MOLECULAR CLONING AND CHARACTERIZATION OF THE GENE SEQUENCE CODING FOR PORCINE RELAXIN

Abstract: Genes and DNA transfer vectors for the expression of por­ cine preprorelaxin; sub-units thereof, including genes and transfer vectors for expression of porcine prorelaxin and the individual A, B and C peptide chains thereof; and equivalents of all such genes. Methods for synthesis of the peptides tech­ niques involving recombinant DNA.

Description: "IIOLECULAR CLONING AND CIIARACTERlSATION OF TItt GENE SEQUENCE CODING FOR PORCINE RELXINl? This invention relates to the molecular cloning and characterization of gene sequence codings for porcine relaxin and sub-units and equivalents thereof. The invention is also concerned with recombinant DNA techniques for the preparation of porcine relaxin, prorelaxin and preprorelaxin. [Note literature references cited herein are given in full at the end of the specification.] Relaxin is a peptide horIaone produced by the corpora lutea of ovaries during pregnancy. It is of interest in mammalian reproductive physiology for its critical role in the softening of the cervix and pubic symphysis prior to parturiticn (Hisaw, 1926). Amino acid sequence analysis has demonstrated the presence of relaxin in the ovaries of the rat (John et al., 1981), pig (Schwabe et al.; 1977; James et al., 1977) and curiously, in shark (Schwabe et al., 1978). Like insulin, relaxin has a primary structure of two peptide chains linked by disulfide bonds, and is derived from a single chain precursor (preprorelaxin) by several processing steps. Rat preprorelaxin has been shown to contain a 105 amino acid connecting peptide linking the A and B chains (Hudson et al., 1981). Cell translations of mRNA isolated from the ovaries of pregnant pigs and immunoprecipitation of the protein products with relax in antisera also sested a large relaxin precursor of about 23,000 daltons as compared to 11,000 daltons for preproinsulin (Gast et al., 1980). It thus appeared likely that the pig relaxin precursor would be similar in structure to that of the rat with a large connecting peptide joining the A and B chains. To study the structure of pig preprorelaxin we have used recombinant DNA techniques to construct E. coli cDNA clones containing the total mRNA population of pig ovarian corpora lutea. However, limited amino acid sequence homology exists between rat and pig relaxins (John et al., 1981) and we have found that no significant cross-hybridization occurs between the rat relaxin cDNA, available frora earlier studies (Hudson et al., 1981) and the pig cDNA clones. Consequently, we have used chemically-synthesized oligonucleotide primers to prepare pig relaxin-specific cDNA which in turn was used as a probe to isolate cDNA clones containing pig preprorelaxin coding sequences. The primary structure of the entire pig preprorelaxin coding sequence was determined from analysis of the recomMinant cDMA clones. The following abbreviations are used in this description. DNA - deoxyribonucleic acid A Adenine RNA - ribonucleic acid T - Thymi ne cDNA - complementary DNA G - Guanine (enzymatically C - Cytosine synthesized U - Uracil from an mRNA sequence) mRNA - messenger RNA The coding relationships between nucleotide sequence in DNA and amino acid sequence in protein are collectively known as the genetic code, which is set out below. First Third position Second position position (5' end) (3' end) U C A G Phe Ser Tyr Cys U Phe Ser Tr Cys C U Leu Ser Stop Stop A Leu Ser Stop Trp G Leu Pro His Arg U Leu Pro His Arg C C Leu Pro Gln Arg A Leu Pro Gln Arg G Ile Thr Asn Ser U Ile Thr Asn Ser C A Ile Thr Lys Arg A et Thr Lys Arg G Val Ala Asp Gly U Val Ala Asp Glv C G Val Ala Glu Gly A Val Ala Glu G1 G The abbreviations used for the amino acids in the table are identified as follows. Phenylalanine (Phe) Histidine (His) Leucine (Leu) Glutamine (Gln) Isoleucine (Ile) Asparagine (Asn) Methionine (Met) Lysine (Lys) Valine (Val) Aspartic acid (Asp) Serine (Ser) Glutamic acid(Glu) Proline (Pro) Cysteine (Cys) Threonine (Thr) Tryptophan (Try) Alanine (Ala) Arginine (Arg) Tyrosine (Tyr) Glycine (Gly) Each 3-lettr codon rspresented in the table, e.g., AUG, CAU (otherwise known as a decsynucleotide triplet or nucleotide triplet) corresponds to a trinucleotide of mZlA, having a 5'-end on the left and a 3'-end on the right. The letters stand for the purine or pyrimidine bases forming the nucleotide sequence. All DIE sequences referred to herein are those of the strand whose sequence corresponds to the mRNA sequences described, with thyrnine (T) substituted for uracil (U). The present invention has as one of its main objectives, the provisicn of recombinant cDNA clones containing the structural gene sequence coding for pig preprorelaxin and peptide sub-units thereof. Such clones will enable the production of porcine preprorelaxin and, hence, prorelaxin and relaxin itself, and peptide sub-units thereof, by recombinant DNA techniques. Apart from the potential usefulness of porcine relaxin in veterinary and, possibly, human medicine, the full characterization and availability of the cDNA structure for porcine relaxin will facilitate future studies on human relaxin. Present sources of human