CLONING OF DNA ENCODING HUMAN MOTILIN PRECURSOR AND EXPRESSION OF THE PRECURSOR

Abstract: A cloned single-strand DNA comprising nucleotide sequence which encodes human motilin precursor, a cloned double-strand DNA consisting of the single-strand DNA and its complementary single-strand DNA, a fragment of the single- or double-strand DNA, a plasmid, in which the double-strand DNA or its fragment is integrated , as well as a process for the preparation of the single- or double-strand DNA.

Description: The present invention relates to motilin and more particularly to a cloned single-strand DNA comprising nucleotide sequence which encodes human motilin precursor, a cloned double-strand DNA consisting of the single-strand DNA and its complementary single-strand DNA, a fragment of the single- or double-strand DNA, and a plasmid, in which the double-strand DNA or its fragment is integrated , as well as a process for the preparation of the single- or double-strand DNA. The motilin is one of peptide hormones, firstly isolated from mucous membrane of porcine upper small intestine and determined its amino acid sequence by Brown, J. C. et al ["Gastroenterology" Vol. 62, pages 4010 - 4040 (1972) and "Can. J. Biochem." Vol. 52, pages 7 - 10 (1974)]. The porcine motilin disclosed in such literatures is one extracted from the animal tissue, consists of 22 amino acids and has molecular weight of about 2700. It has been well known that the porcine motilin has a hypermotility action of digestive tract and contracting action of gastroduodinal and colonic smooth muscle therein, as physiological actions. As the hypermotility action, it has been reported that a staying period of time in stomach is shortened [ "Gastroenterology" Vol. 80, pages 456 - 460 (1981)] and as the contracting action of smooth muscle in the digestive tract, it has been known that the motilin shows a strong contracting action to rabbit and human gastrointesinal tract, independing from a neuro system. Therefore, it has been considered that the motilin is useful for curing gastro-enteropathys at the period of post-operation and for diagnosis thereof. The motilin according to the prior arts has been obtained through extraction from porcine organ and thus it was quite difficult to obtain the same in a large amount. Further, a structure of human motilin has not yet been determined. Namely, the motilin has not actually been applied for clinical use, due to its poor productivity, in spite of that an effectiveness thereof as the agent for curing gastro-enteropathy has been expected. A basic object of the invention lies in developping an industrially acceptable process for the preparation of human motilin and its related substances, by utilizing so-called --Bio-Technologies--. A principal object of the invention is to provide a cloned DNA comprising a human motilin precursor, or a fragment thereof. An additional object of the invention is to provide a process for the preparation of the cloned DNA encoding the human motilin precursor, or the fragment thereof. A further object of the invention is to provide a plasmid, wherein the cloned DNA or its fragment, for instance the human motilin per se or another physiologically or biologically active portion in the human motilin precursor, is integrated, so that a micro-organism or animal cell transformed by such plasmid expresses the human motilin or other biologically active substances to allow a large scale production thereof. The inventors have carefully studied and investigated to finally obtain the cloned DNA for the human motilin precursor and determine its structure, whereby the basic object has been attained. According to the invention, the principal object is attained by a cloned single-strand DNA comprising about 550 nucleotides which encodes therein an amino acid sequence for human motilin precursor. The DNA according to the invention has the following nucleotide sequence or any other nucleotide sequence same with the former in biological view point. wherein A, C, G and T are, respectively, a oligodeoxyribonucleotide having adenine, cytosine, guanine or thymine base and said sequence is given as that of each codon corresponding to a specified amino acid. The term of --nucleotide sequence same in biological view point--means a case of that even if, kinds or arrangement of nucleotides constituting codons, as --TTA-- and --CTG-- are different but each codon designates same amino acid (in the exemplar case, each of the codons designates same amino acid of --leucine). In this case, therefore, the term means various nucleotide sequences encoding the following amino acid sequence which is designated by the nucleotide sequence shown hereinabove. According to the DNA for human motilin precursor having said nucleotide and amino acid sequences, it is found that the human motilin is encoded in a region of the 26th to 47th counting from methionine (Met) residue designated by the initiative codon of ATG and partitioned at both ends by alanine (Ala) residue designated by the codon of GCC and lysine (Lys) residue designated by the codon of AAG. It can be considered that the firstly arranged about 25 amino acids beginning from the initiative Met are those corresponding to a signal peptide which concerns to secretion, in view of its structure. According to a process of the invention, the cloned double-strand DNA encoding human motilin precursor can be prepared by converting RNA extracted from upper small intestine (duodenum) into poly(A)RNA, constructing cDNA library with use of the poly(A)RNA and vector/primer DNA to carry out a transformation of Escherichia coli (E. coli), while previously synthesizing a mixture of 24 tricosamers, each consisting of 23 origodeoxyribonucleotides of the formula wherein I is inosine and employed for the position in the nucleotide sequence, where any of A, G, T and C possibly occupies, which is complementary to mRNA corresponding to Phe-Val-Pro-Ile-Phe-thr-Tyr-Gly of 1st to 8th amino acids from N-terminal in known amino acid sequence for porcine motilin, laveling each synthesized oligodeoxyribonucleotide shown by said formula (A) at N-terminal, screening said transformed E. coli by a hybridization with use of said labeled oligodeoxyribonucleotides as probes to obtain a positive clone which hybridize with the probes. The ground that the 1st to 8th amino acids from N-terminal in the amino acids for porcine motilin is selected for the oligodeoxylibonucleotide (A) as the probe and that inosine is employed in each position, where any of A, G. T and C may occupies, lies in making possible to respond also to the case that there is some difference in sequence between porcine and human motilins, and to be minimum kinds of mRNA corresponding to such amino acids. A cloned single-strand DNA encoding human motilin can be prepared by denaturing the resulting cloned double-strand DNA with use of a method known per se, for instance treating same at 90°C for about 3 minutes and then cooling with an ice bath. The cloned single- or double-strand DNA encoding human motilin precursor can be made into a fragment or fragments of various length, by treating same with a suitable restriction enzyme(s), binding fragments, synthesizing a region not obtaining through the cleaving technique and binding the synthesized region to the cleaved fragment to make the desired fragment of human motilin region only or another region showing a physiological or biological activity. Moreover, the cloned double-strand DNA encoding human motilin precursor or any fragment thereof may be integrated into a plasmid with a technique known per se, for instance taking out a plasmid from E. coli, purifying the plasmid, treating the plasmid with a restriction enzyme to cut the same at a specified base position, and ligating with a DNA ligase the cloned DNA to the cleavage site of the cut plasmid to re-construct a plasmid with the recombinant DNA. The human motilin precursor, human motilin per se or other biologically active substance(s) can be prepared in a large amount by transforming a microorganism or eukaryotic cell with the plasmid integrated therein the double-strand DNA or a fragment thereof and culturing the microorganism of eukaryotic cell. Further, the DNA fragment encoding an amino acid sequence other than that for motilin may also be employed for investigating novel biologically active substance(s). The invention will now be further explained with reference to Examples for preparing a cloned human motilin precursor and a plasmid integrating the precursor therein, respectively as well as a Test Example for determining a structure of the precursor, which will refer to drawings, wherein Fig. 1 is an illustration showing cDNA region comprising cloned human motilin precursor in accordance with the invention, as well as a strategy and a restriction enzyme map for determining a nucleotide sequence in the human motilin precursor region; Fig. 2 shows a determined nucleotide sequence of cloned cDNA encoding human motilin precursor as well as an amino acid sequence corresponding to the nucleotide sequence in the human motilin precursor region; and Fig. 3 is an illustration showing steps to prepare a plasmid, wherein the human motilin is integrated. Example 1 a) Preparation of poly(A)RNA comprising mRNA of human motilin It has been known through a radio immunoassay and others that porcine motilin presents at an upper small intestine and other organs [ "Scand. J. Gastroenterol." Vol. 11, pages 47 - 52(1976)]. Therefore, a human duodinum obtained from a patient during pancreato-duodenectomy for pancreatic cancer was treated, in accordance with the method as disclosed by Chirgwin, J. M. et al [ "Biochemistry" Vol. 18, pages 5294 - 5299 (1979)]. Namely, the total RNA (1.2mg) was extracted from the tissue by 4M-guanidium thiocyanate, bonded to an oligo(dT)cellulose column with use of 10mM-Tris hydrochloride buffer containing 0.5M-KCl, as a binding buffer, and eluted with use of a buffer free from KCl to obtain about 150µg of the desired poly(A)RNA. It has been confirmed through an electrophoresis that this RNA is not degradated, in view of RNA bands at 18s and 28s. b) Synthesis of oligodeoxyribonucleotides complementary to mRNA which corresponds to an amino acid sequence for human motilin A mixture of 24 tricosamers, each consisting of 23 deoxyribonucleotides of the formula wherein I is inosine and employed for the position in the nucleotide sequence, where any of A, G, T and C possibly occupies, which is complementary to mRNA corresponding to Phe-Val-Pro-Ile-Phe-thr-Tyr-Gly of 1st to 8th amino acids from N-terminal in known amino acid sequence for porcine motilin were synthesized with use of a DNA synthesizer marketed as --Gene Assembler-- from Pharmacia, Sweden, It is to be noted that these oligodeoxyribonucleotides include all of possible mRNA sequences corresponding to said amino acid sequence. c) Construction of cDNA library A cDNA library was constructed with use of about 30µg of said poly(A)RNA and 4.3µg of vector/primer DNA and in accordance with the method disclosed by Okayama, H and Berg, P. ["Mol. Cell. Biol." Vol. 2, pages 161 - 170 (1982)] and E. coli (HB101 strain) was transformed in accordance with the method disclosed by Morrison, D. A. [ "Methods in Enzymol." Vol. 68, pages 326 - 331, (1979)]. Through a screening with use of LB-agar plate containing 50µg/ml of ampicillin, ampicillin resistant transformants were obtained by 10,000 cells per 1µg of poly(A)RNA, namely 300,000 cells in total. (d) cDNA cloning The cloning was carried out in accordance with the method as disclosed by Hanahan, D. et al [ "Gene" Vol. 10, pages 63 - 67 (1980)]. Namely, about 50000 cells among said about 300,000 ampicillin resistant transformants as described in said Item (c) were replicated on nitrocellulose filter, cultured for 3 to 4 hours on a agar plate containing 50µg/ml of ampicillin and transferred on LB-agar plate containing 500µg/ml of chloramphenicol to further culture the same at 37°C for one overnight. A colony formed on the filter was subjected to bacteriolysis by treatment of 0.5N sodium hydroxide for 5 minutes, namely a double-strand DNA was made into a single-strand DNA, fixed on a plate, neutralized to make pH to 7.5 and dipped the filter for 5 minutes in Tris hydrochloride buffer (pH 7.5) containing 1.5M-NaCl to remove bacterial fragments and the like other than DNA. Thereafter, the filter was air dried and baked for 2 hours at 80 °C to obtain a testing filter for screening. While, the screening of the transformants was carried out as follows, in accordance with the method as disclosed by Grunstein, M. et al [ "Proc. Natl. Acad. Sci. U.S.A." Vol. 72, pages 3961 - 3965 (1975)]. Namely, each of the oligodeoxyribonucleotides synthesized by the method as stated in said Item (b) and shown by said formula (A) was labeled at 5′-end with [γ - ³²P]ATP (Amersham, 5,000 Ci/mmol) and T₄-polynucleotide kinase (Toyobo Co., Ltd., Osaka, Japan) to make the same into probe for screening. A relative activity of each probe was 1 - 2 x 10⁶ cpm/pmol. The transformants on the testing filter were screened by a hybridization at 50°C and with use of the probes (labeled oligodeoxyribonucleotides) and judged by a conventional autoradiogram method to find that only one transformant among about 50,000 transformants is hybridizing positive clone. This positive clone, namely that comprising human motilin precursor was analized by an electrophoresis to find that it has cDNA insert region of about 650bp including poly(dA)(dT) tails and poly(dG)(dC) tracts. This cloned DNA is double-strand one and nucleotide sequence thereof had been elucidated as disclosed in the Test Example to be stated below. Therefore, the DNA can be made into a fragment(s) in various length, by treating same with a suitable restriction enzyme(s) and if necessary, binding a synthetic DNA to the fragment to obtain a desired DNA fragment consisting of the motilin precursor region, motilin region only or other region showing a certain biological activity. Further, the double-strand DNA in full length, fragment or piece thereof can be made into corresponding single-strand one by treating same at 90°C for about 3 minutes and then ice cooling to cause a denaturation thereof. Test Example (Determination of nucleotide sequence for cloned human motilin precursor and corresponding amino acid sequence) A nucleotide sequence of cDNA insert region for the cloned human motilin precursor obtained in Example 1 was determined by sub-cloning to a pUC19 plasmid, in accordance with the method disclosed by Maxam, A. M. et al [ "Methods in Enzymol." Vol. 65, pages 499 - 560 (1980)] and the dideoxy method disclosed by Hattori, M. et al [ "Anal. Biochem." Vol. 152, pages 232 - 238 (1980)], who employs a modified plasmid. In Fig. 1, there are given a cDNA region of the cloned human motilin precursor, restriction enzymes selected for determining a nucleotide sequence in the region and a strategy for sequencing the cloned cDNA. The numerals at uppermost portion in the Figure are nucleotide number given as from the human motilin precursor region boxed at intermediate portion. The --Pvull-- and the like are names of the restriction enzymes as selected and a numeral given in parentheses shows the numbers indicating the 5′-terminal nucleotide generated by cleavage. Among the boxed region for the human motilin precursor, a region (S) shows putative signal peptides, and another region (Motilin) shows that to be estimated as corresponding to human motilin per se. In the lower portion in Fig. 1, each of horizontal arrow shows a direction and limit of the nucleotide sequence to be determined, due to the respective restriction enzyme. Each of the determination results obtained in accordance with the method disclosed by Maxam A. M. et al isshown by an arrow with a short vertical line which shows an isotope labeled position at 5′-terminal. While, each determination results obtained in accordance with the method disclosed by Hattori, M. et al isshown by adding a small circle at an end of the arrow which indicates a starting position for nucleotide sequencing. In Fig. 2, there are given thus determined nucleotide sequence of the cloned cDNA encoding the human motilin precursor as well as the amino acid sequence corresponding to the nucleotide sequence in the human motilin precursor region. In the Figure, the numeral given at upper sideis number of nucleotides similar to Fig. 1 and another numeral given at lower side is number of amino acid residues in the human motilin precursor region. The region boxed with a solid line shows the putative motilin region, and the region boxed with broken line shows the location of the oligonucleotide probe for screening the transformants in Example 1. As seen from Fig. 2, the human motilin precursor region lies as an open reading frame begining from the initiation codon of ATG and ending by the terminal codon of TGA, which comprises therein the nucleotide sequence encoding the amino acid sequence same with the known porcine motilin. As boxed with the solid line, the nucleotide sequence occupies such a position in the amino acid number counting from first methionine (Met) as from 26th phenylalanine (Phe) to 47th glutamine (Gln). The begining portion in the human motilin precursor region, namely the 1st Met to 25th Ala seems to be a signal sequence ( S region in Fig. 1) which may generally be found on secretory proteins, in view of its structure consisting of relatively hydrophobic amino acids as from 6th Ala to 25th Ala. In view of that end portion in the motilin region is partitioned by neighboring two lysines (Lys) of basic amino acid, it seems to be that the motilin will be secreted in the form of the precursor and then cleaved by a protease in blood or the like to form the mature motilin. Example 2 (Preparation of plasmid integrating human motilin precursor) This example will be explained with reference to the Figures and more particularly Fig. 3. In the first place, the cloned cDNA for the human motilin precursor as shown in Figs. 1 and 2 was treated with restriction enzymes of Hinfl and Ball to obtain a fragment of about 490bp. On the other hand, Nhel and Xhol linkers were prepared with use of a DNA synthesizer marketed by Pharmacia, Sweden. The linkers were joined to the Hinfl and Ball sites of the fragment, respectively. While, a commercially available plasmid (--pMSG-- marketed by Pharmacia, Sweden) was treated with the restriction enzymes of Nhel and Xhol. To the resulting ends of the plasmid fragment, said fragment with linkers was joind to re-construct into a plasmid, in which human motilin precursor is integrated. As shown in the last portion in Fig. 3, the resulting plasmid has DNA clone of the human motilin precursor at down-stream of a powerful promoter of a long terminal repeat on mouse mammary tumor virus (MMTV-LTR). If L or CHO cells are transformed in a conventional manner but with use of such plasmid and cultured same, therefore, the human motilin can be prepared in a large amount.

Claims: A cloned single-stranded DNA encoding a human motilin precursor and having a nucleotide sequence of a single-stranded DNA having a nucleotide sequence which has codons same with che above in degeneracy, or a double-stranded DNA consisting, of said single-stranded DNA and its complementary single-stranded DNA. A cloned single-stranded or double-stranded DNA as claimed in Claim 1, wherein said DNA encodes an amino acid sequence of A plasmid comprising a double-stranded DNA encoding a human motilin precursor and having a single-strand which comprises nucleotides encoding an amino acid sequence of A process for the preparation of a cloned single-stranded DNA encoding a human motilin precursor and a double-stranded DNA consisting of said single-stranded DNA and its complementary single-stranded DNA, which comprises steps of converting mRNA extracted from human upper small intestine into poly(A)RNA, constructing cDNA library from said poly(A)RNA and vector/primer DNA, transforming colonies of Escherichia coli with said cDNA library, screening the transformants with synthetic and labeled probes comprising a mixture of 24 tricosamers, each consisting of 23 deoxyribonucleotides of the formula wherein I is inosine and each tricosamer is complementary to mRNA coding for corresponding to Phe-Val-Pro-Ile-Phe-thr-Tyr-Gly of the 1st to 8th amino acid sequence from N-terminal of the amino acid sequence of motilin, labeling each synthesized oligodeoxyribonucleotide shown by said formula (A) at N-terminal, screening said transformed Escherichia coli by picking clones which hybridize with said probes, and if necessary denaturing the double-stranded DNA into the single-stranded DNA.

Inventors: IMURA, HIROO, KURONO, MASAYASU SANWA KAGAKU KENKYUSHO CO., LTD, MITANI, TAKAHIKO SANWA KAGAKU KENKYUSHO CO., LTD, SAWAI, KIICHI SANWA KAGAKU KENKYUSHO CO., LTD, SEINO, YUTAKA, TAKAHASHI, HARUO SANWA KAGAKU KENKYUSHO CO., LTD, TAKEDA, JUN, TANAKA, KENICHI SANWA KAGAKU KENKYUSHO CO., LTD
Applicants: SANWA KAGAKU KENKYUSHO CO., LTD.
Application year: 1988
Main classification: C12N15
IPC codes: C12N15, C07H21, C12P21, C07K14