From the Laboratory of Viral Oncology CNRS UPR 9045, IFC1, 94801 Villejuif cedex, France, the ¶ Laboratoire de
Neuropathologie Raymond Escourolle, Groupe Hospitalier
Pitié-Salpêtrière, INSERM U360, Association Claude
Bernard, 75651 Paris cedex 13, France, the
Laboratoire de
Virologie, Hôpital Saint Vincent de Paul, 75674 Paris cedex 14, France, and the ** Laboratoire de Neurovirologie CEA, 92265 Fontenay aux
Roses cedex, France
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ABSTRACT |
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We have recently described a novel mRNA denominated ScRG-1, the level of which is increased in the brains of Scrapie-infected mice (Dandoy-Dron, F., Guillo, F., Benboudjema, L., Deslys, J.-P., Lasmézas, C., Dormont, D., Tovey, M. G., and Dron, M. (1998) J. Biol. Chem. 273, 7691-7697). The increase in ScRG-1 mRNA in the brain follows the accumulation of PrPSc, the proteinase K-resistant form of the prion protein (PrP), and precedes the widespread neuronal death that occurs in late stage disease. In the present study, we have isolated a cDNA encoding the human counterpart of ScRG-1. Comparison of the human and mouse transcripts firmly established that both sequences encode a highly conserved protein of 98 amino acids that contains a signal peptide, suggesting that the protein may be secreted. Examination of the distribution of human ScRG-1 mRNA in adult and fetal tissues revealed that the gene was expressed primarily in the central nervous system as a 0.7-kilobase message and was under strict developmental control.
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INTRODUCTION |
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The transmissible spongiform encephalopathies (TSE)1 are in a group of progressive neurodegenerative diseases that includes human pathologies such as Creutzfeldt-Jakob disease (CJD), Gerstman-Sträussler-Scheinker syndrome and Kuru, and animal diseases such as scrapie and bovine spongiform encephalopathy (1).
To identify the genes the altered expression of which is associated with or may even be responsible for the neurodegenerative changes observed in TSE, we have systematically analyzed modifications of gene expression in scrapie-infected mouse brain using "mRNA differential display" (2). This approach has led to the detection of an increased level of expression of eight cellular genes. One of these genes, denominated scrapie-responsive gene 1 (ScRG-1), previously unrecognized, is expressed principally in the brain. Enhanced expression of ScRG-1 in the brain of scrapie-infected mice occurs concomitantly with increased expression of GFAP mRNA, a marker of astrocytosis (3). Moreover, ScRG-1 mRNA was found to be preferentially expressed in cells of glial origin and to encode a protein with a putative signal peptide (2). These observations suggest that ScRG-1 may play a role in the host response to prion-associated infections. Previous reports have suggested that certain molecules enhanced in TSE may be detrimental to neurone survival (4-6). However, the role of overexpressed proteins (7-9), including ScRG-1, in the pathogenesis of TSE remains to be determined. We report herein the nucleotide sequence, size characterization, and tissue distribution of human ScRG-1 mRNA.
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EXPERIMENTAL PROCEDURES |
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RNA Extraction and Northern Blot Hybridization-- RNA was extracted from the brains of either mock infected C57Bl/6 mice or mice infected with the C506M3 strain of scrapie, as described previously (2). Total RNA was extracted by the method of Chirgwin et al. (10) from the frontal cortex obtained at autopsy from a patient free from any neurological disease (patient 941005, 46 years old) and from a patient with typical neuropathological findings of sporadic Creutzfeldt-Jakob disease (patient 93005, 59 years old). The diagnosis was confirmed by the presence of the proteinase K-resistant form of PrP in the sample (data not shown). Samples of normal human brain and of the brain from a patient diagnosed with CJD were obtained by informed consent, under the auspices of the Program de Recherche sur les Encéphalopathies Spongiformes Sub-aigües Transmissibles et les Prions (CNRS, France). Human poly(A)+ mRNA was obtained as described previously (2). Northern blots were performed using glyoxal denaturation, and the blotted membranes were hybridized using probes radiolabeled to a specific activity of at least 1 × 109 cpm/mg, as described previously (2). The blots were first exposed to autoradiography and then quantified using a PhosphorImager (Molecular Dynamics). The multiple human tissue Northern blot and the membrane containing the RNA dots from different human tissues were from CLONTECH laboratories.
Cloning and Sequencing of the Human cDNA-- 1 µg of poly(A)+ mRNA from the human control sample was primed with oligo(dT) and converted into double strand cDNA using standard procedures. One-twentieth of the cDNA synthesized was amplified by polymerase chain reaction using specific forward (5'-TAAGGGAAAATCACGCTGTG-3') and reverse (5'-CTTTTATTACTACTTGTTTAACAC-3') primers and Taq DNA polymerase. The amplified product was purified, sequenced using the Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech), and further cloned in the pCR2.1 Topo plasmid vector from Invitrogen.
Cloning and Sequencing of Mouse ScRG-1 cDNA--
Mouse
ScRG-1 cDNA was isolated by screening a library of whole
BALB/c adult brain cDNA cloned in gt11, with the ScRG-1 cDNA clone 24 previously isolated by RACE (2) used as a probe. The cDNA
inserted in the selected lambda (clone 1) was isolated and further
sequenced.
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RESULTS AND DISCUSSION |
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Isolation of Human ScRG-1 cDNA-- To isolate the human counterpart of the mouse ScRG-1 cDNA, the murine sequence (2) was compared with the randomly isolated human cDNA sequences reported as expressed sequence tags. The sequences potentially related to the mouse cDNA were combined in a contig and used to establish a human consensus sequence corresponding to a putative 902-bp cDNA. To ascertain the existence of the human ScRG-1 mRNA, primers derived from the 5' and 3' ends of the consensus sequence were used to synthesize cDNA from human brain mRNA by specific reverse transcription and polymerase chain reaction amplification. An unique cDNA fragment of the predicted size was obtained, cloned, and sequenced (Fig. 1A). Mapping using expressed sequence tag cDNA related to ScRG-1 indicated that the human gene was located on the long arm of chromosome four (4q31-4q32).
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Characterization of Human Brain ScRG-1 mRNA--
Northern blot
analysis was carried out using RNA from both human and mouse brain to
determine the size of the transcripts in the two species. 14 individual
RNA species were used as molecular weight markers. The blot was split
in two parts to separate the mouse and human samples, and each part was
hybridized under stringent conditions with a radiolabeled probe derived
from murine and human ScRG-1 cDNA, respectively (Fig.
2, A and B). One
band corresponding to 0.7 kb in size was detected in the human samples
and as expected, two bands of 2.6 and 0.7 kb were detected in the
murine samples. A very faint band of 2.6 kb was also revealed upon
overexposure of the autoradiograms of the human blot (data not shown).
The faster migrating band was relatively broad, with an estimated size
of between 0.66 and 0.82 kb (mean size of 0.74 kb) in both species.
Quantification by PhosphorImager indicated that the 0.7-kb message
represented at least 75-80% of the ScRG-1 transcripts in murine brain
and about 98% of the transcripts in human brain. The relative
abundance of the ScRG-1 mRNA in this organ was about 40 times less than the level of -actin mRNA in both species.
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Tissue Distribution and Developmental Expression of Human ScRG-1 mRNA-- Although mouse ScRG-1 mRNA was found to be expressed preferentially in brain tissue (2), the expressed sequence tags related to ScRG-1 and used to define primers to isolate the human cDNA (see above) were recovered from various tissues including brain, testis, aorta, and pregnant uterus. It was of interest therefore to determine the specificity of expression of the gene in different tissues, using a membrane to which poly(A)+ RNA from 50 human tissues have been immobilized in separate dots (Fig. 3). ScRG-1 is abundantly expressed in the central nervous system of the adult, in all the areas of brain investigated, and in spinal cord but is poorly or not expressed at all in fetal brain, indicating marked developmental regulation of the gene. A high level of ScRG-1 transcripts was also observed in testis and aorta, organs with low specificity of expression, which is in agreement with the report of expressed sequence tags from these tissues. We also analyzed, by Northern blotting, mRNA from various human tissues, heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas for the presence of ScRG-1 mRNA (Fig. 2C). The ScRG-1 message was highly expressed in the brain as expected and also to a 6-fold lesser extent in the heart. A faint signal was also detected upon overexposure of the blot in almost all the organs examined, indicating that the low level of hybridization observed in most of the human organs and shown in Fig. 3 corresponds to a real albeit low level of expression (data not shown).
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Conclusions-- The increased level of expression of ScRG-1 transcripts in scrapie-infected mouse brain (2) and probably also in the brain of CJD-infected individuals suggests that ScRG-1 may be involved in the neurodegenerative process in TSE. Indeed, the ScRG-1 protein may play an important physiological role in the central nervous system, as indicated by the high degree of conservation of its amino acid sequence in both man and mouse, the presence of a cleavable signal peptide indicating that the ScRG-1 protein is secreted outside the cell, and its high level of expression in the central nervous system. The potential importance of the gene is also emphasized by the observation that its expression is under developmental regulation.
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FOOTNOTES |
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* This work was supported by grants from CNRS (Programme de Recherche sur les Encéphalopathies Spongiformes Sub-aigües Transmissibles et les Prions, Action Concertée Coordonnée Number 2), from INSERM, and from the Association Nouvelles Recherches Biomédicales.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) AJ223206 and AJ224677.
§ Recipient of a fellowship from the Association Recherche et Partage (Paris, France).
To whom correspondence should be addressed: Lab. of Viral
Oncology, 7 rue Guy Moquet, BP8, 94801 Villejuif, France. Tel.: 33-1-49-58-34-22; Fax: 33-1-49-58-34-44; E-mail:
mdron{at}infobiogen.fr.
1 The abbreviations used are: TSE, transmissible spongiform encephalopathies; CJD, Creutzfeldt-Jakob disease; PrP, prion protein; RACE, rapid amplification of cDNA ends; ORF, open reading frame; kb, kilobase(s); contig, group of overlapping clones; bp, base pair(s).
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REFERENCES |
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