Evolutionary replacement of components in a salamander pheromone signaling complex: more evidence for phenotypic-molecular decoupling

In this article we explore the evolutionary history of a functional complex at the molecular level in plethodontid salamanders. The complex consists of a proteinaceous courtship pheromone, a pheromone-producing gland on the male's chin, and a set of behaviors for delivering the pheromone to the female. Long-term evolutionary stasis is the defining feature of this complex at both the morphological and behavioral levels. However, our previous assessment of the pheromone gene, plethodontid receptivity factor (PRF), revealed rapid evolution at the molecular level despite stasis at higher levels of organization. Analysis of a second pheromone gene, sodefrin precursor-like factor (SPF), now indicates that evolutionary decoupling in this complex is pervasive. The evolutionary profiles of SPF and PRF are remarkably similar in that: (a) both genes exhibit high levels of sequence diversity both within and across taxa, (b) genetic diversity has been driven by strong positive selection, and (c) the genes have evolved heterogeneously in different salamander lineages. The composition of the pheromone signal as a whole, however, has experienced an extraordinary evolutionary transition. Whereas SPF has been retained throughout the 100 MY radiation of salamanders, PRF has only recently been recruited to a pheromone function (27 million years ago). When SPF and PRF coexist in the same clade, they show contrasting patterns of evolution. When one shows rapid evolution driven by positive selection, the other shows neutral divergence restrained by purifying selection. In one clade, the origin and subsequent rapid evolution of PRF appear to have interfered with the evolution and persistence of SPF, leading to a pattern of evolutionary replacement. Overall, these two pheromone genes provide a revealing window on the dynamics that drive the evolution of multiple traits in a signaling complex.     

A mitochondrial stratigraphy for island southeast Asia

Island Southeast Asia (ISEA) was first colonized by modern humans at least 45,000 years ago, but the extent to which the modern inhabitants trace their ancestry to the first settlers is a matter of debate. It is widely held, in both archaeology and linguistics, that they are largely descended from a second wave of dispersal, proto-Austronesian-speaking agriculturalists who originated in China and spread to Taiwan approximately 5,500 years ago. From there, they are thought to have dispersed into ISEA approximately 4,000 years ago, assimilating the indigenous populations. Here, we demonstrate that mitochondrial DNA diversity in the region is extremely high and includes a large number of indigenous clades. Only a fraction of these date back to the time of first settlement, and the majority appear to mark dispersals in the late-Pleistocene or early-Holocene epoch most likely triggered by postglacial flooding. There are much closer genetic links to Taiwan than to the mainland, but most of these probably predated the mid-Holocene "Out of Taiwan" event as traditionally envisioned. Only approximately 20% at most of modern mitochondrial DNAs in ISEA could be linked to such an event, suggesting that, if an agriculturalist migration did take place, it was demographically minor, at least with regard to the involvement of women.     

Role of the N-terminal hydrophilic domain of acyl-coenzyme A:cholesterol acyltransferase 1 on the enzyme's quaternary structure and catalytic efficiency

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an enzyme involved in cellular cholesterol homeostasis and atherosclerosis. ACAT1 is an allosteric enzyme responding to its substrate cholesterol in a sigmoidal manner. It is a homotetrameric protein that spans the membrane multiple times, with its N-terminal 131 hydrophilic amino acids residing at the cytoplasmic side of the endoplasmic reticulum. This region contains two closely linked putative alpha-helices. Our current studies show that this region contains a dimer-forming motif. Adding this motif to the bacterial glutathione S-transferase (GST) converted the homodimeric GST to a tetrameric fusion protein. Conversely, deleting this motif from the full-length ACAT1 converted the enzyme from a homotetramer to a homodimer. The dimeric ACAT1 remains enzymatically active. Its biochemical characteristics, including the sigmoidal response to cholesterol, the IC(50) value toward a specific ACAT inhibitor, and sensitivity toward heat inactivation, are essentially unaltered. On the other hand, the dimeric ACAT1 exhibits a 5-10-fold increase in the V(max) of the overall reaction and a 2.2-fold increase in the K(m) for oleoyl-coenzyme. Thus, deleting the dimer-forming motif near the N-terminus changes ACAT1 from its tetrameric form to a dimeric form and increases its catalytic efficiency.     

Simultaneous suppression of multiple genes by single transgenes. Down-regulation of three unrelated lignin biosynthetic genes in tobacco

Many reports now describe the manipulation of plant metabolism by suppressing the expression of single genes. The potential of such work could be greatly expanded if multiple genes could be coordinately suppressed. In the work presented here, we test a novel method for achieving this by using single chimeric constructs incorporating partial sense sequences for multiple genes to target suppression of two or three lignin biosynthetic enzymes. We compare this method with a more conventional approach to achieving the same end by crossing plants harboring different antisense transgenes. Our results indicate that crossing antisense plants is less straightforward and predictable in outcome than anticipated. Most progeny had higher levels of target enzyme activity than predicted and had lost the expected modifications to lignin structure. In comparison, plants transformed with the chimeric partial sense constructs had more consistent high level suppression of target enzymes and had significant changes to lignin content, structure, and composition. It was possible to suppress three target genes coordinately using a single chimeric construct. Our results indicate that chimeric silencing constructs offer great potential for the rapid and coordinate suppression of multiple genes on diverse biochemical pathways and that the technique therefore deserves to be adopted by other researchers.     