relaxin are limited to extracts from human tissue source material, e.g., ovaries of pregnant women and foetal membranes. The structure of human relax in is not known but is known to differ substantially from animal relaxins. Determination of, and molecular cloning of the cDNA sequence for human relaxin is thus an important research target. Gene probes based on some or all of porcine relaxin cDNA sequence may well be of value in such a determination. In pursuance of the above objectives, we have analysed the nucleotide sequence of cloned cDNA's containing the structural gene sequence coding for pig preprorelaxin. The results allow us to predict the amino acid sequence of the signal peptide and of the connecting (C) peptide and to confirm the previously reported sequence of the B and A chains of pig reiasin. (Schwabe et al., 1977; James et al, 1977; iall et al., 1980). In the following description reference will be made to the accompanying drawings, in which Figure 1 shows a segment of the amino acid sequence of the B-chain of porcine relaxin, with the corresponding m-RNA sequence and the synthetic primer described hereinafter; Figure 2 shows autoradiographs of gel chromatograms of cDNA sequences; Figure 3 is the restriction endonuclease map of porcine relaxin cDNA; Figure 4 shows the mR0JA and peptide sequences of rat and pig preprorelaxin; Figure 5 shows the mRNA and amino acid sequence for porcine preprorelaxin; and Figure 6 shows double stranded DNA fragments used as nucleotide primers. According to one aspect of the present invention, there is provided a gene for the expression of porcine preprorelaxin. More specifically, this aspect of the invention provides a double-stranded DNA fragment for the expression of porcine preprorelaxin, which comprises a coding strand and a complementary strand corresponding to the mRNA sequence shown in Figure 5 of the accompanying drawings. Those skilled in the art will appreciate that polymorphic forms of the gene may exist. Such forms are included in the present invention. The invention also includes any sub-unit of the said sequence described herein, or any equivalent of the said sequence or sub-unit. Among the sub-units to be included by this statement are genes which exclude non-coding regions, such as that shown in Figure 5, genes containing the individual structural gene coding for the signal peptide chain and the A, B and C chains of porcine preprorelaxin (see Figure 4) and any combinations of these chains or sub-units thereof, e.g. the genes for expressing the A and B peptide chains, separately or as prorelaxin (with the C chain). The invention further includes the complements of the above sequences, sub-units or equivalents, and the corresponding RUA sequences, sub-units or equivalents. The invention also includes synthetic primers suitable for screening human or other primate relax in genes. These double-stranded DNA fragments which are set out in Figure 6 of the accompanying drawings, correspond to the most homologous regions between the pig and rat cDNA sequences (c.f. Figure 4). The bottom strands of the sequences have been synthesized for use as nucleotide primers to screen human or other primate relaxins. The number ranges in Figure 6 indicate the corresponding sequence regions in Figure 5. Further aspects of the invention are described hereinafter. The basis of the invention is set out in the following description of our experimental results. Polyadenylated RNA was isolated from corpora lutea dissected from the ovaries of pigs in late pregnancy. Since cell-free translation studies of this material had readily demonstrated a product immunoprecipitable by anti-relaxin antisera it was expected that relaxin-specific mIIA would be present in abundant quantities. Hence in initial studies, cDNA from 'pregnant' and 'non-pregnant' pig ovaries was digested with restriction endonucleases in an attempt to find predominant pregnancy-specific fragments which might derive from relaxin cDNA. As this approach was unsuccessful, it appeared that the level of relaxin mRNA was likely to be less than 1% of the total ovarian mRNA. This assumption was subsequently confirmed when it was found that the proportion of colonies containing relaxin sequences in a clone bank obtained from total pregnant corpora lutea was of the order of 0.3%. Another approach used to identify pig relaxin cDNA clones was to screen bacterial colonies containing recombinant plasmids with a probe made from cloned rat preprorelaxin cDNA. However, specific cross-hybridization was not observed even under conditions of relatively low stringency of hybridization. This result can now be interpreted in terms of the nucleotide sequence homology between rat and pig preprorelaxin cDITA's. While the overall homology (65%) might have allowed cross-hybridization it is now known that the longest region of nucleotide sequence identity uninterrupted by mismatches is only 13 bases. FIGURE 4 of the accompanying drawings shows a comparison of the porcine preprorelaxin amino acid and mRNA sequences (top) with the corresponding rat relaxin sequences (bottom). Asterisks indicate nucleotide homology, while the boxed-in areas mark amino acid homologies. Amino acids are