Journal:

Article Title: The expression of the KAI1 gene, a tumor metastasis suppressor, is directly activated by p53

doi:

Figure Lengend Snippet: p53 activates the KAI1 gene promoter. (A) A phage clone containing the 5′ upstream region of the KAI1 gene was obtained by screening a lambda phage human genomic library. The PstI-XbaI fragment (2,900 bp) containing the immediate upstream region of the KAI1 gene was subcloned into the CAT reporter plasmid pBLCAT3 in both ordered (KAI-2900) and reverse orientations (KAI-2900R). (B) KAI-2900 (lanes 1, 3, 5, and 7) or KAI-2900R (lanes 2, 4, 6, and 8) were transfected to various human prostate cell lines (ALVA, PC3, PPC-1, and DU145) by using Lipofectin. After 48 hr, cell extracts were prepared and assayed for their CAT activities. (C) The dose-dependent response of the KAI1 promoter to p53 was examined. KAI-2900 was cotransfected with various amount of the p53 expression plasmid or its deletion mutant together with the control plasmid, pGL3, to human prostatic tissue cells, ALVA. After 48 hr, cell extracts were prepared and assayed for the CAT activities. The indicated numbers of the fold induction were calculated as means ± SE of at least three independent experiments. (D) The activation of the KAI1 promoter by p53 was examined in various human prostate cells including PC3, PPC-1, and DU145 as described in C.

Article Snippet: A human placenta genomic library in the EMBL-3 Sp6/T7 lambda phage vector (CLONTECH) was screened by using a 32 P-labeled synthetic 58-mer oligonucleotide, which corresponds to nucleotide number 1–58 of the previously published KAI1 cDNA sequence ( 2 ).

Techniques: Plasmid Preparation, Transfection, Expressing, Mutagenesis, Activation Assay