Molecular analysis of two PR-1 pseudogenes from tobacco

Two independent PR-1 lambda genomic clones (W38/1 and W38/3) were isolated and characterized from a tobacco (Nicotiana tabacum cv. Wisconsin 38) library. Neither clone is identical to the previously described PR-1 cDNA clones, and both clones carry mutations within the highly conserved PR-1 protein coding region. For example, clone W38/1 has a GAA Glu codon instead of the translation stop codon thus harbouring an open reading frame extended by 16 additional amino acids. Furthermore, both clones display considerable variations in the genomic flanking sequences when compared to the PR-1a gene. In order to test whether the encoded genes are active, their upstream sequences were fused to the E. coli beta-glucuronidase (GUS) reporter gene. While significant GUS activities as compared to the 35S RNA promoter from cauliflower mosaic virus (CaMV) were obtained with the W38/1 and W38/3 sequences in transient gene expression assays, no transcriptional activities could be observed upon stable transformation of the same constructs. In addition, the protein coding region of W38/1 was joined to the CaMV 35S RNA promoter and transgenic tobacco plants were generated. However, neither transcripts nor a protein could be detected deriving from the W38/1 structural gene with this chimaeric construct in the transformants. Taken together, these data indicate that the genes contained in lambda clones W38/1 and W38/3 are not active in planta.