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• W2000620535 abstract "Here we present the first description of the genomic organization, transcriptional regulatory sequences, and adult and embryonic gene expression for the mouse p97(CDC48) AAA ATPase. Clones representing two distinct p97 genes were isolated in a genomic library screen, one of them likely representing a non-functional processed pseudogene. The coding region of the gene encoding the functional mRNA is interrupted by 16 introns and encompasses 20.4 kilobase pairs. Definition of the transcriptional initiation site and sequence analysis showed that the gene contains a TATA-less, GC-rich promoter region with an initiator element spanning the transcription start site. Cis-acting elements necessary for basal transcription activity reside within 410 base pairs of the flanking region as determined by transient transfection assays. In immunohistological analyses, p97 was widely expressed in embryos and adults, but protein levels were tightly controlled in a cell type- and cell differentiation-dependent manner. A remarkable heterogeneity in p97 immunostaining was found on a cellular level within a given tissue, and protein amounts in the cytoplasm and nucleus varied widely, suggesting a highly regulated and intermittent function for p97. This study provides the basis for a detailed analysis of the complex regulation of p97 and the reagents required for assessing its functional significance using targeted gene manipulation in the mouse. Here we present the first description of the genomic organization, transcriptional regulatory sequences, and adult and embryonic gene expression for the mouse p97(CDC48) AAA ATPase. Clones representing two distinct p97 genes were isolated in a genomic library screen, one of them likely representing a non-functional processed pseudogene. The coding region of the gene encoding the functional mRNA is interrupted by 16 introns and encompasses 20.4 kilobase pairs. Definition of the transcriptional initiation site and sequence analysis showed that the gene contains a TATA-less, GC-rich promoter region with an initiator element spanning the transcription start site. Cis-acting elements necessary for basal transcription activity reside within 410 base pairs of the flanking region as determined by transient transfection assays. In immunohistological analyses, p97 was widely expressed in embryos and adults, but protein levels were tightly controlled in a cell type- and cell differentiation-dependent manner. A remarkable heterogeneity in p97 immunostaining was found on a cellular level within a given tissue, and protein amounts in the cytoplasm and nucleus varied widely, suggesting a highly regulated and intermittent function for p97. This study provides the basis for a detailed analysis of the complex regulation of p97 and the reagents required for assessing its functional significance using targeted gene manipulation in the mouse. ATPases associated with diverse cellularactivities base pair(s) kilobase pair(s) p97 belongs to the family of ATPasesassociated with diverse cellular activities (AAA)1 occurring in eubacteria, archaebacteria, and eukaryotes (1Confalonieri F. Duguet M. Bioessays. 1995; 17: 639-650Crossref PubMed Scopus (313) Google Scholar). The AAA motif is defined by a conserved sequence of 200 amino acids including the Walker type A and B cassettes, which are important in ATP binding and hydrolysis (2Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4234) Google Scholar). AAA family members include proteins involved in vesicle and organelle biogenesis (3Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2001) Google Scholar, 4Rabouille C. Levine T.P. Peters J.M. Warren G. Cell. 1995; 82: 905-914Abstract Full Text PDF PubMed Scopus (311) Google Scholar), components of the 26 S proteasome (5Dubiel W. Ferrell K. Rechsteiner M. Mol. Biol. Rep. 1995; 1: 27-34Crossref Scopus (123) Google Scholar), metalloproteases (6Tomoyasu T. Yamanaka K. Murata K. Suzaki T. Bouloc P. Kato A. Niki H. Hiraga S. Ogura T. J. Bacteriol. 1993; 175: 1352-1357Crossref PubMed Scopus (143) Google Scholar), cell cycle regulators (7Frohlich K.-U. Fries H.