From the Cell Biology Laboratory, Imperial Cancer
Research Fund, Lincoln's Inn Fields, London WC2A 3PX,
Developmental Neurobiology, National Institute of Medical
Research, The Ridgeway, Mill Hill NW7 1AA, and the ** Department of
Histopathology, Imperial College School of Medicine, Hammersmith
Hospital London, Ducane Road, London W12 0HS, United Kingdom
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ABSTRACT |
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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.
p97 belongs to the family of ATPases
associated with diverse cellular activities
(AAA)1 occurring in
eubacteria, archaebacteria, and eukaryotes (1). 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 (2). AAA family members include proteins involved in vesicle
and organelle biogenesis (3, 4), components of the 26 S proteasome (5),
metalloproteases (6), cell cycle regulators (7), and transcription
factors (8).
Mammalian p97 (first termed VCP, for valosin-containing protein) was
originally described as a precursor protein containing the biologically
active peptide valosin (9). Subsequently, in Xenopus laevis,
a 14 S, homo-oligomeric ATPase was identified as the homologue of
mammalian VCP (10), and in Saccharomyces cerevisiae, genetic
alterations in the p97 homologue (CDC48) were shown to underlie a
mitotic-arrest phenotype (7, 11). More recently, highly conserved p97
homologues have been identified in a diverse range of experimental
organisms, including Drosophila melanogaster (TER94) (12),
Arabidopsis thaliana (AtCDC48) (13), and the
archaebacterium Sulfobus acidocaldarius (SAV) (14), 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 (10, 15).
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, named cdc48-1, arrest in mitosis as large budded cells with
elongated nuclei spanning the mother-daughter junction (11). p97 has
been proposed to function in homotypic membrane fusion events,
including fusion of the endoplasmic reticulum (16) and the reassembly of Golgi cisternae from vesicles and tubules generated by treatment with specific drugs (17) or mitotic cytosol (4). 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 (18). Protein interaction studies have
also revealed that p97 binds to clathrin in a stoichiometric complex,
suggesting a role for p97 in the endocytic cycle (19). p97 has been
shown to physically associate with Ufd3p, a protein that is involved in
the regulation of free cellular ubiquitin in yeast (20), and to
copurify with the mammalian 26 S proteasome (21). Furthermore, in these
same experimental systems, immunodepletion or inactivation of p97
severely compromised ubiquitin-dependent proteolysis of
experimental substrates in vitro and in vivo (20, 21).
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.
Gene Isolation and Physical Mapping--
A Fluorescence in Situ Hybridization--
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.
DNA Sequencing--
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 (22). Alignment of cDNA and genomic DNA sequence was
performed by using the GCG analysis suite (version 9.0).
Southern Blot Analysis--
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.
Primer Extension Analysis--
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) Construction of Promoter-Luciferase Fusion Vectors--
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.
Cell Culture and Transient Transfection--
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 (23). Alkaline phosphatase activity was determined
as described previously (24) 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).
Immunostaining of Mouse Tissues--
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.
mRNA in Situ Hybridization--
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 (25), with modifications (26). The presence of
hybridizable mRNA in all compartments of the tissues studied was
established in near serial sections using an antisense Identification of Two Different p97 Loci in M. musculus--
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
The presence of two mouse p97 loci had recently been proposed based on
interspecies backcross mapping and polymerase chain reaction analysis
(27). Fluorescence in situ hybridization using the isolated
Genomic Organization of the p97 Genes--
The genomic structure
of both genes was further characterized by nucleotide sequence
analysis. Analysis of the gene present in
Alignment of nucleotide sequences of
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 (
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) Characterization of the Promoter of p97--
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
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 Distribution of p97 in Mammalian Tissues--
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. 6
and 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.2
An example of the differential expression of p97 could be observed in
the intestinal epithelium (Fig. 6a). 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).
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. 6b). 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.
6c), 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. 6d).
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 (data not shown). Furthermore, as seen in
adult tissue specimens, p97 expression within many embryonic tissues
exhibited distinct cell-to-cell heterogeneity. Examples of this
heterogeneity are shown for the condensing mesenchyme of the kidney
anlagen (10.5 dpc; Fig. 7f) and the Wolffian duct epithelium
(10.5 dpc; Fig. 7g), and at a later stage of development within a subset of chondrocytes in the hypertrophic zone of developing limb cartilage (13.5 dpc; Fig. 7h). Heterogeneous staining
was also observed within the spinal cord (data not shown). In the embryonic tissues analyzed, increased expression of p97 was associated with an apparent accumulation of the protein in the nucleus.
Finally, mRNA expression in situ was examined to
determine if the tissue-specific expression of p97 protein was
associated with similar alterations in mRNA levels. Shown in Fig.
8 is an example of results from a
comparison of p97 and Tissue-specific and Heterogeneous p97 Expression in the
Mouse--
p97 is a highly conserved and ancient protein with
homologous counterparts found in organisms ranging from archaebacteria to man (13). Evidence from several different organisms and experimental systems has suggested disparate roles for this protein in cellular physiology. Based on the evolutionary conservation and widespread abundance of p97, it had earlier been suggested that it is a
ubiquitously expressed component (10), and several of the currently
proposed cellular functions for p97 fall into the category of
"housekeeping" tasks, implying a constitutive role for p97 in all
cells (10, 15, 19, 20). However, our current findings, as well as
recent work from other investigators, support the prospect that p97 may perform a more regulated, rather than constitutive function in cells.
Mouse p97 protein and mRNA is indeed found widely in both
developing embryos and the adult, but appears to be regulated in a cell
type- and cell differentiation-dependent manner within a
given tissue. For example, p97 was enriched in embryos within the
myotome region of the somites, was differentially expressed within
regions of the adult small intestine, and varied among cell types and
developmental stages in the testis. Likewise, in Drosophila,
the p97 homologue has been recently reported to undergo temporal and
tissue-specific regulation, being undetectable in larvae and found
predominantly in the brain and gonads of the imago and adult fly
(12).
The most surprising finding to come from the immunohistochemical
analyses was that p97 levels varied greatly within neighboring cells of
both established and developing tissues, such as hepatocytes, kidney
epithelium, and embryonic chondrocytes. This pattern was not found in
control stainings for the Golgi apparatus protein giantin. Thus, these
findings suggest a specific cell-to-cell regulation of p97 protein
levels, which one could envision as a cell autonomous response to
alterations in subcellular physiology, or a single cell's response to
local micro-environmental cues. Whether this cell-to-cell
heterogeneity, as seen for the tissue-specific regulation of p97,
results from regulation at the mRNA level is still unknown.
However, the variable degree of p97 nuclear staining found in our
studies, as well as independent work showing cell cycle regulation of
p97/Cdc48p nuclear import (31) and inducible tyrosine phosphorylation
of p97 (32), together point toward a significance for
post-translational regulation in p97 function.
p97 Gene Structure and Identification of the Promoter--
The
molecular cloning and analysis of the p97 gene and regulatory sequences
should facilitate dissection of the molecular basis for p97's complex
expression pattern and the functional relevance. Characterization of
the genomic structure of the p97 locus demonstrated that the gene
encoding the functional mRNA encompasses 20.4 kb and is interrupted
by 16 introns. The two AAA domains of p97 are encoded within 6 (N-terminal AAA) and 4 exons (C-terminal AAA). The gene is flanked by a
TATA-less/GC-rich upstream region and is likely to utilize an initiator
element, spanning the mRNA start site, to direct basal
transcription. Transient transfection assays in HeLa cells provide
direct evidence that this region is sufficient for a relatively robust
level of basal transcription. Cis-acting elements necessary for basal
transcription activity seem to reside within 410 bp of the 5'-flanking
region of the gene. Correspondingly, this upstream segment contains
consensus binding sites for several transcriptional activators which
have been implicated in basal gene transcription and also growth
factor/cytokine, developmental, or stress-induced expression, including
CBF, Sp1, AP-2, junB, Ets-1, and Y-box proteins.
Analysis of the phage clone encoding a second p97 gene, termed
In contrast to Mus musculus, p97 is thought to be encoded by
a single, intron-containing gene in humans and Mus spretus
(27). Together with the lack of evidence of a function for pseudogenes and the apparent absence of a cDNA corresponding to the Functional Clues from p97 Expression in Vivo--
The
tissue-specific and heterogeneous expression of p97 in vivo
provides evidence for a regulated or intermittent role for p97. Based
on previous investigations, it could be suggested that the protein's
regulated role is in proliferating cells, particularly during mitosis.
Yeast cdc48-1 is a mutant arresting in mitosis with undivided nuclei
(7), and the CDC48 protein is a nuclear and peripheral endoplasmic
reticulum/nuclear envelope protein, which has been speculated to
regulate the nuclear fusion step required for the completion of mitosis
(16). In mammalian systems, p97 has also been implicated in the fusion
step required for reassembling mitotic Golgi fragments into stacks at
telophase (4). Also, the results obtained from our analysis of p97 in
embryonic mouse tissues and in work on developing
Arabidopsis (13) are consistent with a role for p97 in
expanding/proliferating tissues, and the heterogeneous p97 levels
observed in the embryo could potentially reflect an increase in p97
protein in actively cycling cells.
However, our data and recent data from other investigators do not
support a sole function for p97 in dividing cells. p97 levels appeared
to be most abundant in the majority of highly differentiated, non-proliferating cells throughout the adult. In fact, p97 was significantly less abundant within proliferative zones, including the
crypts of the small intestine, the hair bulb, and the epidermis. Similarly, the Drosophila homologue of p97 is abundant in
non-proliferating cells, predominantly in the nervous system and in the
reproductive organs (12). Moreover, recent biochemical and genetic data
point to a role for p97 in intracellular signaling (22) and targeted proteolysis (21), which are obviously prevalent in both proliferating and differentiated tissues. Finally, the findings presented here, as
well as those discussed above emphasize the necessity of accounting for
p97's complex regulation in vivo in any future models of
p97 function and provide the initial framework for a relatively
unexplored experimental approach to address the cellular and organismal
role(s) of this multifaceted protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
DASH 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.
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.
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
phage library. Southern blot analysis on
digested phage DNA using probes spanning different regions of the
published cDNA (22) was performed to further characterize the
isolated clones. Two clones, designated
1 and
2, extended 20 and
15.6 kb, respectively, and overlapped by 3.7 kb.
2 spanned the 5'
end and
1 the 3' end of the p97 cDNA sequence (Fig.
1a). A third clone, designated
3, 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
3 and
1/2 differed (Fig. 1a). 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
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
1/2, while the second fragment corresponded to a fragment in
3, strongly suggesting the presence of
two different p97 loci (results for
2 versus
3 are
shown in Fig. 1b).
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Fig. 1.
Genomic structure of the mouse p97
genes. a, physical map of the mouse p97 genes as
established from 1/2 and
3; sites are marked for the enzymes
BamHI (B), EcoRI (E),
HindIII (H), and XhoI (X).
The open reading frame of p97 is indicated in black shading
and the untranslated regions in gray shading. b,
Southern blot analysis of phage and genomic DNA. DNA was digested (10 µg) with the indicated enzymes and analyzed in Southern blot using a
mouse cDNA probe covering the base pairs 14-159 of the published
mouse cDNA.
clones as probes confirmed this genetic analysis. The overlapping
clones
1 and
2 reside on mouse chromosome 4 at position B2, while
clone
3 resides on chromosome X at position C-D (data not shown).
1/2 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.
2a). Exons were on average 150 bp in size, and both nucleotide-binding cassettes (2) 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. 1a).
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Fig. 2.
Nucleotide sequence analysis of genomic
regions encoding p97. a, exon-intron organization of
the mouse p97 gene. Exon and intron sizes were established by dideoxy
sequencing and polymerase chain reaction analysis using subcloned
fragments of 1/2. Nucleotide sequence of exon-intron borders are
indicated. b, alignment of homologous p97 cDNA sequences
from different organisms to amino acid positions 568-573 of the mouse
p97 cDNA. All data base cDNAs encode the amino acids AP at
positions 571-572, which are absent in the potential coding region of
p97.
3 to the published mouse
cDNA (22) 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
3 had all the hallmarks expected of a processed pseudogene
(designated herein as
p97) (28), implying that the gene does not
encode a functional product, as reported for many other pseudogenes
(29).
1/2), but which was deleted in
p97
(Fig. 2b). Therefore, we tentatively concluded that
p97 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
1/2, which appeared to encode the
functional p97 mRNA.
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).
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Fig. 3.
Primer extension analysis of the p97
transcription start site. Primer extension was carried out using
poly(A)+ and poly(A) RNA from mouse embryonic
fibroblasts using primer 1 (see "Materials and Methods"). A
sequencing ladder of the corresponding genomic fragments primed with
the same oligonucleotide was electrophoresed in parallel. A single
major extension product was identified, indicating the transcription
initiation site (the complementary nucleotide is shown by an
asterisk); nucleotides encompassing the initiator element
are shown.
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) (30).
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Fig. 4.
Nucleotide sequence of the 5'-flanking region
of the p97 gene. The transcription start site is marked with an
asterisk and designated +1. The initiator element and the
translation start are indicated. Consensus binding sites for several
transcription factors (CBF, Sp1, AP-2, junB, Ets-1, and Y-box proteins)
are indicated.
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.
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Fig. 5.
Functional analysis of the mouse p97
promoter. A series of constructs of different sized p97 promoter
fragments fused to a luciferase reporter were tested for their ability
to initiate transcription in transiently transfected HeLa cells. The
luciferase activities were normalized for transfection efficiency using
placental alkaline phosphatase activity, and are expressed as the level
of luciferase activity relative to a promoterless control plasmid. A
reporter construct driven by the SV40 promoter/enhancer was used as
positive control.
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Fig. 6.
Immunohistological localization of p97 in
adult mouse tissues. The brown reaction product produced by DAB
immunohistochemistry denotes positively stained cells. Sections were
counterstained with hematoxylin. a, villi and crypts in the
small intestine; a', higher magnification of the border
between positive and negative (arrow) epithelium.
b, detail of liver tissue near bile duct (arrow);
b', hepatocytes. Note negative nuclei of some hepatocytes
(arrowhead) and Kupffer cells (arrow).
c, cross-section of seminiferous tubule with interstitial
cells in testis. c', staining was uniform in the cytoplasm
of interstitial cells (filled arrowhead), absent in myoid
cells (empty arrowhead), and the majority of leptotene
spermatocytes (arrow); strong cytoplasmic and nuclear
staining was present in basal and suprabasal layers, which primarily
consist of type A spermatogonia (asterisk). Spermatids and
spermatozoa nuclei were relatively weakly stained. d, kidney
shows little staining within a glomerulus and strong staining of
various parts of the uriniferous tubules of the renal cortex;
d', a cross-section of a proximal tubule in which the
majority of cells exhibited a strong cytoplasmic and nuclear staining.
Occasionally, however, negative nuclear staining (arrowhead)
and negative cells were observed (arrow).
Summary of the localization of p97
) to (+++); cell-to-cell
heterogeneity of p97 levels was observed within given tissue, herein
indicated with (+/
). Subcellular p97 levels are indicated.
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Fig. 7.
Immunohistological localization of p97 in
mouse embryos. The brown reaction product produced by DAB
immunohistochemistry denotes positively stained cells. a-c,
whole mount immunohistochemical analysis of 9.5 dpc (a) and
10.5 dpc (b and c) mouse embryos using
affinity-purified p97 antisera (a and b), or as a
negative control, staining with secondary antibody alone
(c). Staining was observed throughout the embryo, but was
highest in limb buds (a and b; empty
arrowheads), tail bud (a; large black
arrowhead), branchial arches (b; black
arrowhead), and somites (a; small
arrowheads). d-h, immunohistochemical staining of
paraffin sections. Low level p97 staining (arrows) was
observed throughout tissue sections, but staining was elevated in
certain regions (arrowheads). Within the 10.5 dpc somites
(d; asterisk denotes the spinal cord),
examination at higher magnification (e) revealed high levels
of p97 in the myotome region. In addition to elevation in defined
tissues, cell-to-cell heterogeneity in p97 staining was observed within
the 10.5 dpc kidney anlagen (f; asterisk denotes
the dorsal aorta) and Wolffian duct (g), and the
hypertrophic zone (h; black arrowhead) and to a
lesser extent the peripheral regions (h; empty arrowhead) of
13.5 dpc limb cartilage.
