Ras Proteins Have Multiple Functions in Vegetative Cells of Dictyostelium

During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.The Ras subfamily proteins are monomeric GTPases that act as molecular switches, cycling between an active GTP-bound and an inactive GDP-bound state (17). Activation is controlled by guanine nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP, and inactivation regulated by GTPase-activating proteins (GAPs) that stimulate the hydrolysis of bound GTP to GDP (17). Activated Ras proteins stimulate numerous downstream signaling pathways that regulate a wide range of cellular processes, including proliferation, cytoskeletal function, chemotaxis, and differentiation (4). The complexity of this regulation has been emphasized by the discovery of the presence of a large number of Ras subfamily homologues in metazoan organisms (19) and elucidation of the roles played by each protein remains a formidable challenge. An important approach to this problem is an analysis of Ras protein function in organisms amenable to genetic analysis.The Dictyostelium genome encodes 14 Ras subfamily members, an unusually large number for such a relatively simple organism (6, 25). Six of these have been partially characterized and have been shown to be involved in a wide variety of processes, including cell movement, polarity, growth, cytokinesis, chemotaxis, macropinocytosis, and multicellular development (5, 15, 23, 25). They exhibit considerable functional specificity, and even the two highly related proteins, RasD and RasG, perform different functions (23, 26). RasC and RasG are the best characterized of these proteins, and both are activated in response to cyclic AMP (cAMP) during aggregation (11). Although both proteins are involved in aggregation, signaling through RasC is more important for the regulation of the cAMP relay, whereas signaling through RasG is more important for cAMP-dependent chemotaxis, but there is some overlap of function (2, 3). Disruption of both the rasC and rasG genes results in a total loss of cAMP-mediated signaling, suggesting that all cAMP signal transduction in early development is partitioned between pathways that use either RasC or RasG (2, 3).In addition to their roles in early development, both RasG and RasC have vegetative cell functions. Cells with a disrupted rasG gene were found to exhibit a reduced growth rate, which was most apparent when cells were grown in suspension, and were multinucleate, indicating a defect in cytokinesis (13, 23). In addition, rasG⁻ cells exhibited reduced motility and polarity and an altered actin distribution. Vegetative rasC⁻ cells had a less pronounced phenotype: changes in actin distribution and motility but normal growth and cytokinesis (16). Given that there was evidence for some overlap of function between RasG and RasC during early development, it was important to determine the extent of their functional overlap in vegetative cells.In the present study, we have compared the potential overlap of RasG and RasC requirements for vegetative cell function in the recently generated isogenic rasC⁻, rasG⁻, and rasC⁻/rasG⁻ strains (2, 3). In addition, the availability of stable rasG⁻ and rasC⁻/rasG⁻ strains has enabled us to determine to what extent RasD, a protein that is highly related to RasG but not present in wild-type vegetative cells, can substitute for loss of function of RasG.     

A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells

The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.     

Crassulacean acid metabolism in the ZZ plant, Zamioculcas zamiifolia (Araceae)

Zamioculcas zamiifolia (Araceae), a terrestrial East African aroid, with two defining attributes of crassulacean acid metabolism (CAM) (net CO(2) uptake in the dark and diel fluctuations of titratable acidity) is the only CAM plant described within the Araceae, a mainly tropical taxon that contains the second largest number of epiphytes of any vascular plant family. Within the Alismatales, the order to which the Araceae belong, Z. zamiifolia is the only documented nonaquatic CAM species. Zamioculcas zamiifolia has weak CAM that is upregulated in response to water stress. In well-watered plants, day-night fluctuations in titratable acidity were 2.5 μmol H(+)·(g fresh mass)(-1), and net CO(2) uptake in the dark contributed less than 1% to daily carbon gain. Following 10 d of water stress, net CO(2) uptake in the light fell 94% and net CO(2) uptake in the dark increased 7.5-fold, such that its contribution increased to 19% of daily carbon gain. Following rewatering, dark CO(2) uptake returned to within 5% of prestressed levels. We postulate that CAM assists survival of Z. zamiifolia by reducing water loss and maintaining carbon gain during seasonal droughts characteristic of its natural habitat.     

