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Proc Natl Acad Sci U S A. Jan 7, 2003; 100(1): 211–216.
Published online Dec 27, 2002. doi:  10.1073/pnas.0135557100
PMCID: PMC140929
Medical Sciences

Absence of p16INK4a and truncation of ARF tumor suppressors in chickens

Abstract

The INK4b-ARF-INK4a locus on human chromosome 9p21 (Human Genome Organization designation CDKN2B-CDKN2A), and the corresponding locus on mouse chromosome 4, encodes three distinct products: two members of the INK4 cyclin-dependent kinase inhibitor family and a completely unrelated protein, ARF, whose carboxyl-terminal half is specified by the second exon of INK4a but in an alternative reading frame. As INK4 proteins block the phosphorylation of the retinoblastoma gene product and ARF protects p53 from degradation, the locus plays a key role in tumor suppression and the control of cell proliferation. To gain further insights into the relative importance of INK4a and ARF in different settings, we have isolated and characterized the equivalent locus in chickens. Surprisingly, although we identified orthologues of INK4b and ARF, chickens do not encode an equivalent of INK4a. Moreover, the reading frame for chicken ARF does not extend into exon 2, because splicing occurs in a different register to that used in mammals. The resultant 60-aa product nevertheless shares functional attributes with its mammalian counterparts. As well as indicating that the locus has been subject to dynamic evolutionary pressures, these unexpected findings suggest that in chickens, the tumor-suppressor functions of INK4a have been compensated for by other genes.

The INK4a/ARF locus has an important role in the control of cell proliferation and in tumor suppression (1, 2) and is incapacitated in a variety of familial and sporadic cancers (3, 4). The INK4a product, p16INK4a, functions as an inhibitor of cyclin-dependent kinases (Cdks) 4 and 6 (5), hence the official designation CDKN2A. These Cdks, along with D-type cyclins, regulate the phosphorylation of the retinoblastoma protein (pRb) in the late G1 phase of the cell cycle (6). Interestingly, p16INK4a is the prototype of a family of INK4 proteins, each comprising between 3 and 5 ankyrin repeats (3, 4), orthologues of which have been identified in a variety of mammals as well as in Fugu and Xiphophorus fish (7, 8). All members of the gene family isolated thus far show the same exon 1–exon 2 splice junction and, in mammals, INK4b and INK4a occur in a conserved tandem arrangement on human chromosome 9p21 and syntenic regions on mouse chromosome 4 and rat chromosome 5, suggesting that they evolved by a gene-duplication event (3). Because there is no evidence for such a duplication in Fugu (8), it appears that INK4b was the primordial gene at this locus.

However, the INK4a locus has the highly unusual capacity to encode two structurally and functionally different proteins. Two transcripts, designated α and β, are produced; they initiate at separate promoters and incorporate different first exons (1α and 1β) spliced to a common second exon (1, 2). Whereas the α-transcript specifies p16INK4a, the β-transcript encodes p14ARF (p19ARF in mouse), so-called because the second exon is translated in the −1 (alternative) reading frame to that used to generate p16INK4a. As exon 1β is poorly conserved and has no obvious relatives in the current databases, its evolutionary origins remain unknown. For example, did exon 1β originally belong to a different gene and move to the INK4a/b locus at some point after the INK4a/b duplication?

Whereas INK4a operates upstream of pRb (3, 4), the ARF protein functions upstream of p53 by binding directly to MDM2 and protecting p53 from MDM2-mediated degradation (1, 2). Current thinking is that the INK4a/ARF locus plays a key role in cellular defenses against hyperproliferative signals and stress. For example, INK4a accumulates in human diploid fibroblasts (HDFs) that undergo replicative senescence, either as a consequence of telomere attrition or in response to oncogenic Ras (911). Similarly, ARF accumulates as mouse embryo fibroblasts (MEFs) approach their replicative limits and in response to a variety of oncogenes (1214). However, there are clear differences in the relative importance of INK4a and ARF in cells from different lineages or species (1417) and in the way they are regulated. For example, Ras induces ARF in MEFs but not in HDFs (15, 18, 19), whereas pRb represses INK4a in HDFs but not in MEFs (10, 20). There have also been suggestions that the sequences encoded by exon 2 make different contributions to the intracellular localization and function of ARF in the two species (21, 22).

