A strategy to study tyrosinase transgenes in mouse melanocytes.

BACKGROUND: A number of transgenic mice carrying different deletions in the Locus Control Region (LCR) of the mouse tyrosinase (Tyr) gene have been developed and analysed in our laboratory. We require melanocytes from these mice, to further study, at the cellular level, the effect of these deletions on the expression of the Tyr transgene, without potential interference with or from the endogenous Tyr alleles. It has been previously reported that it is possible to obtain and immortalize melanocyte cell cultures from postnatal mouse skin.
RESULTS: Here, we describe the efforts towards obtaining melanocyte cultures from our Tyr transgenic mice. We have bred our Tyr transgenic mice into Tyr c-32DSD mutant background, lacking the endogenous Tyr locus. In these conditions, we failed to obtain immortalized melanocytes. We decided to include the inactivation of the Ink4a-Arf locus to promote melanocyte immortalisation. For this purpose, we report the segregation of the Ink4a-Arf null allele from the brown (Tyrp1b) mutation in mice. Finally, we found that Ink4a-Arf +/- and Ink4a-Arf -/- melanocytes had undistinguishable tyrosine hydroxylase activities, although the latter showed reduced cellular pigmentation content.
CONCLUSION: The simultaneous presence of precise genomic deletions that include the tyrosinase locus, such as the Tyr c-32DSD allele, the Tyr transgene itself and the inactivated Ink4a-Arf locus in Tyrp1B genetic background appear as the crucial combination to perform forthcoming experiments. We cannot exclude that Ink4a-Arf mutations could affect the melanin biosynthetic pathway. Therefore, subsequent experiments with melanocytes will have to be performed in a normalized genetic background regarding the Ink4a-Arf locus.

Background

Eukaryotic genes are organised on chromosomes in units known as expression domains, that are believed to include all regulatory elements required for correct gene expression [1]. We use the mouse tyrosinase locus (Tyr) as an experimental model to study mammalian expression domains [2,3]. The mouse Tyr gene is located in chromosome 7 [4], encodes the rate-limiting enzyme in melanin biosynthesis and is tightly regulated during development, being exclusively expressed in neural crest-derived melanocytes and optic cup-derived retinal pigment epithelium (RPE) cells [5,6].

Classically, the approach used to functionally identify regulatory elements has been testing a series of DNA constructs containing different amounts of regulatory sequences in transgenic animals. Minigene Tyr constructs were able to rescue the albino phenotype of recipient animals, but displayed variability in pigmentation [7,8]. In contrast, the generation of transgenic mice with a 250 kb yeast artificial chromosome (YAC) covering the entire mouse Tyr locus completely rescued the albino phenotype, resulting in mice that were indistinguishable from agouti wild-type pigmented mice [9,10]. These results pointed to the existence of important regulatory elements, absent in previous standard constructs, such as the locus control region (LCR), identified 15 kb upstream of the mouse Tyr promoter [11,12]. The LCR is necessary to establish the proper expression pattern of the mouse tyrosinase gene. The absence of the LCR resulted in weaker pigmentation, variegated expression in the melanocytes and RPE cells and delayed retinal pigmentation in transgenic mice [13]. Moreover, two binding boxes for nuclear factors within the LCR core, known as boxes A and B, were identified by in vitro analysis [14] and, recently, have been incorporated into a new boundary activity associated within the LCR region [15]. We have generated transgenic mice with new YAC Tyr transgenes carrying a range of specific mutations within the LCR region [10].

To address a more detailed study, both at the functional and structural level (using biochemical and cellular approaches), of the role of LCR-variants in these different transgenes, a number of problems had to be solved, including: (1), the dispersed nature of melanocytes which prevented us from direct analysis of relevant tissues, such as skin, where many other unrelated cell types are found; (2), the tyrosinase albino allele (Tyr ), present in all recipient mouse strains used for the generation of transgenic mice, carrying a reported point-mutation within the coding region that results in a non-functional protein, without the transcriptional status of the locus being affected [16-18], and, [3], although it has been demonstrated that it is possible to obtain mouse melanocyte immortal cell lines from postnatal skins [19-22], often it becomes difficult to overcome the senescence period that all primary cell cultures undergo.

In this study we describe our efforts and the strategy to obtain melanocyte cell cultures from YAC Tyr transgenic mice in a genetic background lacking the endogenous mouse tyrosinase gene, and the effect of the inactivation of the Ink4a-Arf locus [23] on proliferation, senescence and tyrosinase activity of established melanocyte cell lines.