High levels of variation despite genetic fragmentation in populations of the endangered mountain pygmy-possum, Burramys parvus, in alpine Australia

In endangered mammals, levels of genetic variation are often low and this is accompanied by genetic divergence among populations. The mountain pygmy-possum (Burramys parvus) is an endangered marsupial restricted to the alpine region of Victoria and New South Wales, Australia. By scoring variation at eight microsatellite loci, we found that B. parvus populations exhibit high levels of genetic divergence and fall into three distinct groups from the northern, central and southern areas of the distribution of this species, consistent with previous assessments of mitochondrial DNA variation. F(ST) values between populations from these regions ranged from 0.19 to 0.54. Within the central area, there was further genetic fragmentation, and a linear association between genetic and geographical distance. This pattern is likely to reflect limited dispersal across barriers despite the fact that individual B. parvus can move several kilometres. Levels of genetic variation within populations were high with the exception of a southern population where there was evidence of inbreeding. From a conservation perspective, all three areas where B. parvus are found should be considered as separate gene pools; management of populations within these areas needs to take into account the low gene flow between populations, as well as threats posed by roads, resorts and other developments in the alpine region. The low genetic variability and inbreeding in the southern population is of particular concern given the high levels of variability in other B. parvus populations.     

Larval dispersal and movement patterns of coral reef fishes,and implications for marine reserve network design

Well‐designed and effectively managed networks of marine reserves can be effective tools for both fisheries management and biodiversity conservation. Connectivity, the demographic linking of local populations through the dispersal of individuals as larvae, juveniles or adults, is a key ecological factor to consider in marine reserve design, since it has important implications for the persistence of metapopulations and their recovery from disturbance. For marine reserves to protect biodiversity and enhance populations of species in fished areas, they must be able to sustain focal species (particularly fishery species) within their boundaries, and be spaced such that they can function as mutually replenishing networks whilst providing recruitment subsidies to fished areas. Thus the configuration (size, spacing and location) of individual reserves within a network should be informed by larval dispersal and movement patterns of the species for which protection is required. In the past, empirical data regarding larval dispersal and movement patterns of adults and juveniles of many tropical marine species have been unavailable or inaccessible to practitioners responsible for marine reserve design. Recent empirical studies using new technologies have also provided fresh insights into movement patterns of many species and redefined our understanding of connectivity among populations through larval dispersal. Our review of movement patterns of 34 families (210 species) of coral reef fishes demonstrates that movement patterns (home ranges, ontogenetic shifts and spawning migrations) vary among and within species, and are influenced by a range of factors (e.g. size, sex, behaviour, density, habitat characteristics, season, tide and time of day). Some species move <0.1–0.5 km (e.g. damselfishes, butterflyfishes and angelfishes), <0.5–3 km (e.g. most parrotfishes, goatfishes and surgeonfishes) or 3–10 km (e.g. large parrotfishes and wrasses), while others move tens to hundreds (e.g. some groupers, emperors, snappers and jacks) or thousands of kilometres (e.g. some sharks and tuna). Larval dispersal distances tend to be <5–15 km, and self‐recruitment is common. Synthesising this information allows us, for the first time, to provide species, specific advice on the size, spacing and location of marine reserves in tropical marine ecosystems to maximise benefits for conservation and fisheries management for a range of taxa. We recommend that: (i) marine reserves should be more than twice the size of the home range of focal species (in all directions), thus marine reserves of various sizes will be required depending on which species require protection, how far they move, and if other effective protection is in place outside reserves; (ii) reserve spacing should be <15 km, with smaller reserves spaced more closely; and (iii) marine reserves should include habitats that are critical to the life history of focal species (e.g. home ranges, nursery grounds, migration corridors and spawning aggregations), and be located to accommodate movement patterns among these. We also provide practical advice for practitioners on how to use this information to design, evaluate and monitor the effectiveness of marine reserve networks within broader ecological, socioeconomic and management contexts.     

Pause point spectra in DNA constant-force unzipping

Under constant applied force, the separation of double-stranded DNA into two single strands is known to proceed through a series of pauses and jumps. Given experimental traces of constant-force unzipping, we present a method whereby the locations of pause points can be extracted in the form of a pause point spectrum. A simple theoretical model of DNA constant-force unzipping is presented, which generates theoretical pause point spectra through Monte Carlo simulation of the unzipping process. The locations of peaks in the experimental and theoretical pause point spectra are found to be nearly coincident below 6000 basepairs for unzipping the bacteriophage lambda-genome. The model only requires the sequence, temperature, and a set of empirical basepair binding and stacking energy parameters, and the good agreement with experiment suggests that pause point locations are primarily determined by the DNA sequence. The model is also used to predict pause point spectra for the bacteriophage phi X174 genome. The algorithm for extracting the pause point spectrum might also be useful for studying related systems which exhibit pausing behavior such as molecular motors.     

