A New Vector Set for GAL4-Dependent Transactivation Assay in Plants.

Abstract

To study transcriptional regulators, an in vivo functional assay is indispensable. Here we report a new convenient vector set for a transactivation assay in plants. The system consists of a luciferase reporter controlled by a synthetic promoter with GAL4-binding sites and an effector to express any fusion protein with the GAL4 DNA binding domain. Co-transfection of the two plasmids causes transactivation of the reporter by the expressed GAL4-effector fusion if the effector exhibits transcriptional activation activity. A multicloning site was introduced into the effector vector which should facilitate the construction of GAL4 -effector fusions . In tobacco transient assay by microprojectile bombardment, a 27-fold transactivation for the GAL4-B17 fusion as an effector was demonstrated with this system.

Characteristics of the Nuclear Form of theArabidopsisTranscription Factor AtbZIP60 during the Endoplasmic Reticulum Stress Response

“…For transactivation assay, plasmids yy64 and yy96 were used as effector and reporter, respectively. 24) Each cDNA fragment of AtbZIP60 was amplified by PCR using following primers:…”

CIP4, a New COP1 Target, Is a Nucleus-Localized Positive Regulator of Arabidopsis Photomorphogenesis

“…The fact that antisense CIP4 only partially suppressed the cop1 mutations (Table 1) suggests the presence of a downstream pathway that bypasses CIP4. Among the identified downstream targets of COP1 (HY5 and CIP7), only HY5 is known to be involved in the regulation of hypocotyl elongation (Koornneef et al, 1980;Yamamoto et al, 1998). Therefore, HY5 might be involved in the predicted bypass pathway that acts in conjunction with CIP4.…”

“…Therefore, CIP4 could be a transcriptional coactivator that does not recognize DNA directly but rather works together with another specific DNA binding factor. This would put CIP4 and CIP7 in the same class (Yamamoto et al, 1998), whereas HY5 would represent another group of transcription factors that bind promoter elements directly (Ang et al, 1998). Puente et al (1996) reported that combinatorial interactions of two distinct light-responsive promoter elements constitute an autonomous light-responsive determinant in CIP4: A Regulator of Photomorphogenesis 407 the promoter.…”

A comparative analysis of basic helix-loop-helix proteins, AtPTF1 and NtWIN4, with reference to plastid localization

“…For DNA staining, samples were incubated with 1 mg/ml 4Ј,6-diamino-2-phenylindole (DAPI) solution before observation. The GAL4BD-PTF1 effector plasmid was constructed by fusing a cDNA encoding AtPTF1 with the GAL4 DNA binding domain in a yy64 vector, a derivative of pMA560 (Yamamoto and Deng 1998). AtPTF1 was subcloned into the BglII/SalI sites and yy64 vector alone was used as a control.…”

“…AtPTF1 was subcloned into the BglII/SalI sites and yy64 vector alone was used as a control. The reporter plasmid, yy96, contained a luciferase gene placed under control of the GAL4 binding site (Yamamoto and Deng 1998). An internal control plasmid, containing a Renilla luciferase (R-Luciferase) gene placed under control of the 35S-CaMV promoter was used to normalize for differences in bombardment efficiency.…”