Renaturation kinetics of labeled Agrobacterium tumefaciens DNA are not influenced by addition of 104-fold excess of crown gall tumor DNA. Reconstruction experiments demonstrated that 0.01% added bacterial DNA produces a detectable increase in rate of renaturation of labeled DNA. Crown gall tumor DNA therefore cannot contain as much as 0.01% A. tumefaciens DNA, (one entire bacterial genome per three diploid tumor cells).By estimated levels range from 0.2% (6, 7) to 0.9% (8). DNA from A. tumefaciens bacteriophage PS8 has been reported present in tumor DNA at a level of 1.8% (8), despite the fact that neither viable PS8 phage nor PS8 DNA could be detected in the bacterial strain which incited the tumor examined. Unfortunately, most of these nucleic acid hybridization studies (5-7, 9) failed to include thermal dissociation profiles of the duplexes, an essential control to support the conclusion that authentic phage or bacterial nucleic acids were detected. Thermal dissociation profiles of bacterial or phage cRNA hybridized to filter-bound tumor DNA (8) have been reported (18)

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The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37 C is shown to be due to loss of a large plasmid (1.2 X 10-8 daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6 is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 X A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

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Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.

Homology between the large plasmids of 15 pathogenic Agrobacterium strains isolated from various parts of the world has been measured and was found to vary over a wide range, from 3 to 100%. Two genetically distinct groups of plasmids can be identified: one closely related to the plasmid of A. tumefaciens A6, an octopine-utilizing strain, and the other closely related to the plasmid of A. tumefaciens C-58, a nopaline-utilizing strain. The plasmids of four Agrobacterium strains do not belong to either group. One of these four strains utilizes octopine, one utilizes nopaline, and two utilize neither. Three strains contained two large plasmids. In one of these strains, the two plasmids were not homologous to one another. Chromosomal homologies for the Agrobacterium strains surveyed also vary over a wide range, but do not correlate with plasmid homologies. Neither do plasmid homologies correlate with any numerical classification scheme. The significance of these plasmid homology studies for crown gall tumorigenesis is considered.

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Auxotrophic mutants of the filamentous cyanobacterium Anabaena variabilis were isolated by a method in which, after mutagenesis and before penicllin enrichment, mutant and wild-type cells were separated by cavitation. Auxotrophs were identified by their inability to grow on minimal medium, and they were partially characterized by replica plating to media supplemented with single nutrients or specific groups of nutrients. Of the 83 auxotrophs isolated, 65 required an inorganic source of nitrogen for growth. In addition, auxotrophs were isolated that required methionine (six), uracil (two), adenine (one), biotin (two), and nicotinic acid (two). (The number of isolates of each type is indicated in parentheses.) The nutrient requirements of five auxotrophs appeared complex and were not determined. A large proportion of the mutants requiring inorgainic fixed nitrogen was altered in the differentiation of heterocysts. The following morphological aberrancies were observed: abnormally high and abnormally low frequencies of heterocysts; thick, uneven heterocyst envelopes; incompletely developed pore regions; very distinct pore regions; and protoplasts separated from the envelope of the heterocyst. Spontaneously occurring, N2-fixing, prototrophic revertants of mutants with aberrant heterocysts have been isolated at a frequency of 2 X 10(-8) to 4 X 10(-8) of the cells plated. That most such revertants produced morphologically normal heterocysts is consisten with the idea that heterocysts play an essential role in aerobic N2 fixation.