Lateral gene transfer and the complex distribution of insertions in eukaryotic enolase

Insertions and deletions in protein-coding genes are relatively rare events compared with sequence substitutions because they are more likely to alter the tertiary structure of the protein. For this reason, insertions and deletions which are clearly homologous are considered to be stable characteristics of the proteins where they are found, and their presence and absence has been used extensively to infer large-scale evolutionary relationships and events. Recently, however, it has been shown that the pattern of highly conserved, clearly homologous insertions at positions with no other detectable homoplasy can be incongruent with the phylogeny of the genes or organisms in which they are found. One case where this has been reported is in the enolase genes of apicomplexan parasites and ciliates, which share homologous insertions in a highly conserved region of the gene with the apparently distantly related enolases of plants. Here we explore the distribution of this character in enolase genes from the third major alveolate group, the dinoflagellates, as well as two groups considered to be closely related to alveolates, haptophytes and heterokonts. With these data, all major groups of the chromalveolates are represented, and the distribution of these insertions is shown to be far more complicated than previously believed. The incongruence between this pattern, the known evolutionary relationships between the organisms, and enolase phylogeny itself cannot be explained by any single event or type of event. Instead, the distribution of enolase insertions is more likely the product of several forces that may have included lateral gene transfer, paralogy, and/or recombination. Of these, lateral gene transfer is the easiest to detect and some well-supported cases of eukaryote-to-eukaryote lateral transfer are evident from the phylogeny.     

A role for hTRPC1 and lipid raft domains in store-mediated calcium entry in human platelets

We have previously suggested that store-mediated Ca2+ entry (SMCE) in human platelets may be activated by a secretion-like coupling model, involving de novo coupling of the type II inositol 1,4,5-trisphosphate receptor (IP(3)RII) to the putative Ca2+ entry channel, hTRPC1. In other cells, hTRPC1 has been reported to be associated with cholesterol-rich lipid raft domains (LRDs) in the plasma membrane. Here we have shown that hTRPC1 is largely associated with detergent-resistant platelet membranes, from which it is partially released when the cells are depleted of cholesterol by treatment with methyl-beta-cyclodextrin (MBCD). MBCD treatment inhibited thapsigargin (TG)-evoked SMCE in a concentration-dependent manner, reducing it to 38.1+/-4.1% at a concentration of 10mM. Similarly, the Ca2+ entry evoked by thrombin (1unit/ml) was reduced to 48.2+/-4.5% of control following MBCD (10mM) treatment. Thrombin- and TG-evoked coupling between IP(3)RII and hTRPC1 was also reduced following cholesterol depletion. These results suggest that hTRPC1 is associated with LRDs in human platelets and that these domains are important for its participation in SMCE.     

P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis

Arabidopsis thaliana has eight genes encoding members of the type 1_B heavy metal–transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.     

Microsatellite markers to assess the influence of population size, isolation and demographic change on the genetic structure of the UK butterfly Polyommatus bellargus

Five microsatellite DNA markers were isolated and used to quantify population genetic structure among a subset of UK populations of the Adonis blue (Polyommatus bellargus Rottemburg). Specifically, whether population size, degree of isolation or history of bottlenecking in 1976-1978 can explain current patterns of genetic variation. The butterfly is at its northern range limit in the UK, where it exists as a highly fragmented metapopulation on isolated pockets of calcareous grassland. Most populations were affected by a severe bottleneck in the late 1970s, when a drought caused the host plant (Hippocrepis comosa) to wilt. Mantel tests and spatial autocorrelation analysis indicated a significant effect of isolation by distance among the UK populations, a relationship that broke down at greater geographical scales (> 23.85 km), probably because of large areas of unsuitable habitat presenting barriers to gene flow. Similarly, amova revealed that variation among geographical regions was almost double that observed within regions. Larger populations were found to support significantly higher levels of genetic diversity, suggesting that small populations may lose genetic diversity through drift. If, as in other butterfly species, low genetic diversity increases the probability of population extinction, then these populations are likely to be under threat. Neither isolation nor a history of bottlenecks appeared to influence genetic diversity. The results indicate that adequate population size a crucial factor in the conservation of genetic diversity in P. bellargus in the UK.     

Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis

Lysosome-related organelles are cell type-specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell-specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates.     

Aquatic biodiversity and saline lakes: Lake Bogoria National Reserve,Kenya

Lake Bogoria, in the Rift Valley of Kenya is an extreme saline lake (conductivity 40–80 mS cm^–1, alkalinity 1500 m equ l^–1). It is hydrologically more stable than the other, endorheic lakes in Kenya, because it is deep – maximum depth at present just over 10 m in an area of 3000 ha – and so does not have periods when it is dry. It is ecologically simple, with only one species dominating the phytoplankton – the cyanobacterium `spirulina', Arthrospira fusiformis. Its biomass and productivity were very high – biomass between 38 and 365 g l<sup>–1</sup> chlorophyll `a' and 3.4–21 × 10³ coils ml^–1 and net production between 0.24 and 1 gm C m³ h, the latter in a narrow zone of less than a metre. There were no macro-zooplankton in the plankton and the only grazer of A. fusiformis was the lesser flamingo, Phoeniconaias minor,which occurred irregularly in very high concentrations (in excess of 1 × 10⁶). Detritivory in the benthos was effected by a single chironomid species, Paratendipes sp., at a maximum density of 4 × 10⁴ m^–2. The mean daily emergence of adult chironomids was estimated to be 1 × 10³ m^–2, the maximum 3. There was no littoral plant community within the lake but 44 dicotyledonous and 31 monocotyledonous plant species in the drawn-down zone and adjacent to it. A diverse draw-down terrestrial invertebrate fauna, only superficially described here, processed the flamingo feathers and carcasses, with other detritus such as chironomid pupal exuviae and decaying A. fusiformis scum. About 50 bird species depended upon the chironomids, either as they emerged through the water column as flying adults or later on the shoreline as floating pupal exuvia and dead adults. The lake has high conservation value because of three bird species in particular – lesser flamingo, Cape teal and black-necked grebe. The former provides real economic value in a region otherwise impoverished, because of the spectacle of tens of thousands of flamingos set against the landscape of hot springs and fumaroles at the lake edge, which draws 15000 visitors per annum. P. minor has experienced three periods during the past ten years when major mortalities have occurred, the last of which killed 700 birds day^–1. This could have involved as many as 200000 birds (about 1/5th of the maximum population at this lake) if mortality was at a constant rate for the nine months it was observed. Causes of mortality have been suggested as avian tuberculosis, poisoning from cyanobacterial toxins or from heavy metal contamination at Lake Nakuru, but it is still not yet clear what contribution each makes to the problem.     

Eye size in birds and the timing of song at dawn

Why do different species of birds start their dawn choruses at different times? We test the hypothesis that the times at which different species start singing at dawn are related to their visual capability at low light intensities. Birds with large eyes can achieve greater pupil diameters and hence, all other things being equal, greater visual sensitivity and resolution than birds with small eyes. We estimated the maximum pupil diameter of passerine birds by measuring the diameter of the exposed eye surface, and measured the times of the first songs at dawn of songbirds present in different bird communities, and the light intensities at these times. Using phylogenetic comparative analyses, we found that songbirds with large eyes started to sing at lower light intensities (and therefore earlier) than species with smaller eyes. These relationships were stronger when differences in body size were controlled for statistically, and were consistent between two phylogenies and when species were treated as independent data points. Our results therefore provide robust support for the hypothesis that visual capability at low light levels influences the times at which birds start to sing at dawn.     

Diagnostic techniques for clinical investigation of laboratory zebrafish

Unexpected morbidity and mortality of aquatic animal models represent a significant problem for researchers. The authors outline the basic procedures used to diagnose disease outbreaks in laboratory zebrafish colonies, and provide a basic framework for initiating clinical investigations.