To gain further insight into these questions, we sought to isolate the equivalent locus from chicken, both because it represents an intermediate between fish and man in evolutionary terms and because of the relative resistance of chicken cells to immortalization in tissue culture, similar to HDFs. After characterizing 18 kb of genomic DNA and two groups of cDNA clones from late-passage chicken embryo fibroblasts (CEFs), we conclude that the chicken INK4b-ARF-INK4a locus is able to encode an equivalent of p15INK4b and a truncated yet functional version of ARF specified only by exon 1β. Surprisingly, a partial duplication of exon 1β has replaced exon 1α, and we find no evidence that chicken cells contain a p16INK4a orthologue.

Materials and Methods

Cells.

Primary CEFs were grown at 37°C in DMEM supplemented with heat-treated 10% (vol/vol) FCS and 2% chicken serum (GIBCO/BRL). The U20S human osteosarcoma cell line and the NARF-2 derivative line in which human p14ARF is expressed from an isopropyl-β-d-thiogalactoside (IPTG)-regulated promoter were cultured as described (23). Cells were transiently transfected by calcium phosphate precipitation and harvested after 48 h. Retroviral infection of the TIG3 strain of HDFs expressing the ecotropic virus receptor was as described (24).

Bacterial Artificial Chromosome (BAC) and cDNA Libraries.

The chicken BAC library was obtained from the UK Human Genome Mapping Project Resource Center. The cDNA library was constructed from CEFs at ≈50 population doublings by using the λZAP-II cDNA synthesis kit according to the manufacturer's protocols (Stratagene). After screening of 1.35 × 106 recombinant phages (unamplified), 170 plaques showed positive signal, of which 50 were purified and analyzed further.

DNA and RNA Analyses.

CEF genomic DNA was isolated by using the Easy-DNA kit (Invitrogen) and analyzed by Southern hybridization performed at either 60°C (normal stringency) or 55°C (low stringency) following standard procedures (25). Total and polyadenylated RNA was prepared from late passage CEFs by using the RNeasy kit (Qiagen, Chatsworth, CA) and the Poly(A) Pure kit (Ambion, Austin, TX), respectively. RNA blots were hybridized at 42°C in ULTRAhyb buffer (Ambion) and washed according to the manufacturer's protocol. The PCR primer sequences used to generate the probes described in Fig. Fig.22A and the conditions for the touch-down RT-PCR of methylthioadenosine phosphorylase gene (MTAP) are detailed in Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org.

Figure 2
Northern and Southern blotting of chicken INK4/ARF. (A) The structure of full-length chicken INK4b and ARF cDNAs are shown schematically with relevant nucleotide numbers. The arrows indicate splice junctions. The shaded regions depict the respective ...

Plasmid Construction.

The longest chicken ARF cDNA was used as a template for PCR by using primers that introduced a BglII site at the presumed 5′ end of chicken ARF (excluding the ATG) and an XbaI site downstream of potential termination codons in all three reading frames of exon 2 (nucleotides 150–816 in the sequence deposited under accession no. AY138245). The resultant 683-bp product was cloned into the pEGFP-C3 vector (CLONTECH) to create an in-frame fusion protein with GFP at its N terminus, designated GFP-ARFCh. A second plasmid was generated comprising nucleotides 147–326 of chicken ARF cDNA flanked by a 5′ BglII site and a 3′ KpnI site. The 196-bp PCR product was cloned into the pEGFP-N1 vector (CLONTECH) in which the initiation codon for the GFP has been eliminated, as described (26). This procedure created a fusion protein, designated Ex1βCh-GFP, in which the 60 amino acids encoded by chicken exon 1β were fused in-frame to a C-terminal GFP tag. All of the recombinant constructs were validated by DNA sequencing. Expression vectors for human p53, MDM2, and a variety of human ARF-GFP fusion proteins have been described (26). The retroviruses were constructed by cloning the relevant coding domains into the pBABE-puro vector containing two tandem copies of the hemagglutinin (HA) tag (27).

