Quantitative genomics of aggressive behavior in Drosophila melanogaster

Aggressive behavior is important for animal survival and reproduction, and excessive aggression is an enormous social and economic burden for human society. Although the role of biogenic amines in modulating aggressive behavior is well characterized, other genetic mechanisms affecting this complex behavior remain elusive. Here, we developed an assay to rapidly quantify aggressive behavior in Drosophila melanogaster, and generated replicate selection lines with divergent levels of aggression. The realized heritability of aggressive behavior was approximately 0.10, and the phenotypic response to selection specifically affected aggression. We used whole-genome expression analysis to identify 1,539 probe sets with different expression levels between the selection lines when pooled across replicates, at a false discovery rate of 0.001. We quantified the aggressive behavior of 19 mutations in candidate genes that were generated in a common co-isogenic background, and identified 15 novel genes affecting aggressive behavior. Expression profiling of genetically divergent lines is an effective strategy for identifying genes affecting complex traits.     

Epimutation of the TNDM locus and the Beckwith–Wiedemann syndrome centromeric locus in individuals with transient neonatal diabetes mellitus

Transient neonatal diabetes mellitus (TNDM) is characterised by intra-uterine growth retardation, while Beckwith–Wiedemann syndrome (BWS) is a clinically heterogeneous overgrowth syndrome. Both TNDM and BWS may be caused by aberrant loss of methylation (LOM) at imprinted loci on chromosomes 6q24 and 11p15.5 respectively. Here we describe two patients with a clinical diagnosis of TNDM caused by LOM at the maternally methylated imprinted domain on 6q24; in addition, these patients had LOM at the centromeric differentially methylated region of 11p15.5. This shows that imprinting anomalies can affect more than one imprinted locus and may alter the clinical presentation of imprinted disease.     

Development and Application of a Predictive Model of Freshwater Fish Assemblage Composition to Evaluate River Health in Eastern Australia

Multivariate predictive models are widely used tools for assessment of aquatic ecosystem health and models have been successfully developed for the prediction and assessment of aquatic macroinvertebrates, diatoms, local stream habitat features and fish. We evaluated the ability of a modelling method based on the River InVertebrate Prediction and Classification System (RIVPACS) to accurately predict freshwater fish assemblage composition and assess aquatic ecosystem health in rivers and streams of south-eastern Queensland, Australia. The predictive model was developed, validated and tested in a region of comparatively high environmental variability due to the unpredictable nature of rainfall and river discharge. The model was concluded to provide sufficiently accurate and precise predictions of species composition and was sensitive enough to distinguish test sites impacted by several common types of human disturbance (particularly impacts associated with catchment land use and associated local riparian, in-stream habitat and water quality degradation). The total number of fish species available for prediction was low in comparison to similar applications of multivariate predictive models based on other indicator groups, yet the accuracy and precision of our model was comparable to outcomes from such studies. In addition, our model developed for sites sampled on one occasion and in one season only (winter), was able to accurately predict fish assemblage composition at sites sampled during other seasons and years, provided that they were not subject to unusually extreme environmental conditions (e.g. extended periods of low flow that restricted fish movement or resulted in habitat desiccation and local fish extinctions).     

Quantitative trait loci for locomotor behavior in Drosophila melanogaster

Locomotion is an integral component of most animal behaviors and many human diseases and disorders are associated with locomotor deficits, but little is known about the genetic basis of natural variation in locomotor behavior. Locomotion is a complex trait, with variation attributable to the joint segregation of multiple interacting quantitative trait loci (QTL), with effects that are sensitive to the environment. We assessed variation in a component of locomotor behavior (locomotor reactivity) in a population of 98 recombinant inbred lines of Drosophila melanogaster and mapped four QTL affecting locomotor reactivity by linkage to polymorphic roo transposable element insertion sites. We used complementation tests of deficiencies to fine map these QTL to 12 chromosomal regions and complementation tests of mutations to identify 13 positional candidate genes affecting locomotor reactivity, including Dopa decarboxylase (Ddc), which catalyzes the final step in the synthesis of serotonin and dopamine. Linkage disequilibrium mapping in a population of 164 second chromosome substitution lines derived from a single natural population showed that polymorphisms at Ddc were associated with naturally occurring genetic variation in locomotor behavior. These data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in locomotor reactivity.     

