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Human Molecular Genetics Pages 229-236  

Expression of the von Hippel-Lindau-binding protein-1 (Vbp1) in fetal and adult mouse tissues
Introduction
Results
   Analysis of Vbp1 expression in mouse embryogenesis
   Vbp1 expression in adult mouse tissues
   VBP1 expression in adult human cerebellum and VHL-associated tumours
   Chromosomal localization of the Vbp1 gene
Discussion
Materials And Methods
   cDNAs used for in situ and northern analysis
   Northern blot analysis
   In situ hybridization
   Chromosomal mapping
Acknowledgements
References

Expression of the von Hippel-Lindau-binding protein-1 (Vbp1) in fetal and adult mouse tissues

Expression of the von Hippel-Lindau-binding protein-1 (Vbp1) in fetal and adult mouse tissues

Myriam Hemberger1,2, Heinz Himmelbauer1, Hartmut P. H. Neumann3, Karl H. Plate4, Georg Schwarzkopf5 and Reinald Fundele1,*

1Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin-Dahlem, Germany, 2Fakultät für Biologie III, Universität Freiburg, Freiburg, Germany, 3Neurozentrum, Ateilung Neuropathologie, Albert Ludwigs Universität Freiburg, Breisacherstrasse 64, Freiburg, Germany, 4Klinikum der Albert Ludwigs Universität Freiburg, Abteilung Innere Medizin IV, Hugstetter Strasse 55, D-79106 Freiburg, Germany and 5Institut für Pathologie der Albert Ludwigs Universität Freiburg, Albertstrasse 19, D-79104 Freiburg, Germany

Received April 1, 1998; Revised and Accepted November 5, 1998

The von Hippel-Lindau (VHL) tumour suppressorgene product is believed to be involved in the down-regulation of transcriptional elongation by preventing the association of elongin B and C with the catalytic subunit elongin A. Alterations in the human VHL gene lead to VHL disease which is associated with various rare neoplasias, including haemangioblastoma of the central nervous system, retinal angioma, clear cell renal carcinoma and pheochromocytoma. Recently, a protein (VBP1) was isolated that was found to bind to the VHL protein in vivo. We have used the murine Vbp1 homologous cDNA to investigate the expression of the Vbp1 mRNA in the mouse by in situ hybridization and northern blot analysis. In fetal stages between days 9 and 18 of gestation, Vbp1 was expressed mainly in the central nervous system, retina and liver. In addition, at day 12, high expression was observed in the labyrinthine region of the placenta. In later stage placentas, Vbp1 expression was, however, considerably reduced. Northern blot analysis of adult mouse tissues showed that Vbp1 was ubiquitously expressed. In situ analysis on several adult tissues showed that in most tissues, transcripts were evenly distributed. In brain, eye, kidney and intestine, however, Vbp1 was expressed in specific cell types. Moreover, expression of the human VBP1 gene was investigated in cerebellum and invarious tumours of VHL patients encompassinghaemangioblastomas, renal cell carcinomas and pheochromocytomas. In all of these tissues, VBP1 was ubiquitously expressed at low levels. However, no consistent differences in VBP1 expression levels could be detected between tumours and normal tissue. Mapping of the murine Vbp1 gene revealed conserved chromosomal localization between mouse and human in a region homologous to human Xq28.

INTRODUCTION

von Hippel-Lindau (VHL) disease is a rare hereditary disorder that is characterized by the occurrence of multiple neoplasias, including haemangioblastomas of the retina (1) and cerebellum (2), renal cell carcinomas, pheochromocytomas and cysts in liver, pancreas and epididymis (3,4). A few years ago, the human VHL tumour suppressor gene was isolated by a positional cloning approach (5). Subsequently, it was possible to characterize the germline mutations present in VHL families (6). Loss of the wild-type VHL allele is necessary for tumorigenesis in familial VHL disease (7), and somatic inactivation of both VHL alleles occurs in ~80% of sporadic clear cell renal carcinomas, the most common malignancy in the human kidney (8-10). VHL disease-associated tumours are characterized by a well-vascularized phenotype and they have been shown to overexpress vascular endothelial growth factor (VEGF) and its receptors (11,12). The VHL protein acts as a negative regulator of transcription by associating with elongins B and C (13-15). This inhibits binding of elongins B and C to elongin A. These subunits form the heterotrimeric transcription factor elongin which increases RNA transcription by suppressing polymerase II pausing (13). Regulation at the transcriptional level has also been proposed for VEGF expression via direct binding of the VHL protein to the SP1 transcription factor (16). However, the product of the VHL tumour suppressor gene was also shown to regulate VEGF post-transcriptionally (17,18).

Several studies have characterized expression of the VHL gene in fetal and adult human tissues. As assessed by mRNA in situ hybridization, VHL was expressed in derivatives of all three germ layers in human fetuses; however, expression was strongest in the central nervous system (CNS), kidneys, testis and lung (19). A similar expression pattern was also detected in a study of Vhl expression in murine embryogenesis (20). Of the adult human organs, VHL is expressed in all tissues that are target sites for VHL disease, i.e. the CNS, kidney tubules, adrenal, pancreas and liver (21,22). However, VHL transcripts were also detected in tissues that are not considered to be at risk for VHL disease, such as small and large intestine, prostate, lung and placenta (10,20,23). Inactivation of the VHL gene was observed recently in sporadic colorectal cancer. However, VHL gene deletion seems to be a late event in colorectal tumour development (24).


Figure 1. Northern analysis of Vbp1 expression. (A) Vbp1 expression determined at different developmental stages both in mouse placenta and embryo. The control hybridization with actin indicates the RNA amounts in each lane. (B) Mouse adult multiple tissue northern blot indicating ubiquitous expression. The strongest signals are detected in skeletal muscle, when RNA amounts are quantitated against actin hybridization signals. In brain, a second transcript of ~4 kb is detectable.


Figure 2. In situ analysis of Vbp1 expression in mouse placenta and embryo of different developmental stages. (A and B) Brightfield (A) and darkfield (B) illumination of an e12 placenta after in situ hybridization with the Vbp1 probe (bar: 200 µm). Expression is visible in the darkfield in decidual tissue and in the labyrinthine trophoblast, but not in the spongiotrophoblast. (C and D) In situ hybridization against Vhl transcripts on a section nearly adjacent to that in (A) and (B). Expression of Vhl is detected mainly in the spongiotrophoblast layer (bar: 200 µm). (E and F) Overview of an e12 fetus in brightfield (E) and darkfield (F). The strongest Vbp1 expression is detectable in brain, spinal cord and liver (bar: 500 µm). (G and H) Magnification of the head of an e15 fetus, showing high amounts of Vbp1 transcripts in the telencephalon, facial ganglia and nasal epithelium. The arrowhead points to the epithelium of the mouth (bar: 500 µm). (I and J) Close-up of the eye of an e16 embryo. Highest expression levels are found in the inner neuroblastic cell layer. The arrowhead shows the pigment layer of the retina (bar: 100 µm).(K and L) Brightfield (K) and darkfield (L) illumination showing the paraspinal ganglia of an e15 fetus with very dense hybridization signals. Vbp1 expression is also visible in the perichondrium of vertebrae, but not in cartilage (bar: 200 µm). (M and N) Heart ventricle and atrium of an e15 embryo. Vbp1 is expressed in both structures as well as in the diaphragm (bar: 200 µm). (O and P) Liver, kidney and adrenal gland of an e15 fetus in brightfield (O) and darkfield (P; bar: 200 µm). a, descending aorta; ag, adrenal gland; at, atrium; d, diaphragm; de, decidua; dr, dorsal root ganglion; he, heart; in, inner neuroblast layer of retina; ir, intra-retinal space; IV, fourth ventricle; ki, kidney; la, labyrinthine trophoblast; le, lens; li, liver; lv, lateral ventricle; m, mandible; me, metencephalon; mu, muscle; mv, mesencephalic ventricle; oe, olfactory epithelium; on, outer neuroblast layer of retina; ov, otic vesicles; ri, cartilage primordium of rib; sc, spinal cord; sp, spongiotrophoblast; v, trigeminal (V. cranial) ganglion; vb, cartilage primordium of vertebral body; ve, heart ventricle; ys, yolk sac.

Recently, several proteins that associate with the VHL protein were identified in a yeast two-hybrid screen (25). One of these proteins, VHL-binding protein 1 (VBP1), was shown to be present in various cell lines that also express the VHL gene (25). Interestingly, it was also shown that both elongin C and VBP1 bind to the C-terminal 26 amino acids of VHL. In the absence of VHL, the VBP1 is localized in the perinuclear region of the cytoplasm of COS-7 cells (25). However, when VHL and VBP1 were co-expressed in COS-7 cells, the VBP1 protein was localized mainly in the nucleus (25). From these findings, the authors concluded that the localization of VBP1 is controlled by VHL. Although the precise role of VBP1 is at present not known, it may predispose different organs specifically to develop VHL tumours.

To provide insight into the possible role of VBP1 in the formation of VHL disease, we have performed in situ hybridization studies to assess the sites of Vbp1 expression in fetal and adult murine tissues, in human VHL-associated tumours and in human cerebellum. Our main interest was to determine whether the risk of VHL-expressing tissues, in patients with inherited VHL disease, developing tumours or not is correlated with the expression of the VBP1 gene.

RESULTS

Analysis of Vbp1 expression in mouse embryogenesis

Using a 374 bp fragment spanning the region between nucleotides 119 and 492 of the murine Vbp1 sequence (26), we have investigated Vbp1 expression during both fetal and placental development between e9 and e18 by northern analysis (Fig. 1A). A single transcript of ~1.7 kb was detected in all tissues assessed. Because of addition of the poly(A) tail to the mature mRNA, this is in agreement with the published size of 1525 bp for the full-length murine transcript. In the placenta, expression of Vbp1 was strongest on e9 and e12 and declined thereafter (Fig. 1A). In the fetus, expression was strong until e14, but then decreased between e16 and e18 (Fig. 1A).

Sections of mouse embryos and placentas were analysed on fetal days e9, e12, e13, e14, e15, e16 and e17. In e9 and e12 placentas, strong Vbp1 expression was observed in the labyrinthine region and in the chorionic plate (Fig. 2A and B). In contrast, the spongiotrophoblast cells and secondary giant cells exhibited no significant Vbp1 expression (Fig. 2A and B). Analysis of expression of the Vhl gene on consecutive sections revealed predominant expression in the spongiotrophoblast layer (Fig. 1C and D). Thus, Vhl and Vbp1 transcripts are distributed reciprocally in the placenta. Expression levels of Vbp1 in the labyrinth and in the chorionic plate declined after e12; however, transcripts could still be detected in e14 and in e16 placentas (data not shown).

No major changes in tissue specificity of Vbp1 expression were detected between the different stages of fetal development. Vbp1 transcripts were detected, albeit at different levels, in most tissues of the fetal mouse. Vbp1 was highly expressed in the brain (Fig. 2E-H), the spinal cord (Fig. 2E and F), the neuroblastic layers of the retina (Fig. 2I and J), the facial and paraspinal ganglia (Fig. 2G, H, K and L) and in the nasal epithelium (Fig. 2G and H). In the brain, Vbp1 transcripts were not evenly distributed. In two regions, encompassing on the one hand medulla, pons, cerebellum and midbrain, and on the other hand lateral cortex, striatum and thalamus, signal density seemed to be elevated when compared with other brain areas. Moderate levels of Vbp1 expression were detected in liver (Fig. 2M and N), adrenal gland (Fig. 2O and P), intestine (Fig. 2E and F), the vibrissae, pancreas and heart muscle (Fig. 2M and N). Remarkably, although fetal kidney clearly exhibited Vbp1 expression, transcripts seemed to be less abundant than in the adrenal gland (Fig. 2O and P). Basal expression levels were also observed for instance in lung (Fig. 2E and F) and bladder. The most notable exception was cartilage, which showed very little expression except in the perichondrium (Fig. 2K and L). Hybridization with the sense probe produced no unspecific binding above background levels (data not shown).

Vbp1 expression in adult mouse tissues

To gain a first overview of the Vbp1 expression in adult murine tissues, northern blot analysis was carried out (Fig. 1B). A 1.7 kb Vbp1 transcript was found in all tissues that were analysed. Strongest expression was observed in skeletal muscle and cardiac muscle. In the brain, a weak second transcript of ~4.0 kb was detected. To assess the cellular specificity of Vbp1 expression, RNA in situ hybridization was performed on sections of several adult mouse tissues, including heart, intestine, lung, kidney, brain, liver and eye. In most tissues, Vbp1 transcripts seemed to be evenly distributed (data not shown). In contrast, intestine, brain, kidney and eye exhibited cell-specific expression patterns.

In the small intestine, hybridization signals were localized mostly in the basal region of the crypts (Fig. 3A and B). This localization argues for a Paneth cell-specific expression of Vbp1. This is supported by the lack of Vbp1 expression in the crypts of the large intestine that do not contain Paneth cells. In the large intestine, highly specific expression was seen in the luminal epithelial lining (Fig. 3C and D). In kidney, signals were conspicuously absent from the glomeruli. Mesenchymal and tubular cells showed high expression. Whereas in most areas of the brain, expression levels were uniform, in the hippocampus (Fig. 3E and F) Vbp1 transcripts were enriched in the subcortical layer and in the granular cell layer of the gyrus dentatus. The adult eye exhibited the same expression pattern as described for the fetal eye structure (data not shown).

VBP1 expression in adult human cerebellum and VHL-associated tumours

To determine VBP1 expression in human tissues and in VHL disease-associated tumours, normal human cerebellum, five haemangioblastomas, two pheochromocytomas and three renal cell carcinomas of VHL patients were investigated. The VHL gene is known to be expressed abundantly in the Purkinje cells and in the dentate nucleus (20) (Fig. 3G and H). In contrast to this and to the observed expression pattern in the mouse (Fig. 3E and F), human VBP1 transcripts were evenly distributed over the whole cerebellum. Elevated expression levels could not be seen in Purkinje cells or in the dentate nucleus (Fig. 3I). Very low levels of VBP1 transcripts were detected in the neoplastic tissues. In addition, only marginal differences between hybridization of sense and antisense probes were seen. No cell-specific alterations of expression levels could be observed (data not shown).


Figure 3. Vbp1 in situ hybridization on adult murine and human tissues. (A and B) Brightfield (A) and darkfield (B) illumination of the small intestine. The arrowheads point towards the base of the crypts, where distinct hybridization signals are visible (bar: 100 µm). (C and D) Epithelial lining of the large intestine. Silver grains indicating Vbp1 expression are located in the epithelial cells (bar: 50 µm). (E and F) Hippocampal region with expression detectable in the cortical cell layer and the granular layer of the gyrus dentatus (bar: 400 µm). (G and H) Adult human cerebellum after in situ hybridization with the VHL probe. Darkfield illumination (H) shows the strikingly high expression levels of VHL in Purkinje cells (arrowheads) and the dentate nucleus (arrow). (I) Expression of VBP1 on a section adjacent to that in (G) and (H). In contrast to VHL and to murine Vbp1 expression, no elevated VBP1 transcript levels can be detected in specific cell types (bar: 400 µm). ep, epithelial cells; g, goblet cell; gd, gyrus dentatus; hi, hippocampus; lu, lumen; m, muscle; me, mesenchyme.

Chromosomal localization of the Vbp1 gene

Vbp1 sequences were amplified by PCR and the 465 bp products were treated with several restriction enzymes. SspI digestion revealed a restriction fragment length polymorphism (RFLP) that allowed differentiation between the Vbp1 alleles of the two species. The Mus musculus PCR product was digested into two fragments of 442 and 23 bp, the M.spretus allele was not cut (data not shown). PCR amplification and SspI digestion were performed with 80 DNA samples derived from the EUCIB (M.musculus × M.spretus) × M.spretus backcross panel (27). This analysis showed clear linkage of the murine Vbp1 gene with the X chromosome. However, segregation distortion against the M.musculus-derived X chromosome in this backcross (28,29) interfered with precise mapping results. Therefore, genotyping was also carried out on the Jackson Laboratory (M.musculus × M.spretus) × M.musculus backcross panel. Unambiguous genotyping information could be derived from 93 backcross mice, and a perfect match (100%) to the allele pattern of marker DXMit60 was observed. Markers flanking Vbp1/DXMit60, map distances in centiMorgans and calculated standard errors were: centromere-DXMit25 (3.2 ± 1.7 cM)-Vbp1/DXMit60 (2.2 ± 1.4 cM)-DXMit7/Bir6/DXHun19-telomere (Fig. 4).

DISCUSSION

The product of the Vbp1 gene is of interest, as it was recently discovered in a yeast two-hybrid approach as a protein that specifically interacts with the VHL tumour suppressor gene product (25). While no indication exists at present concerning the functional role of the VBP1 protein in vivo, co-expression of both genes would have to be predicted if protein-protein interactions detected in the yeast two-hybrid system are functional. We have therefore performed a study using northern blot analysis and mRNA in situ hybridization to describe expression of the Vbp1 gene in fetal and adult murine tissues. By northern analysis, we showed Vbp1 expression both in the fetus and placenta that moderately declined in later stages of development. In adult tissues, a ubiquitous expression could be detected. In addition to the main transcript of ~1.7 kb, a band of ~4 kb was detected in brain only. A band of similar size was also described for human adult tissues, probably indicating differential splicing within the >25 kb of the human VBP1 genomic sequence (26). During fetal development, RNA in situ hybridization revealed expression of Vbp1 in derivatives of all three germ cell layers and in the labyrinthine layer of the placenta. Overall, the Vbp1 expression pattern during embryogenesis agrees very well with that described for the murine Vhl gene (20). Both genes are highly expressed in the retina, the CNS, the facial and paraspinal ganglia and the heart. Thus, the plausible assumption of coordinated expression of the Vhl and Vbp1 genes in vivo seems to be largely supported by our results. However, in some tissues, the relative expression levels were obviously quite different. For instance, Vhl seems to be expressed in the fetal pancreas at very high levels (20). In comparison, Vbp1 transcripts are present in this tissue at moderate levels only. Also, Vhl is expressed more strongly in the kidney than in the adrenal gland, whereas Vbp1 exhibits the opposite expression pattern. However, both Vhl and Vbp1 exhibit no expression differences between adrenal cortex and medulla (20). It is also of interest that in the cerebellum, differences in Vbp1 expression patterns between mouse and human could be established. The significance of this result is at present not clear.

Embryonic lethality around mid-gestation has been described recently in Vhl-deficient mice caused by failure of placental vasculogenesis (30). A common feature in the Vhl-/- mice was a lack of embryonic endothelium and embryonic blood vessels in the placental labyrinth. The Vhl gene was described to be expressed in the labyrinthine trophoblast, the allantoic mesoderm and parts of the embryonic endothelium at e10.5-e12.5 (30). In contrast to these results, we observed the highest levels of Vhl transcripts in the spongiotrophoblast between e12 and e18 of development. The expression level in this tissue was higher than in any other tisue either of embryonic or extraembryonic origin. Since we demonstrate confinement of Vbp1 expression to the decidual and labyrinthine layer of placentas of the same developmental stages, Vhl and Vbp1 seem to exhibit reciprocal expression patterns in the placenta. This observation is of interest especially in light of the fact that the products of two other genes, Vegf and Flt1, are distributed in a similar pattern. VEGF is restricted to the labyrinthine layer, whereas its receptor, FLT1, was detected only in the spongiotrophoblast (31). Several studies have investigated the putative role of VEGF in VHL disease and have shown that VEGF expression is regulated negatively by VHL in renal carcinoma cell lines (17,18,32,33). However, in placentas of VHL-deficient mice, VEGF protein levels were greatly reduced in the labyrinthine trophoblasts (30), indicating that the regulation of Vegf expression by VHL is not straightforward. In this context, the co-expression of Vegf and Vbp1 could indicate that VBP1 is involved in regulation of VEGF levels at least in extraembryonic tissues.

Our results do, however, argue against an intrinsic role for VBP1 in the specificity of tumour generation characteristic of VHL disease; i.e. the decision as to whether or not a tissue that has lost Vhl expression forms tumours, as for instance kidney or intestine, seems not to be dependent on the absence or presence of VBP1. Thus, Vhl and Vbp1 are co-expressed in the adrenal where VHL patients may develop tumours. In contrast, both genes are also strictly co-expressed in the Paneth cells of the small intestine, a tissue not at risk in VHL patients. To examine VBP1 expression in VHL-associated tumours, we also performed in situ hybridizations on haemangioblastomas, renal cell carcinomas and pheochromocytomas. In these neoplastic tissue samples, no consistent tumour-specific alterations of VBP1 expression were observed. Again, this argues against a critical role for VBP1 in the generation of these tumours. To establish fully the role of VBP1 in tumorigenesis of VHL disease and in spontaneous clear cell renal cancer, it will be necessary to determine the cellular effects of VHL-VBP1 interaction. Our results provide evidence that the VHL gene product is not involved in regulation of VBP1 transcription levels, as VBP1 transcript levels are not significantly altered in the absence of functional VHL protein.

In the current work, we have mapped the mouse Vbp1 locus genetically to the X chromosome using an interspecific mouse backcross. Zero recombinants were observed between Vbp1 and DXMit60 in 93 meioses tested. The human homologue of Vbp1 had been mapped previously to the human X chromsome, band Xq28, in the vicinity of the coagulation factor VIII gene locus (26). On the mouse consensus map provided by the Committee for the Mouse X-Chromosome, the mouse homologue of the factor VIII locus (C8f) is assigned to centiMorgan position 30.5. DXMit60 is placed at position 30.7 (Fig. 4). The genetic mapping information obtained for Vbp1, therefore, is in excellent agreement with the existing human-mouse synteny data.


Figure 4. Genetic mapping of the murine Vbp1 gene on an interspecific mouse backcross. (A) Position of Vbp1 on the X chromosome of the Jackson Laboratory BSB backcross. Flanking markers are anonymous markers DXMit25 (proximal) and DXMit7, DXBir6 and DXHun19 (distal). The numbers of recombination events and genetic distances in centiMorgans are indicated. (B) Consensus map according to the 1998 Report of the Committee for the Mouse X-Chromosome. Cf8 is the mouse homologue of the coagulation factor VIII gene. On human Xq28, VBP1 and the factor VIII gene are physically linked within a YAC contig (26).

MATERIALS AND METHODS

cDNAs used for in situ and northern analysis

Primers were designed to span a 374 bp stretch between bp 119 and 492 of the VBP1 sequence (26). The forward primer used was 5[prime]-TGAGACTGCAGATACAGTGT; the reverse primer was 5[prime]-GATCTCGAAGAAAGTCAAGG. For the PCR, cDNA isolated from an e12 mouse embryo was used. PCR conditions were 35 cycles at 94°C for 40 s, 53°C for 40 s and 72°C for 40 s, using buffer A of the Stratagene PCR optimizing kit. The resulting fragment was cloned into the pUAg vector (Ingenius). After verification of the cloned PCR product by sequencing on a LiCor DNA4200 sequencer, the construct was used for in situ and northern hybridization.

As probe for the murine Vhl transcript, clone IMAGp998L-101382 was obtained from the Resource Centre of the German Human Genome Project at the Max-Planck-Institut forMolecular Genetics. For human VHL and VBP1 probes, clones IMAGp998P19186 and IMAGp998E161160 were used, respectively, which were obtained from the same source. Inserts of all clones were checked by sequencing.

Northern blot analysis

Analysis of Vbp1 expression in adult tissues was performed using mouse and human poly(A)+ multiple tissue northern blots. The filters were hybridized with the 32P-labelled PCR product according to the manufacturer’s (Clontech) protocol. Application to Fuji medical X-ray films was carried out for 2-5 days. For analysis of prenatal Vbp1 expression, total RNA was extracted as per Chomczynski and Sacchi (34). A 20 µg aliquot of total RNA was electrophoresed in a 1.5% formaldehyde agarose gel, blotted on to Hybond N+ (Amersham) membrane and UV cross-linked. Hybridization with the 32P-labelled PCR product and filter washings were carried out as described elsewhere (35).

In situ hybridization

Murine tissues were fixed either in Carnoy’s or Serra’s fixatives, and human tissues were fixed in formalin and subsequently processed for routine paraffin histology; 5-7 µm sections were produced, deparaffinized and used for in situ hybridization according to a standard protocol used in our laboratory (36). The Vbp1 probe was linearized with XbaI for antisense and KpnI for sense probes. IMAGp998L101382 was linearized with HindIII for antisense and XhoI for sense probes, IMAGp998E161160 was cut with XhoI for antisense and SalI for sense probes. The NotI-EcoRI insert of clone IMAGp998P19186 was recloned into the SmaI site of vector pBSKS+ (Stratagene) and then linearized with SacII for antisense and XhoI for sense probes. All probes were labelled with [35S]UTP. After hybridization, sections were dipped in Ilford K.5 photoemulsion and exposed for 2 weeks. Counterstaining was performed with haemalaun.

Chromosomal mapping

PCR primers were designed in the 3[prime] region of the murine Vbp1 sequence. The forward primer used was 5[prime]-GCAGGAAAGACATACAGTTCC; the reverse primer was 5[prime]-CACAGTCAGTACCAATGGCT. Reactions were carried out using 50 ng of genomic mouse DNA as template. PCR conditions were 45 cycles at 94°C for 40 s, 57°C for 40 s and 72°C for 60 s. Digestion of the PCR products with SspI allowed the M.musculus strain C57BL/6 (465 bp) and M.spretus (442 + 23 bp) DNA to be distinguished. Genotyping was carried out on the EUCIB (M.musculus C57BL/6 × M.spretus LS) × M.spretus LS backcross panel and the Jackson Laboratory (C57BL/6 × M.spretus) × C57BL/6 backcross panel (37). Information on markers flanking Vbp1 on the mouse X chromosome were obtained from the Jackson Laboratory website (http://www.jax.org/resources/documents/cmdata/bkmap/BSB.html ). Genotyping data have been submitted to the Mouse Genome Database (MGD).

ACKNOWLEDGEMENTS

We are grateful to Drs H. Hameister, B. Royer-Pokora, E. Tais and U. Zechner for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Fu188/2-3 and PI158/3-2), by grant C4 from the Center for Clinical Research I and by the Max-Planck-Gesellschaft.

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*To whom correspondence should be addressed. Tel: +49 308 4131214; Fax: +49 308 4131383; Email: fundele@mpimg-berlin-dahlem.mpg.de

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