Significance and context
Approximately 84% and 55% of the genome sequence and genome annotation, respectively, of Arabidopsis thaliana is publicly available in GenBank as of January 2000, and this has already fostered a number of genome-wide studies on gene expression and gene function. To facilitate the isolation of null alleles, many groups have created lines carrying T-DNA or transposable elements throughout the Arabidopsis genome. Meissner et al. survey the practical advantages and disadvantages of five of these 'knock-out' collections in an attempt to find lines that have insertions in the R2R3 subfamily of MYB genes, which encode transcription factors. MYB genes are involved in the regulation of cell proliferation and differentiation in animals. In plants, they are involved in a number of processes, including the regulation of secondary metabolism, abiotic stress, pathogen defense, and cellular morphogenesis. Finding mutants in this gene family presents a particular challenge because of sequence similarity among the large number of family members, potential redundancy of function, and the relatively small size of the genes (1.6 kilobases, where the expected average size of an Arabidopsis gene size is 4.8 kilobases).
The five 'knock-out' collections screened are: En/Spm (for Enhancer or Suppressor-mutator) transposon insertional populations from AMAZE lines (Max Planck Inst. Zuchtungsforsch, Cologne, Germany); En/Spm-I/dSpm transposon system from SLAT (Sainsbury Laboratory Arabidopsis thaliana) and from the Centre for Plant Breeding and Reproduction Research, Wageningen, The Netherlands; and T-DNA insertional lines from the Laboratoire de Biologie Cellulaire, Versailles, France and Consejo Superior de Investigaciones Cientificas (CSIC), Madrid, Spain. The SLAT and T-DNA populations have stable insertions at low copy numbers (1-3 per line), whereas the AMAZE and Wageningen lines have mobile transposons at higher copy numbers (approximately 2-30 per line). The general strategy for identifying lines with insertions in the R2R3 MYB genes was to use gene-specific primers and primers against the insertion elements in a semi-nested or nested PCR. The AMAZE population gave the highest rate of insertion into the genes that were screened (54% insertion per gene screened from 8000 lines); in the other populations, insertions in MYB genes screened were detected at a rate of about 20%. The multicopy, mobile transposon lines generally had a higher rate of insertion in the MYB genes than the populations with non-mobile insertions, but the presence of other insertions and the mobile nature of the insertion will complicate the further characterization of these genes. On the other hand, SLAT populations (with stable transposons) are better suited for characterization of gene function, but isolating the individual lines carrying the insertion requires much more effort, as each DNA pool tested comes from a population of 50 independent insertional lines. Most of the lines with insertions in a gene of interest had reduced amounts of the gene transcript, but these lines need to be characterized further to identify the consequence of the insertion. None of the lines homozygous for an insertion in the gene of interest gave an obvious visible phenotype under normal growth conditions.
The advantages and disadvantages of searching this set of insertionally mutagenized populations to find insertions in a gene of interest are clearly presented by the authors. Although no single population has reached saturation for insertional mutagenesis, Meissner et al. were able to find insertions in had an approximately 50% of the genes screened, by screening approximately 52,000 lines derived using a variety of mutagenesis methods. It is also clear that finding a homozygous line with an insertion in a gene of interest is a long way from finding a biological role for the gene, as not all these lines lacked the transcript or gave a clear phenotype. Determining the expression pattern of a gene and its protein in wild-type plants, as well as studying the global expression pattern of other genes in a line lacking the relevant protein, and studying lines carrying multiple null alleles of members of the gene family, may help us to understand the roles of the MYB family of transcription factors in Arabidopsis.