-W. Rudiger M. Erdmann R. Botstein D. Mecke D. J. Cell Biol. 1991; 114: 443-453Crossref PubMed Scopus (245) Google Scholar), and transcription factors (8Shibuya H. Irie K. Ninomiya-Tsuji J. Goebl M. Taniguchi T. Matsumoto K. Nature. 1992; 357: 700-702Crossref PubMed Scopus (142) Google Scholar). Mammalian p97 (first termed VCP, for valosin-containing protein) was originally described as a precursor protein containing the biologically active peptide valosin (9Koller K.J. Brownstein M.J. Nature. 1987; 325: 542-545Crossref PubMed Scopus (100) Google Scholar). Subsequently, in Xenopus laevis, a 14 S, homo-oligomeric ATPase was identified as the homologue of mammalian VCP (10Peters J.M. Walsh M.J. Franke W.W. EMBO J. 1990; 9: 1757-1767Crossref PubMed Scopus (233) Google Scholar), and in Saccharomyces cerevisiae, genetic alterations in the p97 homologue (CDC48) were shown to underlie a mitotic-arrest phenotype (7Frohlich K.-U. Fries H.-W. Rudiger M. Erdmann R. Botstein D. Mecke D. J. Cell Biol. 1991; 114: 443-453Crossref PubMed Scopus (245) Google Scholar, 11Moir D. Stewart S.E. Osmond B.C. Botstein D. Genetics. 1982; 100: 547-563Crossref PubMed Google Scholar). More recently, highly conserved p97 homologues have been identified in a diverse range of experimental organisms, including Drosophila melanogaster (TER94) (12Pinter M. Jekely G. Szepesi R.J. Farkas A. Theopold U. Meyer H.E. Lindholm D. Nassel D.R. Hultmark D. Friedrich P. Insect Biochem. Mol. Biol. 1998; 28: 91-98Crossref PubMed Scopus (36) Google Scholar),Arabidopsis thaliana (AtCDC48) (13Feiler H.S. Desprez T. Santoni V. Kronenberger J. Caboche M. Traas J. EMBO J. 1995; 14: 5626-5637Crossref PubMed Scopus (100) Google Scholar), and the archaebacterium Sulfobus acidocaldarius (SAV) (14Confalonieri F. Marsault J. Duguet M. J. Mol. Biol. 1994; 235: 396-401Crossref PubMed Scopus (37) Google Scholar), thus demonstrating that p97 is an ancient protein and implying a fundamental role(s) for this protein within cells. Purified p97 is a soluble, ringlike hexameric complex with each monomeric subunit containing two copies of the AAA domain. This complex is the active Mg2+-dependent ATPase, which is sensitive to the alkylating agent N-ethylmaleimide (10Peters J.M. Walsh M.J. Franke W.W. EMBO J. 1990; 9: 1757-1767Crossref PubMed Scopus (233) Google Scholar, 15Zhang L. Ashendel C.L. Becker G.W. Morre D.J. J. Cell Biol. 1994; 127: 1871-1883Crossref PubMed Scopus (73) Google Scholar). The significance of these structural and biochemical features for the cellular function of p97 has not yet been firmly established. Data from diverse origins have implicated p97 in a remarkable number of cellular processes. In S. cerevisiae, genetic analysis has shown that conditional mutants in the yeast CDC48 gene, namedcdc48–1, arrest in mitosis as large budded cells with elongated nuclei spanning the mother-daughter junction (11Moir D. Stewart S.E. Osmond B.C. Botstein D. Genetics. 1982; 100: 547-563Crossref PubMed Google Scholar). p97 has been proposed to function in homotypic membrane fusion events, including fusion of the endoplasmic reticulum (16Latterich M. Frohlich K.U. Schekman R. Cell. 1995; 82: 885-893Abstract Full Text PDF PubMed Scopus (334) Google Scholar) and the reassembly of Golgi cisternae from vesicles and tubules generated by treatment with specific drugs (17Acharya U. Jacobs R. Peters J.M. Watson N. Farquhar M.G. Malhotra V. Cell. 1995; 82: 895-904Abstract Full Text PDF PubMed Scopus (185) Google Scholar) or mitotic cytosol (4Rabouille C. Levine T.P. Peters J.M. Warren G. Cell. 1995; 82: 905-914Abstract Full Text PDF PubMed Scopus (311) Google Scholar). Using the Golgi reassembly assay, p97's fusion-promoting activity has recently been shown to be dependent upon a stoichiometric association with a cytosolic protein termed p47 (18Kondo H. Rabouille C. Newman R. Levine T.P. Pappin D. Freemont P. Warren G. Nature. 1997; 388: 75-78Crossref PubMed Scopus (363) Google Scholar). Protein interaction studies have also revealed that p97 binds to clathrin in a stoichiometric complex, suggesting a role for p97 in the endocytic cycle (19Pleasure I.T. Black M.M. Keen J.H. Nature. 