Figure 3.

Relative merBpe mRNA and MerB protein levels in independent transgenic plant lines. (A) merBpe mRNA levels. Five plant lines were tested for relative expression of merBpe mRNA by Northern blotting and probing with a ³²P-labeled BamHI–XhoI merBpe fragment. Approximately 10–15 μg of total RNA prepared from >30 seedlings (2- to 3-cm plants) was loaded for each sample. merB mRNA was visualized, and the relative band intensities were quantified using a Molecular Dynamics PhosphorImager. To control for variation in loading and transfer, these values were normalized to levels of 18S rRNA (data not shown), determined by reprobing the filter with a oligonucleotide encoding the soybean 18S rRNA gene (42). An 11-fold difference was detected between the highest expressing line, B1, and the lowest expressing line, B5. (B) MerB protein levels. Western blots were performed on total protein extracted from six merBpe plant lines using the MerB specific mAb, Mab2H8 (see Materials and Methods). Fifteen 3- to 4-cm seedlings were harvested for each sample. The protein–antibody complex was visualized on x-ray film using a horseradish peroxidase-based chemoluminescent detection system (Amersham Pharmacia). Relative amounts of MerB were measured by scanning the film with a Molecular Dynamics densitometer, integrating the intensities of the bands over their respective areas, and normalizing to total protein. Relative amounts of total protein were determined by quantifying the band intensities of a parallel gel stained with Coomassie blue (data not shown) on a densitometer. Values recorded for MerB are the averages of three experiments while those for total protein are the averages of two. All data reported for quantitative experiments described here are measurements of band intensities and reflect relative differences in expression between merBpe lines. The B1 sample was shown to have ≈14-fold more steady-state organomercurial lyase than the lowest expressing line, B3. (C) Relative mRNA and protein levels. A linear regression demonstrates that steady-state levels of mRNA and protein are correlated, confirming differences in merBpe gene expression and indicating that gene regulation is straightforward. The relative merBpe mRNA and MerB protein levels in independent transgenic lines (see Materials and Methods) were normalized to the level in the lowest expressing line in each category (B5, mRNA; B3, protein). The relevance of MerB expression levels to organomercurial resistance is discussed in the text.