Cetus Corporation

Hoffmann-La Roche Inc.

Roche Molecular Systems, Inc.

Frances C Lawyer

David H Gelfand

Susanne Stoffel

Randall K Saiki

Lawrence I Greenfield

Recombinant DNA sequences encoding the DNA polymerase activity of Thermus thermophilus can be used to construct recombinant vectors and transformed host cells for production of the activity. T. thermophilus DNA polymerase is an .about.94 kDa protein especially useful in the DNA amplification procedure known as the polymerase chain reaction.

Recombinant expression vectors and purification methods for thermus

A purified thermostable enzyme is derived from the eubacterium Thermotoga maritima. The enzyme has a molecular weight as determined by gel electrophoresis of about 97 kilodaltons and DNA polymerase I activity. The enzyme can be produced from native or recombinant host cells and can be used with primers and nucleoside triphosphates in a temperature-cycling chain reaction where at least one nucleic acid sequence is amplified in quantity from an existing sequence.

Purified thermostable nucleic acid polymerase enzyme from Thermotoga

Recombinant expression vectors and purification methods for Thermus

Mutated thermostable nucleic acid polymerase enzyme from thermotoga

A purified thermostable enzyme is derived from the eubacterium Thermotoga maritima. The enzyme has a molecular weight of about 97 kilodaltons and DNA polymerase I activity. The enzyme can be produced from native or recombinant host cells and can be used with primers and nucleoside triphosphates in a temperaturecycling chain reaction where at least one nucleic acid sequence is amplified in quantity from an existing sequence.

DNA encoding a thermostable nucleic acid polymerase enzyme from

A novel a selectable fusion protein having aminoglycoside phosphotransferase activity is disclosed. The marker comprises the coding sequences for aminoglycoside phosphotransferase I (APH-I) which has been modified and truncated so as to render its use in recombinant vectors more convenient. The modified, truncated sequence (mtAPH-I) gene is capable, upon expression, of conferring resistance to a number of antibiotics on the host. One of these antibiotics, G418, is toxic to eucaryotic as well as procaryotic hosts. Also disclosed are methods of constructing fusion proteins having N-terminal sequences corresponding to a desired peptide sequence, and C-terminal sequences comprising the amino acids encoded by mtAPH-I. The preferred N-terminal sequences are the first 11 amino acids of .beta.-isopropyl malate dehydrogenase, and the first 7 amino acids of yeast enolase.

Selectable fusion protein having aminoglycoside phosphotransferase

Recombinant DNA vectors that encode a thermostable DNA polymerase are useful in the recombinant production of thermostable DNA polymerase. The recombinant thermostable polymerase is preferred for use in the production of DNA in a polymerase chain reaction. Especially useful vectors encode the .about.94,000 dalton thermostable DNA polymerase from thermus aquaticus.

Purified thermostable enzyme

A purified thermostable enzyme is obtained that has unique characteristics. Preferably the enzyme is isolated from the Thermus aquaticus species and has a molecular weight of about 86,000-90,000 daltons. The thermostable enzyme may be native or recombinant and may be used in a temperature-cycling chain reaction wherein at least one nucleic acid sequence is amplified in quantity from an existing sequence with the aid of selected primers and nucleotide triphosphates. The enzyme is preferably stored in a buffer of non-ionic detergents that lends stability to the enzyme.

Expression vectors containing coding sequences under the control of SV40 early and RSV promoters are disclosed as useful in producing proteins in saccharomyces yeasts. Construction of such vectors, and their use in yeast transformations are described.

SV40 early and RSV promoters useful in saccharomyces expression

Recombinant vectors which are effective in expressing DNA sequences encoding specific fragments of diphtheria toxins at high levels in recombinant host cells are disclosed. Both a fragment consisting of the enzymatically active A chain of diphtheria toxin and a fragment consisting of both the A portion and a B portion partial sequence are constructed by use of this recombinant vector.

Recombinant diphtheria toxin fragments

A novel universal dominant selectable marker cassette is disclosed. The marker comprises the coding sequences for aminoglycoside phosphotransferase I (APH-I) which has been modified and truncated so as to render its use in recombinant vectors more convenient. The modified, truncated sequence (mtAPH-I) gene is capable, upon expression, of conferring resistance to a number of antibiotics on the host. One of these antibiotics, G418, is toxic to eucaryotic as well as procaryotic hosts. Also disclosed are methods of constructing fusion proteins having N-terminal sequences corresponding to a desired peptide sequence, and C-terminal sequences comprising the amino acids encoded by mtAPH-I.

Universal dominant selectable marker cassette

Two types of convenient portable control cassettes for the expression of protein encoding sequences in procaryotes are disclosed. Both cassettes comprise the P.sub.L promoter from lambda phage, which is controllable by means of a temperature sensitive repressor, operably linked to the ribosome binding site for N-gene (N.sub.RBS). In one embodiment, this cassette is bordered by restriction sites upstream of the P.sub.L promoter and immediately downstream from the N.sub.RBS permitting the insertion of a desired sequence containing its own start codon downstream from the cassette. The other embodiment contains an ATG start codon within the cassette and has a restriction site immediately 3' of the start codon.

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Oakland, CA, United States of America

Frances C Lawyer