Method of detecting small cell carcinoma and use of acylpeptide hydrolase encoding sequences therefor

Abstract: The invention relates to the use of the Acyl-Peptide Hydrolase-encoding sequences in the detection of cancer.

Description: The invention is directed toward the diagnosis of small cell carcinoma through the use of a gene sequence which encodes the rat liver acyl-peptide hydrolase. Since the discovery of an acetyl group at the amino-terminus of tobacco mosaic virus coat protein, a number of N α -acetylated proteins have been found in animals, plants, and their viruses, and also in bacteria and fungi. N α -acetylation is therefore considered one of the typical modifications of proteins in living organisms. Moreover, in some eukaryotic cells, it has been suggested that more than 80% of the intracellular soluble proteins are N α -acetylated (Brown, J.L., J. Biol. Chem. 254 :1447-1449 (1979)). The biological significance of N α -acetylation of proteins is still an open question (see Tsunasawa et al ., Method Enzymol. 106 :165-170 (1984)). It has been proposed that this post-translational modification protects intracellular proteins from proteolysis. However, this does not hold true for all proteins. In the case of actin from slime mold, proteolytic degradation becomes slower when the protein is N α -acetylated. In contrast, cat hemoglobin is degraded at the same rate irrespective of N α -acetylation (Tsunasawa et al .,1984). Recent results from DNA sequencing have shown that in structural genes for the secretory proteins that are N α -acetylated, the codon for the acetylated amino-terminal residue is directly preceded by the initiation codon without the insertion of additional codons for amino acids (Tsunasawa et al ., 1984). Little effort has been made to understand the relationship between N α -acetylation and the transport of secretory proteins across biological membranes. To understand completely the function of N α -acetylation, it will be important to identify the N α -acetylated amino acids in proteins and peptides on a microanalytical scale. For this purpose, removal of the N α -acetyl group or the N α -acetyl amino acid must be efficiently achieved. Acyl-Peptide Hydrolase (APH) has been successfully used for the hydrolysis of N α -acylated peptides. One such enzyme, which was purified from animal liver, can liberate the N α -acetyl amino acid from rather short peptides derived from N α -acetylated proteins (Tsunasawa et al ., 1984). The substrate specificity is broad for the amino terminal residue. APH cleaves the N α -terminal acetylated or formylated amino acid from a blocked peptide (Jones et al ., BBRC 126 :933 (1985)). This enzyme catalyzes the hydrolysis of a diverse number of peptides and displays different pH optima for certain substrates in doing so. This enzyme may also play a pivotal role in the processing of polypeptide chains during biosynthesis. APH has been purified from rat liver (Tsunasawa et al ., J. Biochem . 77 :89-102 (1975)); [from bovine liver (Gade et al ., Biochim. Biophys. Acta 662 :86-93 (1981))]; from porcine liver (Tsunasawa et al ., J. Biochem. 93 :1217-1220 (1983)); from rat brain (Marks et al ., J. Neurochem. 41 :201-208 (1983)); and from human erythrocytes (Jones et al ., Biochem. and Biophys. Res. Comm . 126 :933-940 (1985)). A rat liver acyl-peptide hydrolase (APH), which catalyzes the hydrolysis of the acetylated residue from N α -acetylated peptides was recently purified to homogeneity, and various inhibition experiments indicated that it was likely a serine protease, utilizing a charge relay system involving serine, histidine, and probably a carboxyl group (Kobayashi, K. and Smith, J.A., J. Biol. Chem. 262 :11435-11445 (1987)). However, it is not yet clear whether acyl-peptide hydrolase is a unique serine protease. In order to facilitate a more complete understanding of the regulation of rat acyl-peptide hydrolase in vivo, it is, therefore, desirable to clone and sequence the rat acyl-peptide hydrolase gene. The rat liver Acyl-Peptide Hydrolase and methods of production and use are disclosed in EP 0,303,997 A2 which falls under article 54(3) EPC. Acyl-peptide hydrolase catalyzes the hydrolysis of an N α -acetylated amino acid residue from an N α -acetylated peptide. Two overlapping, degenerate oligonucleotide probes based on the sequence of a tryptic peptide, derived from purified rat acyl-peptide hydrolase, were synthesized and used to screen a rat liver λgt11 cDNA library. A 2.5 kb cDNA was cloned and sequenced. This clone contained 2364 bp of rat acyl-peptide hydrolase sequence but lacked an initiation codon. Using a 220 bp probe derived from the 5'-end of this nearly full-length cDNA to rescreen the library, full-length clones were isolated, which contained an in-frame ATG codon at nucleotides 6-8 and encoded the NH₂-terminal sequence, Met-Glu-Arg-Gln···. The DNA sequence encoded a protein of 732 amino acid residues, 40% of which is confirmed by protein sequence data from 19 CNBr or tryptic peptides. The isolated enzyme is NH₂-terminally blocked (Kobayashi, K., and Smith, J.A. (1987) J. Biol. Chem. 262:11435-11445), and based on the NH₂-terminal protein sequence deduced from the DNA sequence and the sequence of the most NH₂-terminal CNBr peptide, it is likely that the NH₂-terminal residue is an acetylated methionine residue, since such residues are frequently juxtaposed to glutamyl residues (Persson, B., et al. , (1985) Eur. J. Biochem. 152 , 523-527). The RNA blot analysis revealed a single message of 2.7 kb in various rat tissues examined. Although this enzyme is known to be inhibited by diisopropyl fluorophosphate and acetylalanine chloromethyl ketone (Kobayashi, K., and Smith, J.A. (1987) J. Biol. Chem. 262 :11435-11445), no strong similarity in protein sequence has been found with other serine proteases. This result suggests that acyl-peptide hydrolase may be a unique serine protease. The invention pertains to a method of detecting small cell carcinoma which comprises: a. incubating a nucleic acid sample from a patient suspected of having small cell carcinoma, in the presence of a nucleic acid molecule having a sequence selected from the group consisting of: a. a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; and b. a sequence which is complementary to a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; the incubation being under conditions sufficient to permit nucleic acid hybridization to occur between the nucleic acid sample and the nucleic acid molecule, and to thereby form a hybridized molecule; and b. detecting, such as by an analysis of restriction fragment length polymorphisms, small cell carcinoma by determining whether the hybridized molecule differs in sequence from a reference molecule, the reference molecule comprising a nucleic acid sample from a normal individual hybridized to a nucleic acid molecule which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1. The invention further includes a two stranded nucleic acid molecule comprising: A. a first strand having a sequence selected from the group consisting of: a. a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; and b. a sequence which is complementary to a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; the first strand being hybridized to: B. a second strand, the second strand having a sequence which is substantially complementary in sequence to the sequence of the first strand, the complementary sequence of the second strand being derived from an individual suspected of having small cell carcinoma. DESCRIPTION OF THE FIGURES Figure 1 illustrates the amino acid sequence of APH. The protein sequence deduced from the cDNA sequence (Figure 3) is indicated by the one letter code for the amino acids. The bracket lines indicate the termini of the CB, CB-R, and CB/R peptides. The arrows pointing right indicate that the corresponding amino acid residue was identified as the Pth-amino acid residue during automated Edman degradation (Table 1). A blank indicates that a Pth amino acid was not identified in this degradative cycle. An asterisk indicates that a Pth-Trp together with an unidentified late-eluting Pth-derivative was identified instead of Pth-Lys during this degradative cycle. Cysteine residues were identified as Pth-derivatives of [¹⁴C] S-carboxymethylcysteine. The active serine is shown at positions 