Guido Jach

An improved mRFP1 adds red to bimolecular fluorescence complementation

Protein-protein interactions are fundamental to virtually every aspect of cellular functions. Blue, green and yellow bimolecular fluorescence complementation (BiFC) systems based on GFP and its variants allow the investigation of protein-protein interactions in vivo. We have developed the first red BiFC system based on an improved monomeric red fluorescent protein (mRFP1-Q66T), expanding the range of possible applications for BiFC.

Expression of a Ribosome Inhibiting Protein (RIP) or a Bacterial Chitinase Leads to Fungal Resistance in Transgenic Plants

In order to protect plants against fungal attack, two genes (RIP, ChiA) encoding proteins with in vitro antifungal activity were expressed in transgenic tobacco plants. 1. A barley derived cDNA clone (RIP) encoding a ribosome inhibiting protein. RIP inhibits protein synthesis in fungi by specific RNA N-glycosidase modification of the 28S RNA. 2. A chitinase gene (ChiA) derived from the bacterium Serratia marcescens with the ability to destroy hyphal tips of growing fungi.

Use of fluorescent proteins as reporters.

Fluorescent proteins (FPs) have established themselves as valuable reporter proteins in plant molecular biology. Beside general background information about spectral properties, protein structure and maturation of different commonly used FPs, this chapter provides detailed protocols about cloning of suitable expression cassettes for GFP and DsRED and detection of FPs in Arabidopsis protoplasts and plants transiently or stably expressing these constructs by fluorescence microscopy.

Increased vegetative development and sturdiness of storekeeper-transgenic tobacco

The STOREKEEPER (STK) family of DNA-binding proteins work as transcription factors and the ectopic expression of two stk-like genes from Arabidopsis thaliana, stk01 (At1g61730) and stk03 (At4g00238), in tobacco increased the number of vegetative internodes and promoted plant and leaf size, stem diameter and sturdiness. The development of these plants started with rosette formation while pronounced shoot elongation and flowering was delayed. Moreover, when the STK01 and STK03 proteins were fused to the Herpes Simplex Virus VP16 transcriptional activation domain and expressed in tobacco the vigorous storekeeper-phenotype did not appear indicating that transgenic STK-like proteins in part worked as repressors of tobacco reproductive development. Furthermore, Yeast Two-Hybrid screenings proved that STK01 and STK03 can form homodimers and heterodimers with further members of the STKlike family. Therefore, we assume that interactions between transgenic Arabidopsis STKs and resident tobacco STKs could have contributed to the observed developmental changes in transgenic tobacco. Our findings open up promising applications for overexpression of stk-like genes in crops that benefit from increased sturdiness and vegetative organ development, such as tobacco in molecular farming approaches, biomass-based energy crops and medicinal plants that produce bioactive compounds in leaves.

Focus on Fluorescent Proteins

ABSTRACT: A number of fluorescent proteins have been discovered in marine organisms with the green-fluorescent protein (GFP) from Aequorea victoria representing the first member of this family being isolated and well characterized. These polypeptides show marked differences in their spectral properties. Today, blue-, yellow-, cyan- and red-light emitting proteins are known in addition to GFP. These natural products possess the unique ability to autocatalytically form a cyclized p -hydroxybenzylidene-imidazolidinone structure acting as a light emitting chromophore in the presence of the suitable environment provided by s-can 3D-structure of the proteins. Fluorescence actually represents the biological activity of these protein. In fact, this intrinsic protein property allows for their non-invasive non-destructive detection in living cells, an ability that has caused enormous interest on all areas of molecular biology employing reporter proteins for gene expression and protein localization studies. Today, GFP is widely used and well accepted as valuable reporter protein. However, during the last decade a number of GFP mutants were described showing altered spectral properties and/or improved solubility upon expression in heterologous systems. In addition, numerous other naturally occurring fluorescent proteins were described such as the red-fluorescent protein from Discosoma sp. (DsRED). A comprehensive description of the available fluorescent proteins is given including the spectral properties, amino-acid sequence alignments, comparisons of the secondary and tertiary structures of the proteins. The mechanisms for this self-catalyzed amino-acid modifications leading to the chromophore formation are best characterized for the GFP although some work was carried out on other proteins as well. This review describes the current knowledge about maturation and (possible) oligomerization of the proteins.