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:3.1.31.1 (micrococcal nuclease)
2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chemical methods for the synthesis of short deoxyribooligonucleotides containing methyl and phenylphosphonodiester linkages have been developed. The interaction of two such nonionic dinucleotide analogs, T(pCH3)T and T(pC6H5)T, with several enzymes has been investigated. Because of the phosphonate linkage each dinucleotide exists as a diastereomeric pair as shown by thin layer chromatography and enzymatic studies. Both isomers of each dinucleotide can be phosphorylated by T4-polynucleotide kinase in the presence of [gamma-32P]ATP. Only one of the diastereoisomers of each dinucleotide is slowly hydrolyzed by snake venom phosphodiesterase and acts as an inhibitor of the enzyme-catalyzed hydrolysis of 5'-labeled oligothymidylic acid. Both isomers of each dinucleotide analog are completely resistant to hydrolysis by spleen phosphodiesterase.
...
PMID:Synthesis and enzymatic properties of deoxyribooligonucleotides containing methyl and phenylphosphonate linkages. 22 43

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.
...
PMID:Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA. 78 11

Reactivation of chicken erythrocyte nuclei for DNA replication in Xenopus egg extracts involves two phases of chromatin remodelling: a fast decondensation leading to a small volume increase and chromatin dispersion occurring within a few minutes (termed stage I decondensation), followed by a slower membrane-dependent decondensation and enlargement of up to 40-fold from the initial volume (stage II decondensation). Chromatin decondensation as measured by nuclear swelling and micrococcal nuclease digestion required ATP. We observed a characteristic change in the phosphorylation pattern of erythrocyte proteins upon incubation in egg extract. While histones H5, H2A, and H4 became selectively phosphorylated during decondensation, the phosphorylation of histone H3 and of several nonhistone proteins was prevented. Furthermore, histone H5 was selectively released from erythrocyte nuclei in an energy-dependent reaction. These molecular changes already occurred during stage I decondensation and they persisted during stage II decondensation. DNA replication was confined to nuclei of stage II decondensation which incorporated lamin LIII from the egg extract. These results show that initiation of DNA replication in chicken erythrocytes requires in addition to ATP-dependent chromatin remodelling (stage I), further changes in chromatin structure that correlates with lamin LIII incorporation, and stage II decondensation.
...
PMID:Reactivation of DNA replication in erythrocyte nuclei by Xenopus egg extract involves energy-dependent chromatin decondensation and changes in histone phosphorylation. 139 77

32P-Postlabeling was employed for analysis of DNA adducts produced in mouse skin following topical administration of enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol). Deoxynucleoside 3'-monophosphates were isolated by digestion of epidermal DNA with micrococcal endonuclease and spleen phosphodiesterase and phosphorylated with [gamma-32P]ATP. 32P-Labeled deoxynucleoside 3',5'-bisphosphate adducts to diastereomeric benzo[a]pyrene dihydrodiol epoxides (BPDE) were separated by four-directional thin-layer chromatography on poly(ethylenimine)-cellulose plates using a recently described solvent system [Reddy, A. P., Pruess-Schwartz, D., and Marnett, L. J. (1992) Chem. Res. Toxicol. (preceding paper in this issue)]. When (+)-BP-7,8-diol was topically administered, a major adduct spot was detected that cochromatographed with a standard produced by reaction of 7(S),8(R)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-syn-BPDE] with DNA. The level of this adduct increased in a dose- and time-dependent fashion and was elevated in animals pretreated with beta-naphthoflavone. Relatively small amounts of radioactivity cochromatographed with standards of deoxynucleoside 3',5'-bisphosphate adducts derived from 7(S),8(R)-dihydroxy-9(R),10(S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-anti-BPDE]. Following topical administration of (-)-BP-7,8-diol, a single adduct spot was detected that cochromatographed with a standard of the major deoxyguanosine adduct derived from 7(R),8(S)-dihydroxy-9-(S),10(R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. The stereochemistry of epoxidation of the enantiomers of BP-7,8-diol indicates that cytochrome P-450 catalyzes the terminal activation step of benzo[a]pyrene activation to an ultimate carcinogen in mouse skin, a target organ for its carcinogenic activity.
...
PMID:32P-postlabeling analysis of DNA adduction in mouse skin following topical administration of (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. 158 33

Northern blot analysis of HeLa cell nuclear extract following electrophoresis in nondenaturing gels revealed that a small proportion of U2 small nuclear ribonucleoprotein (snRNP) displays a low mobility, in confirmation of previous reports. This low mobility form of U2 snRNP (termed LMC, for low mobility complex) also formed in vitro when U2 snRNP present in HeLa cytoplasmic S100 was added to a micrococcal nuclease-treated nuclear extract. Of greater experimental value, we found that the LMC also formed when a T7 U2 RNA transcript was assembled into U2 snRNP in a HeLa cytoplasmic S100 system, followed by its incubation in micrococcal nuclease-treated nuclear extract. LMC formation was ATP-dependent and was specific for U2 snRNP since it was not observed with S100-assembled U1 or U4 snRNPs. RNase H cleavage of U2 snRNP in the nuclear extract with an oligonucleotide complementary to nucleotides 28-42 of U2 RNA, as opposed to micrococcal nuclease treatment, rendered the extract competent to form the LMC, indicating that the nuclear factors responsible for LMC formation reside on endogenous U2 snRNP. LMC formation was not competed by excess U2 RNA but was competed by partially purified native U2 snRNP, providing further evidence that the LMC represents an interaction of nuclear factors with already assembled U2 snRNP. LMC formation did not take place on a mutant U2 snRNP lacking the binding site for the two U2-specific proteins, A' and B", nor on mutant U2 snRNPs lacking nucleotides 34-37 or nucleotides 46-49. Further results revealed that nucleotides 35 and 36 of U2 RNA, but not 34 and 37, are required for LMC formation. These experiments demonstrate a nucleotide sequence-specific interaction of U2 snRNP with nuclear factors in the absence of pre-mRNA. Among the U2 RNA nucleotides involved in the formation of this complex are ones previously implicated in base pairing between U2 RNA and the pre-mRNA lariat branch site. These findings are discussed in the context of the possibility that the LMC is on the spliceosome assembly pathway.
...
PMID:The U2 small nuclear ribonucleoprotein particle associates with nuclear factors in a pre-mRNA independent reaction. 165 22

Assembly of nucleosomes on relaxed, covalently closed DNA has been studied in a nuclear extract of Xenopus laevis oocytes. Nucleosomes containing the four histones H3, H4, H2A and H2B but lacking histone H1 are readily assembled on the DNA. The pattern of micrococcal nuclease digestion shows that the nucleosomes assembled in the absence of ATP and Mg (II) are closely packed, with a periodicity of 150 base pairs (bp). In contrast, in the presence of ATP and Mg (II) the spacing of the nucleosomes is 180 bp, similar to that observed for nucleosomes assembled on DNA microinjected into oocyte nuclei. The ATP and Mg (II) requirements for the assembly of correctly spaced nucleosomes are unrelated to the activity of the ATP and Mg (II) dependent DNA topoisomerase II in the extract; addition of specific inhibitors of eukaryotic DNA topoisomerase II has no effect on the spacing of the reconstituted nucleosomes. The ATP requirement in the assembly of correctly spaced nucleosomes can be substituted by adenosine 5'-O-3'-thiotriphosphate (gamma-S-ATP) but not by adenyl-5'-yl imidodiphosphate (AMP-P-(NH)-P).
...
PMID:Assembly of correctly spaced chromatin in a nuclear extract from Xenopus laevis oocytes. 217 Sep 36

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.
...
PMID:PRP18, a protein required for the second reaction in pre-mRNA splicing. 240 39

We have evidence for the formation of a stable preelongation complex during the replication of simian virus 40 (SV40) origin containing DNA (ori+ DNA) in vitro. Preincubation of ori+ DNA with HeLa cytosolic extracts and SV40-encoded large tumor antigen (T antigen) in the absence of deoxynucleoside triphosphates eliminates a lag that normally precedes replication. This effect requires ATP and is inhibited by RNase A; subsequent elongation is inhibited by aphidicolin but not by RNase A. A T antigen and SV40 origin-dependent complex can be isolated by gel-filtration chromatography of preincubation reaction mixtures. In both cases, the products formed by replication after complex formation resemble those formed during in vitro replication reactions described previously. HeLa cytosolic extract was separated into two ammonium sulfate fractions: a 0-40% fraction (AS 40) that shows low levels of DNA synthesis and a 40-65% fraction (AS 65) that is inactive by itself but stimulates synthesis when added to the AS 40 fraction. DNA synthesis by these combined fractions has the same requirements as crude extract, occurs in two stages as described above, and is sensitive to RNase A. Pretreatment of both fractions with micrococcal nuclease eliminated replication activity, whereas the combination of a pretreated fraction (either AS 40 or 65) with an untreated fraction was active. A heat-inactivated (55 degrees C, 5 min) AS 65 fraction restored replication activity to the combination of micrococcal nuclease-treated AS 40 and AS 65 fractions.
...
PMID:Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains. 242 51

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.
...
PMID:o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase. 247 84

Cytoplasmic extracts made from HeLa cells that have been harvested late after infection with vaccinia virus are capable of specifically transcribing templates containing vaccinia virus late-gene promoters. We applied such an extract to a phosphocellulose column and eluted the proteins with a series of buffers containing successively higher concentrations of NaCl. None of three column fractions alone was capable of specific transcription of a late-gene template. However, specific transcriptase activity could be reconstituted by mixing column fractions, with maximal activity seen when all three fractions were present. The activities present in all fractions were heat labile, resistant to micrococcal nuclease, and present only in extracts from vaccinia virus-infected cells. A quantitative complementation assay was used to further purify one factor, named VLTF-1, over subsequent columns of DEAE-cellulose and hydroxylapatite. VLTF-1 was separated from endogenous RNA polymerase, was a late-promoter-specific transcription factor, and had a sedimentation rate consistent with an apparent Mr of 45,000. The RNA polymerase-containing fraction was not only necessary for transcription with a late-promoter template but alone was capable of specifically transcribing a vaccinia virus early-gene promoter. A further difference between early and late gene transcription in this system was in the ability of the ATP analog beta-8-imidoadenosine-5'-triphosphate (AMP-PNP) to substitute for ATP in supporting specific transcription of only the late-promoter template. The system reconstituted from the various fractions retained the ability to produce the novel poly(A) sequence found on the 5' end of vaccinia virus late messages.
...
PMID:Identification of factors specific for transcription of the late class of vaccinia virus genes. 247 68


1 2 3 4 5 6 7 Next >>

---