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:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The distribution of mRNA with high sequence homology to somatostatin mRNA within the periventricular hypothalamus of rat was assessed using in situ hybridization techniques with synthetic oligodeoxyribonucleotide probes, complementary to the 3' coding region of rat somatostatin mRNA. The probes (22- and 24-mers) were 5'-end labeled using T4 polynucleotide kinase and gamma-32P-ATP. They were used either individually or after ligation with T4 DNA ligase to form a 46-mer. Serial tissue sections (less than 10 microns) were taken from the level of the preoptic/anterior hypothalamus through the paraventricular hypothalamus. In situ hybridizations were conducted at room temperature in hybridization buffer. Neurons immunoreactive with antiserum raised against somatostatin were identified in alternate sections using standard immunocytochemical procedures. The anatomical location of the hybridization signal was determined by autoradiography. Our results show that the peri- and paraventricular hypothalamus is rich in transcripts putatively coding for somatostatin and that these transcripts are co-distributed with neurons immunoreactive with antisomatostatin immunoglobulin.
...
PMID:In situ hybridization of putative somatostatin mRNA within hypothalamus of the rat using synthetic oligonucleotide probes. 286 Jan 16

We have developed a technique for synthesis of single stranded complementary DNA (ss cDNA) using specifically designed phage ssDNA as vector primer. This vector (pPBS27) was constructed by introducing a poly(dT) tail adjacent to the XbaI site of pTZ18R, which can exist either as a plasmid in Escherichia coli or as a ssDNA phage. The pPBS27 phage vector is linearized with XbaI using a restriction-site-directed fragment and used to anneal a mixture of poly(A) + RNA for cDNA synthesis by reverse transcriptase. The RNA is then hydrolysed with NaOH and a poly(dG) tail added to the 3' end of the vector-cDNA with terminal transferase. The linear hybrid ssDNA is then closed by annealing with a 15-mer site-directed fragment oligodeoxynucleotide molecule and ligated with T4 DNA ligase. Almost 10(5) E. coli transformants per microgram of vector primer can be obtained in two days.
...
PMID:Use of a phage vector for rapid synthesis and cloning of single-stranded cDNA. 303 56

The partial self-complementary 24-mer oligodeoxynucleotide d(C-G)5T4(C-G)5 forms a hairpin which can be enzymatically dimerized to a dumbbell structure. The blunt-ended nature of the hairpin is indicated by its ability to inhibit the T4 DNA ligase catalyzed joining of phi X174 HaeIII fragments. The hairpin monomer and dimer (dumbbell) undergo a reversible B to Z transition as shown by ultraviolet, circular dichroism, and 31P NMR spectroscopy. The Z form of the hairpin monomer and dimer is supported by monovalent ions (Na+), divalent ions (Mg2+ but not Mn2+), and dehydrating (ethanol) conditions. The conformational transition of d(C-G)5T4(C-G)5 monomer requires higher ionic or dehydrating conditions than those necessary for the corresponding linear oligomer d(C-G)5. The contribution of the loop (-T4-) of the hairpin to the apparent free energy change for the B to Z conformational transition at the midpoint was calculated to be 3.8 kJ mol-1.
...
PMID:Right- and left-handed (Z) helical conformations of the hairpin d(C-G)5T4(C-G)5 monomer and dimer. 408 87

Six oligodeoxyribonucleotides ranging from 9-mer to 13-mer were synthesized in solution by the phosphotriester approach and enzymatically joined by T4 DNA ligase. The obtained double-stranded DNA (32 b.p.) with protruding 5'-ends corresponding to the recognition sites for restrictases EcoRI and BamHI represents an oligonucleotide template coding for the modified amino acid sequence 4-10 of the adrenocorticotropic hormone, [Pro8,Gly9,Pro10]ACTH-(4-10).
...
PMID:[Chemico-enzymatic synthesis of an oligonucleotide template for an analog of ACTH (4-10) fragment]. 609 15

