Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism

Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastasterone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 microM, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants.     

High vaccine efficacy against shigellosis of recombinant noninvasive Shigella mutant that expresses Yersinia invasin

Live attenuated Shigella vaccines elicit protective immune responses, but involve a potential risk of inducing a strong inflammatory reaction. The bacterial invasiveness that is crucial for Ag delivery causes inflammatory destruction of infected epithelial cells and proinflammatory cell death of infected macrophages. In this study, the noninvasive Shigella mutant DeltaipaB was equipped with Yersinia invasin protein, which has been shown to mediate bacterial invasion and targeting to M cells located in follicle-associated epithelium. Invasin-expressing DeltaipaB (DeltaipaB/inv) was internalized into epithelial cells and retained in the intraphagosomal space. DeltaipaB/inv did not induce necrotic cell death of infected macrophages nor cause symptomatic damage after intranasal vaccination of mice. DeltaipaB/inv was safer and more effective than the conventional live vaccine, DeltavirG. Infection by DeltaipaB/inv caused polymorphonuclear neutrophil infiltration in the lung, but did not induce production of large amounts of proinflammatory cytokines. We concluded that the low experimental morbidity and high vaccine efficacy of DeltaipaB/inv are primarily based on high protective immune responses, which may be enhanced by the polymorphonuclear neutrophil infiltration unaccompanied by tissue injury.     

Genetic relationships between Indians and their neighboring populations

Using gene frequency data for 18 protein and blood group loci, we studied the genetic relationships of four Indian subcontinent populations (peoples from Punjab, Gujarati, Andhra Pradesh, and Bangladesh) with their neighboring populations (Iranians, Afghans, Sinhalese in Sri Lanka, Nepalese, Bhutanese, Malays, Bataks in northern Sumatra, and Chinese). The results obtained indicate that the four Indian subcontinent populations and the Sinhalese are genetically closer to Iranians and Afghans (Caucasoid) than to the other neighboring Mongoloid populations. Genetic distance analysis shows a clear-cut dichotomy between the Caucasoid and Mongoloid populations.     

Relative efficiencies of the maximum parsimony and distance-matrix methods in obtaining the correct phylogenetic tree

The relative efficiencies of the maximum parsimony (MP) and distance-matrix methods in obtaining the correct tree (topology) were studied by using computer simulation. The distance-matrix methods examined are the neighbor-joining, distance-Wagner, Tateno et al. modified Farris, Faith, and Li methods. In the computer simulation, six or eight DNA sequences were assumed to evolve following a given model tree, and the evolutionary changes of the sequences were followed. Both constant and varying rates of nucleotide substitution were considered. From the sequences thus obtained, phylogenetic trees were constructed using the six tree-making methods and compared with the model (true) tree. This process was repeated 300 times for each different set of parameters. The results obtained indicate that when the number of nucleotide substitutions per site is small and a relatively small number of nucleotides are used, the probability of obtaining the correct topology (P1) is generally lower in the MP method than in the distance-matrix methods. The P1 value for the MP method increases with increasing number of nucleotides but is still generally lower than the value for the NJ or DW method. Essentially the same conclusion was obtained whether or not the rate of nucleotide substitution was constant or whether or not a transition bias in nucleotide substitution existed. The relatively poor performance of the MP method for these cases is due to the fact that information from singular sites is not used in this method. The MP method also showed a relatively low P1 value when the model of varying rate of nucleotide substitution was used and the number of substitutions per site was large. However, the MP method often produced cases in which the correct tree was one of several equally parsimonious trees. When these cases were included in the class of "success," the MP method performed better than the other methods, provided that the number of nucleotide substitutions per site was small.     

Evolution of the major histocompatibility complex: independent origin of nonclassical class I genes in different groups of mammals

The class I major histocompatibility complex genes are composed of classical and nonclassical genes, the latter being largely nonfunctional. To understand the evolutionary relationships of the two groups of class I genes, a phylogenetic analysis of DNA sequences was conducted using 45 genes from six mammalian and one avian species. The results indicate that nonclassical genes in one species are more closely related to classical genes from the same species than to nonclassical genes from a species belonging to a different order or family. This indicates that the differentiation of classical and nonclassical genes occurs rather rapidly in the genome. Classical genes are apparently duplicated with a high frequency in the evolutionary process, and many of the duplicated genes seem to degenerate into nonclassical genes as a result of deleterious mutation. The nonclassical Qa genes in the mouse have sequences homologous to regulatory sequences involved in the universal expression of classical class I genes, but they have accumulated numerous nucleotide substitutions in these sequences. The pattern of nucleotide substitution in nonclassical genes is different from that in classical genes. In nonclassical genes, the rate of nonsynonymous substitution is higher in the antigen recognition site than in other gene regions, as is true of classical genes. However, unlike the case of classical genes, the nonsynonymous rate does not always exceed the synonymous rate in the antigen recognition site. Nonclassical proteins further differ from classical proteins in having amino acid replacements in conserved antigen recognition site positions. These observations are consistent with the hypothesis that nonclassical genes have originated from classical genes but have lost classical class I function because of deleterious mutation.     

Accuracy of estimated phylogenetic trees from molecular data

The accuracies and efficiencies of three different methods of making phylogenetic trees from gene frequency data were examined by using computer simulation. The methods examined are UPGMA, Farris' (1972) method, and Tateno et al.'s (1982) modified Farris method. In the computer simulation eight species (or populations) were assumed to evolve according to a given model tree, and the evolutionary changes of allele frequencies were followed by using the infinite-allele model. At the end of the simulated evolution five genetic distance measures (Nei's standard and minimum distances, Rogers' distance, Cavalli-Sforza's f theta, and the modified Cavalli-Sforza distance) were computed for all pairs of species, and the distance matrix obtained for each distance measure was used for reconstructing a phylogenetic tree. The phylogenetic tree obtained was then compared with the model tree. The results obtained indicate that in all tree-making methods examined the accuracies of both the topology and branch lengths of a reconstructed tree (rooted tree) are very low when the number of loci used is less than 20 but gradually increase with increasing number of loci. When the expected number of gene substitutions (M) for the shortest branch is 0.1 or more per locus and 30 or more loci are used, the topological error as measured by the distortion index (dT) is not great, but the probability of obtaining the correct topology (P) is less than 0.5 even with 60 loci. When M is as small as 0.004, P is substantially lower. In obtaining a good topology (small dT and high P) UPGMA and the modified Farris method generally show a better performance than the Farris method. The poor performance of the Farris method is observed even when Rogers' distance which obeys the triangle inequality is used. The main reason for this seems to be that the Farris method often gives overestimates of branch lengths. For estimating the expected branch lengths of the true tree UPGMA shows the best performance. For this purpose Nei's standard distance gives a better result than the others because of its linear relationship with the number of gene substitutions. Rogers' or Cavalli-Sforza's distance gives a phylogenetic tree in which the parts near the root are condensed and the other parts are elongated. It is recommended that more than 30 loci, including both polymorphic and monomorphic loci, be used for making phylogenetic trees. The conclusions from this study seem to apply also to data on nucleotide differences obtained by the restriction enzyme techniques.     

Sampling Variances of Heterozygosity and Genetic Distance

Mathematical formulae for the sampling variances of average heterozygosity and Nei's genetic distance are developed. These sampling variances are decomposed into their two components, i.e. the inter-locus and intra-locus variances. The relationship between the number of loci and the number of individuals per locus to be examined for estimating average heterozygosity and genetic distance is also discussed. The utility of the inter-locus variance of heterozygosity for studying the mechanism of maintenance of genetic variability in populations is indicated.     

Maximum Likelihood Estimation of the Number of Nucleotide Substitutions from Restriction Sites Data

A simple method of the maximum likelihood estimation of the number of nucleotide substitutions is presented for the case where restriction sites data from many different restriction enzymes are available. An iteration method, based on nucleotide counting, is also developed. This method is simpler than the maximum likelihood method but gives the same estimate. A formula for computing the variance of a maximum likelihood estimate is also presented.     

Byssochlamyopeptidase A, a Rennin-like Enzyme Produced by Byssochlamys fulva

The production of a rennin-like enzyme by Byssochlamys fulva varied considerably with the isolates tested. Among the seven isolates tested, NRRL 2260, IMI 83277, and N.Y. 1 were good enzyme producers. The enzyme produced by isolate IMI 83277 was purified approximately 20-fold after (NH(4))(2)SO(4) precipitation, diethylaminoethyl-cellulose chromatography and Sephadex G-100 gel filtration. The partially purified enzyme has a pH optimum at 2.9 and a temperature optimum around 60 C. The enzyme appeared to be relatively stable at 40 C between pH 3.0 and pH 6.85. A name, byssochlamyopeptidase A, was proposed for this new enzyme. The milk-clotting activity of byssochlamyo-peptidase A is dependent on pH and appeared to be minimal at pH 6.2 or above. No extensive proteolysis has been observed during the milk-clotting process. The non-trichloroacetic acid-precipitable nitrogen titration curve on skim milk was comparable to that catalyzed by animal rennet.