Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

The genetic variability of the red king crab, <Emphasis Type="Italic">Paralithodes camtschatica</Emphasis> (Tilesius, 1815) (Anomura,Lithodidae) introduced into the Barents Sea compared with samples from the Bering Sea and Kamchatka region using eleven microsatellite loci

The intentional introduction of red king crab, Paralithodes camtschatica (Tilesius, 1815) in the Barents Sea represent one of a few successful cases and one that now supports a commercial fishery. Introductions of alien species into new environments are often associated with genetic bottlenecks, which cause a reduction in the genetic variation, and this could be important for the spreading potential of the species in the Atlantic Ocean. Red king crab samples collected in the Varangerfjord located on the Barents Sea (northern Norway) were compared with reference crab samples collected from the Bering Sea and Kamchatka regions in the Pacific Ocean. All samples were screened for eleven microsatellite loci, based on the development of species-specific primers. The observed number of alleles per locus was similar, and no reduction in genetic variation, including gene diversity and allelic richness, was detected between the Varangerfjord sample and the reference sample from Okhotsk Sea near Kamchatka, indicating no genetic bottlenecking at least for the microsatellite loci investigated. The same results were found in comparison with the sample from Bering Sea. The level of genetic differentiation among the samples, measured as overall F _ST across all loci, was relatively low (0.0238) with a range of 0.0035–0.1000 for the various loci investigated. The largest pairwise F _ST values were found between the Bering Sea and Varangerfjord/Barents Sea samples, with a value of 0.0194 across all loci tested. The lowest value (0.0101) was found between the Varangerfjord and Kamchatka samples. Genetic differentiation based on exact tests on allele frequencies revealed highly significant differences between all pairwise comparisons. The high level of genetic variation found in the Varangerfjord/Barents Sea sample could be of significance with respect to further spreading of the species to other regions in the North Atlantic Ocean.     

Development and application of genomic tools to the restoration of green abalone in southern California

Due to severe declines in abundance throughout southern California, the green abalone (Haliotis fulgens Philippi 1845) became protected under a state-sponsored fishery moratorium in 1997 and was declared a NOAA NMFS Species of Concern in 2004. Recently, H. fulgens was chosen for possible stock restoration via translocation of wild adults to depleted habitat and supplementation through releasing cultured individuals. Before a management plan could be developed, however, an understanding of the species’ natural population genetic structure was needed. We used a genomic technique called restriction site associated DNA sequencing (RADSeq) to address the issue. RADSeq enabled discovery of 1,209 single nucleotide polymorphisms theoretically spread genome-wide in H. fulgens. Analyses suggested the species may be panmictic throughout our sampled range, with an effective population size (N_e) of 1,100–3,600. Hence, limitations to management, such as requiring local broodstock and restricting translocation potential, might be unnecessary. Sites with larger populations may be suitable sources for restoration of depleted sites (e.g. the Palos Verdes Peninsula), although the extent of local adaptation remains unknown. Despite this potential for restoration, results gathered on a sample of cultured H. fulgens illustrated how quickly genetic diversity can be lost through captive breeding. To help mitigate a drop in N_e due to hatchery supplementation, we recommend collection and replacement of ≥100 wild abalone per generation for broodstock and close management of the proportion of cultured individuals in the wild. Successful implementation will depend on operational capacity and the resilience of the source populations to broodstock collection.     

Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics

During eukaryotic cell division, the sister chromatids of duplicated chromosomes are pulled apart by microtubules, which connect via kinetochores. The kinetochore is a multiprotein structure that links centromeres to microtubules, and that emits molecular signals in order to safeguard the equal distribution of duplicated chromosomes over daughter cells. Although microtubule‐mediated chromosome segregation is evolutionary conserved, kinetochore compositions seem to have diverged. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined orthologs of 70 kinetochore proteins in 90 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that current‐day kinetochores differ substantially. These kinetochores diverged through gene loss, duplication, and, less frequently, invention and displacement. Various kinetochore components co‐evolved with one another, albeit in different manners. These co‐evolutionary patterns improve our understanding of kinetochore function and evolution, which we illustrated with the RZZ complex, TRIP13, the MCC, and some nuclear pore proteins. The extensive diversity of kinetochore compositions in eukaryotes poses numerous questions regarding evolutionary flexibility of essential cellular functions.     

Gene mapping of isozyme loci in chum salmon

Recombination values were used to calculate the gene-centromere map distances for four electrophoretically detected loci, Aat3, Idh1, Idh4, and Mpi, in chum salmon (Oncorhynchus keta). We also report the results from 39 pairwise examinations for joint segregation for 10 loci in nine testcross families. Only two loci assorted nonrandomly--either Aat1 or Aat2 with Gpt. Gene-centromere distances for Aat3 and Mpi differed significantly from those reported previously for rainbow trout (Salmo gairdneri), a closely related species. This difference indicates either the presence of chromosome rearrangements or a different rate of recombination between the species. These results contrast with the conservation of linkage distances previously reported within and between other salmonid genera.