caa a22 4500 477119352 CHVBK 20180405111631.0 cr unu---uuuuu 170330e19960301xx s 000 0 eng 10.1007/BF00240055 doi (NATIONALLICENCE)springer-10.1007/BF00240055 Molecular cloning, sequencing and expression analysis of a fatty acid transport gene in rat heart induced by ischemic preconditiong and oxidative stress [Elektronische Daten] [Nilanjana Maulik, Dipak Das] In this study, ischemia and oxidative stress-inducible gene expression in heart was examined by subtractive hybridization technique. Total RNA was isolated from ventricular muscle fragments of normal and oxidative stress-induced hearts. Poly (A)+ RNA was purified followed by the construction of a plasmid cDNA library. This was followed by the subtractive screening of oxidative stress-induced cDNA library. The positive colonies were amplified and the plasmid isolated. An aliquot was subjected to restriction cutting with Bam H1 and EcoRl; the fragments corresponding to cDNA insert were separated by electrophoresis, radiolabeled by random-primed DNA synthesis, and used as probes in standard Northern blotting experiments. An aliquot containing the plasmid from the confirmed positives was then subjected to bidirectional partial DNA sequencing (using M13 and T7/T3α primers) by the chain-extension/chain termination method. These sequences were subjected to a computerized search for homologies against all sequences in the updated worldwide Gen Bank and EMBL sequence databases followed by restriction mapping and reading frame identification. Out of 24 putative positive colonies screened, one clone was matched with > 97% homology with FAT gene that has been implicated in binding or transport of long chain fatty acids. cDNA probe synthesized from this clone identified two major transcripts of 4.8 and 2.9 kb. Additional experiments were then performed where isolated perfused rat hearts were subjected to the following treatments: (1) 5 min ischemia; (2) 10 min ischemia; (3) 20 min ischemia; (4) 5 min ischemia followed by 10 min reperfusion (ischemic preconditioning); and (5) 5 min ischemia followed by 10 min reperfusion, repeated four times (4 × preconditioning). RNAs were extracted from these hearts and hybridized with the FAT cDNA probe. The results indicated that FAT gene was induced by oxidative stress, ischemic preconditioning, but not by ischemia. (Mol Cell Biochem 160/161: 241-247, 1996) Kluwer Academic Publishers, 1996 fatty acid transport protein nationallicence gene expression nationallicence subtractive hybridization nationallicence oxidative stress nationallicence ischemia/reperfusion nationallicence ischemic preconditioning nationallicence Maulik Nilanjana Cardiovascular Division, Department of Surgery, University of Connecticut School of Medicine, CT 06030, Farmington, USA aut Das Dipak Cardiovascular Division, Department of Surgery, University of Connecticut School of Medicine, CT 06030, Farmington, USA aut Molecular and Cellular Biochemistry Kluwer Academic Publishers; http://www.wkap.nl 160-161/1(1996-03-01), 241-247 0300-8177 160-161:1<241 1996 160-161 11010 https://doi.org/10.1007/BF00240055 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00240055 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Maulik Nilanjana Cardiovascular Division, Department of Surgery, University of Connecticut School of Medicine, CT 06030, Farmington, USA aut NATIONALLICENCE 700 1- Das Dipak Cardiovascular Division, Department of Surgery, University of Connecticut School of Medicine, CT 06030, Farmington, USA aut NATIONALLICENCE 773 0- Molecular and Cellular Biochemistry Kluwer Academic Publishers; http://www.wkap.nl 160-161/1(1996-03-01), 241-247 0300-8177 160-161:1<241 1996 160-161 11010 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer