Synthesis, stability and reactivity of palladium(0) olefin complexes bearing labile or hemi-labile ancillary ligands and electron-poor olefins

An overview of the general features of electron-poor olefin stabilized palladium(0) complexes bearing labile and hemi-labile ancillary ligands is presented. In particular, we have summarized the synthetic methodologies, the ligands commonly used, and the characterization of such complexes. The behavior of these species in solution is also described with particular attention to their fluxional rearrangements and reactivity. Thus, olefin exchange reactions are described and a comprehensive order of coordinative capability of the most widely used electron-poor alkenes is presented. The reactions of the title complexes dealing with olefin isomerization, oxidative addition, and formation of palladacyclopentadiene derivatives are eventually reported together with their main structural characteristics.     

Gene Genealogies Strongly Distorted by Weakly Interfering Mutations in Constant Environments

Neutral nucleotide diversity does not scale with population size as expected, and this “paradox of variation” is especially severe for animal mitochondria. Adaptive selective sweeps are often proposed as a major cause, but a plausible alternative is selection against large numbers of weakly deleterious mutations subject to Hill–Robertson interference. The mitochondrial genealogies of several species of whale lice (Amphipoda: Cyamus) are consistently too short relative to neutral-theory expectations, and they are also distorted in shape (branch-length proportions) and topology (relative sister-clade sizes). This pattern is not easily explained by adaptive sweeps or demographic history, but it can be reproduced in models of interference among forward and back mutations at large numbers of sites on a nonrecombining chromosome. A coalescent simulation algorithm was used to study this model over a wide range of parameter values. The genealogical distortions are all maximized when the selection coefficients are of critical intermediate sizes, such that Muller''s ratchet begins to turn. In this regime, linked neutral nucleotide diversity becomes nearly insensitive to N. Mutations of this size dominate the dynamics even if there are also large numbers of more strongly and more weakly selected sites in the genome. A genealogical perspective on Hill–Robertson interference leads directly to a generalized background-selection model in which the effective population size is progressively reduced going back in time from the present.OBSERVED levels of apparently neutral nucleotide diversity (π_n) are typically lower than expected under the assumptions of standard equilibrium theories, and they vary much less among species than do estimates of long-term effective population sizes (Nei and Grauer 1984; Bazin et al. 2006; Nabholz et al. 2008). Many explanations have been proposed for the apparent shortfalls and the lack of proportionality with population size, including (1) complex demographic histories (e.g., recurring population bottlenecks), (2) adaptive selective sweeps (Maynard Smith and Haigh 1974; Gillespie 1999), and (3) selection against deleterious mutations (Charlesworth et al. 1993, 1995; McVean and Charlesworth 2000; Comeron et al. 2008). Of these three possibilities, bottlenecks and sweeps are by far the most frequently mentioned, even though deleterious mutations occur at high rates in all species, regardless of ecological circumstances (Eyre-Walker and Keightley 2007). Here we show that weakly deleterious mutations can distort genealogies in three different ways and dramatically reduce nucleotide diversities in large populations of nonrecombining chromosomes. The mitochondrial genealogies of several species of whale lice (Kaliszewska et al. 2005) are distorted in exactly these ways, and several lines of evidence suggest that bottlenecks and adaptive sweeps are not likely to be the primary causes.Mitochondria have been proposed to be especially sensitive to selective sweeps. Animal mitochondrial genomes contain more than three dozen essential protein and structural RNA genes, so they are large targets for both mutation and selection (Ballard and Whitlock 2004). They do not undergo sexual recombination, so every advantageous mutation that fixes will reduce variation throughout the genome. Mitochondrial nucleotide diversity therefore could depend strongly on rates of environmental change, which could be similar for species with very different population sizes. Indeed, if rates of mitochondrial adaptation were mutation limited, then larger populations might actually experience higher rates of adaptive substitution and as a result show lower average levels of neutral diversity than smaller populations (Gillespie 2000, 2001). This idea was recently invoked to explain the remarkable similarity of average levels of mitochondrial nucleotide diversity among the major animal classes which appear to have very different average population sizes and substantially different average levels of nuclear nucleotide and amino acid diversity (Bazin et al. 2006).Unconditionally deleterious mutations can also depress linked neutral diversity by reducing the effective population size either through (1) background selection against relatively strongly selected mutations (Charlesworth et al. 1993, 1995) or (2) Hill–Robertson interference (Hill and Robertson 1966) among large numbers of relatively weakly selected mutations (reviewed by Comeron et al. 2008). The second of these processes, called “weak-selection Hill–Robertson interference” (wsHRi) by