Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters

Abstract

Forty-five individually transformed clonal tobacco callus lines were simultaneously assayed for both chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) activity resulting from expression of introduced reporter genes driven by the adjacent and divergent mannopine (mas) promoters. Excluding lines in which one or both of the enzyme activities was essentially zero, the activities of the reporter genes varied by as much as a factor of 136 (CAT) and 175 (GUS) between individual transformants. Superimposed upon the high degree of inter-clonal expression variability was an intra-clonal variability of 3-4-fold. The observed degree of intra-clonal reporter gene activity may be more extreme because of the regulatory characteristics of the mannopine promoters, but must still be addressed when considering the limitations of reporter gene-based analysis of transgene function and structure. There was no consistent correlation between the expression levels of the introduced CAT and GUS genes since the ratio of GUS to CAT activities (nmol min-1 mg-1) within individual lines varied from 0.05 to 49. Even divergent transcription from two directly adjacent promoter regions (both contained within a 479 bp TR-DNA fragment) is insufficient to guarantee concurrent expression of two linked transgenes. Our quantitative data were compared to published data of transgene expression variability to examine the overall distribution of expression levels in individual transformants. The resulting frequency distribution indicates that most transformants express introduced transgenes at relatively low levels, suggesting that a potentially large number of Agrobacterium-mediated transformation events may result in silent transgenes.

The forever young gene encodes an oxidoreductase required for proper development of the Arabidopsis vegetative shoot apex

“…First, the FEYgene may be normally subject to tight regulation and the abnormal position of the introduced genomic fragment could result in abnormal regulation of FEY. Positional effects on introduced genes have been reported often (Blundy et al, 1991;Dean et al, 1988;Peach and Velten, 1991). Alternatively, although the fey mutant has been backcrossed two times, it is still possible that a second, tightly linked mutation is present.…”

Site‐specific integration of DNA into wild‐type and mutant lox sites placed in the plant genome

“…Studies of gene expression involving transformation of plants with modified regulatory elements are often confounded by the variability of gene expression among independent transformants. This variability could be caused by chromosomal position effects at the sites of transgene integration (An, 1986;Odell et aL, 1987;Peach and Velten, 1991). Inserting test constructs into the same genomic target might control this variable and facilitate valid comparisons of expression levels.…”

The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2.

“…The insertion of new genetic material into plant genomes can alter the expression of both newly introduced and endogenous genes (Matzke and Matzke, 1995). In addition, the position of the inserted transgene in the plant genome can affect the level of transgene expression (Dean et al, 1988;Peach and Velten, 1991). Potentially, either or both of these effects could be responsible for insufficient levels of the ANT gene product in the complemented plants to restore wild-type levels of fertility.…”