620-627 of Figure 1 (diagonal-line filled box). The identification of peptides shown here is defined in Table 3. Figure 2 illustrates the cloning and sequencing of the cDNA encoding rat acyl-peptide hydrolase. (A) Oligonucleotide probes used for the initial screening of the rat liver λgt11 cDNA library. The amino acid sequence of an RPLC-purified tryptic peptide (CB18-R11-13-c; Table 3) was used as the basis for the synthesis of two overlapping degenerate oligonucleotides, YS17.2 and YS20.1. (B) Restriction map and DNA sequencing strategy of the clones. Using the degenerate oligonucleotides in Figure 2A, APH5.2 was obtained from a rat liver λgt11 cDNA library, as described below. The arrows indicate the direction and extent of DNA sequence determination for each fragment. DNA sequence analysis for this clone revealed the expected hybridization site near its 5′ end (open region in bold line), a poly(A) sequence at its 3′ end, and an unrelated sequence at its 3′ end (cross-hatched box). After rescreening the rat liver λgt11 cDNA library with the XmnI-KpnI fragment derived from APH5.2 (longer open box), APH36.1 lacked an ATG initiation codon and also contained a 120 base pair fragment encoding rat serum albumin (box with diagonals). After rescreening the library with a 220 bp BanII-PstI fragment derived from APH36.1 (shorter open box), APH2.7 was cloned, which was subsequently subcloned into Bluescript plasmid (Stratagene) and sequenced in part. Abbreviations: B, BanI; P, PstI; X, XmnI; and K, KpnI. Figure 3 shows the nucleotide sequence and deduced amino acid sequence of rat liver acyl-peptide hydrolase. The complete cDNA encoding rat liver acyl-peptide hydrolase was derived by combining the DNA sequence data from APH36.1 and APH2.7 (Figure 2B). The deduced protein sequence is indicated by the one-letter code for the amino acids. Figure 4 shows the nucleotide sequence of the rat acyl-peptide hydrolase gene and its flanking region. The transcriptional initiation site of the gene is indicated by vertical arrow. The nucleotide at this position is assigned at number 1. The intronic DNA sequence is shown in lowercase letters and the exonic DNA sequence is shown in uppercase letters. The beginning and end of each intron are marked by vertical lines. The translational initiation site is located at nucleotides 625-627. The polyadenylation signal is located at nucleotides 9708-9713. The "TATA" box-like sequence (nucleotides -24 to -30) and the "CAAT box"-like sequence (nucleotides -95 to -99) are boxed. The GC repeats are underlined. Tandem 200 bp repeats are indicated by a dashed underline. Figure 5 shows a structural organization of the rat acyl-peptide hydrolase gene. Figure 5A shows overlapping λ recombinant phages containing the acyl-peptide hydrolase gene. The overlapping genomic clones, APHE5 and APHH6, together containing the entire acyl-peptide hydrolase gene, are indicated by solid horizontal lines. Figure 5B shows the restriction map of the acyl-peptide hydrolase gene and its flanking regions. The EcoRI (E), BamHI (B), HindIII (H), and PstI (P) sites are indicated by vertical bars. The 5′ (left) to 3′ (right) transcriptional orientation of this gene is shown. Figure 5C shows the exon-intron organization of rat acyl-peptide hydrolase gene. The location of the 23 exons within the rat acyl-peptide hydrolase gene are indicated by filled boxes. The locations of the translational initiation codon, ATG, and the polyadenylation signal, AATAAA, are marked by vertical lines. Figure 6 shows a comparison of the amino acid sequences of acyl-peptide hydrolase and the DNF 15S2 protein. In the Figures, the amino acids have been designated by single letters of the alphabet such that: A = Alanine, B = Aspartic Acid or Asparagine, C = Cysteine, D = Aspartic Acid, E = Glutamic Acid, F = Phenylalanine, G = Glycine, H = Histidine, I = Isoleucine, K = Lysine, L = Leucine, M = Methionine, N = Asparagine, P = Proline, Q = Glutamine, R = Arginine, S = Serine, T = Threonine, V = Valine, W = Tryptophan, Y = Tyrosine, Z = Glutamine or Glutamic Acid. Definitions To aid in the understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. Transcription . The process of producing mRNA from a structural gene. Translation . The process of producing a polypeptide from mRNA. Expression . The process undergone by a structural gene to produce a polypeptide. It is a combination of transcription and translation. Plasmid . A circular double-stranded DNA molecule that is not a part of the main chromosome of an organism containing genes that convey resistance to specific antibiotics. When the plasmid is placed within a unicellular organism, the characteristics of that organism may be changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (Tet R ) transforms a cell previously sensitive to tetracycline into one which is resistant to it. A cell transformed by a plasmid is called a "transformant." Cloning Vehicle . A plasmid, phage DNA or other DNA sequences which are able to replicate in a host cell. The cloning vehicle is characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the DNA, which may contain a marker suitable for use in the identification of transformed cells. Markers, for example, are tetracycline resistance or ampicillin resistance. A cloning vehicle is often called a vector. Recombinant DNA Molecules or Hybrid DNA . A molecule consisting of segments of DNA from different genomes which have been joined end-to-end outside of living cells and have the capacity to infect some host cell and be maintained therein. Operator . A DNA sequence capable of interacting with the specific repressor, thereby controlling the transcription of adjacent gene(s). Promoter . A DNA sequence in which RNA polymerase binds and initiates transcription of an adjacent gene(s). Acyl-Peptide Hydrolase (APH) . This term is meant to include an acyl-peptide hydrolase(s) from any species, which has the activity of releasing the N α -terminal acylated amino acid from any protein or peptide in an in vivo or in vitro system. The term acyl-peptide hydrolase is also used in this invention to include any analogue, homologue, mutant or derivative of a naturally occurring acyl-peptide hydrolase, which cleaves the N α -acetylated amino acid from the N α -terminal portion of a peptide or a protein. The term is also meant to include fragments having less than the naturally-occurring number of amino acids, such as partial fragments of natural acyl-peptide hydrolases which retain the activity of cleaving the acylated amino acid from the N-terminal end of a protein or peptide. The term is also used to include any product which comprises the sequence of a naturally occurring acyl-peptide hydrolase or analogue thereof, together with one or more flanking amino acids, which show acyl-peptide hydrolase activity. The term acyl-peptide hydrolase also includes synonyms such as acyl-amino acid releasing factor, acyl-amino acid releasing enzyme, acyl-amino peptide hydrolase and acetylaminoacyl-p-nitroanilidase. Substantially Pure Form . As used herein, the term "substantially pure" or "substantially purified" is meant to describe the protein which is substantially free of any compound normally associated with the factor in its natural state. The term is further meant to describe the factor which is homogeneous by one or more purity or homogeneity characteristics used by those of ordinary skill in the art. For example, a substantially pure factor will show constant and reproducible characteristics within standard experimental deviations for parameters such as the following: molecular weight, chromatographic techniques and such other parameters. The term, however, is not meant to exclude artificial or synthetic mixtures of the factor with other compounds. The term is also not meant to exclude the presence of minor impurities which do not interfere with the biological activity of the factor, and which may be present, for example, due to incomplete purification. The molecular weight of rat liver APH, as estimated by gel filtration, is 290,000-320,000. There appear to be four identical subunits, with one active serine per subunit. The N α -terminus of the APH is acylated. APH appears to be a serine protease, with a charge relay system involving serine, histidine and carboxyl groups. The active serine is shown at positions 620-627 of Figure 1 (diagonal-line filled box). The amino acid sequence of this site is MGGSHGGF. The environment of the active site differs from other proteases of the trypsin family, due to the presence of histidine, and the lack of aspartic acid. Although APH displays broad specificity for substrates, it cleaves Ac-Ala-, Ac-Ser-, and Ac-Met- containing peptides (the most common N-terminal acetylated residues) more effectively than other acylated dipeptides. APH has very low or no activity toward Ac-Trp-, Ac-Asp-, Ac-Glu, Ac-Arg-, Ac-Phe, and Ac-Pro- containing peptides. Acyl-Peptide Hydrolase (APH) should be distinguished from N α -acetyltransferase, which catalyzes the reaction in which a protein accepts the acetyl group from an acetyl-CoA (Tsunasawa et al ., Methods in Embryology 106 :165-170 (1984)). Acyl-Peptide Hydrolase should also be distinguished from Aminoacylase (Szajani, Acta Biochim . et Biophys . Acad . Sci . Hung . 15 :223-228 (1980)) [also known as α-N-Acylamino acid hydrolase (Gade et al. , Biochim. Biophys.Acta 662 :86-93 (1981))]. The DNA sequence coding for APH may be derived from a variety of sources. For example, mRNA encoded for APH may be isolated from the tissues of any species that produces APH, by using the Northern blot method (Alwine et al. , Method Enzymol . 68 :220-242 (1979)), and labeled oligonucleotide probes. The mRNA may then be converted to cDNA by techniques known to those skilled in the art. The probes may be synthesized based on the known amino acid sequence of APH peptides. Alternately, degenerative DNA probes may be used to screen a DNA library of a species that produces APH, thereby isolating a clone that contains the DNA sequence encoding APH. The DNA library is created by the fragmentation, using one or more restriction endonucleases of the genomic DNA, followed by incorporation into vectors, and use thereof to transform host cells, which are then plated and screened. The DNA probe may be labeled with a detectable group. Such detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of immunoassays and in general most any label useful in such methods can be applied to the present invention. Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem. 22 :1243 (1976)), enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Pat. Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (see U.S. Pat. No. 4,134,792); fluorescers (see Clin. Chem . 25 :353 (1979)); chromophores; luminescers such as chemiluminescers and bioluminescers (see Clin. Chem. 25 :512 (1979)); specifically bindable ligands; proximal interacting pairs; and radioisotopes such as ³H, ³⁵S, ³²P, ¹²⁵I and ¹⁴C. Such labels and labeling pairs are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors). For example, a cofactor-labeled probe can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme. For example, one can use an enzyme which acts upon a substrate to generate a product with a measurable physical property. Examples of the latter include, but are not limited to β-galactosidase, alkaline phosphatase and peroxidase. A DNA sequence encoding APH may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. A prokaryotic host with a plasmid containing the cDNA encoded for APH has been deposited on August 21, 1987 at the American Type Culture Collection, Rockville, MD, USA, and given accession number ATCC 67504. Having now generally described this invention, the same will be better understood by reference to specific examples, which are included herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified. EXAMPLES Example 1 --Extraction and Purification of Acyl-Peptide Hydrolase (APH) Materials -- DEAE Sepharose® CL-6B, FPLC columns (Mono Q HR5/5, and Mono S HR5/5), Sephacryl® S-300 superfine, Octyl-Sepharose®, and Polybuffer 74 were from Pharmacia. Spherogel® CAA-HIC column (0.46 x 10 cm) was from Beckman. Hydroxylapatite (Biogel HT) was from Bio-Rad. Glycerol was from BRL. Reactigel 6X was from Pierce. Amino acids (Ac-L-Ala) were from Sigma. All other chemicals were reagent grade or better. Enzyme Purification -- APH was purified from 300 g of rat liver (male, CD strain) as described by Tsunasawa et al ., J. Biochem. (Tokyo) 77 :89-102 (1975), except for the substitution of DEAE-Sepharose CL-6B and Sephacryl S-300 for DEAE cellulose and Sepharose 6B, respectively. The column sizes and gradients were also changed. For hydroxylapatite chromatography, the starting gradient was 5 mM phosphate buffer instead of 20 mM phosphate, and 10% glycerol was used in the gradient. Four mg of purified enzyme were obtained. During DEAE-Sepharose CL-6B chromatography, an increase in total activity was observed. In order to confirm the homogeneity of the protein from the Sephacryl S-300, additional chromatography was carried out: (i) ion-exchange chromatography with Pharmacia FPLC system on Mono Q and Mono S with various buffers at pH's between 5 and 8; (ii) hydrophobic interaction chromatography on Octyl-Sepharose and Spherogel CAA-HIC; (iii) chromatofocusing on Mono P with Polybuffer 74; and (iv) affinity chromatography using Ac-L-Ala-- Sepharose, prepared from Reacti-Gel 6X (Pierce) and acetyl-L-alanine. In no case was further separation or increased activity observed. The purification is summarized in Table 1. Purification of Acyl-peptide Hydrolase from Rat Liver Step Activity (unit) Protein (mg) Specific Activity (unit/mg) Yield (%) Purification 1 Homogenate 194 44200 0.00439 100 1.00 2 12000 x g Supernatant 194 39400 0.00492 100 1.12 3 Ammonium Sulfate (20-50%) 150 25400 0.00591 77 1.35 4 Heat Treatment 139 2520 0.0552 65 11.5 5 DEAE-Sepharose 208 29.3 7.10 108 1630 6 Hydroxylapatite 148 5.90 25.1 76 5780 7 Sephacryl S-300 118 4.04 29.2 61 6090 Example 2 --Amino Acid Sequencing of Tryptic and Cyanogen Bromide Fragments of APH Materials --APH was purified as in Example 1. Purity was confirmed by SDS polyacrylamide gel electrophoresis by the method of Laemmli, Nature 227 :680-685 (1970). UV measurements were obtained using a Hewlett-Packard 8450A UV Spectrophotometer. The amount of protein was determined by the method of Bradford, M.M. ( Anal. Biochem . 72 :248-254 (1976)) using bovine serum albumin as a standard and expressed in nmol of rat liver acyl-peptide hydrolase subunlt, assuming that 1 nmol of enzyme refers to 1 nmol of the M r = 80,000 subunit of the enzyme (Kobayashi, K. and Smith, J.A., J. Biol. Chem. 262 :11435-11445 (1987)) Radioactive samples were counted on a Beckman LS 3801 scintillation counter. Cyanogen bromide, guanidine-HCl, 2-mercaptoethanol, trifluoroacetic acid (TFA), were obtained from Pierce. Acetonitrile (HPLC grade UV cut-off 188 nm) was from J.T. Baker. Trypsin treated with N-tosyl-PheCh₂Cl was purchased from Worthington. Bradford protein assay reagent and electrophoresis reagents were obtained from Bio-Rad, except for molecular weight markers and Tris, which were purchased from Sigma. Zwittergent 3-14 was from Calbiochem and [¹⁴-C] iodoacetic acid (9.8 mCi/ mmol) was from New England Nuclear. All other reagents were the purest grade that was commercially available. Amino Acid Analysis --The acyl-peptide hydrolase was dialyzed extensively against 0.1 M acetic acid, lyophilized, and hydrolyzed at 110°C for 24 hr and 48 hr in 6 M HCl containing 0.1% phenol. The amino acid composition was determined using a Beckman Amino Acid Analyzer (see Moore, S., In: Chemistry and Biology of Peptides , (Meinhofer, J., Ed.), pp.629-652, Ann Arbor Science, Ann Arbor, MI (1972)) (Table 2). Reduction of Disulfide Bonds and Alkylation with Iodoacetic Acid -- Purified rat APH (3 nmol) was dissolved in 0.5 M Tris-HCl (pH 8.5) containing 7 M guanidine HCl/2 mM EDTA, and reduced with 8-10 mM 2-mercaptoethanol under argon at a room temperature for 12 hr or at 37°C for 3 hr. To the mixture (0.19 ml), [¹⁴C] iodoacetic acid (2.6 µmol in 30 µl 0.5 M Tris-HCl (pH 8.5)/7 M guanidine HCl/2 mM EDTA) was added and the reaction was carried out for 1 hr at 37°C in the dark. 2-Mercaptoethanol was then added to a final concentration of 0.2 M. The protein was desalted either by precipitating with for volumes of acetone/methanol (3:1 :v/v) or by a dialysis against 0.1 M acetic acid and lyophilized in a Savant concentrator/evaporator. Cyanogen Bromide Cleavage --The carboxymethylated protein was dissolved in 70% formic acid (0.1-0.2 ml), to which 10-15 µl CNBr solution (100 mg/ml in 70% formic