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PubMed1978

Isolation and analysis of recombinant DNA molecules containing yeast DNA.

2500 recombinant plasmids containing insertions of yeast nuclear DNA have been cloned in Escherichia coli. It can be calculated that about 85% of the yeast genome is represented in this collection. The clones have been characterized by hybridization to purified RNA species. Of the 2000 clones examined, 75 contain insertions of yeast ribosomal DNA, 201 contain insertions of yeast tRNA genes, and 26 contain DNA sequences that are complementary to abundant mRNA species.

DNA↗
PubMed1993

[The characteristics of homologous DNA recombination in somatic cells. II. The characteristics of the extrachromosomal recombination of plasmid DNA in cultured cells differing in their tumor potential].

The frequencies of homologous extrachromosomal recombination, following plasmid transfection into cell lines differing in their tumour potential were measured. A 10-fold increase in extrachromosomal plasmid recombination has been reported in tumour cells (Ra-2, Jf-1) relative to immortal (Rat-2) and normal (REF). The differences between the normal and tumour cells are preserved in the in vitro recombination experiments (the recombination in the cell nuclei extracts). However, the recombination activity of nuclear extracts from the immortality transformed cells (Rat-2) is similar to that of nuclear extracts from the tumour cells.

Animals↗
PubMed1991

Diagnosis of genetic disease using recombinant DNA. Third edition.

Recombinant DNA methodology has greatly increased our knowledge of the molecular pathology of the human genome at the same time as providing the means to diagnose inherited disease at the DNA level. Direct detection and analysis of a range of genetic defects are now possible using cloned gene or oligonucleotide probes or by direct sequencing of the disease gene(s). In addition, the use of restriction fragment length polymorphisms (RFLPs) within and around these genes as indirect genetic markers has not potentiated the tracking of disease alleles in affected pedigrees in cases where direct analysis was not feasible. RFLPs associated with linked anonymous segments may also be used not only to diagnose hitherto undetectable disease states, but also for chromosomal localization of the loci responsible. We present here an updated list of reports describing both the direct and the indirect analysis/diagnosis of human inherited disease; it is intended to serve as a guide to current molecular genetic approaches in diagnostic medicine.

DNA, Recombinant↗
PubMed1993

Diagnosis of human genetic disease using recombinant DNA. Fourth edition.

Recombinant DNA methodology has greatly increased our knowledge of the molecular pathology of the human genome at the same time as providing the means of diagnosing inherited disease at the DNA level. Direct detection and analysis of a wide range of genetic lesions are now possible using cloned gene or oligonucleotide probes or by direct sequencing of the disease gene(s). In addition, the use of restriction fragment length polymorphisms (RFLPs) within and around these genes as indirect genetic markers has potentiated the tracking of disease alleles in affected pedigrees in cases where direct analysis is not yet feasible. RFLPs associated with linked anonymous DNA segments may also be used not only to diagnose hitherto undetectable disease states, but also for the chromosomal localization of the loci responsible. We present here an update to our previous list of reports describing the direct and indirect analysis/diagnosis of human inherited disease. This compilation is intended to serve as a guide to current molecular genetic approaches in diagnostic medicine.

DNA, Recombinant↗
PubMed1989

Diagnosis of genetic disease using recombinant DNA. Second edition.

Recombinant DNA methodology has greatly increased our knowledge of the molecular pathology of the human genome at the same time as providing the means to diagnose inherited disease at the DNA level. Direct detection and analysis of a range of genetic defects are now possible using cloned gene or oligonucleotide probes or by direct sequencing of the disease gene(s). In addition, the use of restriction fragment length polymorphism (RFLPs) within and around these genes as indirect genetic markers has now potentiated the tracking of disease alleles in affected pedigrees in cases where direct analysis was not feasible. RFLPs associated with linked anonymous segments may also be used not only to diagnose hitherto undetectable disease states, but also for chromosomal localization of the loci responsible. We present here an up-to date list of reports describing both the direct and the indirect analysis/diagnosis of human inherited disease, which is intended to serve as a guide to current molecular genetic approaches in diagnostic medicine.

DNA, Recombinant↗
PubMed1980

Cytoplasmic processing of myosin heavy chain messenger RNA: evidence provided by using a recombinant DNA plasmid.

