Evolution of clonality and polyploidy in a weevil system

The increased interest in asexual organisms calls for in-depth studies of asexual complexes that actively give rise to new clones. We present an extensive molecular study of the Otiorhynchus scaber (Coleoptera, Curculionidae) weevil system. Three forms have traditionally been recognized: diploid sexuals, triploid, and tetraploid parthenogens. All forms coexist in a small central area, but only the polyploid parthenogens have colonized marginal areas. Analyzing the phylogenetic relationship, based on three partial mitochondrial genes, of 95 individuals from 19 populations, we find that parthenogenesis and polyploidy have originated at least three times from different diploid lineages. We observe two major mitochondrial lineages, with over 2.5% sequence divergence between the most basal groups within them, and find that current distribution and phylogenetic relationships are weakly correlated. Quite unexpectedly, we also discover diploid clones that coexist with, and are morphologically indistinguishable from, the diploid sexual females. Our results support that these diploid clones are derived directly from the diploid sexuals. We also find that it is mainly an increase in ploidy level and not the benefits of asexual reproduction that confers to polyploid parthenogens the advantage over their diploid sexual relatives.     

SC1/hevin. An extracellular calcium-modulated protein that binds collagen I

SC1, a member of the BM-40 family of extracellular matrix proteins, was recombinantly expressed in a eukaryotic expression system. The full-length protein as well as truncated versions were purified to homogeneity under non-denaturing conditions. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of full-length SC1 revealed a mass of 87.8 kDa of which 16.8 kDa is contributed by posttranslational modifications. In electron microscopy, after negative staining, SC1 was revealed as a globule attached to a thread-like structure. A calcium dependence of the SC1 conformation could be demonstrated by fluorescence spectroscopy. In the extracellular matrix of cultured osteosarcoma cells SC1 was found associated with collagen I-containing fibrils, and binding of SC1 to reconstituted collagen I fibrils could be demonstrated by immunogold labeling and electron microscopy. SC1 showed a broad expression in a variety of tissues.     

Design of a monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein

By the introduction of 10 site-specific mutations in the dimer interface of human glutathione transferase P1-1 (GSTP1-1), a stable monomeric protein variant, GSTP1, was obtained. The monomer had lost the catalytic activity but retained the affinity for a number of electrophilic compounds normally serving as substrates for GSTP1-1. Fluorescence and circular dichroism spectra of the monomer and wild-type proteins were similar, indicating that there are no large structural differences between the subunits of the respective proteins. The GSTs have potential as targets for in vitro evolution and redesign with the aim of developing proteins with novel properties. To this end, a monomeric GST variant may have distinct advantages.     

Implications of shoot structure on the rate of photosynthesis at different levels in a coniferous canopy using a model incorporating grouping and penumbra

1. The connection between high leaf area index (LAI) and photosynthetic production with two attributes of coniferous canopy structure: small leaf size and grouping of needles on shoots, was analysed using a simulation model.
2. The small size of conifer needles gives rise to penumbras, which even out the distribution of direct sunlight on the leaf area and thereby act to increase the rate of canopy photosynthesis per unit of LAI.
3. Grouping, by producing a non-uniform distribution of leaf area, causes a decrease in total canopy light interception at any given LAI, but improves the photosynthetic light capture by shoots in the lower canopy.
4. Application of the model on a case study showed that: (a) grouping had a negative effect on the rate of photosynthesis in the upper canopy, but deeper down in the canopy the situation was reversed; (b) in the lower canopy, photosynthetic rates were up to 10 times higher as a result from the combined effect of grouping and penumbra; (c) grouping did not improve the rate of canopy photosynthesis per unit of LAI, however, it can have a positive effect on the total photosynthetic production by allowing a higher productive LAI to be maintained; (d) penumbra, on the other hand, increased the rate of canopy photosynthesis by as much as 40% for moderate values of the LAI.     

Use of recombinant virus to assess human cytomegalovirus early and late promoters in the context of the viral genome.

We have developed a system to study human cytomegalovirus (HCMV) cis-acting promoter elements within the context of the viral genome. A recombinant HCMV (RV134) containing a marker gene (beta-glucuronidase) was used to insert HCMV promoter-chloramphenicol acetyltransferase gene constructs into the viral genome between open reading frames US9 and US10. Using this system, we have studied the promoters for the early DNA polymerase gene (UL54), the early-late lower matrix phosphoprotein gene (pp65, UL83), and the true late 28-kDa structural phosphoprotein gene (pp28, UL99). Transient-expression assays demonstrated that the pp65 and pp28 promoters are activated earlier and to higher levels than typically observed with the endogenous gene. In contrast, insertion of these promoters into the viral genome resulted in kinetics which mimicked that of the endogenous genes. In addition, we have also tested a variant of the pp28 promoter (d24/26CAT) which is deleted from -609 to -41. This promoter behaved similarly to the wild-type pp28 promoter, indicating that sequences from -40 to +106 are sufficient for conferring true late kinetics. Taken together, these data demonstrate that the viral genome affords a level of regulation on HCMV gene expression that has been previously unrealized. Therefore, these experiments provide a model system for the analysis of cis-acting promoter regulatory elements in the context of the viral genome.     

