Abstract: 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.

Abstract: 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 86,000-95,000 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 containing non-ionic detergents that lends stability to the enzyme.

Abstract: Methods are provided for the replication and amplification of RNA sequences by thermostable DNA polymerases. Reverse transcription of RNA is catalyzed by, for example, 92 kDa Taq, 64 kDa Taq, and Tth DNA polymerase. Reverse transcription is coupled to PCR amplification in a one enzyme procedure using a thermostable polymerase.

Abstract: Methods are provided for the replication and amplification of RNA sequences by thermoactive DNA polymerases. In a preferred embodiment, high temperature reverse transcription is coupled to nucleic acid amplification in a one tube, one enzyme procedure using a thermostable DNA polymerase. Methods for eliminating carry over contamination of amplifications due to prior reverse transcription reactions are also provided.

Abstract: Nucleic acid sequences encoding the bacterial cellulose synthase operon derived from Acetobacter are disclosed. Methods for isolating the genes, vectors containing the genes, and transformed hosts useful for the expression of recombinant bacterial cellulose synthase or production of cellulose are also described.

Abstract: The present invention is directed to a process of detecting a target nucleic acid using labeled oligonucleotides. This process uses the 5' to 3' nuclease activity of a nucleic acid polymerase to cleave annealed labeled oligonucleotide from hybridized duplexes and release labeled oligonucleotide fragments for detection. This process is easily incorporated into a PCR amplification assay.

Abstract: 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.

Abstract: 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.

Abstract: Dideoxynucleotide DNA sequencing methods can be dramatically improved by utilizing the DNA polymerase from Thermus aquaticus to catalyze the primer extension reactions.

Abstract: A gene having a DNA sequence complementary to that of the glucoamylase polypeptide mRNA from a fungal species, preferably Aspergillus awamori, is prepared. The mRNA is an approximately 2.2 kilobase poly A RNA obtained from fungal cells grown under conditions of glucoamylase induction. Reverse transcription of the mRNA provides a glucoamylase probe used to identify genomic digest fragments containing glucoamylase gene regions, which are sequenced to locate the introns and exons. The genomic fragments are spliced together to form a gene having a DNA sequence with altered or deleted introns which codes for fungal glucoamylase protein and is capable, when correctly combined with a cleaved DNA expression vector, of expressing a non-native protein having glucoamylase enzyme activity upon transformation of a host organism by the vector. The host is preferably bacteria or yeast. The transformed yeast host may be used to produce ethanol.

Abstract: DNA plasmids are described which are selected mutants in which an altered repressor gene leads to high copy number replication. Elements in the plasmids are modified in such a way that readthrough expression of heterologous DNA inserted in the plasmid will not continue into the replication primer strand. Deletions resulting from interference with replication primer strand transcription are thereby avoided.

Abstract: A process for amplifying any target nucleic acid sequence contained in a nucleic acid or mixture thereof comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers and extending the primers with a thermostable enzyme to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The amplified sequence can be readily detected. The steps of the reaction can be repeated as often as desired and involve temperature cycling to effect hybridization, promotion of activity of the enzyme, and denaturation of the hybrids formed.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: The invention is a plasmid vector in which a first DNA sequence expressionable for a selected gene product is ligated to a positive retroregulatory element in a relationship thereto whereby expression of the selected gene product is enhanced. The first DNA sequence is furthermore operably linked to a second and third DNA sequence operably linked to one another which are respectively the P.sub.L promoter and a DNA sequence corresponding to an N gene ribosome binding site. Microorganisms transformed with the plasmid are also claimed.

Abstract: DNA plasmids are described which are selected mutants in which an altered repressor gene leads to high copy number replication. Elements in the plasmids are modified in such a way that readthrough expression of heterologous DNA inserted in the plasmid will not continue into the replication primer strand. Deletions resulting from interference with replication primer strand transcription are thereby avoided.