DNA Sequence Analysis.

All sequence information was generated by the automated ABI PRISM 377 DNA sequencer using Big Dye Terminator Cycle Sequencing (Applied Biosystems). DYEnamic ET Terminator cycle sequencing kit (Amersham Pharmacia) was used to overcome sequencing-compression problems. Random and directional BAC sequencing was performed by Seqlab (Göttingen, Germany). Random sequences of the 15-kb XbaI fragment were generated by using the Tn7 transposon-based Genome Priming System (New England Biolabs). The computational programs used for analysis and assembly of DNA sequences were MACVECTOR V.6.5 (Oxford Molecular, Oxford, U.K.), SEQUENCHER V.4.1.2 (Gene Codes, Ann Arbor, MI), GAP and BESTFIT (Genetics Computer Group, Madison, WI).

Protein Analyses.

Cell lysate preparation, immunoblotting, and immunoprecipitation were performed as described (26). The antibodies used for immunoblotting were 3E1 for GFP, SMP14 for MDM2 (MS-291, NeoMarkers, Union City, CA), DO-1 for p53 (sc-126, Santa Cruz Biotechnology), DCS35 for Cdk4 (MS-299, NeoMarkers), K6.83 for Cdk6, (MS-398, NeoMarkers), and F-7 for HA (sc-7392, Santa Cruz Biotechnology). For immunoprecipitation, we used SMP14 for MDM2, Y-11 for HA-tagged proteins (sc-805, Santa Cruz Biotechnology), and H-22 for Cdk4 (sc-601, Santa Cruz Biotechnology). Methods used to visualize human ARF and the various GFP fusion proteins by direct and indirect immunofluorescence, and to assay p53 stabilization by ARF, were described (26).

Results

Isolation and Characterization of a BAC Containing the Chicken INK4a/b Locus.

Repeated efforts to identify a chicken orthologue of p16INK4a by low stringency hybridization of genomic libraries or by PCR amplification with degenerate primers were unsuccessful, even using conditions and primers that allowed the isolation of INK4 orthologues from fish (7, 8). Therefore, we exploited the close linkage between INK4a and the evolutionarily conserved MTAP. In humans, MTAP is ≈100–150 kb distal to INK4a in the opposite transcriptional orientation (2830), and the same organization is preserved in other species (Fig. (Fig.11A) including Fugu, where there is no tandem duplication of INK4a/b (8). By using degenerate primers, based on the consensus sequence of human, mouse, and Xenopus MTAP, we isolated a 163-bp RT-PCR product whose sequence showed 85% identity with human MTAP at the amino acid level. Further details of the chicken MTAP gene will be described elsewhere (M.M., S.-H.K., G.P., and J. Sgouros, unpublished work).

Figure 1
Organization of the INK4b/ARF/INK4a locus in different species. (A) Schematic representations (not to scale) of the INK4b, ARF, INK4a, and MTAP genes in Fugu, humans, and chickens. INK4b exons are shown as black, ARF as cross-hatched, ...

When this PCR product was used to screen a BAC genomic library, prepared from white leghorn chicken blood DNA, a single positive clone was identified (91-M20). On further analysis, the BAC was shown to include specific restriction fragments that hybridized, albeit at low stringency, to a human p16INK4a cDNA probe. The organization of the chicken genomic locus is depicted schematically in Fig. Fig.11A and as a restriction map in Fig. Fig.11B. Importantly, two different NotI fragments, 0.3 and 2.3 kb in size (Fig. (Fig.11B), contained sequences with strong similarity to exon 2 of human and mouse INK4a/b, suggesting that the BAC included the second exons of both INK4a and INK4b. We could not, however, identify which was which because of the common exon 2-homology region.