Dynamic genetic interactions determine odor-guided behavior in Drosophila melanogaster

Understanding the genetic architecture of complex traits requires identification of the underlying genes and characterization of gene-by-gene and genotype-by-environment interactions. Behaviors that mediate interactions between organisms and their environment are complex traits expected to be especially sensitive to environmental conditions. Previous studies on the olfactory avoidance response of Drosophila melanogaster showed that the genetic architecture of this model behavior depends on epistatic networks of pleiotropic genes. We performed a screen of 1339 co-isogenic p[GT1]-element insertion lines to identify novel genes that contribute to odor-guided behavior and identified 55 candidate genes with known p[GT1]-element insertion sites. Characterization of the expression profiles of 10 p[GT1]-element insertion lines showed that the effects of the transposon insertions are often dependent on developmental stage and that hypomorphic mutations in developmental genes can elicit profound adult behavioral deficits. We assessed epistasis among these genes by constructing all possible double heterozygotes and measuring avoidance responses under two stimulus conditions. We observed enhancer and suppressor effects among subsets of these P-element-tagged genes, and surprisingly, epistatic interactions shifted with changes in the concentration of the olfactory stimulus. Our results show that the manifestation of epistatic networks dynamically changes with alterations in the environment.     

Quantitative trait loci for thermotolerance phenotypes in Drosophila melanogaster

For insects, temperature is a major environmental variable that can influence an individual's behavioral activities and fitness. Drosophila melanogaster is a cosmopolitan species that has had great success in adapting to and colonizing diverse thermal niches. This adaptation and colonization has resulted in complex patterns of genetic variation in thermotolerance phenotypes in nature. Although extensive work has been conducted documenting patterns of genetic variation, substantially less is known about the genomic regions or genes that underlie this ecologically and evolutionarily important genetic variation. To begin to understand and identify the genes controlling thermotolerance phenotypes, we have used a mapping population of recombinant inbred (RI) lines to map quantitative trait loci (QTL) that affect variation in both heat- and cold-stress resistance. The mapping population was derived from a cross between two lines of D. melanogaster (Oregon-R and 2b) that were not selected for thermotolerance phenotypes, but exhibit significant genetic divergence for both phenotypes. Using a design in which each RI line was backcrossed to both parental lines, we mapped seven QTL affecting thermotolerance on the second and third chromosomes. Three of the QTL influence cold-stress resistance and four affect heat-stress resistance. Most of the QTL were trait or sex specific, suggesting that overlapping but generally unique genetic architectures underlie resistance to low- and high-temperature extremes. Each QTL explained between 5 and 14% of the genetic variance among lines, and degrees of dominance ranged from completely additive to partial dominance. Potential thermotolerance candidate loci contained within our QTL regions are identified and discussed.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Alendronate promotes plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin degradation sites within the MMP-9 catalytic domain

Irreversible MMP-9 inhibition is considered a significant therapeutic goal in inflammatory, vascular and tumour pathology. We report that divalent cation chelators Alendronate and EDTA not only directly inhibited MMP-9 but also promoted irreversible plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin-degradation sites within the MMP-9 catalytic-domain and producing an inhibitory hemopexin-domain fragment. This effect was also observed using MDA-MB-231 breast cancer cells, which activated exogenous plasminogen to degrade endogenous proMMP-9 in the presence of Alendronate or EDTA. Degradation-mediated inactivation of proMMP-9 occurred in the absence of transient activation, attesting to the incapacity of plasmin to directly activate proMMP-9 and direct MMP-9 inhibition by Alendronate and EDTA. Our study provides a novel rational for therapeutic Alendronate use in MMP-9-dependent pathology characterised by plasminogen activation.