Genetic approaches to understanding sugar-response pathways

Plants as photoautotrophic organisms are able to produce the carbohydrates they require and have developed mechanisms to co-ordinate carbohydrate production and its metabolism. Carbohydrate-derived signals regulate the expression of genes involved in both photosynthesis and metabolism, and control carbohydrate partitioning. A number of genetic approaches have been initiated to understand sugar-response pathways in plants and identify the components involved. Screening strategies to date have been based on the effects of high sugar media on early seedling development or on changes in the enzyme activity or expression of sugar-responsive genes. These screens have established roles for plant hormones in sugar-response pathways, in particular for abscisic acid. The present emphasis on the role of plant hormones in sugar responses is due to the fact that mutants could be readily identified as belonging to these established pathways, but also results from the nature of the mutant screens in use. Progress is being made on the identification of mutants and genes that may be specific to sugar-signalling pathways. It is also expected that the modification of existing screens may target sugar-signalling pathways more directly. Genetic approaches may be especially useful in identifying components of novel signalling pathways unique to plants, and their combination with genomic and molecular approaches will guide future research.     

Alzheimer's associated variant ubiquitin causes inhibition of the 26S proteasome and chaperone expression

Intracellular protein inclusions in Alzheimer's disease and progressive supranuclear palsy contain UBB+1, a variant ubiquitin. UBB+1 is able block the 26S proteasome in cell lines. Proteasome inhibition by drug action has previously been shown to induce a heat-shock response and render protection against stress. We investigated UBB+1 by developing a stable, conditional expression model in SH-SY5Y human neuroblastoma cells. Induction of UBB+1 expression caused proteasome inhibition as was confirmed by reduced ability to process misfolded canavanyl proteins, accumulation of GFPu, a proteasome substrate, and reduced cleavage of a fluorogenic substrate. We show that expression of UBB+1 induces expression of heat-shock proteins. This priming of the chaperone system in these cells promotes a subsequent resistance to tert-butyl hydroperoxide-mediated oxidative stress. We conclude that although UBB+1-expressing cells have a compromised ubiquitin-proteasome system, they are protected against oxidative stress conditions.     

Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines,a novel class of herpesvirus antiviral agents

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.     

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.     

Bone marrow transplantation reveals the in vivo expression of the mitochondrial uncoupling protein 2 in immune and nonimmune cells during inflammation

The mitochondrial uncoupling protein 2 (UCP2) is expressed in spleen, lung, intestine, white adipose tissue, and immune cells. Bone marrow transplantation in mice was used to assess the contribution of immune cells to the expression of UCP2 in basal condition and during inflammation. Immune cells accounted for the total amount of UCP2 expression in the spleen, one-third of its expression in the lung, and did not participate in its expression in the intestine. LPS injection stimulated UCP2 expression in lung, spleen, and intestine in both immune and non-immune cells. Successive injections of LPS and dexamethasone or N-acetyl-cysteine prevented the induction of UCP2 in all three tissues, suggesting that oxygen free radical generation plays a role in UCP2 regulation. Finally, both previous studies and our data show that there is down-regulation of UCP2 in immune cells during their activation in the early stages of the LPS response followed by an up-regulation in UCP2 during the later stages to protect all cells against oxidative stress.     

Sequence and structure determinants for the self-aggregation of recombinant polypeptides modeled after human elastin

Elastin is a polymeric structural protein that imparts the physical properties of extensibility and elastic recoil to tissues. The mechanism of assembly of the tropoelastin monomer into the elastin polymer probably involves extrinsic protein factors but is also related to an intrinsic capacity of elastin for ordered assembly through a process of hydrophobic self-aggregation or coacervation. Using a series of simple recombinant polypeptides based on elastin sequences and mimicking the unusual alternating domain structure of native elastin, we have investigated the influence of sequence motifs and domain structures on the propensity of these polypeptides for coacervation. The number of hydrophobic domains, their context in the alternating domain structure of elastin, and the specific nature of the hydrophobic domains included in the polypeptides all had major effects on self-aggregation. Surprisingly, in polypeptides with the same number of domains, propensity for coacervation was inversely related to the mean Kyte-Doolittle hydropathy of the polypeptide. Point mutations designed to increase the conformational flexibility of hydrophobic domains had the unexpected effect of suppressing coacervation and promoting formation of amyloid-like fibers. Such simple polypeptides provide a useful model system for understanding the relationship between sequence, structure, and mechanism of assembly of polymeric elastin.     