Isolation and Characterization of cDNAs from the Chicken INK4a/b Locus.

To facilitate the characterization of the genomic locus, a 1.6-kb XhoI fragment encompassing one of the presumptive INK4 exons (Fig. (Fig.11B) was used to probe a cDNA library constructed from late passage CEFs. Two major groups of cDNAs (with 13 and 22 members, respectively) were identified based on the unique sequences flanking the common exon 2-homology region. The longest cDNA from the first group (accession no. AY138247) was 1,899 bp, with an ORF capable of encoding a 139-aa protein (discussed in more detail below). BLASTX searches indicated strong similarity to human INK4a and INK4b (E values 8e-32 and 4e-33, respectively). The longest cDNA from the second group (accession no. AY138245) was 1,523 bp, and notional translation showed no evidence for sequences resembling the amino terminal region (encoded by exon 1) of either INK4a or INK4b. Although BLASTX searches also failed to reveal similarity to ARF, there was a presumptive initiation codon and reading frame that had some of the hallmarks of exon 1β (discussed in more detail later). Therefore, we suspected and subsequently confirmed that most of the cDNAs in this second group corresponded to the chicken equivalent of the β-transcript.

Based on the mammalian model, the α- and β-transcripts should have the same 3′ UTR, yet the two families of chicken cDNAs had different 3′ UTRs, implying that the first group must correspond to INK4b. As the cDNA library yielded multiple full-length clones, it was puzzling that we did not find a cDNA capable of encoding INK4a. A trivial explanation would be that the reverse transcripts simply failed to read into exon 1α, although 12 of the 22 cDNAs in this group did read into exon 1β. Alternatively, the relevant RNA may not have been present in late-passage CEFs. To distinguish between these possibilities, selected regions of the cDNAs were used to probe a Northern blot of CEF RNA. A probe containing the exon 2 sequences common to both cDNAs (the 1.6-kb XhoI fragment shown in Fig. Fig.11B) identified two prominent transcripts of ≈2.0 and 1.6 kb (Fig. (Fig.22B, lane 1). Allowing for polyadenylation, these sizes would be in agreement with the longest cDNAs, implying that the latter were full-length clones. Significantly, probes representing the presumptive first exon and 3′ UTR of chicken INK4b detected the same 2.0-kb RNA (Fig. (Fig.22B, lanes 2 and 3), whereas probes representing the presumptive exon 1β and 3′ UTR of ARF detected the 1.6-kb RNA (Fig. (Fig.22B, lanes 4 and 5). With the caveat that INK4a and ARF transcripts could be similar in size, the data are more consistent with the idea that CEFs do not express an RNA that encodes INK4a.

Absence of INK4a Exon 1α in the Chicken Genome.

In the anticipation that we would locate the elusive first exon of INK4a, we determined the sequence of the entire genomic locus. Over 1,500 random sequences were generated from two independent sources. The 15-kb XbaI fragment that was known to encompass both second exons (Fig. (Fig.11B) was subjected to random transposon insertions (see Materials and Methods). We also contracted Seqlab (Göttingen, Germany) to conduct random sequencing of the entire BAC. The locus contains extensive regions of repetitive sequence as well as duplications of unique sequence but, by combining the information from the cDNAs and further directional sequencing, we were able to assemble a single contig of 18 kb, representing the entire INK4b-ARF-INK4a locus (accession no. AY138246). A restriction map of this region is depicted in Fig. Fig.11B, together with the positions of the exon–intron boundaries.

Two surprising features emerged from these data. First, we only found a first exon for chicken INK4b, not INK4a. This result would explain the absence of a recognizable INK4a transcript. The second feature was a duplication of the genomic sequences within and adjacent to the putative exon 1β of ARF. As depicted in Fig. Fig.11B (and Fig. 6A, which is published as supporting information on the PNAS web site), there is a repeat of two blocks of almost identical sequence. Interestingly, this duplication of exon 1β-like sequences occurs in the region in which we expected to find exon 1α of INK4a.

