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Mi-1, a Lycopersicon peruvianum gene conferring resistance to the agricultural pests, root-knot nematodes, and introgressed into tomato, has been cloned using a selective restriction fragment amplification based strategy. Complementation analysis of a susceptible tomato line with a 100 kb cosmid array yielded a single cosmid clone capable of conferring resistance both to the root-knot nematode Meloidogyne incognita and to an unrelated pathogen, the potato aphid Macrosiphum euphorbiae. This resistance was stable. The Mi-1 gene encodes a protein sharing structural features with the nucleotide-binding site leucine-rich repeat-containing type of plant resistance genes.

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S-Adenosylmethionine serves as a methyl group donor in numerous transmethylation reactions and plays a role in the biosynthesis of polyamines and ethylene. We have cloned and sequenced an S-adenosylmethionine synthetase gene (sam-1) of Arabidopsis thaliana. The deduced polypeptide sequence of the enzyme has extensive homology with the corresponding enzymes of Escherichia coli and yeast. Genomic hybridization indicates the presence of two adenosylmethionine synthetase genes per haploid Arabidopsis genome. RNA gel blot analysis shows that adenosylmethionine synthetase mRNA levels are high in stems and roots, correlating well with the higher enzyme activity in stems, compared with leaves. Histochemical analysis of transgenic Arabidopsis plants transformed with a chimeric beta-glucuronidase gene, under the control of 748-base pair 5' sequences of the sam-1 gene, demonstrates that the gene is expressed primarily in vascular tissues. In addition, high expression was observed in sclerenchyma and in the root cortex. A hypothesis for the strong cellular preference in the expression of the sam-1 gene is presented.

Although DNA microarrays are currently the standard tool for genome-wide expression analysis, their application is limited to organisms for which the complete genome sequence or large collections of known transcript sequences are available. Here, we describe a protocol for cDNA-AFLP, an AFLP-based transcript profiling method that allows genome-wide expression analysis in any species without the need for prior sequence knowledge. In essence, the cDNA-AFLP method involves reverse transcription of mRNA into double-stranded cDNA, followed by restriction digestion, ligation of specific adapters and fractionation of this mixture of cDNA fragments into smaller subsets by selective PCR amplification. The resulting cDNA-AFLP fragments are separated on high-resolution gels, and visualization of cDNA-AFLP fingerprints is described using either a conventional autoradiography platform or an automated LI-COR system. Observed differences in band intensities between samples provide a good measure of the relative differences in the gene expression levels. Identification of differentially expressed genes can be accomplished by purifying cDNA-AFLP fragments from sequence gels and subsequent sequencing. This method has found widespread use as an attractive technology for gene discovery on the basis of fragment detection and for temporal quantitative gene expression analysis. The protocol can be completed in 3-4 d.