ®rst demonstrated by Fakharzadeh et al. (1991) and is now recognized as an important factor in human cancer. Gene ampli®cation and overexpression of mdm2 protein is common in soft tissue sarcomas (20%), osteosarcomas (16%) and oesophageal cancer (13%) (see Momand et al., 1998). One consequence of mdm2 overexpression is predicted to be the inhibition of p53 function, and this would contribute towards genomic instability and oncogenic transformation. Abnormal transcripts of mdm2 in tumours have also been reported (Haines et al., 1994; Maxwell, 1994; A novel mdm2 exon modulates translation N Veldhoen et al Sigalas et al., 1996; Matsumoto et al., 1998; Kraus et al., 1999) but are not evident in normal cells (Montes de Oca Luna et al., 1996). In the present study we have identi®ed a novel exon within the mdm2 gene which is expressed in normal tissue and is 99% conserved between human and canine mdm2. It is located between the previously identi®ed exons 4 and 5 of mdm2 and is ¯anked by conventional intron/exon splicing boundaries. This unique exon (termed exon a) disrupts translation of the p53 binding domain of mdm2 and may represent a mechanism for uncoupling mdm2-p53 interaction in certain normal cell types and/or under speci®c conditions of cell growth. approximate intronic distances of exon a from exons 4 and 5 are shown in Figure 2b. DNA sequencing within this region of the human and canine mdm2 gene also indicated that the exon-intron boundaries ¯anking the a exon conform to the GT ± AG rules required for accurate mRNA splicing (Table 1) (Padgett et al., 1986). These results demonstrate that both mdm2 and the mdm2a splice variant are highly conserved between human and canine species. For simplicity, in the text these two cDNAs and their related proteins are Results Isolation of canine mdm2 cDNA reveals a novel exon This study forms part of a systematic analysis of cell growth control and spontaneous tumour development in the dog. Having cloned and sequenced canine p53 (Veldhoen and Milner, 1998) we next sought to characterize canine mdm2 and its interaction with p53. A canine homologue of mdm2 cDNA was isolated by reverse transcriptase-PCR (RT ± PCR) using mRNA derived from canine mammary tumour tissue (Materials and methods). The predicted canine mdm2 (cMDM2) protein shares 93% amino acid identity with human mdm2 (hMDM2) and 81% identity with murine mdm2 (Figure 1). A second mdm2 cDNA, called c-mdm2a, was isolated from total RNA derived from canine peripheral blood leukocytes (PBLs). The cmdm2a cDNA is unusual in that it contains an additional 87 base pair sequence which encodes 29 in-frame codons, including a UGA codon (Figures 1 and 2a). This additional sequence, termed the a exon, lies within the sequence encoding the p53 binding domain of mdm2. The a exon is highly conserved We next asked if human cells also express a mdm2a transcript and screened for the presence of mdm2a mRNA in human lymphocytes. cDNA ampli®cation using total RNA isolated from human blood lymphocytes lead to the identi®cation of a h-mdm2a splice variant (Figure 1). The a exon sequence is highly conserved between canine and human mdm2 genes, with only a single base pair dierence between the two species (see Figure 2a). Such a high level of evolutionary conservation suggests that the a exon may represent a functionally important region of the mdm2 gene. The a exon of both canine and human mdm2 are located at identical sites within their respective cDNAs (see Figure 1). This site corresponds to an exon-intron boundary in the murine mdm2 gene (between exons 4 and 5; Montes de Oca Luna et al., 1996; Jones et al., 1996). To characterize the position of the a exon in more detail we performed genomic PCR analysis using oligonucleotide primers that anneal to the a exon and to exons 4 and 5 (Materials and methods). The ampli®ed DNA products served to localize the a exon within the human and canine mdm2 genes and the Figure 1 Alignment of mdm2-related protein sequences. The coding potential of canine mdm2 and mdm2a, and human mdm2a is depicted along with previously published sequences for murine mdm2 (Cahilly-Snyder et al., 