As the characterization of the genomic locus relied on a single BAC clone, we performed comparative Southern blot analysis of the BAC and CEF DNA by using probes described in Fig. Fig.22A. With most probes, the BAC and cellular DNA gave identical results (data not shown). Importantly, probe 4, encompassing exon 1β, detected two sets of restriction fragments, one corresponding to the bona fide exon and the other derived from the duplicated region (Fig. (Fig.22C). The different hybridization intensity reflects the fact that the distal copy has less extensive homology with the probe. We suspect, but have not proved, that odd additional bands in the genomic DNA (e.g., the SfiI fragments in Fig. Fig.22C) or slight differences in size reflect polymorphisms between the BAC (haploid) and the CEF (diploid) DNAs. Importantly, we have no reason to suspect gross rearrangements or deletions in the BAC that could account for the absence of exon 1α. The chicken exon 1β probe also detected two sets of restriction fragments in quail cell DNA (data not shown), indicating that the duplication of exon 1β−like sequences and concomitant loss of exon 1α may pertain in other avian species.

The Chicken INK4b Protein Binds Cdk4 and Cdk6 and Causes Cell-Cycle Arrest.

The 2.0-kb cDNA is capable of encoding a 139-aa protein, with a predicted molecular weight of 14,504 Da and a pI of 11.3. The alignment of the amino acid sequence with those of human and mouse INK4b and INK4a is shown in Fig. Fig.33A. A phylogenetic tree of all known INK4 proteins places chicken INK4b between the mammalian INK4a and INK4b proteins and the more primitive INK4b orthologue in fishes, but clearly distinct from INK4c and INK4d (Fig. (Fig.33B).

Figure 3
Sequence and functional evaluation of chicken INK4b. (A) Amino acid sequence alignment of chicken p15INK4b with mouse and human INK4a and INK4b. The vertical lines delineate ankyrin repeats, and the asterisks identify conserved residues. (B) Phylogenetic ...

To confirm that the chicken INK4b product is functional, the coding domain was transferred into a retrovirus vector containing an amino-terminal 2xHA tag and transduced into HDFs. A similarly tagged version of human p16INK4a and the empty vector provided positive and negative controls. After drug selection, cell lysates were subjected to immunoprecipitation with an anti-HA antibody. As shown in Fig. Fig.33C human Cdk4 and Cdk6 both coprecipitated with the HA-tagged chicken p15INK4b as well as with human p16INK4a. In reciprocal immunoprecipitations, Cdk4 antiserum coprecipitated chicken p15INK4b (not shown). To assess the effect of chicken INK4b on the cell cycle, the drug-resistant cell pools were labeled for 2 h with 5 mM BrdUrd at day 6 after infection, and the proportion of BrdUrd+ cells was determined by immunohistochemistry. Both chicken INK4b and human INK4a caused a substantial inhibition of cell proliferation, as judged by the reduced incorporation of BrdUrd (Fig. (Fig.33D). In preliminary experiments, we have confirmed that chicken p15INK4a will also arrest CEFs (S.-H.K. and G.P., unpublished work).

The Chicken ARF Protein Does Not Have an Alternative Reading Frame.

The second cDNA, corresponding to the 1.6-kb transcript, has a methionine codon at nucleotide 147 that could serve as an initiation site for a protein of 60 amino acids and a predicted pI of 13.8 (Fig. (Fig.44A). With a molecular weight of 7,234 Da, this product would be less than half the size of mammalian ARF. In mammals, the splice from exon 1β to exon 2 enables ARF translation to continue in the −1 reading frame relative to that of p16INK4a, whereas the corresponding splice in chickens puts exon 1β in register with the +1 reading frame (Fig. (Fig.44B). As this frame specifies a stop codon at the beginning of exon 2, the chicken ARF protein is encoded entirely by exon 1β. As exon 1β sequences do not show a high degree of conservation (only 45% identity between the human and mouse), alignment of the chicken sequence with other species is not straightforward (Fig. (Fig.44A). Although there is ≈35% identity between the chicken protein and the first 64 residues of human ARF encoded by exon 1β, computer alignments identify additional matches between chicken exon 1β and human exon 2 (not shown), the significance of which is unclear.

