Structure and organization of the bovine beta-globin genes

Genomic clones spanning the entire cow beta-globin gene locus have been isolated and characterized. These clones demonstrate that the linkage of embryonic-like (epsilon) genes and pseudogenes (psi) to the previously described fetal (gamma) and adult (beta) genes is as follows: 5'-epsilon 3-epsilon 4-psi 3-beta-epsilon 1-epsilon 2-psi 1- psi 2-gamma-3'. Present data indicate that, like that of the goat, the fetal and adult genes arose via block duplication of an ancestral four- gene set: epsilon-epsilon-psi-beta. This duplication event preceded the divergence of cows and goats, which occurred greater than or equal to 18-20 Myr ago. However, cows do not have the additional four-gene block containing a preadult/stress globin gene (beta C). Furthermore, the cow fetal cluster contains an extra beta-like pseudogene, which apparently arose by a small-scale duplication. The fixation of this duplication may indicate a possible evolutionary role for pseudogenes.      

Microsatellite allele frequencies in humans and chimpanzees, with implications for constraints on allele size

The distributions of allele sizes at eight simple-sequence repeat (SSR) or microsatellite loci in chimpanzees are found and compared with the distributions previously obtained from several human populations. At several loci, the differences in average allele size between chimpanzees and humans are sufficiently small that there might be a constraint on the evolution of average allele size. Furthermore, a model that allows for a bias in the mutation process shows that for some loci a weak bias can account for the observations. Several alleles at one of the loci (Mfd 59) were sequenced. Differences between alleles of different lengths were found to be more complex than previously assumed. An 8-base-pair deletion was present in the nonvariable region of the chimpanzee locus. This locus contains a previously unrecognized repeated region, which is imperfect in humans and perfect in chimpanzees. The apparently greater opportunity for mutation conferred by the two perfect repeat regions in chimpanzees is reflected in the higher variance in repeat number at Mfd 59 in chimpanzees than in humans. These data indicate that interspecific differences in allele length are not always attributable to simple changes in the number of repeats.      

Experimental evaluation of the relationship between lethal or non-lethal virulence and transmission success in malaria parasite infections

Background  

Evolutionary theory suggests that the selection pressure on parasites to maximize their transmission determines their optimal host exploitation strategies and thus their virulence. Establishing the adaptive basis to parasite life history traits has important consequences for predicting parasite responses to public health interventions. In this study we examine the extent to which malaria parasites conform to the predicted adaptive trade-off between transmission and virulence, as defined by mortality. The majority of natural infections, however, result in sub-lethal virulent effects (e.g. anaemia) and are often composed of many strains. Both sub-lethal effects and pathogen population structure have been theoretically shown to have important consequences for virulence evolution. Thus, we additionally examine the relationship between anaemia and transmission in single and mixed clone infections.     

A novel thermosensitive escape behavior in Drosophila larvae

We describe a novel thermosensitive escape behavior in Drosophila larvae and a simple assay to accurately define the response temperature. When a larva is placed in a droplet of water that is subsequently heated, a stereotypical escape response is robustly elicited at 29°C. Larvae defective for the painless TRP receptor, or blocked in the function of class IV multi-dendritic sensory dendrites respond to this stimulus at reproducibly higher temperature (34°C). The escape response has novel behavioral components and a lower temperature threshold in comparison with the responses to touch with a hot needle. Furthermore the assay minimizes operator bias that is present in current tests of thermosensitive nociception and generates a precise determination of temperature at the point of response. This response is highly reproducible and directly applicable to genetic and neural circuit analysis of a simple escape behavior.     

Loss of phylogenetic information in chorion gene families of Bombyx mori gene conversion

The silkmoth chorion has provided a stimulating model for the study of evolution and developmental regulation of gene families. Previous attempts at inferring relationships among chorion sequences have been based on pairwise comparisons of overall similarity, a potentially problematic approach. To remedy this, we identified the alignable regions of low sequence variability and then analyzed this restricted database by parsimony and neighbor-joining methods. At the deepest level, the chorion sequence tree is split into two branches, called "alpha" and "beta." Within each branch, early- and late-expressing genes each constitute monophyletic groups, while the situation with middle-expressing genes remains uncertain. The HcB gene family appears to be the most basal beta-branch group, but this conclusion is qualified because the effect of gene conversion on branching order is unknown. Previous studies by Eickbush and colleagues have strongly suggested that ErA, HcA, and HcB families undergo gene conversion within a gene family, whereas the ErB family does not. The occurrence of conversion correlates with a particular tree structure; namely, branch lengths are much greater at the base of the family than at higher internodes and terminal branches. These observations raise the possibility that chorion gene families are defined by gene conversion events (reticulate evolution) rather than by descent with modification (synapomorphy).      

Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.The cytotoxic cell granule-secretory pathway depends on perforin (PFN) to deliver granzyme (Gzm) proteases to the cytosol of target cells where they induce apoptosis and other biological effects, such as inflammation.¹ Ring-shaped transmembrane PFN pores hereafter called ‘cylindrical pores'', are presumed to act as the gateway for cytosolic entry, either at the plasma membrane or after endocytosis.^{2, 3, 4} In either case the highly cationic Gzms are thought to diffuse through these cylindrical pores formed by poly-PFN. Nevertheless, a mechanistic understanding of the phenomenon (how the cationic globular protein exchanges from its carrier proteoglycan, serglycin, to the pore and crosses the plasma and/or vesicular membranes) has been lacking due to limitations in imaging technology and in our detailed understanding of the molecular forms that PFN may adopt following interaction with a target cell plasma membrane.Here we show under conditions where cylindrical pore formation is minimal,⁵ that granzyme B (GzmB) translocation readily occurs. We previously demonstrated that a prelude to granzyme translocation is PFN-mediated, Ca-independent phosphatidylserine (PS) externalization (flip-flop) measured by annexin-V and lactadherin binding.⁶ This rapid PS flip-flop also occurs when mouse CD8 cells contact antigen-pulsed target cells. Inasmuch as the proteinaceous cylinders offer a formidable barrier to lipid flow, we have speculated that the observed movement of anionic phospholipids to the external leaflet is due to the formation of proteo-lipidic structures, which consists of oligomerized PFN monomers bearing an arc morphology and plasma membrane lipids.^{6, 7, 8}In the work reported here, the topology of PFN embedded into homogeneous planar bilayers and tumor cell plasma membranes was imaged by atomic force microscopy (AFM) and deep etch electron microscopy (DEEM), respectively. Further, the influence of an anti-human PFN mAb (pf-80) that rescues target cells from necrosis,⁹ was examined. The AFM data show that PFN forms arcs as well as rings in planar bilayers, while conductance measurements across equivalent membranes in parallel experiments measured functional pore sizes consistent with these varied structures. The pf-80 mAb increased the frequency of arc formation and reduced conductance values. Interestingly, PS flip-flop and granzyme delivery were both increased in target cells after PFN oligomerization was interrupted by the pf-80 mAb. A similar effect was seen in T24 bladder carcinoma cells imaged by DEEM. Treatment with PFN leads to deposition of rings (barrel stave pores) and arcs and the pf-80 mAb increased the ratio of arcs to rings on the surface of these cells. We suggest that the observed protein arcs function as toroidal pores in whole cells, explaining PS flip-flop, and act as focal points for granzyme translocation across lipid bilayer.     

Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.