1987), human mdm2 (Oliner et al., 1992), and Xenopus mdm2 (MareÂchal et al., 1997). The presence of canine and human mdm2a transcripts were con®rmed in PBLs isolated from ®ve dierent dogs and seven separate human individuals. Highly conserved regions are shaded while proposed nuclear localization signals (NLS) and a nuclear export signal (NES) are boxed (Piette et al., 1997; Roth et al., 1998). Amino acid residues that contribute to p53 protein binding are identi®ed above the sequence alignment by black circles (Kussie et al., 1996; BoÈttger et al., 1997; Freedman et al., 1997). The positions within mdm2a of the UGA codon and the downstream AUG (corresponding to AUG62 in mdm2) are shown by an asterisk and an open triangle, respectively 7027 A novel mdm2 exon modulates translation N Veldhoen et al 7028 referred to as mdm2 and mdm2a, with the species of origin given in the relevant ®gures. Translation of mdm2a protein initiates from an internal AUG codon The a exon contains an in-frame UGA codon, the context of which indicates an ecient stop signal for translation (UGAG, Figure 2a; McCaughan et al., 1995). We were therefore surprised when in vitro translation of mdm2a cRNA yielded detectable protein (Figure 3a, lane 3). Translational readthrough seemed unlikely since this would generate an mdm2a protein product containing 29 additional amino acids, whereas the in vitro translated mdm2a protein had a lower apparent molecular weight than mdm2 (Figure 3a, lane 3 compared with lanes 1 and 2). The above results might be explained if translation of mdm2a cRNA begins at the proximal AUG1, stops at the UGA codon within exon a, and subsequently reinitiates at a downstream AUG. Alternatively, translation of mdm2a RNA may be accomplished by internal ribosome entry to the transcript and translation initiation at an AUG codon located downstream of the UGA stop codon. These alternative mechanisms are represented schematically in Figure 3b. Both alternatives would generate an mdm2 protein product with a lower molecular weight than full length mdm2, consistent with the observed results (Figure 3a). AUG62 (codon 62 of mdm2) is the ®rst start codon downstream of the UGA stop signal and we constructed an N-terminal deletion mutant of mdm2, mdm2DN, which is predicted to initiate translation at AUG62 (Figure 3c). When translated in vitro, the truncated mutant comigrated with mdm2a suggesting that the N-terminus of the putative mdm2a protein is equivalent to AUG62 of mdm2 (Figure 3a, lanes 3 and 4). Mdm2a translation in vitro occurs through a re-initiation mechanism We next sought to discriminate between the two models presented in Figure 3b. Does translation of mdm2a start at AUG62 following internal ribosome entry? Alternatively, does translation initiation begin at AUG1 and subsequently re-initiate at AUG62 due to the presence of the stop codon in exon a? If the latter alternative applies we reasoned that removal of the stop signal would permit translational read-through of the a exon open reading frame. The resulting protein would contain 29 additional residues compared with mdm2 (since this represents the in-frame coding capacity of the a exon) and migrate slower on a polyacrylamide gel. When the in-frame UGA stop codon was mutated to CGA (alanine) the mutant mdm2a protein displayed a slightly larger apparent molecular weight than full-length mdm2 (Figure 3a, lane 5). This is consistent with translation initiation at AUG1 and read through of the a exon coding sequence. Overall, these results suggest that primary initiation of translation of mdm2a mRNA occurs at AUG1 and subsequently re-initiates at an internal AUG codon (corresponding to position 62 in the mdm2 sequence) due to the presence of the UGA stop codon in exon a. This re-initiation model is represented in Figure 3b, and the various mdm2 constructs used in the experiment are shown in Figure 3c. Exon a regulates mdm2a translation eciency Figure 2 Characterization of the a exon sequence within human and canine mdm2 genes. (a) The RNA sequence of the a exon from canine and human mdm2a transcripts. The potential amino acid sequence encoded by each a exon is shown above (canine) or below (human) the RNA. The single nucleotide dierence between the two exons is depicted by an open circle. The UGA codon within each a exon is identi®ed by an asterisk. (b) Schematic diagram showing the location of the a exon within the human and canine mdm2 genes. Approximate base pair (bp) lengths are indicated Translation eciency of an open reading frame can be down-regulated by the presence of upstream stop codons within bicistronic mRNA (for examples see Kaufman et al., 1987; Luukkonen et al., 1995). The organization of mdm2a mRNA suggests that translation eciency may be similarly aected by the UGA stop codon in exon a. This would be consistent with the lower amount of in vitro translated mdm2a protein which was routinely observed compared with mdm2 (see Figure 3a). In detailed time course studies we compared the eciency of translation of mdm2 and mdm2a mRNAs using rabbit reticulocyte lysate for Table 1 Exon-intron boundaries within the region of the human and canine mdm2 genes containing the a exon Species Exon DNA Sequence of exon-intron boundaries Exon Canine 4 canine a 4 human a AAAGAGgtaagctaaa ± ND ATAGAGgtgagctgct ± tctatttcagGTGATA AAAGAGgtaagctgaa ± tctatccaagGACTTC ATAGAGgtgagctgat ± tgtatttcagGTTCTT canine a 5 human a 5 Human Exon DNA sequence is shown in uppercase while intron DNA sequence is depicted in lowercase. Invariant dinucleotides used in RNA splicing are shaded. ND, not determined A novel mdm2 exon modulates translation N Veldhoen et al translation (Materials and methods). The rate of accumulation of radiolabelled mdm2 protein was rapid over the ®rst 10 min of translation, and began to plateau after 20 ± 30 min (Figure 4). No further increase in the rate of protein accumulation was observed after 40 min (data not shown). In contrast, radiolabelled mdm2a protein showed a threefold slower rate of accumulation within the ®rst 10 min of translation (Figure 4), with a much lower overall yield. Mutation of the UGA stop codon to GCA restored the eciency of translation of mdm2a mRNA Figure 3 Translation of canine mdm2-related proteins. (a) Migration of human and canine mdm2-related proteins on a 12% SDS-polyacrylamide gel. Protein of human origin is shown by `h' and canine proteins are identi®ed by `c'. (b) Two alternative models for the translation of mdm2a depicting a re-initiation process and internal ribosome entry to the RNA message. (c) Schematic representation of the canine cDNA constructs encoding mdm2, mdm2a, mdm2DN, and mdm2RT. Regions encoding functional protein domains are shaded. The location of translation initiation and termination codons described in the text are identi®ed to that observed for mdm2 (data not shown). We conclude that the stop codon in exon a reduces the rate of translation of mdm2a protein in vitro. Mdm2a lacks an intact p53 binding domain and fails to bind p53 Re-initiation of mdm2a translation at AUG62 would result in disruption of the p53 binding site of mdm2 (residues 15 ± 125; Chen et al., 1993; Kussie et al., 1996; BoÈttger et al., 1997; Freedman et al., 1997). To determine if this crucial binding domain is lost, we characterized the immunoprecipitation pro®le of the mdm2 and mdm2a proteins. Controls showed that human and canine mdm2 share six epitopes (Table 2), including the epitopes for antibodies 4B2 (amino acids 19 ± 50; Chen et al., 1993) and 3G5 (amino acids 66 ± 69; BoÈttger et al., 1997). These two epitopes are located within the p53 binding domain of mdm2. The mdm2a and mdm2DN proteins were recognized by all antibodies except 4B2 and 3G5 (Table 2) thus con®rming that these proteins lack an intact Nterminus and an intact p53 binding domain. To assess the interaction between mdm2a and p53 the two proteins were co-translated in vitro and analysed by co-immunoprecipitation, using PAb421 (directed towards the C-terminus of p53) and 4B11 (directed towards the C-terminus of mdm2). Canine mdm2 and the mdm2DN mutant, which lacks the ®rst 61 residues of mdm2, were included as controls. The results of the co-immunoprecipitation experiments are summarized in Table 2. Complexes between canine mdm2 and canine p53 were observed