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Jon G Hall CITP FBCS says cash and data are important but not as critical as the ability to tell a good story.

ABSTRACr.-An 888-base-pair segment of the mitochondrial cytochrome-b gene was sequenced for New World barbets, Old World barbets, toucans, and several outgroup taxa. Toucans were consistently identified as the sister taxon of the New World barbets by a variety of analyses. These data are fundamentally different from earlier morphological analyses and the DNA-DNA hybridization study of these same taxa. Our results provide an independent confirmation of relationships proposed in both the morphological and molecular studiesthat New World barbets and toucans are sister taxa with respect to Old World barbets. Received 19 June 1992, accepted 25 November 1992. TRADITIONAL MORPHOLOGICAL analyses have disagreed on the phylogenetic relationships among the barbets (Capitonidae), a pantropical assemblage of frugivorous birds, traditionally placed in the Piciformes with woodpeckers

A rapid nonradioactive sandwich immunoassay which utilizes biotin-streptavidin mediated filtration capture of immune complexes in conjunction with a silicon sensor was developed for the detection of virus. Using purified Newcastle disease virus as a model, the lower limits of detection (LOD) were determined for a number of immunoassay configurations employing both monoclonal and polyclonal antibodies. The LODs ranged from 1.3 ng/ml (sample volume of 100 μl) for an incubation of 60 min to 400 ng/ml for a 1 min incubation. The sandwich immune complexes were formed from one-step incubation of antibody and antigen. No ‘hook’ effects were observed over a wide range of analyte concentrations. The assays were easy to perform and required a total time equal to the incubation period plus about 5 min. The assay format is suitable for virus, bacteria and protein antigens. New assays can be developed and optimized readily, often within 1 day.

The concentrations and enantiomeric purity of the major monoterpene constituents in a number of Melaleuca alternifolia (tea tree) oils have been determined by enantioselective gas chromatography. Consrstent enantiomeric ratios of 65:35 (+:-) for terpinen-4-ol and 76:24 (+:-) for a-terpineol were observed for the range of oils analyzed. Attempts to validate these ratios in the intact oil by 1 H NMR together with chiral lanthanide shift reagents have not yet been successful. 1 H NMR was, however, successful in confirming the enantiomeric purity determined by gas chromatography of standard terpinen4-ol [79:21 (+:-)] and α-terpineol [35:65 (+:-)] samples

Genetic analyses, involving backcross and F2 matings, demonstrate that the type I hyperprolinemia of PRO/Re mice is caused by an abnormal allele at a single locus designated pro-1. Mice homozygous for this allele (pro-1b/pro-1b) possess a deficiency in the activity of component 1 of mitochondrial proline dehydrogenase. In liver mitochondria of normal C57BL/6J mice, two proline dehydrogenase activity components are demonstrable by electrophoretic resolution of Triton X-100 solubilized extracts. In mitochondria of PRO/Re mice, the activity of component 1 is not readily detectable. Residual proline dehydrogenase activity in PRO/Re mitochondria appears, therefore, to be due in large measure to activity component 2 which is more stable to incubation at 40 C, exhibits slower electrophoretic mobility, and is less reactive to menadione. Kinetic analyses demonstrate a Km(proline) for the Triton X-100 solubilized enzyme activities of PRO/Re and C57BL/6J liver mitochondria of 0.4 M and 2.9×10−3M, respectively. C57BL/6J enzyme activity is inhibited by high substrate concentration. The activity of component 1 was not detected in other subcellular fractions of PRO/Re liver obtained by differential centrifugation. Abnormal control of respiratory chain function in PRO/Re mitochondria appears to involve primarily proline oxidation, as indicated by the level of activity of several inner membrane enzymes.

There is significant differentiation at five polymorphic loci ofMytilus edulis among certain geographical areas of the Atlantic coast of North America. Non-metric multidimensional numerical methods distinguished three population groups: (I) populations south of Cape Cod, (II) populations throughout the Gulf of Maine, Gulf of St. Lawrence, areas of both southern and northern Newfoundland, and southern Hudson Bay, and (III) populations in southeastern Nova Scotia, northern Newfoundland and Hudson Strait, Quebec. Each subset consists of populations that exhibit characteristic multilocus, multiple allele genotypes. Populations in Groups II and III are spatially interdigitated among each other. At least one geographical area of mixing between genetically distinct populations occurs in northeastern Newfoundland. There is no evidence for interbreeding among genetically distinct individuals in mixed populations, suggesting the possibility that populations in the Atlantic Canadian Provinces and areas of northern Canada may consist of two distinct species.

Two glucosephosphate isomerase (GPI;D-glucose-6-phosphate ketolisomerase; EC 5.3.1.9) alleloenzymes from the blue mussel,Mytilus edulis, were purified to homogeneity. The steady-state kinetic properties of GPI1.00 and GPI.96, which exhibit latitudinal clines in frequency along the Atlantic coast of North America, were determined in both the glycolytic and the gluconeogenic reaction directions at physiological temperatures and pH levels. The two alleloenzymes are catalytically similar at low temperatures (5–10°C), while GPI1.00 diverges to become more efficient at higher physiological temperatures (15–25°C). This pattern of differentiation is consistent with the latitudinal distributions of the alleloenzymes and is due to the greater temperature sensitivities of GPI1.00 V max /K m values of the two alleloenzymes are virtually the same over the physiological range of temperatures. The observed pattern of catalytic differentiation is similar to that seen for interspecific GPI variants.

