Changes in Chemical Constituents of Tobacco Leaves Infected with Corynespora cassiicola and Colletotrichum nicotianae

The following smut fungi reported from Hyparrhenia hirta were studied: Sporisorium andropogonis, S.bardnonense, S. puellare, and S. transfissum. Two new combinations are proposed: Sporisorium barcinonense (Riofrio) Vanky, and S. puellare (H. Sydow) G. Deml. S. andropogonis and S. puellare have morphologically identical spores but differ in the type of spore germination. While S. puellare germinates by a ramified mycelium, S. andropogonis produces four-celled basidia forming basidiospores laterally and apically. It appears that S. andropogonis is restricted to Dichanthium ischaemum, and S. puellare to Hyparrhenia hirta. S. harcinonense and S. transfissum, both producing the same type of floral infection, are differentiated by their spore measurements.     

Estimating synonymous and nonsynonymous substitution rates

Partitioning the total substitution rate into synnonymous and nonsynonymous components is a key aspect of many analyses in molecular evolution. Numerous methods exist for estimating these rates. However, until recently none of the estimation procedures were based on a sound statistical footing. In this paper, the evolutionary model of Muse and Gaut (1994) is used as the basis for two sets of parameters quantifying silent and replacement substitution rates. The parameters are shown to be equal when the four nucleotides are equally frequent and unequal otherwise. Maximum-likelihood estimation of these parameters is described, and the performance of these estimates is compared to that of existing estimation procedures. It is shown that the estimates of Nei and Gojobori (1986) are not unbiased for either set of parameters, although they provide very good estimates for one set as long as sequence divergence is not too high. However, some disturbing properties are found for the Nei and Gojobori estimates. In particular, it is shown that the expected value of the Nei and Gojobori estimate of silent substitution rate is a function of both the silent and replacement substitution rates. The maximum-likelihood estimates have no such problems.      

Testing for Equality of Evolutionary Rates

A likelihood ratio test is presented for comparing rates of evolutionary change in the paths of descent leading to two species. The test is compared to previous relative rate tests based on variances of estimated numbers of base substitutions. The likelihood approach allows for different transversion and transition rates, and when these rates are actually different, the likelihood ratio test can be much more powerful than the variance-based tests. For single-parameter mutation models, however, the two tests have similar power. The tests are applied to a set of chloroplast sequences from several species of grasses, and additional indications of significantly different rates leading to barley were found with the likelihood ratio test.     

Relative rates of nucleotide substitution at the rbcl locus of monocotyledonous plants

Summary We subjected 35 rbcL nucleotide sequences from monocotyledonous taxa to maximum likelihood relative rate tests and estimated relative differences in rates of nucleotide substitution between groups of sequences without relying on knowledge of divergence times between taxa. Rate tests revealed that there is a hierarchy of substitution rate at the rbcL locus within the monocots. Among the taxa analyzed the grasses have the most rapid substitution rate; they are followed in rate by the Orchidales, the Liliales, the Bromeliales, and the Arecales. The overall substitution rate for the rbcL locus of grasses is over 5 times the substitution rate in the rbcL of the palms. The substitution rate at the third codon positions in the rbcL of the grasses is over 8 times the third position rate in the palms. The pattern of rate variation is consistent with the generation-time-effect hypothesis. Heterogenous rates of substitution have important implications for phylogenetic reconstruction.Offprint requests to: M.T. Clegg     

Comparisons of the Nucleotide Substitution Process Among Repetitive Segments of the α- and β-Spectrin Genes

The actin–cross-linking protein spectrin is a prominent component of the membrane cytoskeleton. Spectrin is a tetramer of two antiparallel αβ-dimers which share a unique and ancient gene structure. The α-spectrin and β-spectrin genes are composed primarily of tandemly repeated 106-amino-acid segments, each of which forms a triple α-helical coiled coil. Both the genes and the repeats themselves are homologous. The two genes are thought to be the result of a gene duplication event, and each gene is the product of duplications of the 106-amino-acid repeats. In this work we compare the process of molecular evolution across the repeated segments of the α- and β-spectrin genes. We find that the α-spectrin segments have, for the most part, evolved in a homogeneous fashion, while considerable heterogeneity is found among β-spectrin segments. Several segments with unique known functions are found to have evolved differently than the others. On the basis of heterogeneity of the evolutionary process, we suggest that at least one repeat has a unique function that has yet to be documented. We also present new statistical methods for comparing the evolutionary process between different regions of DNA sequences. Received: 27 March 1996 / Accepted: 21 October 1996     

Evolutionary Analyses of DNA Sequences Subject to Constraints on Secondary Structure

Evolutionary models appropriate for analyzing nucleotide sequences that are subject to constraints on secondary structure are developed. The models consider the evolution of pairs of nucleotides, and they incorporate the effects of base-pairing constraints on nucleotide substitution rates by introducing a new parameter to extensions of standard models of sequence evolution. To illustrate some potential uses of the models, a likelihood-ratio test is constructed for the null hypothesis that two (prespecified) regions of DNA evolve independently of each other. The sampling properties of the test are explored via simulation. The test is then incorporated into a heuristic method for identifying the location of unknown stems. The test and related procedures are applied to data from ribonuclease P RNA sequences of bacteria.     

Chaperone activity with a redox switch

Hsp33, a member of a newly discovered heat shock protein family, was found to be a very potent molecular chaperone. Hsp33 is distinguished from all other known molecular chaperones by its mode of functional regulation. Its activity is redox regulated. Hsp33 is a cytoplasmically localized protein with highly reactive cysteines that respond quickly to changes in the redox environment. Oxidizing conditions like H2O2 cause disulfide bonds to form in Hsp33, a process that leads to the activation of its chaperone function. In vitro and in vivo experiments suggest that Hsp33 protects cells from oxidants, leading us to conclude that we have found a protein family that plays an important role in the bacterial defense system toward oxidative stress.     

Bacteroid formation in the Rhizobium-legume symbiosis

During the Rhizobium-legume symbiosis, bacteria enter the cells of host plants and differentiate into nitrogen-fixing bacteroids. Recent mutant screens and expression studies have revealed bacterial genes involved in the developmental pathway and demonstrate how the genetic requirements can vary from one host-microbe system to another. Whether bacteroids are terminally differentiated cells is an ongoing debate and new experimental systems are required to address this issue.     

A chemical approach for detecting sulfenic acid-modified proteins in living cells

Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO2H) and sulfonic acids (Cys-SO3H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acid-modified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues.