Proc. Nati. Acad. Sci. USA Vol. 89, pp. 9097-9101, October 1992 Genetics

Molecular cloning of a candidate chicken prion protein JEAN-MARC GABRIEL*, BRUNO OESCH*t, HANS KRETZSCHMARt, MICHAEL SCOTT*, AND STANLEY B. PRUSINER*§¶ Departments of *Neurology and §Biochemistry and Biophysics, University of California, San Francisco, CA 94143; and tNeuropathology Institute, University of Munich, Munich, Federal Republic of Germany

Communicated by Gerald D. Fischbach, May 12, 1992

ABSTRACT Fractions enriched for acetylcholine receptor-inducing activity from chicken brain were found to contain a protein that was -30% homologous with mmalian prion proteins [Harris, D. A., Falls, D. L., Johnson, F. A. & Fischbach, G. D. (1991) Proc. NatI. Acad. Sci. USA 88, 7664-76681. To extend these observations, we recovered genomic clones encoding a putative chicken prion protein (PrP). Like mammalian PrP molecules, the candidate chicken PrP Is encoded by a single-copy gene and the entire open reading frame is found within a single exon. All of the structural features of mammalian PrP were found in the chicken protein. When the N-terminal repeats of PrP were not considered, the chicken and mamalian proteins were -55% homologous, allowing for conservative substitutions. Screening of a chicken genomic DNA library failed to identify a more closely related chicken PrP homologue. These findings argue that the protein which purifies with acetylcholine receptor-inducing activity is chicken PrP.

the putative ORF of ARIA and probe cP2 (5'-GTGCATGGCAAAAGGGGTGGA-3') to the last five codons of the ORF. Probes were purified by HPLC before use. Plaque Screening and Purification. In experiment 1, about 600,000 phages of a male Leghorn chicken genomic library constructed in AEMBL3 (Clontech) were screened overnight at 650C with 5 pmol of probe cP1 end-labeled with [y-32P]ATP (Amersham). Filters were washed in 2x standard saline citrate/0.05% NaDodSO4 for 1 hr at room temperature and then twice at 650C for 10 min and air-dried. After autoradiography, positive plaques were further purified two more times as described above with the exception that the stringent posthybridization temperature was 680C. A total of 8 independent clones were isolated. In experiment 2, the same number of phages was screened, except that the stringent temperature was 680C for the three rounds of plaque purification. Ten independent clones were isolated. The 18 recombinant clones were plated at a density of 2000 plaques per small Petri dish. After plaque-lifting on small nitrocellulose filters (Schleicher & Schuell), each filter was cut in half before overnight hybridization at 530C with the cP1 or cP2 32P-labeled probe. After rinsing for 1 hr at room temperature, each half-filter was cut in eight pieces, and two pieces of each of the 18 filters hybridized with either of the probes were washed for 10 min in 3 M tetramethylammonium chloride (12)/2 mM EDTA/50 mM Tris'HCl, pH 8.0/0.2% NaDodSO4 successively at 55, 60, 65, and 70'C. Plate lysate stocks were prepared (12) by infection at high multiplicity, and phages were purified by polyethylene glycol-8000 precipitation, chloroform extraction, and ultracentrifugation. Subcloning and Sequencing. Purified DNA of clone A7AB.1 was digested to completion with Sal I and HindIII, and the resulting 2670-base-pair (bp) fragment containing the ORF was purified by gel electrophoresis and ligated into pBluescript KS(+) vector (Stratagene). After plasmid minipreps were obtained by alkaline lysis, three positive clones were identified by restriction enzyme analysis and Southern blot hybridization with cP1 probe. DNA of clone A8AA.1 was digested to completion with HindIll, and the resulting 2970-bp fragment was purified by gel electrophoresis and ligated to the same vector. After alkaline denaturation of plasmid maxipreps, the resulting clones (pB7HS.10-1 and pB8H.8) were used for supercoil plasmid dideoxy sequencing with Sequenase version 2.0 (United States Biochemical) and [a-[35S]thio]ATP (Amersham). Southern Blots. Ten milligrams of chicken genomic DNA was digested with BamHI, Bgl II, EcoRI, HindIII, or Pst I

