CA1340264C - Recombinant dna expression of novel diuretic/vasodilator compounds - Google Patents

Abstract

Methods and compositions are provided for inducing natriuresis, diuresis and vasodilatation in mammalian hosts by administering pre-proauriculin to said host.
Also provided are methods and compositions for the production of auriculin, proauriculin and pre-pro-auriculin utilizing DNA sequences.

Description

4D~4 Description RECOMBINANT DNA EXPRESSION OF NOVEL
DIURETIC/VASODILATOR COMPOUNDS

Technical Field 5The present invention relates generally to poly-peptide compounds capable of regulating sodium excre-tion and blood pressure in mammals. More particularly, the present invention is directed to methods and compo-sitions corresponding to such polypeptides isolated from atrial tissue, and particularly to the precursor forms of such polypeptides, and the applications of recombinant DNA technology to the large scale produc-tion of such precursors and polypeptides.

Backqround Art 15Most multi-cellular organisms are organized into tissues and organs which perform specialized functions.
Thus, a system has evolved to transport materials between them. In higher animals, including mammals, this circulatory system is closed to improve the efficiency of transport. The flow of blood fluid through this closed cardiovascular system requires that the fluid be maintained under pressure and the regula-tion of the systemic arterial blood pressure requires a complex interaction of numerous factors including, e.g., fluid volume, vascular elasticity and vascular caliber.
The maintenance of normal extracellular fluid volume depends primarily on the excretion of sodium (natriuresis) and water (diuresis) by the kidneys.
This is determined by (1) the rate at which plasma is filtered at the glomerulus (glomerular filtration rate, or GFR) and (2) the degree to which sodium is actively reabsorbed along the renal tubule (with water following - 13~02~

passively). The latter process is in part requlated by the adrenal steroid hormone aldosterone. It has been long believed that~ in addition to GFR and aldosterone, there must be a "third factor" which also regulates sodium reabsorption. It is now apparent that many of the phenomena which required the po.stulation of a "third factor" can be explained by the effects of physical forces (e.g. blood pressure, red blood cell concentation and plasma viscosity) on sodium reabsorp-tion. ~onetheless, the search continues for a "natri-uretic hormone" which miqht directly inhibit tubular reabsorption.
There are several candidates for such a hormone, among which are included the natriuretic factor(s) recently isolated ~rom atrial muscle cells. A natri-uretic effect has ~een demonstrated by crude extracts of rat atrial tissue but not ventricular tissue.
ne Bold, A.~. et al., Life Sciences, 28:89-94 (1981), Garcia, R., Experientia, 38:1071-73 (1982), Currie, M.G. et al.. Science 221:71-73 (1983). Various pep-tides, differing in size, with diuretic and natriuretic properties have been isolated from atrial tissue and sequenced. Flynn, T.G. et al., Biochem. Biophys. Res.
Commun. 117:859-865 (1983), Currie, M.G. et al., Science 223:67-69 (1984), Kangawa, K. et al., Biochem.
Biophys. Res. Commun. 118:131-139 (1984). The exis-tence of these atrial natriuretic factors strengthens the long-held suspicion that the heart, aside from its obvious influence on renal perfusion, may play an important role in regulating renal sodium and water excretion. Stretching of the atria is known to induce diuresis and natriuresis, and this is possibly mediated by increased release of these factors.
A number of clinically imPortant disease states are characterized hy abnormal ~luid volume retention.
Congestlve heart failure, cirrhosis of the liver and , the nephrotic syndrome each lead to excessive fluid accumulation on the venous side of the circulation, the presumed common mechanism being under-perfusion of the kidneys leading to a fall in GFR. In addition the reduced renal perfusion stimulates excessive secretion of renin, a proteolytic enzyme whose action in the circulation leads to the formation of angiotensin.
Angiotensin is a powerful constrictor of arterioles (which helps to maintain arterial pressure) and also stimulates release of the sodium-retaining hormone aldosterone by the adrenal gland (which further worsens fluid retention). These mechanisms do not, however, fully account for the fluid retention of the so-called "edematous states", and additional factors are likely to be involved. One important possibility is that a relative or absolute deficiency of an atrial natri-uretic factor, caused either by chronic over-stretchinq of the atrium (e.q., heart failure) or by inadequate stimulation of the atrium (e.g., cirrhosis and nephro-tic syndrome), might contribute to the fluid retention.

An increase in extracellular fluid volume is alsothought to contribute to the development of hyperten-sion in many instances. Hypertension, or chronically elevated blood pressure, is one of the major causes of illness and death worldwide. It is estimated that more than 20 million Americans suffer from this disease whose complications include heart failure, heart attack, stroke and kidney failure. The major observed hemodynamic abnormality in chronic hypertension is increased resistance to the flow of blood through the arterioles. The mechanisms which lead to this in-creased "peripheral resistance" are, however, incom-pletely understood. In some cases inappropriate activity of the renin-angiotensin system or sympathetic nervous system may lead to excessive constriction of . .

1~0264 the arterioles, by "inappropriate" it is meant that the unknown signal(s) leading to this activity are not based upon a physiological need of the organism and thus lead to elevated blood pressure (whereas, in the example cited earlier, the increased renin secretion in the edematous states is a response to reduced arterial pressure and thus helps to restore or maintain normal pressure). In a substantial fraction of hvpertensives however, inappropriate sodium and volume retention by the kidnev is felt to either initiate or contribute to the elevated blood pressure. The responsible defect in kidney function and the mechanism whereby fluid reten-tion leads to increased peripheral resistance are both unknown. It is certainly possible that deficiency of a natriuretic hormone could be responsible for these observations, particularly if the same substance also normally exerted a relaxant effect on arterioles.
Diuretic therapy is currently a mainstay in the treatment of hypertension, renal failure and the various edematous states (heart failure, etc.). Cur-rently available pharmacological preparations have, however, several imPortant limitations and undesirable effects. While their us'e may be directed at a specific abnormality (i.e. volume expansion), their multiple actions are undoubtedly not physiological, leading for instance to potassium depletion, increased retention of uric acid and abnormal glucose and lipid metabolism.
In addition, all known diuretics profoundly stimulate the renin-angiotensin-aldosterone system, which coun-teracts their volume-depleting and blood pressure-lowering effects and leads to other unwanted effects.
It would be desirable to provide a pharmacologically effective compound which can regulate blood pressure by providing a complete but controlled range of physio-logical responses.

, , . .. , .~ . . . . ..

However, the isolation of such compounds fromatrial tissue is typically a cu.mbersome process and requires substantial substrate tissue to produce minute quantities of the compounds. It was considered desir-able to apply recombinant deoxyribonucleic acid (DNA)and related technoloqies to the production of larger quantities of such compounds to provide material for clinical and therapeutic applications.
Proceeding from the seminal work of Cohen & Boyer, U.S. Patent No. 4,237,224, recombinant DNA technoloqy has become useful to provide novel DNA sequences and produce large amounts of heteroloqous proteins in transformed cell cultures. In general, the joining of DNA from different organisms relies on the excision of DNA sequences usinq restriction endonucleases. These enzymes are used to cut donor DNA at very specific locations, resulting in gene fragments which contain the nNA sequences of interest. These D~A fraqments usually contain short single-stranded tails at each end, termed "sticky-ends". These sticky-ended fraq-ments can then be ligated to complementary fragments in expression vehicles which have been prepared, e.g., by digestion with the same restriction endonucleases.
Havinq created an expression vector which contains the structural gene of interest in proper or~ientation with the control elements! one can use this vector to transform host cells and express the desired gene product with the cellular machinery available. Once expressed, the gene product is generally recovered by lysinq the cell culture, if the product is expressed intracellularly, or recovering the product from the medium if it is secreted by the host cell.
Recombinant DNA technology has been used to express entirely heterologous gene products, termed direct expression, or the gene product of interest can be expressed as a fusion protein containing some parts ~ ~ .. . . . . .. . . .. ... .
. . .

13~02~

of the amino acid sequence of a homologous protein.
This fusion protein is generally Processed post trans-lationally to recover the native gene product. Many of the techniques useful in this technology can be found in Maniatis, T., et al., Molecular Cloning: A
Laboratory Manual, Cold Sprin~ Harbor Laboratory, New York (1982).
However, while the general methods are easy to summarize, the construction of an expression vector containing a desired structural gene is a difficult process and the successful expression of the desired gene product in significant amounts ~hile retaininq its bioloqical activity is not readily predictable.
Frequently qene products are not biologically active when expressed in yeast, bacteria or mammalian cell systems. In these cases, post-translational processing is required to produce biological activitv.
Accordinaly, it is the principal object of the present invention to provide methods and compounds for influencing fluid volume and blood pressure homeostasis in mammals.
It is another object of the present invention to provide methods and compounds which mimic the physio-loqical regulation of fluid volume and blood pressure in mammals.
It is yet another object of the present invention to employ recombinant DNA technology to provide methods and compositions which enable the large scale produc-tion of these compounds and their precursors.

Disclosure of the Invention The obtainment of these and other objects of the invention is provided by methods and compositions of the present invention which include pre-proauriculin substantially free of unrelated atrial tissue or products.

..

~7~ 13~26~
Compositions of the present invention useful as precursors of compounds which find use as natriuretics, diuretics, vasodilators and modulators of the renin-an~iotensin-aldosterone system include polypeptide compounds identified by the amino acid sequence ~escribed 1 below and mature polypeptides derived therefrom.
Another aspect of the present invention provides deoxyribonucleic acid (D~A) sequences, and methods for their use, which are capable of directing the synthesis and expression of compounds of the present invention, which sequences are identified by the DNA sequences described-below, together with sequences substi-tuting codons to produce related amino acids in the peptide sequence. The entire sequences of rat and human pre-proauriculin DNA are disclosed which provides the means to direct the synthesis of fragments of any desired length.
Also provided are methods for using compounds and precursors of the present invention as diaanostic and therapeutic aaents.

Brief DescriPtion of Drawinas Fiaure I provides the deoxyribonucleic acid (DNA) sequence of one embodiment of the present invention, namely DNA encodinq rat pre-proauriculin~ together with the amino acid sequence of the polvpeptide synthesis directed by this DNA-Fiaure 2 portrays the sequences of oligonucleotideprobes used to identify comPlementary DNA (cDNA) clones containin~ nucleic acid compositions of the present invention, Figure 3 depicts the sites at which specific restriction endonucleases cleaved the deoxyribonucleic acid (DNA) encodina rat pre-proauriculin to provide D~A
fragments for dideoxynucleotide sequence analysis;

. .~_ .. , . _ . . _ .. _ __ . .

13~0264~

Fi~ure 4 maps the amino acid se~uence of rat pre-proauriculin and outlines boundaries of the signal peptide and biologically active fra~ments described previously, Figure 5 (a) shows the results of Northern blot analysis of atrial and ventricular m~NA in which lane 1 depicts RNA isolated from rat atrial tissue and lane 2 depicts RNA isolated from rat ventricular tissue:
Figures 5 (b), (c), (d) and (e) show the results of two dimensional ael fractionation of cell-free translation products encoded by poly A RNA where (b) shows 3 S proteins encoded by atrial poly A RNA and (c) shows 35S proteins encoded by ventricular poly A
RNA. In vitro translations of poly A ~NA specifically hybridizinq to and eluting from DNA encoding rat pre-proauriculin is shown, where (d) depicts poly A RNA
derived from atrial tissue and (e) depicts poly A RNA
derived from ventricular tissue, Figure 6 shows sites at which specific restriction endonucleases cleaved human qenomic deoxyribonucleic acid (DNA) encoding human pre-proauriculin to provide DNA fraqments for dideoxynucleotide sequence analysis;
Figure 7 provides the deoxyribonucleic acid (DNA) se~uence of one embodiment of the present invention, namely the gene encodin~ human pre-proauriculin toge-ther with the amino acid sequence of the polypeptide synthesis directed by this DNA, Figure 8 outlines the construction and describes novel features of the pKT52 expression vector, Figure 9 depicts a modification of the pKT52 expression vector to create pRNF-6852, in which DNA
encoding amino acids 87-152 of rat pre-proauriculin was inserted to afford expression of this fragment of rat pre-proauriculin;
Figure 10 is a photoqraphic representation of an SDS polyacrylamide gel showing proteins labelled with 13~0261 S-cysteine in which E. coli in lane 1 contained the pKT52 expression vector, lane 2 contained pRNF-~852 (pKT52 modified to contain DNA encoding pre-proauricu-lin) expression product, lane 3 the products from lane 2 that were immunoprecipitated with a specific anti-auriculin antiserum, lane 4 the products from lane 2 that were immunoprecipitated with a control non-immune antiserum and lane 5 the products from lane 1 which were immunoprecipitated with a specific anti-auriculin antiserum;
Figure 11 depicts a construction for expressinq rat pre-proauriculin and related polypeptide fraqments in Saccharomyces cerevisiae usinq a specific vector and the yeast ~-factor secretion signal;
Fiqure 12 is a photographic representation of an SDS polyacyrylamide gel showing S. cerevisiae secreted proteins labeled with ~S-methionine(A) or 5S-cvsteine and 3 S-methionine(B). In A, S. cerevisiae in lanes 1 and 2 contained the YEp-~-8 shuttle vector, lanes 3 and 4 contained YEp-~-NF-9, lanes 5 and contained YEp-~-NF-12. In B, the secreted proteins were acetone and methanol extracted where lanes 1 and 2 represent S.
cerevisiae containing YEp-~-NF-7 and YEp-~-NF-5 lanes 3 and 4 represent the proteins from these preparations after immunoprecipitation with specific anti-auriculin IgG, lanes 5 and 6 contained YEp-~-NF-12 and YEp-~-NF-9, respectively, and show the proteins specifically immunoprecipitated by anti-auriculin IgG;
Fiqure 13 depicts an expression vector construc-3n tion for expressing rat and human pre-proauriculin in Chinese hamster ovary cells; and Figure 14 is a photographic representation of an SDS-polyacrylamide gel of S-methionine labeled protein from Chinese hamster ovary cell media in which lane 1 shows 35s-labeled proteins from C~ cells transformed with pMT-NF1-10, which were .,.,~.