Adducts of O6-alkylguanine in DNA that are induced by cytotoxic, carcinogenic or mutagenic alkylating agents can be removed by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Human tumor cell lines that do not express this enzyme (Mer-) are hypersensitive to the effects of such alkylating agents, although the molecular basis of MGMT gene suppression is not yet understood. Previous studies suggested that Mer- cells deficient in this enzyme lack neither the gene nor the trans-acting factors necessary for normal transcription. Methylation of CpG dinucleotides is an attractive mechanism to account for suppression of the MGMT gene; however, there have been reports of both direct and inverse correlations between methylation and MGMT expression. We previously demonstrated an inverse correlation between methylation at a single SmaI site in the human MGMT promoter and gene expression. To substantiate this observation, we examined additional CpGs in the promoters of three Mer+ and three Mer- cell lines, using rare methylation-sensitive restriction sites, and then sought to identify the region where methylation correlated with gene expression. Six CpGs in the region from -245 bp to +225 bp (relative to the transcription start site) were completely unmethylated in all Mer+ cells, whereas in Mer- cells were at least partially methylated. The methylation status of CpGs further upstream did not correlate with MGMT expression. We conclude, therefore, that the association between CpG methylation and suppressed MGMT gene activity extends to sites other than SmaI but is limited to a core region of the promoter.
...
PMID:Localization of methylation sites in the human O6-methylguanine-DNA methyltransferase promoter: correlation with gene suppression. 778 59

We have measured the fluorescence of the DNA repair enzyme endonuclease III to discover perturbation to its tryptophans by undamaged DNA and AP (apyrimidinic or apurinic) DNA and to estimate binding affinity for intact and AP DNAs. Endonuclease III has two tryptophans, Trp132 in a helix-hairpin-helix region of possible flexibility near the active site for AP lyase activity and Trp178 in the domain containing the iron-sulfur center of endonuclease III; Trp132 is the more solvent-accessible tryptophan [Kuo, C.-F., McRee, D. E., Fisher, C. L., O'Handley, S. F., & Cunningham, R. P. (1992) Science 258, 434-440]. The fluorescence emission peak wavelength near 350 nm (excitation at 290 nm) indicated an exposure of the fluorescing tryptophans to a polar environment. Quenching of tryptophan fluorescence by iodide demonstrated that there are indeed two tryptophans which are differently accessible to anionic quencher. Significant (approximately 60%) fluorescence quenching occurred when endonuclease III was titrated with high molecular weight duplex undamaged poly(dAdT). The apparent second-order nonspecific binding constant to poly(dAdT) was 4 x 10(7) M-1, and there were approximately 12 base pairs per endonuclease III binding site for binding to poly(dAdT). This nonspecific binding to duplex DNA had ionic character, and there was no fluorescence quenching brought on by single-stranded DNA. A comparison between fluorescence quenching titrations of high molecular weight duplex DNA and undamaged duplex 19-mer oligonucleotide showed that the binding constant to the high molecular weight DNA was approximately 400-fold larger than to the undamaged 19-mer.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Endonuclease III interactions with DNA substrates. 2. The DNA repair enzyme endonuclease III binds differently to intact DNA and to apyrimidinic/apurinic DNA substrates as shown by tryptophan fluorescence quenching. 787 34

Approx. 20% of human tumor cell lines (termed Mer-) are deficient in the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT; E.C.2.1.1.63). Such cells possess the MGMT gene and promoter sequences but have virtually no mRNA or protein. Cytosine methylation of gene sequences has been proposed as a mechanism by which MGMT could be suppressed in Mer- cells; however, the experimental evidence does not uniformly support this idea. We therefore investigated the effect of in vitro methylation of the MGMT promoter in a reporter gene construct transfected into cultured human cells. DNA methylation by HpaII or HhaI methylases suppressed the activity of the promoter, although the effect was not absolute. The occurrence of partial intracellular demethylation of promoter sequences may account for the incomplete inhibition of transcription. A model that attempts to reconcile the opposing views on the role of cytosine methylation in MGMT gene expression is presented.
...
PMID:In vitro methylation of the human O6-methylguanine-DNA methyltransferase promoter reduces transcription. 811 Aug 28

Elucidation of the structure of DNA by Watson and Crick [Nature 171 (1953) 737-738] has led to many crucial molecular experiments, including studies on DNA replication, transcription, physical mapping, and most recently to serious attempts directed toward the sequencing of large genomes [Watson, Science 248 (1990) 44-49]. I am totally convinced of the great importance of the Human Genome Project, and toward achieving this goal I strongly favor 'top-down' approaches consisting of the physical mapping and preparation of contiguous 50-100-kb fragments directly from the genome, followed by their automated sequencing based on the rapid assembly of primers by hexamer ligation together with primer walking. Our 'top-down' procedures totally avoids conventional cloning, subcloning and random sequencing, which are the elements of the present 'bottom-up' procedures. Fragments of 50-100 kb are prepared in sufficient quantities either by in vitro excision with rare-cutting restriction systems (including Achilles' heel cleavage [AC] or the RecA-AC procedures of Koob et al. [Nucleic Acids Res. 20 (1992) 5831-5836]) or by in vivo excision and amplification using the yeast FRT/Flp system or the phage lambda att/Int system. Such fragments, when derived directly from the Escherichia coli genome, are arranged in consecutive order, so that 50 specially constructed strains of E. coli would supply 50 end-to-end arranged approx. 100-kb fragments, which will cover the entire approx. 5-Mb E. coli genome. For the 150-Mb Drosophila melanogaster genome, 1500 of such consecutive 100-kb fragments (supplied by 1500 strains) are required to cover the entire genome. The fragments will be sequenced by the SPEL-6 method involving hexamer ligation [Szybalski, Gene 90 (1990) 177-178; Fresenius J. Anal. Chem. 4 (1992) 343] and primer walking. The 18-mer primers are synthesized in only a few minutes from three contiguous hexamers annealed to the DNA strand to be sequenced when using an over 100-fold excess of hexamers and T4 DNA ligase at room temperature, preferably in the presence of the single-strand-binding (SSB) protein of E. coli. These 18-nt primers are immediately extended by the DNA polymerase, Sequenase 2.0, in the dideoxy sequencing reaction. Very high quality sequencing ladders are obtained for single-stranded DNA or denatured double-stranded approx. 50-kb fragments, as exemplified by phage lambda DNA.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:From the double-helix to novel approaches to the sequencing of large genomes. 827 70

Chloroethylnitrosoureas (CENUs) alkylate DNA at specific sites and inhibit DNA replication in tumor cells. O6-Alkylguanine moieties resulting from alkylation of guanine bases are thought to be one of most lethal adducts in living cells. Effectiveness of CENUs is known to relate well with an enzymic activity of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), which recognizes and removes O6-alkylguanine. To improve therapeutic results of CENUs, we have measured MGMT activity of human brain tumors and studied the relationship between MGMT activity and clinical responsiveness to I-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). Thirty-seven patients with brain tumors were entered into the study. The neoplasms included gliomas, non-glial tumors, and brain metastases. The MGMT activity of gliomas was significantly lower than that of non-glial tumors and brain metastases. No significant difference in the enzyme activity was noted between low- and high-grade gliomas. Out of the 22 gliomas 5 tumors indicated a value below 60 fmol/mg, suggestive of a methyl excision repair minus (Mer-) tumor. Two out of 3 evaluable patients with a Mer- tumor responded well to post-operative ACNU adjuvant chemotherapy. Our results suggest that brain tumors include a certain percentage of Mer- phenotype tumors, and that CENUs such as ACNU should be applied selectively on tumors with a low MGMT activity in order to increase the therapeutic effectiveness.
...
PMID:Influence of O6-methylguanine-DNA methyltransferase activity on chloroethylnitrosourea chemotherapy in brain tumors. 839 42

A method is described for the ordered ligation of hexanucleotides (hexamers) in solution to produce unique longer oligonucleotides. To form an 18-mer, for example, six different hexamers are selected that can base pair unambiguously to form a double-stranded complex of indefinite length. In the most efficient arrangement, each hexamer forms three complementary base pairs with two other hexamers, generating complementary chains of contiguous hexamers with strand breaks staggered by three bases. Two adjacent hexamers in one chain contain 5' phosphate groups and the others are unphosphorylated. Both T4 and T7 DNA ligase can ligate the phosphorylated hexamers to their neighbors in such a complex at hexamer concentrations in the 50-100 microM range, producing an 18-mer and leaving three unphosphorylated hexamers. Twenty-nine of 34 complexes that satisfied the requirements for unambiguous ligation generated the desired 18-mers, which could be used directly for cycle sequencing or, after removal of the unreacted hexamers, for polymerase chain reactions (PCR). Comparable ligation reactions also produced 12-, 24-, and 30-mers. With a library of all 4096 possible hexamers, unambiguous ligation has the potential to produce more than 82% of all possible 18-mers and could readily supply the oligonucleotides needed for DNA sequencing by primer walking, for PCR, or for gene synthesis.
...
PMID:Ligation of hexamers on hexamer templates to produce primers for cycle sequencing or the polymerase chain reaction. 857 93


<< Previous 1 2 3 4 Next >>

---