Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Characterization and environmental regulation of outer membrane proteins in Xenorhabdus nematophilus.

We have examined the production of the outer membrane proteins of the primary and secondary forms of Xenorhabdus nematophilus during exponential- and stationary-phase growth at different temperatures. The most highly expressed outer membrane protein of X. nematophilus was OpnP. The amino acid composition of OpnP was very similar to those of the porin proteins OmpF and OmpC of Escherichia coli. N-terminal amino acid sequence analysis revealed that residues 1 to 27 of the mature OpnP shared 70 and 60% sequence identities with OmpC and OmpF, respectively. These results suggest that OpnP is a major porin protein in X. nematophilus. Three additional proteins, OpnA, OpnB, and OpnS, were induced during stationary-phase growth. OpnB was present at a high level in stationary-phase cells grown at 19 to 30 degrees C and was repressed in cells grown at 34 degrees C. OpnA was optimally produced at 30 degrees C and was not present in cells grown at lower and higher temperatures. The production of OpnS was not dependent on growth temperature. In contrast, another outer membrane protein, OpnT, was strongly induced as the growth temperature was elevated from 19 to 34 degrees C. In addition, we show that the stationary-phase proteins OpnA and OpnB were not produced in secondary-form cells.     

Molecular analysis of the two-component genes, ompR and envZ, in the symbiotic bacterium Xenorhabdus nematophilus

In Escherichia coli the histidine kinase sensor protein, EnvZ, undergoes autophosphorylation and subsequently phosphorylates the regulatory protein, OmpR. Modulation of the levels of OmpR-phosphate controls the differential expression of ompF and ompC . While the phosphotransfer reaction between EnvZ and OmpR has been extensively studied, the domains involved in the sensing function of EnvZ are not well understood. We have used a comparative approach to study the sensing function of EnvZ. During our search of numerous bacteria we found that the symbiotic/pathogenic bacterium Xenorhabdus nematophilus contained the operon encoding both ompR and envZ . Nucleotide sequence analysis revealed that EnvZ of X. nematophilus (EnvZ^X.n.) is composed of 342 amino acid residues, which is 108 residues shorter than EnvZ of E. coli (EnvZ^E.c.). Amino acid sequence comparison showed that the cytoplasmic domains of the EnvZ moleculsshared 57% sequence identity. In contrast, the large hydrophilic periplasmic domain of EnvZ^E.c. was absent in EnvZ^X.n., and was replaced by a shorter hydrophobic region. Although the periplasmic domains had diverged extensively, envZ^X.n. was able to complement a Δ envZ strain of E. coli . OmpF and OmpC were differentially produced in response to changes in medium osmolarity in this strain. Further genetic analysis established that heterologous phosphorylation between EnvZ^X.n. and OmpR of E. coli (OmpR^E.c.) accounted for the complementation of the Δ envZ strain. In addition we show that the OmpR molecules of X. nematophilus and E. coli share 78% amino acid sequence identity. These results indicate that the EnvZ protein of X. nematophilus was able to sense changes in the osmolarity of the growth environment and properly regulate the levels of OmpR-phosphate in E. coli .     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane: III. Discrimination between the causal rôle of split products and of lecithin removal

Summary Cleavage of 55% of the lecithin in intact human erythrocytes by phospholipase A₂ (bee venom) markedly inhibits the mediated transport ofl-lactate (via the monocarboxylate carrier) and ofl-arabinose (via the monosaccharide carrier), while the major anion exchange system (probed by oxalate) and diffusion via the lipid domain (probed by erythritol) remain essentially unaltered. The causal role of the split products, unsaturated fatty acids and saturated lysolecithin, and of lecithin removal were now studied by sequential extraction of split products with serum albumin and by their controlled insertion into normal membranes. Careful choice of the albumin-to-cell ratio allowed the extraction of more than 95% of the fatty acids and up to 80% of the lysolecithin without hemolysis.Extraction of fatty acids abolished inhibition of lactate and arabinose transfer, but induced inhibition of anion exchange and translipid permeation. Subsequent extraction of lysolecithin produced no further effects except on lactate transfer, which was inhibited.Exogenous oleic and linoleic acid, at intramembrane concentrations equal to those produced by phospholipase A₂, inhibit lactate and arabinose transfer, while accelerating oxalate and erythritol movements, in agreement with effects of endogenous fatty acids. Exogenous lysolecithin inhibits all mediated transfer processes but does not alter translipid permeation. This pattern differs from that obtained for endogenous lysolecithin.The action of exogenous lysolecithin can be suppressed by loading of the cells with cholesterol. Insertion of exogenous lysolecithin into cells depleted of endogenous lysolecithin does not restore the functional state before depletion, indicating that exogenous and endogenous lysolecithin may act differently.Modification of membrane phospholipids by cleavage with phospholipases has been used by many investigators to study the relevance of lipids for protein-related functions of biomembranes. In many instances pronounced effects could be demonstrated. With the exception, however, of electrical characteristics of neurons [21] and axons [39], the properties investigated only comprised the binding of toxins, drugs [4, 28], transmitters [1], and hormones [2, 48] to their receptors, or enzymatic reactions [5, 10, 11, 13, 36, 37, 43].In previous investigations [49, 50] of this series we have analyzed the effect of enzymatic cleavage of exofacial membrane phospholipids (phosphatidylcholine, sphingomyelin) on simple translipid, and on facilitated, protein-mediated diffusion processes across the human erythrocyte membrane. Rates of nonelectrolyte movements via the lipid domain and of mediated exchange of inorganic anions remained essentially unaltered after hydrolysis of up to 60% of the phosphatidylcholine, corresponding to about 18% of the membrane phospholipids or 36% of those in the outer leaf of the lipid bilayer. In contrast, the movements ofl-arabinose, catalyzed by the monosaccharide carrier system, and ofl-lactate, transported by a specific monocarboxylate carrier, were markedly inhibited by phospholipid cleavage. In similar studies, inhibition of the active extrusion of Na⁺ has recently been demonstrated in human erythrocytes treated with phospholipase A₂ [14]. These results obtained on erythrocytes provided first evidence for effects of phospholipid cleavage on solute translocation across biomembranes in intact cells.Inhibitory effects of phospholipid cleavage can in principle be due either to the production of the split products, lysolecithin and fatty acid, which remain bound to the membrane, or to the disappearance of a particular phospholipid. In order to distinguish between these possible mechanisms, two procedures can be used. First, the split products of lecithin, although tightly bound to the membrane core, can be removed by treatment with serum albumin. Second, split products can be introduced into the membrane of normal cells. If the former procedure abolishes and the latter one mimics the effects of phospholipase A₂ treatment, split products are likely to be responsible for the effects of phospholipase A₂. Otherwise, the disappearance of a native phospholipid has to be considered.Testing the removal of split products is easily accomplished in isolated membranes [10, 11, 13, 37, 43], but has met problems in intact erythrocytes, which lysed after extraction of part of the split products in earlier studies [17]. Comparisons between the actions of exogenous and endogenous fatty acid and lysolecithin, on the other hand, were mostly qualitative as yet, since effects were related to bulk concentrations of the exogenously added substances and not to thosewithin the membrane.The following attempt to further clarify the effects of phospholipase A₂ treatment on erythrocytes is based on a stepwise, controlled extraction of endogenous split products and a quantitative evaluation of the action of exogenous split products. From the results it will become evident that transport processes in the same membrane may differ markedly with respect to the mechanisms by which cleavage of phosphatidylcholine exerts its effects.     

The early life history of two sympatric New Zealand octopuses: eggs and paralarvae of Octopus huttoni and Pinnoctopus cordiformis

This study combined morphological and morphometric information on egg clutches, egg capsules and paralarvae of two sympatric coastal octopuses from New Zealand waters, Octopus huttoni and Pinnoctopus cordiformis, to provide species-specific traits to identify their early life stages obtained from field surveys. Eggs of O. huttoni (2.5 mm length; 1 mm width) were entwined with one another forming strings that ranged from 11 to 25.8 mm in length. Eggs of P. cordiformis (6.4 mm length; 1.5 mm width) were significantly bigger than those of O. huttoni and were grouped in small clusters of about seven eggs. Paralarvae O. huttoni and P. cordiformis differed in hatching size (1.4 mm versus 3.1 mm mantle length), number of suckers per arm (four versus eight), number of lamellae per outer demibranch (five versus ten) and arrangements of chromatophores in the body surface (29 to 59 versus 91 to 179), respectively. The morphological traits described in hatchlings from the laboratory allowed comparisons with field-collected paralarvae, suggesting that such characters were reliable species-specific patterns to enable a consistent differentiation between the early life stages of these two sympatric species, even in the absence of the brooding female.