134026~
, o--immunoprecipitated with anti-aurieulin IgG, lane 2 S-labeled proteins from non-transformed CHO cells that were immunoprecipitated with anti-auriculin IgG, lane 3 S-labeled proteins from CHO cells transformed with pMT-~F1-10, which were immunopreeipitated with eontrol IgG and lane 4 S-labeled proteins from non-transformed CHO eells that were immunoprecipitated with eontrol IgG.

Best Mode for Praetieing the Invention In aceordanee with the present invention methods and eompositions are provided for produeing eompounds useful for the reaulation of fluid volume and blood pressure in mammals, in which aspeets of the invention provide pre-proauriculin and proaurieulin, and provides these compounds substantially free of unrelated atrial tissue or products.
Another aspect of the invention provides polypep-tide compounds comprising the amino acid sequence disclosed in Fiaure 1.
A further aspect of the invention provides nucleic acid sequences capable of directing the synthesis of pre-proauriculin, and fraqments derived therefrom, such nucleic acid sequences comprising the DNA sequence of Figure 1, including oligonueleotide sequenees contained therein, and allowing for the replacement of codons with other codons eapable of directing the synthesis of related amino aeid sequenees.
The nomenelature used to deseribe polyPeptides of the present invention follows the eonventional practice of usinq the first three letters of the trivial name of the amino aeid and wherein the L form of any amino aeid having an optieal isomer is intended unless otherwise expressly indieated.
As used herein, pre-proaurieulin is defined as the amino acid sequenee of the precursor of the compound auriculin together with the siqnal peptide, which .. ~ . . . .

13~26~
, 1 precursor and compound are shown to be useful as natriuretic, diuretic and vasorelaxant compounds.
Proauriculin is defined as the amino acid sequence of the precursor of auriculin without the signal peptide.
Auriculin is defined as the biologically active com-pound of pre-proauriculin which compound is shown to be useful as a natriuretic, diuretic and vasorelaxant compound. The pre-proauriculin amino acid sequence produced in accordance with the present invention has been determined in part by sequencing of cDNA clones and deducing the resultant amino acid sequence. It will be understood that natural allelic variations can exist between various individuals of a species, and that minor variations can occur between species while preserving the usefulness of the resultant compounds.
These variations may appear as amino acid substitutions in the overall sequence or by deletions, insertions, extensions or substitutions of amino acids in the amino acid sequence as described.
Compounds within the scope of the present inven-tion can also be obtained by modifying the above recited formula in numerous ways while preserving the activity of the polypeptides thus obtained. For example, while the amino acids of these polypeptide compounds are normally in the natural L form, one or more, usually two or less and preferably one amino acid may be replaced with the optical isomer D form. Amino acid residues contained within the polypeptide com-pounds can also be modified by acetvlation or substi-tuted with other chemical groups which can, for exam-ple, change the solubility of the compounds without effecting their activity.
More particularly, modifications in the amino acid sequence of the various forms of pre-proauriculin can be effected by various chanqes in the nucleotide sequence of the cloned structural qene used to direct 13~026~

the synthesis of pre-proauriculin. Included within such modification of the DNA sequence are the replace-ment of various codons with other codons which, due to the degeneracy of the genetic code, direct the synthe-sis of the same amino acid.
In addition, by codon substitution, one or more amino acid residues can be replaced by functionally equivalent residues; for example basic amino acids can be replaced with other basic amino acids and acidic amino acids can be replaced with other acidic amino acids. However, the replacement of hydrophobic amino acids, particularly cysteine, are considered less desirable due to the likelihood of interfering with a presumptive disulfide bridqe.
Further modifications are possible by extending or decreasinq, preferably extending, the compounds' amino acid sequence by the addition of amino acids or oligo-peptides on the N-terminal or C-terminal end of the sequence disclosed above. In particular, numerous N-terminal extensions of auriculin are ~ossible within the scope of the disclosed precursor sequences. Fur-thermore, compounds of the present invention can be bonded to or conjugated with compounds having the same range of activities to obtain the benefits of the present invention.
Polypeptide compounds of the present invention which are shown to have the above recited physiological effects can find use in numerous therapeutic applica-tions such as, e.g., inducing natriuresis, diuresis, and vasodilatation. Thus these compounds can find use as therapeutic agents in the treatment of various ede~atous states such as, for example, congestive heart failure, nephrotic syndrome and hepatic cirrhosis, in addition to hypertension and renal failure due to ineffective renal perfusion or reduced glomerular filtration rate.

-13~26~

These compounds can be administered to mammals for veterinary use such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents, that is in a physiologically acceptable carrier. In general the dosage will range from about 0.01 to 100 ~g/kg, more usually 0.1 to 10 ~g/kg of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained.
These compounds can be administered neat, as mixtures with other physiologically acceptable active or inactive materials, or with physiologically suitable carriers such as, for example, water or normal saline.
The compounds can be administered orally or paren-terally, for example, by injection or nasal inhalation.
Injection can be subcutaneous, intravenous, or by intramuscular in~ection.
These compounds are desirably administered in pharmaceutically effective amounts and often as pharma-cologically acceptable salts such as acid addition salts. Such salts can include, e.g., hydrochloride, hydrobromide, phosphate, sulphate, acetate, benzoate, malate, among others.
Compounds produced in accordance with the present invention can also be used for preparing antisera for use in immunoassays employing labelled reaqents, usually antibodies. Convenientlv, the smaller poly-peptide fragments can be con~uqated to an antigen bymeans of dialdehydes, particularly from 4 to 6 carbon atoms and aliphatic, or carbodiimide. These compounds and immunologic reagents may be labelled with a variety of labels such as chromophores, fluorophores such as, e.g., fluorescein or rhodamine, radioisotopes such as .. ~ . .. . . . ... .. . . ... .

13402~

I, 3 S, C, or H, or magnetized particles, by means well known in the art.
These labeled compounds and reagent.s, or labeled reagents capable of recognizing and specifically binding to them, can find use as, e.g., diagnostic reagents. Samples derived from biological specimens can be assayed for the presence or amount of substances having a common antigenic determinant with com~ounds of the present invention. In addition, monoclonal anti-bodies can be Prepared by methods known in the art,which antibodies can find both diaanostic and thera-peutic use, e.q., to neutralize overproduction of immunologically related compounds in vivo. In addi-tion, the DNA seauences encodinq portions of pre-proauriculin can find use as hiqhly specific probes for nucleic acid nybridization diaanostic assavs. These assays are constructed by techniques well known in the art, includinq for example, ~ilson, J.T., et al., Proc.
Natl. Acad. Sci. USA 79:3628-3631 (1982).
The following examples are provided by wav of illustration, rather than implyinc anv limitat on of the subject invention.

EXAMPLES
In the examples that follow, deoxyribonucleic acid seauences encoding rat and human pre-proauricu'in are described. E. coli, Saccharomyces cerevisiae and Chinese hamster ovary cell host cultures were employed to express these DNA sequences. However, it will be readily understood by one having ordinary skill in the art that other procaryotic and eu~aryotic cells will provide suitable hosts for various aspects of the present invention.

-15- 1~026~
A. Clonina of Rat Pre-proauriculin cDNA.
1. Isolation of mRNA
Total RNA was isolated from rat atria by the method of Chirgwin, J.M. et al. Biochemistry 18:5294-5299 (1979). The tissue was homogenized in a solutionof 6 M guanidine thiocyanate, 0.005 M sodium citrate, pH 7.0, 0.1 M 2-Mercaptoethanol, 0.5% Sarcrosyl. This hdmogenate was made 2.0 M in CsCl and layered over a 5.7 M CsCl cushion in 0.1 M ethylenediaminetetraacetic acid (EDTA). The RNA was pelleted throuqh this cushion by centrifugation at 115,000 xq for 16 hours, thereby separating it from the cellular DNA and protein which do not sediment through the higher density CsCl solu-tion. The RNA was then dissolved in o.n1 M Tris, pH
7.4, 0.005 M EDTA, 1.0% sodium dodecylsulfate (SDS), extracted with a 4:1 mixture of chloroform and 1-butanol, and precipated from 70~ ethanol. The poly-adenylated RNA (poly A RMA) fraction was obtained by affinity chromatography through oliao (dT) cel~ulose as described by Aviv, H. and P. Leder, Proc. Natl. Acad.
Sci. USA 69:1408-1412 (1972). The polyadenylated RNA
was bound to the cellulose matrix in a solution of 0.02 ,~ Tris, pH 7.6, 0.001 M EDTA, 0.1~ SDS, containinq 0.5 M NaCl. The non-polyadenylated RNA, which does not bind under these conditions, was removed by extensive washing of the chromatography column with this solu-tion. The polyadenylated RNA was then eluted in the same solution without NaCl, and precipated from 70%
ethanol. With these techniques, 100 g of polyadenyl-ated RNA was isolated from 10 am of atrial tissue.
2. Generation of rat atrial cDNA library Double-stranded cDNA was synthesized and prepared for insertion into the plasmid vector pUC8 (Vieira, J.
and J. ~essinq, Gene 19:259-268, 1982) using the sequential addition of EcoRI and SalI oligonucleotide linkers as described by Helfman, D.M. et al., Proc.

.~, .. .. . ..

13~026~1-Natl. Acad. Sci. USA 80:31-35 (1983). First strand cDNA was synthesized by the RNA-dependent DNA polymer-ase from Avian Myeloblastosis Virus, by priming with oligo(dT)12 18 The RNA template was then removed by base hydrolysis. Second strand DNA was synthesized by use of RNA-dependent DNA polymerase, relying on self-priming at the 3'-end of the first strand molecule, thereby forming a double-stranded hairpin DNA. These molecules were blunt-ended at the open-ended termini using the large fraqment of DNA polymerase I of E. coli to fill in single-stranded regions. EcoRI oligonucleo-tide linkers were added to the open-end using T4-DNA
ligase. The hairpin loop was cleaved open with S1 nuclease from ~spergillus orvzae and the termini of the molecules were again blunt-ended as before. SalI
oliqonucleotide linkers were then added, again using T4-DNA liqase. SalI and EcoRI "stickv ends" were -released by cleavage with these restriction encio-nucleases. These double-stranded double-linkered cDNA
molecules were then Iigated into EcoRI and SalI-digested pUC8 and transferred into E. coli MC1061 by the CaCl2-treatment described by Casabaden, M. and S.
Cohen, J. Mol. ~iol. 138:179-207 (1980).
Five ~g of rat atrial poly A RNA yielded about 25 nq of cDNA, size selected to greater than 300 base pairs, and gave a library of about 200,00n independent recombinants. These were plated on nitrocellulose filters, replica plated and the library stored frozen on glycerol impregnated filters at -70~C with the protocol of Hanahan, D. and M. Meselson, Gene 10:63-67 (1980) and Hanahan, D. and M. Meselson, Methods in ~nzymoloqy, Academic Press, New York, pp. 333-342.

3. Screening of the rat atrial cDMA library Amino acid sequences for rat atrial auriculin were used to design the oligonucleotide probes shown in Figure 2 which were used to screen the rat atrial cDNA
i . ~ . .. . ..

134026~

library. The use of short synthetic oligonucleotides based on amino acid sequence information has been described (see, e.g., Wallace, R.B. et al., Nucleic Acids Res. 9:879-894 (1981)). Due to the degeneracy of the codons, two oligonucleotide pools were synthesized for each region. Region 1 was covered by two tetra-decamer oligonucleotide pools, probe a and probe b, each consisting of 64 sequences. Region 2 was covered by another two tetradecamer pools, probe c and probe d, each consisting of 72 sequences. The sequence and location of these oligonucleotide probes are shown in Figure 2. The sequence of amino acids 4-13 of rat auriculin is shown alonq with the seauence of the four oligonucleotide mixtures, probes a and b for region 1, and probes c and d for region 2, wherein R=A or G, Y=T
or C, N=A,G,T or C. Each oligonucleotide mixture was synthesized on a Biosearch SAM I oligonucleotide synthesizer (~iosearch, Inc., San Rafael, Cal.) by a modification of the standard phosphotriester method usinq mesitylenesulfonyl chloride in the presence of N-methylimidazole as condensing reagents as described by Efimov, V.A. et al., Nuc. Acids Res. 10:6875-6894 (1982) and purified by polyacrylamide ael electro-phoresis.
The cDNA library was then screened usinq these oligonucleotide probes by colony hybridization. Four replica filters were prepared from each filter, so that each colony could be screened with each oligonucleotide probe pool.
The filters were baked for 2 hrs. at 80~C under vacuum and then washed overnight at 68~C with shakinq in a large volume of 3X S.SC (where 1X SSC is 0.15 M
NaCl, 0.15 M sodium citrate, pH 7.5) 0.1% SDS. The filters were prehybridized in 6X SSC, 0.1% SDS, 1 mM
EDTA, 5x Denhardt's solution, (0.1~ Ficoll, 0.1~

13~026~
, ~
polyvinylpyrrolidone, 0.1~ bovine serum albumin) 0.05 sodium pyrophosphate at 50~C for a minimum of 2 hrs.
Filters were then hybridized with 2.5 X 10 cpm 32P-labeled oligonucleotide probe mixture (phosphoryl-ated in accordance with Maniatis, T. et al., MolecularCloninq, Cold Spring Harbor Laboratories, 1982, pp.
122-123) per filter in 10 ml hybridization solution containing 10n ~g/ml tRNA at 45~C in a shaking water bath. After 1 hr., the thermostat was lowered to 25~C
and the bath allowed to equilibrate for 12 hrs. The filters were washed twice in 6X SSC, 0.1% SDS at room temperature for 15 mins., then washed in 6X SSC, 0.1%
SDS at 35~C tfor probes C and D) or 39~C (for probes A
and 3) for 1-2 mins. The final washing temperature was obtained from the empirical formula of Suqgs, S.V. et al., Developmental Bioloav Usina Purified Genes (ed.
D.D. Brown and C.F. Fox) Academic Press, Mew York pp.
683-693, that is Td = 4(G + C) + 2(A + T). The hvbrid-ized filters were then dried and autoradiographed on Kodak~ XAR film with Dupont~ Cronex intensifying screens until comPlete exposures were obtained.
A colony was considered positive if it hybridized with one probe from reaion number 1 and one probe from region number 2. One colony was chosen which hy-bridized strongly to the oliqonucleotide probes (pools A and C) and hybridized to a random primed atrial cDMA
probe but not a ventricular cDNA probe. Sequencing of this clone demonstrated that it encoded rat pre-proauriculin. This clone is referred to as pNFl.

4. Complete sequencing of the rat pre-proauriculin cDNA.
The purified DNA insert, obtained from pNFl, was prepared using small miniprep methods (Maniatis et al., supra at p. 366) and was isolated on acrylamide gels.

. .

........ ............. . . ... .... . ... ....... . .........

_ _ _ _ _ . .
13~026~
, g The intact DN~ insert was then subcloned into bacterio-phage M13 (a single stranded phage desiqned specific-ally for DNA sequencing using the dideoxynucleotide method as described by Messing J. and J. Vieira, Gene _ :259-258 (1982)), via the EcoRI and SalI sites on the 5' and 3' ends, respectively (Figure 3). An initial reading of the entire seauence was then obtained from these clones usinq the Sanqer dideoxynucleotide se-~uencing technique, Sanger, F. et al., Proc. Nat. Acad.
Sci. USA 74:5463-5469 (1977). In order to confirm this initial sequence, a separate reading of the other DNA
strand was necessary. For this, the HincII site at base 340 was used. The prepared insert was cleaved with endonuclease HincII, and the resulting digest was cloned into M13 mp9 cleaved with SmaI plus EcoRI (arrow 5) and M13 mp8 diqested with SalI plus SmaI (arrow 6).
A similar approach was taken using the PstI site at base 647 to obtain additional confirmation (arrows 3 and 4). Althouqh the initial clone used for sequencing (pNFl) terminated at base 78a of the sequence (see fiqure 1), another clone (pMF4) extended further 3', containing the final 22 bases plus the 3' poly A tail.
The sequence of the 3' end of this clone was obtained using M13 clones containing the PstI to SalI portion of the insert (arrow 7) and is shown in Figure 1 as bases 785-806. Finally, the very 5'-terminal nucleotides of the DNA were determined by Maxam and Gilbert sequencing (Maxam, A. and W. Gilbert, Proc. Nat. Acad. Sci. USA
74:560-564 (1977)) of a 32P-labelled single stranded DNA made complementary to the 5' region using the BglII
fraqment spanning bases 1-186. The sequence determined thereby was included in Figure 1 as bases 1-22. Thus, nucleotide seauence analysis confirmed that clone pNFl, which includes bases 23-784 of Figure 1, encodes an auriculin precursor, pre-proauriculin. When the atrial cDNA library was re-screened with the cDNA insert, .. .. . . . . . ... .. .

r ~ r ~ . -. -. , ., ~
. _ .. _ . __ ~ _ . ... ___ ... ~, . __ .. _ _ . ...

134026~

approximately 0.5% of the colonies hybridized. This indicates that pre-proauriculin mRNA is a ma~or species in rat atrial mRNA population.
The amino acid sequence of pre-proauriculin was determined from the cDNA nucleotide sequence. A single open readinq frame encodinq a 152 amino acid sequence was disclosed, extending from thè initiation codon ATG
at base 85 to the termination codon TAA at position 541. Biologically active auriculin (see Figure 4) can be identified in the amino acid sequences of r~t and human pre-proauriculin tsee Figure 1 and Figure 7, respectively)).
5. Determination of atrial specificitv Atrial and ventricular poly A RNA was subject to Northern blot analysis after fractionation by electro-phoresis on a 1.4~ aqarose qel containinq methvl-mercuric hydroxide by the method of Bailey, J.M. and ~.
Davidson, Anal. Biochem. 7n 75-85 (1976). Nor~hern blot analysis results, using nick translated pi~lFl D~A, are shown in Figure 5a where lane 1 contains atrial poly A RNA and lane 2 ventricular poly A RNA. As indicated in Figure 5a, pNFl hybridizes to an atrial mRNA of ap~roximately 800-900 nucleotides in length.
It does not hybridize with ventricular mRNA.
The cDNA sequence for pre-Proauriculin determined above indicates that pre-proauriculin has a molecular weight of approximately 16,500 daltons. To determine the actual precursor size, atrial mRNA encoding pre-proauriculin was purified by hybrid selection (Goldberg, M.L. et al., Methods in Enzymology 68:206-220, Academic Press, New York), by immobilizing 5 ~ug pNFl DNA on 1 cm nitrocellulose discs and hybridizing with 5 ~g of poly A RNA for 3 hrs. at 50 C in 20 mM
PIPES, pH 6.4, lmM EDTA, 65% formamide, 5X SSC, 0.1%
SDS. The filters were washed extensively with 10 mM
Tris-YCl, pH 7.5, 0.15 M NaCl, lmM EDTA, 0.1% SDS at . , " " " ~. ,.,, , . ~,, . . . " .. .. .

13402~ 1 70~C Thereafter filters were washed in the same buffer but without SDS. Hybridized RNA was eluted in H20 at 100~C in the presence of 50 ~g yeast tRNA for l min.
and quickly frozen at -70~C. After thawing, the RNA
was ethanol precipitated using 2 volumes of absolute ethanol.
Hybrid selected RNA and total poly A RNA was translated using a rabbit reticulocyte lysate system (Bethesda Research Labs, Gaithersburg, Maryland) in the presence of 250 ~uCi/ml [ S]-methionine. Translation products were fractionated by 2-dimensional gel electrophoresis by loading 1 X 10 cpm of acid-precipi-table radioactivity per sample. The first dimension was an isoelectric focusing gel using a gradient from 15 pH3.5-10, O'Farrell, P.Z. et al., Cell 12:113-1142 (1977). The results of the isoelectric focusing were sub1ect to electroPhoresis in an SDS-PA~E using a 15%
gel. Following sodium salicylate equilibration, the gel was dried and then fluorgraphed at 70~C for 24 hrs.
The results were as indicated in Figures 5b and 5c, where the position of several atrial-specific translation products having molecular weiahts between 12,000 and 30,000 daltons are marked by arrows. Trans-lation products encoded by pNFl hybrid selected atrial RNA are indicated in Figure 5d which shows at least 3 related protein species having molecular weight between 18,000 and 20,000 daltons which are major atrial-specific species. Figure 5e shows that hybrid selec-tion does not recognize any ventricular-specific proteins. Because the proteins in Figure 5d were hybrid selected, are atrial specific and are of the correct molecular weight range, they are pre-proauricu-lin.

... .... .

' 13~02~1 B. Cloninq of the Human Gene Encoding Pre-proauriculin 1. Isolation of the human pre-proauriculin gene The rat cDNA (isolated from pNFl) encoding pre-proauriculin provided us with a probe for the identifi-cation of the human gene. A human genomic clone library in bacteriophage Charon 4A (Lawn, R.M. et al., Cell 15:1157-1174 (1978)) was obtained from Dr. T.
Maniatis, Harvard University. Approximately 10 phage were grown on E. coli K803, and plaque lysates were transferred to nitrocellulose filters as described by Benton, ~.D. and R. W. Davis, Science 196:180-182 (1977). These filters were hybridized with the rat cDNA which had been radioactively labeled with 3 P by the nick-translation method of Riaby, P. W. J. et al., J. Mol. Biol. 113:237-251 (1977). Filters were pre-washed in hybridization buffer (0.75M NaCl, 0.75M
sodium nitrate, 40~ formamide, 0.05~ SDS, 0.02~ bovine serum albumin, 0.02% Ficoll - 400,000, 0.02% polyvinyl Pyrollidone~ 0.1~ sodium pyrophosphate, 20 ma/ml denatured sheared salmon sperm DNA) at 42~C for 1 hr.
5 x 10 cpm of 32P-labelled boiled rat pre-proauriculin cDNA was added per ml fresh hybridization buffer and the filters were incubated in this buffer at 42~C for 16 hrs. They were then washed in 0.3 M NaCl and 0.3 M
sodium nitrate and .05% SDS three times at 50~C, and exposed for autoradiography overnight. Six clones containing sequences hybridizing to rat pre-proauricu-lin cDNA were purified.
Meanwhile, the size of the human pre-proauriculin gene was determined for the purpose of identifying a full length clone. Two mg of high-molecular weight DNA
was prepared from 20 g of rat liver by the method of Blin, N. and ~. Stafford, (Nuc. Acid Res. 3:2303-2308 (1976)). This DNA was diqested with the restriction endonucleases BamHI, BalII, K~nI, and SacI, alone and 13~026 i in combination with EcoRI, electrophoresed on 1~
agarose gels, and transferred to nitrocellulose filters by the method of Southern, E. M., J. Mol. Biol.
98:503-517 t1975). These filters were probed for sequences homologous to rat pre-proauriculin by the same conditions used to identify the clones. In this manner we identified a unique 2,600 base pair EcoRI -BamH1 DNA fragment which appeared to span the entire gene.
The six human genomic clones that hybridized to rat pre-proauriculin cDNA were then analyzed for the presence of a similarly sized fragment and one of them, designated HG6, contained such a fragment.
~G6 DNA was then digested with EcoRl and BamHl and DNA fraqments were ligated into pBR322 previously digested with the same endonucleases. Ligation prod-ucts were transfered into E. coli MC1061 cells as previously described. Plasmid pHGRBl was thusly generated among the clones to the other fragments, and identified by the colony hybridization procedure of Grunstein, M. and D. Hogness, Proc. Natl. Acad. Sci.
USA 72:3961-3965 (1975). Hybridizations were performed as described above. pHGRBl was then sequenced and shown to contain the entire gene sequence for human pre-proauriculin 2. Se~uencinq of the human pre-proauriculin gene.
For the human gene, the 2589 base pair fragment shown to hybridize with the rat cDNA was prepared from a larqe-scale plasmid prep by 4% polyacrylamide gel electrophoresis. Before sequencing could proceed, the large size of the DNA segment dictated that several useful restriction endonuclease cleavaae sites be determined which would break the sequence up into smaller pieces. Particularly useful sites were found at positions 586 tSstI~, 984 and 1839 (AvaI), and 1581 .. . . . . . . . ... .... . . .. ..

-24- 13~61 and 2374 (PstI). These sites are shown in Figure 6 which portrays the human ~ene sequencing strateay consistent with methods described for rat cD~A in Section A.4. Several M13 subclones were prepared spanning the DNA segments between these sites in order -~ to cover these regions on both DNA strands. The DNA
fragments generated by restriction endonuclease cleav-age and M13 subcloning are indicated in Figure 6 by arrows 1-10. ~The resulting sequence is shown in Figure 7. The sequence information obtained was analyzed ~ using various Intelligenetics*(Palo Alto, California) computer programs in accordance with the instructions of the manufacturer. The regions containina the si~nal peptide, precursor sequence and mature peptide were identified by comparison to the rat pre-proauriculin cDNA. The entire coding region is contained within the BamHI to EcoRI fraqment, and the coding reaion for the qene contains 2 introns of 122 and 1095 bases, and 3 exons spanning approximately bases 477-696, 819-1145 and 2241-2536. Putative control signals for both transcriptional initiation (bases 347-354 and 446-~52) and termination (bases 2515-2520) were also localized within the fragment.

C. Expression of proauriculin and related fracments in Escherichia coli 1. Construction of pKT52 bacterial expression plasmid.
a) Generation of the trc promoter One ~q of plasmid ~EA300 ~Figure 8) (Amman, E. et al., Gene 25:167-178, 1983) was digested in 10 ~l accordinq to the manufacturer's instructions with PvuII
and ClaI, Purchased from New En~land Biolabs, Inc.
-Beverly, Massachusetts. The diqest was electro~horesedin a 0.8% agarose gel as described by Maniatis, T. et al. supra at p. 157-160. The large ~raqment containing * Trade Mark .. . . . , . . .. _, .. .... .

-25- 13402G~
the -35 nucleotide region of the trp promoter near the ClaI site was detected by UV-shadowing as described by Maniatis et al., supra at p. 157, and eluted from the gel slice in 500 ~l of gel elution buffer overnight at 37~ as described by Maxam, A. and ~. Gilbert, ~ethods in Enzymoloqy, 6~:449-560 (1980). The ClaI site of the large fragment (50 na) was filled in with 5n ~M dCTP in a 10 ~l volume as described in Maniatis et al., supra at p. 394, and the remainina single-stranded 5' over-hang removed by digestion with mung bean nuclease(Pharmacia P-L Biochemicals, Inc.) as described by Kroeker, W. et al., Biochemistry 17:3236-3239 (1978).
One ~g of plasmid pGL101 (Figure 8) (Lauer, G. et al., J. Mol. Appl. Genet. 1:139-147, 1981) was diqested with PvuII and HpaII (New England Biolabs) as described and the digested fragments filled in by the method of Maniatis et al., supra at p. 394, with 5 units of E.
coli polymerase I, Klenow fragment (Boehrinqer-Mannheim, Inc., E~annheim, FRG) and the addition of 1 ~Ci [~- 2P]-dCTP (Amersham, Chicago, Illinois, 800 Ci/mM) for 15 minutes at 37 C followed by the addition of dCTP and dGTP to 50 M for 30 minutes at 37~C. The labeled, blunt fragments were electrophoresed on a 12%
polyacrylamide gel by the method of Maniatis et al., supra at pp. 174-175, exposed at 4~C for 30 minutes to Kodak~ XAR X-ray film, and the 55 base pair blunted HpaII-PvuII fragment cut out of and eluted from the gel as described. The two isolated fragments were liaated in 20 ~l as described in Maniatis et al., supra at p.
392, and used to transform E. coli strain RB791 (R.
Brent and M. Ptashne, Proc. Natl. Acad. Sci. USA
78:4204-4208, 1981) as described in Maniatis, et al., supra at p. 250-251. The resulting plasmid, pKK10-0 (Figure 8) contains the modified promoter, called the trc promoter, and was isolated by the rapid boiling method as described in Maniatis, et al., supra at pp.

, . , . ,, .. , . ~ ......

-~ --26- 13~026~
366-367. Fifty ng of pKK10-0 was digested in 20 ~
with EcoRI (Bethesda Research Labs, Inc.), and used to transform E. coli RB791 as described above. This plasmid, pKK10-1 (Figure 8), was isolated as described and 50 ng digested with PvuII (New England Biolabs) according to the manufacturer's instructions. The PvuII diqested plasmid was ligated to 10 ng of NcoI
linker (dACCATGGT, Creative Biomolecules, Inc. Foster City, California), digested with NcoI (New England Biolabs), filled in with dATP, dCTP, dGTP, and dTTP, and ligated as described to a linker containing PstI
and HindIII sites svnthesized as two complementary oligonucleotides (5' -dGCTGCAGCCAAGCTTGG-3' and 5'dCCAAGCTTGGCTGCAGC-3') on a Biosearch Sam I DNA
Synthesizer (3iosearch, Inc.) according to the manu-facturer's instructions. The ligation mixture was digested with 3amHI and HindIII (~ew England Biolabs), electrophoresed on a 5~ polyacrylamide gel, anc the small BamHI - HindIII fragment eluted as described above. This fragment contains the trc oromoter.
b) Construction of the trc promoter plasmid, pKT52 50 nq of pKK10-2 (Figure 8, Brosius, J., Gene 27:161-172, 1984) was digested with BamHI and H.indIII.
The large fragment was isolated from a 0.8% agarose gel and ligated to the trc promoter fragment described above. The ligation was used to transform E. coli RB791 and the new plasmid, pKK233-1 (Figure 8), iso-lated as described. One ~g of pKK233-1 was digested to completion with PvuI (New England Biolabs) and par-tially digested with BqlI (New England Biolabs) in accordance with Maniatis et al., supra at p. 381. At the same time, 10 ~g of pUC8 (Vieira, J. and J.
Messing, Gene 19, supra) was digested with PvuI and BglI and the 360 base pair PvuI-BglI fragment from the ampicillin resistance qene ~that no longer contains a 13~n2fi~

PstI site) was isolated from a 5~ polyacrylamide ael.
500 ng of this fragment was mixed with 50 ng of the PvuI-~I partial digestion mix of p~K233-1, ligated and used to transfor~ E. coli RB791. Transformants were screened for the presence of only one PstI site ' and checked with a EcoRI-PstI digestion that the remaininq PstI site was next to the trc promoter, generating plasmid pKK233-2 (Figure 8). 20 nq of plasmid pKK233-2 was digested with EcoRI and PvuII, filled in with dATP andJTTP, ligated, and transformed - into E. coli RB791. The resulting vector is pKT52 (Figure 8).
2. Expression of rat proauriculin fracments a) Construction of plasmid pRNF-6852 The construction of the plasmid which allowed the --expression of proauriculin fragments in E. coli is schematically represented in Figure 9 and detailed below. All restriction endonuclease enzymes and T4-DNA
liqase were purchased from New England Biolabs and used according to the manufacturers specification. Five ~g of plasmid pMFl (see above) were di~ested to comPletion with HincII for 2 hrs. at 37~C. Following digestion, the mixture was extracted with phenol:chloroform:
ethanol precipitated dried in vacuo and resuspended in water. An NcoI decamer linker (dAGCCATGGCT) was synthesized on a SAM I DNA Synthesizer*(Biosearch Inc.
and purified by preparative gel electrophoresis as described by the manufacturer's instructions. The synthetic NcoI linker (0.5 ng) was phosphorylated at it's 5' end with T4-polynucleotide kinase (P-L
Biochemicals) using the procedure of Maniatis et al., supra at p. 396 and attached to 3 ng of HincII digested pNFl by blunt-end ligation with T4-DNA ligase in a 20 ~l reaction volume at 12.5~C for t6 hrs.

* Trade Mark 1 3 ~

Following an incubation at 65~C for 5 min. the liqation mixture was adjusted to 100mM NaCl and incu-bated for 2 hrs. at 37~C with NcoI and PstI. The mixture was submitted to gel electrophoresis on a non-denaturing 5% polyacrylamide gel (Maniatis, et al.,supra at pp. 174-177) until the bromphenol blue dye was at the bottom of the gel. The separated DMA was visualized by autoradiography followed by excision of a 316 bp band. The DNA was eluted overnight at 37~C as described (Maxam, A. and W. Gilbert, supra) followed by ethanol precipitation; drying in vacuo and resuspen-sion in water.
Two ~g of the expression plasmid, pKT52 were digested to completion with NcoI and PstI followed b~
t5 treatment with calf intestinal phosphatase (Boehringer Mannheim, Mannheim, FRG) in accordance with Maniatis, et al., supra at pp. 133-134. Fifty nanograms of the purified 316 bp NcoI-PstI fragment derived from pNF1, were mixed with 10 ng of NcoI-PstI digested pKT52 and incubated with T4-DNA-ligase in a total volume of 20 ~l for 30 min. at 25 C and 4 hours at 12.5~C. E. coli strain JA221 (1pp , hsd M , trpE5, leuB6, lacY, recA1/F', lacIq, lacZ , proA , proB , Nakamura, K. et al., J. Mol. Appl. Genet. 1:289-239 (1982)) was made competent for transformation by the CaCl2 method and transformed with the ligation mixture as described in Maniatis et al., supra at pp. 250-251. Resulting ampicillin resistant colonies were grown overnight in 1 ml of L-Broth from which plasmid DNA was prepared by the alkaline lysis method (Maniatis, et al., supra at pp. 368-369). Plasmids were screened f9r the correct insert by digestion with first HindIII followed by KpnI
or NcoI. A plasmid having both HindIII-KpnI and HindIII-NcoI fragments of approximately 120 bp and 320 bp respectively, was chosen and designated pRNF-6852 (Figure 9).

-- 131026~

To confirm that the reading frame of the cloned proauriculin sequence in pKT-52 was correct, pRNF-6852 was digested with EcoRI and PstI followed by purifica-tion of a band of approximately 509 bp by 5% polyacryl-amide gel electrophoresis as described above. The EcoRI-PstI fraament was cloned in plasmids M13mp8 and M13mp9 (Messing, J. and J. Vieria, supra) and submitted to dideoxynucleotide sequence analysis (Sanger et al., supral.
As shown in Figure 9, plasmid pRNF-6852 was designed to express a fragment of the rat proauriculin - cDNA which encodes a protein from amino acids 87 to 152 ' (see Figure 1~. Because a synthetic decamer N~oI
linker was used to allow cloning of the proauriculin cDN~ into the expression vector pKT52, the first two ~ amino terminal amino acids of the expressed fragment are NH2- Met-~la followed by amino acids 87 through 152 of the rat proauriculir. precursor ~Figure 9).
b) Ex~ression of cloned rat Proauriculin fragment (~7-152~ cDNA in plasmid pRNF-6852 E. coli JA221 lpp /F' lacIq containin~ pRNF-6852 or pKT52 were grown at 37~C in media containing M9 minimal salts ~Miller, J., Experiments in Molecular Genetics, Cold Spring ~arbor Laboratory, Cold Spring ~arbor, New York) supplemented with glucose (4 mg/ml), thiamine (2 ~g/ml), MqS04 7H20 (200 ~g/ml) leucine (20 ~g/ml), tryotophan (20 ~g/ml), ampicillin (100 ~g/ml), and isopropyl-1-thio-~-D-galactopyranoside (2 ~M). At a cell density of approximately 2.5 x 10 cellsjml, L-[35S~-cysteine (100~Ci/ml culture (Amersham Corp., Chicago, Illinois 930 Ci/mmole)), was added. Following 30 sec of incubation, 1 ml of culture was removed and added to 0.34 ml of ice-cold 20% trichloroacetic acid in a 1.5 ml Eppendorf centrifuge tube, vortexed and allowed to stand at 0~C for 30 min. The mixture was then centrifuged at 4~C for 15 min in an Eppendorf*

* Trade Mar~

,. . . . . . .

