Commercial sealing in the 18th and 19th centuries had a major impact on the Antarctic and subantarctic fur seal populations (Arctocephalus gazella and A. tropicalis) in the Southern Ocean. The intensive and unrestricted nature of the industry ensured substantial reductions in population sizes and resulted in both species becoming locally extinct at some sites. However, both species are continuing to recover, through the recolonization of islands across their former range and increasing population size. This study investigated the extent and pattern of genetic variation in each species to examine the hypothesis that higher levels of historic sealing in A. gazella have resulted in a greater loss of genetic variability and population structure compared with A. tropicalis. A 316-bp section of the mitochondrial control region was sequenced and revealed nucleotide diversities of 3.2% and 4.8% for A. gazella and A. tropicalis, respectively. There was no geographical distribution of lineages observed within either species, although the respective PhiST values of 0.074 and 0.19 were significantly greater than zero. These data indicate low levels of population structure in A. gazella and relatively high levels in A. tropicalis. Additional samples screened with restriction endonucleases were incorporated, and the distribution of restriction fragment length polymorphism (RFLP) and sequence haplotypes were examined to identify the main source populations of newly recolonized islands. For A. tropicalis, the data suggest that Macquarie Island and Iles Crozet were probably recolonized by females from Marion Island, and to a lesser extent Ile Amsterdam. Although there was less population structure within A. gazella, there were two geographical regions identified: a western region containing the populations of South Georgia and Bouvetoya, which were the probable sources for populations at Marion, the South Shetland and Heard Islands; and an eastern region containing the panmictic populations of Iles Kerguelen and Macquarie Island. The latter region may be a result of a pronounced founder effect, or represent a remnant population that survived sealing at Iles Kerguelen.

During λ infections, the holin S105 accumulates harmlessly in the membrane until, at an allele-specific time, suddenly triggering to form irregular holes of unprecedented size (>300 nm), releasing the endolysin from the cytoplasm, resulting in lysis within seconds. Here we used a functional S105-GFP chimera and real-time deconvolution fluorescence microscopy to show that the S105-GFP fusion accumulated in a uniformly distributed fashion, until suddenly, within 1 min, it formed aggregates, or rafts, at the time of lethal triggering. Moreover, the isogenic fusion to a nonlethal S105 mutant remained uniformly distributed, whereas a fusion to an earlylysing mutant showed early triggering and early raft formation. Protein accumulation rates of the WT, early, and nonlethal alleles were identical. Fluorescence recovery after photobleaching (FRAP) revealed that the nonlethal mutant and untriggered WT hybrids were highly mobile in the membrane, whereas the WT raft was essentially immobile. Finally, an antiholin allele, S105 ΔTMD1 -mcherryfp, in the product of which the S105 sequence deleted for the first transmembrane domain was fused to mCherryFP. This hybrid retained full antiholin activity, in that it blocked lethal hole formation by the S105-GFP fusion, accumulated uniformly throughout the host membrane and prevented the S105-GFP protein from forming rafts. These findings suggest that phage lysis occurs when the holin reaches a critical concentration and nucleates to form rafts, analogous to the initiation of purple membrane formation after the induction of bacteriorhodopsin in halobacteria. This model for holin function may be relevant for processes in mammalian cells, including the release of nonenveloped viruses and apoptosis.bacteriophage | latent period | peptide linker T he programmed formation of nonspecific, lethal membrane lesions, or "holes," is featured in many cytocidal phenotypes, including Bax-mediated apoptosis, virion release in infections of nonenveloped mammalian viruses, and the dissemination of important human toxins from bacteria (1-6). The most genetically tractable hole-formation process is host lysis in double-strand DNA bacteriophage infections (7,8). In the infection cycle of phage λ, lysis is a precisely timed event controlled by the holin, S105, a 105-aa product of the S gene (Fig. 1). S105 accumulates in the membrane throughout the morphogenesis period of the infection cycle. This accumulation has no effect on membrane integrity or the proton-motive force (PMF), as shown by noninvasive assays measuring the flagellar rotation speed (9), until suddenly triggering to form irregular holes of unprecedented size (>300 nm) (10). These holes allow release of the phage endolysin, R, from the cytoplasm, resulting in destruction of the cell wall within seconds (9). The holin triggering time is allele specific (11,12), in that it can be advanced or retarded by missense mutations throughout all three transmembrane domains (TMDs) of S105. Holins can be triggered prematurely by energy poisons; for S10...

This paper investigates the production technology facing computerized credit unions in Canada. A full system of translog cost equations is estimated in order to test for economies of scale, economies of scope, and other production characteristics in a multiproduct context. The regression results indicate that most of the credit unions in our sample experience significant increasing returns to scale as they expand their level of output. There is also evidence of cost complementarity or economies of scope in their mortgage and other lending activities. As a result, legislation which limits the ability of credit unions to grow and diversify will likely raise the operating costs of this important group of financial institutions. Additional structural tests of the most general translog specification suggest that none of the restrictive production conditions commonly imposed by other researchers using Cobb-Douglas and CES specifications provide a valid representation of credit union technology. The results of many earlier studies are therefore open to question. THIS PAPER PRESENTS A number of structural tests of the production technology facing large computerized credit unions in Canada. A full system of translog cost equations is estimated using detailed cross-sectional data on British Columbia credit unions. The parameter estimates obtained from our multiproduct specification allow us to test for economies of scale, economies of scope, and factor substitution.Unlike many earlier studies, the present paper introduces the various outputs as separate variables instead of crudely proxying them with a single output measure such as total assets, the total number of loans, or the number of depositors. By explicitly recognizing the multiproduct nature of the intermediation process, we are able to address more interesting production questions and remove some of the bias caused by misspecification and measurement error in simpler Approaches. The flexible translog specification allows us to estimate the sensitivity of costs to proportionate increases in all outputs (economies of scale), as well as the cost savings firms realize by producing several outputs jointly rather than specializing in the production of one (economies of scope).The test results reported in the latter part of the paper suggest that increasing returns to scale exist in almost all of the credit unions in our sample. Though

Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or "holes." In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.bacteriophage | cryoelectron tomography | Escherichia coli | holin | lambda B acteriophage lysis, the most frequent cytolethal event in the biosphere, is a precisely scheduled process controlled by proteins of the holin family (1). Holins are an extremely diverse class of small phage-encoded membrane proteins (2). The best studied holin is S105, a 105-residue polypeptide with three transmembrane domains (TMDs) encoded by the S gene of phage λ (3). Throughout the period of late gene expression and particle assembly, S105 accumulates in the cytoplasmic membrane of Escherichia coli without any effect on its integrity (4). Suddenly, at a programmed time, S105 triggers to form a lesion, or hole, in the membrane; this allows the λ-endolysin, R, to escape from the cytoplasm and attack the cell wall (2). In phages of Gram-negative hosts, there is a third step to complete the lysis pathway involving a protein or protein complex, the spanin, which connects the cytoplasmic and outer membranes (5, 6). In λ, the spanin complex consists of the cytoplasmic membrane protein, Rz, and the outer membrane lipoprotein, Rz1. This complex is essential for lysis in media containing millimolar concentrations of divalent cations, and thus is thought to act by disrupting the outer membrane, possibly by fusion with the inner membrane (6).Although the S105 holin has been extensively studied using genetic and biochemical approaches (3, 4, 7-9), nothing is known about the membrane holes except that they are nonspecific and large enough to allow escape of fully folded tetrameric R-β-galactosi...

Methods are given for efficient calculation of the likelihood for multilocus linkage in families comprised of grandparents, parents, and children. Such families are being used in large-scale cooperative efforts to build a detailed linkage map of the human genome. The methods are illustrated by an application to loci on chromosome 13.

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Summary Holins control the length of the infection cycle of tailed phages (the Caudovirales) by oligomerizing to form lethal holes in the cytoplasmic membrane at a time dictated by their primary structure. Nothing is currently known about the physical basis of their oligomerization or the structure of the oligomers formed by any known holin. Here we use electron microscopy and single-particle analysis to characterize structures formed by the bacteriophage λ holin (S105) in vitro. In non-ionic or mild zwitterionic detergents, purified S105, but not the lysis-defective variant S105A52V, forms rings of at least two size classes, the most common having inner and outer diameters of 8.5 and 23 nm respectively, and containing approximately 72 S105 monomers. The height of these rings, 4 nm, closely matches the thickness of the lipid bilayer. The central channel is of unprecedented size for channels formed by integral membrane proteins, consistent with the non-specific nature of holin-mediated membrane permeabilization. S105 present in detergent-solubilized rings and in inverted membrane vesicles showed similar sensitivities to proteolysis and cysteine-specific modification, suggesting that the rings are representative of the lethal holes formed by S105 to terminate the infection cycle and initiate lysis.

Affinity-matured, functional anti-pathogen antibodies are present at low frequencies in natural human repertoires. These antibodies are often excellent candidates for therapeutic monoclonal antibodies. However, mining natural human antibody repertoires is a challenge. In this study, we demonstrate a new method that uses microfluidics, yeast display, and deep sequencing to identify 247 natively paired anti-pathogen single-chain variable fragments (scFvs), which were initially as rare as 1 in 100,000 in the human repertoires. Influenza A vaccination increased the frequency of influenza A antigen-binding scFv within the peripheral B cell repertoire from <0.1% in non-vaccinated donors to 0.3-0.4% in vaccinated donors, whereas pneumococcus vaccination did not increase the frequency of antigen-binding scFv. However, the pneumococcus scFv binders from the vaccinated library had higher heavy and light chain Replacement/Silent mutation (R/S) ratios, a measure of affinity maturation, than the pneumococcus binders from the corresponding non-vaccinated library. Thus, pneumococcus vaccination may increase the frequency of affinity-matured antibodies in human repertoires. We synthesized 10 anti-influenza A and nine anti-pneumococcus full-length antibodies that were highly abundant among antigen-binding scFv. All 10 anti-influenza A antibodies bound the appropriate antigen at KD<10 nM and neutralized virus in cellular assays. All nine anti-pneumococcus full-length antibodies bound at least one polysaccharide serotype, and 71% of the anti-pneumococcus antibodies that we tested were functional in cell killing assays. Our approach has future application in a variety of fields, including the development of therapeutic antibodies for emerging viral diseases, autoimmune disorders, and cancer.