Evidence for multiple paternity in the school shark Galeorhinus galeus found in New Zealand waters.

This study assessed the levels of relatedness of Galeorhinus galeus of progeny arrays using six microsatellite DNA markers. A parentage analysis from five families (mother and litter) from the North Island of New Zealand suggested the occurrence of genetic polyandry in G. galeus with two of the five litters showing multiple sires involved in the progeny arrays. This finding may be consistent with the reproductive characteristics of G. galeus, in which females can potentially store sperm for long periods of time after the mating season.

Rates of evolution in ancient DNA from Adélie penguins.

Well-preserved subfossil bones of Adélie penguins, Pygoscelis adeliae, underlie existing and abandoned nesting colonies in Antarctica. These bones, dating back to more than 7000 years before the present, harbor some of the best-preserved ancient DNA yet discovered. From 96 radiocarbon-aged bones, we report large numbers of mitochondrial haplotypes, some of which appear to be extinct, given the 380 living birds sampled. We demonstrate DNA sequence evolution through time and estimate the rate of evolution of the hypervariable region I using a Markov chain Monte Carlo integration and a least-squares regression analysis. Our calculated rates of evolution are approximately two to seven times higher than previous indirect phylogenetic estimates.

Time-dependent inhibition of protein farnesyltransferase by a benzodiazepine peptide mimetic.

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase-I (GGTase-I) catalyze the prenylation of proteins with a carboxy-terminal tetrapeptide sequence called a CaaX box, where C refers to cysteine, "a" refers to an aliphatic residue, and X typically refers to methionine, serine, or glutamine (FTase), or to leucine (GGTase-I). Marsters and co-workers [(1994) Bioorg. Med. Chem. 2, 949--957] developed inhibitors of FTase with cysteine and methionine attached to an inner hydrophobic benzodiazepine scaffold. We found that the most potent of these compounds (BZA-2B) resulted in the time-dependent inhibition of FTase. The K(i) of BZA-2B for FTase, which is the dissociation constant of the initial complex, was 79 +/- 13 nM, and the K(i)*, which is the overall dissociation of inhibitor for all enzyme forms, was 0.91 +/- 0.12 nM. The first-order rate constant for the conversion of the initial complex to the final complex was 1.4 +/- 0.2 min(-1), and that for the reverse process was 0.016 +/- 0.002 min(-1). The latter rate constant corresponds to a half-life of the final complex of 45 min. Our experiments favor the notion that the inhibitor binds to the FTase--farnesyl diphosphate complex which then undergoes an isomerization to form a tighter FTase*--farnesyl diphosphate--BZA2-B complex. Diazepam, a compound with a benzodiazepine nucleus but lacking amino acid extensions, was a weak (K(i) = 870 microM) but not time-dependent inhibitor of FTase. Cys-Val-Phe-Met and Cys-4-aminobenzoyl-Met were instantaneous and not time-dependent inhibitors of FTase. Furthermore, BZA-4B, with a leucine specificity determinant, was a classical competitive inhibitor of GGTase-I and not a time-dependent inhibitor.

Gene flow on the ice: genetic differentiation among Adélie penguin colonies around Antarctica.

Each summer Adélie penguins breed in large disjunct colonies on ice-free areas around the Antarctic continent. Comprising > 10 million birds, this species represents a dominant feature of the Antarctic ecosystem. The patchy distribution within a large geographical range, natal philopatry and a probable history of refugia, suggest that this species is likely to exhibit significant genetic differentiation within and among colonies. We present data from seven microsatellite DNA loci for 442 individuals from 13 locations around the Antarctic continent. With the exception of one locus, there was no significant genic or genotypic heterogeneity across populations. Pairwise FST values were low with no value > 0.02. When all colonies were compared in a single analysis, the overall FST value was 0.0007. Moreover, assignment tests were relatively ineffective at correctly placing individuals into their respective collection sites. These data reveal a lack of genetic differentiation between Adélie penguin colonies around the Antarctic continent, despite substantial levels of genetic variation. We consider this homogeneity in terms of the dispersal of individuals among colonies and the size of breeding groups and discuss our results in terms of the glacial history of Antarctica.

A repeat complex in the mitochondrial control region of Adélie penguins from Antarctica.

We have determined the nucleotide sequence of the entire mitochondrial control region (CR) of the Adélie penguin (Pygoscelis adeliae) from Antarctica. Like in most other birds, this CR region is flanked by the gene nad6 and transfer (t)RNA trnE(uuc) at the 5' end and the gene rns and trnF(gaa) at the 3' end. Sequence analysis shows that the Adelie penguin CR contains many elements in common with other CRs including the termination associated sequences (TAS), conserved F, E, D, and C boxes, the conserved sequence block (CSB)-1, as well as the putative light and heavy strand promoters sites (LSP-HSP). We report an extraordinarily long avian control region (1758 bp) which can be attributed to the presence, at the 3' peripheral domain, of five 81-bp repeat sequences, each containing a putative LSP-HSP, followed by 30 tetranucleotide microsatellite repeat sequences consisting of (dC-dA-dA-dA)30. The microsatellite and the 81-bp repeat reside in an area known to be transcribed in other species.

Molecular phylogenetics and the evolution of antarctic notothenioid fishes.

The monophyly of the antarctic fish suborder Notothenioidei and the monophyly of its earliest family the Bovichtidae have been investigated with 12S and 16S mitochondrial DNA sequences. New data from Cottoperca, Pseudaphritis, Harpagifer and several outgroups, in addition to available sequences, show that the bovichtids are paraphyletic. Pseudaphritis is the sister group of all the non-bovichtid notothenioids. The same results are found from two independent genetic markers, the nuclear 28S rDNA and the 12S and 16S mitochondrial rDNA. This reliably refutes a previous hypothesis that placed Pseudaphritis as the sister group of all the remaining notothenioids (including Cottoperca and Bovichtus). Bootstrap analyses show that the Notothenioidei are monophyletic (although members of the suborder Trachinoidei have not been surveyed). Subsequent data from hemoglobin composition confirm the present relationships. After discussions between members of the European Science Foundation (ESF) network during its last two meetings, we point out here some fundamental aspects of comparative biology to improve understanding between the physiologist community and phylogeneticists. The most important points are differences in how the concept of homology is used and differences in the consideration of adaptation. When adaptation is evoked or questioned, endless speculations and untestable scenarios are often developed. We strongly advocate the use of phylogenetic trees for testing hypotheses of adaptation (through multiple character mapping). Such a "research program" in comparative biology has the power to improve knowledge because it can potentially lead to new experiments for testing adaptive hypotheses.

Mitochondrial phylogeny of trematomid fishes (Nototheniidae, Perciformes) and the evolution of Antarctic fish.

The subfamily of fishes Trematominae is endemic to the subzero waters of antarctica and is part of the longer notothenioid radiation. Partial mitochondrial sequences from the 12S and 16S ribosomal RNA (rRNA) genes and a phylogeny for 10 trematomid species are presented. As has been previously suggested, two taxa, Trematomus scotti and T. newnesi, do not appear to be part of the main trematomid radiation. The genus Pagothenia is nested within the genus Trematomus and has evolved a unique cyropelagic existence, an association with pack ice. Using a mitochondrial rRNA molecular clock rate of 0.14% transversion changes per million years, the average age of the trematomids is estimated at 3.4 million years (MY). If the age of the trematomids is approximately 3.4 MY, this group could have speciated during the period of deglaciation in Antarctica 2.5-4.8 million years ago. This era was marked by significant changes on the Antarctic shores, such as the opening of fjords, which might have provided a stimulus for specification.

Molecular evolution at subzero temperatures: mitochondrial and nuclear phylogenies of fishes from Antarctica (suborder Notothenioidei), and the evolution of antifreeze glycopeptides.