Results and discussion

Transfer of YAC Tyr transgenes from albino outbred NMRI mice (Tyr / Tyr ) to a Tyr / Tyr background

Previous Tyr transgenic mice have been generated in albino outbred NMRI mice [9,10,12]. All observed pigmentation is due to the expression of the Tyr transgene but the presence of mutant albino Tyr protein and mRNA from the host Tyr allele [16-18] could interfere in subsequent cellular, biochemical and genomic analyses. The albino 32DSD mutant mouse (Tyr ) carries a deletion of the entire Tyr locus, encompassing ~200 kb of mouse chromosome 7 [24]. Therefore, a breeding program was established between Tyr transgenic and albino 32DSD mice in order to obtain Tyr transgenes in animals lacking the endogenous Tyr alleles. Genotype analysis of the resulting mice was carried out by Southern blot. An EcoR I restriction fragment length polymorphism (RFLP) in exon 2 detected by the RFLP probe allows the identification of the transgenic Tyr allele (17 kb), derived from the mouse C3H strain, and the endogenous counterpart albino Tyr NMRI allele (12 kb) (9) (Fig. 1).

Figure 1

Transfer of YAC Southern blot analysis of representative mice obtained during the breeding program, using the RFLP [9] and p19E1 probes. Lanes: (M) 1 kb-ladder (Invitrogen), (1) YAC Tyr YRT2/∅ [9]; Tyr / Tyr , (2) YRT2/∅ ; Tyr / Tyr , (3) ∅/∅ ; Tyr / Tyr , and (4) ∅/∅ ; Tyr / Tyr . Note: ∅ indicates the absence of the transgene. Tg: YAC Tyr transgene derived signal. Tyr : endogenous albino tyrosinase locus. p19: The p19Arf E1 probe (kindly provided by M. Malumbres), detecting exon 1 of the single-copy p19arf gene, was employed as an internal control for DNA loading and comparisons.

Melanocyte primary cultures of YAC Tyr/∅ ; Tyr / Tyr

To gain further insight on a series of YAC Tyr transgenic mice carrying a range of deletions around the LCR [10,12], we decided to prepare cell lines that could be representative of these animals. Chromatin analyses cannot be done in tissue samples obtained directly from transgenic animals, due to the low number of cells expressing the Tyr gene (RPE cells and melanocytes) and the complexity of the tissues or organs containing these cells (eye and skin, respectively). In addition, the presence of the mutated, but transcriptionally active [16-18], albino Tyr locus in all transgenic mice generated to date could interfere with the interpretation and the acquisition of experimental data. To avoid this problem we mobilised the YAC Tyr transgenes to a genetic background lacking the endogenous mouse gene, as shown in Fig. 1, and then we tried to establish melanocyte cultures from these mice.

Mouse melanocyte immortal cell lines can be derived from postnatal skins [19-22]. Mouse crosses were established with parental lines to obtain pigmented pups with the desired genotype (YAC Tyr / ∅ ; Tyr / Tyr ). Genotype analysis was not necessary to distinguish between transgenic and non transgenic pups, because melanin, clearly visible in the eye and in the skin at this stage, could only derive from the YAC Tyr transgene expression. Melanocyte primary cultures from dorsal skin of heterozygous YAC Tyr transgenes in homozygous Tyr background were prepared as described [19]. Individual melanocytes and small melanocytes colonies appeared at culture day 10. The number of cells increased slowly until day 25–35, when melanocytes showed signs of senescence. Most cells died by day 85–90 and no immortal cell lines could be established (Fig. 2A–C). Similar results were obtained from all YAC Tyr derivative transgenes bred to homozygous Tyr mice.

Figure 2

The absence of at least one . Melanocytes from heterozygous YAC Tyr transgenic in homozygous Tyr background became large, flat, vacuolated and highly pigmented (A, B). No surviving melanocytes were detected in the culture dishes after the senescence step (C). Melanocytes from Ink4a-Arf +/- (G, H, I) and Ink4a-Arf -/- (J, K, L) mutant mice in a Tyrp1background are black, small, bipolar, pale and without significant sings of senescence, as compared to wild type Ink4a-Arf +/+ melanocytes (D, E, F). A: culture at day 10; B: culture at day 18; C: culture at day 48; D, G, J: cultures at day 43; E, H, K: cultures at day 57; and, F, I, L: cultures at day 82. Scale ebars in C, D, G, J = 200 μm and in A, B, E, F, H, I, K, L = 150 μm.

Mouse melanocyte primary cultures and their corresponding immortalised cell lines have been established from a number of mutant mice [25-29], although this type of cell lines can be sometimes difficult to achieve, as it may be inferred from these listed publications, in which reported cell lines have been generated by the same laboratory. A number of parameters can influence the success in obtaining immortalised melanocytes from mice. First, skins from mouse pups are used as starting material, carrying bacteria and other microorganisms that can contaminate the cultures. Second, and most important, melanocytes, as any somatic cell line in culture, undergo a senescence step previous to their immortalisation. Due to the low number or surviving cells after this senescence step, cells need to be cultured continuously during a minimum of 3–6 months to obtain an immortal cell line [19,20]. In most of the cultures we did not observe melanocytes after the senescence step (Fig. 2A–C). These results were obtained with all different primary cultures, regardless of their genotype, indicating a problem at the immortalisation step.

Segregation of the Ink4a-Arf locus from the Tyrp1 locus

It has been reported that melanocyte immortal cell lines (i.e. melan-a and melan-c) lack the p16 protein, most likely due to the lost of the Ink4a-Arf locus during the culture process [30]. Melanocytes from Ink4a-Arf (-/-) null mice proliferate exponentially without showing any signs of senescence, thus it has been proposed that the generation of melanocyte immortal cell lines in an Ink4a-Arf null background would be much easier [30]. Comparable results had been obtained before with fibroblast cultures from Ink4a-Arf homozygous mutant mice [31]. The absence of p16 leads to the inhibition in the inactivation of CDK4 and CDK6. These kinases inactivate the retinoblastoma pathway, promoting the proliferation of the cells [32,33]. Therefore, we decided to mobilise the Ink4a-Arf null allele into our YAC Tyr transgenic mice.

First, we had to remove the brown mutation co-segregating with the Ink4a-Arf null allele. The murine brown locus corresponds to the gene encoding the Tyrosinase-related protein 1 (Tyrp1), an enzyme which is also implicated in the melanin biosynthetic pathway [34]. It was originally noted that the coat colour of Ink4a-Arf null mice was paler than their wild-type littermates [23]. Indeed, biochemical evidence was presented that was highly suggestive of defective Tyrp1 activity in Ink4a-Arf null melanocytes [35]. Homozygous Tyrp1 mutant mice display less tyrosinase activity than wild type, because mutated forms of the Tyrp1 protein affect tyrosinase processing [36]. Notably, the Tyrp1 locus, is close to the Ink4a-Arf locus, in mouse chromosome 4, at a distance of 8.7 Mb (mouse ENSMBL, build 32). Most inbred laboratory strains with defective Tyrp1 activity carry the recessive brown allele, Tyrp1 that contains a single amino acid change at a critical residue in the Tyrp1 protein [37]. To test directly whether the Ink4a-Arf null allele was linked to the Tyrp1 allele, two single nucleotide polymorphisms (SNPs) that characterize the Tyrp1 allele and that can be diagnosed by subsequent restriction enzyme digestion [37] were used. In particular, a Tyrp1 -linked SNP at exon 4 eliminates a Taq I restriction site and, similarly, another Tyrp1 – linked SNP at exon 5 eliminates an Hga I restriction site. Using these two markers, we directly proved that the original Ink4a-Arf null mice were indeed homozygous for the Tyrp1 allele, thus explaining their paler coat colour (Fig. 3). The Tyrp1 recessive allele linked to the mutated Ink4a-Arf allele was probably derived from the ES cell line originally used for targeting the Ink4a-Arf locus, namely WW6 ES cells, which had a complex genetic background [38].

Figure 3

Generation of an inbred . Illustrative examples of the genotyping of the Tyrp1 locus by PCR and digestion with Taq I (A) or Hga I (B). Gel lanes correspond to: (1) Ink4a/Arf -/- ; Tyrp1 / Tyrp1 , (2) Ink4a/Arf +/- ; Tyrp1 / Tyrp1 , (3) Ink4a/Arf -/- ;Tyrp1 / Tyrp1 and (4) Ink4a/Arf -/- ; Tyrp1 / Tyrp1 .

