Microbial rhodopsins: functional versatility and genetic mobility

The type 1 (microbial) rhodopsins are a diverse group of photochemically reactive proteins that span the three domains of life. Their broad phylogenetic distribution has motivated conjecture that rhodopsin-like functionality was present in the last common ancestor of all life. Here, we discuss the evolution of the type 1 microbial rhodopsins and document five cases of lateral gene transfer (LGT) between domains. We suggest that, thanks to the functional versatility of these retinylidene proteins and the relative ease with which they can complement the existing energy-generating or photosensory repertoires of many organisms, LGT is in fact the principal force that determines their broad but patchy distribution.     

Phenotypic characterization of the Ath-1 gene controlling high density lipoprotein levels and susceptibility to atherosclerosis

The Ath-1 gene determines the levels of high density lipoprotein (HDL) lipid in response to a high fat diet challenge as well as susceptibility to diet-induced atherosclerosis in mice (Paigen et al. 1987. Proc. Natl. Acad. Sci. USA. 84: 3763-3767). As yet, the identity of the Ath-1 gene and how it acts to affect HDL levels are completely unknown. In an effort to clarify the nature of the gene, we have examined HDL phenotypes in strains carrying either the susceptible or resistant alleles. When challenged with a high fat diet, the susceptible strain C57BL/6 exhibited a marked decrease in the levels of HDL cholesterol and apolipoprotein A-I (apoA-I), the major protein of HDL, whereas the resistant strains C3H and BALB/c maintained high levels of both. Separation of HDL subfractions by polyacrylamide gradient gel electrophoresis revealed that the decrease was particularly striking among the larger HDL species. The rates of synthesis of apoA-I in liver and intestine were similar in the strains and were unaffected by the high fat diet. Although the rates of synthesis of apoA-II and the levels of apoA-II mRNA were decreased in response to the high fat diet, similar decreases were observed in both the susceptible and resistant strains. We conclude that the Ath-1 gene results in a rapid decrease in both HDL lipid and HDL apolipoprotein levels in the susceptible strain in response to the high fat diet and that this is mediated primarily at the level of HDL catabolism.     

Alternative methods for concatenation of core genes indicate a lack of resolution in deep nodes of the prokaryotic phylogeny

It has recently been proposed that a well-resolved Tree of Life can be achieved through concatenation of shared genes. There are, however, several difficulties with such an approach, especially in the prokaryotic part of this tree. We tackled some of them using a new combination of maximum likelihood-based methods, developed in order to practice as safe and careful concatenations as possible. First, we used the application concaterpillar on carefully aligned core genes. This application uses a hierarchical likelihood-ratio test framework to assess both the topological congruence between gene phylogenies (i.e., whether different genes share the same evolutionary history) and branch-length congruence (i.e., whether genes that share the same history share the same pattern of relative evolutionary rates). We thus tested if these core genes can be concatenated or should be instead categorized into different incongruent sets. Second, we developed a heat map approach studying the evolution of the phylogenetic support for different bipartitions, when the number of sites of different phylogenetic quality in the concatenation increases. These heatmaps allow us to follow which phylogenetic signals increase or decrease as the concatenation progresses and to detect emerging artifactual groupings, that is, groups that are more and more supported when more and more homoplasic sites are thrown in the analysis. We showed that, as far as 7 major prokaryotic lineages are concerned, only 22 core genes can be said to be congruent and can be safely concatenated. This number is even smaller than the number of genes retained to reconstruct a "Tree of One Per Cent." Furthermore, the concatenation of these 22 markers leads to an unresolved tree as the only groupings in the concatenation tree seem to reflect emerging artifacts. Using concatenated core genes as a valid framework to classify uncharacterized environmental sequences can thus be misleading.     

Lipase Maturation Factor LMF1, Membrane Topology and Interaction with Lipase Proteins in the Endoplasmic Reticulum

Lipase maturation factor 1 (LMF1) is predicted to be a polytopic protein localized to the endoplasmic reticulum (ER) membrane. It functions in the post-translational attainment of enzyme activity for both lipoprotein lipase and hepatic lipase. By using transmembrane prediction methods in mouse and human orthologs, models of LMF1 topology were constructed and tested experimentally. Employing a tagging strategy that used insertion of ectopic glycan attachment sites and terminal fusions of green fluorescent protein, we established a five-transmembrane model, thus dividing LMF1 into six domains. Three domains were found to face the cytoplasm (the amino-terminal domain and loops B and D), and the other half was oriented to the ER lumen (loops A and C and the carboxyl-terminal domain). This representative model shows the arrangement of an evolutionarily conserved domain within LMF1 (DUF1222) that is essential to lipase maturation. DUF1222 comprises four of the six domains, with the two largest ones facing the ER lumen. We showed for the first time, using several naturally occurring variants featuring DUF1222 truncations, that Lmf1 interacts physically with lipoprotein lipase and hepatic lipase and localizes the lipase interaction site to loop C within DUF1222. We discuss the implication of our results with regard to lipase maturation and DUF1222 domain structure.     

Genetic toxicology studies comparing the activity of sidestream smoke from cigarettes which burn or only heat tobacco

The results of in vitro genetic toxicology studies of sidestream cigarette smoke (SSCS) from cigarettes which heat but do not burn tobacco were compared to those of sidestream smoke from cigarettes which burn tobacco. SSCSs from 5 cigarettes were compared. Three of the cigarettes, the Kentucky reference research cigarette (1R4F), a commercially available ultra-low-tar brand (ULT) and a commercially available ultra-low-tar menthol brand (ULT-menthol) burn tobacco while two of the cigarettes, a regular (TEST) and a menthol (TEST-menthol) heat tobacco. SSCSs from all cigarettes were prepared by identical techniques, which involved collecting sidestream smoke particulate matter on Cambridge filter pads and combining the particulate matter with the vapor-phase materials collected by bubbling the smoke exiting the Cambridge pad through DMSO. The SSCSs obtained (equivalent to 0.4 cigarettes/ml DMSO) were evaluated at identical concentrations in an in vitro genetic toxicology test battery. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in Ames bacterial strains TA98, TA100, TA1537 and TA1538 in the presence of metabolic activation (S9 from Aroclor-induced rat liver) but negative results in strain TA1535. In the absence of metabolic activation, 1R4F, ULT and ULT-menthol SSCSs were not significantly mutagenic. TEST and TEST-menthol SSCSs produced negative results in all 5 bacterial strains, both with and without metabolic activation. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in the CHO chromosomal aberration assay and in the CHO sister-chromatid exchange assay both with and without metabolic activation while TEST and TEST-menthol SSCSs produced negative results in both assays, either with or without metabolic activation. The SSCSs from 1R4F, ULT and ULT-menthol cigarettes were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST and TEST-menthol SSCSs were negative in this assay. All 5 SSCSs were nonmutagenic in the CHO-HGPRT assay both with and without metabolic activation. SSCSs from the 1R4F, ULT and ULT-menthol cigarettes were cytotoxic in the CHO-HGPRT assay, both with and without metabolic activation, while TEST and TEST-menthol SSCSs were not cytotoxic under either condition. These results demonstrate that sidestream smoke from cigarettes which heat but do not burn tobacco (TEST and TEST-menthol) was neither genotoxic nor cytotoxic under conditions where sidestream smoke from cigarettes which burn tobacco (1R4F, ULT and ULT-menthol) was genotoxic and/or cytotoxic in a concentration-dependent manner.