The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton

In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B" regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.     

Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo.

The mouse develops five pairs of mammary glands that arise during mid-gestation from five pairs of placodes of ectodermal origin. We have investigated the molecular mechanisms of mammary placode development using Lef1 as a marker for the epithelial component of the placode, and mice deficient for Fgf10 or Fgfr2b, both of which fail to develop normal mammary glands. Mammary placode induction involves two different signaling pathways, a FGF10/FGFR2b-dependent pathway for placodes 1, 2, 3 and 5 and a FGF10/FGFR2b-independent pathway for placode 4. Our results also suggest that FGF signaling is involved in the maintenance of mammary bud 4, and that Fgf10 deficient epithelium can undergo branching morphogenesis into the mammary fat pad precursor.     

Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Semi-continuous scale-down models for clone and operating parameter screening in perfusion bioreactors

Perfusion cell culture, confined traditionally to the production of fragile molecules, is currently gaining broader attention in the biomanufacturing of therapeutic proteins. The development of these processes is made difficult by the limited availability of appropriate scale-down models. This is due to the continuous operation that requires complex control and cell retention capacity. For example, the determination of an optimal perfusion and bleed rate for continuous cell culture is often performed in scale-down bioreactors and requires a substantial amount of time and effort. To increase the experimental throughput and decrease the required workload, a semi-continuous procedure, referred to as the VCD_max (viable cell density) approach, has been developed on the basis of shake tubes (ST) and deepwell plates (96-DWP). Its effectiveness has been demonstrated for 12 different CHO-K1-SV cell lines expressing an IgG1. Further, its reliability has been investigated through proper comparisons with perfusion runs in lab-scale bioreactors. It was found that the volumetric productivity and the CSPR_min (cell specific perfusion rate) determined using the ST and 96-DWP models were successfully (mostly within the experimental error) confirmed in lab-scale bioreactors, which then covered a significant scale-up from the half milliliter to the liter scale. These scale-down models are very useful to design and scale-up optimal bioreactor operating conditions as well as screening for different media and cell lines.     

Testing the impact of calibration on molecular divergence times using a fossil-rich group: the case of Nothofagus (Fagales)

Although temporal calibration is widely recognized as critical for obtaining accurate divergence-time estimates using molecular dating methods, few studies have evaluated the variation resulting from different calibration strategies. Depending on the information available, researchers have often used primary calibrations from the fossil record or secondary calibrations from previous molecular dating studies. In analyses of flowering plants, primary calibration data can be obtained from macro- and mesofossils (e.g., leaves, flowers, and fruits) or microfossils (e.g., pollen). Fossil data can vary substantially in accuracy and precision, presenting a difficult choice when selecting appropriate calibrations. Here, we test the impact of eight plausible calibration scenarios for Nothofagus (Nothofagaceae, Fagales), a plant genus with a particularly rich and well-studied fossil record. To do so, we reviewed the phylogenetic placement and geochronology of 38 fossil taxa of Nothofagus and other Fagales, and we identified minimum age constraints for up to 18 nodes of the phylogeny of Fagales. Molecular dating analyses were conducted for each scenario using maximum likelihood (RAxML + r8s) and Bayesian (BEAST) approaches on sequence data from six regions of the chloroplast and nuclear genomes. Using either ingroup or outgroup constraints, or both, led to similar age estimates, except near strongly influential calibration nodes. Using "early but risky" fossil constraints in addition to "safe but late" constraints, or using assumptions of vicariance instead of fossil constraints, led to older age estimates. In contrast, using secondary calibration points yielded drastically younger age estimates. This empirical study highlights the critical influence of calibration on molecular dating analyses. Even in a best-case situation, with many thoroughly vetted fossils available, substantial uncertainties can remain in the estimates of divergence times. For example, our estimates for the crown group age of Nothofagus varied from 13 to 113 Ma across our full range of calibration scenarios. We suggest that increased background research should be made at all stages of the calibration process to reduce errors wherever possible, from verifying the geochronological data on the fossils to critical reassessment of their phylogenetic position.     

Development of sensory innervation in chick skin: comparison of nerve fibre and chondroitin sulphate distributions in vivo and in vitro

In bird skin, nerve fibres develop in the dermis but do not enter the epidermis. In co-cultures of 7-day-old chick embryo dorsal root ganglia and epidermis, the neurites also avoid the epidermis. Previous studies have shown that chondroitin sulphate proteoglycans may be involved. Chondroitin sulphate has therefore been visualized by immunocytochemistry, using themonoclonal antibody CS-56, both in vivo and in vitro using light and electron microscopy. Its distribution was compared to those of 2 other chondroitin sulphate epitopes and to that of the growing nerve fibres. In cultures of epidermis from 7-day-old embryonic chicks, immunoreactivity is found uniformly around the epidermal cells while at 7.5 days the distribution in dermis is heterogeneous, and particularly marked in feather buds. In vivo, chondroitin sulphate immunoreactivity is detected in the epidermis, on the basal lamina, on the surfaces of fibroblasts and along collagen fibrils. This localization is complementary to the distribution of cutaneous nerves. Chondroitin sulphate in the basal lamina could prevent innervation of the epidermis and the dermal heterogeneities could partly explain the nerve fibres surrounding the base of the feathers. Chondroitin sulphate could therefore be important for neural guidance in developing chick skin.