Abstract: A signal amplification system comprises a bacterial multi-hybrid system, and more preferably a two-hybrid system, of at least two chimeric polypeptides containing a first chimeric polypeptide corresponding to a first fragment of an enzyme and a second chimeric polypeptide corresponding to a second fragment of an enzyme or a modulating substance capable of activating said enzyme. The first fragment is fused to a molecule of interest and the second fragment or the modulating substance is fused to a target ligand. The activity of the enzyme is restored by the in vivo interaction between the molecule of interest and the target ligand. Signal amplification is generated and, for example, triggers transcriptional activation. The signal amplification system is useful in a method of selecting a molecule of interest, which is capable of binding to target ligand, wherein the interaction between the molecule of interest and the target ligand is detected with the signal amplification system as a kit therefor.

Abstract: Methods for the controllable expression in high yields of heterologous proteins under the control of a modified tryptophan promoter-operator system are disclosed. The system employed involves sequential resort to tryptophan repression and derepression, as well as deletion of an attenuator function native to the tryptophan operon. Means are also disclosed for effecting deletions at any given point within a gene fragment and for the ready identification of transformant bacteria via triple ligation procedures.

Abstract: The present invention cloned a cDNA clone encoding isopentenyl diphosphate (hereafter “IPP”) isomerase (EC 5.3.3.2) from a cDNA library of Hevea brasiliensis latex. The clone has a continuous open reading frame encoding a peptide of 234 amino acids with a predicted molecular mass of 26.7 kDa. The deduced protein is acidic with an isoelectric point of 4.7 and shows high sequence identity with other IPP isomerases. The recombinant protein expressed in Escherichia coli showed IPP isomerase activity. In vitro rubber biosynthesis assays using washed rubber particle (WRP) deprived of initiating allylic diphosphates were performed with the addition of IPP isomerase in the reaction mixture. Results revealed that the recombinant IPP isomerase is catalytically active in catalyzing the conversion of IPP to DMAPP, a key activation step of the basic five-carbon isoprene unit in rubber biosynthesis. Southern analysis indicated that the IPP isomerase is encoded by two genes in Hevea rubber tree.

Abstract: The subject invention relates to &bgr;-casein expressing constructs which have significant stability when introduced into host cells. These constructs, for purposes of the present invention, have been designated as pRAB-84-69 and pRSB-14. Each construct comprises an isolated DNA sequence comprising i) a nucleotide sequence encoding a protein, wherein said nucleotide sequence is operably linked to a promoter, ii) a nucleotide sequence encoding a first subunit of a kinase, iii) a nucleotide sequence encoding a second subunit of a kinase, iv) a nucleotide encoding a peptidase and v) a nucleotide sequence encoding a bacterial resistance marker. These constructs, once introduced into a host cell, may be used in the production of, for example, recombinant human beta-casein.

Abstract: The invention concerns bacterial strains capable of enhanced transformation efficiencies that are produced by the introduction of the F′ genetic material. The invention also concerns processes for producing transformable competent bacteria with enhanced transformation efficiencies.

Abstract: Introducing sucrose phosphorylase activity into plants by transformation with a gene for the enzyme increases the rate of sucrose hydrolysis, leading to increased starch, oil, and/protein levels. Sucrose phosphorylase genes from Streptococcus mutans and Leuconostoc mesenteroides have been found particularly advantageous for use in the present invention. Surprisingly, in potatoes transformed to express these genes in tubers, reduced bruise discoloration susceptibility and increased uniformity of starch deposition throughout the tuber are achieved.

Abstract: A new recombinant form of the plasmid-encoded protein pgp3 from C. trachomatis, serotype D, was purified by ion exchange column chromatography and shown to be suitable for quantitative immunoassy on clinical samples in an ELISA format.

Abstract: This invention relates to an isolated nucleic acid fragment encoding a glycolysis or respiration protein. The invention also relates to the construction of a chimeric gene encoding all or a portion of the glycolysis or respiration protein, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the glycolysis or respiration protein in a transformed host cell.

Abstract: The present invention relates to a process for integration of a chosen gene or of a specific DNA sequence in a DNA sequence such as the chromosome or episome of a bacterium, wherein: a) the said chosen gene or the chosen DNA sequence is cloned inside a defective transposon outside the essential parts of the transposon, b) the said transposon is integrated in the DNA sequence such as the chromosome or the episome of the said bacterium, and also the bacterium strains obtained by implementation of this process.

Abstract: A cloning and/or sequencing vector enables recombinant clones to be selected directly. The vector encodes a fusion protein which includes a protein poison.

