Workshop: Gene and protein function

Workshop 2 : Gene and Protein Function

Chair: Ottoline Leyser, University of York, UK. and Malcolm Bennett, University of Warwick, UK.
email: Ottoline Leyser (hmol1@unix.york.ac.uk) and Malcolm Bennett (bt@dna.bio.warwick.ac.uk)
The main aim of this workshop was to highlight the range of methods available for determining the biological and biochemical functions of cloned genes and their products. The first half dealt with methods based on a genetic approach, whilst the second half dealt with protein level approaches.
The first 2 speakers addressed targeted gene disruption. Andy Pereira from (CPRO-DLO, The Netherlands) described how the maize transposon En-1 could be used as a powerful insertional mutagen. Transposition can be replicative, so lines with a multiple but manageable number of inserts can be generated. A constitutively expressed transposase can drive highly active En-1 transposition which occurs preferentially to linked sites. This system has already been used successfully as a random mutagen, to target know mutationally defined genes, and to target ORFs of unknown function.
Eric Lam (Rutgers University, USA) then described progress toward developing an efficient system for homologous gene disruption, allowing the replacement of any cloned gene with other desired sequences. Homologous recombination is clearly possible and its efficiency is improving with the introduction of vacuum infiltration for transformation and new selectable markers to weed out illegitimate recombination events.
Nigel Kilby (Cambridge University, UK) described the transfer of FLP/FRT site-specific recombinase system from yeast to Arabidopsis, where it is clearly able to function. This system can be used to generate genetic mosaics which would be very useful in cell autonomy studies and in allowing the study of post-embryonic functions of genes absolutely required for embryo development.
In the final talk before tea, Mark Estelle (Indiana University, USA) described how yeast homologues were helping to determine the biochemical function of the AXR1 protein. This gene, known to be involved in auxin signalling, has homology to the amino-terminal half of ubiquitin activating enzyme, but the significance of this homology is unclear. With the yeast genome completely sequenced, anyone lucky enough to find a yeast homologue for their gene can bring the power of yeast genetics to bare on the problem. In this case, a yeast AXR1-like protein was shown to interact genetically with a ubiquitin conjugating enzyme, CDC34 which is thought to be involved in cyclin degradation.
The second half of the workshop addressed the topic of protein function. The first 3 speakers described a selection of heterologous expression systems which have proved useful experimental systems to study plant protein function. Dan Bush (University of Illinois, USA) summarised his latest work with plant amino acid permeases which went far "beyond functional complementation in yeast". He outlined his yeast-based mutational approach which has started to yield detailed information about the residues within the plant amino acid permeases which determine various transport parameters.
Richard Napier (HRI Wellesborne, UK) detailed the latest work on auxin binding protein 1 (ABP1), describing how insect cells have proved to be the ideal expression system to compare and contrast the intracellular targeting characteristics of wildtype and KDEL-sequence variants of ABP1. In addition, he presented experimental data about the heterologous expression of the membrane-localised AUX1 polypeptide, a putative IAA uptake carrier. Freddy Theodoulou (IACR-Rothamstead, UK) provided an excellent overview of the Xenopus oocyte expression system, highlighting its practical advantages and disadvantages. Freddy illustrated her talk with examples of soluble and membrane-localised plant proteins which have been successfully studied in oocytes, concluding with results she has obtained using an electrophysiology-based study of plant sugar carriers.
The workshop was concluded by Jim Hasseloff (MRC-LMB, Cambridge, UK) who provided a beautifully illustrated video presentation of the applications of Green Fluorescent Protein (GFP)-based imaging to study protein intracellular targeting. Jim described his lab's work to improve GFP's characteristics for expression in transgenic Arabidopsis. GFP has been targeted to various organelles in order to reduce its cytotoxicity, plus novel GFP spectral variants have been selected in E.coli which feature altered excitation spectra and improved light emission.