Cloned tobacco crown gall tissue induced by the Agrobacterium
tumefaciens C58 T‐DNA mutant pGV3132, defective for the T‐DNA‐encoded amihydrolase (iaaH), accumulates about 1000‐times more indole‐3‐acet‐amide (IAM) when compared to untransformed tobacco callus and crown gall tissue induced by a T‐DNA mutant defective for gene 1. In vitro experiments demonstrated that this IAM accumulation is correlated with the active conversion of Trp into IAM. The results presented in this report provide biochemical evidence that the T‐DNA gene 1 encodes a tryptophan 2‐monooxygenase (iaaM) activity in transformed plant cells. This gene cooperates with the gene 2‐encoded amidohydrolase (iaaH) in the T‐DNA‐controlled indole‐3‐acetic acid (IAA) biosynthesis pathway in crown gall cells.
The T‐DNA genes 1 and 2 of the Ti plasmid of Agrobacterium tumefaciens are involved in the biosynthesis of IAA in transformed plant cells. Previously, it has been shown that gene 2 codes for an amidohydrolase able to convert IAM into IAA. We have isolated Nicotiana tabacum regenerates transformed with either gene 1 or genes 6a and 6b of the T‐DNA. The tobacco plants transformed with gene 1 contain 500–1000‐times more IAM as compared to plants transformed with genes 6a and 6b, and as compared to untransformed control plants. No drastic differences in endogenous IAA concentrations were observed between the three plant types analyzed.
Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.
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