numbered from 49 the start of the B chain. The asterisk beneath Ala denotes the putative intron processing site in rat relaxin (Hudson et al., 1981). Little nucleotide homology occurs in the 3'-untranslated region between procine and rat relaxin mRNA's ; consequently the rat nucleotide sequence is omitted from all but the boxed areas after the termination codon and surrounding the AAUAAUA sequence. In a third approach a series of oligodeoxynucleotides were synthesized on the basis of the known amino acid sequence of pig relaxin (Scanlon, 1981). FIGURE 1 of the drawings shows (a) amino acids 15 to 23 of the porcine relaxin B chain sequence which formed the basis for the synthesis of eight oligonucleotide primers which were used to hybridize specifically to porcine relaxin mRNA; (b) the possible nucleotide sequences of the mRNA corresponding to the amino acid sequence, where X represents any base; (c,) the primer actually used to initiate relaxin specific cDNA synthesis. The eight 11-base primers represented by (b) for the Trp-Val-Glu-Ile region were all synthezised. Several of the primers initiated pregnancy specific cDNA products, but these proved not to contain sequences coding for porcine relaxin. However, one of the synthetic oligonucleotides [ ATCTCCACCRA3 ] was found to initiate a pregnancy specific cDNA of roughly 300 nucleotides whose sequence encoded the NH2-terminal region of the relaxin B chain. Using this primer cDNA was synthesized with pregnant and non-pregnant ovarian itiRNA and analyzed on 6% polyacrylamide 7M urea gels. Figure 2 of the drawings shows autoradiographs of the gel columns. The arrow indicates the (approx.) 300 nucleotide 32P-labelled cDNA which was found to contain the coding sequence for the procine relaxin B chain and signal peptide. This relaxin specific cDNA was used to probe a cDNA clone bank obtained by recombinant DNA techniques from the total mRNA isolated (as described above) from pig ovaries. Approximately 5000 E.coli ovarian cDNA clones were screened by in situ filter hybridization with the relaxin-specific cDNA probe and 15 colonies proved positive on rescreening (0.3% of the total clones). Their plasmid inserts were isolated and sized on 1% agarose yels. Several different Pst insert sizes were found, presumably caused by variation in either reverse transcription or in the S1 nuclease digestion of the "hairpin" loop during the double stranding reaction. Three clones designated as pRLX2, pRLX13 and pRLX 14 were selected for DNA sequence analysis. The restriction endonuclease map of relaxin cDNA determined from analysis of these three clones appears in Figure 3 of the drawings, along with the sequencing strategy. The map also indicates the length of each clone relative to the composite structure. Group A represents those sequences derived by the Maxam and Gilbert method (the asterisk marks the position of the 32P-end label). a. *PstI-HpaII; b. *HpaII-BglII; c. PstI-HpaII*; d. *HpaII-PstI ; e. PstI-TaqI* ; f. *I-Pst ; g. PstI-HinfI* ; h. *HinfI-PstI ; i. HinfI-PstI* ; j. HinfI-HpaII*. Group B represents those sequences derived by subcloning restriction fragments into the appropriate sites of bacteriophage M13 mp 7.1 and mp 9 ; (k,l,m,p,q) were PstI fragments, (n,o) were HpaII-TaqI fragments subcloned intQ the AccI site. Recombinant plaques were sequenced using an M13-specific primer by the dideoxy procedure of. Sanger et al (1980) except (1) which was primed with the relaxin-specific B-chain primer [ ATCTCCACCCA ]. Each of the inserts contained single Hpa II and Hinf I restriction sites; cleavage at these sites provided fragments for end-labelling and Maxam and Gilbert sequencing. For one of the clones, pRLX2, both DNA strands were sequenced with the exception of the A chain region and the 3' untranslated region where clear sequence was obtained using two separate restriction fragments from the same strand. The 3' untranslated region is 206 nucleotides in length beginning with the UGA termination codon at the end of the A chain. The DNA sequence of all three clones confirmed that the nucleotide sequence encoded the known A and B chain primary structure of porcine relaxin (Fig. 4.). Regions corresponding to a C or connecting peptide and a signal peptide could be identified, giving an overall structure of the form: signal peptide - B chain - C peptide - A chain. In another aspect, the present invention also provides a method for the isolation of a porcine chromosomal relaxin gene, which comprises using as a probe a molecule comprising a sub-unit of the above-described gene or an equivalent thereof. By virtue of the ow known nucleotide sequence homology between the porcine relax in gene and the genes of humans, other primates, or related species, similar probes can be used to identify and isolate such genes from other species. The present invention further provides a process for the production of a gene or an equivalent thereof or a sub-unit thereof according to any one of the above-mentioned embodiments, which comprises using recombinant DNA techniques to obtain a cDNA clone bank from total mRNA, isolated from porcine corpora lutea, and screening the clone bank using a cDNA probe initiated by the synthetic