Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a simple and rapid procedure for cloning and sequencing of DNA fragments separated by gel electrophoresis, using novel hydrophilic gels, Clearose BG, Spreadex, and Poly(NAT), that do not melt at 95 degrees C. For cloning, a band of interest is excised precisely and incubated in an extraction buffer containing 5-10 mM MgCl2 at 70 degrees C for 15-45 min. The eluted DNA is added directly to the plasmid solution. Using a topoisomerase-based ligation system, we were able to transform bacteria with a few picograms of DNA and isolate recombinant clones. For in situ sequencing, the DNA in the gel serves as the template. No treatment before cycle sequencing is necessary for fragments up to 500 bp.
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PMID:Picogram cloning and direct in situ sequencing of DNA from gel pieces. 1168 Jul 11

We recently reported a diurnal and norepinephrine (NE) -induced expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the rat pineal gland and postulated that this MKP-1 expression might impact adrenergic-regulated arylalkylamine-N-acetyltransferase (AA-NAT) activity via modulation of MAPKs. In this study, we investigated the effect of depletion of MKP-1 expression by using doxorubicin, a topoisomerase inhibitor that suppresses the expression of MKP-1 in other cell types and small interfering RNA targeted against Mkp1 in NE-stimulated pinealocytes. We found that both treatments were effective in inhibiting NE induction of MKP-1 expression. Moreover, both treatments also resulted in a prolonged activation of p42/44MAPK and an increase in AA-NAT induction by NE. In contrast, treatment of pinealocytes with PD98059, an inhibitor of MAPK kinase, reduced NE-stimulated AA-NAT activity. Interestingly, suppressing MKP-1 expression had no effect on the time profile of NE-stimulated p38MAPK activation. These results indicate that MKP-1 modulates the profile of AA-NAT activity by selectively shaping the activation profile of p42/44MAPK but not that of p38MAPK.
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PMID:Mitogen-activated protein kinase phosphatase-1 (MKP-1) preferentially dephosphorylates p42/44MAPK but not p38MAPK in rat pinealocytes. 1743 49