RNA structures facilitate recombination-mediated gene swapping in HIV-1

Many viruses, including retroviruses, undergo frequent recombination, a process which can increase their rate of adaptive evolution. In the case of HIV, recombination has been responsible for the generation of numerous intersubtype recombinant variants with epidemiological importance in the AIDS pandemic. Although it is known that fragments of genetic material do not combine randomly during the generation of recombinant viruses, the mechanisms that lead to preferential recombination at specific sites are not fully understood. Here we reanalyze recent independent data defining (i) the structure of a complete HIV-1 RNA genome and (ii) favorable sites for recombination. We show that in the absence of selection acting on recombinant genomes, regions harboring RNA structures in the NL4-3 model strain are strongly predictive of recombination breakpoints in the HIV-1 env genes of primary isolates. In addition, we found that breakpoints within recombinant HIV-1 genomes sampled from human populations, which have been acted upon extensively by natural selection, also colocalize with RNA structures. Critically, junctions between genes are enriched in structured RNA elements and are also preferred sites for generating functional recombinant forms. These data suggest that RNA structure-mediated recombination allows the virus to exchange intact genes rather than arbitrary subgene fragments, which is likely to increase the overall viability and replication success of the recombinant HIV progeny.     

Compartmentalization directs assembly of the signal recognition particle

Many ribonucleoprotein complexes assemble stepwise in distinct cellular compartments, a process that usually involves bidirectional transport of both RNA and proteins between the nucleus and cytoplasm. The biological rationale for such complex transport steps in RNP assembly is obscure. One important example is the eukaryotic signal recognition particle (SRP), a cytoplasmic RNP consisting of one RNA and six proteins. Prior in vivo studies support an "SRP54-late" assembly model in which all SRP proteins, except SRP54, are imported from the cytoplasm to the nucleus to bind SRP RNA. This partially assembled complex is then exported to the cytoplasm where SRP54 binds and forms the SRP holocomplex. Here we show that native SRP assembly requires segregated and ordered binding by its protein components. A native ternary complex forms in vitro when SRP19 binds the SRP RNA prior to binding by SRP54, which approximates the eukaryotic cellular pathway. In contrast, the presence of SRP54 disrupts native assembly of SRP19, such that two RNA-binding loops in SRP19 misfold. These results imply that SRP54 must be sequestered during early SRP assembly steps, as apparently occurs in vivo, for proper assembly of the SRP to occur. Our findings emphasize that spatial compartmentalization provides an additional level of regulation that prevents competition among components and can function to promote native assembly of the eukaryotic SRP.     

The Khatri Sikh Diabetes Study (SDS): study design, methodology, sample collection, and initial results

Non-insulin-dependent diabetes mellitus, or type 2 diabetes (T2DM), has become a major public health problem in India. The high prevalence, relatively young age of onset, and strong familial aggregation of T2DM in some Indian communities remains to be explained. Many of the traditional risk factors established for European populations do not appear to be present in Asian Indians. Phase I of the Sikh Diabetes Study (SDS) was launched to build the population resources required to initiate a large-scale genetic epidemiological study of diabetes in an Asian Indian population. The SDS is focused on the Khatri Sikh population of North India. In all, 1,892 subjects were enrolled to participate in the family-based study; 1,623 of these subjects belong to 324 families, each of which has at least 2 siblings affected with T2DM. The sample included 1,288 individuals affected with T2DM (siblings, parents, or relatives) and 335 unaffected siblings, parents, or relatives. The remaining 269 subjects were unrelated nondiabetic control subjects, including unaffected spouses of probands or siblings. This primarily nonvegetarian, nonsmoking endogamous caste group has presented an unusual clinical picture of uneven distribution of adiposity and a high rate of T2DM with secondary complications. Such well-characterized population isolates may offer unique advantages in mapping genes for common complex diseases.