Yeast promoters of glycolytic enzymes are modified by isolating a fragment encompassing the RNA polymerase binding site and joining to the 5' end of this fragment a DNA sequence providing for enhanced inducible or constitutive transcription of a structural gene. Constructs are prepared for efficient expression of foreign genes in yeast. Yeast strains 2150-2-3 (pC1/1GAPSOD) and AB110 (pC1/1GAPATi9), producing human .alpha..sub.1 -antitrypsin and superoxide dismutase, were desposited at the A.T.C.C. on May 9, 1984 and given Accession Nos. 20708 and 20709, respectively; and 2150-2-3 (GAP5), 2150-2-3 (Pyk5) and 2150-2-3 (PHO5GAP1), expressing Hepatitis B surface antigen, were deposited at the A.T.C.C. on May 9, 1984 and given Accession Nos. 20705, 20706 and 20707, respectively.
This application is a continuation of application Ser. No. 073,381, filed July 13, 1987 which is a continuation of U.S. Ser. No. 609,540 filed May 11, 1984, both now abandoned.
A hybrid promoter for controlling exogenous gene transcription is constructed by insertion of a UAS.sub.G into a GPD portable promoter. The resulting hybrid promoter is placed upstream of an exogenous gene in a hybrid yeast-bacterial plasmid, which is used to transform yeast cells. Due to the regulation imparted to the GPD promoter by the UAS.sub.G, transcription of the exogenous gene, and hence production of the exogenous gene product, may be regulated by controlling the composition of the carbon source in the yeast culture medium. Specifically, glucose is used to repress transcription and galactose is used to induce transcription.
A hybrid upstream regulatory sequence including an upstream regulatory sequence of E. coli origin and yeast upstream regulatory sequence, which hybrid upstream regulatory sequence can function in a yeast cell; a hybrid promoter including the regulatory sequence of E. coli origin and a TATA region of a yeast promoter, which hybrid promoter can also function in a yeast cell; a hybrid promoter including the upstream regulatory sequence of E. coli origin and a yeast promoter, which hybrid promoter can function in a yeast cell; and a plasmid useful for a test of an upstream regulatory sequence, including a TATA region of a yeast promoter, a structural gene containing a translation start codon, and a yeast origin of replication.
An isolated DNA segment is disclosed which is derived from a Saccharomyces cerevisiae sorbitol dehydrogenase gene and which functions to increase expression of an associated foreign polypeptide when the DNA segment and the gene coding for the foreign polypeptide are operably linked in a vector in such a manner that the vector is replicated and carried by a host yeast cell. The functionally active portion of the segment is under the control (i) a transcriptional regulatory sequence of the sorbitol dehydrogenase gene of Saccharomyces cerevisiae, (ii) a translation initiation regulatory sequence of the sorbitol dehydrogenase gene of Saccharomyces cerevisiae, and (iii) a termination regulatory sequence of the sorbitol dehydrogenase gene of Saccharomyces cerevisiae. Yeast cells containing these regulatory sequences linked to a foreign DNA sequence are grown in medium containing sorbitol under conditions permitting a foreign polypeptide to be expressed.
The present invention relates to a process for producing recombinant desulphatohirudin by means of culturing microorganisms. Concerning the codon usage of microorganisms the synthesized nucleotide sequences were joined downstream of and in reading frame with isolated promoters and signal sequences, subsequently the expression/secretion cassettes comprising the foregoing elements were inserted into plasmid DNAs allowing the cultivation of cells under selective culture conditions. E. coli, Saccharomyces and Streptomyces species were transformed with the said recombinant plasmids to biosynthesize the thrombin inhibitor desulphatohirudin HV-1 which was then isolated and identified. The thus-produced desulphatohirudin can be used to inhibit blood coagulation.
An isolated DNA segment is disclosed which is derived from a Saccharomyces cerevisiae sorbitol dehydrogenase gene and which functions to increase expression of an associated foreign polypeptide when the DNA segment and the gene coding for the foreign polypeptide are operably linked in a vector in such a manner that the vector is replicated and carried by a host yeast cell. The functionally active portion of the segment is under the control (i) a transcriptional regulatory sequence of the sorbitol dehydrogenase gene of Saccharomyces cerevisiae, (ii) a translation initiation regulatory sequence of the sorbitol dehydrogenase gene of Saccharomyces cerevisiae, and (iii) a termination regulatory sequence of the sorbitol dehydrogenase gene of Saccharomyces cerevisiae. Yeast cells containing these regulatory sequences linked to a foreign DNA sequence are grown in medium containing sorbitol under conditions permitting a foreign polypeptide to be expressed.