- ~ 3 4~

centrifuge at 15,000 x g. The supernatant was dis-carded and the pellet washed with 1 ml of ice-cold acetone followed by centrifugation and drying of the resulting pellet in vacuo.
An IqG fraction was prepared from 1 ml of non-immune serum or anti-serum (raised against a chemically synthesized rat auriculin peptide~ using Protein A-Sepharose~ 4B ~Pharmacia Fine Chemicals, Uppsala, Sweden) chromatography as described in the manufac-turer's specifications and collected in a total volume of 4 ml.
The dried TCA pellet was resuspended in 40 tl of 50 mM Tris-Cl, pH 8.0, 1 mM EDTA, and 1~ SDS and incubated at 100~C for 5 min. Ten ~l of this mixture (representing total bacterial protein) was diluted to 20 ~l with 20 mM Tris-~Cl, pH 6.8, 22~ glycerol, 2%
S~S, 2~ 2-mercaptoethanol, ana 0.1~ bromphenol blue, followed by incubation at 100~C for 5 min. The remain-in~ 30 ~1 (used for immunoabsorption) of the mixture was diluted to 1 ml with 50 m-'~ Tris-HCl, p~ 8.0, 1 mM
EDTA, 0.15 M NaCl, and 2~ Triton-X100, followed by the addition of 40 ~l of purified IgG derived from non-immune serum or antiserum raised aaains~ rat auriculin.
The mixture was incubated at room temperature for 30 min and 4~C overnight.
Following the overnight incubation, 50 ~ul of Protein A-sepharose~ 4B (10% suspension in 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.15 M NaCl, 0.5% Nonidet P-40*
(NP-40) and 1 mg/ml ovalbumin) was added to the mixture and incubated at 4~C for 1 hr with gentle agitation.
Following centrifugation at 4 C, the supernatant was discarded and the Protein A-Sepharose~ pellet resus-pended in 0.5 ml of 50 mM Tris-HCl, pH7.5, 5 mM EDTA, 0.5 M NaCl, 0.5% NP-40, and 1 mg/ml ovalbumin. The pellet was washed by vigorous vortexing, followed by centrifugation and removal of the supernatant.

* Trade Mark , . .

~31- 13~2~
This procedure was repeated four additional times.
The Protein A-Sepharose~ pellet was washed an addi-tional two ti~es with 5n m~ Tris-HCl, pH 7.5, 5 mM
EDTA, 0.15 M NaCl and 0.5% NP-4G*, followed by one wash with 10 mM Tris-HCl, pH 7.5. Following drying in vacuo, the pellet was resuspended in 60 1 of 10 mM
Tris-HCl, pH 6.8, 1% glycerol, 1% SDS, 1% 2-mercapto-- ethanol, and 0.05% bromphenol blue, followed by incuba-tion at 100~C for 10 min.
The total and immunoabsorbed samples were sub-jected to discontinuous SDS-polyacrylamide gel electro-phoresis as described by Anderson, C.W. et al., J.
Virol. 12:241-252 (1973) on a 130 x 200 x 0.8 mm polyacrylamide slab gel containing 17.5% acrylamide, i 15 0.0735~ bis-acrylamide, 0.335 M Tris-HCl, pH 8.7, 0.04 M NaCl, 0.1~ SDS, 0.05% ammonium persulfate, and 0.05%
TEMED. The samples were run at 30 mA constant current until the bromphenol blue dye reached the bottom of the gel. The separated proteins were fixed in the gel by shaking in a solution of 25% isopropyl alcohol, l9~
acetic acid, and 0.12 mg/ml Coomassie Brilliant Blue (Sigma Chemicals, St. Louis, Missouri) R-250 for l hr at room temperature, followed by overnight incubation in a solution of 10% isopropyl alcohol, 10% acetic acid, and 0.12 mq/ml Coomassie Blue. Foilowing de-staining with 10% acetic acid, over a period of 3 hours with several changes, the gel was treated with En Hance (trade mark ) (New England Nuclear, Boston, Massachusetts) according to the manufacturer's direc-tions, followed by drying and fluoroqraphy at -70~C
using Kodak~ XAR-5 x-ray film.
A comparison of the polypeptide patterns from cells containing plasmids pKT-52 or pRNF-6852, labeled with L-[35S]-cysteine as described above, is shown in Figure 10. A polypeptide with an approximate molecular size of 6200 daltons appears uniquely in lane 2, which * Trade Mark ~ 13~026~

represents the total polypeptides derived from pRNF-6852. This polypeptide is specifically immunoreactive only to anti-auriculin IgG and not non-immune IgG
(compare lane 3 with lane 4 in Figure 10). In addi-tion, there was no detectable reaction of immune IgGwith any polypeptide derived from pKT-52 (lane 5, Figure 10). Thus, it was concluded that the predicted fragment of proauriculin was expressed in cells con-taining the specific plasmid pRNF-fi852.
E. coli strain JA221 1pp F' lacIq containing pRNF-6852 was deposited with the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, MD
20852 on May 31, 1984 and accorded the accession number 39720.
3. Expression of the full-length rat proauriculin In a manner similar to that described in Section C.2, full length proauriculin is expressed. To accomplish this, plasmid pNFl is digested to completion with AccI, followed by phenol:chloroform extraction and ethanol precipitation. The AccI-digested DNA is treated with E.coli DNA polymerase (Klenow fragment) (Boehringer Mannheim, Mannheim, FRG) followed by extraction and ethanol precipitation. The synthetic NcoI linker (dACGGGAGCCATGGCTCCCGT) is synthesized, purified, and phosphorylated and attached to the AccI
digested pNFl DNA via blunt-ended ligation as described in Section C.2.
Digestion of the ligation mixture with NcoI and PstI yields a 487 bp DNA fragment which is purified by 5% polyacrylamide gel and eluted. Fifty ng of the purified 487 bp NcoI-PstI-fragment are mixed with 10 ng of NcoI-PstI diqested pKT52 and incubated with T4-DNA
ligase.
Following transformation of JA221 1pp /F'lacIq with the ligation mixture, mini-preps of plasmids -33- 13~26~
derived from the resulting ampicillin resistant colonies, are screened for the correct insert by digestion with HindIII followed by KPnI or HincII
.
digestion.
A plasmid having both a HindIII - KpnI and HindIII
- HincII fragments of approximately 120 bp and 312 bp respectively, is chosen and designated pRNF-12852. The reading frame of the cloned full length proauriculin sequence in pKT52 can be confirmed by dideoxynucleotide sequence analysis (Sanger, supra).
Plasmid pRNF-12852 will encode a protein from residues 26 through 152 of the rat pre-proauriculin precursor (see Section A.4.).
Because a synthetic NcoI linker is used to allow cloning of the proauriculin cDNA into the expression vector pKT52, the first two amino-terminal amino acids of the expressed fragment are NH2-Met-Ala followed by amino acids 26 through 152 of the rat proauriculin.
4. Expression of human proauriculin The plasmid pHGRBl containing the human genomic DNA can be digested to completion with ApaI, followed by T4-DNA polymerase treatment (Maniatis et al., supra at p. 395) to repair the 3'-extended termini. A
synthetic HindIII linker (pCAAGCTTG, Collaborative Research Inc., Lexington, MA) was attached to the blunt-ended human genomic DNA through blunt-end liqa-tion as described above. The ligation mixture is then digested with HindIII and NcoI, followed by the isola-tion of a 272 bp HlndIII-NcoI fragment using 5% poly-acrylamide gel electrophoresis. The 272bp HindIII-NcoI
fragment is mixed with ~indIII-NcoI digested pBR329 (Covarrubias, L. and F. Bolivar, Gene 17:79-89 (1982)) and treated wtih T4-DNA li~ase. The resulting plasmid pHNF-298 is digested with BamHI and NcoI and the resulting 620 bp NcoI-BamHI fragment purified by agarose gel electrophoresis. The 620 bp NcoI-BamHI

02~4 fragment is digested to completion with MspI followed by repair of the 5'extended termini by E. coli DNA
polymerase I (Klenow fragment). The synthetic HindIII
linker pTTACTAAGCTTAGTAA is synthesized, purified and phosphorylated and attached to the MspI digested NcoI-BamHI fragment through blunt-end ligation.
The ligation mixture is digested with HindIII, followed by the isolation of an 156 bp HindIII fragment by 5~ polyacrylamide gel electrophoresis. The 156 bp HindIII fragment is attached to pKT52, which had been digested with HindIII and treated with calf int:estinal alkaline phosphatase using T4-DMA ligase.
Following transformation of JA221 1pp /F'lacIq with the ligation mixture, mini-preps of plasmids derived from the resulting ampicillin resistant, colonies, are screened for the correct insert by digestion with NcoI followed by ClaI digestion.
A ~lasmid having an NcoI-ClaI insert of 150 bp is chosen and designated pHNF-5752. The reading frame of the cloned human proauriculin sequence in pKT52 is confirmed by DNA seauence analysis as described.
Because a synthetic HindIII 8-mer linker is used to allow cloning of the proauriculin cDNA fragment into the HindIII site of pKT52, the amino acids preceding the proauriculin sequence are l~et-Ala-Ala-Ala-Lys-Leu-Ala. In addition the svnthetic HindIII 16-mer linker is used to reconstruct the carboxy terminal amino acid residue Arg and Tyr. Therefore, the sequence of the expressed human proauriculin fragment is: NH2 Met Ala Ala Ala Lys Leu Ala Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr COOH.

, . ... ~ .~ ........................... .. . . . . . . .

-35- 13~02~
D. Expression of Proauriculin and Pre-proauriculin in Saccharomyces cerevisiae 1. Intracellular expression Two procedures are disclosed for the preparation of vectors for intracellular expression in the yeast Saccharomyces cerevisiae of cDNA encoding pre-proauriculin, proauriculin and fragments of proauriculin containing mature auriculin. Each utilizes the strong promoter sequence found in front of the yeast phosphoglycerate kinase (PGK) gene. For the first Procedure~ the plasmid pNFl was digested with HincII (New England Biolabs). BamHI linker oliqo-nucleotides (8 nucleotides in length Collaborative Research, Inc.) were ligated onto the diqestion prod-ucts, and the resulting molecules were digested withBamHI. The 454 b~ fraqment from this di~est containing the mature auriculin sequence was then purified by 5~
polyacrylamide ~el electrophoresis and ligated into the BamHI site of the yeast - E. coli vector pYPGK2. This vector was constructed by digesting the yeast - E. coli shuttle vector YEpl3 (J. Broach et al., Gene 8:121-133 (1979)) with the restriction enzymes BamHI and HindIII, and then ligating the largest of the restricticn fragments obtained to a restriction fragment spanning the promoter region from the yeast PGK gene. Ihe PGK-promoter-containing fragment extends from a HindIII
restriction site approximately 1500 base pairs upstream from the ATG start codon of PGK, to a BamHI linker oligonucleotide (8 base pairs in lenqth, Collaborative Research,. Inc.) inserted 28 base pairs downstream from the ATG start codon after BAL-31 digestion from within the PGK coding region.
Using this vector, any sequence of DNA in the pre-proauriculin sequence can be inserted and used to express a desired fragment of pre-proauriculin. For example, insertion of the 454 bp auriculin-containing 134~64 fragment into the BamHI linker site in this vector in the correct orientation allows the synthesis of a 78-amino acid-long fusion protein from the PGK promoter (consisting of 9 amino acids from the amino terminus of the PKG gene, 3 amino acids coded for by the linker oliaonucleotide, 39 amino acids of the pro-auriculin reqion, 25 amino acids of the mature auriculin sequence, and the two arginine residues of the carboxy terminus of the auriculin precursor).
A second procedure for intracellular expression of pre-proauriculin also allows extracellular secretion of proauriculin and fragments thereof. In the second procedure, a restriction fraqment containing the entire pre-proauriculin precursor coding region is isc>lated from the plasmid pNF4 by first digesting the plasmid with the restriction enzyme SalI (Mew England E~iolabs).
The single-stranded regions on the ends of the result-inq linear length plasmid molecules are made double-stranded by treatment with DNA polymerase I (Klenow fragment), and Ba~HI linkers (8 nucleotides in length, Collaborative Research, Inc.) are then liqated on to these blunt ends. The linear-length plasmid molecules are then di~ested with BamHI and EcoRI, and the approx-imately 900 bp Bam~I (SalI) - EcoRI fragment containing the pre-proauriculin sequence is isolated. The frag-ment is ligated into a vector identical to the pYPGK2 vector described above, except for two modifications:
(1) the BamHI linker oliaonucleotide lies 23 bp up-stream from (5' to) the ATG codon of PGK, and (2) the cloned cDNA fraqment is followed by the transcription termination region of the PGK gene (EcoRI - HindIII
fragment containing the 3' end of the PGK locus, plus the 346 bp HindIII - BamHI fragment from pBR322 as a 3' linker. Expression of the inserted pre-proauriculin cDNA from the PGK promoter results in the synthesis of pre-proauriculin. The pre-proauriculin that is ex-13~2~

