A bi-directional promoter, DP, was cloned by PCR amplification using the genomic DNA of melon as template. Analysis of its cis-acting elements in both directions revealed a series of inducible ...regulatory elements and some enhancer elements. To evaluate its transcriptional activity, DP in both directions was then cloned into vector pBI121 to replace the CaMV 35S promoter. DP in both directions also was inserted downstream of CaMV 35S to investigate whether the double promoter might affect expression of the uidA reporter gene at higher levels. Transient expression in cucumber leaves, stems, and fruits as well as in tobacco leaves and stems showed that DP in both directions drove transcription to much higher levels than did the single promoter CaMV 35S. However, activity of the double promoter was lower than the corresponding activity of the single promoter DP in both directions. These results demonstrate that DP is a natural bi-directional promoter, with much more activity than is found with the CaMV 35S promoter. Furthermore, in cucumber and tobacco, it is not suitable to insert DP in either direction downstream of the CaMV 35S promoter to form a double promoter.
The aim of this work was to design strong transcriptional activators that can be used to regulate plant gene expression. The contribution of different components in a transcription factor and target ...gene system was assayed by measuring transcriptional activation. Each component was optimised to achieve maximal reporter gene expression in transient protoplast transformation assays. The DNA-binding domain of the yeast transcriptional activator GAL4 was studied in the context of fusion proteins with activation domains of the herpes simplex virus protein VP16 or the tomato Myb-like activator THM18. Multimerisation of the activation domain and insertion of a homopolymeric glutamine stretch was used to increase transcription factor potency. Evidence is presented that these modifications can result in even more active transcription factors when they are combined. Finally, it was demonstrated using competition experiments that transcription factors with acidic activation domains can mutually suppress their activation potentials when expressed at high levels.
In the past few years, methods have been developed which allow the introduction of exogenous DNA into the human malaria parasite Plasmodium falciparum. This important technical advance known as ...parasite transfection, provides powerful new tools to study the function of Plasmodium proteins and their roles in biology and disease. Already it has allowed the analysis of promoter function and has been successfully applied to establish the role of particular molecules and/or mutations in the biology of this parasite. This review summarises the current state of the technology and how it has been applied to dissect the function of the P. falciparum genome.
The promoter region flanking the 5' CAZFP1 coding region was isolated from the genomic DNA of Capsicum annuum. To identify the upstream region of the CAZFP1 gene required for promoter activity, a ...series of CAZFP1 promoter deletion derivatives was created. Each deletion construct was analyzed by Agrobacterium-mediated transient transformation in tobacco leaves after infection by Pseudomonas syringae pv. tabaci, or treatment with methyl jasmonate (MeJA), ethylene, abscisic acid (ABA), salicylic acid (SA), cold and wounding.
Pollen tube development Johnson, Mark A; Kost, Benedikt
Methods in molecular biology (Clifton, N.J.),
01/2010, Volume:
655
Journal Article
Pollen tubes grow rapidly in a strictly polarized manner as they transport male reproductive cells through female flower tissues to bring about fertilization. Vegetative pollen tube cells are an ...excellent model system to investigate processes underlying directional cell expansion. In this chapter, we describe materials and methods required for (1) the identification of novel factors essential for polarized cell growth through the isolation and analysis of Arabidopsis mutants with defects in pollen tube growth and (2) the detailed functional characterization of pollen tube proteins based on transient transformation and microscopic analysis of cultured tobacco pollen tubes.
During maize seed development, endosperm cells synthesize large amounts of storage proteins, alpha-, beta-, and gamma-zeins, which accumulate within endoplasmic reticulum (ER)-derived protein bodies. ...The absence of lysine in all zein polypeptides results in an imbalance in the amino acid composition of maize seeds. We modified the maize gamma-zein gene through the introduction of lysine-rich (Pro-Lys)n coding sequences at different sites of the gamma-zein coding sequence. Maize endosperms were transiently transformed by biolistic bombardment with Lys-rich gamma-zein constructs under the control of the 1.7 kb gamma-zein seed-specific promoter and the cauliflower mosaic virus (CaMV) 35S promoter. When (Pro-Lys)n sequences were inserted contiguous to or in substitution of the Pro-Xaa region of the gamma-zein, high levels of protein were observed. In contrast, when (Pro-Lys)n sequences were inserted five residues from the C-terminal, the transcript was present but modified protein was not detected. These results suggest that only an appropriate positioning of Lys-rich inserts leads to the modified molecule displaying correct folding and stability. Subcellular localization analyses and immunoelectron microscopy studies on isolated protein bodies demonstrated that modified gamma-zeins accumulate within these organelles and co-localized with endogenous alpha- and gamma-zeins. The studies reported here show the feasibility of manipulating the gamma-zein gene in order to obtain stable and correctly targeted Lys-rich zeins in maize seeds.
Production and detection of beta glucuronidase (GUS) protein in fruit tissue of papaya (Carica papaya L.) and banana (Musa acuminata L.) using Agrobacterium transient transformation Sunsanee Nacharoen(Kasetsart University. Kamphaeng Saen Campus, Nakhon Pathom (Thailand). Center for Agricultural Biotechnology) E-mail:nongsunsa@gmail.com; Ratree Koohapitakthum(Thailand Science Park, Pathum Thani (Thailand). National Center for Genetic Engineering and Biotechnology. Plant Research Laboratory); Anjana Bhunchoth(Thailand Science Park, Pathum Thani (Thailand). National Center for Genetic Engineering and Biotechnology. Plant Research Laboratory) ...
Warasān wičhai Mō̧.Khō̧. = KKU research journal,
Mar-Apr 2013
Journal Article
The high mobility group (HMG) proteins represent a class of chromosomal non-histone proteins with an assumed influence on transcription. In this context, the effect of the maize HMGa protein on ...reporter gene expression was examined. Transient co-transformation experiments in maize protoplasts with plasmid constructs directing the synthesis of the maize HMGa protein and with a luciferase reporter plasmid demonstrated a stimulatory effect of the HMGa protein on the reporter gene expression. Additional experiments with HMGa deletion constructs indicated that the HMG-Box DNA-binding motif is important for the observed effect, while the acidic carboxy-terminal domain of the HMGa protein appears to be dispensable.
PDF