following reciprocal immunoprecipitations with PAb421 and 4B11 (Table 2). No complex formation was observed between canine p53 and mdm2DN protein (Table 2). Figure 4 Translation kinetics of mdm2-related proteins. The rate of protein accumulation from human mdm2 (closed circle), canine mdm2 (open circle), canine mdm2a (closed square), and mdm2DN (open square) are shown. Radioactive label incorporation (c.p.m.) for each protein sample was adjusted for methionine content. The rate of protein accumulation was observed to plateau after 40 min (data not shown) 7029 A novel mdm2 exon modulates translation N Veldhoen et al 7030 Table 2 Protein h mdm2 mdm2c mdm2ac mdm2DNc mdm2RTc Immunoprecipitation pro®les and p53 binding activities of mdm2-related proteins p53 protein binding 4B2 3G5 + + 7 7 ND + + 7 7 + + + 7 7 + Antibody binding 2A9 SMP14 + + + + + + + + + + 2A10 4B11 + + + + + + + + + + Comparison of antibody reactivity and p53 protein binding of mdm2-related proteins. Strong antibody binding is shown by`+',while reduced or no antibody binding is depicted by `+'and `7' respectively. Association with p53 protein is shown by `+' and no p53 binding by `7'. Human mdm2 protein labelled with an `h' and canine mdm2 proteins with a `c'. ND, not determined Co-translation of p53 with mdm2a also yielded little, if any detectable p53-mdm2a complexes (Table 2) demonstrating that incorporation of the a exon of mdm2 disrupts protein ± protein interaction between p53 and the mdm2a isoform. Mdm2a is highly expressed in leukocytes and testicular tissue Given the potential functional signi®cance of the a exon (see Discussion) we next asked if mdm2a mRNA is similarly expressed in all tissues, or is expressed in a tissue-speci®c manner. For this purpose, we investigated the expression pattern of mdm2a mRNA in a number of dierent tissue types using a nonquantitative nested RT ± PCR (Materials and methods). Controls showed that mdm2 mRNA was detectable in all tissues examined (Figure 5, upper panel). An oligonucleotide primer speci®c for the a exon was used in the second round of DNA ampli®cation to detect the presence of mdm2a mRNA. Most tissues showed little, if any mdm2a expression (Figure 5, lower panel). The exceptions were lymphoid and testicular tissues in which mdm2a transcripts were clearly detectable. These observations were extended to include normal lymphocytes from a total of seven human individuals and normal leukocytes from ®ve individual dogs: all gave results similar to those presented for canine peripheral blood leukocytes (see Figure 5, lower panel for canine leukocytes). These results indicate that mdm2a mRNA can be expressed in a tissue-speci®c manner and is present in lymphoid and testicular tissues. Discussion In the process of studying malignant transformation in the dog we have now cloned and sequenced canine p53 and mdm2 cDNAs (Veldhoen and Milner, 1998 and this paper). This systematic approach has revealed the presence of a novel `a exon' in the canine mdm2 gene and an almost identical a exon in human mdm2. The a exon exhibits a number of remarkable properties and represents a novel mechanism for the determination of protein structure by causing re-initiation of translation with consequent loss of the N-terminus of mdm2 protein. Modi®cation of protein structure and function by alternative mRNA splicing is not unusual. For Figure 5 Expression of mdm2a mRNA in dierent tissues of the dog. Ampli®ed mdm2 control and mdm2a cDNA derived from each tissue type were run on a 1% agarose gel example, expression of the alternatively spliced exon 5 of the Wilms tumour suppressor gene leads to the incorporation of an additional 17 amino acid sequence that disrupts the regulatory domain of the WT1 protein (Bruening and Pelletier, 1996). Mechanistically, however, this modi®cation of protein structure is distinct from the system we describe for mdm2a since exon 5 of WT1 (i) does not contain an in-frame stop codon and (ii) does not aect translation of the Nterminus of the protein product. A mechanism much closer to the one we describe for mdm2a is indicated for the expression of the cAMP response modulator (CREM). Two CREM transcripts, CREM 23 and CREM 24, contain a 100 bp exon (exon c) with an in-frame stop codon (Gellerson et al., 1997). The mRNA is nevertheless translated into functional CREM isoforms and, signi®cantly, both protein isoforms lack the N-terminus and appear to arise by initiation at an internal AUG. However, further studies of CREM 23 and CREM 24 are required to support a re-initiation model for translation and to determine the signi®cance of the stop codon in exon c. Thus, we believe that the mdm2a exon is the ®rst example of a mammalian exon with the capacity to `reprogram' translation initiation of protein synthesis. The remarkable conservation of the a exon suggests that it comprises a functionally important component of the mdm2 gene, and our results suggest that the a exon may regulate mdm2 expression at the level of translation. Thus, in vitro, exon a causes translation of the mdm2a transcript to stop, due to the presence of an in-frame stop codon, and then re-initiate at a down- A novel mdm2 exon modulates translation N Veldhoen et al stream AUG codon (which corresponds to position 62 in mdm2) (see Figure 3). The resulting protein product lacks the N-terminus of mdm2 and the p53 binding domain is disrupted (Table 2). The biological consequences of this eect are likely to be profound. It is well established that interaction with p53 is a vital function of mdm2, and that mdm27/7 mouse embryos are non-viable (Montes de Oca Luna et al., 1995). Therefore any mechanism which disrupts mdm2-p53 interaction must be under stringent control. This would be consistent with the observed reduced rate of translation and cell type speci®c expression of mdm2a mRNA (see Figures 3a and 5). Studies are in progress to evaluate the translation potential of mdm2a in vivo, using cells of dierent tissue types in case translational control is also cell type speci®c. The expression of the mdm2 gene has previously been shown to involve a combination of dierential mRNA splicing and internal translation initiation (Olson et al., 1993; Barak et al., 1994; Gudas et al., 1995; Kraus et al., 1999). Codons AUG50 and AUG62 within mdm2 mRNA can be used as sites of translation initiation (Barak et al., 1994; Saucedo et al., 1999). However, the precise factors that modulate selection of these internal translation start sites remains poorly de®ned. In the case of the alternatively spliced mdm2a transcript, the presence of the a exon may serve to redirect translation initiation to AUG62 (see Figure 3). Mdm2 proteins derived from these internal initiation events, such as the mdm2a isoform, lack a functional p53 binding domain and may be involved in p53independent activities within the cell (Saucedo et al., 1999). For example, N-terminally truncated mdm2 protein can rescue TGFb-induced growth arrest by aecting the Rb/E2F pathway (Sun et al., 1998). Thus, expression of the mdm2a protein isoform could serve as a proliferative signal in certain normal cell types. Preliminary results using speci®c primers to compare mdm2 and mdm2a mRNA levels in normal canine testicular tissue indicate that mdm2a mRNA is approximately an order of magnitude lower than mdm2. Interestingly, in canine testicular tumour mdm2 expression was two orders of magnitude higher than mdm2a (Watterson and Milner, unpublished observations). This would be consistent with abnormal down-regulation of p53 (by mdm2) in the tumour cells whilst retaining putative proliferative functions of mdm2a. Tissue-speci®c expression of mdm2a mRNA in normal testicular tissue and peripheral blood lymphocytes is intriguing. Several lines of evidence indicate that p53 may function in the maturation of spermatocytes and of lymphoid cells (Rotter et al., 1993, 1994; Shick et al., 1997). Moreover there is evidence that spatial and cyclical expression of p53 in the testis plays a role in the meiotic process of spermatogenesis (Almon et al., 1993). In order to allow cell-type speci®c functions it may be necessary to uncouple p53 from mdm2-mediated nuclear export and degradation. This could be achieved by expression of mdm2a mRNA since it contains the a exon and would encode mdm2 