Polygenic Control of Carotid Atherosclerosis in a BALB/cJ x SM/J Intercross and a Combined Cross Involving Multiple Mouse Strains

Atherosclerosis in the carotid arteries is a major cause of ischemic stroke, which accounts for 85% of all stroke cases. Genetic factors contributing to carotid atherosclerosis remain poorly understood. The aim of this study was to identify chromosomal regions harboring genes contributing to carotid atherosclerosis in mice. 228 female F2 mice were generated from an intercross between BALB/cJ (BALB) and SM/J (SM) apolipoprotein E-deficient (Apoe^-/-) mice and fed 12 weeks of western diet. Atherosclerotic lesion sizes in the left carotid artery were quantified. 149 genetic markers were genotyped across the entire genome. Quantitative trait locus (QTL) analysis revealed 8 loci for carotid lesion sizes, located on chromosomes 1, 5, 12, 13, 15, 16, and 18. Combined cross linkage analysis using data from this cross and two previous F2 crosses derived from BALB, C57BL/6J and C3H/HeJ strains identified 5 significant QTLs on chromosomes 5, 9, 12 and 13 and 9 suggestive QTLs for carotid atherosclerosis. Of them, the QTL on chromosome 12 had a high LOD score of 9.95. Bioinformatic analysis prioritized Arhgap5, Akap6, Mipol1, Clec14a, Fancm, Nin, Dact1, Rtn1, and Slc38a6 as probable candidate genes for this QTL. Atherosclerotic lesion sizes were significantly correlated with non-HDL cholesterol levels (r=0.254; p=0.00016) but inversely correlated with HDL cholesterol levels (r=-0.134; p=0.049) in the current cross. Thus, we demonstrated the polygenic control of carotid atherosclerosis in mice. The correlations of carotid lesion sizes with non-HDL and HDL suggest that genetic factors exert effect on carotid atherosclerosis partially through modulation of lipoprotein homeostasis.

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Genome-Wide Screen for Haploinsufficient Cell Size Genes in the Opportunistic Yeast Candida albicans

One of the most critical but still poorly understood aspects of eukaryotic cell proliferation is the basis for commitment to cell division in late G1 phase called Start in yeast and the Restriction Point in metazoans. In all species, a critical cell size threshold coordinates cell growth with cell division and thereby establishes a homeostatic cell size. While a comprehensive survey of cell size genetic determinism has been performed in the saprophytic yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, very little is known in pathogenic fungi. As a number of critical Start regulators are haploinsufficient for cell size, we applied a quantitative analysis of the size phenome, using elutriation-barcode sequencing methodology, to 5,639 barcoded heterozygous deletion strains of the opportunistic yeast Candida albicans. Our screen identified conserved known regulators and biological processes required to maintain size homeostasis in the opportunistic yeast C. albicans. We also identified novel C. albicans-specific size genes and provided a conceptual framework for future mechanistic studies. Interestingly, some of the size genes identified were required for fungal pathogenicity suggesting that cell size homeostasis may be elemental to C. albicans fitness or virulence inside the host.

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Highly-Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection

Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet or even surpass Cas9 targeting rates (approaching 100% targeting) but require higher concentrations of mRNA and guide. We also demonstrate that co-injecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.

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Genome Report: Identification and Validation of Antigenic Proteins from Pajaroellobacter abortibovis Using De Novo Genome Sequence Assembly and Reverse Vaccinology

Epizootic bovine abortion (EBA), or "foothill abortion", is the leading cause of beef cattle abortion in California and has also been reported in Nevada and Oregon. In the 1970's, the soft-shelled tick Ornithodoros coriaceus, or "pajaroello tick", was confirmed as the disease-transmitting vector. In 2005, a novel Deltaproteobacterium was discovered as the etiologic agent of EBA (aoEBA), recently named Pajaroellobacter abortibovis. This organism cannot be grown in culture using traditional microbiological techniques; it can only be grown in experimentally-infected severe combined immunodeficient (SCID) mice. The objectives of this study were to perform a de novo genome assembly for P. abortibovis and identify and validate potential antigenic proteins as candidates for future recombinant vaccine development. DNA and RNA were extracted from spleen tissue collected from experimentally-infected SCID mice following exposure to P. abortibovis. This combination of mouse and bacterial DNA was sequenced and aligned to the mouse genome. Mouse sequences were subtracted from the sequence pool and the remaining sequences were de novo assembled at 50X coverage into a 1.82 Mbp complete closed circular Deltaproteobacterial genome containing 2,250 putative protein coding sequences. Phylogenetic analysis of P. abortibovis predicts that this bacterium is most closely related to the organisms of the order Myxococcales, referred to as Myxobacteria. In silico prediction of vaccine candidates was performed using a reverse vaccinology approach resulting in the identification and ranking of the top ten candidate proteins that are likely to be antigenic. Immunologic testing of these candidate proteins confirmed antigenicity of seven of the nine expressed protein candidates using serum from P. abortibovis immunized mice.

