T-strand integration in maize protoplasts after codelivery of a T-DNA substrate and virulence genes

We describe a plant protoplast transformation method that provides transformants with a simple pattern of integration of a foreign gene. The approach is to deliver into plant protoplasts by direct gene transfer the Agrobacterium virulence genes virD1 and virD2 with or without virE2, together with a target plasmid containing a gene of interest flanked by Agrobacterium T-DNA border repeat sequences of 25 bp. We present evidence of T-DNA formation in maize protoplasts and its integration into the maize genome. The frequency of VirD1-VirD2-mediated integration events was about 20–35% of the total number of transformants. The addition of virE2 doubled the transformation efficiency. The method described here is of sufficient efficiency and simplicity to be useful for the production of transgenic plants with single-copy well-defined transgenic inserts.

Designing conditions for in vitro formation of amyloid protofilaments and fibrils

We have been able to convert a small α/β protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30–50 Å in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive β-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions.

Functional transitions in myosin: Formation of a critical salt-bridge and transmission of effect to the sensitive tryptophan

For analyzing the mechanism of energy transduction in the “motor” protein, myosin, it is opportune both to model the structural change in the hydrolytic transition, ATP (myosin-bound) + H2O → ADP⋅Pi (myosin-bound) and to check the plausibility of the model by appropriate site-directed mutations in the functional system. Here, we made a series of mutations to investigate the role of the salt-bridge between Glu-470 and Arg-247 (of chicken smooth muscle myosin) that has been inferred from crystallography to be a central feature of the transition [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960–8972]. Our results suggest that whether in the normal, or in the inverted, direction an intact salt-bridge is necessary for ATP hydrolysis, but when the salt-bridge is in the inverted direction it does not support actin activation. Normally, fluorescence changes result from adding nucleotides to myosin; these signals are reported by Trp-512 (of chicken smooth muscle myosin). Our results also suggest that structural impairments in the 470–247 region interfere with the transmission of these signals to the responsive Trp.

Feature integration in pattern perception

The human visual system is able to effortlessly integrate local features to form our rich perception of patterns, despite the fact that visual information is discretely sampled by the retina and cortex. By using a novel perturbation technique, we show that the mechanisms by which features are integrated into coherent percepts are scale-invariant and nonlinear (phase and contrast polarity independent). They appear to operate by assigning position labels or “place tags” to each feature. Specifically, in the first series of experiments, we show that the positional tolerance of these place tags in foveal, and peripheral vision is about half the separation of the features, suggesting that the neural mechanisms that bind features into forms are quite robust to topographical jitter. In the second series of experiment, we asked how many stimulus samples are required for pattern identification by human and ideal observers. In human foveal vision, only about half the features are needed for reliable pattern interpolation. In this regard, human vision is quite efficient (ratio of ideal to real ≈ 0.75). Peripheral vision, on the other hand is rather inefficient, requiring more features, suggesting that the stimulus may be relatively underrepresented at the stage of feature integration.

A novel structure involved in the formation of liver endothelial cell fenestrae revealed by using the actin inhibitor misakinolide

Hepatic endothelial fenestrae are dynamic structures that act as a sieving barrier to control the extensive exchange of material between the blood and the liver parenchyma. Alterations in the number or diameter of fenestrae by drugs, hormones, toxins, and diseases can produce serious perturbations in liver function. Previous studies have shown that disassembly of actin by cytochalasin B or latrunculin A caused a remarkable increase in the number of fenestrae and established the importance of the actin cytoskeleton in the numerical dynamics of fenestrae. So far, however, no mechanism or structure has been described to explain the increase in the number of fenestrae. Using the new actin inhibitor misakinolide, we observed a new structure that appears to serve as a fenestrae-forming center in hepatic endothelial cells.

Human fatty acid synthase: Role of interdomain in the formation of catalytically active synthase dimer

The human and animal fatty acid synthases are dimers of two identical multifunctional proteins (Mr 272,000) arranged in an antiparallel configuration. This arrangement generates two active centers for fatty acid synthesis separated by interdomain (ID) regions and predicts that two appropriate halves of the monomer should be able to reconstitute an active fatty acid synthesizing center. This prediction was confirmed by the reconstitution of the synthase active center by using two heterologously expressed halves of the monomer protein. Each of these recombinant halves of synthase monomer contains half of the ID regions. We show here that the fatty acid synthase activity could not be reconstituted when the ID sequences present in the two recombinant halves are deleted, suggesting that these ID sequences are essential for fatty acid synthase dimer formation. Further, we confirm that the ID sequences are the only regions of fatty acid synthase monomers that showed significant dimer formation, by using the yeast two-hybrid system. These results are consistent with the proposal that the ID region, which has no known catalytic activity, associates readily and holds together the two dynamic active centers of the fatty acid synthase dimer, therefore playing an important role in the architecture of catalytically active fatty acid synthase.

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at λ >495 nm. Subsequent irradiation of the complex at λ310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with Tα. For identification of the sites of crosslinks in Tα, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the Tα species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting Tα derivatives and isolation of Tα peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in Tα containing the amino acid sequences 310–313 and 342–345.

Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: Use of a chemically preactivated reagent

Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at λ > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the Tα subunit. Crosslinking to Tα was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in Tα were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization–time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19–Arg-28 at the N-terminal region of Tα. The total results show that both the N and the C termini of Tα are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.

Star scientists and institutional transformation: Patterns of invention and innovation in the formation of the biotechnology industry

The most productive (“star”) bioscientists had intellectual human capital of extraordinary scientific and pecuniary value for some 10–15 years after Cohen and Boyer’s 1973 founding discovery for biotechnology [Cohen, S., Chang, A., Boyer, H. & Helling, R. (1973) Proc. Natl. Acad. Sci. USA 70, 3240–3244]. This extraordinary value was due to the union of still scarce knowledge of the new research techniques and genius and vision to apply them in novel, valuable ways. As in other sciences, star bioscientists were very protective of their techniques, ideas, and discoveries in the early years of the revolution, tending to collaborate more within their own institution, which slowed diffusion to other scientists. Close, bench-level working ties between stars and firm scientists were needed to accomplish commercialization of the breakthroughs. Where and when star scientists were actively producing publications is a key predictor of where and when commercial firms began to use biotechnology. The extent of collaboration by a firm’s scientists with stars is a powerful predictor of its success: for an average firm, 5 articles coauthored by an academic star and the firm’s scientists result in about 5 more products in development, 3.5 more products on the market, and 860 more employees. Articles by stars collaborating with or employed by firms have significantly higher rates of citation than other articles by the same or other stars. The U.S. scientific and economic infrastructure has been particularly effective in fostering and commercializing the bioscientific revolution. These results let us see the process by which scientific breakthroughs become economic growth and consider implications for policy.

The chaperone/usher pathways of Pseudomonas aeruginosa: Identification of fimbrial gene clusters (cup) and their involvement in biofilm formation

Pseudomonas aeruginosa, an important opportunistic human pathogen, persists in certain tissues in the form of specialized bacterial communities, referred to as biofilm. The biofilm is formed through series of interactions between cells and adherence to surfaces, resulting in an organized structure. By screening a library of Tn5 insertions in a nonpiliated P. aeruginosa strain, we identified genes involved in early stages of biofilm formation. One class of mutations identified in this study mapped in a cluster of genes specifying the components of a chaperone/usher pathway that is involved in assembly of fimbrial subunits in other microorganisms. These genes, not previously described in P. aeruginosa, were named cupA1–A5. Additional chaperone/usher systems (CupB and CupC) have been also identified in the genome of P. aeruginosa PAO1; however, they do not appear to play a role in adhesion under the conditions where the CupA system is expressed and functions in surface adherence. The identification of these putative adhesins on the cell surface of P. aeruginosa suggests that this organism possess a wide range of factors that function in biofilm formation. These structures appear to be differentially regulated and may function at distinct stages of biofilm formation, or in specific environments colonized by this organism.

Role of the iroquois3 homeobox gene in organizer formation

In zebrafish, the organizer is thought to consist of two regions, the yolk syncytial layer (YSL) and the shield. The dorsal YSL appears to send signals that affect formation of the shield in the overlying mesendoderm. We show here that a domain of dorsal deep cells located between the YSL and the shield is marked by expression of the iro3 gene. As gastrulation proceeds, the iro3 positive domain involutes and migrates to the animal pole. Iro3 expression is regulated by Nodal and bone morphogenic protein antagonists. Overexpression of iro3 induced ectopic expression of shield-specific genes. This effect was mimicked by an Iro3-Engrailed transcriptional repressor domain fusion, whereas an Iro3-VP16 activator domain fusion behaved as a dominant negative or antimorphic form. These results suggest that Iro3 acts as a transcriptional repressor and further implicate the iro3 gene in regulating organizer formation. We propose that the iro3-expressing dorsal deep cells represent a distinct organizer domain that receives signals from the YSL and in turn sends signals to the forming shield, thereby influencing its expansion and differentiation.

Auxin as a positional signal in pattern formation in plants.

By using a novel, extremely sensitive and specific gas chromatography-mass spectrometry technique we demonstrate in Pinus sylvestris (L.) trees the existence of a steep radial concentration gradient of the endogenous auxin, indole-3-acetic acid, over the lateral meristem responsible for the bulk of plant secondary growth, the vascular cambium. This is the first evidence that plant morphogens, such as indole-3-acetic acid, occur in concentration gradients over developing tissues. This finding gives evidence for a regulatory system in plants based on positional signaling, similar to animal systems.

Mapping rat megalin: the second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis.

Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.

Chemotactic signal integration in bacteria.

Chemotactic signaling in Escherichia coli involves transmission of both negative and positive signals. In order to examine mechanisms of signal processing, behavioral responses to dual inputs have been measured by using photoactivable "caged" compounds, computer video analysis, and chemoreceptor deletion mutants. Signaling from Tar and Tsr, two receptors that sense amino acids and pH, was studied. In a Tar deletion mutant the photoactivated release of protons, a Tsr repellent, and of serine, a Tsr attractant, in separate experiments at pH 7.0 resulted in tumbling (negative) or smooth-swimming (positive) responses in ca. 50 and 140 ms, respectively. Simultaneous photorelease of protons and serine resulted in a single tumbling or smooth-swimming response, depending on the relative amounts of the two effectors. In contrast, in wild-type E. coli, proton release at pH 7.0 resulted in a biphasic response that was attributed to Tsr-mediated tumbling followed by Tar-mediated smooth-swimming. In wild-type E. coli at more alkaline pH values the Tar-mediated signal was stronger than the Tsr signal, resulting in a strong smooth-swimming response preceded by a diminished tumbling response. These observations imply that (i) a single receptor time-averages the binding of different chemotactic ligands generating a single response; (ii) ligand binding to different receptors can result in a nonintegrated response with the tumbling response preceding the smooth-swimming response; (iii) however, chemotactic signals of different intensities derived from different receptors can also result in an apparently integrated response; and (iv) the different chemotactic responses to protons at neutral and alkaline pH may contribute to E. coli migration toward neutrality.

Adeno-associated virus (AAV) site-specific integration: Formation of AAV–AAVS1 junctions in an in vitro system

An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo.