Purification and properties of the urea amidolyase from Candida utilis.

Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis. The molecular weight of urea amidolyase, as determined by gel filtration and disc-gel electrophoresis, is between 500,000 and 520,000. Treatment with sodium dodecyl sulfate results in two peptides with molecular weights of 70,000 and 170,000. The urea carboxylase and allophanate hydrolase activities of urea amidolyase may be distinguished from one another on the basis of (a) the effect of the stabilizers, urea and glycerol, (b) the effect of storage pH on activity, and (c) selective inhibition by sulfhydryl reagents.     

Plant self-incompatibility systems: a molecular evolutionary perspective

Incompatibility recognition systems preventing self-fertilization have evolved several times in independent lineages of Angiosperm plants, and three main model systems are well characterized at the molecular level [the gametophytic self-incompatibility (SI) systems of Solanaceae, Rosaceae and Anthirrhinum, the very different system of poppy, and the system in Brassicaceae with sporophytic control of pollen SI reactions]. In two of these systems, the genes encoding both components of pollen-pistil recognition are now known, showing clearly that these two proteins are distinct, that is, SI is a lock-and-key mechanism. Here, we review recent findings in the three well-studied systems in the light of these results and analyse their implications for understanding polymorphism and coevolution of the two SI genes, in the context of a tightly linked genome region.     

Pseudomonas aeruginosa 1244 pilin glycosylation: glycan substrate recognition

The pilin of Pseudomonas aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Comparative structural analysis of O subunits that had been previously shown to be compatible with the 1244 glycosylation machinery revealed similarities among sugars at the presumed reducing termini of these oligosaccharides. We therefore hypothesized that the glycosylation substrate was within the sugar at the reducing end of the glycan precursor. Since much is known of PA103 O-antigen genetics and because the sugars at the reducing termini of the O7 (strain 1244) and O11 (strain PA103) are identical (beta-N-acetyl fucosamine), we utilized PA103 and strains that express lipopolysaccharide (LPS) with a truncated O-antigen subunit to test our hypothesis. LPS from a strain mutated in the wbjE gene produced an incomplete O subunit, consisting only of the monosaccharide at the reducing end (beta-d-N-acetyl fucosamine), indicating that this moiety contained substrate recognition elements for WaaL. Expression of pilAO(1244) in PA103 wbjE::aacC1, followed by Western blotting of extracts of these cells, indicated that pilin produced has been modified by the addition of material consistent with a single N-acetyl fucosamine. This was confirmed by analyzing endopeptidase-treated pilin by mass spectrometry. These data suggest that the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O repeat and that structures of the remaining sugars are irrelevant.     

High paternal diversity in the self-incompatible herb Arabidopsis halleri despite clonal reproduction and spatially restricted pollen dispersal

The number of sires fertilizing a given dam is a key parameter of the mating system in species with spatially restricted offspring dispersal, since genetic relatedness among maternal sibs determines the intensity of sib competition. In flowering plants, the extent of multiple paternity is determined by factors such as floral biology, properties of the pollen vector, selfing rate, spatial organization of the population, and genetic compatibility between neighbours. To assess the extent of multiple paternity and identify ecological factors involved, we performed a detailed study of mating patterns in a small population of a self-incompatible clonal herb, Arabidopsis halleri . We mapped and genotyped 364 individuals and 256 of their offspring at 12 microsatellite loci and jointly analysed the level of multiple paternity, pollen and seed dispersal, and spatial genetic structure. We found very low levels of correlated paternity among sibs ( P _full-sib = 3.8%) indicating high multiple paternity. Our estimate of the outcrossing rate was 98.7%, suggesting functional self-incompatibility. The pollen dispersal distribution was significantly restricted (mean effective pollen dispersal distance: 4.42 m) but long-distance successful pollination occurred and immigrating pollen was at most 10% of all pollination events. Patterns of genetic structure indicated little extent of clonal reproduction, and a low but significant spatial genetic structure typical for a self-incompatible species. Overall, in spite of restricted pollen dispersal, the multiple paternity in this self-incompatible species was very high, a result that we interpret as a consequence of high plant density and high pollinator service in this population.     

Hitch-hiking to a locus under balancing selection: high sequence diversity and low population subdivision at the S-locus genomic region in Arabidopsis halleri.

Hitch-hiking to a site under balancing selection is expected to produce a local increase in nucleotide polymorphism and a decrease in population differentiation compared with the background genomic level, but empirical evidence supporting these predictions is scarce. We surveyed molecular diversity at four genes flanking the region controlling self-incompatibility (the S-locus) in samples from six populations of the herbaceous plant Arabidopsis halleri, and compared their polymorphism with sequences from five control genes unlinked to the S-locus. As a preliminary verification, the S-locus flanking genes were shown to co-segregate with SRK, the gene involved in the self-incompatibility reaction at the pistil level. In agreement with theory, our results demonstrated a significant peak of nucleotide diversity around the S-locus as well as a significant decrease in population genetic structure in the S-locus region compared with both control genes and a set of seven unlinked microsatellite markers. This is consistent with the theoretical expectation that balancing selection is increasing the effective migration rate in subdivided populations. Although only four S-locus flanking genes were investigated, our results suggest that these two signatures of the hitch-hiking effect are localized in a very narrow genomic region.     

