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

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

A characteristic of alphaherpesviruses, including pseudorabies virus (PRV), is that the acute phase of the disease is followed by lifelong latency. Latently infected animals are asymptomatic but can transmit reactivated virus. Corticosteroid administration, tissue explanation, blot- and in situ hybridizations have been used to demonstrate the presence of latent PRV infections. The use of blot hybridization as a convenient method for defining the incidence of PRV infections in swine herds has been hampered by the detection limit of this method. The objective of this study was to increase this sensitivity of blot hybridization by polymerase chain reaction (PCR) amplification of target sequences. Two sets of 20-mer primers were synthesized and used to amplify gX and gII glycoprotein gene sequences in two different strains of PRV. The specificity of the amplification was verified by Southern blot hybridization and restriction endonuclease analysis of the amplified fragments. Amplification of target sequences by PRC increased their detection limit by a factor of at least 10(5). Porcine ganglion samples, in which latency had been demonstrated by in vitro explanation, were analyzed by PCR together with positive and negative controls. Duplicate slot blot analyses of a portion of the amplified products were used to demonstrate latency in seven of eight samples. It was concluded that blot hybridization of PCR amplified DNA appears to be both a sensitive and convenient method for the detection of PRV induced latency.
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PMID:Polymerase chain reaction amplification of pseudorabies virus DNA from acutely and latently infected cells. 217 26

In restriction enzyme digested Xenopus laevis erythrocyte chromatin, the 5S oocyte genes were specifically labeled with colloidal gold so they could be identified in an electron microscopic study. Nuclei were digested first with HindIII restriction endonuclease followed by T7 exonuclease to leave single-stranded 3' tails on the fragments. A biotinylated 15-mer oligonucleotide complementary to the tail on the 3' end of the 5S gene was annealed to the chromatin, and streptavidin complexed colloidal gold was bound to it. In a spread for electron microscopy the labeled fragments were readily recognized.
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PMID:Gene-specific labeling of chromatin for electron microscopy. 247 79

Under certain circumstances sequence-specific inhibition of gene expression may be achieved in intact cells using exogenous anti-sense oligodeoxynucleotides. The efficacy of this approach to investigating gene function is limited in part by the rapid serum nuclease mediated degradation of oligodeoxynucleotides in culture media. In order to determine the relative contributions of 3'-exonuclease, 5'-exonuclease and endonuclease activity in fetal calf serum to oligodeoxynucleotide destruction, we have tested chimeric N-ras anti-sense sequence molecules protected against exonuclease attack with terminal methylphosphonate diester linkages. An 18-mer with two methylphosphonate diester linkages at the 3'-terminus, a 20-mer with two methylphosphonate diester groups at both ends, and the 16-mer 3'-methylphosphonate monoester components of their respective piperidine hydrolysates were totally resistant to venom phosphodiesterase, whereas the 16-mer 3'-hydroxyl components of the hydrolysates were rapidly degraded. Both the chimeric oligodeoxynucleotides and 3'-methylphosphonate monoesters were considerably more stable than normal 3'-hydroxyl oligodeoxynucleotides at 37 degrees C in McCoy's 5A medium containing 15% heat inactivated fetal calf serum. Typically 20-30% of the former (initial concentration 10-100 microM) remained intact at 20 h as compared to the latter which were 88-100% degraded in 4 h and undetectable at 20 h. We conclude that a 3'-phosphodiesterase activity is a predominant nuclease responsible for oligodeoxynucleotide degradation by fetal calf serum, and that for cell culture studies, significant protection of oligodeoxynucleotides may be achieved by incorporating 3'-terminal methylphosphonate diester or even monoester end groups.
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PMID:Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups. 255 58