-actin mRNA expression in the testes.
Unlike
-actin mRNA, which was abundant and widespread across
most cellular regions of the seminiferous tubules (the exception being
the mature sperm), p97 mRNA was differentially expressed within
cells of the tubule (arrow). Similar to our observations for
p97 protein (see Fig. 7), elevated mRNA levels were highest in
basal and suprabasal cells (arrowheads) located 1-2 cell
layers from the basement membrane. A comparison of actin and p97
mRNA expression in several other adult and embryonic mouse tissues (data not shown) consistently yielded a similar contrast in the distribution pattern of mRNAs from the "housekeeping" gene
-actin and p97, and thus suggested a general role for
tissue-specific regulation, rather than constitutive production of p97
mRNA. These results also suggest that a contributing factor to the
tissue-specific control of p97 protein expression is the regulation of
mRNA levels.
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Fig. 8.
Analysis of p97 mRNA expression in adult
testis. Isotopic in situ hybridization of mouse testis
using p97 (a, b, e, and f)
and -actin (c and d) antisense riboprobes.
Conventional lightfield (a and c) and darkfield
(b and d) microscopic detection of riboprobe
distribution in a cross-section of seminiferous tubules demonstrates
the restricted distribution of p97 mRNA to the basal and near-basal
layers of the tubule (arrow), and for comparison, the
relatively widespread localization of
-actin transcripts.
e and f, light- and darkfield views of p97
riboprobe distribution from a magnified region show suprabasal cells
with elevated mRNA levels (arrowheads).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
p97,
revealed that the region features all the hallmarks of a processed
pseudogene, which are thought to evolve by random insertion of a
reverse transcript of the mRNA into the genome (28). Since most
processed pseudogenes derive from integration of reverse transcribed
mRNA generated by RNA polymerase II, the sequence homology between
pseudogenes and their functional counterpart ceases at the points
corresponding to the beginning and the end of the transcript. However,
p97 contained an additional 15 bp of 5'-flanking genomic sequence
that was identical to sequences found in the corresponding
untranscribed region of the functional p97 gene
counterpart.3 Other cases
have been reported in which the homology between processed pseudogenes
and their counterparts extends beyond the transcription start site, and
it has been suggested that these pseudogenes may result from
transcription via RNA polymerase III (28).
p97
sequence, it therefore seems unlikely that
p97 is functionally
significant. However, since the pseudogene has an intact open reading
frame, and thus the potential to encode for a nearly intact p97
protein, further studies are required to confirm that
p97 is
functionally inactive. We are currently in the initial stages of
analyzing conditional gene disruption mutants of p97 that have been
engineered in mouse embryonic stem cells. Our observations suggest
that, similar to yeast, p97 is an essential gene in the
mouse,4 thus demonstrating
that the p97 gene and pseudogene are not functionally redundant members
of a multi-gene family.
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ACKNOWLEDGEMENTS |
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We thank Jill Williams for fluorescence in situ hybridization analysis; Graham Clark and Andrew Davies for DNA sequencing assistance; George Elia and the ICRF Histology Unit for their contributions to the immunohistochemistry; Richard Poulsom, Rosemary Jeffery, and Jan Longcroft for skilled assistance with the in situ hybridization studies; Richard Mortenson for providing the genomic library; and Lawrence Samelson for the kind gift of the mouse p97 cDNA.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF122047 and AF122048.
§ Supported by the Boehringer Ingelheim Fund.
¶ Supported by an Imperial Cancer Research Fund (ICRF) fellowship and the Deutsche Forschungsgemeinschaft.
Supported by the ICRF and a Hitchings-Elion fellowship from the
Burroughs Wellcome Fund. To whom correspondence should be addressed:
Cell Biology Laboratory 624, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, England. Tel.: 44-0171-269-2880; Fax: 44-0171-269-417; E-mail:
shima{at}icrf.icnet.uk.
2 D. T. Shima and C. Ruhrberg, unpublished observation.
3 J. M. M. Müller and D. T. Shima, unpublished observation.
4 J. M. M. Müller and D. T. Shima, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: AAA, ATPases associated with diverse cellular activities; bp, base pair(s); kb, kilobase pair(s).
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REFERENCES |
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