acid) was added. The mixture was incubated at room temperature for 24 hr and vacuum dried after the dilution with water. The CNBr-cleaved peptide fragments were purified by reversed-phase HPLC (RPLC) or by lyophilization in a Savant concentrator/evaporator or further fragmented by tryptic digestion. Digestion with Trypsin --The crude mixture of CNBr peptides (3 nmol) were dissolved in 0.2 ml of 0.2 M ammonium bicarbonate containing 0.2% Zwittergent 3-14 and digested with trypsin (50 pmol) treated with N-tosyl-PheCH₂Cl for 20 hr at 37°C. The digest was vacuum dried and dissolved in 6 M guanidine HCl in 0.1% TFA for RPLC purification. Purification of Peptide Fragments by Reversed-Phase HPLC --The peptides were purified by RPLC on a Beckman HPLC system 344, using a C₄ column (Vydac, 0.46 x 25 cm, 10 micron particle with 300 Å pore size) for CNBr fragments or a Phenyl column (Vydac, 0.46 x 25 cm, 5 micron particle with 300 Å pore size) for tryptic fragments. The crude peptide mixture was applied to the column equilibrated with 0.1% TFA and eluted with 0-80% linear gradient of acetonitrile in 0.1% TFA (for CB-R and CB peptides) or with 0-60% acetonitrile in 0.1% TFA (for CB/R peptides) in 160 min at a flow rate of 1 ml/min. A mixture of tryptic peptides derived from a crude mixture of CNBr peptides was applied as described above, and eluted with a 0-60% linear gradient of acetonitrile in 0.1% TFA in 180 minutes at a flow rate of 1 ml/min. The elutions were monitored both by 214 nm and 280 nm absorbance. Each peak was collected manually, and, if necessary, further purified by isocratic RPLC using the same column after being dried and redissolved in 0.2 ml of 6 M guanidine HCl-0.1% TFA. The optimum concentration of acetonitrile for separating the peptides each fraction was estimated from the elution pattern of the first HPLC (see equation of Wong et al. , Proc. Nat'l Acad. Sci . USA 82 :7711-7715 (1985)). Peptide Sequencing --Peptide sequence analyses were carried out using an Applied Biosystems 470A Protein Sequencer and an Applied Biosystems 120A Pth Analyzer (see Hewick et al ., J. Biol. Chem. 256 :7900-7997 (1981)) (Table 3). Example 3 --Preparation of Probes Construction of Probes --Two overlapping, degenerate oligonucleotide probes, YS20.1 and YS17.2 (Figure 2A), derived from the amino acid sequence of peptide CB18-R11-13c (Table 3) were synthesized and used to screen a rat liver λgt11 cDNA library. The oligonucleotide probes and primers were synthesized with an Applied Biosystems 380A DNA synthesizer using β-cyanoethyl phosporamidites (Sinha, N.D. et al. , Nucl. Acid Res 12 :4539-4557 (1984)) and purified by polyacrylamide gel electrophoresis according to the Applied Biosystems Manual or by ethanol precipitation from a solution of oligonucleotide containing 10 mM MgCl₂. YS17.2 and YS20.1 represent pools of 128-fold degenerative oligonucleotides. The YS17.2 and YS20.1 pools were 17 and 20 nucleotides in length, respectively. The two probes overlap by 12 nucleotides, such that sequential use of the probes to screen a DNA library would effectively screen for a 25 nucleotide piece of APH encoded DNA. Example 4 --Creation and Screening of the cDNA Library and Sequencing APH Encoded cDNA. Preparation of RNA --Strain CD rat liver was quick-frozen in liquid N₂ and thawed in guanidine isothiocyanate, and the RNA was purified by centrifugation through CsCl (Chirgwin et al. , Biochem 18 :5294-5299 (1979)). The yield was 600 µg of RNA. Poly(A)+ RNA was selected by passage of the total RNA through an oligo(dT) cellulose column (Aviv, H. et al. , Proc. Natl. Acad. Sci. (U.S.A.) 69 :1408-1412 (1972)). Forty five µg of poly(A)+ RNA were obtained and shown not to be degraded by RNA blot analysis of 1 µg of RNA by hybridizing with an action cDNA probe (Spiegelman, B.M. et al. , J. Biol. Chem. 258 :10083-10089 ((1983)). Preparation of cDNA Library --Complementary DNA was synthesized from 10 µg poly(A)+ RNA by the method of Gubler, U. et al. ( Gene 25 :263-269 (1983)), and cloned into the λgt11 (Young, R.A. et al. , Proc. Natl. Acad. Sci. (U.S.A.) 80 :1194-1198 (1983)), as described by Klickstein, L.B. (In: Current Protocols in Molecular Biology (Ausubel, F.M. et al. , Eds.) pp 5.8.1. - 5.8.4., Wiley-Interscience and Greene Publishing Associates, New York, NY (1987)). The yield of recombinants was 4 million from 100 ng of cDNA and 10 µg of λgt11 vector DNA. The library was amplified in E. coli strain Y1088 (δ lacU169 , supE , supF , HsdR ⁻, HsdM ⁺, metB , trpR , tonA21 , proC::Tn5 (pMC9)) and stored at 4°C. Isolation of CDNA Clones --The library was plated at 25,000 plaques per 150 mm plate (for screening 10⁶ or fewer plaques) or at 10⁶ plaques per 225 mm x 225 mm plate (for screening more than 10⁶ plaques), and duplicate filters were lifted from each ((Maniatis, T. et al. , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)). For screening with oligonucleotides, the oligonucleotides were 5′ end-labeled to a specific activity of 2-8 x 10⁸ cpm/µg with [γ-³²P]-ATP and T4 polynucleotide kinase (Zoller, M. et al. , DNA 3 :479-488 (1985)). The filters were hybridized with oligonucleotide in 6xSSC, 0.1% SDS,0.1%SDS, 0.05% sodium pyrophosphate, 1x Denhardt's solution and 100 µg/ml salmon sperm DNA at 65°C overnight. The T d max and T d min were calculated for each mixture of oligonucleotides with the formula: T d = 4(G+C)+2(A+T) , as previously described for short sequences (Suggs, S.V. et al. , In: Developmental Biology Using Purified Genes (Brown, D., Ed.), pp. 683-693, Academic Press, New York, NY (1981)). The sequences were washed at progressively higher temperatures in 6xSSC, 0.05% sodium pyrophosphate, and 0.1% SDS until non-specific binding was reduced. For screening with cDNA probes, (XmnI-KpnI fragment from APH5.2 or BanII-PstI fragment from APH36.1; Figure 2B) the filters were hybridized overnight with nick-translated probes in 50% formamide, 5xSSC, 5x Denhardt's solution, 10 mM sodium phosphate, 0.1% SDS, 1 mM EDTA and 50 µg/ml sonicated, denatured salmon sperm DNA at 42°C. Filters were washed in 0.2xSSC, 0.1% SDS, 1 mM sodium phosphate, pH 7.0, and 1 mM EDTA at 55°C. The washed filters were exposed to Kodak XAR film with an intensifying screen at -70°C. Phage yielding duplicate signals were plaque-purified by additional rounds of screening. DNA Sequence Analysis --Restriction fragments from APH5.2 and APH36.1 were subcloned into M13mp18 or M13mp19 and sequenced by the dideoxynucloetide chain termination method (Sanger, F. et al. , J. Molec. Biol. 94 :441-448 (1975)). The sequence of some clones was obtained by first constructing deletion mutants using exonuclease III (Henikoff, S., Gene 28 :351-356 (1984)). The cDNA Insert of APH2.7 was subcloned into Bluescript plasmid (Stratagene) and sequenced by the dideoxy chain termination method, modified for double-stranded sequencing by Guo et al. ( Nucl. Acids Res. 12 :387-394 (1983)). The DNA sequence data were analyzed with the University of Wisconsin Genetics Computer User Group programs (Devereux, J. et al. , Nucl. Acid Res. 12 :387-395 (1984)). Cloning and Sequencing of cDNA Encoding Rat Liver Acyl-Peptide Hydrolase --Twenty-Seven out of 450,000 recombinant clones were found to hybridize with probe YS20.1. Twelve of these clones were rescreened with the probe, YS17.2 to yield a single clone, APH5.2, containing a 1.3 kb insert (Figure 2B). The DNA sequence of APH5.2 encoded the entire peptide sequence of the tryptic peptide CB18-R11-13-c confirming that APH5.2 was an authentic clone. Since APH5.2 contained a poly(A)+ sequence at its 5′ end (Figure 2B, cross-hatched box), probably artifactually created during the construction of the cDNA library, an XmnI-KpnI fragment of APH5.2 was used to rescreen one million clones from the same cDNA library, and the APH36.1 clone containing a 2.5 kb insert was obtained (Figure 2B). The protein sequence deduced from the DNA sequence of APH36.1 contained all of the protein sequences in Table 3, except for the amino terminal three residues of peptide CB17-R13. However, its 5′ end contained a 120 bp fragment encoding rat serum albumin (Figure 2B, box with diagonals). In order to obtain the missing 5′ sequence data, a 220 bp BanII-PstI fragment (Figure 2B) was used to rescreen the same cDNA library. Five positive clones with different length Poly(A) tails were obtained from 5 million recombinants. Four cDNA clones, including APH2.7, started with the same nucleotide sequence and contained an in-frame ATG codon at nucleotides 6-8, while the 5′ end of the fifth cDNA clone lacked 18 base pairs. A polyadenylation signal, AATAAA, was found at nucleotides 2444-2349. Figure 3 illustrates the complete cDNA sequence for APH, as derived from APH5.2, APH36.1 and APH2.7. Primary structure of acyl-peptide hydrolase deduced from cDNA --The complete DNA sequence was determined by combining the sequences of APH36.1 and APH2.7 (Fig. 3). The DNA sequence encodes a protein containing 732 amino acid residues, assuming that the ATG at nucleotides 6-8 is the translation initiation codon. The deduced protein sequence contains all the peptide sequences in Table 3 (Fig. 1). The protein has a calculated molecular weight of 81,347, and the amino acid composition based on the deduced protein sequence agrees closely with the observed composition (Table 2). As deduced from the DNA sequence, three lysyl residues were identified at amino acid residues 118, 291, and 443, which correspond to the positions where Pth-Trp together with a late-eluting Pth-derivative were observed (Table 3 and Fig. 1). Three N-glycosylation consensus sequences (i.e., Asn-Xxx-Thr/Ser (Parodi, A.J. et al. , Biochim. Biophys. Acta 559 :1-37 (1979)) are identified at residues 134-136, 233-235, and 243-245. Hydrophobicity Analysis --The hydrophobicity profile was determined using the algorithm of Kyte, J. et al. ( J. Molec. Biol. 157 :105-132 (1982)) with a window size of 8. The deduced protein sequence of rat acyl-peptide hydrolase was compared to the National Biomedical Research Foundation and Swiss protein databases using the Wordsearch and Bestfit programs from the University of Wisconsin Genetics Computer User Group programs (Devereux, J. et al. , Nucl. Acid Res. 12 :387-395 (1984)), and the FASTP program based on the algorithm of Lipman, D.J. et al. ( Science 227 :1435-1441 (1985)). In order to identify possible active site regions in rat acyl-peptide hydrolase, its sequence was compared with the peptide sequences, containing the active-site seryl, histidyl or aspartyl residues, derived from known serine proteases. The hydrophobicity plot reveals that the protein contains a hydrophilic region located between residues 80 and 220, but it remains unclear whether this region has a specific role in interactions with other proteins or in catalysis. The computer-based search of the National Biomedical Research Foundation and the Swiss Protein databases revealed no strongly homologous proteins. In addition, the comparison between rat acyl-peptide hydrolase and short active site serine-, histidine-, and aspartic acid-containing peptides, derived from known serine proteases, failed to reveal any significant similarities. Although acyl-peptide hydrolase was previously shown to be serine protease by inhibition experiments, using diisopropyl fluorophosphate, acetylalanine chloromethyl ketone, and other enzyme inhibitors (Kobayashi, K. and Smith, J.A., J. Biol. Chem. 262 :11435-11445 (1987); Tsunasawa et al ., J. Biochem. (Tokyo) 77 :89-102 (1975)), no strong similarity between rat acyl-peptide hydrolase and active-site peptides from other serine proteases were found, suggesting that this enzyme may be a unique serine protease. Example 4 --Cloning of the Entire Rat Acyl-Peptide Hydrolase Gene Materials --Restriction enzymes, T4 ligase, T4 polynucleotide kinase, E. coli DNA polymerase I and its Klenow fragment, AMV reverse transcriptase, exonuclease III, DNase I, RNase H, T4 DNA polymerase, EcoRI methylase, calf intestinal alkaline phosphatase, and nuclease S1 were from Boehringer Mannheim and New England Biolabs. RNase A was from Sigma. The Bluescript plasmid, λgt10 arms and packaging extract were from Stratagene. [γ-³²P]ATP, [α-³²P]dCTP, and Genescreen Plus membrane were purchased from New England Nuclear. [α-³⁵S][dATPαS was from Amersham Corp. Oligo(dT)-cellulose was from Collaborative Research. Synthetic oligonucleotides were synthesized with an Applied Biosystems 380A DNA Synthesizer using the silica-based solid phase (Matteucci, M.D. et al. , J. Am. Chem. Soc. 103 :3185-3191 (1981)) and β-cyanoethyl phosphoramidite method (Sinha, N.D. et al. , Nucleic Acids Res. 12 :4539-4544 (1984)). Preparation of Rat Liver DNA and RNA --The source of rat genomic DNA and liver cytoplasmic RNA is adult Sprague-Dawley rat liver. Liver DNA was prepared as described by Blin and Stafford (Blin, N. et al. , Nucleic Acids Res. 3 :2303-2308 (1976)). Rat liver total RNA was isolated by guanidine thiocyanate method, as described by Chrigwin et al. (Chirgwin, J.M. et al. , Biochemistry 18 :5294-5299 (1979)). Polyadenylated RNA was purified by oligo(dT)-cellulose chromatography (Aviv, H. et al. , Proc. Natl. Acad. Sci. USA 69 :1408-1412 (1972)). RNA Blot Analysis --RNA was purified as described above, denatured at 65 °C and transferred to Zetabind membrane (Thomas, P.S., Proc. Natl. Acad. Sci. (U.S.A.) 77 :5201-5202 (1980)). Blots were hybridized with the nick-translated XmnI-KpnI cDNA fragment from APH5.2 in 50% formamide, 5xSSC, 5X Denhardt's solution, 10 mM sodium phosphate, 0.1% SDS, 1 mM EDTA and sonicated, denatured salmon sperm DNA (50 µg/ml). Filters were washed in 0.2xSSC, 0.1% SDS, 10 mM sodium phosphate pH 7.0 and 1 mM EDTA at 55 °C. The RNA blot analysis of total RNA, using the XmnI-KpnI cDNA fragment derived from APH5.2 as probe (Fig. 2B), revealed that a single mRNA of 2.7 kb in roughly equivalent amounts encodes acyl-peptide hydrolase in various rat tissues (i.e., spleen, muscle, lung, liver, kidney, and brain). Isolation of Genomic Clones --Two rat genomic libraries were used to screened for APH gene. One library was from Clone-tech, which was constructed by partial EcoRI digestion of Sprague-Dawley liver DNA and cloning into λ phage Charon 4A. A 2.4 kb EcoRI restriction fragment encoding rat APH derived from APH36.1 was labeled by nick translation (Sargent, T.D. et al. , Proc. Natl. Acad. Sci. USA 76 :3256-3260 (1979)) with [α-³²P]dCTP to a specific activity of 10⁸ cpm/µg and used as a probe for screening this genomic library. The other library which is constructed by partial HaeIII digestion and cloning into λ phage Charon 4A was a generous gift from Professor James Bonner of California Institute of Technology (Church, G.M. et al. , Proc. Natl. Acad. Sci. USA 81 :1991-1995 (1984)). A 200 bp BanII-PstI fragment of APH36.1 labeled by random-priming (Feinberg, A. et al. , Anal. Biochem. 132 :6-13 (1983)) with [α-³²P]dCTP to a specific activity of 10⁹ cpm/µg, was used to screen this library. Approximately 1 x 10⁶ phages from each library were screened by plaque hybridization (Church, G.M. et al. , Proc. Natl. Acad. Sci. USA 81 :1991-1995 (1984)). Positive plaques were purified, and phage DNA was isolated (Maniatis, T. et al. , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)). Analysis of DNA by Restriction Mapping and DNA Hybridization -- The restriction map of the cloned gene was constructed by digestion of phage DNA with various restriction endonucleases (Maniatis, T. et al. , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)). For DNA blot analysis, DNA restriction fragments were separated in an agarose gel, blotted onto GeneScreen Plus membrane, and hybridized to ³²P-labeled probe according to manufacturer's recommendations. Probes are three ³²P-labeled rat APH cDNA fragments of APH36.1: a 5′ 200 bp BanII-PstI fragment, a 420 bp Kpn-EcoRI fragment, and a 2.4 kb EcoRI fragment. For genomic DNA blot hybridization, the 2.4 kb EcoRI fragment of APH2.7 was used as the probe. DNA Sequencing --Restriction fragments of the rat genomic clones were subcloned into Bluescript plasmid. Both orientations of the complete sequence, as well as upstream and downstream regions, of the rat APH gene were determined by Sanger's dideoxy chain termination method (Sanger, F. et al. , Proc. Natl. Acad. Sci. USA 74 :5463-5467 (1977)), modified for double-stranded sequencing (Guo, L.-H. et al. , Nucleic Acids Res. 11 :5521-5539 (1983)), employing sequencing strategies of the DNase I deletion method (Lin, H.-C. et al. , Anal. Biochem. 147 :114-119 (1985)), exonuclease III deletion method (Henikoff, S., Gene 28 :351-359 (1984)), and synthetic oligonucleotide primers. Nucleotide sequence data were compiled and analyzed by the Genetics Computer Group Sequence Analysis Software Package, version 5.0 (Devereux, J. et al. , Nucleic Acids Res. 12 :387-395 (1984)). Preparation of a cDNA Library Containing 5′-Untranslated Region of APH mRNA --A 17 bp synthetic oligonucleotide, 5′ GTGACCTCCGGACCCAG 3′, complementary to nucleotides 95-112 of the APH2.7 was used as a specific primer to construct a cDNA library in λgt10. The synthetic oligonucleotide (1 µg) was annealed to 10 µg of poly(A)⁺RNA, and the first and second strand synthesis of the cDNA was performed by the method of Gubler and Hoffman (Gubler, U. et al. , Gene 25 :263-269 (1983)). The ends of cDNAs were blunted with T4 DNA polymerase, and internal EcoRI sites were methylated. The blunt-ended cDNAs were ligated to EcoRI linkers, and following EcoRI digestion the cDNAs were size-fractionated on a CL-4B column (Wong, W.W. et al. , Proc. Natl. Acad. Sci. USA 82 :7711-7715 (1985)). Then the cDNAs were ligated to λgt10 arms, and the recombinant phage DNA was packaged according to the manufacturer's (Stratagene) recommendations. Recombinant phages were screened with 2 synthetic oligonucleotides, 5′ AAGTCCCGGAAGTGAGG 3′ and 5′ CTGACGCTCCATAGTCG 3′, whose sequences were derived from genomic (nucleotides 586-592, Fig. 4) and cDNA sequence (nucleotldes 1-17 of APH2.7) respectively. The phage DNA with the largest insert was purified (Maniatis, T. et al. , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982)), and the insert was subcloned into Bluescript plasmid. Primer Extension --An oligonucleotide, 5′ TAGGAGTGAGAAAATCA 3′, complementary to nucleotide sequence in the first exon (nucleotides 44-60, Fig. 4) was labeled at 5′ end with [γ-³²P]ATP and T4 polynucleotide kinase and then hybridized to 10 µg of rat liver poly(A)⁺RNA in a solution of 0.1 M KCl, 5 mM EDTA,a nd 5 mM sodium phosphate, pH 6.8. For hybridization, the temperature of the solution was raised to 90°C for 5 min and returned gradually to 42°C. The RNA-DNA hybrids were then subjected to reverse transcriptase reaction. After RNase A digestion, phenol:chloroform extraction, and ethanol precipitation, the primer extension product was then electrophoresed on an 8% polyacrylamide sequencing gel. Isolation and DNA Sequence of the Rat Acyl-peptide Hydrolase Gene --Initially, a rat genomic library, constructed in Charon 4A from a partial EcoRI digest of Sprague-Dawley rat liver DNA was screened using the ³²P-labeled rat liver APH cDNA insert of APH36.1, as the probe. One million plaques were screened, and twenty-eight hybridized with the probe. All twenty-eight plaques were isolated and characterized by restriction enzyme mapping and were found to be identical. DNA blots of the restriction endonuclease digests derived from each recombinant DNA were probed with the cDNA inserts of APH36.1, the 200 bp BanII-PstI fragment (5′ end of APH36.1), and the 420 bp KpnI-EcoRI fragment (3′end of APH36.1), and each contained a 9.7 kb EcoRI fragment, corresponding to the 3′ end of the cDNA. One of these clones, APHE5 (Fig. 5A), was restriction enzyme-mapped, subcloned into Bluescript, and sequenced. A second rat genomic library, constructed by partial HaeIII digestion of rat liver DNA and cloned into Charon 4A (generously provided by Professor J. Bonner, California Institute of Technology), was screened with the 200 bp BanII-PstI fragment of APH36.1. Eight of one million plaques hybridized with the probe, and their phage DNAs were isolated. After restriction enzyme-mapping and DNA hybridization, one clone, APHH6 (Fig. 5A), overlapping with clone APHE5 and extending further in the 5′ direction was analyzed. The combined restriction map of APHE5 and APHH6 is shown in Fig. 5B. DNA blot analysis of rat genomic DNA revealed two bands corresponding to BamHI restriction fragments of about 4 and 9.4 kb. The size of the larger fragment agrees with the size calculated from the restriction map, and the smaller fragment extends beyond the 3′ end of the map (Fig. 5B). Two bands corresponding to EcoRI restriction fragments of about 7.5 and 9.7 kb were observed. The sizes of both fragments agree with the sizes determined from the restriction map (Fig. 5B). This indicates that APH gene is present in a single form in the rat genome. For sequence analysis, the individual EcoRI, PstI or BaMHI fragments were subcloned into Bluescript, and individual subclones were either subjected to limited unidirectional digestion with exonuclease III followed by S1 nuclease digestion or subjected to random, limited nicking with DNase I followed by restriction enzyme digestion to generate a nested set of deletions. Double-stranded plasmid DNA was prepared from each deletion and sequenced by the dideoxy chain termination method. For certain regions, the sequence was determined by using specific synthetic oligonucleotides as the sequencing primers. These two genomic clones were sequenced in both orientations. The complete nucleotide sequence is shown in Fig. 4. For simplicity in discussing the genomic sequence, a numbering system is used in which position +1 denotes the transcriptional initiation site of the APH gene. As shown in Fig. 5C and Table 4, the precise locations of each of the 5′ and 3′ exon-intron boundaries were defined by aligning the genomic sequence with the cDNA sequence. Analysis of the 5′-Untranslated Region of Rat Acyl-peptide Hydrolase mRNA --Because the cDNA sequence lacked about 300 base pairs of the 5′-untranslated region, estimated by comparing the size of APH mRNA and the size of cDNA, a cDNA library containing the 5′-untranslated region of APH mRNA was constructed as described above. This library was screened with two ³²P-labeled oligonucleotide probes with sequences complementary to the first seventeen bases of the cDNA clone APH2.7 or to nucleotides 586-602 of the genomic DNA (Fig. 4). A 466 bp cDNA insert was isolated, subcloned into Bluescript, and sequenced by the dideoxy chain termination method. This 5′-extension sequence contained the nucleotide sequence corresponding to nucleotides 37 to 262, 492 to 636, and 711 to 805 of the genomic DNA (Fig. 4). Therefore, the translational initiation codon, ATG, is located at nucleotides 625-627 (Fig. 4) since it is preceded in frame by a termination codon, TAG, at nucleotides 568-570 and since there is no other ATG codon in between. Determination of the Translational Initiation Site of the Rat Acyl-peptide Hydrolase Gene --The 5′ end of the APH mRNA was mapped by primer extension analysis. A ³²P-labeled oligonucleotide, 5′ TAGGAGTGAGAAAATCA 3′, complementary to nucleotides 44-60 (Fig. 4), was hybridized with rat liver poly(A)⁺ RNA. This primer was extended with reverse transcriptase in the presence of deoxynucleotides, and the length of the extended product, determined as described above, was 60 nucleotides. Furthermore, if yeast poly(A)⁺ RNA was substituted for rat liver poly(A)⁺ RNA, no extended product was observed. Transcription was found to begin with a T residue on the DNA template, which corresponds to an A residue at position 1 in Fig. 4. This A residue is situated 395 bp 5′ of the ATG translational initiation codon. In summary, these examples show that the rat APH gene is present in a single form. The complete sequence of the rat APH gene was determined, including 2.58 kb of 5′ flanking DNA, 2.75 exonic DNA, 6.94 kb of intronic DNA, and 1 kb of 3′ flanking DNA (Fig. 4). The exonic DNA data unequivocally identified the translational initiation site, corresponding the codon encoding the methionine at residue 1 of rat acyl-peptide hydrolase, since there was no ATG codon positioned between the in frame stop coding (Fig. 4, nucleotides 568-570) and the translational initiation codon in exon 2 (Fig. 4). These results also indicate that APH is not synthesized as a precursor protein, since the protein sequence of APH following the NH₂-terminal Met could be identified by automated Edman degradation following CNBr cleavage. As shown in Fig. 5C, rat APH gene, spanning 9.69 kb is divided into 23 exons. The individual exons vary in size between 41 and 262 bp (Table 4). The first intron interrupts the 5′-untranslated sequence; all of the other introns were within the protein coding region of the gene. Table 4 lists the sequences at the 5′ and 3′ junctions of each intron, and these sequences are consistent with the consensus sequences for intron-exon junctions of other eukaryotic genes (Sharp, P.A., Cell 23 :643-646 (1981); Breathnach, R. et al. , Annu. Rev. Biochem. 50 :349-383 (1981); Mount, S.M., Nucleic Acids Res. 10 :459-472 (1982)). All introns begin with the sequence GT at the 5′ boundary and end with the sequence AG to the 3′ boundary, and in all cases the intron sequences flanking the 5′ and 3′ boundaries are purine and pyrimidine-rich, respectively. The exon-intron organization of APH gene, presumably encoding a protease with active-site serine residue (Kobayashi, K. and Smith, J.A., J. Biol. Chem. 262 :11435-11445 (1987)), is much more complex than either trypsin or chymotrypsin, which contain five and seven exons, respectively (Rogers, J., Nature 315 :458-459 (1985)). The residues of the charge relay systems of these enzymes are known to be encoded by separate exons, but the distribution of the corresponding residues in APH awaits additional studies. Based on an extensive search of the National Biomedical Research Foundatlon and Swiss Protein databases, as well as a comparison of exon-intron organization of other serine proteases (e.g., trypsin, chymotrypsin, elastase, urokinase, kallikrein, adipsin), acyl-peptide hydrolase is not clearly similar to any of these serine proteases. Analysis of the 5′ flanking DNA of the APH gene revealed a number of conserved sequences. These is a sequence 5′ TGATAAA 3′, which could be a variant sequence for a "TATA" box, located at nucleotides -24 to -30. This is a customary location for the TATA box, which is typically found 26-34 nucleotides upstream from the transcriptional initiation site (Cordon, J. et al. , Science 209 :1406-1414 (1980)). Another sequence, 5′-TCAAT-3′ (nucleotides -95 to -99), is found 95 nucleotides upstream from the transcriptional initiation site and is similar to the "CCAAT" box sequence, which is usually found 70-80 bases from the transcriptional initiation site. In addition, a 6 bp sequence, GGGCGG, is repeated three times. One repeat is located at the positions -81 to -76 and is presumed to be within the promoter region of the APH gene. The other two repeats, present in the reverse orientation as CCGCCC, are located 5 nucleotides and 31 nucleotides downstream from the transcriptional initiation site. It is reported that all the Sp1-binding regions contain one or more exact copies of this GGGCGG sequence, which may be present in either orientation with respect to transcription (Dynan, W.S. et al. , Nature 316 :774-448 (1985)). A 200 bp sequence appears tandemly starting at 917 bp upstream from the transcriptional initiation site. Compared with other sequences in the gene bank, the 3′ two-thirds of this sequence is similar to the mouse type 2 Alu repeat (80% in similarity) (Kominami, R. et al. , J. Mol. Biol. 165 :209-228 (1983)). A similar sequence, but only as one copy, is present in the junction of SV40 and Fisher rat DNA (Hasson, J.-F. et al. , J. Mol. Biol. 177 :53-68 (1984)) and several other genes (Min, H.Y. et al. , Nucleic Acids Res. 14 :8879-8892 (1986); Osumi, T. et al. , J. Biol. Chem. 262 :8138-8143 (1987); Corden, L.J. et al. , Proc. Natl. Acad. Sci. USA 82 :7934-7938 (1985); Phillips, M. et al. , J. Biol. Chem. 261 :10821-10827 (1986)). This repeat may have a regulatory role. This gene may be specifically regulated by either cis-and/or trans-acting regulatory factors. Such regulation may be associated with protein synthesis or degradation. Acyl-peptide hydrolases have been isolated from various mammalian tissues, and their molecular properties and reaction mechanism have been partially characterized. One aspect of the present invention concerns the primary structure of rat liver acyl-peptide hydrolase which has been deduced from the nucleotide sequence of two cDNA clones isolated from a rat liver λgtll library (Fig. 3). This cDNA encodes a protein of 732 amino acid residues, and protein sequence analyses derived from 19 CNBr and tryptic peptides confirmed the identity of 292 residues. This enzyme has been shown to consist of 4 subunits of identical size based on estimations of M r for the native protein and its subunits by gel filtration and SDS-PAGE, respectively (Kobayashi, K. and Smith, J.A., J. Biol. Chem. 262 :11435-11445 (1987)). Since all the peptide sequences obtained were found in the deduced protein sequence, it is likely that the four subunits are identical in their primary structure. A comparison of the deduced protein sequence (Fig. 1) and the amino acid sequences derived from automated Edman degradation (Table 3) reveals that the protein contains three equivalently modified lysyl residues (residues 118, 291, 443), although the chemical nature of this modification has not yet been determined. There are three indirect lines of evidence that suggest that the methionine residue at position 1 is indeed the NH₂-terminus of the protein. First, the calculated molecular weight agrees closely with the subunlt M r estimated by SDS-PAGE (81,347 versus 80,000 (Kobayashi, K. and Smith, J.A., J. Biol. Chem. 262 :11435-11445 (1987)), respectively). Second, the theoretical amino acid composition is similar to the observed amino acid composition of purified acyl-peptide hydrolase (Table 2). Third, the initiation codon, ATG, corresponding to this methionine is in the right context for an initiation codon, as described by Kozak, M. ( Cell 44 :283-292 (1986)). The NH₂-terminal sequence of the deduced primary structure of the enzyme is Met-Glu-Arg-Gln···. However, previous protein sequence analysis indicated that the NH₂-terminus of the protein is blocked (Kobayashi, K. and Smith, J.A., J. Biol. Chem. 262 :11435-11445 (1987)). If the methionine were removed during translation by a methionine-specific aminopeptidase, a residue located more C-terminally would be expected to be blocked, in which case the sequence of peptide CB17-R13 (Glu-Arg-Gln···) could not be obtained. Therefore, the methionine residue remains on the polypeptide chain and undergoes an NH₂-terminal modification. Although the chemical nature of this blocking group has not yet been established, the well-documented occurrence of glutamyl residues, as well as aspartyl and asparaginyl residues adjacent to acetylated methionines suggests that the protein probably is N α -acetylated. If this is the case, the Ac-Met of acyl-peptide hydrolase is apparently not cleaved in vitro or in vivo by itself or other NH₂-terminal processing enzymes during its processing or intracellular sorting. It has been demonstrated that acyl-peptide hydrolase does not effectively remove acetylated amino acid from native or denatured proteins in vitro (Kobayashi, K. and Smith, J.A., J. Biol. Chem. 262 :11435-11445 (1987); Gade et al. Biochim . et Biophys . Acta 662 :86-93 (1981)), although such residues are effectively cleaved from N α -acetylated peptides (< 20 residues). Therefore, it seems likely that the in vivo substrates for this enzyme may be short N α -acetylated peptides resulting from protein degradation. However, a role for this enzyme in the removal of N α -acetylated amino acids from other polypeptide chains during co-translational processing cannot be ruled out (Rubenstein, P.A. et al. , J. Biol. Chem. 258 :11354-11360 (1983)). RNA blot analysis indicates that a single 2.7 kb RNA encodes acyl-peptide hydrolase in all the rat tissues examined. Further, the amount of mRNA detected in these tissues appears to be roughly equivalent, suggesting that there are no tissue-specific regulation of acyl-peptide hydrolase mRNA levels. Example 5 -- Detection and Diagnosis of Small Cell Carcinoma Four major types of lung neoplasms -- small cell carcinoma (also referred to as "oat cell" carcinoma), squamous carcinoma (also referred to as epidermoid carcinoma), adenocarcinoma and large cell carcinoma -- account for 95% of all primary lung neoplasms. Small cell carcinoma is of substantial medical importance. It accounts for about 25% of all lung neoplasms. Whereas other forms of lung cancer have 27-37% 5-year survival times, less than 1% of patients suffering from small cell carcinoma survive 5 years from the time of diagnosis (See, for example, Harrison's Principles of Internal Medicine, 11th Ed., Braunwald, E. et al ., eds. (1987), pp.1115-1123. Presently, small cell carcinoma is generally detected through routine chest radiograph; as many as 5-15% of such cancers are asymptomatic at the