oligonuleotide 51 3' ATCTCCACCCA ]. The invention also provides a DNA transfer vector comprising the cDNA deoxynucleotide sequences corresponding to any gene as defined above. The Invention further provides a gene or DNA transfer vector as defined above in which one or more natural codons or their cDNA equivalents are replaced by another codon which codes for the same amino-acid, or in which one or more of the natural codons are deleted and/or are replaced by codons which code for amino acids other than that code by the natural codon, and/or further codons are added to the natural sequence. The DNA transfer vector may be a bacterial plasmid bacteriophage DNA. Thus, in one specific aspect the invention may provide a plasmid recombinant which comprises a plasmid vector having inserted therein at an insertion site a gene or a sub-unit thereof or an equivalent thereof, according to the above-described embodiments, the plasmid recombinant enabling translation in the correct phase for the mBlA corresponding to the inserted gene or sub-unit thereof or equivalent thereof and having a bacterial promoter upstream of and adjacent to the insertion site such that the inserted gene or sub-unit thereof or equivalent thereof is under bacterial promoter control. The production of such a recombinant molecule b a process which comprises inserting DNA corresponding to the desired structural gene for relaxin at the insertion site of an appropriate vector also constitutes an embodiment of the present invention. The present invention further provides a transformed cell which comprises inserted therein such a gene or a sub-unit thereof or an equivalent thereof or such a plasmid recombinant. The production of such a cell by a process which comprises inserting the DNA via a suitable vector into a cell also constitutes an embodiment of the present invention. In a further embodiment, the present invention provides a process for the production of porcine preprorelaxin, or a protein having properties resembling those of porcine preprorelaxin, which comprises culturing such a cell and recovering expressed protein. The invention also includes the production of porcine prorelaxin or the relaxin A, B and C peptides by analogous methods to those described above, which may also involve synthetic modification of the cDNA, acording to methods and practices already known in the art. The invention also extends to synthetic preprorelaxin, prorelaxin and the A, B, and C peptide chains, especially when prepared by the above-described processes. The invention further includes the production of relaxin from preprorelaxin or prorelaxin, or by joining of the A and B chains by methods analogous to those used for the production of insulin from its respective precursors. The materials and procedures used in the methods associated with or forming part of the invention are further illustrated by the following descriptions. Materials Terminal transferase was purchased from Ratcliff Biochemicals (Los Alamos, N.M.) Pst I, Bgl II, DNA polymerase (Klenow fragment) and S1 nuclease were from Boehringer Mannheim. Hinf I, TaQ I, Hae III, Alu I, and T4 DNA ligase were from New England Biolabs (Beverly, tMA). Oligo-dT cellulose type 7 was obtained from PL Biochemicals (Milwaukee, Wis). 32P-labelled nucleoside triphosphates and 35S-labelled amino acids were from Amersham. messenger RNA isolation Ovaries from pigs in late pregnancy were obtained by collection at abattoirs and quickly frozen on dry ice. The corpora lutea were dissected free of other tissue, the RNA isolated in SM guanidinium thiocyanate (Merck) (Chirgwin et al., 1979) and the poly A+ containing RNA purified by oligo - dT cellulose affinity chromatography (Aviv and Leder, 1972). + Yields were approximately 10pg poly A RNA per 100 mg tissue. To assay mRNA for relaxin-containing message, cell free translations were carried out employing rabbit reticulocyte lysate (Pelham and Jackson, 1976) and immunoprecipited proteins (Kessler, 1975) were electrophoresed or SDS polyacrylamide gels (Studier, 1973). cDNA was prepared using reverse transcriptase and an oligo - dT primer (slickers et al., 1978), the RNA hydrolyzed in alkali, and the cDNA made double-stranded (Ullrich et al., 1977) with DNA polymerase I (lenow fragment). The single-stranded hairpin loop was removed with 10 units of S1 nuclease at 200C for 5' (Vogt, 1973). omopolymeric deoxycytidine extensions were enzymatically added using terminal transferase (10 units in 50 Nil reaction volume at 370 for 10') in a controlled reaction designed to add approximately 30 C-residues to the 3'-OH ends (Roychoudbury et al., 1976). This 'tailed' cDNA was annealed to the plasmid pBR322 which had been cleaved with PstI and similarly 'tailed' with G-residues. The recombinant plasmids were used to transform E. coli RRI (efficiency = 105 clones per Vg plasmid) and clones containing ovarian cDNA were selected on the basis of tetracycline resistance (orrison, 1979). Selection of specific cDNA clones To screen the ovarian cDNA clone bank for relaxin specific sequences, eight ll-base primers were chemically synthesized by the phosphotriester method (Itakura et al., 1973) to make all possible complementary sequences