A 12-mer oligodeoxynucleotide containing 10 methylphosphonate bonds and 1 phosphodiester bond was shown to bind specifically to the restriction endonuclease fragment containing complementary DNA in a Southern blot. This 12-mer as well as 14-mer oligodeoxynucleotides containing 3 methylphosphonate and 10 phosphodiester bonds was used to examine the effect of reduced charge on the thermodynamics of binding to complementary DNA or complementary oligodeoxynucleotides with additional nucleotides overlapping both the 3' and 5' ends. The 14-mer oligodeoxynucleotides were synthesized with one methylphosphonamidite (A, C, G, or T). Melting profiles were examined by spectrophotometry for the 14-mers and by a gel-shift assay for the 12-mer. Nearest-neighbor free energy values were compiled for predicting concentration-dependent melting temperatures for all oligodeoxynucleotide hybridizations, including those involving adjacent dG residues. The free energy contribution to duplex formation from the dangling ends was about 1 kcal/mol. The free energy decrement due to introduction of each methylphosphonate linkage was -0.75 kcal/mol in high salt independent of the methylphosphonamidite used for synthesis of the oligodeoxynucleotide. However, the change in charge per nearest-neighbor base pair decreased from 0.26 to 0.0 when the nearest-neighbor base pair contained one methylphosphonate. Thus at very low salt, methylphosphonate-substituted oligodeoxynucleotides form more stable hybrids than analogous phosphodiester sequences. The 12-mer with 10 methylphosphonate bonds outcompetes the analogous phosphodiester 12-mer below 0.01 M NaCl. The temperature of 50% dissociation of bound oligodeoxynucleotide after being washed for 30 min was measured with a dot-blot assay. These results, together with the thermodynamic results, indicate that the substitution of methylphosphonate linkages at high salt only affects the reverse rate constant.
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PMID:Effect of ionic strength on the hybridization of oligodeoxynucleotides with reduced charge due to methylphosphonate linkages to unmodified oligodeoxynucleotides containing the complementary sequence. 271 56

Three tandem repeats of a 13-mer in the AT-rich region are essential to the unique replication origin of E. coli and of remotely related Enterobacteriaceae. These iterated sequences are identified by deletion analysis and sensitivities to endonucleases as the site for initial duplex opening by the initiator dnaA protein. This "open complex" requires ATP and 38 degrees C for optimum formation and stability. The subsequent dnaC-dependent entry of dnaB helicase to form a "prepriming complex" stabilizes the open structure, blocks cleavages by a restriction endonuclease in the 13-mer region, and broadens the endonuclease cutting pattern. We propose that dnaA protein recognizes and successively opens the 13-mer sequences, thereby guiding the entry of dnaB helicase into the duplex preparatory to priming of replication.
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PMID:Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. 283 Sep 93

The normal M2 variant of alpha 1-antitrypsin (alpha 1AT) was cloned from a genomic DNA library of an individual homozygous for this allele. Sequencing of all coding exons of the M2 gene revealed it was identical to the common M1(Val213) gene except for two bases (M1(Val213) CGT Arg101, M2 CAT His101; M1(Val213) GAA Glu376 M2 GAC Asp376). Analysis of the sequence of the M1(Val213) and M2 genes around residue 101 revealed the M1 Arg101----M2 His101 caused a loss of the cutting site for the restriction endonuclease RsaI. Using this enzyme, as well as 19-mer oligonucleotides probes centered at residues 101 and 376, evaluation of genomic DNA from 22 M1 alleles and 14 M2 alleles revealed that residue 101 was Arg in all M1 alleles and His in all M2 alleles, while residue 376 was Glu in all M1 alleles and Asp in all M2 alleles. Despite the differences in sequence at two amino acids, the M1(Val213) and M2 proteins function similarly as assessed by quantification of the association rate constant of each for their natural substrate neutrophil elastase. In the context that there are two mutations separating the M1(Val213) and M2 alleles, it is likely that there is another alpha 1AT variant that was an intermediate in the evolution of these genes.
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PMID:Characterization of the gene and protein of the common alpha 1-antitrypsin normal M2 allele. 290 Dec 26