1993; 365: 459-462Crossref PubMed Scopus (111) Google Scholar). p97 has been shown to physically associate with Ufd3p, a protein that is involved in the regulation of free cellular ubiquitin in yeast (20Ghislain M. Dohmen R.J. Levy F. Varshavsky A. EMBO J. 1996; 15: 4884-4899Crossref PubMed Scopus (236) Google Scholar), and to copurify with the mammalian 26 S proteasome (21Dai R.M. Chen E. Longo D.L. Gorbea C.M. Li C.C. J. Biol. Chem. 1998; 273: 3562-3573Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Furthermore, in these same experimental systems, immunodepletion or inactivation of p97 severely compromised ubiquitin-dependent proteolysis of experimental substrates in vitro and in vivo (20Ghislain M. Dohmen R.J. Levy F. Varshavsky A. EMBO J. 1996; 15: 4884-4899Crossref PubMed Scopus (236) Google Scholar,21Dai R.M. Chen E. Longo D.L. Gorbea C.M. Li C.C. J. Biol. Chem. 1998; 273: 3562-3573Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). Despite the abundance of clues provided by these investigations, a common functional link relating these varied observations remains to be established, and thus the precise cellular role(s) of p97 is still obscure. To begin to understand the physiological role of p97 in mammals, we have initiated an analysis of p97 structure and function in mouse. Here we present the first description of the genomic structure of the p97 gene and a likely processed pseudogene. We also identify an upstream region that acts as a functional basal promoter and potential regulatory elements, and describe a pattern of p97 expression in mouse embryos and adults that suggests an unexpected degree of regulated expression and subcellular localization in both proliferating and differentiated tissues. A λDASH II genomic library (kindly provided by R. Mortenson, Harvard Medical School, Boston, MA) prepared from 129 SVJ mouse spleen was screened with a random primed 32P-labeled probe corresponding to the mouse p97 coding region spanning amino acids 2–141. Approximately 9 × 105 plaques were screened on charged nylon filters by hybridizing the p97 probe overnight at 42 °C in 1% SDS, 2× SSC, 10% dextran sulfate, 50% deionized formamide, 2.5× Denhardt's, and 0.1 mg/ml denatured calf thymus DNA. Several positive clones were identified and rescreened in two additional rounds. Phage DNA was isolated and analyzed by restriction digestion and Southern blot. Restriction fragments spanning the relevant phage clones were subloned into pBSII (Stratagene) for further analysis. Purified phage DNA from three unique clones was used to determine the chromosomal location of the mouse p97 gene by fluorescence in situ hybridization. Briefly, metaphase spreads were prepared using standard cytogenetic techniques from mouse diploid cultures and cell lines. Phage DNA was labeled with biotin dUTP by nick translation (Bionick, Life Technologies, Inc.). The labeled probe was combined with mouse Cot-1 DNA and hybridized to metaphase chromosomes in a solution containing 50% formamide, 10% dextran sulfate, 2× SSC, and 1% Tween 20, pH 7.0. Specific hybridization signals were detected by incubating slides in fluorescein isothiocyanate-conjugated avidin. Slides were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (Sigma) and analyzed with a Zeis Axioscop microscope. p97 subclones were sequenced with gene-specific primers using the ABI dye termination kit (Perkin Elmer). Primers were designed on the basis of the published mouse p97 cDNA sequence (22Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samuelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar). Alignment of cDNA and genomic DNA sequence was performed by using the GCG analysis suite (version 9.0). Genomic DNA was prepared from mouse spleen using standard protocols. Restriction-digested DNA (10 μg) was electrophoresed, depurinated in 0.25 m HCl, denatured, and transferred in 0.4 m NaOH to charged nylon (GeneScreen Plus, NEN Life Science Products). Membranes were rinsed in 2× SSC, dried, and hybridized according to the manufacture's protocol. Blots were washed with 1% SDS and 0.5× SSC at 55 °C and visualized by autoradiography. The transcription start site of the p97 gene was determined by primer extension assay using two different end-labeled antisense oligonucleotides (primer 1: 5′-CCGGGGCTGGACTCGCTGAAGCGG-3′; primer 2: 5′-CTCTCGCTTCCTCCCAGGGGCACC-3′). Total RNA from mouse embryonic fibroblasts was isolated with RNAzol (Biogenesis) and divided into poly(A)+ and poly(A)− pools using oligo(dT) affinity beads (Oligotex, Qiagen). The assay was performed using standard protocols. Briefly, radiolabeled oligonucleotide was mixed with approximately 10 μg of each RNA pool and denatured for 90 min at 65 °C in 0.15 m KCl, 1 mm EDTA, and 10 mm Tris-Cl, pH 8.3. The mixture was cooled to room temperature and primer extension was initiated by addition of 10 mm MgCl2, 5.5 mm dithiothreitol, 150 μg/ml actinomycin D, 0.15 mm dNTPs, and 150 units of Superscript II (Life Technologies, Inc.) at 42 °C for 60 min. Products were separated on 8 m urea, 8% polyacrylamide gels and visualized by autoradiography. The precise location of the transcription start site was determined by comparison to dideoxy sequencing reactions carried out using the same oligonucleotides. Three fragments containing 147 bp of the 5′-untranslated region and additionally either 410, 1434, or 3000 bp of 5′-flanking sequence were obtained by polymerase chain reaction and ligated in both orientations with respect to the predicted transcription start site into a promoterless luciferase reporter plasmid (pGL2-basic, Promega). The pGL2-control plasmid (Promega), which utilizes the SV 40 promoter/enhancer to initiate luciferase transcription, was used as a positive control. HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The day before transfection, cells were split into six-well plates. On the next day, cells were transfected in triplicate with 5 μg of test plasmid using SuperFect (Qiagen). To control for transfection efficiency, an expression plasmid containing a secreted alkaline phosphatase reporter gene was cotransfected (2 μg) with test plasmids (23Shima D.T. Kuroki M. Deutsch U. Ng Y.S. Adamis A.P. D'Amore P.A. J. Biol. Chem. 1996; 271: 3877-3883Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Alkaline phosphatase activity was determined as described previously (24Berger J. Hauber J. Hauber R. Geiger R. Cullen B.R. Gene (Amst.). 1988; 66: 1-10Crossref PubMed Scopus (579) Google Scholar) and used to normalize luciferase assay values. Luciferase activity in cell extracts was assayed 48 h after transfection according to the manufacturer's protocol (Promega). Anti-p97 antibodies N2 and N5 were raised in rabbit against two bacterially expressed fusion proteins consisting of glutathione S-transferase (GST) fused to fragments of mouse p97 (N2: amino acids 2–186; N5: amino acids 200–461). The antibodies were affinity-purified using full-length recombinant His-tagged p97 and were shown to uniquely recognize p97 in Western blot analyses of rat liver homogenate (data not shown). Immunohistochemical results obtained using N2 or N5 were indistinguishable. For analyses, samples were fixed in 3% formaldehyde, embedded in paraffin, sectioned, and dewaxed. Endogenous peroxidase activity was quenched, and sections were incubated with purified antibodies (0.5 μg/ml). Binding was detected by subsequent incubation with biotinylated secondary antibody and streptavidin-coupled peroxidase, followed by development with diaminobenzidine. In adult tissues, counterstaining was performed with hematoxylin. Samples were fixed and prepared as for immunostaining. Specific localization of p97 mRNA was accomplished by in situ hybridization using an antisense riboprobe synthesized with SP6 RNA polymerase using 35S-UTP (∼ 800 Ci/mmol; Amersham, UK). The linearized template consisted of a 500-bp fragment of the mouse p97 3′-untranslated region within the plasmid backbone of pSP73. The 3′-untranslated region of sequence used to produce the riboprobe did not show significant homology to any other known gene sequences in the nucleotide data base (GenBank version 109.0). The methods for pretreatment, hybridization, washing, and dipping of slides in Ilford K5 for autoradiography were basically as described by Senior et al., for formalin-fixed paraffin-embedded tissue (25Senior P.V. Critchley D.R. Beck F. Walker R.A. Varley J.M. Development. 1988; 104: 431-446Crossref PubMed Google Scholar), with modifications (26Poulsom R. Longcroft J.M. Jeffery R.E. Rogers L. Steel J.H. Eur. J. Histochem. 