Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.
...
PMID:Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA. 617 25

A general method has been developed for the recovery of any DNA fragment inserted into a cloning vehicle containing a single endonuclease PstI site. Endonuclease PstI sites are regenerated by the addition of one or more deoxyguanosine residues to the 3' termini of the PstI-cleaved vehicle by terminal deoxynucleotidyl transferase. Chain elongation by terminal deoxynucleotidyl transferase is then continued with dITP, dATP or dGTP. A plasmid vehicle, pAO1, containing a single PstI site has been constructed. Insertional (foreign) DNA fragments that were "tailed" with dCTP have been annealed to PstI-cleaved pAO1 that was "tailed" with dGTP. When the annealed fragments were used to transform competent Escherichia coli cells, the single-stranded DNA gaps in the recombinant plasmids were repaired. Plasmids recovered from transformed bacteria could be cleaved by PstI into the insertional DNA with dG:dC tracts and linear pAO1 molecules.
...
PMID:Recovery of DNA fragments inserted by the "tailing" method: regeneration of PstI restriction sites. 626 21

Aleutian disease virus (ADV) of mink is a nondefective parvovirus with a single-stranded DNA genome. We characterized the viral DNA forms found in infected cells prepared by a modified Hirt extraction procedure. Double-stranded DNA molecules corresponding in size to 4.8-kilobase-pair duplex monomers and 9.6-kilobase-pair duplex dimers were identified in agarose gels by blot hybridization to 32P-labeled ADV DNA. A rapidly reannealing ADV duplex monomer was isolated on a preparative scale and physically mapped with a series of restriction endonucleases. The map derived was similar to one derived from double-stranded ADV DNA produced by self-primed synthesis on virion DNA, but differed from restriction endonuclease maps reported for other parvovirus DNAs. The purified duplex monomer could be labeled with [32P]dCTP by nick translation and used as a probe in blot hybridization to detect ADV sequences in DNA from small numbers of infected cells. Additional studies indicated that double-stranded ADV DNA could first be detected at 24 h after infection.
...
PMID:Characterization of the Aleutian disease virus genome and its intracellular forms. 630 Apr 67

Three nonoverlapping segments representing approximately 80% of the 4.8-kilobase pair Aleutian disease virus (ADV-G) duplex genome were molecularly cloned into either bacteriophage M13mp9 (M13bm2 = 0.07 to 0.15 map unit; M13bm1 = 0.15 to 0.54 map unit) or plasmid pUC8 (pBM1 = 0.54 to 0.88 map units). In addition the 0.54- to 0.88-map unit segment of a Danish isolate of ADV (DK ADV) was also cloned into pUC8 (pBM2). The recombinant plasmids pBM1 and pBM2 induced expression of several polypeptides in Escherichia coli JM103 that were specifically recognized by sera from mink infected with ADV. The same three proteins with approximate molecular weights of 55,000, 34,000, and 27,000 were detected both by immune blotting and by immunoprecipitation of [35S]methionine-labeled JM103 (pBM1). None of these proteins were recognized in JM103 or JM103 (pUC8), nor were they detected by sera from normal mink. Purified pBM1 and pBM2 DNA appeared identical in size by gel analysis and contour length measurement, and electron microscopic heteroduplex mapping revealed no visible areas of heterology. However, restriction endonuclease mapping showed that pBM2 was different from pBM1, indicating that this segment of the ADV genome was similar but not identical for two strains of ADV (ADV-G and DK ADV). Furthermore, when cloned DNA from ADV-G was labeled with [32P]dCTP by nick translation, DNA relatedness to several field strains of ADV (Utah I, Pullman, and DK), but not to mink enteritis virus or cellular DNA, was shown by Southern blot hybridization.
...
PMID:Molecular cloning of the Aleutian disease virus genome: expression of Aleutian disease virus antigens by a recombinant plasmid. 631 59