corresponding to the peptide sequence Trp-Val-Glu-Ile in the pig relaxin B chain (Fig. 1). These primers were used to select specific cDNA products for use as hybridization probes to the cDNA clone bank. Oligonucleotide primers were used to initiate reverse transcription of mRNA under conditions designed for the synthesis of high specific activity and yield full length cDNA. 2pg of mRNA was added to between 0.1 and 1 Vg of primer, heated to 90 0C for 2 minutes, slowly cooled, and the reverse transcriptase reaction carried out at 37 C in 50mM Tris pH8.0, 5024 liaCl, 10mM MgS04, 0.1mM DTT with 600pM dATP, dTTP, dGTP, 30pM dCTP, 30 Ci ss- P-dCTP, and ib units of reverse transcriptase. Reacticn products were alkali treated (100m1 NaOH, 800C, 10 min) to remove RNA and analyzed on 6% acrylamide 7M urea gels for the presence of pregancy-specific cDNA's (Fig. 2). In order to identify which of the cDNA clones contained relaxin-specific sequences in a separate experiment the primer (lacking the 5' phosphate) was radiochemically end-labelled with T4 polynucleotide kinase and y32P-ATP (100 Ci per pg primer) and the cDNA synthesized in the absence of ss-32p-deoy- nucleotide (Noyes et al. 1979). This yielded end-labelled DNA suitable for sequencing by the chemical degradation technique (Maxam and Gilbert, 1977). The regions of the gel corresponding to specific cDNA bands were excised, placed in dialysis membrane bags and the DNA electrophoretically removed from the gel matrix in 50razz Tris-borate, lrMl EXTA (pH 8.1). Eluted DNA samples were precipitated with 2 volumes of ethanol in 0.3DI Na acetate (pH5.5) at -200C for 4 hours. The relaxin-specific cDNA fragment identified by the methods described above was synthesized to a specific activity greater than 108 cprl/pg, purified on polyacrylamide gels, and used to screen the cDNA clone banks (Thayer, 1979). Colonies which hybridized to this probe were rescreened in duplicate in order to grade the positives as to their rate of specific hybridization. The strongest hybridizing clones were screened by a rapid plasmid isolation procedure (Homes and Quigley, 1981) and those containing the largest recombinant plasmids were grown in one litre cultures for large scale plasmid isolation (Tanaka and Weisblum, 1975) yielding about 700pg of plasmid. Sequence analysis of recombinant plasmids Restriction digests were carried out unde the conditions specified by New England Biolabs. Purified DNA fragments with 5' protruding ends were end-labelled using reverse transcriptase and the appropriate ss-labelled deoxynucleotide triphosphase (dCTP for HpaII and TaqI; dATP for Hindi). 3'-protruding ends generated with PstI were labelled using terminal transferase and [32P]-cordycepin triphosphate. Fragments were separated by electrophoresis on 8% polyacrylamide gels and sequenced by chemical degradation method (Maxam and Gilbert, 1977). The cloned cDNA inserts released by Pst I digestion were also ligated into bactericphase M13 mp9 or mp7.1 and the single-stranded template DNA isolated and sequenced using the dideoxy procedure (Sanger et al. 1980). The M13 recombinants were sequenced using either the Ml 3-specific primer or with specific primers chosen over particular regions of the internal relaxin sequence. Expression of porcine preprorelaxin The expression vector TrpED5-1 (Hallewell, R.A. and Emtage, S., 1980, Gene 9, 27) contains the replicative properties of pBR322 with the tryptophan promoter, and TrpE and TrpD sequences. The preprorelaxin gene may be introduced into the TrpD sequence via a unique Hind III restriction endonuclease site, producing a fusion TrpD/preprorelaxin protein regulated by the inducible Trp promoter. TrpED5-1 was cleaved with Hind III 370C for 3 hours, (50mM Tris C1 pH7.4, 10mM MgCl2, 50 mDI NaCl) and the enzyme inactivated at 700C for 10 minutes, and the staggered ends filled in with T4 Polymerase (200 mM dATP, dCTP, dGTP, dTTP; 110C 3 hrs). The preprorelaxin sequence (Haley, J.D. et al (1982) DA 1, 155-162) was cleaved with Xba to remove the TAG termination codon in the 5' non-translated sequences. The 3' end was cleaved with Pst and was full length. The staggered termini were removed with 10 units of S1 nuclease 200C 5 minutes (0.3M NaCl, 20mM NaAc pH 7.6, 3mD4 ZnAc) to produce blunt ends. The reaction was stopped by phenol/chloroform extraction, ether extracticn and ethanol precipitation of the supernatent. Equimolar amounts of processed TrpED5-1 vector and preprorelaxin were joined with T4 ligase (100C 24 hours, 50mM Tris.Cl pH7.4, 10mM MgC12, 1mM DTT, 1mM ATP) E.coli 294 grown to ED600=0.3 were made competent by CaCl2 treatment (75mM CaCl2, 10mI Tris.Cl pH7.4, 40C 1 hour). Joined TrpED5-1/preprorelaxin DNA was added. After 3 hours on ice the cell/DNA mix was heat shocked (450C 90 sec) and the mix returned to the ice for an additional 30 minutes. Cells were plated on L broth plates supplemented with 20pg/ml Ampicillin, and recombinant bacteria were screened with ss-32P-dATP labelled preprorelaxin by in situ filter hybridization (Thayer, RE. (1979), Anal.Biochem. 98, 60-63). Recombinant bacteria produced in this manner are capable of producing TrpD/preprorelaxin fusion protein inducible with 3-indolacrylic acid (Martial, J.A. et al (1979) Science 205, 602-6-6). REFERENCES Aviv, H. and Leder, P. (1972). Purification of biologically active glob in messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Nat. Acad. Sci. USA 69, 1408-1412. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem. 18, 5294-5299. Gast, M.J., Mercado-Simmen, R., Niall,H. and Boime, 1. (1980) Cell-free synthesis of a high molecular weight relaxirr-related protein. Ann. N.Y. Acad. Sci. 343, 148-154. Hackenthal, E., Hackenthal, R. and Hilgenfeldt, U. (1978) Isorenin, pseudorenin, cathepsin D and renin : a comparative enzymatic study of angiotensin-forming enzymes. Biochim. Biophys. Acta 522, 574-588. Hisaw, F.L. (1926) Experimental relaxation of the pubic ligament of the guineapig. Proc. Soc. Exp. Biol. Med. 23, 661-663 Holmes, D.S. and Quigley, M. (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 114, 193-197. Hudson, P., Haley, J., Cronk, M., Shine, J. and Niall, H. (1981) Molecular cloning and characterization of cDNA sequences coding for rat relaxin. Nature 291, 127-131. Itakura, K., Bahl, C.P., Katagiri,N-, Michniewicz, J.J., Wrightman, R.H. and Narang, S.A. (1973) A modified triester method for the synthesis of deoxypolynucleotides. Can. J. Chem. 51, 3649-3651. James, R., Niall, H., Kwok, S. and Bryant-Greenwood, G. (1977) Primary structure of porcine relaxin : homology with insulin and related growth factors. Nature 267, 544-546 John, M.J., Walsh, J.R., Borjesson, B.W. and Niall, H.D. (1981) Limited sequence homology between porcine and rat relaxin implications for physiological studies. Endocrinology 108, 726-729 Kessler, S.W. (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbant: parameters of the interaction of antibodrantigen complex with protein A./ J. Immunol. 115, 1617-1624. Maxam, A.M. and Gilbert, W. (1977) A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA 74, 560-564. Mercier, J < ., Brignon, G. and Pvibadeau-Dumas B. (1973) Primary structure of bovine kB-casein. Eur. J. Biochem 35, 222-235. McDonald, J.K., Callahan, P.X., Ellis, S. and Smith, R.E. (1977) In Tissue Proteinases. A.J. Barret and J.T. Dingle, eds. (Amsterdam : North-Holland Publishing Co.) pp. 69-107. Morrison, D A. (1979) In : Methods in Enzymology. R. Wu, ed. (New York : Academic Press) pp. 326-331. Niall, H.D., James, R., John, M-, Walsh, J., Kwok, S., Bryant-Greenwood, G.D., Tregear, G.W. and Bradshaw, R.A. (1980) Proceedings of the 15th Midwest Conference on Endocrinology and Metabolism (New York : Plenum Publishing) (in press). Noyes, B.E., Mevarech, M., Stein, R. and Agarwal, K.L. (1979) Detection and partial sequence analysis of gastrin mRNA by using an oligonucleotide probe. Proc. Natn. Acad. Sci. 76, 1770-1774. Pelham, H.R.B. and Jackson, R.J. (1976) An efficient mRN & dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247-256. Proudfoot, N.J. and Brownlee, G.G. (1976) 3 non-coding region sequences in eukaryotic messenger RNA. Nature 263, 211-214. Roychoudbury, R. Jay, E. andwu, R. (1976) Terminal labelling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase. Nucleic Acid Res. 3, 863-877. Sanger, F., Coulson, A.R., Barrell, B.G.. Smith, A.J H. and Roe B.A. (1980) Cloning in single stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143, 161-178. Schwabe, C., McDonald, J.K. and Steinetz, B C. (1977) Primary structure of the B-chain of porcine relaxin- Biochem. biophys. Res. Commun, 75, 503-510. Schwabe, C., Steinetz, B., Weiss, G., Segaloff, A. McDonald, J.K., O Byrne, E., Hochman, J., Carrier, B. and Goldsmith, L. (1978) Relaxin. Rec. Progr. Horm. Res. 34, 123-211. Sherwood, O.D. and O Byrne, E.M. (1974) Purification and characterization of porcine relaxin. Arch. Biochem. Biophys. 160, 185-196. Steiner, D F., Quinn, P.S., Chan, S.J., Marsh, J. and Tager, H.S. (1980) Processing mechanisms in the biosynthesis of proteins. Ann N.Y. Acad. Sci. 343, 1-16. Studier, F.W. (1973) Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol.Biol. 79, 237-248. Tager, H.S., Emdin, S.0., Clark, J.L. and Steiner, D.F. (1973). Studies on the conversion of proinsulin to insulin. J. Biol. Chem. 248, 3476-3482. Tanaka, T. and Weisblum, B. (1975) Construction of a colicin E1-R factor composite plasmid in vitro : means for amplification of deoxy-ribonucleic acid. J. Bacteriol. 121, 354-362. Thayer, R.E. (1979) An improved method for detecting foreign DNA in plasmids of Escherichia coli Anal. Biochem. 98, 60-63. Thibault, G. and Genest, J. (1981) Tonin, an esteroprotease from rat submaxillary glands. Biochimica et Biophysica Acta 660, 23-29. Ullrich, A., Shine, J., Chirgwin, J., Picket, R., Tischer, E.,Rutter, W.J. and Goodman, H.M. (1977) Rat insulin genes : construction of plasmids containing the coding sequences. Science 196, 1313-1319. Vogt, V.M. (1973) Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae. Eur. J.Biochem. 33, 192-200. Walsh, J.R. and Niall, H.D. (1980) Use of an octadecylsilica purification method minimizes proteolysis during isolation of porcine and rat relaxins. Endocrinolovy 107, 1258-1260. Wickers, M.P., Buell, G.N. and Schimke, R.T. (1978) Synthesis oE double-stranded DNA complementary to lysozyme, ovomucoid, and ovalbumin mRNAs. J. Biol. Chem. 253, 2483-2495.

Claims: CLAIMS: 1. A gene for the expression of porcine preprorelaxin, or a sub-unit thereof or an equivalent of such a gene or sub-unit. 2. A double-stranded DNA fragment for the expression of porcine preprorelaxin, characterized in that it comprises a coding strand and a complementary strand corresponding to the following complete mRNA sequence: AUG CCG CGC CUG UUC UCC UAC CUC CUA GGU GUC UGG CUG CUC CUG AGC CAA CUU CCC AGA GAA AUC CCA GGC CAG AGU ACG AAC GAU UUU AUU AAG GCA UGC GGC CGA GAA UUA GUC CGU CUG UGG GUG GAG AUC UGU GGC UCC GUC UCC UGG GGA AGA ACU GCU CUC AGC CUG GAA GAG CCU CAG CUG GAA ACU GGA CCC CCG GCA GAA ACC AUG CCA UCC UCC AUC ACC AAA GAU GCA GAA AUC UUA AAG AUG AUG UUG GAA UUU GUU CCU AAU UUG CCA CAG GAG CUG AAG GCA ACA UUG UCU GAG AGG CAA CCA UCA CUG AGA GAG CUA CAA CAA UCA GCA UCA AAG GAU UCG AAU CUU AAC UUU GAA GAA UUU AAG AAA AUU AUU CUU AAC AGA CAA AAU GAA GCA GAA GAC AAA AGU CUU UUA GAA UUA AAA AAC UUA GGU UUA GAU AAA CAU UCC AGA AAA AAG AGA CUG UUC CGU AUG ACA CUG AGC GAG AAA UGU UGU CAA GUA GGU UGU AUC AGA AAA GAU AUU GCU AGA UUA UGC UGAAAAGGAGCUAAUGAUAUAUUUUAUAUAAUGUUCACAUGUAUUCUCAGUGACA UAUUCACUUAUGCCUCUGUCACCCACUGAUUAUUAGCACUGUUUAGUGUUUAGGU UUUUCAUUUUAUGUGUAAGAAAAUGUCCCUUGCAUUAAUGUAGUUUCUGCUAAUA AUAUUUUUUUAAACUAAAAGGUAAAIAAZLAAAAAAAA or a sub-unit thereof or an equivalent of such a gene or sub-unit. 3. A sub-unit of the gene claimed in Claim 1 or Claim 2. 4. A gene for the expression of porcine prorelaxin, or a sub-unit thereof or an equivalent of such a gene or sub-unit. 5. A double-stranded DNA fragment for the expression of porcine prorelaxin, characterized in that it comprises a coding strand and a complementary strand corresponding to the following mRNA sequence: CAG AGU ACG AAC GAU UUU AUU AAG GCA UGC GGC CGA GAA UUA GUC CGU CUG UGG GUG GAG AUC UGU GGC UCC GUC UCC UGG GGA AGA ACU GCU CUC AGC CUG GAA GAG CCU CAG CUG GAA ACU GGA CCC CCG GCA GAA ACC AUG CCA UCC UCC AUC ACC AAA GAU GCA GAA AUC UUA AAG AUG AUG UUG GAA UUU GUU CCU AAU UUG CCA CAG GAG CUG AAG GCA ACA UUG UCU GAG AGG CAA CCA UCA CUG AGA GAG CUA CAA CAA UCA GCA UCA AAG GAU UCG AAU CUU AAC UUU GAA GAA UUU AAG AAA AUU AUU CUU AAC AGA CAA AAU GAA GCA GAA GAC AAA AGU CUU UUA GAA UUA AAA AAC UUA GGU UUA GAU AAA CAU UCC AGA AAA AAG AGA CUG UUC CGU AUG ACA CUG AGC GAG AAA UGU UGU CAA GUA GGU UGU AUC AGA AAA GAU AUU GCU AGA UUA UGC or a sub-unit thereof or an equivalent of such a gene or sub-unit. 6. A gene for the separate expression of the signal, A, B or C peptide chains of porcine relaxin or any combination of two or more of the said chains. 7. A double-stranded DNA fragment for the expression of the signal peptide chain of porcine preprorelaxin, characterized in that it comprises a coding strand and a complementary strand corresponding to the following mRNA sequence: AUG CCG CGC CUG UUC UCC UAC CUC CUA GGU GUC UGG CUG CUC CUG AGC CAA CUU CCC AGA GAA AUC CCA GGC or a sub-unit thereof or an equivalent of such a gene or sub-unit. 8. A double-stranded DNA fragment for the expression of the A peptide chain of porcine preprorelaxin, characterized in that it comprises a coding strand and a complementary strand corresponding to the following mRNA sequence: CGU AUG ACA CUG AGC GAG AAA UGU UGU CAA GUA GGU UGU AUC AGA AAA GAU AUU GCU AGA UUA UGC or a sub-unit thereof or an equivalent of such a gene or sub-unit. 9. A double-stranded DNA fragment for the expression of the B peptide chain of porcine preprorelaxin, characterized in that it comprises a coding strand and complementary strand corresponding to the following mRNA sequence: CAG AGU ACG AAC GAU UUU AUU AAG GCA UGC GGC CGA GAA UUA GUC CGU CUG UGG GUG GAG AUC UGU GGC UCC GUC UCC UGG GGA AGA ACU GCU CUC or a sub-unit thereof or an equivalent of such a gene or sub-unit, 10. A double-stranded DNA fragment for the expression of the C peptide chain of porcine preprorelaxin, characterized in that it comprises a coding strand and complementary strand corresponding to the following mRNA sequence: AGC CUG GAA GAG CCU CAG CUG GAA ACU GGA CCC CCG GCA GAA ACC AUG CCA UCC UCC AUC ACC AAA GAU GCA GAA AUC UUA AAG AUG AUG UUG GAA UUU GUU CCU AAU UUG CCA CAG GAG CUG AAG GCA ACA UUG UCU GAG AGG CAA CCA UCA CUG AGA GAG CUA CAA CAA UCA GCA UCA AAG GAU UCG AAU CUU AAC UUU GAA GAA UUU AAG AAA AUU AUU CUU AAC AGA CAA AAU GAA GCA GAA GAC AAA AGU CUU UUA GAA UUA AAA AAC UUA GGU UUA GAU AAA CAU UCC AGA AAA AAG AGA CUG UUC or a sub-unit thereof or an equivalent of such a gene or sub-unit. 11. A double-stranded DNA fragment for the expression of a peptide consisting of a combination of any two or more of the signal, A, B and C peptide chains of porcine preprorelaxin characterized in that it comprises a coding strand and a complementary strand corresponding to the appropriate combination of the mRNA sequences given in the preceding Claims 7 to 10. 