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At least 109 records · Page 6Linked to original sources
PubMed1988

Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1.

The Cre protein encoded by the coliphage P1 is a 38-kDa protein that efficiently promotes both intra- and intermolecular synapsis and recombination of DNA both in Escherichia coli and in vitro. Recombination occurs at a specific site, called lox, and does not require any other protein factors. The Cre protein is shown here also to be able to cause synapsis of DNA and site-specific recombination in a mammalian cell line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallothionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. One such substrate is a circular plasmid with two directly repeated lox sites (lox2) flanking a marker gene and was introduced into cells by Ca3(PO4)2 transformation. As a second substrate we used a pseudorabies virus (a herpesvirus) containing a lox2 insertion designed to provide a sensitive detection system for recombination. In both cases, site-specific recombination in vivo is dependent on the presence of the Cre protein and occurs specifically at the 34-base-pair lox sites. These results demonstrate the controlled site-specific synapsis of DNA and recombination by a prokaryotic protein in mammalian cells and suggest that Cre-mediated site-specific recombination may be a useful tool for understanding and modulating genome rearrangements in eukaryotes.

Animals↗
PubMed1983

Use of recombinant DNA procedures to study mechanisms controlling synthesis of hypophyseal hormones.

Recombinant DNA technology has provided a useful new tool for analysis of the molecular mechanisms regulating pituitary hormone biosynthesis. Application of recombinant DNA technique to the study of prolactin production has provided new insights into the structure and regulation of the prolactin gene. In the course of these studies a recombinant DNA plasmid containing a DNA copy of prolactin mRNA was constructed and analyzed. This prolactin cDNA clone has provided a homogeneous hybridization probe for subsequent studies of the prolactin gene. Studies utilizing this probe have provided evidence that the dopaminergic and estrogenic regulation of prolactin mRNA levels involves changes in the transcription of the prolactin gene. Recombinant DNA techniques have also been utilized to isolate a clone containing the chromosomal prolactin gene. Future studies of the regulation of prolactin gene expression will undoubtedly rely on recombinant DNA techniques.

Animals↗
PubMed1987

Sedimentation equilibrium measurements of recombinant DNA derived human interferon gamma.

Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation. Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight. Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500. Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+. No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1. Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1. At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer. Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+. The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components. A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.

DNA, Recombinant↗
PubMed1983

Recombinant DNA technology and laboratory medicine.

Recombinant DNA technology has already had a major impact on our understanding of microbiology, cell biology, and genetic diseases and it will certainly have extensive applications in laboratory medicine. The techniques of restriction endonuclease analysis of DNA, nucleic acid hybridization after electrophoretic separation of nucleic acid fragments, and molecular cloning of bacterial, viral, and human genes are already being used in epidemiologic studies and the prenatal diagnosis of certain genetic diseases, such as sickle cell anemia and the thalassemias. New insights into genes that may be involved in human cancer are being developed and may lead to improved methods for diagnosis and classification of tumors.

Animals↗
PubMed2001

Managing DNA polymerases: coordinating DNA replication, DNA repair, and DNA recombination.

Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes.

Animals↗
PubMed1988

Recombinant DNA techniques in medicine.

Medical applications of recombinant DNA technology are reviewed. The main impact of these techniques has been in the diagnosis of genetic disease and analysis of the underlying mutations. Two different methods can be used for the diagnosis of genetic disease. When the gene defect is known and DNA probes are available, it may be possible to establish the diagnosis directly. In many cases, however, the gene is unknown or the mutation cannot be detected easily. In these situations, it may still be possible to make a diagnosis. The genotype can be determined indirectly by linkage analysis using sequence variations which alter restriction sites (restriction fragment length polymorphisms) and DNA probes for loci close to the disease locus. Recombinant DNA techniques have also led to a process of 'reverse' genetics for identification and analysis of the causes of genetic disease. Genes can be located, isolated and characterized without any prior knowledge of their function. The nature of the gene product can then be determined and its role in the disease process defined.

DNA Probes↗
PubMed1987

Control of therapeutic goods produced by recombinant DNA technology.