A recombinant DNA plasmid, designated pMHC25, has been constructed that contains structural gene sequences for rat skeletal muscle myosin heavy chain (MHC). The identity of the MHC sequence insert in pMHC25 was determined by muscle-tissue specificity, inhibition of MHC protein synthesis in vitro by hybrid-arrested translation, purification of mRNA that directs the synthesis of MHC protein in vitro, and hybridization to a 33S cytoplasmic mRNA found only in differentiated muscle cells. pMHC25-DNA-excess filter hybridizations were used to show that more than 90% of the newly synthesized MHC mRNA that appears in the cytoplasm of differentiated L6E9 myotubes contains a long 3' poly(A) tail. In contrast, 90% of the MHC mRNA that accumulates in the cytoplasm of these same cells during myogenic differentiation lacks this long 3' poly(A) tail. These results suggest the occurrence of a posttranscriptional event in differentiated L6E9 myotubes that involves the cytoplasmic processing of poly(A)+ MHC mRNA to poly(A)- or poly(A)-short MHC mRNA.

Animals↗
PubMed1993

Introduction to recombinant-DNA technology.

Recombinant-DNA technology is now commonly used in virtually every aspect of the biological sciences. The purpose of this brief exposition is to provide an outline of the approaches used to identify genes, to isolate the gene of interest, to amplify the gene if necessary, and to clone genes. A short introduction to the principles of separating very large genes is provided, along with a description of an approach to propagating and cloning these large genes.

Animals↗
PubMed1983

The recombinant DNA technology.

The recombinant DNA technology or DNA cloning permits the isolation, amplification, and precise manipulation of specific DNA fragments. This is generally accomplished by linking or recombining the desired DNA fragment with a DNA molecule, termed the vector, which is capable of directing the replication of itself in a suitable host cell and any DNA segment covalently attached to it. Using this and associated technologies, it is possible to produce large amounts of specific proteins and to modify cell types by introducing the genes for proteins that are otherwise absent. Moreover, it is now possible to construct variants of naturally-occurring proteins with improved biological or physical properties.

Animals↗
PubMed1982

Analysis of myogenesis with recombinant DNA techniques.

Recombinant phages containing the rat skeletal muscle alpha-actin gene and the cytoplasmic beta-actin gene were isolated and the structure of these genes was determined. Both genes contain a large intron in the 5' untranslated region and smaller introns at codons 41, 267 and 327. In addition, the alpha-actin contains introns at codons 150 and 204 not present in the beta-actin gene, whereas the beta-actin gene contains an intron at codon 121. The evolutionary aspects of these findings are discussed. Active genes are organized in chromatin in a conformation which renders them preferentially sensitive to digestion with nucleolytic enzymes. The DNAase I sensitivity of genes programmed to be expressed during myogenesis was tested in a cloned cell population of a myogenic cell line. It was found that these genes are not preferentially sensitive to DNAase I in the chromatin of proliferating mononucleated cells. They become DNAase I sensitive during terminal differentiation.

Actins↗
PubMed1998

A broadening view of recombinational DNA repair in bacteria.

Recombinational DNA repair is both the most complex and least understood of DNA repair pathways. In bacterial cells grown under normal laboratory conditions (without a DNA damaging treatment other than an aerobic environment), a substantial number (10-50%) of the replication forks originating at oriC encounter a DNA lesion or strand break. When this occurs, repair is mediated by an elaborate set of recombinational DNA repair pathways which encompass most of the enzymes involved in DNA metabolism. Four steps are discussed: (i) The replication fork stalls and/or collapses. (ii) Recombination enzymes are recruited to the location of the lesion, and function with nearly perfect efficiency and fidelity. (iii) Additional enzymatic systems, including the phiX174-type primosome (or repair primosome), then function in the origin-independent reassembly of the replication fork. (iv) Frequent recombination associated with recombinational DNA repair leads to the formation of dimeric chromosomes, which are monomerized by the XerCD site-specific recombination system.

Bacteria↗
PubMed1990

[DNA hybridization and identification of Leptospira serogroup icterohaemorrhagiae with Leptospira recombinant DNA probes].

Two recombinant DNA probes-PLIpso1 (15kb) and PLIEc34 (4kb), derived from Leptospira serovaricterohaemorrhagiae genomic libraries, were applied for the hybridization and identification of 13 strains of Leptospira in serogroup icterohaemorrhagiae. Difference in hybridization signal in combination with the banding pattern provide a good way for identification of serovars and strains. The recombinant DNA, specific to L. serogroup icterohaemorrhagiae, hybridized with a limited number of DNA fragments which had been digested by several restriction endonucleases. The less complex banding pattern and higher sensitivity facilitate characterization of various serovars and strains in serogroup icterohaemorrhagiae. In general the DNA patterns recognized by both probes have extensive genomic homology in same serovar (but still can distinguish strains in same serovar by some unique bands) and apparent difference in various serovars (especially serovars naam, nanxi and honghe). The results indicated that Southern blotting with recombinant DNA probe might provide tools for identification, characterization and analysis of leptospira.

Blotting, Southern↗
PubMed1982

Mammalian cells contain a DNA-recombining activity that can be dissociated from topoisomerase activity.

The role of the topoisomerase enzyme in DNA recombination was investigated by extracting chromosomal deoxyribonucleoproteins from a variety of cultured mammalian cells and assaying for the formation of recombinant DNA structures. Although each of the crude deoxyribonucleoprotein preparations contained topoisomerase activity, they did not all contain DNA-recombining activity. A distinct, perhaps novel, enzyme may therefore promote DNA recombination in these cell-free systems.

Animals↗
PubMed1989

A versatile non-radioactive technique for restriction analysis of recombinant DNA.

Restriction analysis of recombinant DNA is most frequently performed according to Smith and Birnstiel by labeling 5'-termini with 32P, followed by partial digestion, separation, and autoradiographic detection of labeled fragments. We describe a rapid, non-radioactive technique for restriction analysis of recombinant DNA which combines Southern blotting of partial restriction digests and hybridization with a vector-specific probe labeled with the steroid-hapten Digoxigenin for immunological detection. This technique has several advantages compared to conventional methods. Labeling with 32P is not necessary and as the labeled DNA-fragment used as probe is vector-specific, it can be applied for numerous constructs using the particular cloning vector (e.g.pBR322). Furthermore, the probe can be stored for several months and can be reused many times.

Blotting, Southern↗
PubMed1978

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.

Chromosome Mapping↗
PubMed1984

Novel identification of differences in the kinetoplast DNA of Leishmania isolates by recombinant DNA techniques and in situ hybridisation.

Kinetoplast DNA (kDNA) was extracted from marker isolates of three 'Old World' cutaneous leishmania, L. tropica, L. aethiopica and L. major. Restriction endonuclease digestion followed by electrophoresis showed that each isolate produced a unique pattern of DNA fragments. The kDNA of each isolate was hybridised to Southern blots of digests of all three kDNAs. This showed that the kDNA of each isolate contained sequences unique to that isolate as well as sequences common to all three isolates. The kDNA sequences of L. tropica and L. aethiopica were more closely related than either were to those of L. major. kDNA of each isolate was cloned and plasmids selected which contained a fragment of DNA unique to the isolate from which the kDNA originated. Whole kDNA was hybridised to organisms fixed on a microscope slide. In each case the kDNA hybridised strongly to the kinetoplast of the organism from which the DNA had been extracted. There was a small amount of cross-hybridisation between L. tropica and L. aethiopica confirming the result of the Southern blot hybridisation. The feasibility of a method of isolate identification using recombinant DNA probes containing sequences unique to a particular isolate or group of isolates is discussed.

Animals↗
PubMed2005

Dual negative regulatory mechanisms of RecX on RecA functions in radiation resistance, DNA recombination and consequent genome instability in Deinococcus radiodurans.

RecA protein plays a central role in homologous recombination and DNA repair. RecX, a gene directly downstream in Escherichia coli and some other bacterial species, down regulates it. However, the precise mechanism of regulation of RecA by RecX is not known. In order to study the function of RecX in the highly radioresistant bacterium Deinococcus radiodurans, null and overexpression strains were constructed. Our data demonstrates that RecX represses radiation resistance, DNA recombination and consequent genome instability in the stationary phase bacteria. Further biochemical analyses reveal that RecX not only down regulates recA transcription, but also directly inhibits RecA activities in vitro. These data suggests a dual negative regulatory control of RecX on RecA functions in D. radiodurans.

Adenosine Triphosphatases↗