12. A double-stranded DNA fragment selected from the following: 5' 3' GAAGAATTTAAGAAAA CTTCTTAAATTCTTTT AGGAGCTGAAGGCA TCCTCGACTTCCGT CAAAGATGCAGA GTTTCTACGTCT TGTCTGAGAGGC ACAGACTCTCCG TGAAGCAGAAGA ACTTCGTCTTCT AGAGCTACAACAA TCTCGATGTTGTT AAAAACTTAGG TTTTTGAATCC GGTTGTATCAGAA CCAACATAGTCTT. 13. A process for the isolation of a chromosomal relaxin gene of a human or other primate or a related species characterized in that it comprises using as a probe a sub-unit of the gene as defined in any one of Claims 1 to 11 or an equivalent of such a sub-unit. 14. A process for the production of a gene or an equivalent thereof or a sub-unit thereof as set out in any one of the preceding claims, characterized in that it comprises using recombinant DNA techniques to obtain a cDNA clone bank from total mRNA, isolated from porcine corpora lutea, and screening the clone bank using a cDNA probe initiated by the synthetic oligonuleotide [5 ATCTCCACerA3']. 15. A DNA transfer vector, characterized in that it contains a cDNA deoxynucleotide sequences corresponding to a gene as defined in any one of Claims 1 to 11 or a sub-unit of such a gene or an equivalent of such a gene or sub-unit. 16. A gene or DNA transfer vector as claimed in any one of Claims 1 to 11 and 15, characterized in that one or more natural codons or their cDNA equivalents are replaced by another codon which codes for the same amino-acid. 17. A gene or DNA transfer vector as claimed in any one of Claims 1 to 11 and 15, characterized in that one or more of the natural codons are deleted and/or are replaced by codons which code for amino acids other than that code by the natural codon, and/or further codons are added to the natural sequence. 18. A DNA transfer vector as claimed in any one of Claims 15 to 17, characterized in that it is a bacterial plasmid. 19. A DNA transfer vector as claimed in any one of Claims 15 to 17 which is bacteriophage DNA. 20. A cell transformed by a transfer vector as claimed in any one of Claims 15 to 17 21. A process for making a DNA transfer vector for use in maintaining and replicating a deoxynucleotide sequence coding for porcine preprorelaxin or a sub-unit thereof, characterized-in that it comprises reacting the appropriate deoxynucleotide sequence of porcine preprorelaxin or the sub-unit with a DNA molecule prepared by cleaving a transfer vector with a restriction enzyme. 22. A process for making a DNA transfer vector for use in maintaining and replicating a deoxynucleotide sequence coding for porcine prorelaxin, characterized in that it comprises reacting the appropriate deoxynucleotide sequence porcine prorelaxin with a DNA molecule prepared by cleaving a transfer vector with a restriction enzyme. 23. A process for making a DNA transfer vector for use in maintaining and replicating a deoxynucleotide sequence coding for the signal peptide or the A or B or C chain peptides of porcine preprorelaxin or any combination of two or more of said peptides characterized in that it comprises reacting the appropriate deoxynucleotide sequence with a DNA molecule prepared by cleaving a transfer vector with a restriction enzyme. 24. A process for making a fusion protein comprising an amino acid sequence consisting of all or part of the amino acid sequence of porcine preprorelaxin as its C-terminal sequence and a portion of a procaryotic protein as its N-terminal sequence, characterized in that it comprises incubating a microorganism transformed by an expression transfer vector comprising the appropriate deoxynucleotide sequence. 25. A process for synthetizing porcine prorelaxin comprising the A and B peptides separated from each other by a C peptide, characterized in that it comprises incubating a microorganism, transformed by an expression transfer vector comprising a deoxynucleotide sequence coding for said porcine prorelaxin under conditions suitable for expression of said sequence coding for porcine prorelaxin, and purifying porcine prorelaxin from the lysate or culture medium of said microorganism. 26. A fusion protein comprising an amino acid sequence characterized in-that it consists of all or part of the amino acid sequence of porcine preprorelaxin as its C-terminal sequence and a portion of a procaryotic protein as its N-terminal sequence. 27. Synthetic porcine preprorelaxin. 28. Synthetic porcine prorelaxin. 29. The synthetic A, B or C peptide chains of porcine prorelaxin.

Inventors: HALEY, JOHN DOUGLAS, HUDSON, PETER JOHN, NIALL, HUGH DAVID, SHINE, JOHN
Applicants: HOWARD FLOREY INSTITUTE OF EXPERIMENTAL PHYSIOLOGY AND MEDICINE
Application year: 1983
Main classification: C12N15
IPC codes: C07K14, C12N15, C07K19, C12R1, C12P21, C12N1, C12P19, C07H21