Structure of the tomato Adh2 gene and Adh2 pseudogenes, and a study of Adh2 gene expression in fruit

A cDNA library was constructed from RNA from the pericarp of ripe tomato fruit and four cDNAs encoding ADH2 were isolated and characterized. The cDNAs encode a peptide 379 amino acids in length. They hybridized strongly with a 1.8 kb RNA species well represented in RNA from ripe, but not from mature, unripe fruit, and strongly to a similar RNA species present in hypoxic, but not in aerobic roots. Northern analysis showed that the mRNA for ADH2 in fruit increased in abundance through ripening, particularly during late ripening. In pericarp tissue of fruit, the Adh2 mRNA level increased to a maximum within 8-16 h of exposure to atmospheres with 3% (v/v) oxygen, and returned to the basal level within 16 h of a return to air. The mRNA level was sensitive to the oxygen level in the atmosphere, increasing 20-fold in 12% (v/v) oxygen and 100-fold in 3% oxygen. The homologous tomato Adh2 gene was isolated from a genomic library. The gene has an overall length of 2334 bp from transcription start site to poly(A) addition site and includes eight introns. Southern blot analysis of tomato genomic DNA identified multiple Adh2-related sequences. Two of these, PSA1 and PSA2, were cloned and found to have 94% similarity with each other and 77% similarity with the tomato Adh2 gene over a 1000 bp region. The homologous regions include introns and exons but the equivalent exons contain frame shifts, deletions and stop codons. The two regions are therefore presumptive pseudogenes.