Population-Genomic Analysis Identifies a Low Rate of Global Adaptive Fixation in the Proteins of the Cyclical Parthenogen Daphnia magna

Daphnia are well-established ecological and evolutionary models, and the interaction between D. magna and its microparasites is widely considered a paragon of the host-parasite coevolutionary process. Like other well-studied arthropods such as Drosophila melanogaster and Anopheles gambiae, D. magna is a small, widespread, and abundant species that is therefore expected to display a large long-term population size and high rates of adaptive protein evolution. However, unlike these other species, D. magna is cyclically asexual and lives in a highly structured environment (ponds and lakes) with moderate levels of dispersal, both of which are predicted to impact upon long-term effective population size and adaptive protein evolution. To investigate patterns of adaptive protein fixation, we produced the complete coding genomes of 36 D. magna clones sampled from across the European range (Western Palaearctic), along with draft sequences for the close relatives D. similis and D. lumholtzi, used as outgroups. We analyzed genome-wide patterns of adaptive fixation, with a particular focus on genes that have an a priori expectation of high rates, such as those likely to mediate immune responses, RNA interference against viruses and transposable elements, and those with a strongly male-biased expression pattern. We find that, as expected, D. magna displays high levels of diversity and that this is highly structured among populations. However, compared with Drosophila, we find that D. magna proteins appear to have a high proportion of weakly deleterious variants and do not show evidence of pervasive adaptive fixation across its entire range. This is true of the genome as a whole, and also of putative ‘arms race’ genes that often show elevated levels of adaptive substitution in other species. In addition to the likely impact of extensive, and previously documented, local adaptation, we speculate that these findings may reflect reduced efficacy of selection associated with cyclical asexual reproduction.     

Livestock trade history, geography, and parasite strains: the mitochondrial genetic structure of Echinococcus granulosus in Argentina

A sample of 114 isolates of Echinococcus granulosus (Cestoda: Taeniidae) collected from different host species and sites in Argentina has been sequenced for 391 bp from the mitochondrial cytochrome c oxidase subunit I gene to analyze genetic variability and population structure. Nine different haplotypes were identified, 5 of which correspond to already characterized strains. Analysis of molecular variance and nested clade analysis of the distribution of haplotypes among localities within 3 main geographic regions indicate that geographic differentiation accounts for the overall pattern of genetic variability in E. granulosus populations. Significant geographic differentiation is also present when the sheep strain alone is considered. Our results suggest that geographic patterns are not due to actual restricted gene flow between regions but are rather a consequence of past history, probably related to the time and origin of livestock introduction in Argentina.     

Functional characterisation of a purified homogeneous Photosystem II core complex with high oxygen evolution capacity from spinach

The functional properties of a purified homogeneous spinach PS II-core complex with high oxygen evolution capacity (Haag et al. 1990a) were investigated in detail by measuring thermoluminescence and oscillation patterns of flash induced oxygen evolution and fluorescence quantum yield changes. The following results were obtained:

1. Depending on the illumination conditions the PS II-core complexes exhibit several thermoluminescence bands corresponding to the A band, Q band and Z_v band in PS II membrane fragments. The lifetime of the Q band (T_max=10°C) was determined to be 8s at T=10°C. No B band corresponding to S₂Q_B ^? or S₃Q_B ^? recombination could be detected.
2. The flash induced transient fluorescence quantum yield changes exhibit a multiphasi relaxation kinetics shich reflect the reoxidation of Q _A ^? . In control samples without exogenous acceptors this process is markedly slower than in PS II membrane fragments. The reaction becomes significantly retarded by addition of 10 μM DCMU. After dark incubation in the presence of K₃[Fe(CN)₆
3. Excitation of dark-adapted samples with a train of short saturating flashes gives rise to a typical pattern dominated by a high O₂ yield due to the third flash and a highly damped period four oscillation. The decay of redox states S₂ and S₃ are dominated by short life times of 4.3 s and 1.5 s, respectively, at 20°C.

The results of the present study reveal that in purified homogeneous PS II-core complexes with high oxygen evolution isolated from higher plants by β-dodecylmaltoside solubilization the thermodynamic properties and the kinetic parameters of the redox groups leading to electron transfer from water to Q_A are well preserved. The most obvious phenomenon is a severe modification of the Q_B binding site. The implications of this finding are discussed.     

Lactobacillus kunkeei sp. nov. : a spoilage organism associated with grape juice fermentations

A Gram-positive rod was isolated from a commercial grape wine undergoing a sluggish/stuck alcoholic fermentation. The organism produced L- lactic acid from glucose, possessed weak catalase activity, and fermented relatively few carbohydrates, i.e. glucose, fructose, sucrose, raffinose (weakly) and mannitol. The 16S rRNA gene sequence analysis revealed that the isolate was phylogenetically a member of the genus Lactobacillus and formed a distinct subline within the Lact. casei cluster of species. Based on phenotypic and phylogenetic evidence, a new species is proposed, Lact. kunkeei. The type strain of Lact. kunkeei is ATCC 700308.     

Genetic identification of multiple loci that control breast cancer susceptibility in the rat.

We have used a rat model of induced mammary carcinomas in an effort to identify breast cancer susceptibility genes. Using genetic crosses between the carcinoma-resistant Copenhagen (COP) and carcinoma-sensitive Wistar-Furth rats, we have confirmed the identification of the Mcs1 locus that modulates tumor number. We have now also identified two additional loci, Mcs2 and Mcs3. These three loci map to chromosomes 2, 7, and 1, respectively, and interact additively to suppress mammary carcinoma development in the COP strain. They are responsible for a major portion of the tumor-resistant phenotype of the COP rat. No loss of heterozygosity was observed surrounding the three loci. A fourth COP locus, Mcs4, has also been identified on chromosome 8 and acts in contrast to increase the number of carcinomas. These results show that mammary carcinoma susceptibility in the COP rat is a polygenic trait. Interestingly, a polymorphism in the human genomic region homologous to the rat Mcs4 region is associated with an increased breast cancer risk in African-American women. The isolation of the Mcs genes may help elucidate novel mechanisms of carcinogenesis, provide information important for human breast cancer risk estimation, and also provide unique drug discovery targets for breast cancer prevention.     

Effects of photoinhibition on the PS II acceptor side including the endogenous high spin Fe2+ in thylakoids,PS II-membrane fragments and PS II core complexes

Effects of photoinhibition at 0 °C on the PS II acceptor side have been analyzed by comparative studies in isolated thylakoids, PS II membrane fragments and PS II core complexes from spinach under conditions where degradation of polypeptide(s) D1(D2) is highly retarded. The following results were obtained by measurements of the transient fluorescence quantum and oxygen yield, respectively, induced by a train of short flashes in dark-adapted samples: (a) in the control the decay of the fluorescence quantum yield is very rapid after the first flash, if the dark incubation was performed in the presence of 300 M K₃[Fe(CN)₆]; whereas, a characteristic binary oscillation was observed in the presence of 100 M phenyl-p-benzoquinone with a very fast relaxation after the even flashes (2nd, 4th. . . ) of the sequence; (b) illumination of the samples in the presence of K₃[Fe(CN)₆] for only 5 min with white light (180 W m^-2) largely eliminates the very fast fluorescence decay after the first flash due to Q_A ^- reoxidation by preoxidized endogenous non-heme Fe³⁺, while a smaller effect arises on the relaxation kinetics of the fluorescence transients induced by the subsequent flashes; (c) the extent of the normalized variable fluorescence due to the second (and subsequent) flash(es) declines in all sample types with a biphasic time dependence at longer illumination. The decay times of the fast (6–9 min) and the slow degradation component (60–75 min) are practically independent of the absence or presence of K₃[Fe(CN)₆] and of anaerobic and aerobic conditions during the photo-inhibitory treatment, while the relative extent of the fast decay component is higher under anaerobic conditions. (d) The relaxation kinetics of the variable fluorescence induced by the second (and subsequent) flash(es) become retarded due to photoinhibition, and (e) the oscillation pattern of the oxygen yield caused by a flash train is not drastically changed due to photoinhibition.Based on these findings, it is concluded that photoinhibition modifies the reaction pattern of the PS II acceptor side prior to protein degradation. The endogenous high spin Fe²⁺ located between Q_A and Q_B is shown to become highly susceptible to modification by photoinhibition in the presence of K₃[Fe(CN)₆] (and other exogenous acceptors), while the rate constant of Q_A ^- reoxidation by Q_B(Q_B ^-) and other acceptors (except the special reaction via Fe³⁺) is markedly less affected by a short photoinhibition. The equilibrium constant between Q_A ^- and Q_B(Q_B ^-) is not drastically changed as reflected by the damping parameters of the oscillation pattern of oxygen evolution.     

