Richard A. Jefferson

GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

We have used the Escherichia coli beta‐glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta‐glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta‐glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

Assaying chimeric genes in plants: The GUS gene fusion system

DeJi~eitio, r Ge~e lrlt.~irm Much of tile attention and interest in modern molecular biology is fi~cussed on the regulation of gene expression. Factors influencing or mediating such regulation are often best studied using gene Alsions. Gene fusions can be defined its DNA constructions (perfi3rmed ill vitro or i~e Hvo) that result in the coding sequences from one gene (r@o,ter) being transcribed and/or translated under the direction of the controlling sequences of another gene (cmltrr Gene fusions can be of two general types, with many wtriatiuns within types. Transcriptional fusions are defined as fusions in which all protein coding sequences are derived from the reporter, with none from the cmm,//e~. Thus, although the m R N A produced may consist of sequences from both control/o and re/;o~ter, the protein synthesized will be encoded only by the reporter. Translational fusions, in contrast, are defined as those in which the polypeptide produced is the result of coding information provided by both copraoiler and reporter.

Gene transfer to plants by diverse species of bacteria

Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology. However, the complexity of the patent landscape has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile ‘open source’ platform for plant biotechnology, may lead to new uses of natural bacteria–plant interactions to achieve plant transformation.

ß-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other Gram-negative bacteria.

Summary: A series of transposons are described which contain the gusA gene, encoding β-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.

Tissue- and cell-specific activity of a phenylalanine ammonia-lyase promoter in transgenic plants.

Phenylalanine ammonia‐lyase (PAL) catalyses the first step in the biosynthesis of phenylpropanoids, which form a wide variety of plant secondary products. The transcription of PAL is regulated in response to various factors that induce the accumulation of flavonoids, lignin and compounds thought to be involved in plant defence reactions. The 5′ upstream sequence of a PAL gene from Phaseolus vulgaris was fused to the coding region of the reporter gene encoding beta‐glucuronidase (GUS), and transformed into potato and tobacco plants. Histochemical analysis of GUS expression showed that the PAL promoter was active in specific cell types that accumulated phenylpropanoid derivatives in response to mechanical wounding, and also during normal development of the xylem and flower. In xylem that had undergone secondary thickening, GUS activity occurred in rays of cells thought to be the xylem parenchyma. It was postulated that PAL activity in these cells could provide intermediates for lignin synthesis in xylem vessels that had terminally differentiated.

Transcriptional regulation of a patatin-1 gene in potato

Patatin is an abundant glycoprotein in the tubers of potato plants that has a lipid acyl hydrolase activity. Fusions of the promoter of patatin genes that are highly expressed in tubers with the reporter gene encoding β-glucuronidase (GUS) have shown that patatin transcription has a high degree of tuber specificity. Patatin transcription was also inducible in other organs of transgenic potato by growth on high concentrations of sucrose. Experiments were conducted to define regions of the patatin promoter that confered tuber specific expression and sucrose inducibility. Sequences between -40 and -400 bp and between -400 and -957 bp of the transcriptional start site were able to confer tuber-specific expression on a heterologous truncated promoter. The cell specificity of GUS transcription in the transformants indicated that organ specificity was possibly determined by source-sink relationships of sucrose, or a metabolite of sucrose, in the whole plant.

Endoplasmic reticulum targeting and glycosylation of hybrid proteins in transgenic tobacco.

The correct compartmentation of proteins to the endomembrane system, mitochondria, or chloroplasts requires an amino-terminal signal peptide. The major tuber protein of potato, patatin, has a signal peptide in common with many other plant storage proteins. When the putative signal peptide of patatin was fused to the bacterial reporter protein beta-glucuronidase, the fusion proteins were translocated to the endoplasmic reticulum in planta and in vitro. In addition, translocated beta-glucuronidase was modified by glycosylation, and the signal peptide was correctly processed. In the presence of an inhibitor of glycosylation, tunicamycin, the enzymatically active form of beta-glucuronidase was assembled in the endoplasmic reticulum. This is the first report of targeting a cytoplasmic protein to the endoplasmic reticulum of plants using a signal peptide.

Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro.

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5-terminal 67-nucleotide sequence (omega) derived from the untranslated 5-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega, this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega sequence. These findings extend the utility of omega as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.

A Versatile and Reliable Two-Component System for Tissue-Specific Gene Induction in Arabidopsis

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein

SummaryWe have constructed chimaeric genes consisting of sequences encoding the transit peptide and 4, 16, 24, 53 or 126 amino-terminal residues of the mature chlorophyll a/b binding (Cab) apoprotein fused to the Escherichia coli gene encoding β-glucuronidase (GUS). These genes were introduced into tobacco plants and the fate of the fusion proteins they encode was analysed. Less than 1% of the total activity of fusion proteins containing the transit peptide and 4 (FP4) or 16 (FP16) amino-terminal amino acids of the mature Cab protein was associated with chloroplasts. Moreover, FP4 appears to be unprocessed. This is in striking contrast to fusion proteins containing the transit peptide and 24 (FP24), 53 (FP53) or 126 (FP126) amino-terminal residues of the mature Cab polypeptide. Approximately 98%, 96% or 75%, respectively, of the total activity of these fusion proteins was associated with purified intact chloroplasts, and protease protection experiments showed that of this, approximately 98%, 87% or 50%, respectively, was located within this organelle. Furthermore, both FP24 and FP53 appear to be processed. However, less than 10% of the activity of those fusion proteins translocated into chloroplasts was associated with thylakoid membranes.