Chromosome flexion: potential for assessing the state of spindle assembly

A novel measurement, that of chromosome flexion, has been used to assess the degree of spindle polymerization at metaphase in human lymphocytes. It was found that this measurement showed a highly repeatable quantitative response to nocodazole exposure. Thus this measurement could be used to assess the potential for chemicals to depolymerize spindles. Under controlled conditions, individual differences were observed between the subjects which might be related to their age. However, the response to nocodazole-induced spindle depolymerization, as measured by flexion, was uniform for all subjects. 4 chemicals reported to induce aneuploidy in mammalian cells were used in a flexion assay. Only substances known to depolymerize microtubules reduced chromosome flexion.     

Conservation of Functionally Important Global Motions in an Enzyme Superfamily across Varying Quaternary Structures

The α‐d‐phosphohexomutase superfamily comprises enzymes involved in carbohydrate metabolism that are found in all kingdoms of life. Recent biophysical studies have shown for the first time that several of these enzymes exist as dimers in solution, prompting an examination of the oligomeric state of all proteins of known structure in the superfamily (11 different proteins; 31 crystal structures) via computational and experimental analyses. We find that these proteins range in quaternary structure from monomers to tetramers, with 6 of the 11 known structures being likely oligomers. The oligomeric state of these proteins not only is associated in some cases with enzyme subgroup (i.e., substrate specificity) but also appears to depend on domain of life, with the two archaeal proteins existing as higher‐order oligomers. Within the oligomers, three distinct interfaces are observed, one of which is found in both archaeal and bacterial proteins. Normal mode analysis shows that the topological arrangement of the oligomers permits domain 4 of each protomer to move independently as required for catalysis. Our analysis suggests that the advantages associated with protein flexibility in this enzyme family are of sufficient importance to be maintained during the evolution of multiple independent oligomers. This study is one of the first showing that global motions may be conserved not only within protein families but also across members of a superfamily with varying oligomeric structures.     

The genetic structure of Australasian green turtles (Chelonia mydas): exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long-term mark-recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Phi = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.     

The [PSI+] prion of Saccharomyces cerevisiae can be propagated by an Hsp104 orthologue from Candida albicans

The molecular chaperone Hsp104 is not only a key component of the cellular machinery induced to disassemble aggregated proteins in stressed cells of Saccharomyces cerevisiae but also plays an essential role in the propagation of the [PSI⁺], [URE3], and [RNQ/PIN⁺] prions in this organism. Here we demonstrate that the fungal pathogen Candida albicans carries an 899-residue stress-inducible orthologue of Hsp104 (CaHsp104) that shows a high degree of amino acid identity to S. cerevisiae Hsp104 (ScHsp104). This identity is significantly lower in the N- and C-terminal regions implicated in substrate recognition and cofactor binding, respectively. CaHsp104 is able to provide all known functions of ScHsp104 in an S. cerevisiae hsp104 null mutant, i.e., tolerance to high-temperature stress, reactivation of heat-denatured proteins, and propagation of the [PSI⁺] prion. As also observed for ScHsp104, overexpression of CaHsp104 leads to a loss of the [PSI⁺] prion. However, unlike that of ScHsp104, CaHsp104 function is resistant to guanidine hydrochloride (GdnHCl), an inhibitor of the ATPase activity of this chaperone. These findings have implications both in terms of the mechanism of inhibition of Hsp104 by GdnHCl and in the evolution of the ability of fungal species to propagate prions.     

The location and the significance of a cross-link between the sarcin/ricin domain of ribosomal RNA and the elongation factor-G

During translocation peptidyl-tRNA moves from the A-site to the P-site and mRNA is displaced by three nucleotides in the 3' direction. This reaction is catalyzed by elongation factor-G (EF-G) and is associated with ribosome-dependent hydrolysis of GTP. The molecular basis of translocation is the most important unsolved problem with respect to ribosome function. A critical question, one that might provide a clue to the mechanism of translocation, is the precise identity of the contacts between EF-G and ribosome components. To make the identification, a covalent bond was formed, by ultraviolet irradiation, between EF-G and a sarcin/ricin domain (SRD) oligoribonucleotide containing 5-iodouridine. The cross-link was established, by mass spectroscopy and by Edman degradation, to be between a tryptophan at position 127 in the G domain in EF-G and either one of two 5-iodouridine nucleotides in the sequence UAG2655U in the SRD. G2655 is a critical identity element for the recognition of the factor's ribosomal binding site. The site of the cross-link provides the first direct evidence that the SRD is in close proximity to the EF-G catalytic center. The proximity suggests that the SRD RNA has a role in the activation of GTP hydrolysis that leads to a transition in the conformation of the factor and to its release from the ribosome.     

Genetic Recombination between Human and Animal Parasites Creates Novel Strains of Human Pathogen

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.     

Sexual dimorphism in the North Island Kaka Nestor meridionalis septentrionalis: selection for enhanced male provisioning ability?

The North Island Kaka Nestor meridionalis septentrionalis , an endemic New Zealand parrot, displays a disproportionate degree of sexual dimorphism in bill-size, males being 13.6% and 12.4% larger than females in culmen-length and -depth respectively, but only 2.0% and 4.2% larger in tarsus-length and cube root of body-mass. Culmen-length provides a reliable means of sexing Kakas if the age-class of each bird is known; all adult and subadult males had exposed culmens ≥ 47 mm long, while those of females were less than this value. Juveniles with culmen-lengths in excess of 44 mm were male. Similar sexual dimorphism in the Kea N. notabilis , the sole extant congener of the Kaka, suggests a phylogenetic basis for this condition. The monogamous mating system of the Kaka and Kea, together with the prolonged provisioning of females and young by males in both species, suggests that selection for enhanced male provisioning ability, rather than sexual selection, could be maintaining sexual bill dimorphism in these species.