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Latrunculin A Induced Perturbation of the Actin Cytoskeleton Mediates Pap1p-Dependent Induction of the Caf5p Efflux Pump in Schizosaccharomyces pombe

As part of an earlier study aimed at uncovering gene-products with roles in defending against latrunculin A (LatA) induced cytoskeletal perturbations, we identified three members of the oxidative stress response pathway: the Pap1p AP-1 like transcription factor, the Imp1p importin-alpha, and the Caf5p efflux pump. In this report, we characterize the pathway further and show that Pap1p translocates from the cytoplasm to the nucleus in an Imp1p-dependent manner upon LatA treatment. Moreover, preventing this translocation -- through the addition of a nuclear export signal – confers the same characteristic LatA-sensitive phenotype exhibited by pap1 cells. Lastly, we show that the caf5 gene is induced upon exposure to LatA and that Pap1p is required for this transcriptional upregulation. Importantly, the expression of trr1, a Pap1p target specifically induced in response to oxidative stress, is not significantly altered by LatA treatment. Taken together, these results suggest a model in which LatA-mediated cytoskeletal perturbations are sensed, triggering the Imp1p-dependent translocation of Pap1p to the nucleus and the induction of the caf5 gene (independently of oxidative stress).

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Genetic Analysis of the Lambda Spanins Rz and Rz1: Identification of Functional Domains

Coliphage lambda proteins Rz and Rz1 are the inner membrane and outer membrane subunits of the spanin complex, a heterotetramer that bridges the periplasm and is essential for the disruption of the outer membrane during phage lysis. Recent evidence suggests the spanin complex functions by fusing the inner and outer membrane. Here we use a genetics approach to investigate and characterize determinants of spanin function. Because Rz1 is entirely embedded in the +1 reading frame of Rz, the genes were dis-embedded before using random mutagenesis to construct a library of lysis-defective alleles for both genes. Surprisingly, most of the lysis-defective missense mutants exhibited normal accumulation or localization in vivo and also were found to be normal for complex formation in vitro. Analysis of the distribution and nature of single missense mutations revealed sub-domains that resemble key motifs in established membrane-fusion systems, i.e. two coiled-coil domains in Rz, a proline-rich region of Rz1, and flexible linkers in both proteins. When coding sequences are aligned respective to the embedded genetic architecture of Rz1 within Rz, genetically silent domains of Rz1 correspond to mutationally-sensitive domains in Rz, and vice-versa, suggesting that the modular structure of the two subunits facilitated the evolutionary compression that resulted in the unique embedded gene architecture.

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Comparative High-Density Linkage Mapping Reveals Conserved Genome Structure but Variation in Levels of Heterochiasmy and Location of Recombination Cold Spots in the Common Frog

By combining 7077 SNPs and 61 microsatellites, we present the first linkage map for some of the early diverged lineages of the common frog, Rana temporaria, and the densest linkage map to date for this species. We found high homology with the published linkage maps of the Eastern and Western lineages but with differences in the order of some markers. Homology was also strong with the genome of the Tibetan frog Nanorana parkeri and we found high synteny with the clawed frog Xenopus tropicalis. We confirmed marked heterochiasmy between sexes and detected non-recombining regions in several groups of the male linkage map. Contrary to the expectations set by the male heterogamety of the common frog, we did not find male heterozygosity excess in the chromosome previously shown to be linked to sex determination. Finally, we found blocks of loci showing strong transmission ratio distortion. These distorted genomic regions might be related to genetic incompatibilities between the parental populations, and are promising candidates for further investigation into the genetic basis of speciation and adaptation in the common frog.

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