Abstract: On the basis of a first repertoire of genes coding for a population of one of two kinds of polypeptides capable of being optionally covalently combined, particularly variable regions of either the antibody light chain type or the antibody heavy chain type, and at least one gene coding for the other type of polypeptide, particularly a variable region of the other type, an antibody chain or preferably a second repertoire of genes coding for a population of said other type, the genes from the first repertoire are inserted into a first vector to form a population of vectors carrying the various genes of said first repertoire, and said gene of said other type or the genes from said second repertoire is/are inserted into a second vector. Both starting vectors have means enabling each to exchange one part by one or more irreversible recombinations to generate recombinant final vectors of which one contains a gene of one of said types and a gene of the other type.

Abstract: The invention concerns mutant microorganisms blocked in a step of Streptogramin biosynthesis and methods for preparing cells blocked in a step of Streptogramin biosynthesis.

Abstract: A method for in vivo production of a library in cells comprising a multitude of mutated genetic elements, wherein an error-prone polymerase is used in each ancestral cell to replicate all or a part of a genetic element independently of the host chromosomal replication machinery. The genetic element comprisesi) an origin of replication from which replication is initiated,ii) optionally a genetic marker, e.g. a gene conferring resistance towards an antibiotic,iii) a gene encoding the polypeptide of interest.Also methods for the generation of a DNA sequence encoding a desired variant of a polypeptide of interest, and for the determination of such a DNA sequence are described.

Abstract: An artificial operon comprising polynucleotides encoding each of chaperones DnaK, DnaJ and GrpE; an expression plasmid carrying the operon; a cotransformant prepared by introducing the expression plasmid into E. coli together with a foreign protein expression vector; and a method for producing a foreign protein comprising using the cotransformant.

Abstract: The present invention provides the complete cDNA sequence of maize acetyl CoA carboxylase and a method introducing and expressing a plant acetyl CoA carboxylase gene in plant cells. The method includes the steps of introducing an expression cassette encoding a plant acetyl CoA carboxylase or an antisense DNA sequence complementary to the sequence for a plant acetyl CoA carboxylase gene operably linked to a promoter functional in plant cells, into the cells of a plant tissue and expressing the plant acetyl CoA carboxylase gene. The expression cassette can also be introduced into other host cells to increase yield of a plant acetyl CoA carboxylase crystallized enzyme.

Abstract: A recombinant vector for cloning a heterologous nucleotide sequence and/or expressing it and/or transferring it to a cell host. The vector includes, at a site which is not essential for replication, the gene coding for a lipoprotein other than E. coli lipoproteins, or a part of said gene which contains the elements required for controlling the expression of said lipoprotein and exposing it on the surface of the outer host cell membrane, so that the heterologous nucleotide sequence can be inserted into the gene or said part thereof under conditions suitable for expressing said heterologous sequence and exposing it on the surface of the cell host.

Abstract: Cloning systems useful for the isolation of recombinant nucleic acid are disclosed in which the recombination of cloning-system nucleic acid and foreign nucleic acid is linked to the expression of a moiety on the surface of a host organism, the moiety being a first member of a binding pair. When recombination occurs between the nucleic acid and the foreign nucleic acid, the moiety is expressed on the surface of the host organism. The isolation of recombinant nucleic acid is then performed by attaching a second member of the binding pair to a solid support and contacting the host organism with the support. When the first member of the binding pair is expressed on the surface of the host organism, the host organism binds to the second member of the binding pair attached to the solid support, thereby selectively isolating those organisms.

Abstract: The subject invention relates to a method of producing and purifying large quantities of a biosynthetic protein.The gene which codes for the protease is placed between the binding domain of a gene which codes for a binding protein and a gene coding for the target protein of interest. The fused gene construct is inserted in an expression vector which is then introduced into a host cell.

Abstract: The present invention relates to surface anchoring vectors, a method for preparation of foreign proteins onto a cell surface and use thereof, which uses outer cell membrane protein, ice nucleation protein (NIP) derived from Pseudomonas syringae, a gram-negative bacterium.

Abstract: The present invention discloses a selection marker free system which can be used to introduce genetic modifications in bacteria, yeasts and fungi. The system can be employed to introduce or delete desired genes or DNA fragments in the genome of the indicated host species without leaving any undesired DNA i.e. the selection marker used for selection of transformants or other DNA used for cloning. In this way strains have been developed containing only desired genes introduced at desired chromosomal sites. Similarly, desired DNA fragments have been deleted or replaced at desired sites.

Abstract: This invention provides improved electroporation methods for transferring nucleic acids of interest into host cells, wherein the host cells are (1) suspended in a substantially non-ionic solution comprising at least one sugar or sugar derivative, (2) mixed with the nucleic acids of interest, and (3) electrically treated. Also, this invention provides for kits used in the method for transferring nucleic acids into host cells.

Abstract: Hybrid and novel polyketide synthases and polyketides are produced by use of a multiple vector system. The combinatorial possibilities offered by placing the various catalytic activities of PKS systems on separate vectors permits the construction of improved libraries of PKS and polyketides. In addition, polyketides can be produced in hosts that ordinarily do not produce polyketides by supplying, along with an expression system for the desired PKS, an expression system for holo ACP synthase.

Abstract: A new method for the identification of useful promoters is disclosed. The method is capable of identifying bacterial promoters sensitive to a particular cellular insult and may be modified to identify promoters sensitive to herbicides and crop protection chemicals. Constructs comprising promoters upstream of a luminescent reporter genes are placed in transformed hosts. Transformants grown in liquid media to a predetermined growth stage and contacted with a cellular insult are assessed for regulatory region activity by measurement of the resulting change in bioluminesence. The method is able to identify promoters undetectable by standard methods.

Abstract: The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated M.sub.r 15,500 protein of Rhodococcus ruber, which is referred to as the GA14-protein, was analysed. The sequence revealed that the corresponding structural gene is represented by the open reading frame 3 encoding a protein with a calculated M.sub.r 14,175 which was recently localized downstream of the PHA synthase gene (Pieper, U., and A. Steinbuchel, 1992. FEMS Microbiol. Lett. 96: 73-80). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10*) with ORF3 overexpressed the GA14-protein. The GA14-protein was subsequently purified in a three-step procedure including chromatography on DEAE-Sephacel, Phenyl-Sepharose CL-4B and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopic studies make a tetrameric structure of the recombinant, native GA14-protein most likely.

Abstract: Provided herein is a method to produce knockout mutations at targeted sites in the genome of Streptococcus pneumoniae.

Abstract: The present invention is directed to a vector for transferring heterologous DNA into a plant cell. The vector is based on the bacterial artificial chromosome (BAC) vector designed for the construction of genomic libraries with large DNA inserts, and the binary (BIN) vector designed for Agrobacterium-mediated plant transformation. The BIBAC vector according to the subject invention allows the construction of plant genomic libraries with large DNA inserts that can be directly introduced into plants by transformation mediated by Agrobacterium.

Abstract: A system is used to express clostridial gene constructions in a clostridial host. A mobilizable transfer plasmid is described which permits the direct transfer of the plasmid, and genes carried on it, from E. coli into Clostridium species. A promoter is described for use in clostridial species. Also, a useful host strain is used which is nontoxigenic and which permits high levels of expression of clostridial genes using the clostridial promoter.

Abstract: Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. This invention describes a strategy which simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, employing homologous recombination in bacteria rather than in eucaryotic cells. Following transfections of such plasmids into a mammalian packaging cell line, viral production can be conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system expedites the process of generating and testing recombinant adenoviruses.

Abstract: A novel uracil DNA glycosylase enzyme (referred to as Bpa UDG) has been identified in Bacillus pallidus and the gene encoding Bpa UDG has been cloned, sequenced and expressed to produce a recombinant UDG protein. The enzyme is thermostable and exhibits reaction kinetics similar to E. coli UDG. It is effectively inhibited by B. subtilis UGI.Bpa UDG may be used to inactivate contaminating amplicons in nucleic acid amplification reactions, particularly at higher reaction temperatures. It may also be used to generate Bpa UDG-specific antibodies for purification of Bpa-UDG or for detecting Bpa UDG in a sample. Certain Bpa UDG antibodies may inactivate the enzyme and may therefore be useful as substitutes for UGI or heat, or in combination with UGI and/or heat, for controlling UDG activity in a reaction.

Abstract: The present invention provides recombinant DNA vehicles which are suitable for the microbial expression of DNA encoding a heterologous polypeptide which comprises a portion of the trp operon having the promoter-operator and leader ribosome binding site, and a restriction site providing an insertion site for the DNA sequences encoding the heterologous polypeptide, wherein the restriction site is located 3' of the leader ribosome binding site as a substitute for the Taq I site of the trp promoter-operator and is selected from the group consisting of Xba I and Eco RI. Also provided are E. coli strains transformed with the above described recombinant DNA vehicles.