Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil bacteria which transfer DNA (T-DNA) to plant cells. Two Agrobacterium strains, each with a different T-DNA, can infect plants and give rise to transformed tissue which has markers from both T-DNAs. Although marker genes from both T-DNAs are in the tissue, definitive proof that the tissue is a cellular clone and that both T-DNAs are in a single cell is necessary to demonstrate cotransformation. We have transferred two distinguishable T-DNAs, carried on binary vectors in separate Agrobacterium rhizogenes strains, into tomato cells and have recovered hairy roots which received both T-DNAs. Continued expression of marker genes from each T-DNA in hairy roots propagated from individual root tips indicated that both T-DNAs were present in a single meristem. Also, we have transferred the two different T-DNAs, carried on identical binary vector plasmids in separate Agrobacterium tumefaciens strains, into tobacco cells and recovered plants which received both T-DNAs. Transformed plants with marker genes from each T-DNA were outcrossed to wild-type tobacco plants. Distribution of the markers in the F1 generation from three cotransformed plants of independent origin showed that both T-DNAs in the plants must have been present in the same cell and that the T-DNAs were genetically unlinked. Cotransformation of plant cells with T-DNAs from two bacterial strains and subsequent segregation of the transferred genes should be useful for altering the genetic content of higher plants.

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We show here the changes in the NMR spectra of the Escherichia coli lac repressor when bound to isolated lac operator DNA. The observations focus on the aromatic residues-four tyrosines and a single histidine-in the amino-terminal DNA binding domain of the lac repressor. There is a good correlation between chemical shift changes seen by '9F NMR when compared with 1H NMR of otherwise identical repressor-DNA complexes. The results suggest that the tyrosines do not intercalate in the DNA. The NMR spectral changes with similarly sized DNA fragments, not containing the lac operator DNA sequence, are different. Thus, the amino-terminal domain of the lac repressor is independently capable of discriminating between lac operator and nonspecific DNA sequences. There can be two amino-terminal fragments per operator in the specific complex.The lac repressor-lac operator interaction is the prototype system in revealing the molecular mechanism involved in gene regulation (1)(2)(3). Extensive and detailed information has been accumulated about the DNA sequence requirements and some of the important features of the individual bases in the DNA sequences necessary for proper lac repressor-lac operator DNA complex formation (4-7). Equivalently detailed information about the involvement of specific sites on the lac repressor protein is lacking. It is normally a tetrameric protein made up of four identical subunits with known sequence of360 amino acids each (8). Limited chymotrypsin digestion of the protein gives a tetrameric core containing amino acid residues 57-360 and four amino-terminal peptides containing residues 1-56 (9). The amino-terminal domain, or headpiece, which appears to be part of the DNA binding site, contains five aromatic residues: four tyrosines and a histidine (9-12). We show here changes in the NMR signals from these residues when it is titrated with a 36-base-pair (bp) double helical DNA fragment containing the lac operator sequence.We exploit the advantages of using lac repressors containing the nuclear spin label, 19F, attached to the 3 position on the tyrosine rings. The substituted repressor binds operator DNA and releases it upon binding the inducer (13,14).Direct spectroscopic observations of lac repressor-nonspecific DNA interactions have been made by using absorption, fluorescence, and circular dichroism (15)(16)(17)(18). Similar methods (19) and NMR (20,21) have been used to look at headpiece nonspecific DNA interaction. The experiments that are described here represent the combination of two systematic efforts to understand the molecular nature of the specific protein-DNA interaction. First, the operator DNA fragment was synthesized as part of a program to explore the recognition sites on the lac operator DNA sequence (6, 7). To obtain enough operator DNA, it was polymerized in tandem and cloned in a plasmid (22). Second, our ability to describe and assign the specific changes in the NMR spectrum of the repressor-lac operator complex comes from a comprehensive genetic study ofthe protein (2...

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