Prions cause degenerative diseases of the central nervous system in both humans and animals and are composed largely, if not entirely, of the scrapie isoform of the prion protein (PrPsc) (1-4). The chromosomal PrP gene encodes both PrPSc and the cellular isoform, PrPC. The entire open reading frame (ORF) of the PrP gene is contained within a single exon, which is separated from one or two exons encoding the 5' untranslated region of PrP mRNAs by a large intron. The cell surface localization of PrPC together with its highly regulated expression in the brains of neonatal hamsters suggested that PrPC might function in cell recognition (5, 6). Of particular interest are studies of a putative chicken brain factor called acetylcholine receptor-inducing activity (ARIA) (7), which shares structural features with mammalian PrP (8, 9). There is a hydrophobic region where 24 consecutive amino acids are identical between the putative ARIA protein and the PrP of the I/Ln mouse (10). From these observations, it was suggested that the putative ARIA protein may represent the chicken homologue of PrP (8, 9). Whether ARIA and a candidate chicken PrP molecule copurify coincidentally or they are one and the same remains uncertain (9). These findings prompted us to search for clones encoding a putative PrP in chicken. We report here studies on a genomic clone encoding a candidate chicken PrP and describe its relationship to the mammalian PrP molecules. 1

METHODS Oligonucleotide Probe Synthesis and Purification. Two synthetic oligonucleotide probes were synthesized according to the reported partial cDNA sequences of a candidate clone encoding ARIA (11): probe cP1 (5'-ATGGCTAGGCTCCTCACCACCTGC-3') corresponds to the first five codons of

Abbreviations: PrP, prion protein; ORF, open reading frame; GPI, glycoinositol phospholipid; ARIA, acetylcholine receptor-inducing activity; PAUP, phylogenetic analysis using parsimony. tPresent address: Brain Research Institute, University of Zurich, Zurich, Switzerland. ITo whom reprint requests should be addressed at: Department of Neurology, HSE-781, University of California, San Francisco, CA 94143-0518. "The sequence presented in this paper has been deposited in the GenBank data base (accession no. M95404).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9097

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and loaded into each slot of a preparative 1.5% agarose gel. After electrophoresis and transfer to nitrocellulose, the Southern blots were processed as described (10), except that the final posthybridization washes were done twice at 420C or 550C for 10 min. The Sal I/HindIII-digested insert fragment of clone A7AB.1 was used as a probe and labeled by randomprimer nucleotide incorporation with the Klenow fragment of DNA polymerase and [a-32P]dATP (Amersham) to a specific activity of 1.5 x 109 dpm/,ug. DNA and Protein Analysis. The EuGene DNA and Protein Analysis software package, version 3.2, of the Molecular Biology Information Resource (Baylor College of Medicine, Houston) was used. PrP amino acid sequences of chicken, bovine six- or five-octapeptide-repeat alleles (13) (M.S., unpublished data), sheep (14) (D. Westaway, personal communication), rat (15), mouse A and B (10), Armenian, Chinese, or Syrian hamster (3, 16), mink (H. Kretzschmar, personal communication), and human (4, 17) were aligned in this order by the Feng and Doolittle algorithm (18). In order to keep gaps where they should be according to the nucleotide sequence, "X" residues were introduced into the amino acid sequence, and phylogenetic trees were built by the progressive alignment program of Feng and Doolittle (19). Phylogenetic analysis using parsimony (PAUP) (20) was then used to select trees satisfying the most parsimonious criterion. Both heuristic and exhaustive search procedures were used with the steepest option in effect, and branch swapping was performed on each minimal-length tree by the tree bisection-reconnection option. Gaps were treated as "21st amino acid" and not as missing data. The missing N terminus of the rat PrP sequence was replaced by the mouse sequence in the final alignment.

RESULTS AND DISCUSSION Isolation of Genomic Clones Encoding Putative PrP. A chicken genomic DNA library was screened with the cP1 oligonucleotide sense probe (see Fig. 2) (11, 12). The initial screening was performed at 130C below the calculated melting temperature in order to detect imperfectly hybridizing DNA sequences corresponding to the first five codons of the signal-peptide sequence. During plaque purification of the clones in the first experiment, the filters of two clones consistently displayed a much weaker hybridizing signal than the filters of the other six clones. In contrast, the autoradiogram of all the clones isolated in a second experiment, where a more stringent temperature was chosen, showed an intense signal. To screen all 18 clones for the presence of DNA sequences homologous with both oligonucleotide probes and to detect the presence of possible mismatches, plaque lifts were washed after hybridization at increasing stringency in the presence of 3 M tetramethylammonium chloride (12). The autoradiogram revealed that 16 clones contained DNA sequences that bound uniformly both probes at a temperature very close to the respective calculated melting temperatures (21, 22). Two clones (A8AA.1 and A18BA.1) from experiment 1 showed hybridization with the 5'-end cP1 oligonucleotide only under conditions of reduced stringency. The purified A phage DNAs of six clones from the first experiment were digested with Sal I and the digestion products were electrophoresed in a 0.35% agarose gel overnight. The insert varied in size from 11 to 20 kilobases (kb). Restriction enzyme analysis with EcoRI or Nco I followed by Southern blotting with either the 5'-end cP1 oligonucleotide sense probe or the 3'-end cP2 oligonucleotide antisense probe (see Fig. 2) showed that five of the clones contained sequences homologous to both probes and revealed a similar restriction pattern. However, another clone (A8AA.1) did not contain a DNA sequence homologous to the 3'-end cP2

Proc. Natl. Acad. Sci. USA 89 (1992)

oligonucleotide probe. It is unlikely that this clone was truncated at the 3' end, because Southern blotting with the 5'-end cP1 probe revealed a distinct restriction pattern. This was also confirmed with the HindIII restriction enzyme pattern as described below. Restriction analyses of clone A7AB.1 and clone A8AA.1 were followed by Southern blotting with the I-L/F antisense oligonucleotide probe, which corresponds to a part of the region where 24 residues are identical in the putative ARIA protein and I/Ln mouse PrP (10). The length of this probe is 19 nucleotides and it has a melting temperature of 580C. Posthybridization washes at 40, 48, and 53TC for 10 min showed binding for clone A7AB.1 but not for clone A8AA.1. Thus, clone A7AB.1 was thought to be related to both the putative chicken ARIA and I/Ln mouse PrP sequences. Chicken PrP Gene Is Single-Copy. Fig. 1 shows a Southern blot with chicken genomic DNA probed with the radiolabeled 2.7-kb insert fragment of one of the 16 candidate chicken PrP clones, A7AB.1. This probe detected a single band migrating at 23, >23, 8.1, or 4.6 kb when hybridized to DNA digested with BamHI, EcoRI, HindIII, or Pst I, respectively. In contrast, digestion with Bgl II gave two bands migrating at 2.7 or 2.3 kb. Identical Southern blots were obtained by washing at either low or high stringency. A Southern blot of 1 of the 16 candidate chicken PrP clones (A1OBA.1) with the 5'-end cP1 probe or the 3'-end cP2 probe revealed an 8-kb HindIII

fragment.

Sequencing of the subclone pB7HS.1 was performed by primer walking and ambiguities were solved by sequencing the corresponding opposite strand. A single ORF of 819 nucleotides (Fig. 2) encodes a protein of 273 amino acids whose calculated molecular mass is 29,912 daltons. Two ATG methionine codons are present at nucleotides 25 and 37; however, only the second conforms to the consensus sequence for initiation of translation (23). By comparing the cDNA sequence (9) with our genomic sequence, we identified a 3' splice acceptor consensus site between nucleotides 28 and 34. In chicken, the coding exon would therefore begin 2 nucleotides before the putative initiator methionine codon, whereas the hamster, mouse, and sheep coding exons begin 10 nucleotides before the initiation codon (10, 14, 24). The protein sequence deduced from the chicken genomic clone is identical to that deduced from the cDNA clone (9) 2

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Proc. Natl. Acad. Sci. USA 89 (1992) 40

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except for two sites. (i) The protein deduced from genomic DNA contains a serine at position 156 (AGC) instead of the arginine (AGA) encoded by the cDNA. (ii) The genomic DNA clone pB7HS encodes nine imperfect hexapeptide repeats instead of the eight encoded by the cDNA clone. At the nucleotide level, only three of the hexapeptide repeats deduced from the genomic DNA clone are identical, whereas two repeats of the cDNA clone are identical. This finding suggests that the extra repeated element, denoted HR3 (see Fig. 4), is located somewhere between residues 54 and 71 of the pB7HS clone. At the amino acid level, six of the hexapeptide repeats are identical, suggesting that these repeated elements have arisen by a duplication phenomenon that may have occurred quite recently in the White Leghorn chicken. The second population of chicken genomic DNA clones was found to be unrelated to the clones described above. Southern blots with the cP1 probe identified one subclone (pB8H.8) displaying a single band migrating at 6 kb. Sequencing primed with the cP1 probe gave a deduced amino acid sequence that was S^GGOWG 4. SH.; P OS v PfROa YPTH C. ovyiHN p

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Molecular cloning of a candidate chicken prion protein.

Fractions enriched for acetylcholine receptor-inducing activity from chicken brain were found to contain a protein that was approximately 30% homologo...
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