Photoinhibition affects the non-heme iron center in photosystem II.

Effects on the PS II acceptor side caused by exposure to strong white light (180 W/m2) of PS II membrane fragments (spinach) at pH 6.5 and 0 degrees C were analyzed by measuring low temperature EPR signals and flash-induced transient changes of the fluorescence quantum yield. The following results were obtained: (a) the extent of the light induced g = 1.9 EPR signal as a measure of photochemical Fe2+QA- formation declines with progressing photoinhibition. The half-life of this effect is independent of the absence or presence of an exogenous electron acceptor during the photoinhibitory treatment; (b) in samples photoinhibited in the absence of an electron acceptor and subsequently incubated with K3[Fe(CN)6] in the dark, the extent of the g = 8 EPR signal (reflecting the oxidized Fe3+ form of the endogenous non-heme iron center) and of the flash-induced change of the fluorescence yield (as a measure of fast electron transfer from QA- to Fe3+ after the first flash; [see (1992) Photosynth. Res. 31, 113-126] exhibits the same dependence on photoinhibition time as the g = 1.9 EPR signal; (c) in samples photoinhibited in the presence of an exogenous electron acceptor, the signals reflecting Fe(3+)-formation and fast electron transfer from QA- to Fe3+ decline faster than the g = 1.9 EPR signal. These results provide for the first time direct evidence that the endogenous non-heme iron center located between QA and QB is susceptible to modifications by light stress. The implications of this finding will be discussed.     

Influence of systematic errors on the evaluation of the S phase portions from DNA distributions of solid tumors as shown for 328 breast carcinomas

The portion of cells in S phase has proved to be a valuable prognostic indicator of early relapse and life expectancy, particularly in breast carcinoma. Comparisons of published data on samples of primary breast carcinoma biopsies showed that the values obtained by analyses of flow cytometric DNA distributions were generally higher than those of determinations based on the tritiated thymidine (3H-ThdR) labeling index (LI). Flow cytometric DNA analyses of 328 biopsy samples of primary breast carcinomas revealed that these differences could be explained by varying contributions of debris background. Since this influence is inversely proportional to the cell counts in each channel, it may cause considerable errors, particularly in the S phase channels, which normally contain the lowest counts of the DNA distributions. Two different mathematical rationales were tested in order to separate DNA distributions from the debris superimposition. No appreciable differences were found with respect to the essential results. After appropriate subtraction of the background levels, the previously reported discrepancies between cytometrically determined S phase portions and 3H-ThdR LI values disappeared, and good agreement was achieved for the comparable tumor samples of the present study. In conclusion, debris background subtractions should generally precede the DNA histogram analyses, particularly of solid tumors, in order to obtain reliable S phase values.     

Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation

ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2-which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation-is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by monocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively.