The gain and loss of genes during 600 million years of vertebrate evolution

Background  

Gene duplication is assumed to have played a crucial role in the evolution of vertebrate organisms. Apart from a continuous mode of duplication, two or three whole genome duplication events have been proposed during the evolution of vertebrates, one or two at the dawn of vertebrate evolution, and an additional one in the fish lineage, not shared with land vertebrates. Here, we have studied gene gain and loss in seven different vertebrate genomes, spanning an evolutionary period of about 600 million years.     

Genotypic diversity and differentiation among populations of two benthic freshwater diatoms as revealed by microsatellites

Given their large population sizes and presumed high dispersal capacity, protists are expected to exhibit homogeneous population structure over large spatial scales. On the other hand, the fragmented and short‐lived nature of the lentic freshwater habitats that many protists inhabit promotes strong population differentiation. We used microsatellites in two benthic freshwater diatoms, Eunotia bilunaris ‘robust’ and Sellaphora capitata, sampled from within a pond and connected ponds, through isolated ponds from the same region to western Europe to determine the spatial scale at which differentiation appears. Because periods of low genotypic diversity contribute to population differentiation, we also assessed genotypic diversity. While genotypic diversity was very high to maximal in most samples of both species, some had a markedly lower diversity, with up to half (Eunotia) and over 90% (Sellaphora) of the strains having the same multilocus genotype. Population differentiation showed an isolation‐by‐distance pattern with very low standardized F_ST values between samples from the same or connected ponds but high values between isolated ponds, even when situated in the same region. Partial rbcL sequences in Eunotia were consistent with this pattern as isolated ponds in the same region could differ widely in haplotype composition. Populations identified by Structure corresponded to the source ponds, confirming that ‘pond’ is the main factor structuring these populations. We conclude that freshwater benthic diatom populations are highly fragmented on a regional scale, reflecting either less dispersal than is often assumed or reduced establishment success of immigrants, so that dispersal does not translate into gene flow.     

Functional analysis of the simian immunodeficiency virus Vpx protein: identification of packaging determinants and a novel nuclear targeting domain

The vpx gene products of human immunodeficiency virus type 2 (HIV-2) and of the closely related simian immunodeficiency viruses from sooty mangabeys (SIVsm) and macaques (SIVmac) comprise a 112-amino-acid virion-associated protein that is critical for efficient virus replication in nondividing cells such as macrophages. When expressed in the absence of other viral proteins, Vpx localizes to the nuclear membrane as well as to the nucleus; however, in the context of virus replication Vpx is packaged into virions via interaction with the p6 domain of the Gag precursor polyprotein (p55(gag)). To identify the domains essential for virion incorporation and nuclear localization, site-directed mutations were introduced into the vpx gene of SIVsmPBj1.9 and functionally analyzed. Our results show that (i) mutation of two highly conserved L74 and I75 residues impaired both virion incorporation and nuclear localization of Vpx; (ii) substitution of conserved H82, G86, C87, P103, and P106 residues impaired Vpx nuclear localization but not virion incorporation; (iii) mutations of conserved Y66, Y69, and Y71 residues impaired virion incorporation but not the translocation of Vpx to the nucleus; and (iv) a mutation at E30 (predicted to disrupt an N-terminal alpha-helix) had no effect on either virion incorporation or nuclear localization of Vpx. Importantly, mutations in Vpx which impaired nuclear localization also reduced virus replication in macaque macrophages, suggesting an important role of the carboxyl terminus of Vpx in nuclear translocation of the viral preintegration complex. Analyzing this domain in greater detail, we identified a 26-amino-acid (aa 60 to 85) fragment that was sufficient to mediate the transport of a heterologous protein (green fluorescent protein [GFP]) to the nucleus. Taken together, these results indicate that virion incorporation and nuclear localization are encoded by two partially overlapping domains in the C-terminus of Vpx (aa 60 to 112). The identification of a novel 26-amino-acid nuclear targeting domain provides a new tool to investigate the nuclear import of the HIV-2/SIV preintegration complex.     

Prevention and reversal of hepatic steatosis with a high-protein diet in mice

The hallmark of NAFLD is steatosis of unknown etiology. We tested the effect of a high-protein (HP)² diet on diet-induced steatosis in male C57BL/6 mice with and without pre-existing fatty liver. Mice were fed all combinations of semisynthetic low-fat (LF) or high-fat (HF) and low-protein (LP) or HP diets for 3 weeks. To control for reduced energy intake by HF/HP-fed mice, a pair-fed HF/LP group was included. Reversibility of pre-existing steatosis was investigated by sequentially feeding HF/LP and HF/HP diets. HP-containing diets decreased hepatic lipids to ~ 40% of corresponding LP-containing diets, were more efficient in this respect than reducing energy intake to 80%, and reversed pre-existing diet-induced steatosis. Compared to LP-containing diets, mice fed HP-containing diets showed increased mitochondrial oxidative capacity (elevated Pgc1α, mAco, and Cpt1 mRNAs, complex-V protein, and decreased plasma free and short-chain acyl-carnitines, and [C₀]/[C₁₆ + C₁₈] carnitine ratio); increased gluconeogenesis and pyruvate cycling (increased PCK1 protein and fed plasma–glucose concentration without increased G6pase mRNA); reduced fatty-acid desaturation (decreased Scd1 expression and [C_16:1n ? 7]/[C_16:0] ratio) and increased long-chain PUFA elongation; a selective increase in plasma branched-chain amino acids; a decrease in cell stress (reduced phosphorylated eIF2α, and Fgf21 and Chop expression); and a trend toward less inflammation (lower Mcp1 and Cd11b expression and less phosphorylated NFκB). Conclusion: HP diets prevent and reverse steatosis independently of fat and carbohydrate intake more efficiently than a 20% reduction in energy intake. The effect appears to result from fuel-generated, highly distributed small, synergistic increases in lipid and BCAA catabolism, and a decrease in cell stress.     

Characterization of CFTR expression and chloride channel activity in human endothelia

The cystic fibrosis transmembrane conductance regulator (CFTR)functions as a low-conductance, cAMP-regulated chloride(Cl) channel in a varietyof cell types, such as exocrine epithelial cells. Our resultsdemonstrate that human primary endothelial cells isolated fromumbilical vein (HUVEC) and lung microvasculature (HLMVEC) also expressCFTR as determined via RT-PCR and immunohistochemical andimmunoprecipitation analyses. Moreover,Cl efflux and whole cellpatch-clamp analyses reveal that HUVEC (n = 6 samples,P < 0.05) and HLMVEC(n = 5 samples,P < 0.05) display cyclicnucleotide-stimulated Cltransport that is inhibited by the CFTR selectiveCl channel blockerglibenclamide but not by the blocker DIDS, indicative of CFTRCl channel activity. Takentogether, these findings demonstrate that human endothelial cellsderived from multiple organ systems express CFTR and that CFTRfunctions as a cyclic nucleotide-regulated Cl channel in human endothelia.

     

Plasmid transfer from Pseudomonas putida to the indigenous bacteria on alfalfa sprouts: characterization,direct quantification,and in situ location of transconjugant cells

The transfer of the plasmids pJKJ5 and TOL (pWWO) from Pseudomonas putida to the indigenous bacterial community on alfalfa sprouts was studied. Tagging with fluorescent protein markers allowed direct quantification of the introduced donor bacteria and of indigenous bacteria that had received the plasmids. The sprouts were observed for 9 days; during this time alfalfa seeds, inoculated with donor bacteria, developed to edible and subsequently decaying sprouts. The first transconjugants were detected on day 6 after donor inoculation and occurred at frequencies of 3.4 x 10(-4) and 2.0 x 10(-6) transconjugant cells per donor cell for pKJK5::gfp and TOL::gfp, respectively. Confocal laser scanning microscopy revealed that the sprouts were heavily colonized with donors and that most transconjugants were located around the hypocotyl and root areas. Randomly selected members of the indigenous bacterial community from both inoculated and uninoculated sprouts, as well as a representative part of the community that had received the plasmids, were characterized by polymorphisms of PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by partial 16S rDNA sequencing. This showed that the initially dominating genera Erwinia and Paenibacillus were gradually replaced by Pseudomonas on the fully developed sprouts. Transconjugants carrying either of the investigated plasmids mainly belonged to the genera Pseudomonas and ERWINIA: The numbers of transconjugant cells did not reach detectable levels until 6 days after the onset of germination, at which point these species constituted the majority of the indigenous bacteria. In conclusion, the alfalfa sprouts provided an environment that allowed noteworthy frequencies of plasmid transfer from P. putida in the absence of selective pressure that could favor the presence of the investigated plasmids.