Natural variability in Drosophila larval and pupal NaCl tolerance.

The regulation of NaCl is essential for the maintenance of cellular tonicity and functionality, and excessive salt exposure has many adverse effects. The fruit fly, Drosophila melanogaster, is a good osmoregulator and some strains can survive on media with very low or high NaCl content. Previous analyses of mutant alleles have implicated various stress signaling cascades in NaCl sensitivity or tolerance; however, the genes influencing natural variability of NaCl tolerance remain for the most part unknown. Here, we use two approaches to investigate natural variation in D. melanogaster NaCl tolerance. We describe four D. melanogaster lines that were selected for different degrees of NaCl tolerance, and present data on their survival, development, and pupation position when raised on varying NaCl concentrations. After finding evidence for natural variation in salt tolerance, we present the results of Quantitative Trait Loci (QTL) mapping of natural variation in larval and pupal NaCl tolerance, and identify different genomic regions associated with NaCl tolerance during larval and pupal development.

Quantitative trait locus mapping of gravitaxis behaviour in Drosophila melanogaster.

Drosophila melanogaster, like other organisms, move and orient themselves in response to the earth's gravitational force. The ability to sense and respond to gravity is essential for an organism to navigate and thrive in its environment. The genes underlying this behaviour in Drosophila remain elusive. Using 88 recombinant inbred lines, we have identified four quantitative trait loci (QTLs) that contribute to adult gravitaxis (geotaxis) behaviour in Drosophila. Candidate genes of interest were selected from the QTLs of highest significance based on their function in chordotonal organ formation. Quantitative complementation tests with these candidate genes revealed a role for skittles in adult gravitaxis behaviour in D. melanogaster.

Natural variation in food acquisition mediated via a Drosophila cGMP-dependent protein kinase.

In natural environments where food abundance and quality can change drastically over time, animals must continuously alter their food acquisition strategies. Although genetic variation contributes to this plasticity, the specific genes involved and their interactions with the environment are poorly understood. Here we report that natural variation in the Drosophila gene, foraging (for), which encodes a cGMP-dependent protein kinase (PKG), affects larval food acquisition in an environmentally dependent fashion. When food is plentiful, the wild-type rover (for(R)) allele confers lower food intake and higher glucose absorption than both the wild-type sitter (for(s)) allele and the mutant for(s2) allele. When food is scarce, for(R), for(s) and for(s2) larvae increase food intake to a common maximal level, but for(R) larvae retain their increased absorption efficiency. Changes in for expression can induce corrective behavioral modifications in response to food deprivation. When reared in environments with low food levels, for(R) larvae have higher survivorship and faster development than for(s) and for(s2) larvae. Together, these results show that natural variation in for has far reaching implications affecting a suite of phenotypes involved in the regulation of food acquisition.

Genetic and behavioral analysis of natural variation in Drosophila melanogaster pupation position.

Drosophila melanogaster pupae are exposed to many biotic and abiotic dangers while immobilized during several days of metamorphosis. As a passive defense mechanism, appropriate pupation site selection represents an important mitigation of these threats. Pupation site selection is sensitive to genetic and environmental influences, but the specific mechanisms of the behavior are largely unknown. Using a set of 76 recombinant inbred strains we identify a single quantitative trait locus, at polytene position 56A01-C11, associated with pupation site variation. We furthermore present a detailed investigation into the wandering behaviors of two strains expressing different pupation position tendencies, and identify behavioral differences. Larvae from a strain that tends to pupate relatively far from the food also tend to travel significantly farther from the media during wandering. We did not observe consistent differences in either the number or duration of wandering forays made by near or far pupating strains. The ability of larvae to integrate several internal and external environmental cues while choosing a contextually appropriate pupation site, and specifically, the variation in this ability, presents a very interesting behavioral phenotype in this highly tractable genetic model organism.

Drosophila soluble guanylyl cyclase mutants exhibit increased foraging locomotion: behavioral and genomic investigations.

Genetic variation in the gene foraging (for) is associated with a natural behavioral dimorphism in the fruit fly, Drosophila melanogaster. Some larvae, called 'rovers', have increased foraging locomotion compared to others, called 'sitters', and this difference is directly related to for-encoded cGMP-dependent protein kinase (PKG) activity. Here we report that larvae with mutations in the gene dgcalpha1, which encodes a soluble guanylyl cyclase (sGC) subunit, have increases in both PKG activity and foraging locomotion. This is contrary to our original prediction that, based on the role of sGC in the synthesis of cGMP, dgcalpha1 mutant larvae would have deficient cGMP production leading to decreased PKG activation and thus reduced larval foraging locomotion. We performed DNA microarray analyses to compare transcriptional changes induced by a dgcalpha1 mutation in both rover and sitter wildtype genetic backgrounds. In either background, we identified many genes that are differentially transcribed, and interestingly, relatively few are affected in both backgrounds. Furthermore, several of these commonly affected genes are enhanced or suppressed in a background-dependent manner. Thus, genetic background has a critical influence on the molecular effects of this mutation. These findings will support future investigations of Drosophila foraging behavior.

Behavioral genetics: guanylyl cyclase prompts worms to party.

When it comes to foraging, there are two types of worm in the world: those who enjoy a party, and those who prefer to dine alone. Two recent reports describe roles for guanylyl cyclase in the neuromolecular signaling systems that effect this natural behavioral dimorphism.

The human oesophageal squamous epithelium exhibits a novel type of heat shock protein response.

The human oesophageal epithelium is subject to damage from thermal stresses and low extracellular pH that can play a role in the cancer progression sequence, thus identifying a physiological model system that can be used to determine how stress responses control carcinogenesis. The classic heat shock protein HSP70 is not induced but rather is down-regulated after thermal injury to squamous epithelium ex vivo; this prompted a longer-term study to address the nature of the heat shock response in this cell type. An ex vivo epithelial culture system was subsequently used to identify three major proteins of 78, 70, and 58 kDa, whose steady-state levels are elevated after heat shock. Two of the three heat shock proteins were identified by mass spectrometric sequencing to be the calcium-calmodulin homologue transglutaminase-3 (78 kDa) and a recently cloned oesophageal-specific gene called C1orf10, which encodes a 53-kDa putative calcium binding protein we have named squamous epithelial heat shock protein 53 (SEP53). The 70-kDa heat shock protein (we have named SEP70) was not identifiable by mass spectrometry, but it was purified and studied immunochemically to demonstrate that it is distinct from HSP70 protein. Monoclonal antibodies to SEP70 protein were developed to indicate that: (a) SEP70 is induced by exposure of cultured cells to low pH or glucose starvation, under conditions where HSP70 protein was strikingly down-regulated; and (b) SEP70 protein exhibits variable expression in preneoplastic Barrett's epithelium under conditions where HSP70 protein is not expressed. These results indicate that human oesophageal squamous epithelium exhibits an atypical heat shock protein response, presumably due to the evolutionary adaptation of cells within this organ to survive in an unusual microenvironment exposed to chemical, thermal and acid reflux stresses.

Synergistic activation of p53-dependent transcription by two cooperating damage recognition pathways.

High level activation of p53-dependent transcription occurs following cellular exposure to genotoxic damaging agents such as UV-C, while ionizing radiation damage does not induce a similarly potent induction of p53-dependent gene expression. Reasoning that one of the major differences between UV-C and ionizing radiation damage is that the latter does not inhibit general transcription, we attempted to reconstitute p53-dependent gene expression in ionizing irradiated cells by co-treatment with selected transcription inhibitors that alone do not activate p53. p53-dependent transcription can be dramatically enhanced by the treatment of ionizing irradiated cells with low doses of DRB, which on its own does not induce p53 activity. The mechanism of ionizing radiation-dependent activation of p53-dependent transcription using DRB is more likely due to inhibition of gene transcription rather than prolonged DNA damage, as the non-genotoxic and general transcription inhibitor Roscovitine also synergistically activates p53 function in ionizing irradiated cells. These results identify two distinct signal transduction pathways that cooperate to fully activate p53-dependent gene expression: one responding to lesions induced by ionizing radiation and the second being a kinase pathway that regulates general RNA Polymerase II activity.

Dephosphorylation of p53 at Ser20 after cellular exposure to low levels of non-ionizing radiation.

Induction of the transactivation function of p53 after cellular irradiation was studied under conditions in which upstream signaling events modulating p53 activation were uncoupled from those regulating stabilization. This investigation prompted the discovery of a novel radiation-responsive kinase pathway targeting Ser20 that results in the masking of the DO-1 epitope in undamaged cells. Unmasking of the DO-1 epitope via dephosphorylation occurs in response to low doses of non-ionizing radiation. Our data show that phosphorylation at Ser20 reduces binding of the mdm2 protein, suggesting that a function of the Ser20-kinase pathway may be to produce a stable pool of inactive p53 in undamaged cells which can be readily activated after cellular injury. Phospho-specific monoclonal antibodies were used to determine whether the Ser20 signaling pathway is coupled to the Ser15 and Ser392 radiation-responsive kinase pathways. These results demonstrated that: (1) dephosphorylation at Ser20 is co-ordinated with an increased steady-state phosphorylation at Ser392 after irradiation, without p53 protein stabilization, and (2) stabilization of p53 protein can occur without Ser15 phosphorylation at higher doses of radiation. These data show that the Ser20 and Ser392 phosphorylation sites are both targeted by an integrated network of signaling pathways which is acutely sensitive to radiation injury.

Novel phosphorylation sites of human tumour suppressor protein p53 at Ser20 and Thr18 that disrupt the binding of mdm2 (mouse double minute 2) protein are modified in human cancers.

The ability to separate the isoforms of human tumour suppressor protein p53 expressed in insect cells using heparin-Sepharose correlates with differences in the isoelectric point of p53, demonstrating that p53 can be heterogeneously modified and providing support for the use of insect cells as a model system for identifying novel signalling pathways that target p53. One p53 isoform that was reduced in its binding to the monoclonal antibody DO-1 could be stimulated in its binding to DO-1 by prior incubation with protein phosphatases, suggesting the presence of a previously unidentified N-terminal phosphorylation site capable of masking the DO-1 epitope. A synthetic peptide from the N-terminal domain of p53 containing phosphate at Ser(20) inhibited DO-1 binding, thus identifying the phosphorylation site responsible for DO-1 epitope masking. Monoclonal antibodies overlapping the DO-1 epitope were developed that are specific for phospho-Thr(18) (adjacent to the DO-1 epitope) and phospho-Ser(20) (within the DO-1 epitope) to determine whether direct evidence could be obtained for novel phosphorylation sites in human p53. A monoclonal antibody highly specific for phospho-Ser(20) detected significant phosphorylation of human p53 expressed in insect cells, whereas the relative proportion of p53 modified at Thr(18) was substantially lower. The relevance of these two novel phosphorylation sites to p53 regulation in human cells was made evident by the extensive phosphorylation of human p53 at Thr(18) and Ser(20) in a panel of human breast cancers with a wild-type p53 status. Phospho-Ser(20) or phospho-Thr(18) containing p53 peptides are as effective as the phospho-Ser(15) peptide at reducing mdm2 (mouse double minute 2) protein binding, indicating that the functional effects of these phosphorylation events might be to regulate the binding of heterologous proteins to p53. These results provide evidence in vivo for two novel phosphorylation sites within p53 at Ser(20) and Thr(18) that can affect p53 protein-protein interactions and indicate that some human cancers might have amplified one or more Ser(20) and Thr(18) kinase signalling cascades to modulate p53 activity.

Expression of reducing sugar accumulation in interspecific somatic hybrids of potato.

A somatic hybridisation programme was undertaken to evaluate the expression of reducing sugar accumulation in potato. Interspecific hybrids created between the Solanum tuberosum cultivar Record and the diploid species Solanum phureja were evaluated at the morphological and molecular levels. These analyses indicated that the protoplast regenerants were partial (asymmetric) hybrids which had undergone elimination of S. phureja chromosomes. Tubers of the parents exhibited significant differences for reducing sugar accumulation during cold storage with S. phureja having lower levels of glucose and fructose than Record. The somatic hybrids resembled the S. phureja parent in terms of reducing sugar accumulation demonstrating that low reducing sugar accumulation is dominant to high reducing sugar accumulation in these particular genotypes. These results are discussed in relation to the exploitation of asymmetric hybridisation for the production of potato genotypes for the potato processing industry.