1998; 42: 121-132PubMed Google Scholar). The presence of hybridizable mRNA in all compartments of the tissues studied was established in near serial sections using an antisense β-actin probe. Sections were examined under conventional or reflected light darkfield conditions (Olympus BH2 with epi-illumination) that allowed individual autoradiographic silver grains to be seen as bright objects on a dark background. To investigate the genomic structure of the mouse p97 gene, we used a probe spanning the N-terminal region of the protein to isolate three unique clones from a λ phage library. Southern blot analysis on digested phage DNA using probes spanning different regions of the published cDNA (22Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samuelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar) was performed to further characterize the isolated clones. Two clones, designated λ1 and λ2, extended 20 and 15.6 kb, respectively, and overlapped by 3.7 kb. λ2 spanned the 5′ end and λ1 the 3′ end of the p97 cDNA sequence (Fig.1 a). A third clone, designated λ3, extended 14 kb and appeared to encompass the whole p97 coding region based on Southern blot analysis; however, initial analyses revealed that restriction patterns within the p97 coding sequence of λ3 and λ1/2 differed (Fig. 1 a). A more detailed physical map of the different p97 clones was assembled by performing single and double digestions with various restriction enzymes, and Southern blot analysis using radiolabeled probes spanning the published cDNA of p97. Comparison of the physical maps of total genomic and λ clone DNA revealed that p97 is not a single copy gene; in Southern blot analyses, we consistently found two restriction fragments in the total genomic DNA hybridizing to a p97 specific probe. One fragment corresponded to a similarly sized fragment in λ1/2, while the second fragment corresponded to a fragment in λ3, strongly suggesting the presence of two different p97 loci (results for λ2 versus λ3 are shown in Fig. 1 b). The presence of two mouse p97 loci had recently been proposed based on interspecies backcross mapping and polymerase chain reaction analysis (27Hoyle J. Tan K.H. Fisher E.M. Mamm. Genome. 1997; 8: 778-780Crossref PubMed Scopus (4) Google Scholar). Fluorescence in situ hybridization using the isolated λ clones as probes confirmed this genetic analysis. The overlapping clones λ1 and λ2 reside on mouse chromosome 4 at position B2, while clone λ3 resides on chromosome X at position C-D (data not shown). The genomic structure of both genes was further characterized by nucleotide sequence analysis. Analysis of the gene present in λ1/2 revealed that it was interrupted by 16 introns, and all exon-intron borders corresponded to consensus splicing signals, except the exon 14/intron 14 boundary (Fig.2 a). Exons were on average 150 bp in size, and both nucleotide-binding cassettes (2Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4234) Google Scholar) were located in single exons, exon 7 and exon 13. Intron sizes were estimated with polymerase chain reaction analysis using exon-specific primers. The location of exons relative to the restriction map was established by nucleotide sequencing of restriction sites proximal to exons, and restriction digestion analysis of polymerase chain reaction-amplified DNA of the subclones (Fig. 1 a). Alignment of nucleotide sequences of λ3 to the published mouse cDNA (22Egerton M. Ashe O.R. Chen D. Druker B.J. Burgess W.H. Samuelson L.E. EMBO J. 1992; 11: 3533-3540Crossref PubMed Scopus (122) Google Scholar) revealed that this gene contained the complete coding region of p97 without disrupting introns. Several mutations were identified, including a substitution of Val207 to Ile and a deletion of Gln569, Ala570, Ala571, Pro572, and Cys573. A polyadenylation signal and a poly(A) stretch were found in the region corresponding to the 3′ end of the published cDNA. Each end of the gene was flanked by a direct repeat of 8 nucleotides. The structure of the gene encoded by λ3 had all the hallmarks expected of a processed pseudogene (designated herein as ψp97) (28Vanin E.F. Annu. Rev. Genet. 1985; 19: 253-272Crossref PubMed Scopus (524) Google Scholar), implying that the gene does not encode a functional product, as reported for many other pseudogenes (29Richardson M.P. Braybrook C. Tham M. Moore G.E. Stanier P. Gene (Amst.). 