time of detection. The disease is said to be in a limited stage when it is confined to one hemithorax and regional lymph nodes; the disease is said to be in an extensive stage when greater involvement is observed. Early detection of small cell carcinoma is associated with a substantial increase in prognosis. Five year cure rates for limited stage disease are potentially 15-25%, however, the potential 5 year cure rate for extensive stage disease is only 1-5%. Small cell carcinoma is treated with intensive chemotherapy and radiotherapy. The initial goal of treatment is to obtain a complete regression of the tumor within 6-12 weeks of therapy. Although the tumor often returns, the extent of regression correlates to both median and long-term survival. Because of its metastatic potential, small cell carcinoma is not generally treatable with surgery. However, if detected at an early stage, surgical resection is possible, and is associated with significantly improved cure rates. Karyotypic studies have revealed a consistent deletion in chromosome 3p (p14-p23) among small cell carcinomas (Whang-Peng, J. et al ., Science 215 :181-182 (1981)). This observation has been supported by polymorphic RFLP marker studies (Naylor, S.L. et al. , Nature 329 :451-454 (1987); Kok, K. et al. , Nature 330 :578-581 (1987); Brauch, H. et al. , N. Engl. J. Med. 317 :1109-1113 (1987); Yakota, J. et al. , Proc. Natl. Acad. Sci. (U.S.A.) 84 :9252-9256 (1987)). The frequency of allele loss indicates that virtually all small cell carcinomas contain a deletion for a portion of chromosome 3 (Naylor, S.L. et al. , Genomics 4 :355-361 (1989). The short arm of chromosome 3 has been implicated in other lung cancers, in renal cell carcinomas, and in von Hippel-Lindau syndrome (Kok, K. et al. , Nature 330 :578-581 (1987); Brauch, H. et al. , N. Engl. J. Med. 317 :1109-1113 (1987); Zbar, B. et al. , Nature 327 :721-724 (1987); Kovacs, G. et al. , Proc. Natl. Acad. Sci. (U.S.A.) 85 :1571-1575 (1988); Seizinger, B.R. et al. , Nature 332 :268-269 (1988). The loss of activity of aminoacylase 1 in small cell carcinoma tumors and the familial association of some of these diseases further supports the correlation between these diseases and a deletion in the small arm of chromosome 3 (Naylor, S.L. et al. , Genomics 4 :355-361 (1989)). Recently, a DNA sequence (designated "DNF 15S2") was cloned, and mapped to chromosome 3 (Gerber, M.J. et al. , Amer. J. Hum. Genet . 43 :442-451 (1988); Naylor, S.L. et al. , Genomics 4 :355-361 (1989)). The cloned DNA was found to be capable of identifying RFLP differences between normal DNA and DNA of small cell carcinomas. In particular, polymorphisms were identified using the TaqI restriction enzyme. The amino acid sequence of the acyl-peptide hydrolase of the present invention is substantially similar to the amino acid sequence encoded by the DNF 15S2 probe sequence. Of the 621 residues of the DNF 15S2 protein, 67.6% were identical to those found in the acyl-peptide hydrolase of the present invention (Figure 6). Thus, nucleotide sequences which encode the acyl-peptide hydrolase of the present invention, or fragments of this enzyme, may be used as probes to detect and identify small cell carcinoma, and other cancers associated with a deletion in chromosome 3. When used as a probe, such sequences are incubated under conditions which permit them to hybridize to DNA or RNA of a patient being tested to determine the presence of small cell carcinoma. Suitable hybridization methods are well-known in the art (see, for example, Hames, B.D. and Higgins, S.J. Nucleic Acid Hybridization, a practical approach , IRL Press, Washington, D.C. (1985). After hybridization has been achieved, well-known methods for detecting polymorphism (preferably restriction fragment length polymorphism ("RFLP") analysis) may be employed to determine whether a nucleic acid-containing sample contains a polymorphism or sequence which is correlated to the presence of small cell or other carcinoma. Many methods for performing polymorphism detection analysis are known, and may be readily adapted to employ the acyl-peptide hydrolase encoding sequences of the present invention in the detection of small cell and other cancers (see, for example, Wainscoat, J.S. et al. , Hum. Genet. 75 :384-387 (1987); Rabin, D. et al. , Hum. Genet. 75 :120-122 (1987); Azuma, C. et al. , Amer. J. Obstet. Gynecol. 160 :734-736 (1989); Pakkala, S. et al. , Leuk. Res. 12 :757-762 (1988); Todd, S. et al. , Genomics 4 :53-59 (1989); Chowdhury, M.K.U. et al. , Theor. Appl. Genet. 76 :25-32 (1988); Yam, P. et al. , Amer. J. Hum. Genet. 41 :867-881 (1987); Freeman, S.M. et al. , Hum. Immunol. 20 :1-12 (1987); Yoffe, G. et al. , Exper. Hematol. 15 :725-728 (1987); Jones, F.S. III et al. , Gene 39 :77-84 (1986); Bernheim, A. et al. , Proc. Natl. Acad. Sci. (U.S.A. ) 80 :7571-7575 (1983). Polymorphism detection assays have been used to detect and identify cancers (Wada, M. et al. , Jpn. J. Canc. Res. 78 :780-784 (1987); Naylor, S.L. et al. , Genomics 4 :355-361 (1989); Gerber, M.J. et al. , Amer. J. Hum. Genet. 43 :442-451 (1988); Kakehi, Y. et al. , Int. J. Cancer 43 :391-394 (1989); Gum, J.R. et al. , J. Biol. Chem. 264 :6480-6487 (1989). Such assays may be used as a general model for the assays of the present invention.

Claims: Claims for the following Contracting States : AT, BE, CH, DE, DK, FR, GB, GR, IT, LI, LU, NL, SE A method of detecting small cell carcinoma which comprises: A. incubating a nucleic acid sample from a patient suspected of having small cell carcinoma, in the presence of a nucleic acid molecule having a sequence selected from the group consisting of: i. a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; and ii. a sequence which is complementary to a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; said incubation being under conditions sufficient to permit nucleic acid hybridization to occur between said nucleic acid sample and said nucleic acid molecule, and to thereby form a hybridized molecule; and B. detecting small cell carcinoma by determining whether said hybridized molecule differs in sequence from a reference molecule, said reference molecule comprising a nucleic acid sample from a normal individual hybridized to a nucleic acid molecule which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1. The method of claim 1, wherein said step B comprises an analysis of restriction fragment length polymorphisms. A two stranded nucleic acid molecule comprising: A. a first strand having a sequence selected from the group consisting of: i. a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; and ii. a sequence which is complementary to a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; said first strand being hybridized to: B. a second strand, said second strand having a sequence which is substantially complementary in sequence to the sequence of said first strand, said complementary sequence of said second strand being derived from an individual suspected of having small cell carcinoma. Claims for the following Contracting State : ES A method of detecting small cell carcinoma which comprises: A. incubating a nucleic acid sample from a patient suspected of having small cell carcinoma, in the presence of a nucleic acid molecule having a sequence selected from the group consisting of: i. a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; and ii. a sequence which is complementary to a sequence which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1; said incubation being under conditions sufficient to permit nucleic acid hybridization to occur between said nucleic acid sample and said nucleic acid molecule, and to thereby form a hybridized molecule; and B. detecting small cell carcinoma by determining whether said hybridized molecule differs in sequence from a reference molecule, said reference molecule comprising a nucleic acid sample from a normal individual hybridized to a nucleic acid molecule which encodes all or part of an acyl-peptide hydrolase enzyme according to Fig. 1. The method of claim 1, wherein said step B comprises an analysis of restriction fragment length polymorphisms.

Inventors: SMITH JOHN A, SMITH, JOHN A.
Applicants: GEN HOSPITAL CORP, THE GENERAL HOSPITAL CORPORATION
Application year: 1990
Main classification: C12Q1
IPC codes: C12N9, C12Q1, A61K49, C07K14, C12N15