The Therapeutics Division of the Commonwealth Department of Health is responsible for ensuring the quality, safety, and efficacy of rDNA products intended for human therapeutic use in Australia. The National Biological Standards Laboratory evaluates information on production and quality control submitted by manufacturers, and the Drug Evaluation Section examines the clinical safety and efficacy aspects of product applications. rDNA production methods are considered to be biological processes requiring control of source materials, production methods, and final product for adequate quality assurance of products. The Department has recently prepared guidelines on appropriate methods of manufacture and testing of rDNA products and the requirements for product applications. The guidelines emphasize several areas of particular regulatory concern with rDNA derived products, including non-identity with the equivalent natural protein and contamination with host cell components.

DNA, Recombinant↗
PubMed1978

[Recombinant DNA studies, a controversial subject which is also promising from the point of view of veterinary medicine (author's transl)].

The hereditary characters of organisms are bound up in the DNA of their genomes. Genetic information is expressed by a complicated mechanism. The process by which this occurs and the control of the degree of expression in bacteria and a number of the other lower organisms are known only in broad outline. There usually is no exchange of DNA between organisms of unrelated species in nature. "Foreign" DNA can be linked to bacterial plasmid DNA and multiplied in the laboratory as a recombinant DNA molecule in bacteria using the recently discovered so-called restriction enzymes. Expression of bacterial DNA can thus be accomplished in a different, unrelated bacterium. In principle, this means that the natural barriers between unrealated species of organisms may be passed by. Human and animal DNA can also be multiplied in bacteria by this method. Previous recombinant DNA studies showed that the mode of expression of genetic information in the higher organisms differs markedly from that in bacteria. The initially sanguine expectations regarding the practical use of recombinant DNA research, for instance in the production of biologically important substances by bacteria, will therefore possibly not be realized at short notice. On the other hand this technique has added considerably to the knowledge of expression of genes within a short space of time. This is particularly true of the mechanisms of pathogenicity of E. coli, the bacterium which plays such an important role in veterinary medicine. The recombinant DNA technique is briefly reviewed in the present paper. The uses are broadly outlined, and one of these, which could be of importance in veterinary medicine, is described in greater detail. International discussions of potential hazards, and therefore also of the admissibility of this type of experiment, are griefly summarized.

Animals↗
PubMed2003

Monitoring the spread of recombinant DNA from field plots with transgenic sugar beet plants by PCR and natural transformation of Pseudomonas stutzeri.

Previous studies had shown that recombinant DNA can be detected for several months in soil after the deposition of litter from transgenic (tg) plants. Here we show by PCR monitoring of field releases of tg sugar beet plants that during the growth of the plants the soil close to the plants and also plant material contains recombinant DNA, in the form of extracellular molecules. Surprisingly, the monitoring also revealed the presence of tg DNA in many field plots (30-70%) in which tg plants were never grown. These studies and the further monitoring during other tg sugar beet release experiments by PCR and a novel bioassay (measuring the transforming potential of recombinant DNA for Pseudomonas stutzeri) indicated that recombinant DNA was only detectable in the surface soil of field plots and their vicinity where flowering of the tg beet plants was allowed. Recombinant DNA was found in soil at a distance of 50 m from pollen-producing plants surrounded by a strip with hemp plants as a containment regime. It is concluded that recombinant DNA is deposited in soil during the growth of tg sugar beets and that a major mechanism of recombinant DNA spread in the environment is the dispersal of pollen which allows recombinant DNA to persist in the field plot for at least a year.

Beta vulgaris↗
PubMed1999

Recombinational DNA repair in bacteria and the RecA protein.

In bacteria, the major function of homologous genetic recombination is recombinational DNA repair. This is not a process reserved only for rare double-strand breaks caused by ionizing radiation, nor is it limited to situations in which the SOS response has been induced. Recombinational DNA repair in bacteria is closely tied to the cellular replication systems, and it functions to repair damage at stalled replication forks, Studies with a variety of rec mutants, carried out under normal aerobic growth conditions, consistently suggest that at least 10-30% of all replication forks originating at the bacterial origin of replication are halted by DNA damage and must undergo recombinational DNA repair. The actual frequency may be much higher. Recombinational DNA repair is both the most complex and the least understood of bacterial DNA repair processes. When replication forks encounter a DNA lesion or strand break, repair is mediated by an adaptable set of pathways encompassing most of the enzymes involved in DNA metabolism. There are five separate enzymatic processes involved in these repair events: (1) The replication fork assembled at OriC stalls and/or collapses when encountering DNA damage. (2) Recombination enzymes provide a complementary strand for a lesion isolated in a single-strand gap, or reconstruct a branched DNA at the site of a double-strand break. (3) The phi X174-type primosome (or repair primosome) functions in the origin-independent reassembly of the replication fork. (4) The XerCD site-specific recombination system resolves the dimeric chromosomes that are the inevitable by-product of frequent recombination associated with recombinational DNA repair. (5) DNA excision repair and other repair systems eliminate lesions left behind in double-stranded DNA. The RecA protein plays a central role in the recombination phase of the process. Among its many activities, RecA protein is a motor protein, coupling the hydrolysis of ATP to the movement of DNA branches.

Bacteria↗
PubMed1982

Recombinant DNA technology for the preparation of subunit vaccines.

Recombinant DNA technology appears to be on the verge of producing safe and effective protein vaccines for animal and human diseases. The procedure is applicable to most viruses because their isolated surface proteins generally possess immunogenic activity. Strategies used for the preparation and cloning of the appropriate genes depend on the characteristics of the viral genomes: whether DNA or RNA; their size, strandedness, and segmentation; and whether messenger RNA are monocistronic or polycistronic. Cloned surface proteins of foot-and-mouth disease and hepatitis B viruses are being tested for possible use as practical vaccines. Two doses of the cloned foot-and-mouth disease viral protein have elicited large amounts of neutralizing antibody and have protected cattle and swine against challenge exposure with the virus. Surface proteins have also been cloned for the viruses of fowl plague, influenza, vesicular stomatitis, rabies, and herpes simplex. Cloning is in progress for surface proteins of viruses causing canine parvovirus gastroenteritis, human papillomas, infectious bovine rhinotracheitis, Rift Valley fever, and paramyxovirus diseases. In addition, advances in recombinant DNA and other facilitating technologies have rekindled interest in the chemical synthesis of polypeptide vaccines for viral diseases. The bioengineering of bacterial vaccines is also under way. Proteinaceous pili of enterotoxigenic Escherichia coli are being produced in E coli K-12 strains for use as vaccines against neonatal diarrheal diseases of livestock.

Animals↗
PubMed1984

Some clinical implications of recombinant DNA technology with emphasis on prenatal diagnosis of hemoglobinopathies.

Recombinant DNA technology has made possible remarkable advances in understanding the molecular genetics of human and other eucaryotic cells. This technology also has clinical applications, some of which may soon involve clinical laboratories. Restriction endonucleases and cloned DNA probes permit the direct analysis of cellular DNA to detect sequence abnormalities associated with particular genetic disorders. Use of this approach in the antenatal diagnosis of hemoglobinopathies is now possible on a routine basis. The principles behind the methods are quite general and may be applied to other hereditary diseases once suitable DNA probes become available. The same approach may be used to detect carriers of recessive gene defects and so improve genetic counselling. Other clinically related applications of recombinant DNA technology include the production of antigens for vaccine preparation and of specific human proteins (e.g. interferon and human growth hormone) for therapeutic use, as well as the use of nucleic acid hybridization for identification of microbial pathogens. It seems likely that recombinant DNA technology will, in the future, play an increasingly important role in the diagnosis, prevention and treatment of human disease.

Alleles↗
PubMed1992

Recombinant DNA approaches for the development of metabolic systems used in in vitro toxicology.

In the past few years there has been considerable progress in the development of mammalian cell systems for use in genetic toxicology by the stable transfer of genes/cDNAs coding for drug metabolizing enzymes directly into the target cell. Alternative approaches have also been developed in which mammalian cells are transiently transfected with cDNAs coding for drug-metabolizing enzymes and S9 preparations expressing a single metabolizing enzyme isolated and used for metabolic activation. Progress in these areas is reviewed here and the relative merits of the different approaches are discussed. Work to date has focused primarily on the cytochrome P450 family of enzymes, although other enzyme systems involved in xenobiotic metabolism have been used. The central theme of this review is the transfer of genetic information to improve the metabolic capability of cell systems used in genetic toxicology. However, a basic philosophy of the review is that genetic manipulation of cultured mammalian cells has the potential for developing systems to be used to better understand chemically induced toxicological effects.

Animals↗