The effects of genetic drift in experimental evolution

Experimental evolution is characterized by exponential or logistic growth and periodic population bottlenecks. The fate of a rare beneficial mutation in these systems is influenced both by the bottleneck effect and by genetic drift. This paper explores the effects of incorporating genetic drift into models of fixation probability in populations with periodic bottlenecks. To model the inherent stochasticity during the growth phase in these populations, we assume a Poisson distribution of offspring. An analytical solution is developed to calculate the fixation probability and a computer simulation is used to verify the results. We find that the fixation rate of a favourable mutant is significantly lower when genetic drift is considered; fixation probability is reduced by over 25% for realistic experimental protocols. Our method is valid for both weak and strong selection; since very large selection coefficients have been reported in the experimental literature, this is an important improvement over previous results.     

Elongation correlates with nutrient deprivation in Pseudomonas aeruginosa-unsaturates biofilms

Bacteria in nature frequently grow as biofilms, yet little is known regarding how biofilm bacteria morphologically adapt to low nutrient availability, which is common in unsaturated environments such as the terrestrial subsurface or on plant leaves. For unsaturated biofilms, in which the substratum may provide all nutrients, what are the relationships between nutrition and cell size and shape-the simplest metrics of cellular morphology? To address this question, we cultured Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium that is environmentally and medically important, on membranes overlaying solid media, and then measured cellular dimensions using atomic force microscopy (AFM). Nutrition was controlled chemically by media composition and physically by stacking membranes to increase the path length for nutrient diffusion. Under conditions of carbon-nitrogen imbalance, low carbon bioavailability, or increased nutrient diffusional path length, cells elongated while maintaining constant width. A mathematical relationship suggests that, by elongating, biofilm bacteria strategically enlarge their nutrient collection surface without substantially changing the ratio of surface area to volume (SA/V). We conclude that P. aeruginosa growing as unsaturated biofilm with a planar nutrient source morphologically adapt to starvation by elongating. This adaptation, if generalizable, differs from a better-understood starvation response (i.e., cell size decreases; thus SA/V in-creases) for planktonic bacteria in well-mixed environments.     

The apomyoglobin folding pathway revisited: structural heterogeneity in the kinetic burst phase intermediate

Extensive analysis of accurate quench-flow hydrogen exchange results indicates that the burst phase kinetic intermediate in the folding of apomyoglobin (apoMb) from urea is structurally heterogeneous. The structural variability is associated with the partial folding of the E helix during the burst phase (<6.4ms) of the folding process. Analysis of the effects of exchange-out of amide proton labels during the labeling pulse ( approximately pH 10) of the quench-flow process indicates that three of the amide protons in the E helix are in fact largely protected in the burst phase of folding, while the remainder of the E helix has a substantial complement of amide protons that show biphasic kinetics, i.e. are protected partly during the burst phase and partly during the slow phase of folding. The locations of these amide protons can be used to map the sites of structural heterogeneity in the kinetic molten globule. These sites include, besides the E helix, the ends of the A and B helices and part of the C helix. Our results give significant support to the hypothesis that the kinetic molten globule intermediate of apoMb is native-like.     

The role of the alpha2 chain in the stabilization of the collagen type I heterotrimer: a study of the type I homotrimer in oim mouse tissues

We have previously reported that the fragility of skin, tendon and bone from the oim mouse is related to a significant reduction in the intermolecular cross-linking. The oim mutation is unlikely to affect the efficacy of the lysyl oxidase, suggesting that the defect is in the molecule and fibre. We have therefore investigated the integrity of both the oim collagen molecules and the fibre by differential scanning calorimetry.The denaturation temperature of the oim molecule in solution and the fibre from tail tendon were found to be higher than the wild-type by 2.6deg.C and 1.9deg.C, respectively. With the loss of the alpha2 chain, the hydroxyproline content of the homotrimer is higher than the heterotrimer, which may account for the increase.There is a small decrease in the enthalpy of the oim fibres but it is not significant, suggesting that the amount of disorder of the triple-helical molecules and of the fibres is small and involves only a small part of the total bond energy holding the helical structure together. The difference in denaturation temperature of the skin collagen molecules (t(m)) and fibres (t(d)) is significantly lower for the oim tissues, 19.9deg.C against 23.1deg.C, indicating reduced molecular interactions and hence packing of the molecules in the fibre. Computation of the volume fraction of the water revealed that the interaxial separation of the oim fibres was indeed greater, increasing from 19.6A to 21.0A. This difference of 1.4A, equivalent to a C-C bond, would certainly decrease the ability of the telopeptide aldehyde to interact with the epsilon -amino group from an adjacent molecule and form a cross-link. We suggest, therefore, that the reduction of the cross-linking is due to increased water content of the fibre rather than a distortion of the molecular structure.The higher hydrophobicity of the alpha2 chain appears to play a role in the stabilisation of heterotrimeric type I collagen, possibly by increasing the hydrophobic interactions between the heterotrimeric molecules, thereby reducing the water content and increasing the binding of the molecules in the fibre.     

Adaptive response induction and variation in human lymphoblastoid cell lines

Adaptive response is a term used to describe the ability of a low, priming dose of ionizing radiation to modify the effects of a subsequent higher, challenge dose, but it has been observed to be highly variable in both presence and magnitude. To examine this variability, 10 human lymphoblastoid cell lines were screened for adaptability to 137Cs radiation by determining the frequency of micronuclei in binucleated cells. Of these, six adapted, three did not adapt and one was synergistic. The assay was then repeated on each of the cell lines to test for reproducibility. Five cell lines showed the same result both times; four of these adapted and one did not.To determine whether fluctuations in the cell cycle distribution in the irradiated population of cells could alter the adaptive response, and therefore explain some of the observed variability, two of the cell lines were tested for adaptation after enriching the population, by synchronization, for a given cell cycle stage. In both cell lines, the direction of the response was altered when the distribution of cells within the cell cycle was changed, suggesting that the adaptive response can be affected by cell cycle stage at the time of irradiation.     

Adenine phosphoribosyltransferase isoforms of Arabidopsis and their potential contributions to adenine and cytokinin metabolism

Adenine phosphoribosyltransferase (APT; EC 2.4.2.7) is a constitutively expressed enzyme involved in the one-step salvage of adenine to AMP. The Arabidopsis thaliana genome contains five sequences annotated as encoding APT or APT-like enzymes. Three of these have now been cloned, over-expressed and compared using kinetic analyses. At a cytosolic pH, all bind adenine efficiently based on their K_m values (0.8–2.6 µ M ), although APT1 metabolizes adenine at a rate 31–53 times faster than APT2 and APT3, respectively. Since APT also has a possible role in the interconversion of cytokinin bases to nucleotides, we characterized the activity of each isoform on zeatin, isopentenyladenine and benzyladenine. Based on their K_m values, APT2 and APT3 had much higher affinities than APT1 for all three cytokinins (15–440 µ M for APT2 and 3 vs. 1.8–2.5 m M for APT1); conversely the V_max values for APT2 and APT3 on these CK substrates showed the opposite trend, being 4- to 19-fold lower than those of APT1. Anti-peptide antibodies for APT1, APT2, and APT3 were prepared and used to examine the subcellular localization of each isoform. Based on these results, APT1 and APT3 appear to be cytosolic, while the localization of APT2 was inconclusive although sequence analysis implies that APT2 is also cytosolic. Each isoform was modelled against the crystal structure of APT from Leishmania donovani , and structural differences in substrate specificity-determining domains have been found. The estimated kinetic activities of these APTs suggest that they contribute primarily to adenine recycling, although an involvement in cytokinin interconversion cannot be discounted.     

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

Functional characterization of a novel mammalian zinc transporter, ZnT6

We describe ZnT6, a new member of the CDF (cation diffusion facilitator) family of heavy metal transporters. The human ZNT6 gene was mapped at 2p21-22, while the mouse Znt6 was localized to chromosome 17. Overexpression of ZnT6 in both wild-type yeast and mutants that are deficient in cytoplasmic zinc causes growth inhibition, but this inhibition is abolished in mutant cells with high cytoplasmic zinc. ZnT6 may function in transporting the cytoplasmic zinc into the Golgi apparatus as well as the vesicular compartment, as evidenced by its overlapping intracellular localization with TGN38 and transferrin receptor in the normal rat kidney cells. We also demonstrate that the intracellular distributions of ZnT6 as well as ZnT4 are regulated by zinc in the normal rat kidney cells. The results from this report, combined with those from other studies, suggest that the intracellular zinc homeostasis is mediated by many ZnT proteins, which act in tissue-, cell-, and organelle-specific manners.