We describe an approach for investigating the protein folding process, using protein fragments as inhibitory probes of the refolding protein. The refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3), reversibly unfolded in 7 M urea, was monitored by the reappearance of enzyme activity after diluting the unfolded enzyme into low urea concentrations (less than or equal to 2 M) in the presence of substrates. Of eight protein fragments produced by limited proteolysis of the 159-residue enzyme, three isolated peptides--Ser-49/Glu-90, Ile-91/Glu-154, and Gln-102/Glu-154--were evaluated for their effects on the recovery of the refolding protein's enzymatic activity. By this criterion, 13 microM peptide Gln-102/Glu-154 inhibits the refolding of 0.015 microM enzyme by approximately 80%, while the related peptide, Ile-91/Glu-154, and peptide Ser-49/Glu-90 at the same concentration inhibit the recoverable activity of the refolding enzyme by less than or equal to 20%. None of these three peptides has any significant effect on the activity of the folded enzyme. Our results indicate that peptides may inhibit refolding differentially and that these effects may be extremely sensitive to fragment sequence and composition. We suggest that peptide specificity in the inhibition of protein folding may be exploited as a structural probe of protein folding mechanisms.

1. 1. Four regions of Urosalpinx cinerea foot muscle esterases were differentiated electrophoretically and partially characterized with the use of inhibitors and substrates. 2. 2. The isozymes of Esterase II are inferred to be that of a dimeric molecule, each subunit encoded at a different locus, on the basis of their electrophoretic variants, tissue distributions and selective inhibition by PMSF. 3. 3. Esterase IV, probably a diallelic polypeptide, is significantly heterogeneous for allele frequencies among the Connecticut and Virginia populations examined; this is probably associated with the limited larval dispersal pattern in this species.

Selection for allyl alcohol resistance in respiratory incompetent yeast is a highly specific method for isolating functional mutations at ADH1, the gene coding for the cytoplasmic alcohol dehydrogenase, ADHI. Because of the nature of this selection scheme, the ADHI activity of such mutants is retained, but the kinetic characteristics of the enzymes are altered. The high specificity for targeting functional mutations at this locus suggested that selection for enzyme variants with more subtle phenotypic effects might be possible. Here, we describe functional ADHI mutants that are temperature-conditional in their allyl alcohol resistance. Haploid cells of one of these mutants grow well on plates at 10 mm allyl alcohol at 19°, but not at 37°, the restrictive temperature. A second mutant grows well at 10 mm at 37°, but its growth is restricted at 19°. What distinguishes these mutants from other temperature-sensitive mutants is that the temperature-conditional growth phenotypes described here must be due to interactions between allyl alcohol levels and ADHI functional properties and cannot be due to lability of the enzyme at the restrictive temperature. This system shows promise for the investigation of functional enzyme variants that differ by only one or two amino acid residues but have significant temperature- and substrate-conditional effects on growth phenotypes in both the haploids and the diploids.

Homologues of glucosephosphate isomerase (GPI, EC 5.3.1.9) were purified to homogeneity and kinetically characterized from Mytilus edulis and Isognomon alatus, two bivalve molluscs experiencing contrasting thermal environments. The enzyme isolated from I. alatus functions at warmer temperatures (25-35 C) than GPI from M. edulis, a species that inhabits colder marine littoral habitats (5-20 C). The former exhibits apparent first-order (with respect to substrate) catalytic rate constants ( Vm,,/KM) in vitro that become progressively greater than the mussel enzyme as the assay temperature is raised. Apparent zero-order catalytic rate constants (I’,,,) are relatively less differentiated. Catalytic efficiency, defined as the rate at which a catalytic event occurs in either reaction direction for reference standard states (substrate concentrations), is greater for the enzyme from the tropical species (I. alatus) at all realistic combinations of temperature and substrate concentration except for the lowest temperatures and highest substrate concentrations, where the GPI from the boreal/temperate M. edulis is more efficient. This pattern of catalytic divergence appears to be due primarily to differentiation in V,,,/K, . These results and other published data are reviewed and shown to be inconsistent with claims that adaptation of enzymes to higher cell temperatures requires a loss in catalytic efficiency.

Documented examples of heterosis attributable to overdominance at specific protein-encoding gene loci have rarely been reported, the association of sickle cell hemoglobin with malarial resistance being the best documented example of this phenomenon. Here we report an example of overdominance that is temperature- and allyl alcohol-dependent and due to heterozygosity at the ADH1 locus, involving two ADHI functional mutants. Overdominance appears to be due in part to an intermediate level of ADHI activity in the heterozygote. Unlike previous work with this system using haploid strains, the NAD+/NADH ratios show no negative correlation with allyl alcohol resistance. This system is formally equivalent to that of sickle cell hemoglobin and shows promise as a tool for investigating the physiological basis for overdominance.

In the last two decades the study of adaptation at the molecular level has become a rapidly growing part of evolutionary biology. Some of the impetus for this interest has come from the largely unconvincing attempts to explain the existence and patterns of structural diversity of homologous proteins, especially enzymes, within and among plant and animal species (Lewontin, 1974; Nei, 1975; Wilson et al., 1977; Ayala et al., 1976). Additional motivation has come from comparative physiology and the need to understand the molecular basis of physiological adaptation to different environments. These two interests have merged in the study of the function and evolution of both interspecific and intraspecific enzyme variants and the possible roles the observed structural and functional variation might play in adaptation.