Origin and diversification dynamics of self-incompatibility haplotypes

Self-incompatibility (SI) is a genetic system found in some hermaphrodite plants. Recognition of pollen by pistils expressing cognate specificities at two linked genes leads to rejection of self pollen and pollen from close relatives, i.e., to avoidance of self-fertilization and inbred matings, and thus increased outcrossing. These genes generally have many alleles, yet the conditions allowing the evolution of new alleles remain mysterious. Evolutionary changes are clearly necessary in both genes, since any mutation affecting only one of them would result in a nonfunctional self-compatible haplotype. Here, we study diversification at the S-locus (i.e., a stable increase in the total number of SI haplotypes in the population, through the incorporation of new SI haplotypes), both deterministically (by investigating analytically the fate of mutations in an infinite population) and by simulations of finite populations. We show that the conditions allowing diversification are far less stringent in finite populations with recurrent mutations of the pollen and pistil genes, suggesting that diversification is possible in a panmictic population. We find that new SI haplotypes emerge fastest in populations with few SI haplotypes, and we discuss some implications for empirical data on S-alleles. However, allele numbers in our simulations never reach values as high as observed in plants whose SI systems have been studied, and we suggest extensions of our models that may reconcile the theory and data.     

Evidence for Fisher's dominance theory: how many 'special cases'?

Dominance, its genetic basis and evolution has been at the heart of one of the most intense controversies in the history of genetics. For more than eighty years the existence of dominance modifiers, genetic elements controlling dominance-recessivity interactions, has been suggested as a theoretical possibility, but the modifier elements themselves have remained elusive. A recent study of the self-incompatibility locus in flowering plants provided the first empirical evidence for such genetic elements: small non-coding RNAs that control dominance-recessivity by mediating methylation of the promoter of the recessive allele. Theory has shown that several biological situations are favorable for the evolution of dominance modifiers. We argue that the elucidation of this mechanism of dominance opens up new research avenues that could lead to uncovering dominance modifiers in other genetic systems, such as genes controlling Batesian and Müllerian mimicry or host-parasite interactions, thereby shedding light on the generality of the proposed mechanism.     

Physiological, Endocrine, and Genetic Bases of Anadromy in the Brook Charr, Salvelinus Fontinalis, of the Laval River (Québec, Canada)

Brook charr, Salvelinus fontinalis, often display alternate life history styles in coastal areas. In the Laval River, some brook charr remain freshwater residents, while others undergo seasonal migrations between freshwater and saltwater environments. In the present paper, we examined physiological (electrolyte concentrations, gill Na⁺, K⁺-ATPase activity, and thyroid hormone levels) as well as genetic differences (neutral genetic markers) between anadromous and river-resident fish from the Laval River. We also examined how artificial rearing conditions affected seasonal variations in the osmoregulatory physiology of a domestic strain derived from wild anadromous fish. Sympatric anadromous and resident forms of brook charr of the Laval River exhibited differences in gill Na⁺, K⁺-ATPase activity, plasma thyroxine (T₄), and triidothyronine (T₃) concentrations. In domestic anadromous charr, rearing conditions during development had no negative impact on osmoregulatory ability or on gill Na⁺, K⁺-ATPase activity. These results argued for an important hereditary component of gill Na⁺, K⁺-ATPase activity. However, the spring increase in T₄ was present only in wild fish. Significant differences observed at microsatellite loci further suggested that at least some level of reproductive isolation may have occurred between anadromous and resident charr in the Laval River.     

Cloning and sequencing of the Pseudomonas aeruginosa 1244 pilin structural gene

The pilin structural gene of Pseudomonas aeruginosa 1244 was cloned in both cosmids and lambda. Expression of the cloned gene was detected in P. aeruginosa strains PAO2003, PA103, and 653A by an immunoblot reaction utilizing monoclonal antibodies. Western blot analysis showed that pilin expressed from the cloned gene was slightly larger than native 1244 pilin when produced in strains PAO2003 and 653A, but distinctly smaller in PA103. Bacteriophages specific for the 1244 pilus did not lyse strain PAO2003 containing the cloned 1244 pilin gene, indicating that functional 1244 pili were not assembled in this recombinant strain. Nucleotide sequencing revealed a coding region which when translated would produce a 15,615 dalton peptide. The amino-terminal region of this peptide is identical with published pilin sequences. While the rest of the peptides are generally dissimilar, common residues are seen within potentially antigenic regions.