Copurification of the Lac repressor with polyhistidine-tagged proteins in immobilized metal affinity chromatography

One of the commonly used resins for immobilized metal affinity purification of polyhistidine-tagged recombinant proteins is TALON resin, a cobalt (II)--carboxymethylaspartate-based matrix linked to Sepharose CL-6B. Here, we show that TALON resin efficiently purifies the native form of Lac repressor, which represents the major contaminant when (His)(6)-tagged proteins are isolated from Escherichia coli host cells carrying the lacI(q) gene. Inspection of the crystal structure of the repressor suggests that three His residues (residues 163, 173, and 202) in each subunit of the tetramer are optimally spaced on an exposed face of the protein to allow interaction with Co(II). In addition to establishing a more efficient procedure for purification of the Lac repressor, these studies indicate that non-lacI(q)-based expression systems yield significantly purer preparations of recombinant polyhistidine-tagged proteins.     

Extra pair paternity in birds: a review of interspecific variation and adaptive function

The application of molecular genetic techniques has revolutionized our view of avian mating systems. Contrary to prior expectations, birds are only very rarely sexually monogamous, with 'extra-pair offspring' found in approximately 90% of species. Even among socially monogamous species, over 11% of offspring are, on average, the result of extra-pair paternity (EPP). Based on over 150 molecular genetic studies of EPP in birds, we review two topical areas: (i) ecological explanations for interspecific variation in the rate of EPP; and (ii) evidence bearing on the adaptive function of EPP. We highlight the remaining challenges of understanding the relative roles of genes and ecology in determining variation between taxa in the rate of extra paternity, and testing for differences between extra-pair offspring and those sired within-pair.     

The 'island rule' in birds: medium body size and its ecological explanation

Do birds show a different pattern of insular evolution from mammals? Mammals follow the ''island rule'', with large-bodied species getting smaller on islands and small-bodied species getting bigger. By contrast, the traditional view on birds is that they follow no general island rule for body size, but that there is an insular trend for large bills. Insular shifts in feeding ecology are, therefore, widely assumed to be the primary cause of divergence in island birds. We use a comparative approach to test these ideas. Contrary to the traditional view, we find no evidence for increased bill size in insular populations. Instead, changes in both bill size and body size obey the ''island rule''. The differences between our results and the traditional view arise because previous analyses were based largely on passerines. We also investigate some ecological factors that are thought to influence island evolution. As predicted by the traditional view, shifts in bill size are associated with feeding ecology. By contrast, shifts in body size are associated with the potential for intraspecific competition and thermal ecology. All these results remain qualitatively unchanged when we use different methods to score the ecological factors and restrict our analyses to taxa showing pronounced morphological divergence. Because of strong covariation between ecological factors, however, we cannot estimate the relative importance of each ecological factor. Overall, our results show that the island rule is valid for both body size and bill length in birds and that, in addition to feeding ecology, insular shifts in the level of intraspecific competition and the abiotic environment also have a role.     

Comparison of tissue culture and animal models for assessment of Cryptospridium parvum infection

The current increased interest for using tissue culture as a surrogate for mouse infection to assess Cryptospridium viability suggests that a comparison of the two models is essential for data interpretation. Therefore, a need remains for a statistical comparison that can demonstrate if infection and inactivation predicted by new tissue culture models are comparable with those predicted by animal models. Data from a total of 31 dose-response trials using both tissue culture and mouse models to assess C. parvum infectivity were compared. The dose needed to infect 50% of the tissue cultures (ID(50)) was also compared to each ID(50) in mice. Average ID(50)s developed using the logit dose-response method for tissue culture and mice were 8 and 107, respectively, suggesting that tissue culture was more sensitive to infection. However, correlation (r) between tissue culture and mouse infectivity was statistically significant (0.9167 [95% CI=0.8428 to 0.9594, p<0.0001]). Comparison of oocyst disinfection by UV and chlorine dioxide showed no significant difference between inactivation predicted by tissue culture and mouse models (p=0.8893; t=0.0141; n=21). These results demonstrate that tissue culture can successfully be used to measure C. parvum infection and can be used for determining inactivation in disinfection studies.     

Developmental neurotransmitters?

Previous studies support an early role for neurotransmitter signaling before synaptogenesis, but puzzlingly, a neurological phenotype is absent in embryonic mice that lack vesicular release. Demarque et al. (in this issue of Neuron) now report that early release of transmitter is unconventional in not requiring action potentials, Ca(2+) entry, or vesicle fusion, thus potentially reconciling the discrepancy.     

Asymmetrical,water-soluble phthalocyanine dyes for covalent labeling of oligonucleotides

Two new water-soluble, porphyrazine (Pz) dyes containing an isothiocyanate function for covalent linking have each been prepared by cross condensation of two different aromatic dinitriles, one containing carboxylates for solubilizing purposes and the other containing a nitro group for conversion into the labeling function. The initial mononitrotricarboxylato Pzs have been purified to homogeneity from the mixture of Pz congeners formed in the condensation reaction by anion exchange chromatography. The phthalocyanine dye 1 has an absorption maxima at 683 nm while the trinaphthoporphyrazine dye 2 has an absorption maxima at 755 nm, due to the increased size of the aromatic system. Both dyes were successfully conjugated to oligonucleotide primers, showing their potential for use in near-infrared-based DNA diagnostic applications.     

Experimental challenge and clinical cases of Bohle iridovirus (BIV) in native Australian anurans

Ranaviruses have been observed with increasing frequency amongst poikilothermic vertebrate hosts. The impact of ranaviruses upon amphibian populations has remained largely unknown. A gene probe for Bohle iridovirus (BIV) based upon primers designed to detect epizootic haematopoietic necrosis virus (EHNV) was constructed. A PCR and dot-blot system was used successfully in screening for the presence of BIV nucleic acid in digested formalin-fixed, paraffin-embedded amphibian tissues. Juvenile frogs were more susceptible to BIV than adults. In experimental challenges and epizootics in captive frogs, juvenile Litoria caerulea, L. alboguttata, Cyclorana brevipes and Pseudophryne coriacea were acutely susceptible. High mortality (at or near 100%) resulted, usually occurring within 5 to 25 d depending on dose and method of exposure. Histopathological changes included mainly hepatic, renal and splenic necroses. Significant haemosiderosis was encountered in more chronically infected frogs. BIV could be reisolated from juvenile L. caerulea >40 d after inoculation, and >200 d after the first mortalities occurred in an epizootic in L. alboguttata. Adult L. rubella, L. inermis, L. caerulea, Cophixalus ornatus and Taudactylus acutirostris were less susceptible in trials ranging from 30 to > 100 d. There was some evidence of chronic infection, and BIV could be detected by PCR. Wild moribund adult L. caerulea from Townsville and captive juvenile Pseudophryne corieacea from Sydney undergoing mortality tested positive with the BIV PCR. PCR and dot blot was more sensitive than viral isolation. PCR could detect BIV in amphibians long after BIV challenge, and in amphibians which appeared healthy. Ranaviruses could be having an impact on Australian herpetofauna.     

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.