pressed will be processed and secreted by the yeast cell if the signal and/or processing sites are recog-nized as such by the cell and acted upon. The material so secreted will be either proauriculin, fragments thereof or auriculin alone. If recognition of the signal sequence does not occur, the full-length pre-proauriculin or fragments thereof will be found intern-ally in the cells.
2. Extracellular Expression a) Construction of YEp~X-8 expression vector A yeast library in the E. coli-yeast shutt:le vector YEp13 (Nasmyth, K. and K. Tatchell, Cel~
19:753-764 (1980)) was screened using a ~5_ 2p end labeled oligodeoxynucleotide (5'-CCTGGCCAACCAATG-3'), (see Maniatis et al., supra at pp. 324-325). E'lasmids containing inserts of yeast DNA hybridizing to this oligonucleotide were subsequently isolated. One of these plasmids contained an insert of apProximately 15kb of yeast DNA, and was shown to contain the 1.7kb EcoRI fragment containing the ~-factor gene as de-scribed by Kurjan, J. and I. Herskowitz, Cell _0:933-943 (1982). The ends of the 1.7kb EcoRI fragment were made blunt bS incubation with DNA polymerase I (Klenow fragment) and BamHI linkers using T4-DNA ligase (Maniatis et al., suPra at pp. 113-114, 116, 392-394).
The BamHI ends were made cohesive by digestion with BamHI restriction endonuclease, and subsequently ligated into the BamHI site of the yeast-E. coli shuttle plasmid pCV7-Hina228. A deletion around the HindIII site of the plasmid C~17 was made by HindIII
diqestion, treatment with exonuclease III, treatment with S1 nuclease, and religation with T4-DNA ligase to generate the plasmid pCV7-Hin~228, all using the method described in Broach, J.R. and J.B. Hicks, Cell 21:501-508 1980. This plasmid containing the yeast ~-factor .. . . . .

- ~ . -13~U261 gene is diagrammed in Figure 11, and henceforth re-ferred to as YEp-~-8.
b) Insertion of cDNA codinq for rat proauriculin into YEp~X-8 Two fragments of DNA from pNFl (Section A.3.) encoding pre-proauriculin were inserted into the unique HindIII site of YEp~-8 (Figure 11) by restriction endonuclease cleavage, filling in the ends of DNA with DNA polymerase I (Klenow fraqment) as necessary and adding HindIII linkers (Maniatis et al., supra at p.
392). The ends of the DNA fraqments were subsequently made cohesive by digestion with HindIII endonuclease, and liqated into ~indIII cleaved YEp~-8, which had been treated with alkaline phosphatase to remove its 5' phosphate moiety (see Maniatis et al., supra at pP.
133-134). Recombinant molecules were transformed into _ coli, and colonies analyzed for plasmid DNA
(Maniatis et al., supra at pp. 366-369).
A HaeIII fragment was generated as shown and size selected from polyacrylamide gels as described in Maniatis et al, supra at pp. 173-175. This fragment of 266 bp was then cloned into YEp~X-8, as described above to generate expression vector YEpt~-NF-5. This insert, in the correct orientation, encodes a 33 amino acid peptide containing the mature auriculin sequence, correspondinq to amino acids 121-152 of the prc-auriculin sequence (Figure 1) with an additional phenylalanine at the amino terminus. As a control, the reverse orientation of the insert was cloned into YEp-~-8 and designated YEp-~-NF-7. This insert wo~ld encode an unrelated protein having a sequence of different amino acid. Similarly, an AccI fragment of 623 bp was isolated and cloned in its correct orienta-tion into YEp-~-8, yielding expression vector YEp-~-NF-9. This insert encodes a 126 amino acid polypeptide comprisin~ almost the entire proauriculin sequence ~39~ 13102~4 (amino acids 28-152J with an additional tyrosine at the NH2 terminus. This insert was also cloned in its inverse orientation to generate control plasmid YEp~-NF12. Insertion of these HaeIII and AccI fragments of rat proauriculin, after the addition of the HindIII
linkers, yields DNA sequences codin~ a chimeric pro-tein. This protein codes for the ~-factor sig-nal/leader peptide, a spacer fraqment and the proper proauriculin fragment.
DNA was prepared from E. coli cultures containing these plasmids (~aniatis et al., supra at pp. :366-369) and was used to transform yeast strain W301-18A (C~ ade 2-1, trp 1-1, leu 2-3, 112, can 1-100, ura 3-1, his 3-11, 5) to Leu 2 prototrophy. Yeast strains were grown on standard media (Sherman et al., Methods in ~'east Genetics, Cold Spring Harbor Press (Cold Spring Harbor, New York)). Plasmid DNA from E. coli was also re-cloned into ~13 for sequencing and confirmation of the ~-factor proauriculin DNA constructions (i~lessinq J. and J. Vieira, supra).
c) Expression and secretion of rat proauriculin seauences in S. cerevisiae The factor proauriculin fragment processing scheme shown in Figure 11. The mRNA transcript: is initiated and terminated from the ~-factor sequences in the vector. This is translated into a chimeric protein and initiated through the yeast secretory process.
Proteolytic processing of this protein occurs both at the Glu-Ala (QA) residues and the Lys-Arg (KR) residues in the ~-factor portion of the molecule (Kurjan J. and I. Herskowitz, supra). The C-terminal portion of this processed protein therefore is the predicted amino acid of rat proauriculin. Cultures of yeast containing these plasmids were maintained in synthetic medium lackinq leucine (see Sherman et al., supra). This selection is necessary, as yeast plasmids are rela-... . .. . . . .

13~02~

tively unstable and lost at approximately 1.0% per generation. Yeast cultures were labeled with 0.1 to 0.5 mCi/ml 3 S-cysteine (approximately 1000 Ci,~mmole) in synthetic medium without leucine for four hours.
a 5 Bovine serum albumin was added at a final concentrationof 100 ~g/ml to prevent possible proteolysis. Samples (1.0 ml) were taken, cells removed by centrifugation, the media proteins concentrated by 10~ TCA precipita-tion at 4~C for 15 minutes and subsequent centrifuga-tion in an Eppendorf~microfuge (15,000 x g). The resulting pellet was washed with acetone, dried under vacuum and resuspended in SDS sample buffer (Laemlli, U.K., Nature 227:680-685 (1970)). These samples were applied directly to an SDS-PAGE gel (17.5~ acrylamide) to examine the pattern of total secreted 5S-met proteins by autoradiography of the dried gel. As can be seen in Figure 12A, culture supernatants from yeast cultures containing YEp-~-NF-9 showed 3 S-met labeled hands at approximately 11.1 and 9.~ kd (lanes 3 and 4) while media from cultures of YEp-~-NF-12 (the inverse construction) showed a 35S-met labeled band at approxi-mately 5 kd (lanes 5 and 6). Neither of these bands were detected in media from cultures containing the plasmid vector YEp-~-~ (lanes 1 and 2).
The molecular weights of the proteins whose synthesis and secretion is directed by YEp-~-NF-9 are not inconsistent with the possibility that an endogen-ous yeast protease cleaves the auriculin peptide from the proauriculin precursor encoded by the AccI fragment in this plasmid. To confirm this possibility, yeast cultures harboring this plasmid, its corresponding inverse orientation (YEp-X-NF-12), and yeast cultures harboring YEp~X-NF-5 and YEp~X-NF-7 (the HaeIII frag-ment encodin5 the small fraament of proauriculin) were labeled as above with both 3 S-Met and 35S-Cys to determine if they expressed s-labeled proteins which * Trade Mark ~41- 134026~

could be specifically immunoprecipitable. The 5S-Met will be incorporated into proauriculin protein but not mature auriculin while S-Cys is selectively incorpor-ated into mature auriculin (see Figure 1). Since control experiments suqgested that some yeast media components prohibited direct immunoprecipitation, a novel partial purification scheme was performed as follows.
Cells were removed by centrifugation and the cell free supernatant used either directly or concentrated by lyophilization. Ten volumes of acetone were added to the aqueous solution and the mixture allowed to precipitate on ice for 10-15 minutes. The precipitate was then pelleted by centrifugation, and the acetone removed. A small amount of water (no more than 1 volume) was added to this pellet to facilitate resus-pension. Ten volumes of methanol were then added to this mixture, extensively mixed, and the precipitate collected by centrifugation. The supernatant was then removed and dried under vacuum. This pellet was resolubilized in 1.0 ml of immunoprecipitation buffer and immunoprecipitated and washed as described in Section C.2.
As shown in Figure 12B, the complexity of proteins as determined after the above extraction proceciure is relatively simple compared with the complexity of total secreted protein. Lanes 1 and 2 show the secreted 35S-Cys labeled protein whose synthesis is directed by YEp-~-NF-7 and YEP-~-NF-5 respectively in the methanol soluble fraction. Lanes 3 and 4 show the same proteins following immunoprecipitation in both cases with anti-auriculin IgG. The antiserum appears to specifically precipitate a 3,000 Dalton protein from YEp-~-NF-5 (lane 4) while no protein was precipitated from the corresponding inverse orientation (YEp-~-NF-7) (lane 3). Lanes 5 and 6 show a similar immunoprecipitation 13~0264 of 35S-Cys label proteins appearing in the methanol soluble fraction of media conditioned by yeast eultures harboring plas~ids YEp-~-NF-12 and YEp-~-NF-9, respee-tively. The result is the same as shown for lanes 3 and 4 and a 3,000 Dalton protein was speeifieally immunopreeipitated from media eonditioned by S.
cerevisiae containing YEp-~-NF-9.
These results suggest that both yeast expression plasmids, YEp-~-NF-5 and YEp-~-NF-9, direct the synthe-sis of a 3 kd 35S-Cys labeled protein (approximately 25-30 amino acids in length) which is immunoprecipi-tated by specific anti-auriculin IgG.
_ cerevisiae strain ~1301-18A eontaining YEp-~-NF-9 was deposited with the ATCC on May 31, 1984 and accorded accession number 20710.
d) Expression and Secretion of human proauriculin and related fraaments in ~S. cerevisiae.
As has been shown in the case of rat proauriculin DNA fragments cloned into the plasmid YEp-~-8, the human proauriculin D~A fraqments can similarly be expressed and the products are secreted. DNA fragments for insertion into YEp-~-8 are prepared as described below. The plasmid pHGRB1 (Section B.1.) containing the human genomic DNA was digested to completion with ApaI followed by T4-DNA-polymerase treatment. A
synthetie HindIII linker (pCCAAGCTTGG) (Collaborative Researeh Ine.) is attached to the blunt-ended human genomic DNA through blunt end ligation. The ligation mixture is digested with HindIII and NcoI and inserted into plasmid pBR329 as described in Section C.2. with the resulting plasmid designated pHNF-2910.

. , . , . . . . . . .. . , . ~ .~ . ...

-43- 13~26~
A 619 bp N I-Bam~I fragment is prepared from pHNF-2910 and attached to the synthetic HindIII linker pTTACTAAGCTTAGTAA as described in section C.2.
A 155 bp _ dIII fraqment is isolated by 5%
acrylamide gel electrophoresis followed by treatment with the DNA polymerase I (Klenow). This fragment is mixed with plasmid YEp-~-8, which had been digested with HindIII and treated with DNA polymerase I (Klenow) and calf intestinal alkaline phosphatase, usin~ T4-DNA
liqase.
Following transformation of E. coli strain JA221 1pp /F'lacIq ~ith the ligation mixture, mini preps of plasmids derived from the resulting ampicillin resis-tant colonies are screened for the correct insert by diqestion with SalI followed by ClaI. A plasmid having a SalI-ClaI insert of 411 bp is chosen and designated YEp- NF-20.
The readinq frame is confirmed by DNA sequence analysis as described and encodes the followinc~:

Ala Glu Ala Ser Phe Ala Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arq Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg l~let Asp Arq Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr COOH

These plasmids are subsequently transformed into W301-1~A, or any other suitable yeast strain, to express human Proauriculin DNA sequences from t:his vector and secrete the products.

