Identification of a nuclear matrix attachment region like sequence in the last intron of PI3Kgamma.
MARs are not only the structure bases of chromatin higher order structure but also have much biological significance. In this study, the whole sequence of about 100 kb in length from BAC clone of GS1-223D4 (GI: 5931478), in which human PI3Kgamma gene is localized, was analyzed by two online-based computer programs, MARFinder and SMARTest. A strong potential MAR was predicted in the last and largest intron of PI3Kgamma. The predicted 2 kb MAR, we refer to PIMAR, was further analyzed through biochemical methods in vitro and in vivo. The results showed that the PIMAR could be associated with nuclear matrices from HeLa cells both in vitro and in vivo. Further reporter gene analysis showed that in the transient transfection the expression of reporter gene linked with reversed PIMAR was repressed slightly, while in stably integrated state, the luciferase reporter both linked with reversed and orientated PIMAR was enhanced greatly in NIH-3T3 and K-562. These results suggest that the PIMAR maybe has the capacity of shielding integrated heterogeneous gene from chromatin position effect. Through combination of computer program analysis with confirmation by biochemical methods, we identified, for the first time, a 2 kb matrix attachment region like sequence in the last intron of human PI3Kgamma.
Dai
Bingbing
B
Department of Biochemistry and Molecular Biology, Shanghai Second Medical University, Shanghai 2000 25, PR China.
Ying
Lei
L
Cai
Rong
R
Li
Ying
Y
Zhang
Xingqian
X
Lu
Jian
J
Qian
Guanxiang
G
D000818:Animals; D001483:Base Sequence; D001671:Biochemistry / Q000379:methods; D002467:Cell Nucleus / Q000378:metabolism; D002874:Chromosome Mapping; D058544:Class Ib Phosphatidylinositol 3-Kinase; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D007438:Introns; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D020014:K562 Cells; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D015530:Nuclear Matrix / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012984:Software; D014162:Transfection
D017931:DNA Primers; D006657:Histones; D007527:Isoenzymes; D008156:Luciferases; D019869:Phosphatidylinositol 3-Kinases; D058544:Class Ib Phosphatidylinositol 3-Kinase; C546760:PIK3CG protein, human; C546764:Pik3cg protein, mouse