Molecular characterization of the virD operon from Agrobacterium tumefaciens.

The Agrobacterium tumefaciens Ti plasmid virulence (vir) region contains at least six transcriptional units required for the efficient transfer of T-DNA to the plant genome (virA, B, C, D, E, and G). We have reported that two proteins encoded by the 5'portion of the virD operon are required for a site-specific endonuclease activity that nicks the direct repeats which flank the T-DNA. We have presented the nucleotide sequence for this portion of the operon. The nucleotide sequence of the remainder of the virD operon essential for virulence has now been determined. Two additional open reading frames encode proteins of 21.3 and 75.8 kilodaltons (kd). Translational fusions between virD2, virD3, and virD4 proteins and trpE produced fusion proteins of the size predicted from the nucleotide sequence data. We have used antisera directed against the trpE-virD2 fusion protein to detect both native virD2 protein and a virD2-lacZ fusion protein in crude extracts from Agrobacterium.

Characterization of new gibberellin-responsive semidwarf mutants of arabidopsis.

Chemical mutagenesis of Arabidopsis thaliana (L.) Heynh. yielded four semidwarf mutants, all of which appeared to be gibberellin (GA)-biosynthesis mutants. All four had atypical response profiles to C20-GAs, suggesting that each had impaired 20-oxidation. One mutant, 11.2, was shown to be allelic to ga5 and has been named ga5-2. It had altered metabolism of [14C]GA15 relative to that in wild-type plants and undetectable levels of C19-GAs in young stems, consistent with the known function of GA5 as a stem-expressed GA 20-oxidase. Two mutants (2.1 and 10.3), which had very short inflorescences and siliques, were allelic to each other but not to the known GA-responding mutants, ga1 to ga5. The locus defined by these two mutations is provisionally named GA6 and is purported to encode an inflorescence- and silique-expressed GA 20-oxidase. A double mutant, ga5-2 ga6-2, had an extreme dwarf phenotype with very short siliques. The fourth mutation, 1.1, gave a phenotype like ga5, but was not allelic to any of the known ga mutations. It has not yet been given a gene symbol pending further studies.

The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease.

Tumor formation by Agrobacterium tumefaciens involves the transfer and integration of a defined segment (T-DNA) of tumor-inducing (Ti) plasmid DNA into the plant nuclear genome. A set of plasmid genes outside the T-DNA, the vir genes, are thought to mediate the transfer process. We report here that the virD operon encodes a site-specific endonuclease that cleaves at a unique site within each of the 24 bp direct repeats that flank the T-DNA. The endonuclease function was further localized to the 5' end of this operon by demonstrating that cleavage does not occur in virD mutant strains of Agrobacterium and that the 5' end of the virD operon is sufficient to direct cleavage in E. coli. Analysis of nucleotide sequence and protein data indicate that two proteins of 16.2 and 47.4 kd are involved.

Cloning, chromosome localization, expression, and characterization of an Src homology 2 and pleckstrin homology domain-containing insulin receptor binding protein hGrb10gamma.

hGrb10alpha (previously named Grb-IR) is a Src-homology 2 domain-containing protein that binds with high affinity to the tyrosine-phosphorylated insulin receptor and insulin-like growth factor-1 receptor. At least two isoforms of human Grb10, (hGrb10alpha and hGrb10beta), which differ in the pleckstrin homology (PH) domain and the N-terminal sequence, have previously been identified in insulin target tissues such as human skeletal muscle and fat cells. Here we report the cloning of the third isoform of the hGrb10 family (hGrb10gamma) from human skeletal muscle and its localization to human chromosome 7. We have also determined the human chromosome localization of Grb7 to 17q21-q22 and Grb14 to chromosome 2. hGrb10gamma contains an intact PH domain and an N-terminal sequence that is present in hGrb10alpha but absent in hGrb10beta. RNase protection assays and Western blot analysis showed that hGrb10alpha and hGrb10gamma are differentially expressed in insulin target cells including skeletal muscle, liver, and adipocyte cells. hGrb10gamma is also expressed in HeLa cells and various breast cancer cell lines. The protein bound with high affinity to the insulin receptor in cells, and the interaction was dependent on the tyrosine phosphorylation of the receptor. hGrb10gamma also underwent insulin-stimulated membrane translocation and serine phosphorylation. hGrb10gamma phosphorylation was inhibited by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, and wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Taken together, our data suggest that hGrb10 isoforms are potential downstream signaling components of the insulin receptor tyrosine kinase and that the PH domain may play an important role in the involvement of these isoforms in signal transduction pathways initiated by insulin and other growth factors.