Interaction of the restriction endonuclease BamHI with a series of synthetic oligodeoxynucleotides containing the restriction site has been studied. The enzyme is shown to specifically cut the BamHI site in hexanucleotide (I) and in non-selfcomplementary deca- and octanucleotides (II)-(IV). The data obtained led to the conclusion that BamHI reacts with duplex structures, while playing an important role in their stabilization. In 14-mer (V) BamHI cuts a non-standard half-site GAA to yield the 5'-terminal tri- (rather than hepta-) nucleotide. Hypothetical mechanisms of the process are discussed basing on conception of the role of higher DNA structures in the interaction with restriction endonucleases.
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PMID:[Characteristics of the interaction of restriction endonuclease BamHI with oligodeoxynucleotides]. 301 53

Species-specific oligonucleotide probes have been constructed for the filarial parasites Brugia malayi and Brugia pahangi. Both parasites contain a 322 base pair repeated DNA sequence that is cleaved once by the restriction endonuclease HhaI. A consensus repeat sequence was determined from the DNA sequence of 15 cloned isolates of each species. Although the two repeats have an average homology of 89%, half the differences are clustered in a region of 66 nucleotides that has a homology of only 72%. Within this region, two probes, a 29-mer that is B. malayi specific and a 21-mer that is B. pahangi specific, were constructed. The sequence of both probes was chosen to obtain the maximum difference between the consensus sequences of the two species. The probes were also selected to be GC rich to increase their stability as a DNA hybrid. In a filter hybridization assay, the B. malayi probe has a 500-fold preference for B. malayi DNA versus B. pahangi DNA and a sensitivity of 200 pg. The B. pahangi probe has similar specificity and sensitivity for B. pahangi DNA. A rapid lysis procedure allows the probes to detect 1-2 third stage larvae of either B. malayi or B. pahangi in a filter hybridization assay.
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PMID:Species-specific oligonucleotide probes for the identification of human filarial parasites. 336 34

Decanucleotide (Ap)6GpTpUpC and undecanucleotide GpApUpCpCp (Up)5U have been synthesised. They constitute 5'- and 3'-parts of a 21-mer which imitates T psi C-arm of yeast tRNA(Val1) and is a potential substrate for m1A-methylases and pseudouridine synthetase. The oligonucleotide blocks, synthesised enzymatically by means of ribonucleases of various substrate specificity and polynucleotide phosphorylases (TpUpC, ApUpCpC, pGpTpUpC, GpApUpCpC) or obtained by hydrolysis of poly(U) and poly(A) with Serratia marcescens endonuclease (hexauridilate and hexaadenilate), were joined by T4 RNA ligase.
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PMID:[Model substrates of enzymes modifying ribonucleic acids. Synthesis of deca- and undecanucleotid-fragments of 21-member oligoribonucleotide simulating T psi C-branch of yeast valine tRNA]. 344 71

Competitive hybridization was used to detect the deletion of chromosomal DNA accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). The method was also used to screen a partial plasmid library of chromosomal HindIII fragments from the MRSA strain. Eight recombinant plasmid clones were identified as containing DNA included in the deletion. These clones were used as probes to screen a phage library of the total DNA of the same MRSA strain, resulting in the isolation of overlapping recombinant phage clones carrying 24 kb of the deleted DNA. Two of the cloned HindIII fragments were associated closely with methicillin resistance, as shown by probing DNA from an independent methicillin-sensitive/resistant transduced strain pair and from two MRSA strains following growth in the presence of high concentrations of methicillin. The endonuclease map of the cloned DNA indicates the presence of four copies of a direct repeat less than 1 kb in size. The map is also consistent with the presence in the chromosome of sequences for mercury resistance (mer A mer B) and for tetracycline-resistance plasmid pT181.
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PMID:The cloning of chromosomal DNA associated with methicillin and other resistances in Staphylococcus aureus. 366 2


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