Ink4a/Arf-null strain in a pure C57BL/6J genetic background and unlinked from the mutant Tyrp1 allele were obtained to avoid the confounding presence of the Tyrp1 allele. Ink4a-Arf +/- mice were backcrossed seven times with wild-type C57BL/6J mice, eventually yielding Ink4a-Arf +/- ; Tyrp1 / Tyrp1 mice. These mice were intercrossed to produce a number of Ink4a-Arf -/- mice that were in their majority Ink4a-Arf -/- ; Tyrp1 / Tyrp1 and, accordingly, had a brown coat. Exceptionally, one mouse (from a total population of 27 Ink4a-Arf -/- mice) was identified as an Ink4a-Arf -/- but had a black coat. This mouse turned out to represent a recombinant with an Ink4a-Arf -/- ; Tyrp1 / Tyrp1 genotype. From this animal, and after the appropriate crosses, a strain of mice in C57BL/6J background that were Ink4a-Arf -/- ; Tyrp1 / Tyrp1 was obtained (Fig. 3), and used for subsequent experiments.

Melanocyte primary cultures from Tyrp1 / Tyrp1 Ink4a-Arf mutant mice

To evaluate the effect of the inactivation of the Ink4a-Arf locus in Tyrp1 / Tyrp1 genetic background on the immortalisation of mouse melanocytes, melanocyte primary cultures from dorsal skin of Ink4a-Arf +/+, Ink4a-Arf +/-, Ink4a-Arf -/- pups in a Tyrp1 background were prepared (Fig. 2D–L), using the same described procedures [19]. Individual melanocytes and colonies appeared at day 10. The number of cells increased slowly in Ink4a-Arf +/+ (Fig. 4A) and, around day 35, melanocytes increased their size and lost their bipolar shape. Few or none Ink4a-Arf +/+ melanocytes survived the senescence step, and most of these melanocytes died by day 85–90 (Fig. 2D–F). In contrast, Ink4a-Arf +/- and Ink4a-Arf -/- kept their bipolar shape and small size, with a limited number of cells showing any sign of senescence (Fig. 2G–L). Proliferation of Ink4a-Arf -/- melanocytes was higher than in Ink4a-Arf +/- melanocytes, and both were much higher than in Ink4a-ARF +/+ melanocytes (Fig. 4A). By day 45 of culture, Ink4a-Arf -/- melanocytes entered the exponential phase of growth. In contrast, Ink4a-Arf +/- melanocytes did not show evidences of exponential growth until day 65 (Fig. 4A). This delay could be explained by LOH (loss of heterozygosity), spontaneously occurring in Ink4a-Arf +/- melanocytes and affecting the remaining wild-type allele of the Ink4a-Arf locus, a common event that is known to take place both in vitro [39] and in vivo [40]. Similar results have been reported [30] using independent mouse colonies.

Figure 4

The effect of (A) Cell growth of Ink4a-Arf -/- (open circles), Ink4a-Arf +/- (closed circles) and Ink4a-Arf +/+ (crosses) melanocytes. Each point represents one subculture step and represents the mean count of cells from three replicates. (B) Tyrosine hydroxylase activity in albino melan-c (white bars), [Ink4a-Arf +/-; Tyrp1 / Tyrp1 ] (dark-grey bars), [Ink4a-Arf -/- ; Tyrp1 / Tyrp1 ] (light-grey bars) and melan-a (black bars) cells. (C) Melanin content in the same cellular types, as in (B). Bars represent the mean value (+/- SD) from three replicates. Tyrosinase activity and melanin content were measured in melanocyte primary cultures at passage 8 (~day 70 of culture). Statistically significant differences (t-Student test) are indicated as follows: * p < 0,1; ** p < 0,01; *** p < 0,001.

Tyrosinase activity and melanin content in the melanocyte cultures from Ink4a-Arf mutant mice

A valid approach to analyse the role of the different regulatory regions of the mouse Tyr gene in the transcription of the locus and, eventually, in the amount of mature protein being made, is the measurement of the enzymatic activity of the derived Tyr protein. We measured the levels of Tyr enzymatic activity and melanin content using reported procedures [41] in melanocyte cultures, to study the influence of the presence or absence of the p16 protein in the expression of the Tyr gene. Tyrosine hydroxylase activity values from Ink4a-Arf -/- and Ink4a-Arf +/- cell extracts were undistinguishable and significantly lower to values obtained in melan-a cells (Fig. 4B). However, the quantity of melanin in the proliferative Ink4a-Arf -/- melanocytes cell cultures was significantly lower than in Ink4a-Arf +/- or melan-a cells (Fig. 4C).

Differences in melanin content could be explained by different individual cell culture response to TPA or/and CT that are present in cell culture medium. However, the observed differences in cellular pigmentation were maintained after removing CT from cell culture medium. In addition, TPA is always required as an additive to maintain cellular proliferation. Increasing the amount of TPA does not result in a parallel increase in pigmentation, opposite to what is observed with CT.

Differences in melanin content could also be explained by the control of the retinoblastoma (RB) protein by p16, and the observation that RB protein interacts with the transcription factor microphthalmia (Mift) [42], that controls the expression of the Tyr gene. Differences between Ink4a-Arf mutant cells and melan-a could be due to the presence of a number of additional alterations in this latter immortal cell line, such as the loss of expression of p16Ink4a [30]. The recent generation of mice with increased gene dosage of Ink4a-Arf will be instrumental to further investigate the influence of this locus on Tyr activity [43].

Conclusion

With all these results we can conclude that the simultaneous presence of: [1] at least one mutant allele of the Ink4a-Arf locus; [2], the Tyr mutant albino allele in homozygosis and; [3], the presence of the relevant Tyr transgene in heterozygosis, are required for the establishment and the study of immortal mouse melanocyte cultures from transgenic mice carrying Tyr constructs. Finally, we cannot exclude that Ink4a-Arf mutations could affect the melanin biosynthetic pathway. Therefore, experiments with mouse melanocytes will have to be performed in a normalized genetic background regarding the Ink4a-Arf locus.

Methods

Animals

Four types of mice were used in this study: YRT2 YAC tyrosinase heterozygous transgenic mice in an albino outbred NMRI background (YRT2/∅ ; Tyr / Tyr ) (line #1999) [9], 32DSD radiation induced albino mutant mice (Tyr / Tyr ) [24], homozygous Ink4a-Arf mutant mice (Tyr + / Tyr + ; Tyrp1 / Tyrp1 ; Ink4a-Arf -/-) in C57BL/6J genetic background [23] and wild-type pigmented C57BL/6J mice. All experiments complied with local and European legislation concerning vivisection and the experimentation and use of animals for research purposes.

Southern blot analysis

The discrimination of the endogenous tyrosinase gene from the YAC-tyrosinase transgenes was performed as previously described, using the RFLP probe, containing exon 2 of the mouse tyrosinase gene [9,12]. To obtain an endogenous internal control, membranes were co-hybridised with a single-copy mouse gene, the p19E1 probe, a EcoR I DNA fragment, 230 bp in length, containing exon 1 of the p19gene (pRSp19arfE1 plasmid, generous gift from M. Malumbres). In brief, genomic DNA was isolated from mice tail tips and prepared for southern blot as described [12]. 15–20 μg of genomic DNA were digested with EcoR I (Roche, Basel, Switzerland), fractionated by horizontal gel electrophoresis in 0,8% agarose and transferred to a Hybond-N nylon membrane (Amersham, Buckinghamshire, UK) by capillary blotting. RFLP and p19E1 DNA probes were labelled with [α32P] dCTP using the High Prime labelling kit (Roche). Membranes were hybridised in Southern hybridisation solution (0.25 M Na2HPO4 pH = 7.2, 7% SDS, 1% BSA) overnight at 65°C, washed at 65°C in 20 mM Na2HPO4 pH = 7.2, 1% SDS, 1 mM EDTA pH = 8 and resulting blots exposed for 1–3 days and scanned with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). Quantification of the hybridisation signals was performed using the ImageQuant v1.2 software (Molecular Dynamics).

Genotyping of the Ink4a-Arf and Tyrp1 loci

The Ink4a-Arf and Tyrp1 loci were genotyped by PCR as follows. The Ink4a-Arf wild-type allele was specifically detected using primers that amplify Ink4a-Arf exon 2: mp16F, 5'-ATGATGATGGGCAACGTTC-3' and mp16R, 5'-CAAATATCGCACGATGTC-3'. The Ink4a-Arf null allele, which has a neo-cassette substituting exons 2 and 3 [23], was genotyped using primers that hybridise, respectively, with the neo-cassette (oligo Neo) and with Ink4a-Arf flanking genomic sequences (oligo R1): Neo, 5'-CTATCAGGACATAGCGTTGG-3' and R1, 5'-AGTGAGAGTTTGGGGACA GAG-3'. To genotype the Tyrp1 locus, two different PCR reactions were performed to amplify, respectively, exons 4 and 5 of the Tyrp1 gene. Amplification of exon 4 was performed using primers: Tyrp1-4F, 5'-CTGCGATGTCTGCACTGATGACTT-3' and Tyrp1-4R, 5'-AGGGTATCGTACTCTTCCAAGGAT-3'. Amplification of exon 5 was performed using primers: Tyrp1-5F, 5'-ACAGCACTGAGGGTGGACCAATC-3' and Tyrp1-5R, 5'-AGGGTATCGTACTCTTCCAAGGAT-3'. After amplification, the product corresponding to exon 4 was digested with Taq I, and the product corresponding to exon 5 was digested with Hga I. Digestion mixtures were separated in standard agarose gels and visualized with ethidium bromide. The Tyrp1 mutant allele does not contain neither of the previous restriction sites, Taq I and Hga I, whereas both enzymes digest the Tyrp1 wild-type allele. PCR reactions had 3 mM MgCl2, 1% DMSO, 2.5 mM dNTPs (Epicentre, Madison, WI, USA), 20 pmol of each primer, and 0.25 μl of Taq-Gold (Applied Biosystems, Foster City, CA, USA). In addition, each reaction had approximately 100 ng of genomic DNA extracted from the tail tips. Annealing temperature was 60°C and PCR reactions were carried on for 30 amplification cycles.

Melanocyte cultures

Melanocyte cultures were prepared, essentially, as previously described [19]. Briefly, dorsal skin biopsies were obtained from pups of all investigated genotypes between +19.5 and +22.5 d.p.c. stages (postnatal P2–P3). Dorsal skin was split in 5 μg/ml trypsin (Sigma, St. Louis, MO, USA) in PBS and the epidermal layer then minced with a pair of surgical blades in 250 μg/ml trypsin and 200 μg/ml EDTA in PBS. Cells were cultured on a feeder layer of mitomycin-treated murine XB2 keratinocytes [44]. The cells were grown in RPMI-1640 medium containing 2 mM glutamine, 10% fetal calf serum, 100000 U/l penicillin, 100 mg/l streptomycin sulphate (all from Invitrogen, Carlsbad, CA, USA), 200 nM tetradecanoyl phorbol acetate (TPA) (Sigma) and 200 pM cholera toxin (CT) (Sigma), at 37°C, 95% humidity and 10% CO2 pressure. Explant cultures from different donor mice were kept separate. Passages were made when cultures became subconfluent, and melanocytes were counted at each passage. Feeder cells were added when necessary. Control melan-a and melan-c cells, derived from inbred C57BL/6J and outbred albino LAC-MF1 mice, respectively, were cultured as previously described [19,20].

Quantification of melanin and tyrosinase enzymatic activities

Melanin contents in whole cell extracts were measured by spectrophotometer essentially as described [45]. In brief, 6 × 106 cells from ~day 70 of culture were collected and homogenised in 300 μl of PBS, 100 μl of homogenate incubated for 14–16 hours at room temperature with 900 μl of 2 M NaOH, 20% DMSO and absorbance measured at 470 nm.

Tyrosinase enzymatic activities were recorded following described assays [41,46,47]. In brief, for tyrosine hydroxylase activity, 6 × 106 cells from ~day 70 of culture were collected and cell extracts were prepared in 10 mM Sodium Phosphate buffer pH = 6.8 to which Tween-20 (Igepal) was added (1% final concentration) prior the assay. Reaction volume included 10 μl of L-DOPA 250 mM, 10 μl of L-[3,5-3H]-Tyrosine mix (450 μl of L-Tyrosine 262 μM in 10 mM Sodium Phosphate buffer pH = 6.8 and 50 μl L-[3,5-3H]-Tyrosine [1 mCi/ml, 46 Ci/mmol, Amersham]), 20 μl of Sodium Phosphate buffer pH = 6.8 and 10 μl of cell extracts, was incubated for 1 hour at 37°C, and stopped by adding 450 μl trichloracetic acid (TCA) 1% (Merck, Darmstadt, Germany). A small amount of absorbing substrate (active carbon [Merck] and Celite 545 [Fluka, St. Gallen, Switzerland], 1:1) was added, mixed for 30 min at room temperature and centrifuged. Radioactivity from clear supernatants (100 μl) was measured in a β-scintillation counter (Beckmann, Fullerton, CA, USA).

Authors' contributions

AL carried out the molecular genetic, cellular, biochemical and statistical studies and drafted the manuscript, MS conceived of the segregation of ink4a-Arf from mutant brown locus, AM carried out the genotype analysis of ink4a-Arf mice. LM conceived of the general study, and participated in its design and coordination, in collaboration with MS, and helped to draft the manuscript. MS also helped to draft the manuscript. All authors read and approved the final manuscript.