Impaired membrane resealing and autoimmune myositis in synaptotagmin VII-deficient mice

Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII-deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. Inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease.     

Estimated frequency of nonrecessive Bt resistance genes in bollworm,Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in eastern North Carolina

In summer 2000, adult female bollworm moths, Helicoverpa zea (Boddie), were collected from light-traps at four locations near the Tidewater Research Station, Plymouth, NC. Female moths were allowed to lay eggs, and at hatch, 72 larvae from each female were screened for growth rate on normal artificial diet and on diets containing 5.0 microg of either Cry1Ac or Cry2Aa Bt toxin per milliliter of diet. The growth rate bioassays were performed to isolate nonrecessive Bt resistance genes present in field populations of bollworm. We found one individual out of 583 screened that appeared to carry a major gene for resistance to Cry1Ac. Assuming four alleles per individual, the gene frequency is 1/2332 or 0.0003. Other females appeared to have minor genes for Cry1Ac resistance or major genes with lower levels of dominance. We also found one individual out of 646 screened that appeared to carry a major gene for resistance to Cry2Aa. The gene frequency for Cry2Aa resistance was estimated at 1/2584 or 0.00039. Again, other females seemed to carry additional minor resistance genes. Along with other results that indicate partially dominant inheritance of Cry1Ac resistance in bollworm, these allele frequency estimates are important for determining the rate of resistance evolution in H. zea to specific Bt toxins.     

Spatial processes in the evolution of resistance in Helicoverpa zea (Lepidoptera: Noctuidae) to Bt transgenic corn and cotton in a mixed agroecosystem: a biology-rich stochastic simulation model

A simulation model is developed to examine the role of spatial processes in the evolution of resistance in Helicoverpa zea populations to Bt corn and Bt cotton. The model is developed from the stochastic spatially explicit Heliothis virescens model described by Peck et al. (1999), to accommodate a spatial mix of two host crops (corn and cotton), and to reflect the agronomic practices, as well as the spatial and temporal population dynamics of H. zea, in eastern North Carolina. The model suggests that selection for resistance is more intense in Bt cotton fields than in Bt corn fields. It further suggests that local gene frequencies are highly dependent on local deployment levels of Bt crops despite the high mobility of the adult insects. Region-wide average gene frequencies depend on the region-wide level of Bt deployment, so incomplete technology adoption slows the rate of resistance evolution. However, on a local scale, H. zea populations in clusters of fields in which Bt use is high undergo far more rapid evolution than populations in neighboring clusters of fields in which Bt use is low. The model suggests that farm-level refuge requirements are important for managing the risk of resistance. The model can be used as an aid in designing plans for monitoring for resistance by suggesting the appropriate distribution of monitoring locations, which should focus on areas of highest Bt crop deployment. The findings need to be placed in the context of the input parameters, many of which are uncertain or highly variable in nature, and therefore, a thorough sensitivity analysis is warranted.     

Sensitivity analysis of a spatially-explicit stochastic simulation model of the evolution of resistance in Helicoverpa zea (Lepidoptera: Noctuidae) to Bt transgenic corn and cotton

The sensitivities of a model simulating the evolution of resistance in Helicoverpa zea to Bt toxins in transgenic crops were investigated by examining effects of each of the model parameters on the frequency of resistance alleles after 8 yr. The functional dominance of resistance alleles and the initial frequency of those alleles had a major impact on resistance evolution. The survival of susceptible insects on the transgenic crops and the population dynamics of the insect, driven by winter survival and reproductive rates, were also important. In addition, agricultural practices including the proportion of the acreage planted to corn, and the larval threshold for spraying cotton fields affected the R-allele frequency. Many of these important parameters are inherently variable or cannot be measured with accuracy, so model output cannot be interpreted as being a forecast. However, this analysis is useful in focusing empirical research on those aspects of the insects' life system that have the largest effects on resistance development, and indicates ways in which to improve products and agricultural practices to increase the expected time to resistance. The model can thus be used as a scientific basis for devising a robust resistance management strategy for Bt crops.