protein products unable to associate with p53. Such a model might explain the high levels of mdm2a mRNA observed in testicular and lymphoid cells compared with other cell types. In addition, mdm2a protein may have intrinsic functions speci®c to these cell types (see above). These considerations lead us to suggest that the a exon of mdm2 may represent an important determinant of mdm2 function in normal testicular and lymphoid cells. Materials and methods RNA isolation and RT ± PCR ampli®cation of mdm2 cDNA sequences Total RNA was prepared from human blood lymphocytes and various canine tissues using the RNeasy kit as per the manufacturer's protocol (Qiagen). In some cases, messenger RNA was further puri®ed using the Oligotex mRNA kit as per the manufacturer's protocol (Qiagen). Ampli®cation of mdm2 cDNA by reverse transcriptase-PCR (RT ± PCR) from human and canine total RNA was carried out using the degenerate mdm2-speci®c primers 5'MDM (5'-GCGGTACCAGGCM AATGTGCAATACCAAC-3') and 3'MDM (5'GCGAATTCAGGTCARCTAGKK GAARTAASTTAG-3') and the Access RT ± PCR kit as per the manufacturer's protocol (Promega). Cloning of mdm2 cDNA sequences from human and canine tissues Human peripheral blood lymphocytes were obtained from blood samples from healthy volunteers. Samples were diluted 1 : 1 in sterile PBS and lymphocytes puri®ed on Ficoll according to standard methods. Canine leukocytes were obtained from pre-operative blood samples and prepared as above. The fractionated lymphoid cells were cryo-preserved in 10% DMSO, 50% FCS and 40% RPMI until required. Solid tissues of canine origin were obtained during routine surgical resection of tumour mass and normal tissue surrounding the tumour material. Canine tissue was stored at 7808C until required for RNA extraction. Mdm2 cDNA ampli®ed from human or canine mRNA was cut with KpnI and EcoRI and ligated into the same sites in pBluescript SK+. The plasmid pHDM2 contained human mdm2 cDNA isolated from blood lymphocytes, the plasmid pCDM2 contained canine mdm2 cDNA derived from a benign mammary tumour, and the plasmid pCDM2a contained the mdm2a cDNA isolated from canine peripheral blood leukocytes. Two canine mdm2a mutants were generated by DNA ampli®cation and cloning. A deletion mutant lacking the ®rst 90 codons, called mdm2DN (plasmid pCDM2DN), was constructed using the primers DH/Cup (5'GGCGGTACCAG TATATTATGACTAAACG-3') and 3'CDM (5'-CGCGGAATTCAGGTCAAC TAGGGGAAATAAGTTAG-3') and cloned into the KpnI and EcoRI sites of pBluescript SK+. A `read-through' mutant, called mdm2RT (plasmid pCDM2RT), was constructed using the primers CDM2RT-(5'-GGCGGATCCCAGGTTAGAACTTCT-CACTAGAGATACAGCAGTACAGTATAC-3') and 3'CDM which contained an alanine codon substitution at UGA71 in mdm2a. Plasmid pK9 contains canine p53 cDNA cloned into pLitmus 29 (Veldhoen and Milner, 1998). All cDNA constructs were con®rmed by manual DNA sequencing using a T7 DNA polymerase-based sequencing kit as per the manufacturer's protocol (Pharmacia). Plasmids were maintained in E. coli XL-1 Blue MRF'. Identi®cation of exon-intron boundaries ¯anking the a exon within mdm2 DNA ampli®cation was performed using genomic DNA isolated from human and canine blood. The canine primer pairs included 98.14 (5'-GGCGGTACCAAAAAG ACACT- 7031 A novel mdm2 exon modulates translation N Veldhoen et al 7032 TATACTATG-3')/cMDM2ad n (5'-GGGATCCATGGATGCCCAAGAAGTC-3') and cMDM2a u p (5'-GGGCATCCATGGATCCCAGGTTAAG-3')/98.10 (5'-GGCAGAATTCGTTTAGTCATAATATACTGG-3'). The human primer pairs included 98.14/hMDM2adn (5'-TTCCTGGGATCCAGGGATGCCCAAG-3') and hMDM2aup (5'-GGGCATCCCTGGATCCCAGGTTAAG-3')/98.10. The 30 ml amplification reactions contained 16PC2 buer (50 mM Tris-HCl (pH 9.1), 16 mM ammonium sulphate, 3.5 mM MgCl2, and 150 mg/ml BSA, 0.25 mM of each primer, 166 mM dNTPs (dATP, dCTP, dGTP, dTTP), 250 ng genomic DNA, and 12.5 units Taq Supreme (Helena Biosciences). The thermocycle program included a denaturation step at 948C (7 min), 40 cycles of 948C (60 s), 588C (60 s), and 688C (3 min) and a ®nal elongation step at 688C (7 min). Ampli®ed DNA products were cloned into pBluescript SK+ and manually sequenced using a T7 DNA polymerase-based sequencing kit (Pharmacia). Detection of mdm2a mRNA expression in dierent tissues Mdm2a expression within various canine tissues was assessed by performing mdm2-speci®c RT ± PCR on total RNA or puri®ed mRNA, as detailed above, followed by secondary nested PCR using the primer 98.4 (5'-GGCGGTACCAATGA AAGAGGACTTCTTGGG-3') and the degenerate primer IIDN (5'-CCAGGCCTYACG AAGGGYCCARCATCTNTTRCA-3'). Mdm2 control reactions were performed using primers 98.5 (5'-TATTATGACTAAACGATTG-3') and IIDN. The 20 ml reactions contained 16PC2 buer, 0.25 mM of each primer, 200 mM dNTPs (dATP, dCTP, dGTP, dTTP), 1 ml initial RT ± PCR sample, and 0.05 units Taq Supreme (Helena Biosciences). The thermocycle program included a denaturation step at 948C (7 min), 35 cycles of 948C (30 s), 588C (30 s), and 688C (30 s) and a ®nal elongation step at 688C (7 min). Expression of mdm2 proteins in vitro Plasmids pHDM2, pCDM2, pCDM2a, pCDM2DN, pCDM2RT, and pK9 were linearized with EcoRI. Each 50 ml transcription reaction contained 40 mM Tris-HCl pH 7.5, 6 mM MgCl2, 2 mM spermidine, 10 mM dithiothreitol, 20 units RNasin (Promega), 0.5 mM each ATP, CTP and UTP, 0.025 mM GTP, 2 mg template DNA, 0.5 mM m7GpppG, and 40 units T7 RNA polymerase (Promega). Transcription was allowed to proceed at 378C for 30 min and GTP was added to a ®nal concentration of 1 mM. A further incubation of 60 min at 378C was performed followed by phenol:chloroform extraction, ethanol precipitation, and resuspension in 40 ml of water. Further puri®cation of mRNA transcripts was performed using the RNeasy kit as per the manufacturer's protocol (Qiagen). Human and canine mdm2 proteins and canine p53 protein were translated in a rabbit reticulocyte lysate system (Promega) as described previously by Gamble and Milner (1998) and Veldhoen and Milner (1998). The rate and eciency of translation was determined by TCA precipitation of 2 ml of each reaction on glass ®lters followed by scintillation counting. Translated protein products were separated on a 12% SDS-polyacrylamide gel and visualized by autoradiography with Fuji RX ®lm at room temperature. Immunoprecipitation Protein conformation was determined using an immunoprecipitation method as described by Cook and Milner (1990). The following anti-mdm2 antibodies were used; 10 ml hybridoma supernatants containing antibodies 4B2, 3G5, 2A9, 2A10, and 4B11 and 1 ml puri®ed SMP14 monoclonal antibody (Oncogene Science). Antibody PAb416 (30 ml), directed towards the large T-antigen of SV40, was used as a negative control. For co-immunoprecipitation experiments, canine p53 protein and canine and human mdm2 proteins were co-expressed in rabbit reticulocyte lysate. Complexes were immunoprecipitated using antibody 4B11 directed to mdm2-related proteins or antibody 421 directed towards canine p53 protein. Immunoprecipitated proteins were resolved by 15% SDS-polyacrylamide gel electrophoresis and visualized by autoradiography with Fuji RX ®lm at room temperature. Acknowledgements We thank Arnold Levine and Stephen Picksley for making available the anti-mdm2 antibodies 4B2, 3G5, 2A9, 2A10, and 4B11. We are also indebted to Andrei Okorokov and Ming Jiang, YCR p53 Research Group, for many helpful discussions. Photographic work was prepared by Meg Stark. This work was supported by a grant from Yorkshire Cancer Research to J Milner. References Almon E, Gold®nger N, Kapon A, Schwartz D, Levine AJ and Rotter V. (1993). Dev. Biol., 156, 107 ± 116. Barak Y, Gottlieb E, Juven-Gershon T and Oren M. (1994). Genes Dev., 8, 1739 ± 1749. BoÈttger A, BoÈttger V, Garcia-Echeverria C, CheÁne P, Hochkeppel H-K, Sampson W, Ang K, Howard SF, Picksley SM and Lane DP. (1997). J. Mol. Biol., 269, 744 ± 756. Bruening W and Pelletier J. (1996). J. Biol. Chem., 271, 8646 ± 8654. Cahilly-Snyder L, Yang-Feng T, Francke U and George DL. (1987). Somat. Cell Mol. Genet., 13, 235 ± 244. Chen J, MareÂchal V and Levine AJ. (1993). Mol. Cell. Biol., 13, 4107 ± 4114. Cook A and Milner J. (1990). Br. J. Cancer, 61, 548 ± 552. Elenbaas B, Dobbelstein M, Roth J, Shenk T and Levine AJ. (1996). Mol. Med., 2, 439 ± 451. Fakharzadeh SS, Trusko SP and George DL. (1991). EMBO J., 10, 1565 ± 1569. Freedman DA, Epstein CB, Roth JC and Levine AJ. (1997). Mol. Med., 3, 248 ± 259. Gamble J and Milner J. (1988). Virology, 162, 452 ± 458. Gellersen B, Kempf R and Telgmann R. (1997). Mol. Endocrin., 11, 97 ± 113. Gudas JM, Nguyen H, Klein RC, Katayose D, Seth P and Cowan KH. (1995). Clin. Cancer Res., 1, 71 ± 80. Haines DS. (1997). Leuk. Lymph., 26, 227 ± 238. Haines DS, Landers JE, Engle LJ and George DL. (1994). Mol. Cell. Biol., 41, 1171 ± 1178. Haupt Y, Maya R, Kazaz A and Oren M. (1997). Nature, 387, 296 ± 299. Honda R, Tanaka H and Yasuda H. (1997). FEBS Lett., 420, 25 ± 27. Honda R and Yasuda H. (1999). EMBO J., 18, 22 ± 27. Jones SN, Ansari-Lari MA, Hancock AR, Jones WJ, Gibbs RA, Donehower LA and Bradley A. (1996). Gene, 175, 209 ± 213. Juven-Gershon T, Shifman O, Unger T, Elkeles A, Haupt Y and Oren M. (1998). Mol. Cell. Biol., 18, 3974 ± 3982. Kaufman RJ, Murtha P and Davies MV. (1987). EMBO J., 6, 187 ± 193. A novel mdm2 exon modulates translation N Veldhoen et al Kraus A, Ne F, Behn M, Schuermann M, Muenkel K and Schlegel J. (1999). Int. J. Cancer, 80, 930 ± 934. Kubbatat MHG and Vousden KH. (1998). Mol. Med., 4, 250 ± 256. Kussie PH, Gorina S, MareÂchal V, Elenbaas B, Moreau J, Levine AJ and Pavletich NP. (1996). Science, 274, 948 ± 953. Leng P, Brown DR, Shivakumar CV, Deb S and Deb SP. (1995). Oncogene, 10, 1275 ± 1282. Lundgren K, Montes de Oca Luna R, McNeill YB, Emerick EP, Spencer B, Bar®eld CR, Lozano G, Rosenberg MP and Finlay CA. (1997). Genes Dev., 11, 714 ± 725. Luukkonen BG, Tan W and Schwartz S. (1995). J. Virol., 69, 4086 ± 4094. MareÂchal V, Elenbaas B, Piette J, Nicolas JC and Levine AJ. (1994). Mol. Cell. Biol., 14, 7414 ± 7420. MareÂchal V, Elenbaas B, Taneyhill L, Piette J, Mechali M, Nicolas J-C, Levine AJ and Moreau J. (1997). Oncogene, 14, 1427 ± 1433. Martin K, Trouche D, Hagemeier C, Sùrenson TS, La Thangue NB and Kouzarides T. (1995). Nature, 375, 691 ± 694. Matsumoto R, Tada M, Nozaki M, Zhang CL, Sawamura Y and Abe H. (1998). Cancer Res., 58, 609 ± 613. Maxwell SA. (1994). Anticancer Res., 14, 2541 ± 2548. McCaughan KK, Brown CM, Dalphin ME, Berry MJ and Tate WP. (1995). Proc. Natl. Acad. Sci. USA, 92, 5431 ± 5435. Momand J, Jung D, Wilczynski S and Niland J. (1998). Nucl. Acids Res., 26, 3453 ± 3459. Momand J and Zambetti GP. (1997). J. Cell. Biochem., 64, 343 ± 352. Momand J, Zambetti GP, Olson DC, George D and Levine AJ. (1992). Cell, 69, 1237 ± 1245. Montes de Oca Luna R, Tabor AD, Eberspaecher H, Hulboy DL, Worth LL, Colman MS, Finlay CA and Lozano G. (1996). Genomics, 33, 352 ± 357. Montes de Oca Luna R, Wagner DS and Lozano G. (1995). Nature, 378, 203 ± 206. Oliner JD, Kinzler KW, Meltzer PS, George DL and Vogelstein B. (1992). Nature, 358, 80 ± 83. Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW and Vogelstein B. (1993). Nature, 362, 857 ± 860. Olson DC, Marechal V, Momand J, Chen J, Romocki C and Levine AJ. (1993). Oncogene, 8, 2353 ± 2360. Padgett RA, Grabowski PJ, Konarska MM, Seiler S and Sharp PA. (1986). Annu. Rev. Biochem., 55, 1119 ± 1150. Piette J, Neel H and MareÂchal V. (1997). Oncogene, 15, 1001 ± 1010. Roth R, Dobbelstein M, Freedman DA, Shenk T and Levine AJ. (1998). EMBO J., 17, 554 ± 564. Rotter V, Aloni-Grinstein R, Schwartz D, Elkind NB, Simons A, Wolkowicz R, Lavigne M, Beserman P, Kapon A and Gold®nger N. (1994). Semin. Cancer Biol., 5, 229 ± 236. Rotter V, Schwartz D, Almon E, Gold®nger N, Kapon A, Meshorer A, Donehower LA and Levine LA. (1993). Proc. Natl. Acad. Sci. USA, 90, 9075 ± 9079. Saucedo LJ, Myers CD and Perry ME. (1999). J. Biol. Chem., 274, 8161 ± 8168. Sigalas I, Calvert AH, Anderson JJ, Neal DE and Lunec J. (1996). Nat. Med., 2, 912 ± 917. Shick L, Carman JH, Choi JK, Somasundaram K, Burrell M, Hill DE, Zeng Y-X, Wang Y, Wiman KG, SalhanyK, Kadesch TR, Monroe JG, Donehower LA and El-Deiry WS. (1997). Cell Growth Di., 8, 121 ± 131. Sun P, Dong P, Dai K, Hannon GJ and Beach D. (1998). Science, 282, 2270 ± 2272. Veldhoen N and Milner J. (1998). Oncogene, 16, 1077 ± 1084. Xiao Z-X, Chen J, Levine AJ, Modjtahedi N, Xing J, Sellers WR and Livingston DM. (1995). Nature, 375, 694 ± 698. 7033