The accuracy with which Escherichia coli DNA polymerase I (Pol I) copies natural DNA in vitro has been determined. When phi X174 viral DNA containing an amber mutation (am3) is primed with a single restriction endonuclease fragment, copied in vitro with Pol I and then expressed in E. coli spheroplasts (Weymout, L. A., and Loeb, L. A. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1924), the reversion frequency of this DNA is greater than that of uncopied DNA. This change in reversion frequency can be increased by selectively increasing the concentration of either dATP or dCTP relative to the other deoxyribonucleotide substrates. DNA sequence analyses of revertants obtained from substrate pool bias experiments demonstrates that the revertants contain the selectively biased nucleotide as an incorrect substitution at position 587 of the am3 codon. We have analyzed the product of the in vitro Pol I reaction using neutral and alkaline sucrose gradients. Fifty per cent of the input phi X174 DNA template molecules are copied past the am3 site. The phenotypic expression of the product (revertant) strand in the spheroplast assay was estimated using a model heteroduplex molecule similar in structure to the product of the reaction and containing a single base mismatch (A:A or A:C) at position 587. Using these data, and by extrapolation from pool bias experiments, we estimate the error rate of Pol I in Mg2+-activated reactions using equimolar concentrations of the four deoxynucleotide substrates is 1/680,000 for an A:C mispair and < 1/6,300,000 for an A:A mispair at position 587 of the am3 codon in phi X174 DNA.
...
PMID:On the fidelity of DNA replication. The accuracy of Escherichia coli DNA polymerase I in copying natural DNA in vitro. 644 43

Strand displacement amplification is an isothermal DNA amplification reaction based on a restriction endonuclease nicking its recognition site and a polymerase extending the nick at its 3' end, displacing the downstream strand. The reaction resembles rolling-circle replication of single-stranded phages and small plasmids. The displaced sense strand serves as target for an antisense reaction and vice versa, resulting in exponential growth and the autocatalytic nature of this in vitro reaction as long as the template is the limiting agent. We describe the optimization of strand displacement amplification for in vitro evolution experiments under serial transfer conditions. The reaction was followed and controlled by use of the fluorescent dye thiazole orange binding to the amplified DNA. We were able to maintain exponential growth conditions with a doubling time of 3.0 min throughout 100 transfers or approximately 350 molecular generations by using an automatic handling device. Homology of in vitro amplification with rolling-circle replication was mirrored by the occurring evolutionary processes. Deletion events most likely caused by a slipped mispairing mechanism as postulated for in vivo replication took place. Under our conditions, the mutation rate was high and a molecular quasi-species formed with a mutant lacking internal hairpin formation ability and thus outgrowing all other species under dGTP/dCTP deficiency.
...
PMID:Strand displacement amplification as an in vitro model for rolling-circle replication: deletion formation and evolution during serial transfer. 805 37

Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcription as well as in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe a novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary epidermolysis bullosa dystrophica associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by EarI endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of alpha 32P-labeled dCTP. The reaction was followed by EarI digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR.
...
PMID:Use of spontaneously mutated human DNA as competitive internal standard for nucleic acid quantification by reverse transcription-polymerase chain reaction (RT-PCR). 874 25