Most fishes of the perciform suborder Notothenioidei are endemic to the subzero marine waters of Antarctica. A number of remarkable physiological attributes allow them to inhabit this extreme environment; for example, the blood of almost all notothenioid species contains antifreeze glycopeptides. To establish a molecular phylogenetic hypothesis for these fishes, DNA sequences from two mitochondrial genes, portions of the 12S and 16S ribosomal genes (928 base pairs [bp]), were determined for 18 species. These belong to 15 genera in five families of the suborder. The DNA data suggest that two of these families are unnatural groups and consequently that the classification and phylogeny of this suborder is in need of revision. In terms of DNA variation, the Bovichtidae are a distantly related sister group to the other families of the suborder that includes the icefishes, the only vertebrates without hemoglobin. The fishes of the suborder (except the Bovichtidae) seem to have speciated rapidly, forming an adaptive radiation in the Antarctic waters. A phylogenetic analysis of published hemoglobin amino acid sequences for other notothenioid fishes supports these results from mtDNA. On the basis of molecular phylogeny, the evolution of antifreeze glycopeptides was studied. The age of the radiation of notothenioid fishes had been estimated to be at least 38 Mya. However, the level of mtDNA variation detected in notothenioid fishes appears to be too low to agree with this date of origin and might instead suggest a younger age (10-15 Mya). Alternatively, the low level of detected mtDNA variation would agree with the traditional old-age estimate if an extremely slow rate of mtDNA evolution is postulated for this group. This slow-rate hypothesis, if true, could be explained by decreased metabolic rates slowing down the tempo of molecular evolution.

Decontamination of halothane from anaesthetic machines achieved by continuous flushing with oxygen.

The contamination of four types of anaesthetic machine with halothane was sequentially sampled by mass spectrometry while the machines were continuously flushed with oxygen 8 litre min-1 for up to 24 h. Contamination decreased in an exponential manner. Machines fitted with Selectatec vaporizer mounting systems and with the vaporizer removed showed contamination less than 0.02 parts per million (p.p.m.) of halothane after 12 h flushing. Machines with cage-mounted vaporizers or with vaporizers left connected to the Selectatec block demonstrated persisting contamination. The Fluotec Mk.4 vaporizer showed an improvement on earlier designs in this respect. Background contamination concentrations of greater than 0.05 p.p.m. were measured in a patient-free recovery area of an operating theatre suite. Concentrations increased to 1 p.p.m. when patients were admitted following halothane anaesthesia. Decontamination of anaesthetic machines to concentrations of halothane below those detected as background contamination within recovery areas may allow such machines to be used safely to anaesthetize patients at risk from halothane.

Conformational transitions in the subfragment-2 region of myosin.

A differential scanning calorimeter was used to observe thermally induced conformational transitions in subfragment 2 (S-2) of myosin. In addition to an endotherm for the major transition which had been observed by several other methods earlier, a small broad endotherm was noted with a Tm of 41 degrees C. By analysis of the heat capacity profiles of long and short S-2, this endotherm was assigned to the hinge region. Comparison of the amino acid compositions of S-2 and tropomyosin showed them to be remarkably similar, and in view of their similar behavior in calorimetric studies, it is apparent that interactions stabilizing the coiled-coil structure of S-2 are a hydrophobic interface supported by charged interaction spanning the groove as was suggested for tropomyosin by McLachlan and Stewart [McLachlan, A. D., & Stewart, M. (1975) J. Mol. Biol. 98, 293-304].

Enthalpy of nucleotides binding to myosin.

The enthalpies of binding adenosine 5'-diphosphate (ADP) and 5'-adenylyl imidodiphosphate [AMP-P(NH)P] to rabbit skeletal myosin have been measured in Pipes and Tris buffers at pH 7.8 and 15 degrees C. For ADP the enthalpy of binding was exothermic, whereas the enthalpy of binding AMP-P(NH)P, a nonhydrolyzable ATP analogue, was small and endothermic. For the reaction of ATP and myosin, the development of enthalpy was resolved into two phases: a fast endothermic phase, which is the summation of binding and hydrolysis, and a slow exothermic phase, which is associated with product-release steps. These results are discussed in terms of their implications for energy transduction.