Decreased serglycin proteoglycan size is associated with the platelet alpha granule storage defect in Wistar Furth hereditary macrothrombocytopenic rats. Serglycin binding affinity to type I collagen is unaltered

The Wistar Furth (WF) rat has a hereditary defect in platelet formation that resembles gray platelet syndrome of man with a large mean platelet volume and platelet alpha granule deficiency. The alpha granule abnormality is suggestive of a defect in granule packaging and/or stability. Proteoglycans are hypothesized to play a role in granule packaging. Therefore, we have analyzed the structure of the platelet proteoglycan, serglycin, in platelets of WF and normal Wistar rats. Normal and Wistar Furth rats were injected with ³⁵S-sulfate to label platelet proteoglycans via synthesis by the megakaryocytes, and platelets were isolated 3 days later. We found that WF rat platelets have only one-third of the normal proteoglycan mass per unit platelet volume, and the proteoglycans are smaller in hydrodynamic size with shorter glycosaminoglycan chains than those of Wistar rats. However, WF rat platelet proteoglycans showed no defect in binding to collagen on affinity coelectrophoresis gels. We conclude that the structure of WF rat platelet proteoglycans is abnormal, and speculate that this abnormality may contribute to abnormal packaging of the alpha granule contents. Leakage of alpha granule contents into the marrow by platelets and megakaryocytes could perturb the marrow matrix, and promote the development of myelofibrosis noted in gray platelet syndrome. J. Cell. Physiol. 172:87–93, 1997. © 1997 Wiley-Liss, Inc.     

Calcium-binding EGF-like modules in coagulation proteinases: function of the calcium ion in module interactions

Epidermal growth factor (EGF)-like modules are involved in protein-protein interactions and are found in numerous extracellular proteins and membrane proteins. Among these proteins are enzymes involved in blood coagulation, fibrinolysis and the complement system as well as matrix proteins and cell surface receptors such as the EGF precursor, the low density lipoprotein receptor and the developmentally important receptor, Notch. The coagulation enzymes, factors VII, IX and X and protein C, all have two EGF-like modules, whereas the cofactor of activated protein C, protein S, has four EGF-like modules in tandem. Certain of the cell surface receptors have numerous EGF modules in tandem. A subset of EGF modules bind one Ca(2+). The Ca(2+)-binding sequence motif is coupled to a sequence motif that brings about beta-hydroxylation of a particular Asp/Asn residue. Ca(2+)-binding to an EGF module is important to orient neighboring modules relative to each other in a manner that is required for biological activity. The Ca(2+) affinity of an EGF module is often influenced by its N-terminal neighbor, be it another EGF module or a module of another type. This can result in an increase in Ca(2+) affinity of several orders of magnitude. Point mutations in EGF modules that involve amino acids which are Ca(2+) ligands result in the biosynthesis of biologically inactive proteins. Such mutations have been identified, for instance, in factor IX, causing hemophilia B, in fibrillin, causing Marfan syndrome, and in the low density lipoprotein receptor, causing hypercholesterolemia. In this review the emphasis will be on the coagulation factors.     

Structures of thermolabile mutants of human glutathione transferase P1-1

An N-capping box motif (Ser/Thr-Xaa-Xaa-Asp) is strictly conserved at the beginning of helix alpha6 in the core of virtually all glutathione transferases (GST) and GST-related proteins. It has been demonstrated that this local motif is important in determining the alpha-helical propensity of the isolated alpha6-peptide and plays a crucial role in the folding and stability of GSTs. Its removal by site-directed mutagenesis generated temperature-sensitive folding mutants unable to refold at physiological temperature (37 degrees C). In the present work, variants of human GSTP1-1 (S150A and D153A), in which the capping residues have been substituted by alanine, have been generated and purified for structural analysis. Thus, for the first time, temperature-sensitive folding mutants of an enzyme, expressed at a permissive temperature, have been crystallized and their three-dimensional structures determined by X-ray crystallography. The crystal structures of human pi class GST temperature-sensitive mutants provide a basis for understanding the structural origin of the dramatic effects observed on the overall stability of the enzyme at higher temperatures upon single substitution of a capping residue.