Changing images of the gene.

During the twentieth century the gene emerged as the major driving force of biology. Initially, even the nature and behavior of gene vehicles, the chromosomes, were subjected to doubts. The basic or standard gene concept, as a unit of function, mutation, and recombination, had to be revised. Half a century was required for reaching a general consensus about the chemical nature of the genetic material, DNA and RNA. The relationship between single genes and individual proteins was a great milestone at the middle of the twentieth century, but within two decades it was realized that the relationship was more complex. Understanding of genetic coding, transcription, and translation during the 1960s laid a firm foundation to the "nucleic doctrine," harking back to the dicta of Lederberg (1959) and meaning that single nucleic acid genes alone were responsible for each separate function within the cell. However, important aspects of gene expression are recognized now as a function of the genome and many genes collaborate in circuits. It has come to light that genes may be mobile, exist in plasmids and cytoplasmic organelles, and can be imported by nonsexual means from other organisms or as synthetic products. Epigenetics has reborn as a new field of developmental genetics. The unorthodox prion proteins can even simulate some gene properties. Genetics was to an extent reincarnated as of the twenty-first century by assimilating the tools of cybernetics and of many formerly distant areas of science. This overview highlights some of the historical milestones that contributed to the development of our image of the gene, extending elements of issues laid down by Rédei (2003).

Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome.

Induction of knockout mutations by T-DNA insertion mutagenesis is widely used in studies of plant gene functions. To assess the efficiency of this genetic approach, we have sequenced PCR amplified junctions of 1000 T-DNA insertions and analysed their distribution in the Arabidopsis genome. Map positions of 973 tags could be determined unequivocally, indicating that the majority of T-DNA insertions landed in chromosomal domains of high gene density. Only 4.7% of insertions were found in interspersed, centromeric, telomeric and rDNA repeats, whereas 0.6% of sequenced tags identified chromosomally integrated segments of organellar DNAs. 35.4% of T-DNAs were localized in intervals flanked by ATG and stop codons of predicted genes, showing a distribution of 62.2% in exons and 37.8% in introns. The frequency of T-DNA tags in coding and intergenic regions showed a good correlation with the predicted size distribution of these sequences in the genome. However, the frequency of T-DNA insertions in 3'- and 5'-regulatory regions of genes, corresponding to 300 bp intervals 3' downstream of stop and 5' upstream of ATG codons, was 1.7-2.3-fold higher than in any similar interval elsewhere in the genome. The additive frequency of insertions in 5'-regulatory regions and coding domains provided an estimate for the mutation rate, suggesting that 47.8% of mapped T-DNA tags induced knockout mutations in Arabidopsis.

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes.

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.