McVean and Charlesworth (2000) and “interference selection” (IS) by Comeron and Kreitman (2002), can shorten genealogies, give them strongly nonneutral branch-length proportions, and skew their topologies (Higgs and Woodcock 1995; Maia et al. 2004).To date, weak interference has mainly been studied by forward simulation, with the aim of assessing its possible effects on patterns of optimal synonymous codon use within eukaryotic nuclear genes and genomes, in the presence of recombination (Comeron and Guthrie 2005; Loewe and Charlesworth 2007; Comeron et al. 2008). In an attempt to understand the striking genealogical distortions seen in whale-louse mitochondria (Kaliszewska et al. 2005), we have developed a structured-coalescent algorithm that accurately models selection of arbitrary strength on a nonrecombining chromosome of finite length. All of the distortions seen in the whale-louse mitochondria are replicated under parameters that might plausibly apply to whale lice and many other animal species, and these distortions scale only weakly with population size.Whale lice are permanent, obligate ectoparasites of cetaceans. They feed on the dead outer surface of their host''s skin, and they appear to be harmless. They are amphipod Crustacea comprising a monophyletic family, Cyamidae, with ∼50 described species in several genera. Three of these species (Cyamus ovalis, C. gracilis, and C. erraticus) occur on right whales (Eubalaena spp.) but not regularly on any other hosts. Most adult right whales carry large populations of all three species.Right whales in the North Pacific, the North Atlantic, and the southern hemisphere have been separated for ∼5 million years, and so have their cyamids (Rosenbaum et al. 2000; Gaines et al. 2005; Kaliszewska et al. 2005). For this reason the right whales in different ocean systems are now considered distinct species (Eubalaena japonica, E. glacialis, and E. australis), and we refer to their cyamids as North Pacific C. ovalis, North Atlantic C. ovalis, southern C. ovalis, and so on, in anticipation that a future revision of the genus Cyamus will recognize them as “triplet” sibling species (3 × 3 = 9 species in all). We studied their mitochondrial population genetics with the initial aim of quantifying patterns of genetic differentiation among the cyamid populations on individual whales within local populations (Kaliszewska et al. 2005).We had reasoned (incorrectly) that the pattern of differentiation among whales might say something about their social interactions, since cyamids can transfer only between whales that are in direct physical contact with each other. We found very low levels of differentiation among whales and to our surprise literally no differentiation among the major southern hemisphere breeding aggregations that calve off the coasts of South America, South Africa, Australia, and New Zealand (Kaliszewska et al. 2005). This absence of population structure seems remarkable by terrestrial standards but is easily explained by modest rates of cyamid exchange among whales within local populations and between the major breeding aggregations, given the enormous sizes of cyamid populations. Right whales are highly gregarious (spending hours per day in social interactions), mobile (traveling thousands of kilometers per year on annual foraging migrations), and mortal (carrying their cyamid populations to the sea floor when they die). Thus cyamids have many opportunities to transfer between whales, and they might be expected to have evolved an inclination to do so when the opportunity presents itself (Hamilton and May 1977).The well-defined ecology of right-whale cyamids allows their population sizes to be estimated directly. The number of adult cyamids per whale (∼500–10,000, varying by species) times the number of whales per ocean (∼50,000–200,000, prior to human exploitation) equals the number of cyamids per species (Kaliszewska et al. 2005). Thus for all three nominal species of right-whale cyamids, long-term census population sizes are expected to have been in the range 2.5 × 10⁷–2 × 10⁹. Given conservative estimates of the per-generation mitochondrial mutation rate, even the lower end of this range predicts levels of synonymous nucleotide diversity at least an order of magnitude larger than those actually seen in the cyamids, which are consistently modest and similar to those seen in typical terrestrial arthropods (Kaliszewska et al. 2005). The three North Atlantic and southern hemisphere sibling-species pairs are strongly reciprocally monophyletic, as illustrated for C. ovalis in Figure 1. This is not expected at mutation–drift equilibrium, given their very large population sizes.Open in a separate windowFigure 1.—Mitochondrial gene genealogies for North Atlantic and southern hemisphere Cyamus ovalis, estimated by UPGMA from partial COI sequences. Left: The intraspecific genealogies coalesce globally at ∼0.5 and 1 MY and share an ancestor at ∼5 MY (Kaliszewska et al. 2005). Center and right: Each intraspecific genealogy is aligned with a generalized skyline plot (Strimmer and Pybus 2001) showing estimates of θ at different times in the past under a piecewise constant model of population size change fit by GENIE 3.0 (Pybus and Rambaut 2002). Three different point estimates of present-day θ are also indicated on the plots (synonymous-site nucleotide diversity π, Watterson''s θ estimated from synonymous sites, and θ estimated jointly with the apparent exponential growth rate by the MCMC coalescent algorithm in LAMARC). Values of Tajima''s D are lower (−1.5 to −1.6) when estimated from the sequences than when estimated from the branch lengths of the trees (−2.1 to −2.4), as expected because multiple substitutions occur at some sites. Similar values of D_T (−2.3 for both species) were obtained from the highest-likelihood trees found by BEAST with a fully parameterized GTR substitution model and a coalescent prior. Those trees have standardized imbalance statistics (−I_S) of −3.7 (southern hemisphere C. ovalis) and −2.2 (North Atlantic C. ovalis). The trees shown here have more extreme values of −I_S (−5.5 and −4.0, respectively), probably as a consequence of artificially pectinate branching orders induced by UPGMA among sets of identical sequences. Slightly different sets of sequences were used to make the two-species genealogy on the left and the single-species genealogies in the center. The long interspecific branches in the two-species tree are based on a multispecies maximum-likelihood analysis involving smaller numbers of much longer (4.1 kb) sequences (Kaliszewskaet al. 2005, Figure 3).These dramatic deficits of variation are not easily explained by population bottlenecks or adaptive selective sweeps. The bottleneck hypothesis is especially problematic because it requires the long-term near extinction of right whales in all three ocean systems. (Short bottlenecks such as those caused by human exploitation of right whales are not expected to have a noticeable effect on cyamid genetic diversity because cyamid populations remain large, and rates of genetic drift low, even when there are few whales.) That all three right whales have survived for millions of years suggests that they have maintained reasonably large population sizes, and the mitochondrial nucleotide diversity of southern right whales is consistent with this assumption (Kaliszewska et al. 2005), as is the nucleotide diversity of a cyamid nuclear gene (described below). Right whales eat copepods and krill, which are relatively close to the base of marine food webs, and right-whale populations are thought to be food limited. Thus a long-term, severe depression of their numbers would also seem to imply a collapse of marine ecosystems worldwide, for which there is no evidence.Several features of cyamid mitochondrial nucleotide diversity are also inconsistent with the bottleneck model and with adaptive sweeps as well. The most obvious of these features is the uniformity of cyamid mitochondrial diversity among species (π = 0.007–0.015 for COI sequences in the seven species surveyed by Kaliszewska et al. 2005). Gene genealogies estimated from these sequences also seem remarkably uniform in total depth, with last common ancestors differing in age by only a factor of 3 and in six of the seven species by less than a factor of 2 (see Kaliszewska et al. 2005, Figure 4). Adaptive sweeps might be expected to occur at roughly random intervals and not to be well coordinated in time among seven species in three different ocean systems. Interspecific coordination of such sweeps (over the whole globe) would seem to be required if they were to be a plausible primary cause of the genealogical shortening.Open in a separate windowFigure 4.—Apparent average effective sizes of ancestral populations, for models with different values of s. Values of N_e are given on the vertical axis (logarithmically scaled). They are estimated from the variance of expected contributions to the present, for the adults of any given generation. The parameters are those of Figure 2 (N = 65,536 = “64k,” μ = 1.5 × 10⁻⁶, L_s = 2048, U = 0.0031). In theory the curve for s = 0 should be perfectly horizontal. The discrepancy appears to be caused by subtle flaws in the shape of the very broad “idealized” simulated distribution that was used in this calculation. The curves for strong-selection cases are perfectly horizontal at times beyond a few thousand generations because the mutation-number distributions are compact, with little stochastic variation. The two lowest curves are those for the selection coefficients (s = 2⁻¹⁰, U/s = 3.2, and s = 2⁻¹¹, U/s = 6.3) that produce the most extreme values of the polymorphism and tree-shape statistics, given the other parameters (Figure 2).In addition to showing too little nucleotide variation, the cyamid mitochondrial genomes show strong and consistent excesses of rare nucleotide states, reflecting the “comb-like” or “star-like” shapes of the genealogies, in which deeper branches tend to be much too short relative to terminal branches (as if the trees had been “squished” from behind). This kind of distortion causes negative values of Tajima''s (1989) D and related statistics. It can be caused by population expansion from a bottleneck or by lineage expansion under positive selection (Kaplan et al. 1989; Slatkin and Hudson 1991; Rogers and Harpending 1992; Bamshad and Wooding 2003). However, the form of branch-length distortion seen in the cyamid genealogies suggests a slow, steady, roughly exponential form of population or lineage growth, not the relatively sudden increases suggested by the bottleneck and selective-sweep hypotheses. Generalized skyline plots (Strimmer and Pybus 2001) describing the histories of population size implied by the shapes of the northern and southern C. ovalis gene genealogies are shown in Figure 1. They are remarkably similar, as are the growth rates and estimates of present-day θ (= 2N_fμ_n) obtained by fitting exponential growth models using the coalescent algorithms in LAMARC (Kuhner et al. 1998, 2004) or BEAST (Drummond and Rambaut 2007).Cyamid populations cannot have grown in numbers as seemingly implied by these analyses. The number of cyamids on each whale appears to be set mainly by microhabitat limitations (e.g., by the area of rough callosity tissue on the head, where C. ovalis and C. gracilis live), and these features of their environment have hardly changed for millions of years, as demonstrated by the strong similarities of northern and southern right whales and their cyamids. Likewise, the numbers of right whales cannot have increased gradually from vanishingly small numbers over several hundred thousand years, for the reasons discussed above.The genealogical signals of “growth” therefore seem likely to be caused by selection. Environmental change is the most obvious potential cause of selection, but the apparent rate of growth seen here is strangely slow—in fact, slower than glacial. The orbitally forced Plio-Pleistocene glacial climate cycles have a major period of ∼100,000 years (Lambert et al. 2008 and references therein), but all seven of the right-whale cyamids for which we have mitochondrial population samples appear to have been “expanding,” more or less continuously, through at least several such cycles. The seemingly fairly consistent rate of branch-length foreshortening seen in the genealogies therefore suggests the action of a process that is relatively homogeneous in time, in addition to being very slow overall.The cyamid mitochondrial genealogies also appear to be topologically skewed, with sister clades too unequal in size, on average. In random bifurcating trees, the distribution of sister-clade sizes is uniform (Yule 1924; Heard 1992; Rogers 1994). Deviations from this null expectation can be quantified by statistics such as Colless''s (1982) index of tree imbalance (Shao and Sokal 1990; Rogers 1996). Our estimates of the cyamid genealogies tend to be excessively imbalanced (Figure 1). Strong topological imbalance is not caused by classic adaptive sweeps or by population growth following a bottleneck, but previous theoretical work has indicated that it can be caused by selection (Higgs and Woodcock 1995; Maia et al. 2004).To summarize, the cyamid mitochondrial genealogies are consistently much too short, too squished, and too skewed, relative to neutral-theory expectations. Owing to several special features of cyamid and right-whale biology, selection seems to be the only plausible explanation for this set of distortions, but conventional adaptive sweeps do not seem likely to be the primary cause. We therefore asked whether weakly deleterious mutations might be sufficient to generate the observed combination of patterns, in the absence of environmental change. Previous work (mentioned above) showed that interference among weak mutations at many sites can strongly affect linked neutral variation, but this work did not fully explore the parameter space relevant to our system or connect all the patterns in a genealogical setting.To address this question we first carried out forward simulations of populations of nonrecombining chromosomes with large numbers of nucleotide positions subject to forward and back mutations with unconditional fitness effects of size s. Large numbers of linked neutral sites were used to estimate genealogies and to calculate population statistics of interest. We found that for a range of intermediate values of s, considerable fitness variation was maintained and all three of the genealogical distortions (and the signal of apparent exponential growth) seen in the cyamid genealogies reached impressively large maxima. However, the computational burden of full forward simulation prevented us from considering realistic parameter values (i.e., large N and small μ), and it was not obvious that extrapolations based on compound parameters (e.g., Nμ and Ns) would work as hoped in all respects (see Comeron et al. 2008). We developed an equivalent coalescent algorithm that accurately reproduces all results of the forward simulations and allows for realistic parameter values. Under parameters relevant to cyamid mitochondria, the distortions of genealogical depth, proportions, and topology can be even more extreme than those seen in the cyamids, and the mean pairwise coalescence times (and resulting neutral nucleotide diversities) associated with maximally distorting intermediate values of s (U_s/s ∼ 5, where U_s is the total genomic mutation rate at sites with selection coefficents of size s) depend only weakly on N.All parameters of this model (including those of the environment) remain constant over time, yet in some respects it displays apparently nonequilibirum behavior. Under weak to intermediate selection (U_s/s > 10), the effective population size appears to become progressively smaller as time recedes into the past, giving rise to the illusion of growth. And in the maximally distorting range of intermediate selection coefficients, the distributions of deleterious mutation numbers and the shapes of genealogies show conspicuous dynamical instability of a form that could be taken to suggest “adaptive evolution” in response to episodes of environmental change. Adaptive mutations contribute importantly to this process, but they are reversions at some of the many sites previously mutated to mildly deleterious states. Subtle patterns of environmental change that converted previously optimal nucleotide states to slightly suboptimal states could give rise to a category of “virtual reversions” that would augment (or even outnumber) simple reversions, and the effects of such a process might well be consistent with the distortions seen in the cyamid mitochondrial genealogies. However, models with no environmental change of any kind appear to explain the observations surprisingly well.     

Long Lasting Effects of Electroconvulsive Seizures on Brain Oxidative Parameters

This work was performed in order to determine the level of oxidative damage and antioxidant enzymes activities late after acute and chronic electroconvulsive shock (ECS) in rats. We measured oxidative parameters in hippocampus, cortex, and striatum, at 45, 60, 90 and 120 days after a single or multiple ECS. We demonstrated an increase in lipid peroxidation after multiple ECS in the hippocampus and striatum. This was also the case for protein carbonyls in the single or multiple protocols. In this way, we demonstrated an increase in catalase in cortex in contrast to striatum and hippocampus, were there were decreases sometimes in chronic ECS. The superoxide dismutase activities decrease in different times after single and multiple ECS in the hippocampus. Our findings demonstrated that there is a delayed increase after ECS in oxidative damage and decrease in antioxidant enzymes activities in hippocampus and striatum.     

Systems biology of circadian rhythms: an outlook

The circadian system in higher organisms temporally orchestrates rhythmic changes in a vast number of genes and gene products in different organs. Complex interactions between these components, both within and among cells, ultimately lead to rhythmic behavior and physiology. Identifying the plethora of circadian targets and mapping their interactions with one another is therefore essential to comprehend the molecular mechanisms of circadian regulation. The emergence of new technology for unbiased identification of biomolecules and for mapping interactions at the genome-wide scale is offering powerful tools to decipher the regulatory networks underpinning circadian rhythms. In this review, the authors discuss the potential application of these genome-wide approaches in the study of circadian rhythms.     

Inhibition of Candida rugosa lipase by saponins, flavonoids and alkaloids

Lipase inhibitors have generated a great interest because they could help in the prevention or the therapy of lipase-related diseases. Therefore, the aim of the work was to evaluate by HPLC, and using Candida rugosa lipase as model, the inhibitory effect of several saponins: β-aescin, digitonin, glycyrrhizic acid (GA) and Quillaja saponin (QS); flavonoids: 3-hydroxyflavone, 5-hydroxyflavone, (±)-catechin and kaempferol; and alkaloids: aspidospermine, papaverine, physostigmine, pilocarpine, raubasine, rescinnamine, reserpine and trigonelline.

The inhibition produced by most of these compounds is described here for the first time. Saponins appeared very active, being β-aescin and digitonin the most active compounds (IC₅₀ = 0.8–2.4 × 10⁻⁵ M). The inhibitory activity of flavonoids was lower than that of saponins (except GA), and (±)-catechin and kaempferol were the most active. Alkaloids was the most heterogeneous group assayed, varying from rescinnamine, with an IC₁₆ similar to that of digitonin, to papaverine and others which showed almost no inhibition.

In conclusion, β-aescin, digitonin, kaempferol or (±)-catechin, strong lipase inhibitors with a low toxicity and present herbal drugs used for lipase-related diseases such as acne or ulcer, are promising candidates for the prevention or the treatment of these diseases.     

Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.     

Key aspects of melatonin physiology: thirty years of research

Numerous studies of melatonin, by now widely acknowledged as a circadian rhythm-affecting neurohormone, also describe its anti-oxidant, anti-cytotoxic or immune-modulating activity. While emphasizing the multifunctional aspect of melatonin action, this review presents the results of our thirty years of research, which point to the following conclusions: melatonin is capable of promoting platelet production by megakaryocytes, of acting on the latter's ion channels by way of the outward currents, and of performing a physiological anti-aggregation function thus lengthening platelet life span. Melatonin can be transported everywhere by platelets and, thanks to its lipophilicity, can cross cellular membranes easily, thus regulating blood-tissue exchanges and ensuring an improved haematic crisis. It interacts with endothelial cells by regulating their release of both relaxing-factor and contracting-factor, and with platelets by affecting their discharge of dense-body components. Finally, platelets could behave as mobile and itinerant serotonergic and/or melatonergic elements, a function comparable to the release of neurotransmitters by neurons of the central nervous system. This dynamism in melatonin physiology could prove to be a key in approaching tumour aetiopathogenesis.     

Tachykinins and the control of prolactin secretion

Tachykinins are present in the pituitary gland and in brain areas involved in the control of the secretion of pituitary hormones. Tachykinins have been demonstrated to stimulate prolactin release acting directly on the anterior pituitary gland. These peptides have also been revealed to be able to act at the hypothalamic level, interacting with neurotransmitters and neuropeptides that have the potential to affect prolactin secretion. Tachykinins seem to act by stimulating or inhibiting the release of the factors that affect prolactin secretion. Among them, tachykinins have been demonstrated to stimulate oxytocin and vasopressin release, which in turn results in prolactin release. Tachykinins also potentiated the response to vasoactive intestinal peptide (VIP) and reinforced the action of glutamate, which in turn result in prolactin release. They have also been shown to interact with serotonin, a neurotransmitter involved in the control of prolactin secretion. In addition, tachykinins have been shown to inhibit GABA release, a neurotransmitter with prolactin-release inhibiting effect. This inhibition may result in an increased prolactin secretion by removal of the GABA inhibition. On the other hand, tachykinins have also been shown to stimulate dopamine release by the hypothalamus, an action that results in an inhibition of prolactin release. Dopamine is a well known inhibitor of prolactin secretion. In conclusion, although tachykinins have been shown to have a predominantly stimulatory effect on prolactin secretion, especially at the pituitary level, under some circumstances they may also exert an inhibitory influence on prolactin release, by stimulating dopamine release at the hypothalamic level.     

Subtelomeric proteins negatively regulate telomere elongation in budding yeast

The Tbf1 and Reb1 proteins are present in yeast subtelomeric regions. We establish in this work that they inhibit telomerase-dependent lengthening of telomere. For example, tethering the N-terminal domain of Tbf1 and Reb1 in a subtelomeric region shortens that telomere proportionally to the number of domains bound. We further identified a 90 amino-acid long sequence within the N-terminal domain of Tbf1 that is necessary but not sufficient for its length regulation properties. The role of the subtelomeric factors in telomere length regulation is antagonized by TEL1 and does not correlate with a global telomere derepression. We show that the absence of TEL1 induces an alteration in the structure of telomeric chromatin, as defined biochemically by an increased susceptibility to nucleases and a greater heterogeneity of products. We propose that the absence of TEL1 modifies the organization of the telomeres, which allows Tbf1 and Reb1 to cis-inhibit telomerase. The involvement of subtelomeric factors in telomere length regulation provides a possible mechanism for the chromosome-specific length setting observed at yeast and human telomeres.     

Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidized crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridization efficiency; parallel imaging of the process by Atomic Force Microscopy

Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).