1998; 206: 145-150Crossref PubMed Scopus (10) Google Scholar). In support of this notion, alignment of data base cDNA sequences for p97 gene counterparts in organisms ranging from archaebacteria to mouse demonstrated that all cDNA encoded the amino acid sequence AP (found at positions 571–572 in the mouse protein), which was present in the discontinuous p97 gene (λ1/2), but which was deleted in ψp97 (Fig. 2 b). Therefore, we tentatively concluded that ψp97 most likely represents a non-functional processed pseudogene, rather than a second, independently evolved, intronless and active p97 gene. Thus, we focused our attention on the further characterization of the p97 gene locus represented by λ1/2, which appeared to encode the functional p97 mRNA. To identify the transcription start site, primer extension assays were performed. Based on the 5′ end nucleotide sequence of the p97 cDNA and the region of deviation between the p97 gene and its pseudogene, two antisense primers were designed to map the 5′ end of the p97 gene. The oligonucleotides were annealed to either purified poly(A)+ RNA or poly(A)− RNA as negative control. After reverse transcription and denaturing gel electrophoresis, a single major product was identified for each primer extension reaction. The precise transcription start site, designated +1, was determined by comparison of the migration of the products with a sequencing reaction using the same primers which were run in parallel on the gel (results for primer 1 are shown in Fig.3). A consensus mRNA-CAP site resided at the identified transcription start, thus supporting the mapping data. Both primers also gave rise to a low abundance extension product, which may correspond to a minor transcription start at position +33 (data not shown). Sequence analysis of the putative transcription initiation region demonstrated the absence of a consensus TATA sequence for RNA polymerase II-initiated transcription. Instead, an initiator element (Inr) TCTGA+1CT surrounding the predicted transcription start site, a CCAAT box at position −40, and GC-rich regions at position −80 to −150 were identified, thus revealing a common core organization found in TATA-less promoters (Fig.4) (30Azizkhan J.C. Jensen D.E. Pierce A.J. Wade M. Crit. Rev. Eukaryotic Gene Exp. 1993; 3: 229-254PubMed Google Scholar). To determine if the 5′-flanking region of the p97 gene can initiate basal transcription, a fragment containing 3000 bp of the flanking nucleotide sequence and additionally 147 bp of the 5′-untranslated region of the gene was inserted in both orientations into a luciferase reporter construct and assayed for activity in transiently transfected HeLa cells. Fusion of p97 sequence to the reporter gene in the appropriate orientation resulted in approximately 75-fold increase of luciferase activity when compared with either promoterless luciferase constructs or constructs in which p97 sequence was fused in the opposite transcriptional orientation (Fig.5). Deletion of 1566 bp from the 5′ end of the promoter fragment resulted in a 1.6-fold increase in reporter activity, whereas deletion of 2590 bp resulted in activity similar to that of the full-length fragment, implying that the minimal sequence necessary for basal transcription of the p97 gene in HeLa cells maps to approximately −1 to −410 with respect to the transcription start site. Comparison of the activity of the p97 promoter fragments to an assayed construct containing the SV 40 promoter/enhancer, which was used as positive control, revealed that the p97 promoter was a relatively powerful transcriptional initiator, with p97 promoter sequences yielding 50–75% of the activity measured for the viral promoter/enhancer. The fragment containing 1434 bp of the 5′-flanking region initiated transcription of the reporter gene more efficiently than the 3000-bp fragment, indicating that potential repressor elements may reside further upstream of the basal promoter. However, more detailed studies will be required to identify the components involved in regulating transcription of the p97 gene. To provide clues about the gene regulation and potential function of p97 in vivo, we investigated the distribution of p97 protein in adult and embryonic mouse tissues by immunohistochemical staining using affinity purified antisera (see “Materials and Methods”). For adult tissue, the immunohistochemical analysis clearly showed that p97 was widespread in most investigated tissues (results from the small intestine, liver, testis, and kidney are shown in Fig. 6and summarized in Table I). However, the degree of staining between different cell types or cell differentiation states within a given tissue showed exceptional heterogeneity. This type of heterogeneity was not observed in an analysis performed in parallel for the Golgi apparatus structural protein giantin, which displayed a relatively uniform distribution in virtually all cells within the tissues examined. 2D. T. Shima and C. Ruhrberg, unpublished observation. An example of the differential expression of p97 could be observed in the intestinal epithelium (Fig. 6 a). Whereas within the villi the epithelial cells were largely positive in the cytoplasm and nucleus, the crypts, including the stem cell-rich proliferative zone, were mostly negative. Interestingly, other tissues rich in mitotic cells, such as the epidermis and the proliferative zone of the hair bulb, often showed little or no staining for p97 (data not shown).Table ISummary of the localization of p97TissueCell typeNucleusCytoplasmSmall and large intestineCryptsProliferative zone−−Bottom+/−+/−Villi++++++TestisInterstitial cells++Basal epithelial cells+/−++Suprabasal epithelial cells+++++Spermatozoa−+/−LiverHepatocytes++/−+Kupffer cells−−Bile duct cells++++KidneyTubule cells++++Glomerula cells+/−+/−SpleenLymphocytes−−Megakaryocytes+++Dorsal skinFibroblasts++Endothelial cells−−Epidermal cells++Hair bulbProliferative zone−−Outer root sheath++++PancreasAcinar cells+/−+/−BrainNeurons+/−+/−Expression levels of p97 in adult mouse tissues examined by immunohistological methods are scored from (−) to (+++); cell-to-cell heterogeneity of p97 levels was observed within given tissue, herein indicated with (+/−). Subcellular p97 levels are indicated. Open table in a new tab Expression levels of p97 in adult mouse tissues examined by immunohistological methods are scored from (−) to (+++); cell-to-cell heterogeneity of p97 levels was observed within given tissue, herein indicated with (+/−). Subcellular p97 levels are indicated. As with the small intestine, cells within the liver exhibited heterogeneity in the levels of p97 protein. Yet, in contrast to the uniform cytoplasmic and nuclear staining observed in the intestinal villi, hepatocytes often showed relatively weak cytoplasmic staining and strongly positive nuclei (Fig. 6 b). Interestingly, however, nuclear staining was not seen in all hepatocytes (arrowhead in b′). Kupffer cells were consistently negative (arrow in b′), whereas the cells of the bile duct were strongly positive in both cytoplasm and nucleus (arrow in b). In testis (Fig.6 c), the interstitial cells were uniformly stained with a relatively strong cytoplasmic and a weak nuclear signal (arrowhead); however, in the seminiferous tubules, differential staining was observed between basal and suprabasal populations, suggesting heterogeneity in p97 expression during different stages of spermatogenesis. Differential levels of p97 were also observed within the kidney (Fig. 6 d). Immunohistochemical analysis of variously staged mouse embryos also revealed a widespread, but non-uniform pattern of p97 protein expression. Wholemount stainings from 9.5 and 10.5 dpc embryos suggested expression of p97 within most regions of the developing embryo, with specific elevations within the emerging limb buds, tail bud, branchial arches, and somites (Fig.7, a and b). Furthermore, immunostaining of sectioned embryos and comparison to parallel hematoxylin and eosin sections revealed that highly elevated levels of p97 were present within one layer of the somites, most likely the myotome (Fig. 7, d and e). Limb buds exhibited moderate levels of p97 throughout the mesenchyme, but higher levels of staining in a subset of cells within the apical ectodermal ridge and surface epithelium (d" @default.
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• W2000620535 title "The Mouse p97 (CDC48) Gene" @default.
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