The Recombinant DNA Controversy: A Contemporary Cautionary Tale

A discussion of the scientific and political aspects of recombinant DNA research

A Highly Sensitive Plant Hybrid Protein Assay System Based on the \u3cem\u3eSpm\u3c/em\u3e Promoter and TnpA Protein for Detection and Analysis of Transcription Activation Domains

TnpA is a multifunctional DNA binding protein encoded by the maize Suppressor-mutator (Spm) transposable element. TnpA is required for transposition and is a repressor of the unmethylated Spm promoter. While analyzing protein domains using a yeast GAL4-based hybrid system in transiently transformed tobacco cells, we found that TnpA represses the \u3e10-fold transcriptional activation observed when the GAL4 DNA-binding domain is used alone. By contrast, compared to the backgroundless TnpA DNA-binding domain alone, 33- to 45-fold activation of the Spm promoter was observed when the VP16 activation domain was fused to it. TnpA-binding sites, but no TATA box, were required for transcription activation. Among the TnpA deletion derivatives tested, those retaining the coding sequences for the DNA-binding and protein dimerization domains gave the highest level of transcription activation when fused with the VP16 activation domain. The TnpA gene and TnpA-binding sites in the short Spm promoter therefore provide a novel, highly sensitive single-hybrid system for identifying and studying plant transcription activation domains in plant cells

Agricultural Biotechnology-An Opportunity to Feed a World of Ten Billion

The latest United Nations population projections predict that the human population will expand from roughly 7.5 billion to between 8.3 and 10.9 billion by mid-century. This presents an acute need to increase agricultural productivity quickly and to do so without unduly damaging the many other kinds of organisms that share our planet. The advances of genetic engineering and genetic modification hold the promise of making it possible for us to grow more food on the same amount of land using less water, energy, and chemicals: critically important objectives if we are to live sustainably within planetary constraints. At the same time, these advances have evoked an almost unprecedented level of societal controversy quite specifically in the realm of food production, resulting in the proliferation of regulatory and legal issues that threaten to block their use in achieving a more sustainable existence for humanity on planet Earth. If modem science is to contribute to the agricultural productivity increases required in coming decades as the climate warms and the human population continues to grow, it is imperative to get beyond the cultural and political biases against molecular crop modification, acknowledge the safety record of GM crops, and ease the regulatory barriers to their development and deployment

Concerted Formation of Macromolecular \u3cem\u3eSuppressor-mutator\u3c/em\u3e Transposition Complexes

Transposition of the maize Suppressor-mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA-DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein-protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range

A case study in pathway knowledgebase verification

BACKGROUND: Biological databases and pathway knowledgebases are proliferating rapidly. We are developing software tools for computer-aided hypothesis design and evaluation, and we would like our tools to take advantage of the information stored in these repositories. But before we can reliably use a pathway knowledgebase as a data source, we need to proofread it to ensure that it can fully support computer-aided information integration and inference. RESULTS: We design a series of logical tests to detect potential problems we might encounter using a particular knowledgebase, the Reactome database, with a particular computer-aided hypothesis evaluation tool, HyBrow. We develop an explicit formal language from the language implicit in the Reactome data format and specify a logic to evaluate models expressed using this language. We use the formalism of finite model theory in this work. We then use this logic to formulate tests for desirable properties (such as completeness, consistency, and well-formedness) for pathways stored in Reactome. We apply these tests to the publicly available Reactome releases (releases 10 through 14) and compare the results, which highlight Reactome's steady improvement in terms of decreasing inconsistencies. We also investigate and discuss Reactome's potential for supporting computer-aided inference tools. CONCLUSION: The case study described in this work demonstrates that it is possible to use our model theory based approach to identify problems one might encounter using a knowledgebase to support hypothesis evaluation tools. The methodology we use is general and is in no way restricted to the specific knowledgebase employed in this case study. Future application of this methodology will enable us to compare pathway resources with respect to the generic properties such resources will need to possess if they are to support automated reasoning

Purification and Properties of Bacteriophage f2 Replicase

The present dissertation concerns the purification and properties of an RNA-dependent ribonucleotide-polymerizing enzyme (RNA replicase) produced during infection of E. coli with the RNA bacteriophage f2. Studies on the RNA replicase of the distantly-related bacteriophage Q3 have established the feasibility of using a simplified assay for replicase activity based on the ability of the enzyme to polymerize GTP in the presence of polycytidylic acid template. A similar poly Cdependent poly G polymerase activity is detectable in E. coli infected with bacteriophage f2. The f2 poly G polymerase has been purified by ion exchange chromatography on DEAE cellulose, affinity chromatography on RNA cellulose and density gradient centrifugation. Highly purified preparations of phage-induced poly G polymerase consist predominantly of four proteins having approximate molecular weights of 75,000, 63,000, 46,000 and 33,000. The 63,000 m.w. protein has been identified as the phage-coded replicase protein based on co-electrophoresis of this polypeptide with replicase protein obtained from phage-infected cells. Partially purified preparations of f2 poly G polymerase exhibit replicase activity. Such preparations catalyze nucleotide polymerization in the presence of single-stranded f2 phage RNA or f2 complementary strand RNA, but are inactive with other viral and bacterial RNAs. The product of the in vitro enzymatic reaction has been analysed by RNA annealing techniques and found to consist entirely of polynucleotides homologous to f2 RNAs. In the presence of single-stranded phage RNA template, the enzyme synthesizes both complementary strand RNA and some product copies of the input template strand. However, the enzyme does not synthesize RNA in excess of the input template amount and is able to release only a small proportion of the product RNA as single-stranded viral RNA. Most of the product RNA remains in partially double-stranded complexes with template RNA. Higihly purified f2 poly G polymerase shows no replicase activity. It has been found that a factor fraction necessary for replicase activity separates from the poly G polymerase during glycerol gradient centrifugation. This fraction, which shows no nucleotide-polymerizing activity and is presumably of bacterial origin, restores replicase activity with both f2 single-stranded viral RNA and f2 complementary strand RNA to the 4-polypeptide poly G polymerase. The f2 replicase and poly G polymerase activities are quite similar with respect to such parameters as stability, temperature optimum, divalent cation requirements, phosphate insensitivity and template saturation kinetics. Template competition experiments further suggest that the synthetic polymer and phage RNA compete for binding to the enzyme. The effect of the ionic strength of the incubation medium on the two activities is quite different, however. Replicase activity is stimulated at ionic strengths up to about 0.1, while the poly G polymerase is markedly inhibited, even at quite low salt concentrations. The results of substrate saturation studies suggest that the affinity of replicase is considerably higher for ATP, CTP and UTP than for the chain-initiator nucleotide GTP. Furthermore, the complex saturation kinetics observed for GTP in both replicase and poly G polymerase reactions indicate the simultaneous interaction of more than one molecule of GTP with the enzyme. On the basis of the present studies with the f2 enz3rme and the considerable literature on other nucleotidepolymerizing enzymes, it is proposed that the replicase has separate active sites for RNA chain initiation and polymerization. It is postulated that the synthetic polymer-dependent reaction occurs primarily or exclusively at the chain initiation site, whose normal function is to recognize the 3\u27 terminus of phage RNA templates and carry out the polymerization of the 5\u27 terminal guanylate residues of phage RNAs. It is further suggested that replication of phage RNAs occurs primarily at the polymerization site, whose activity is governed by specific secondary interactions of the enzyme with natural template RNAs

Inducible DNA Demethylation Mediated by the Maize Suppressor-mutator Transposon-Encoded TnpA Protein

Heritable epigenetic inactivation of the maize Suppressor-mutator (Spm) transposon is associated with promoter methylation, and its reversal is mediated by the transposon-encoded TnpA protein. We have developed an assay that permits demethylation of the Spm sequence to be controlled by inducing the expression of TnpA in plant cells. Using this assay, we show that demethylation is a rapid, active process. TnpA is a weak transcriptional activator, and deletions that abolish its transcriptional activity also eliminate its demethylation activity. We show that cell cycle and DNA synthesis inhibitors interfere with TnpA-mediated Spm demethylation. We further show that TnpA has a much lower affinity for fully methylated than for hemimethylated or unmethylated DNA fragments derived from Spm termini. Based on these observations, we suggest that TnpA binds to the postreplicative, hemimethylated Spm sequence and promotes demethylation either by creating an appropriate demethylation substrate or by itself participating in or recruiting a demethylase

Mendel in the kitchen: a scientist's view of genetically modified foods

While European restaurants race to footnote menus, reassuring concerned gourmands that no genetically modified ingredients were used in the preparation of their food, starving populations around the world eagerly await the next harvest of scientifically improved crops. "Mendel in the Kitchen" provides a clear and balanced picture of this tangled, tricky (and very timely) topic. Any farmer you talk to, could tell you that we've been playing with the genetic makeup of our food for millennia, carefully coaxing nature to do our bidding. The practice officially dates back to Gregor Mendel - who was not a renowned scientist, but a 19th century Augustinian monk. Mendel spent many hours toiling in his garden, testing and cultivating more than 28,000 pea plants, selectively determining very specific characteristics of the peas that were produced, ultimately giving birth to the idea of heredity - and the now very common practice of artificially modifying our food. But as science takes the helm, steering common field practices into the laboratory, the world is now keenly aware of how adept we have become at tinkering with nature - which in turn has produced a variety of questions. Are genetically modified foods really safe? Will the foods ultimately make us sick, perhaps in ways we can't even imagine? Isn't it genuinely dangerous to change the nature of nature itself? Nina Fedoroff, a leading geneticist and recognized expert in biotechnology, answers these questions, and more. Addressing the fear and mistrust that is rapidly spreading, Federoff and her co-author, science writer Nancy Brown, weave a narrative rich in history, technology, and science to dispel myths and misunderstandings. In the end, Fedoroff argues, plant biotechnology can help us to become better stewards of the earth while permitting us to feed ourselves and generations of children to come. Indeed, this new approach to agriculture holds the promise of being the most environmentally conservative way to increase our food supply