The genetic architecture of larval aggregation behavior in Drosophila.

Many insect species exhibit basal social behaviors such as aggregation, which play important roles in their feeding and mating ecologies. However, the evolutionary, genetic, and physiological mechanisms that regulate insect aggregation remain unknown for most species. Here, we used natural populations of Drosophila melanogaster to identify the genetic architecture that drives larval aggregation feeding behavior. By using quantitative and reverse genetic approaches, we have identified a complex neurogenetic network that plays a role in regulating the decision of larvae to feed in either solitude or as a group. Results from single gene, RNAi-knockdown experiments show that several of the identified genes represent key nodes in the genetic network that determines the level of aggregation while feeding. Furthermore, we show that a single non-coding variant in the gene CG14205, a putative acyltransferase, is associated with both decreased mRNA expression and increased aggregate formation, which suggests that it has a specific role in inhibiting aggregation behavior. Our results identify, for the first time, the genetic components which interact to regulate naturally occurring levels of aggregation in D. melanogaster larvae.

Converting GLX2-1 into an active glyoxalase II.

Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) exhibits extensive sequence similarity with GLX2 enzymes but is catalytically inactive with SLG, the GLX2 substrate. In an effort to identify residues essential for GLX2 activity, amino acid residues were altered at positions 219, 246, 248, 325, and 328 in GLX2-1 to be the same as those in catalytically active human GLX2. The resulting enzymes were overexpressed, purified, and characterized using metal analyses, fluorescence spectroscopy, and steady-state kinetics to evaluate how these residues affect metal binding, structure, and catalysis. The R246H/N248Y double mutant exhibited low level S-lactoylglutathione hydrolase activity, while the R246H/N248Y/Q325R/R328K mutant exhibited a 1.5-2-fold increase in k(cat) and a decrease in K(m) as compared to the values exhibited by the double mutant. In contrast, the R246H mutant of GLX2-1 did not exhibit glyoxalase 2 activity. Zn(II)-loaded R246H GLX2-1 enzyme bound 2 equiv of Zn(II), and (1)H NMR spectra of the Co(II)-substituted analogue of this enzyme strongly suggest that the introduced histidine binds to Co(II). EPR studies indicate the presence of significant amounts a dinuclear metal ion-containing center. Therefore, an active GLX2 enzyme requires both the presence of a properly positioned metal center and significant nonmetal, enzyme-substrate contacts, with tyrosine 255 being particularly important.

The metal ion requirements of Arabidopsis thaliana Glx2-2 for catalytic activity.

In an effort to better understand the structure, metal content, the nature of the metal centers, and enzyme activity of Arabidopsis thaliana Glx2-2, the enzyme was overexpressed, purified, and characterized using metal analyses, kinetics, and UV-vis, EPR, and (1)H NMR spectroscopies. Glx2-2-containing fractions that were purple, yellow, or colorless were separated during purification, and the differently colored fractions were found to contain different amounts of Fe and Zn(II). Spectroscopic analyses of the discrete fractions provided evidence for Fe(II), Fe(III), Fe(III)-Zn(II), and antiferromagnetically coupled Fe(II)-Fe(III) centers distributed among the discrete Glx2-2-containing fractions. The individual steady-state kinetic constants varied among the fractionated species, depending on the number and type of metal ion present. Intriguingly, however, the catalytic efficiency constant, k(cat)/K(m), was invariant among the fractions. The value of k(cat)/K(m) governs the catalytic rate at low, physiological substrate concentrations. We suggest that the independence of k(cat)/K(m) on the precise makeup of the active-site metal center is evolutionarily related to the lack of selectivity for either Fe versus Zn(II) or Fe(II) versus Fe(III), in one or more metal binding sites.

Human glyoxalase II contains an Fe(II)Zn(II) center but is active as a mononuclear Zn(II) enzyme.

Human glyoxalase II (Glx2) was overexpressed in rich medium and in minimal medium containing zinc, iron, or cobalt, and the resulting Glx2 analogues were characterized using metal analyses, steady-state and pre-steady-state kinetics, and NMR and EPR spectroscopies to determine the nature of the metal center in the enzyme. Recombinant human Glx2 tightly binds nearly 1 equiv each of Zn(II) and Fe. In contrast to previous reports, this study demonstrates that an analogue containing 2 equiv of Zn(II) cannot be prepared. EPR studies suggest that most of the iron in recombinant Glx2 is Fe(II). NMR studies show that Fe(II) binds to the consensus Zn(2) site in Glx2 and that this site can also bind Co(II) and Ni(II), suggesting that Zn(II) binds to the consensus Zn(1) site. The NMR studies also reveal the presence of a dinuclear Co(II) center in Co(II)-substituted Glx2. Steady-state and pre-steady-state kinetic studies show that Glx2 containing only 1 equiv of Zn(II) is catalytically active and that the metal ion in the consensus Zn(2) site has little effect on catalytic activity. Taken together, these studies suggest that Glx2 contains a Fe(II)Zn(II) center in vivo but that the catalytic activity is due to Zn(II) in the Zn(1) site.

The neural basis for insect pheromonal communication.

Insects rely on chemosensory signals to drive a multitude of behavioral decisions. From conspecific and mate recognition to aggression, the proper detection and processing of these chemical signals - termed pheromones - is crucial for insects' fitness. While the identities and physiological impacts of diverse insect pheromones have been known for many years, how these important molecules are perceived and processed by the nervous system to produce evolutionarily beneficial behaviors is still mostly unknown. Here we present an overview of the current state of research into the peripheral and central nervous system mechanisms that process and drive behavioral responses to diverse pheromonal cues.

Npas4 regulates a transcriptional program in CA3 required for contextual memory formation.

The rapid encoding of contextual memory requires the CA3 region of the hippocampus, but the necessary genetic pathways remain unclear. We found that the activity-dependent transcription factor Npas4 regulates a transcriptional program in CA3 that is required for contextual memory formation. Npas4 was specifically expressed in CA3 after contextual learning. Global knockout or selective deletion of Npas4 in CA3 both resulted in impaired contextual memory, and restoration of Npas4 in CA3 was sufficient to reverse the deficit in global knockout mice. By recruiting RNA polymerase II to promoters and enhancers of target genes, Npas4 regulates a learning-specific transcriptional program in CA3 that includes many well-known activity-regulated genes, which suggests that Npas4 is a master regulator of activity-regulated gene programs and is central to memory formation.