Figure 4
(A) Amino acid sequence alignment of chicken ARF with the equivalent exon 1β regions of human, mouse, rat, and opossum proteins. (B) Schematic representation of the human and chicken INK4a/ARF transcripts with exons shown as boxes and ...

As illustrated in Fig. Fig.44 B and C, the second exon of the chicken ARF transcript retains an ORF capable of encoding 121 residues that could have formed the carboxyl terminus of an INK4a-like product, if exon 1α had existed. Also, the −1 reading frame in this exon is capable of encoding 162 amino acids that could have formed the carboxyl terminus of ARF, if splicing had occurred in the register used in mammals (Fig. (Fig.44D). Although we have confirmed the relevant sequences in multiple cDNA clones and in independent derivations of the genomic sequence, a single error in the nucleotide sequence could alter the reading frame. Therefore, we constructed two types of expression vector: one in which a GFP tag was inserted at the amino terminus of the putative ARF coding domain, and the other in which a GFP tag was inserted in-frame at the end of exon 1β (Fig. (Fig.55A). Equivalent constructs for human ARF have been described (26). After transfection of U20S human osteosarcoma cells, the respective fusion proteins were detected by immunoblotting for GFP. Whereas the human ARF-GFP fusion proteins had the expected molecular weight of ≈43 kDa in SDS/PAGE, both versions of the chicken ARF-fusion proteins were ≈36 kDa (Fig. (Fig.55B). This size would be consistent with natural termination at the end of exon 1β, as predicted from the sequence. Curiously, the fusion protein with human exon 1β has a higher mobility than does the chicken counterpart, despite having more amino acid residues. It is unclear whether this difference reflects posttranslational modification or anomalous mobility caused by the unusual amino acid composition of the proteins.

Figure 5
Functional evaluation of chicken ARF. (A) Two types of chicken ARF–GFP fusion proteins (see text) are depicted schematically. The presumed stop codon is indicated by an asterisk. (B) The various ARF fusion proteins were transiently expressed in ...

Chicken ARF Binds MDM2 and Stabilizes p53.

As the mammalian ARF proteins show distinctive nucleolar localization, it was of interest to determine the subcellular distribution of chicken ARF by using the two GFP fusion proteins. For these studies, we used a derivative of the U20S cell line (NARF) that expresses human p14ARF from an IPTG-inducible promoter. As described (23, 26), induction of human ARF in these cells results in nucleolar accumulation, here detected by immunofluorescence using the monoclonal antibody 4C6 (red signal in Fig. Fig.55C). Interestingly, whereas some of the GFP-ARFCh fusion protein was also in the nucleolus, a substantial proportion was localized in nonnucleolar bodies. A similar distribution was observed when using the chicken E1β-GFP construct (Fig. (Fig.55C), with or without induction of human ARF (not shown). As p53 has been reported to localize in so-called promyelocytic leukemia (PML) nuclear bodies in some circumstances (reviewed in ref. 31), we costained the cells with an antibody against the PML protein. There was no correlation with the ARF speckles (data not shown).

In view of its distinctive nuclear localization, we next asked whether chicken ARF could substitute for human p14ARF in functional assays. The two versions of chicken ARF (GFP-ARFCh and Ε1βCh-GFP) and the corresponding fusion proteins containing 132 or 64 residues of human ARF (GFP-ARFHu and Ε1βHu-GFP) were expressed in U20S cells along with a plasmid encoding human MDM2. Both versions of chicken ARF coimmunoprecipitated with MDM2 as effectively as human ARF (Fig. (Fig.55D). In similar cotransfection assays, MDM2 promotes the ubiquitylation and proteasome-mediated destruction of p53 (32, 33), as shown in Fig. Fig.55E, lanes 2 and 3. In this system, GFP-ARFCh was able to protect p53 from MDM2-mediated destruction (Fig. (Fig.55E, lanes 6 and 7), although not as effectively as human p14ARF (Fig. (Fig.55E, lanes 4 and 5). Thus, the 60-aa chicken ARF protein displays most if not all of the functional attributes of its mammalian counterparts.

Discussion

Our analysis of the chicken INK4/ARF locus revealed a number of unexpected features. First, the chicken locus lacks the capacity to encode p16INK4a, as we found no evidence for an INK4a transcript or exon 1α-related sequence in either BAC or CEF DNA. However, the presence of two copies of exon 2 implies that the tandem duplication of INK4a/b took place before the branch between birds and terrestrial animals, and that exon 1α was subsequently lost. The partial repeat of exon 1β and adjacent sequences exactly where we would have expected to find exon 1α suggests that these events are connected and happened quite recently in evolution. Otherwise, the distal exon 2 sequence should not have been so well conserved. In the absence of a functional INK4a gene, it will be interesting to determine whether INK4b assumes some of its functions. Chicken p15INK4b clearly has the ankyrin repeat, Cdk binding, and cell-cycle arrest properties of a typical INK4 protein (Fig. (Fig.3),3), and as the primordial INK4 gene at this locus, INK4b probably performed physiological roles that were subsequently assumed and perhaps refined by INK4a. A two exon version of INK4a, the vestiges of which remain apparent in chickens, presumably predates the three exon format adopted in mammalian genomes.

A further surprise was that the presumptive ARF protein in chickens terminates abruptly at the end of exon 1β rather than exploiting sequences from exon 2. Because there is no “alternative reading frame,” the ARF acronym is not really appropriate. However, most of the published data on mammalian ARF imply that the amino terminal region encoded by exon 1β is sufficient for the known functions of protein (22, 23, 26, 3438), and the data on chicken ARF concur. Thus, despite comprising only 60 amino acids, of which 22 are arginines, chicken ARF can interact with human MDM2 and protect human p53 from MDM2-mediated degradation (Fig. (Fig.5),5), albeit less effectively than the human p14ARF. This difference could simply reflect reduced affinity between proteins from different species. Our recent data indicate that chicken ARF will stabilize endogenous p53 in CEFs (S.-H.K. and G.P., unpublished work).

We favor the interpretation that, whether or not it was part of a more complex ancestral gene, the original exon 1β was itself capable of specifying a functional protein. Splicing into exon 2 of INK4a might simply have facilitated the production of a polyadenylated transcript. In principle, any 3′ exon would suffice, but the adjacent MTAP gene is in the opposite transcriptional orientation, and we did not find evidence for an intervening gene, such as NTp16 (39). Had the ancestral exon 1β translocated before the INK4 duplication, it is not clear that there would have been a suitable exon and polyadenylation signal available. Therefore, we suspect that exon 1β became transposed between INK4b and INK4a after the duplication. Thus, rather than chicken ARF losing the capacity to exploit exon 2, it seems more likely that mammalian ARF acquired this capacity through an alteration in the splicing register. Of course, this interpretation begs the question whether the exon 2-encoded sequences contribute significantly to ARF function in mammalian cells. Paradoxically, our attempts to clarify such issues by characterizing the chicken locus now suggest that it would be informative to conduct similar investigations in other species. In the short term, however, the data described here will enable us to develop reagents to investigate the relative roles of p15INK4b and ARF in chicken cell senescence and to draw comparisons between the regulatory networks operating in different cell types and species.

Supplementary Material

Supporting Information:

Acknowledgments

We thank John Sgouros, David Ish-Horowicz, and Mike Fried for helpful comments on the manuscript.

Abbreviations

HDF
human diploid fibroblast
CEF
chicken embryo fibroblast
BAC
bacterial artificial chromosome
MTAP
methylthioadenosine phosphorylase
Cdk
cyclin-dependent kinase
HA
hemagglutinin

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AY138245AY138247).

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