E. Expression of rat pre-proauriculin in cultured Chinese hamster ovary cells 1) Expression of rat pre-proauriculin To facilitate the expression of rat pre-proauricu-lin in mammalian cells, a hybrid gene was constructedin which the coding segment for rat pre-proauriculin was fused to a powerful regulated promoter derived from the human metallothionein II (hMTII) gene. This was performed in two steps. First, an exoression vector was prepared. As shown in Figure 13, the expression vector, pHSI, carries 840 nucleotide base pairs of hMTII sequence (Karin, ~. et al., Nature 299:797-802 (1982)) from a naturally occuring HindIII restriction site at base -765 at the start of transcription to base 70, located in the 5' untranslated region adjacent to the coding region. pHSI also carries a region into which coding seauences may he inserted. To construct pE~SI the plasmid o84H, which carries the hMTII gene, was digested to completion with restriction encionu-clease BamHI followed by treatment with exonuc~easeBal-31 to remove terminal nucleotides. Following digestion with HindIII, the products of this reaction were ligated into plasmid pUC8 (Vieira, J. and J.
Messing, Gene 19:259-268 (1982) which had been opened with HindIII and HincII digestion. One of the result-ing plasmid recombinants had the composition of pHSI as determined by nucleotide sequencing.
To complete the construction of the hybrici gene, the EcoRI-SalI rat pre-proauriculin cD~A was isolated from plasmid pNFl (Section A.3) by digestion with EcoRI
and SalI followed by polyacrylamide gel purification.

13~026~

pHSI was opened with EcoRI and ligated to the cDNA
fragment with T4-DNA ligase. The reaction products were then incubated with the four nucleotide triphos-phates and DNA polymerase I (Klenow fragment) in order to create blunt-ended molecules which were subsequently subjected to a second ligation to allow recirculariza-tion. The recombinant plasmid molecules were intro-duced into _ coli MC1061 and screened by restriction endonuclease analysis (Maniatis et al., supra at p.
104). Two recombinants, with the structure shown in Figure 13, pMT-NFl-10 and pMT-NFl-20, were introduced into the chinese hamster ovary (CH0) line of cultured cells by co-transformation with pSV2:NEO (Southern, P.
and P. Berg, J. Mol. Appl. Genet. 1:327-341 (1q82)), a plasmid carrying a functional gene conferring resis-tance to the neomycin analogue G418. 500 ng of pSV2:MEO and 5ug of pMT-NFl-10 or p~T-NFl-20 were applied to a 60 mm dish of cells in a calcium phosphate-DNA coprecipitate according to standard protocols (~igler, M., et al., Cell 16:777-785 (1979)) with the inclusion of a two minute "shock" with 15 glycerol after 4 hours exposure to the DNA. A day later the cells were subjected to exposure to G418 at lmg/ml. This procedure yielded a pool of G418 resis-tant colonies most which had also acquired stableinheritance of pMT-NFl-10 or p~T-MF1-20. Previous experience with CHO cells and other cultured cells (McCormick, F. et al., Molecular and Cellular Biology 4-1 p.166 (1984)) indicates that they are able to cleave the signal peptide from mammalian prehormones and are able to secrete the remainder of the polvpep-tide into the nutrient medium. Accordinaly, the production of pre-proauriculin and related peptides is then examined by incubating the cells with 3 S-met and examining the radio-labeled secreted products by standard protein gel analysis and immunoprecipitation.

13~026~

Autoradiograms of 3 S-Met-labeled prsteins se-creted into the media and immunoprecipitated with anti-auriculin IgG or non-immune IgG are shown in Figure 14. Lane 1 shows inmunoprecipitates of 5S-Met-labeled protein from media of CHO cells containing pMT-NF1-10. The appearance of a 18,000-20,000 Dalton protein that is specifically immunoprecipitated by anti-auriculin IgG is seen in lane 1. This protein is not seen in lane 2 which shows immunoprecipitates of cells containing a control plasmid. Likewise, this band is not seen in lanes 3 or 4 which contain samples identical to lanes 1 and 2, respectively, that were immunoprecipitated with control IgG. Thus CHO cells containing pMT-MF-1-10 secrete pre-proauriculin or a large ( 20,000 Dalton) fragment derived from the precursor into the media of these cells.
Chinese Hamster Ovary (CHO) cells containing pMT-MF1-10 were deposited with the ATCC on May 31, 1984 and accorded accession number CRL 8569.
2) Expression of human pre-proauriculin in cultured mammalian cells Human pre-proauriculin is expressed in a similar manner with appropriate modifications to account for the features of the human genomic clone. Briefly, a plasmid, phANF-B-R, carrying the BamHI to EcoRI human genomic seament spanning the pre-proauriculin gene is constructed, partially digested with restriction endonuclease Ac~I and completely digested with EcoRI.
The resulting ~ EcoRI fragment isolated by poly-acrylamide gel purification. This fragment, whichextends from the 5' untranslated region to a point past the 3' end of the gene, is ligated to the e~pression plasmid pMT401 which is opened with AccI and EcoRI.
Plasmid pMT401 is derived by insertion of the BamHI-bounded polylinker region from M13mp7 (Vieira andMessing, supra) into the BamHI site of pHSI. The 13402~
.

resulting recombinant has the human pre-proauriculin gene positioned 3' from the human metallothionein promoter. The hybrid construction is then introduced into cultured cells for expression in a manner similar to that described above for rat pre-oroauriculin.

F. Biological activitv of exPression products derived from pre-proauriculin and proauriculin Various fragments of proauriculin whose ex?ression and secretion are directed by the yeast ~-factor system described in .Section D.2 and the ~. coli system de-scribed in Section C.2 above were tested and shown to possess biological activity. Yeast cultures (100ml) were grown in synthetic media containing 100 ~g/ml HSA
for 16 hours at 30~C. The cells were removed by centrifugation and the media was lyophilized. The lyophilized powder was reconstituted in 2 ml of dis-tilled H2O and 10 volumes of acetone were added. The solution was thoroughly mixed and then centrifuged at 10,000 x g for 10 minutes in a Sorvall RT6000*centri-fuqe. (Sorvall Instruments, Wilmington, D~). Afterremoval of supernatant, the pellet was resuspended in 1 ml of distilled H2O and 10 volumes of methanol added.
This solution was thorouahly mixed and aaain centri-fuged at 20,000 x g in a Sorvall RCSB centrifuge for 10 minutes. One-half of the solution was dried by rotary evacuation on a Savant-type evaporator ("methanol soluble" fraction; see Table I). The remaining solu-tion was diluted 1:1 with 0.5 M acetic acid and applied to a 3 ml column of SP-Sephadex~ (Sigma Chemical Co.) equilibrated in 0.5 M acetic acid. The column was washed with 15 ml 0.5 M acetic acid and then eluted with 6 ml of 1.0 M ammonium acetate. The eluted material was then dried by lyophilization ("post SP-Sephadex" fraction of Table I). The dried methanol soluble and ammonium acetate eluted material was Trade ~ark ,, ,. , , . ~ . . .. . . . . . ..

13~02~

resolubilized in 0.5 ml of distilled H2O and tested for biological activity using the precontracted rabhit thoracic aortic ring model described in Kleinert, et al., Hypertension 6:Suppl. 1:143-146 (1984~. ~qual volumes of material reconstituted from crude lyophil-ized media, methanol soluble protein or protein eluted from SP-Sephadex~ with 1.0 M ammonium acetate were compared using aortic rings precontracted with 5 ~M
histamine. Material synthesized by yeast cultures whose plasmids encoded proauriculin (amino acids 26-152) (Y~p-~-NF-9) and proauriculin (amino acids 121-152) (YEp-~-NF-5), as well as the corresponding inverse orientations (YEp-~-NF-12 and YEp-~-NF-7, respective-ly), were compared. The results are dePicted in Table I. Si~nificant vasodilatory activity was detected in the methanol soluble material as well as the post SP-SePhadex material in extracts from cells containing YEp-X-NF-5 and YEp-~-NF-9. Extracts from cells con-taining inverse orientation DNA plasmids were not active. This finding, as well as the immunoprecipita-tion data shown in Figure 12, demonstrates that- yeast process full-len~th and smaller proauriculin fragments containing mature auriculin into a form that exhibits potent biological activitv.

.. . .. , . ~ ~ . .....

13i~26~

Table I
Vasorelaxant Properties of Proauriculin and Proauriculin Fragments Expressed by S. cerevisiae -Media Sample% ~elaxation YEp-~-NF-5 (methanol soluble)100.0 YEp-~-NF-5 (post SP-SephadexJ65.5 YEp-~-NF-7 (methanol soluble)11.7 YEp-~-~F-7 (post SP-Sephadex')3.3 ' YEp-~-NF-9 (methanol soluble)71.2 YEp-~-NF-9 (post SP-Sephadex)48.2 - YEP-~-NF-12 (methanol soluble) 13.2 YEp-~-NF-12 (post SP-Sephadex)6.5 Aortic rings were precontracted with 5~M histamine.
The aortic rings were then treated with proteins obtained from S çerevisiae cultures. The proteins . ~.
were purified by acetone/methanol treatment of culture media and the indicated fractions were passed over SP-Sephadex. YEp-~-MF-5 and YEp-~-MF-9 contained pro-auriculin cDNA in its correct orientation and YEp~X-NF-7 and YEp-~-NF-12 contained DNA in an inverse , , orientation. Data are expressed as percent relaxation of the precontracted rings as described in Kleinert, supra.

Samples of the above expressed material were also tested for natriuretic and diuretic activity using the Y isolated perfused rat kidney model as described by Camargo, M. et al., Am. J. Physiol. 246:F447-: F456(1984). As shown in Table II material synthesized and secreted by yeast cultures containing YEp~X-NF-5 and purified as described above through the SP-Sephadex *

* Trade Mark _ . .... . . .

' 50 134026~

step increased urinary Na excretion approximately 3-fold and urinary volume 2-fold as examined during repeated test periods of 10 minutes each. Glomerular filtration also increased in this experiment consistent with the diuretic action. No significant increase occurred in urinary Na excretion, urinary volume, glomerular filtration or renal resistance when the same experiment was performed on material synthesized and secreted by yeast cultures containing the reverse orientation control plasmid YEp-~-NF-7 and purified through SP-Sephadex~.

Table II
Effects of Auriculin Expressed and Secreted by S. cerevisiae on Renal Function in the Isolated Perfused Rat Kidney Control YEp-~-NF-5 YEp-~-NF-7 Urinary Sodium (~Eq/min) 3.56 11.05 3.78 Urinary Volume (rl/min) 6.15 14.0 7.24 Results represent the average of two ten minute control periods followed by the addition of 50 ~1 of SP-Sephadex purified protein. Experimental measurements represent the averaqe of values ohtained during three successive ten minute periods.

As discussed in Section D.2, human proauriculin fragments will be expressed and secreted in S.
cerevisiae and processed bv the cell machinery to a biologically active form as was demonstrated with rat proauriculin. Furthermore, since the amino àcid sequence of human proauriculin is homoloqous to rat proauriculin, the extraction and purification proce-dures described above can be used to produce proteins contained within the human proauriculin sequence.

13~02~

In a manner similar to the yeast-expressed active material, rat and human pre-proauriculin, proauriculin and auriculin expressed by the bacterial and mammalian cell expression systems described in Section C and E, respectively, can be shown to possess biological activity.
The proauriculin fragment whose synthesis was directed by plasmid pRMF-6852 in E. coli was extracted from 1 liter of bacterial cells as follows. The cells were collected by centrifugation at 5,000 x g for 60 minutes and resuspended in 10 ml of 50 mM Tris, pH 7.5.
This suspension was sonicated for 1 minute using a Heat Systems ultrasonic sonicator (Heat Systems, Farmingdale, NY) at setting 4. The sonicate was then centrifuced at 105,000 xg to remove particulate matter and the resultinq supernatent was saved and called crude bacterial extract. A fraction of the crude bacteria extract was subseauently boiled for 5 minutes and lowered to pH 2.5 for 1 hour. The pH was then neutralized and both the resulting boiled-acid extract ; and crude bacterial extract were applied to rabbit thoracic aortic rings as described. As shown in Table III, both the crude bacterial extract and boiled-acid ; extract from extracts containing pRNF-6852 relaxed the precontracted tissue. Control samples from bacteria containing the pKT52 vector without proauriculin DNA
were inactive. Thus, in a manner similar to the yeast expression products, the bacterial proauriculin frag-ments containing mature auriculin were vasodilatory.
Furthermore, since a fraction of this sample was boiled and acid extracted to prevent subsequent processing, I without a loss of biological activity relative to the crude bacterial extract, it was believed that the entire 68 amino acid fragment was biologically active.

* Trade Mark ,, , 13~026~

In addition the bacterial extracts can be prepared in other manners. For example, the soluble protein is further purified by molecular seive and ion exchange chromatography, usin~ methods well known in the art prior to assay of the biological activity. Mature auriculin can also be cleaved from the longer pro-auriculin precursor expressed by the above bacteria using limited proteolysis as described by Currie et al., supra (1984). Proteolytic cleavage can be per-formed either prior to or followinq the purificationprocedure. These processes result in biologically active compounds.

Table III
Vasorelaxant Properties of a Proauriculin Fraqment Expressed in E. coli SamPle ~ Relaxation Crude Bacterial Extract (pRNF-6852) 33 Crude Bacterial Extract (pKT52) o Boiled-Acid Extract (pRMF-6852) 25 Boiled-Acid Extract (pKT52) 0 Rabbit thoracic aortic rin~s were precontracted with 5 ~M histamine. Data are expressed as the ~ relaxation of the precontracted rings.

In the case of mammalian cell expression of full length rat or human pre-proauriculin and proauriculin it may be necessary to cleave the active auriculin protein from this precursor. This can be accomplished by treatinq culture media conditioned by CHO cells harboring plasmids encoding pre-proauriculin (Section E.1) with trypsin under conditions described by Currie, et al., Proc. Natl. Acad. Sci., USA 81:123n-1233 (1984) for the conversion of biologically inactive atrial -13~02~

natriuretic factor (atriopeptin) to biologically active natriuretic factor (atriopeptin).
The proauriculin proteins expressed using the above described recombinant DNA techniques in yeast, bacteria and mammalian cells all contain the common auriculin sequence:
NH2 Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arq Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr25 OH
where amino acid 9 is Met in human auriculin and Ile in rat auriculin. This sequence was also synthesized by automated solid phase methods using a Biosearch SAM II
automated peptide svnthesizer (Biosearch Inc.) accord-ing to the manufact~rer's specifications. The pre-proauriculin fragments expressed in the systems de-scribed above show potent vasorelaxant, natriuretic anddiuretic activities (Tables I and II). These same and related activities have also been demonstrated with a chemically synthesized compound comprising amino acids 126-150 of pre-proauriculin. Thus, these expressed fragments should share all properties with a synthetic auriculin, both on in vitro preparations and when injected in vlvo into humans or animals. The effects of _ vivo administration of a synthetic auriculin on anesthetized dogs are shown in Table IV.

* Trade Mark .

54 1~26~

Table IV
Hemodynamic, Renal and Metabolic Effects Of Synthetic Auriculin in Anesthetized Dogs Control Experimental Recovery MAP (mm Hg) 134 + 5 122 + 4* 136 + 4 GFR (ml/min) 25.5 + 2.7 32.3 + 4.1* 25.4 + 3.3 V (ml/min) 0.21 + 0.03 1.06 + 0.14* 0.37 + 0.05 H2O ( ) 0.9 + 0.2 3.4 + 0.3* 1.5 + 0.2 UNaV (~Eq/min) 38 + 6 187 + 35* 68 + 14 Na ( ) 1.1 + 0.2 4.1 + 0;5* 1.9 + 0.4 UKV (~Eq/min) 15 + 2 36 + 6* 21 + 4 FEK (%) 18 + 1 34 + 6* 21 + 4 PRA (ng/ml/hr) 13 + 2. n 8.3 + 1.8* 14 + 2.5 PA (nq/100 ml) 8.5 + 1.9 5.4 + 0.9* 7.0 + 1.3 MAP, mean arterial pressure (blood pressure)- GFR, glomerular filtration rate; ~.7, urine flow rate; FEH o' fractional water excretion; U~aV, urinary sodium excretion rate; FENa, fractional sodium excretion; UKV, urinary potassium excretion rate; FEK, fractional potassium excretion; PRA, plasma renin activity; PA, plasma aldosterone. *P<0.05 compared to control;
P<0.05 compared to recovery.

As shown, synthetic auriculin lowered mean arter-ial blood pressure, plasma renin activity and plasma aldosterone, and increased urine volume and urine sodium excretion. These are desirable properties for diuretic and antihypertensive a~ents. Furthermore, proauriculin and auriculin act at all major foci of volume and blood pressure requlation. Thus, it can be concluded that pre-proauriculin, proauriculin, and auriculin, when produced by recombinant ONA methods and expressed in yeast, bacteria or mammalian cells in a -13~02~

manner comparable to that described above, will find utility in the acute and chronic treatment of edematous states (i.e. congestive heart failure, nephrotic syndrome, hepatic cirrhosis and ineffective renal perfusion) and in the chronic treatment of renal insufficiencies and hypertension.
As demonstrated in the above examples, the methods and compositions disclosed can find use in expressing pre-proauriculin, proauriculin and fraaments thereof, and auriculin. It is apparent to one having or-dinary skill in the art that any sequence or fragment of polypeptide disclosed herein can be exPressed by employing minor modifications to this disclosure, while remaining within the scope of the invention.
In particular, any fragment of the pre-proauricu-lin amino acid sequence disclosed can be expressed.
Certain of these fragments will not contain the com-plete sequence of auriculin, but may contain portions of auriculin. These fragments can themselves demon-strate biological activity comparable with or comple-mentary to the activities disclosed for the instant compounds.
In addition, human-derived proauriculin, fragments thereof and auriculin contain a single amino acid replacement compared to the bioloaically active rat-derived polypeptides. Therefore yeast, bacterial and mammalian cell expressed human-derived compounds, prepared in the manner previously described, will display similar biological activity.
Although the foregoing invention has been de-scribed in some detail by way of clarity and for purposes of understanding, it will be understood by those skilled in the art that modifications of the invention may be practiced while remaining within the spirit and scope of the appended claims.

~.

.. . ..

Claims (6)

1. A yeast culture transfected or transformed with a DNA sequence comprising DNA encoding a peptide having an amino acid sequence selected from:

H Met Gly Ser Phe Ser Ile Thr Lys Gly Phe Phe Leu Phe Leu Ala Phe Trp Leu Pro Gly His Ile Gly Ala Asn Pro Val Tyr Ser Ala Val Ser Asn Thr Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Val Glu Asp Glu Val Met Pro Pro Gln Ala Leu Ser Glu Gln Thr Asp Glu Ala Gly Ala Ala Leu Ser Ser Leu Ser Glu Val pro Pro Trp Thr Gly Glu Val Asn Pro Ser Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Pro Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Ala Gly Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr R, H Asn Pro Val Tyr Ser Ala Val Ser Asn Thr Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Val Glu Asp Glu Val Met Pro Pro Gln Ala Leu Ser Glu Gln Thr Asp Glu Ala Gly Ala Ala Leu Ser Ser Leu Ser Glu Val Pro Pro Trp Thr Gly Glu Val Asn Pro Ser Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Pro Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Ala Gly Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr R, Claim 1 Cont'd.

h Met Ser Ser Phe Ser Thr Thr Thr Val Ser Phe Leu Leu Leu Leu Ala Phe Gln Leu Leu Gly Gln Thr Arg Ala Asn Pro Met Tyr Asn Ala Val Ser Asn Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser Glu Pro Asn Glu Glu Ala Gly Ala Ala Leu Ser Pro Leu Pro Glu Val Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala Leu GLy Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr OH, H Asn Pro Met Tyr Asn Ala Val Ser Asn Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser Glu Pro Asn Glu Glu Ala Gly Ala Ala Leu Ser Pro Leu Pro Glu Val Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr OH and H Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr OH

wherein R can be OH, Arg OH or Arg Arg OH and including biologically active fragments of said polypeptide or its allelic variants and pharmacologically acceptable salts thereof.

2. The yeast culture of claim 1 which is identified as ATCC accession number 20710.

3. A mammalian cell culture transfected or transformed with a DNA sequence comprising DNA encoding a peptide having an amino acid sequence selected from:

H Met Gly Ser Phe Ser Ile Thr Lys Gly Phe Phe Leu Phe Leu Ala Phe Trp Leu Pro Gly His Ile Gly Ala Asn Pro Val Tyr Ser Ala Val Ser Asn Thr Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Val Glu Asp Glu Val Met Pro Pro Gln Ala Leu Ser Glu Gln Thr Asp Glu Ala Gly Ala Ala Leu Ser Ser Leu Ser Glu Val pro Pro Trp Thr Gly Glu Val Asn Pro Ser Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Pro Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Ala Gly Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr R, H Asn Pro Val Tyr Ser Ala Val Ser Asn Thr Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Val Glu Asp Glu Val Met Pro Pro Gln Ala Leu Ser Glu Gln Thr Asp Glu Ala Gly Ala Ala Leu Ser Ser Leu Ser Glu Val Pro Pro Trp Thr Gly Glu Val Asn Pro Ser Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Pro Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Ala Gly Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr R, Claim 3 Cont'd.

H Met Ser Ser Phe Ser Thr Thr Thr Val Ser Phe Leu Leu Leu Leu Ala Phe Gln Leu Leu Gly Gln Thr Arg Ala Asn Pro Met Tyr Asn Ala Val Ser Asn Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser Glu Pro Asn Glu Glu Ala Gly Ala Ala Leu Ser Pro Leu Pro Glu Val Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr OH, H Asn Pro Met Tyr Asn Ala Val Ser Asn Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser Glu Pro Asn Glu Glu Ala Gly Ala Ala Leu Ser Pro Leu Pro Glu Val Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr OH and H Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr OH

wherein R can be OH, Arg OH or Arg Arg OH and including biologically active fragments of said polypeptide or its allelic variants and pharmacologically acceptable salts thereof.

4. The mammalian cell culture of Claim 3 which is identified as ATCC accession number CRL8569.

5. A process for producing a compound having natriuretic and/or diuretic and/or vasorelaxant activity wherein said compound is a peptide having an amino acid sequence selected from:

H Met Gly Ser Phe Ser Ile Thr Lys Gly Phe Phe Leu Phe Leu Ala Phe Trp Leu Pro Gly His Ile Gly Ala Asn Pro Val Tyr Ser Ala Val Ser Asn Thr Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Val Glu Asp Glu Val Met Pro Pro Gln Ala Leu Ser Glu Gln Thr Asp Glu Ala Gly Ala Ala Leu Ser Ser Leu Ser Glu Val pro Pro Trp Thr Gly Glu Val Asn Pro Ser Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Pro Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Ala Gly Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr R, H Asn Pro Val Tyr Ser Ala Val Ser Asn Thr Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Val Glu Asp Glu Val Met Pro Pro Gln Ala Leu Ser Glu Gln Thr Asp Glu Ala Gly Ala Ala Leu Ser Ser Leu Ser Glu Val Pro Pro Trp Thr Gly Glu Val Asn Pro Ser Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Pro Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Ala Gly Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr R, Claim 5 Cont'd.

H Met Ser Ser Phe Ser Thr Thr Thr Val Ser Phe Leu Leu Leu Leu Ala Phe Gln Leu Leu Gly Gln Thr Arg Ala Asn Pro Met Tyr Asn Ala Val Ser Asn Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser Glu Pro Asn Glu Glu Ala Gly Ala Ala Leu Ser Pro Leu Pro Glu Val Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr OH, H Asn Pro Met Tyr Asn Ala Val Ser Asn Ala Asp Leu Met Asp Phe Lys Asn Leu Leu Asp His Leu Glu Glu Lys Met Pro Leu Glu Asp Glu Val Val Pro Pro Gln Val Leu Ser Glu Pro Asn Glu Glu Ala Gly Ala Ala Leu Ser Pro Leu Pro Glu Val Pro Pro Trp Thr Gly Glu Val Ser Pro Ala Gln Arg Asp Gly Gly Ala Leu Gly Arg Gly Pro Trp Asp Ser Ser Asp Arg Ser Ala Leu Leu Lys Ser Lys Leu Arg Ala Leu Leu Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr OH and H Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr OH

wherein R can be OH, Arg OH or Arg Arg OH and including biologically active fragments of said polypeptide or its allelic variants and pharmacologically acceptable salts thereof, said process comprising the steps of:
(a) culturing the yeast culture of claim 1 or the mammalian cell culture of claim 3 under conditions which permit the expression of a nucleotide sequence encoding peptide so as to produce said peptide; and (b) recovering said peptide from said yeast or mammalian cell culture.

6. A peptide defined by the amino acid sequence:
H-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH.