Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases.

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.

Simple mechanochemistry describes the dynamics of kinesin molecules

Recently, Block and coworkers [Visscher, K., Schnitzer, M. J., & Block, S. M. (1999) Nature (London) 400, 184–189 and Schnitzer, M. J., Visscher, K. & Block, S. M. (2000) Nat. Cell Biol. 2, 718–723] have reported extensive observations of individual kinesin molecules moving along microtubules in vitro under controlled loads, F = 1 to 8 pN, with [ATP] = 1 μM to 2 mM. Their measurements of velocity, V, randomness, r, stalling force, and mean run length, L, reveal a need for improved theoretical understanding. We show, presenting explicit formulae that provide a quantitative basis for comparing distinct molecular motors, that their data are satisfactorily described by simple, discrete-state, sequential stochastic models. The simplest (N = 2)-state model with fixed load-distribution factors and kinetic rate constants concordant with stopped-flow experiments, accounts for the global (V, F, L, [ATP]) interdependence and, further, matches relative acceleration observed under assisting loads. The randomness, r(F,[ATP]), is accounted for by a waiting-time distribution, ψ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{1}^{+}}}\end{equation*}\end{document}(t), [for the transition(s) following ATP binding] with a width parameter ν ≡ 〈t〉2/〈(Δt)2〉≃2.5, indicative of a dispersive stroke of mechanicity ≃0.6 or of a few (≳ν − 1) further, kinetically coupled states: indeed, N = 4 (but not N = 3) models do well. The analysis reveals: (i) a substep of d0 = 1.8–2.1 nm on ATP binding (consistent with structurally based suggestions); (ii) comparable load dependence for ATP binding and unbinding; (iii) a strong load dependence for reverse hydrolysis and subsequent reverse rates; and (iv) a large (≳50-fold) increase in detachment rate, with a marked load dependence, following ATP binding.

Integrated fossil and molecular data reconstruct bat echolocation

Molecular and morphological data have important roles in illuminating evolutionary history. DNA data often yield well resolved phylogenies for living taxa, but are generally unattainable for fossils. A distinct advantage of morphology is that some types of morphological data may be collected for extinct and extant taxa. Fossils provide a unique window on evolutionary history and may preserve combinations of primitive and derived characters that are not found in extant taxa. Given their unique character complexes, fossils are critical in documenting sequences of character transformation over geologic time and may elucidate otherwise ambiguous patterns of evolution that are not revealed by molecular data alone. Here, we employ a methodological approach that allows for the integration of molecular and paleontological data in deciphering one of the most innovative features in the evolutionary history of mammals—laryngeal echolocation in bats. Molecular data alone, including an expanded data set that includes new sequences for the A2AB gene, suggest that microbats are paraphyletic but do not resolve whether laryngeal echolocation evolved independently in different microbat lineages or evolved in the common ancestor of bats and was subsequently lost in megabats. When scaffolds from molecular phylogenies are incorporated into parsimony analyses of morphological characters, including morphological characters for the Eocene taxa Icaronycteris, Archaeonycteris, Hassianycteris, and Palaeochiropteryx, the resulting trees suggest that laryngeal echolocation evolved in the common ancestor of fossil and extant bats and was subsequently lost in megabats. Molecular dating suggests that crown-group bats last shared a common ancestor 52 to 54 million years ago.

Rapid evolution in plant chitinases: Molecular targets of selection in plant-pathogen coevolution

Many pathogen recognition genes, such as plant R-genes, undergo rapid adaptive evolution, providing evidence that these genes play a critical role in plant-pathogen coevolution. Surprisingly, whether rapid adaptive evolution also occurs in genes encoding other kinds of plant defense proteins is unknown. Unlike recognition proteins, plant chitinases attack pathogens directly, conferring disease resistance by degrading chitin, a component of fungal cell walls. Here, we show that nonsynonymous substitution rates in plant class I chitinase often exceed synonymous rates in the plant genus Arabis (Cruciferae) and in other dicots, indicating a succession of adaptively driven amino acid replacements. We identify individual residues that are likely subject to positive selection by using codon substitution models and determine the location of these residues on the three-dimensional structure of class I chitinase. In contrast to primate lysozymes and plant class III chitinases, structural and functional relatives of class I chitinase, the adaptive replacements of class I chitinase occur disproportionately in the active site cleft. This highly unusual pattern of replacements suggests that fungi directly defend against chitinolytic activity through enzymatic inhibition or other forms of chemical resistance and identifies target residues for manipulating chitinolytic activity. These data also provide empirical evidence that plant defense proteins not involved in pathogen recognition also evolve in a manner consistent with rapid coevolutionary interactions.

RNA virus error catastrophe: Direct molecular test by using ribavirin

RNA viruses evolve rapidly. One source of this ability to rapidly change is the apparently high mutation frequency in RNA virus populations. A high mutation frequency is a central tenet of the quasispecies theory. A corollary of the quasispecies theory postulates that, given their high mutation frequency, animal RNA viruses may be susceptible to error catastrophe, where they undergo a sharp drop in viability after a modest increase in mutation frequency. We recently showed that the important broad-spectrum antiviral drug ribavirin (currently used to treat hepatitis C virus infections, among others) is an RNA virus mutagen, and we proposed that ribavirin's antiviral effect is by forcing RNA viruses into error catastrophe. However, a direct demonstration of error catastrophe has not been made for ribavirin or any RNA virus mutagen. Here we describe a direct demonstration of error catastrophe by using ribavirin as the mutagen and poliovirus as a model RNA virus. We demonstrate that ribavirin's antiviral activity is exerted directly through lethal mutagenesis of the viral genetic material. A 99.3% loss in viral genome infectivity is observed after a single round of virus infection in ribavirin concentrations sufficient to cause a 9.7-fold increase in mutagenesis. Compiling data on both the mutation levels and the specific infectivities of poliovirus genomes produced in the presence of ribavirin, we have constructed a graph of error catastrophe showing that normal poliovirus indeed exists at the edge of viability. These data suggest that RNA virus mutagens may represent a promising new class of antiviral drugs.

Evidence from molecular systematics for decreased avian diversification in the pleistocene Epoch.

Pleistocene glaciations have been suggested as major events influencing speciation rates in vertebrates. Avian paleontological studies suggest that most extant species evolved in the Pleistocene Epoch and that species' durations decreased through the Pleistocene because of heightened speciation rates. Molecular systematic studies provide another data base for testing these predictions. In particular, rates of diversification can be determined from molecular phylogenetic trees. For example, an increasing rate of speciation (but constant extinction) requires shorter intervals between successive speciation events on a phylogenetic tree. Examination of the cumulative distribution of reconstructed speciation events in mtDNA phylogenies of 11 avian genera, however, reveals longer intervals between successive speciation events as the present time is approached, suggesting a decrease in net diversification rate through the Pleistocene Epoch. Thus, molecular systematic studies do not indicate a pulse of Pleistocene diversification in passerine birds but suggest, instead, that diversification rates were lower in the Pleistocene than for the preceding period. Documented habitat shifts likely led to the decreased rate of diversification, although from molecular evidence we cannot discern whether speciation rates decreased or extinction rates increased.