Pancreatic adenocarcinoma involves activation of the Ki-ras oncogene, inactivation of the p53 tumor suppressor gene, and dysregulation of growth factors and perhaps metastasis genes. Ki-ras oncogene point mutations are known to be involved in pancreatic oncogenesis. The p53 tumor suppressor gene product plays a critical role in cell cycle regulation and also functions as a nuclear transcription factor. Point mutations in the p53 gene have been observed in a variety of malignancies. We determined the frequency of p53 protein overexpression and p53 point mutations in the conserved and nonconserved domains in pancreatic cancers as well as the coincidence of Ki-ras mutation in pancreatic ductal adenocarcinoma. Genomic DNA was isolated from 20 frozen pancreatic adenocarcinomas (14 primary, six metastases) along with six specimens of control pancreatic tissue and screened by single-strand conformation polymorphism (SSCP) analysis followed by direct genomic sequencing of SSCP variants. SSCP analysis was accomplished by incorporating 32P-dCTP in 12 separate polymerase chain (PCR) amplifications covering the p53 coding exons 2-11. All mobility shifts on SSCP were subjected to direct genomic sequencing by the modified dideoxy method. Immunoperoxidase (IP) staining was also done with a p53 monoclonal antibody. Ki-ras codon 12 mutational analysis was accomplished by incorporating 32P-dCTP by polymerase chain reaction amplification utilizing mismatched primers, which create a BstN1 restriction endonuclease site spanning codon 12; the products were digested by BstN1. Polyacrylamide gel electrophoresis allowed distinction between wild-type and mutant Ki-ras. p53 mutations were found in 5 of 20 pancreatic cancers (three of 14 primary tumors, two of six metastatic tumors). Point mutations were observed in three of 14 primary tumors, and one of six metastases, while a 2-base pair duplication resulting in a premature stop codon in exon 5 was found in one metastatic tumor. Point mutations were noted in conserved domains (exons 4, 5, 8) and in the nonconserved domain (exon 10). IP staining revealed that eight of 14 of the primary tumors and two of six metastases exhibited moderate to strong nuclear staining (> 30%), while no nuclear staining was evident in the controls. Ki-ras codon 12 mutations were found in 14 of 20 (70%) pancreatic cancers (nine of 14 primary tumors, five of six metastatic tumors) and none of the six controls. Fifty percent of the primary pancreatic tumors demonstrated moderate to strong nuclear staining. Extensive genetic analysis demonstrated mutations in 30% of the pancreatic cancers. One cancer had a nonsense mutation not detected by IP. Seven of 19 (37%) pancreatic cancers exhibited both Ki-ras point mutation and p53 protein overexpression or mutation. Both genetic analysis and IP are required to characterize all p53 mutations in pancreatic cancer. Ki-ras codon 12 mutations and p53 protein overexpression are important steps in pancreatic oncogenesis.
...
PMID:Ki-ras and p53 mutations in pancreatic ductal adenocarcinoma. 892 12

An enzyme that plays an important role in the repair of oxidative DNA damage is the 3'-phosphodiesterase. This activity, which repairs damaged DNA 3'-termini,can be detected using several available biochemical assays. We present a method to detect 3'-phosphodiesterase activity of renatured proteins immobilized in polyacrylamide gels. The model substrate, labeled with [alpha-32P]dCTP, contains 3'-phosphoglycolate termini produced by bleomycin-catalyzed cleavage of the self-complementary alternating copolymer poly(dGdC). The DNA substrate is incorporated into the gel matrix during standard SDS-PAGE. Active 3'-phosphodiesterase enzymes are detected visibly by the loss of radioactivity at a position corresponding to the mobility of the enzyme during SDS-PAGE. Using this procedure, two Escherichia coli 3'-phosphodiesterases, exonuclease III and endonuclease IV, are readily detected in crude cell extracts or as homogeneous purified proteins. Extracts of mutant cells lack activity at the positions of exonuclease III and endonuclease IV but retain activity in the position of a much larger protein (Mr approximately 100 kDa). The identification of this novel 100 kDa E.coli 3'-phosphodiesterase demonstrates the potential value of the activity gel method described here.
...
PMID:In situ activity gel for DNA repair 3'-phosphodiesterase. 910 75

A new method for efficiently labeling and amplifying DNA probes from anonymous samples has been developed. The two/three base recognition endonuclease CviJI* restricts DNA to numerous small fragments primarily 20-60 bp in size. Thermal denaturation of these fragments results in sequence-specific oligonucleotides complementary to their cognate template. Repeated cycles of denaturation, annealing, and extension of such a multiprimed template by a thermostable DNA polymerase results in a significant amplification of the starting material. This method of amplification, referred to as thermal cycle labeling (TCL), appears to generate a large fraction of rearranged and presumably branched products. The inclusion of nucleotide analogs in the TCL reaction generates microgram amounts of haptentagged probe with a detection limit of 25 zmol (2.5 x 10(-20) mol). Reactions containing [alpha-33P]dCTP yield high-specific-activity probes (2.6 x 10(9) cpm/microgram) with reduced radiolytic decay and a useful shelf life of 1 month. CviJI* -generated primers circumvent the need for synthetic oligos while providing microgram amounts of amplified and labeled probes using the described TCL protocol.
...
PMID:Thermal cycle labeling: zeptomole detection sensitivity and microgram probe amplification using CviJl* restriction-generated oligonucleotides. 944 52


<< Previous 1 2 3 Next >>

---