Synthesis of potentially cytotoxic steroidal lactones

A review of the biological properties and the synthesis of α-methylene-γ- and -δ-lactones is presented. Cholesterol (201) was converted into 4-oxa-3-oxo-5α-cholestane (203) and the α-methylene moiety was introduced by α-hydroxymethylenation, diethylamination and elimination of diethylamine after hydrogenation to give 2-methylene-4-oxa-3-oxo-5α-cholestane (225). The same sequence of reactions was employed to prepare 17β-hydroxy-2-methylene-4-oxa-3-oxo-5α-androstane (238) and its 17-yl acetate (239) from androst-5-en-3β-ol-17-one (228). Reactions of the above α-methylene lactones with L-cysteine gave the cysteine–lactone adducts in a Michael-type addition. Reaction of the lactone (203) with phenyl magnesium bromide gave 4-oxa-3-phenyl-5α-cholest-2-ene (278). On epoxidation this compound gave an unusual rearranged product. 3-hydroxy-4-oxa-3-phenyl-5α-cholestan-2-one (281) which on treatment with ethanol/hydrochloric acid formed 3-ethoxy-4-oxa-3-phenyl-5α-cholestan-2-one (282). Oxidation of the rearranged product (281) with lead tetra acetate gave 5-benzoyloxy-2,3-seco-5α-cholestan-2-oic acid (299) which on esterification gave the methyl ester (300). The oxidised product (299). after hydrolysis. was cyclised to give A-nor-3-oxa-5α-cholestan-2-one (274). [Continues.

A phylogenomic gene cluster resource: the Phylogenetically Inferred Groups (PhIGs) database

BACKGROUND: We present here the PhIGs database, a phylogenomic resource for sequenced genomes. Although many methods exist for clustering gene families, very few attempt to create truly orthologous clusters sharing descent from a single ancestral gene across a range of evolutionary depths. Although these non-phylogenetic gene family clusters have been used broadly for gene annotation, errors are known to be introduced by the artifactual association of slowly evolving paralogs and lack of annotation for those more rapidly evolving. A full phylogenetic framework is necessary for accurate inference of function and for many studies that address pattern and mechanism of the evolution of the genome. The automated generation of evolutionary gene clusters, creation of gene trees, determination of orthology and paralogy relationships, and the correlation of this information with gene annotations, expression information, and genomic context is an important resource to the scientific community. DISCUSSION: The PhIGs database currently contains 23 completely sequenced genomes of fungi and metazoans, containing 409,653 genes that have been grouped into 42,645 gene clusters. Each gene cluster is built such that the gene sequence distances are consistent with the known organismal relationships and in so doing, maximizing the likelihood for the clusters to represent truly orthologous genes. The PhIGs website contains tools that allow the study of genes within their phylogenetic framework through keyword searches on annotations, such as GO and InterPro assignments, and sequence similarity searches by BLAST and HMM. In addition to displaying the evolutionary relationships of the genes in each cluster, the website also allows users to view the relative physical positions of homologous genes in specified sets of genomes. SUMMARY: Accurate analyses of genes and genomes can only be done within their full phylogenetic context. The PhIGs database and corresponding website address this problem for the scientific community. Our goal is to expand the content as more genomes are sequenced and use this framework to incorporate more analyses

RegPrecise web services interface: programmatic access to the transcriptional regulatory interactions in bacteria reconstructed by comparative genomics.

Web services application programming interface (API) was developed to provide a programmatic access to the regulatory interactions accumulated in the RegPrecise database (http://regprecise.lbl.gov), a core resource on transcriptional regulation for the microbial domain of the Department of Energy (DOE) Systems Biology Knowledgebase. RegPrecise captures and visualize regulogs, sets of genes controlled by orthologous regulators in several closely related bacterial genomes, that were reconstructed by comparative genomics. The current release of RegPrecise 2.0 includes >1400 regulogs controlled either by protein transcription factors or by conserved ribonucleic acid regulatory motifs in >250 genomes from 24 taxonomic groups of bacteria. The reference regulons accumulated in RegPrecise can serve as a basis for automatic annotation of regulatory interactions in newly sequenced genomes. The developed API provides an efficient access to the RegPrecise data by a comprehensive set of 14 web service resources. The RegPrecise web services API is freely accessible at http://regprecise.lbl.gov/RegPrecise/services.jsp with no login requirements

FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments

Background: We recently described FastTree, a tool for inferring phylogenies for alignments with up to hundreds of thousands of sequences. Here, we describe improvements to FastTree that improve its accuracy without sacrificing scalability. Methodology/Principal Findings: Where FastTree 1 used nearest-neighbor interchanges (NNIs) and the minimum-evolution criterion to improve the tree, FastTree 2 adds minimum-evolution subtree-pruning-regrafting (SPRs) and maximumlikelihood NNIs. FastTree 2 uses heuristics to restrict the search for better trees and estimates a rate of evolution for each site (the ‘‘CAT’ ’ approximation). Nevertheless, for both simulated and genuine alignments, FastTree 2 is slightly more accurate than a standard implementation of maximum-likelihood NNIs (PhyML 3 with default settings). Although FastTree 2 is not quite as accurate as methods that use maximum-likelihood SPRs, most of the splits that disagree are poorly supported, and for large alignments, FastTree 2 is 100–1,000 times faster. FastTree 2 inferred a topology and likelihood-based local support values for 237,882 distinct 16S ribosomal RNAs on a desktop computer in 22 hours and 5.8 gigabytes of memory. Conclusions/Significance: FastTree 2 allows the inference of maximum-likelihood phylogenies for huge alignments

Method of converting cholesterol in food to coprostanol

Cholesterol reductase was discovered in certain green plant parts. The enzyme is known to be present in several bacteria that commonly inhabit the digestive tract of animals. Eubacteria species A.T.C.C. 21408 is one such cholesterol reductase-containing bacterium. It is concentrated from a homogenate, preferably of leaves of plants or from bacteria or other organisms to provide a cell-free, cholesterol reductase-enriched preparation that can be used to decrease cholesterol content of food substances

Horizontal gene transfer and the evolution of transcriptional regulation in Escherichia coli

Most Escherichia coli transcription factors have paralogs, but these usually arose by horizontal gene transfer rather than by duplication within the E. coli lineage, as previously believed

Orthologous Transcription Factors in Bacteria Have Different Functions and Regulate Different Genes

Transcription factors (TFs) form large paralogous gene families and have complex evolutionary histories. Here, we ask whether putative orthologs of TFs, from bidirectional best BLAST hits (BBHs), are evolutionary orthologs with conserved functions. We show that BBHs of TFs from distantly related bacteria are usually not evolutionary orthologs. Furthermore, the false orthologs usually respond to different signals and regulate distinct pathways, while the few BBHs that are evolutionary orthologs do have conserved functions. To test the conservation of regulatory interactions, we analyze expression patterns. We find that regulatory relationships between TFs and their regulated genes are usually not conserved for BBHs in Escherichia coli K12 and Bacillus subtilis. Even in the much more closely related bacteria Vibrio cholerae and Shewanella oneidensis MR-1, predicting regulation from E. coli BBHs has high error rates. Using gene–regulon correlations, we identify genes whose expression pattern differs between E. coli and S. oneidensis. Using literature searches and sequence analysis, we show that these changes in expression patterns reflect changes in gene regulation, even for evolutionary orthologs. We conclude that the evolution of bacterial regulation should be analyzed with phylogenetic trees, rather than BBHs, and that bacterial regulatory networks evolve more rapidly than previously thought

The evolutionary dynamics of the Saccharomyces cerevisiae protein interaction network after duplication

Gene duplication is an important mechanism in the evolution of protein interaction networks. Duplications are followed by the gain and loss of interactions, rewiring the network at some unknown rate. Because rewiring is likely to change the distribution of network motifs within the duplicated interaction set, it should be possible to study network rewiring by tracking the evolution of these motifs. We have developed a mathematical framework that, together with duplication data from comparative genomic and proteomic studies, allows us to infer the connectivity of the preduplication network and the changes in connectivity over time. We focused on the whole-genome duplication (WGD) event in Saccharomyces cerevisiae. The model allowed us to predict the frequency of intergene interaction before WGD and the post duplication probabilities of interaction gain and loss. We find that the predicted frequency of self-interactions in the preduplication network is significantly higher than that observed in today's network. This could suggest a structural difference between the modern and ancestral networks, preferential addition or retention of interactions between ohnologs, or selective pressure to preserve duplicates of self-interacting proteins

Systematic mapping of two component response regulators to gene targets in a model sulfate reducing bacterium

BackgroundTwo component regulatory systems are the primary form of signal transduction in bacteria. Although genomic binding sites have been determined for several eukaryotic and bacterial transcription factors, comprehensive identification of gene targets of two component response regulators remains challenging due to the lack of knowledge of the signals required for their activation. We focused our study on Desulfovibrio vulgaris Hildenborough, a sulfate reducing bacterium that encodes unusually diverse and largely uncharacterized two component signal transduction systems.ResultsWe report the first systematic mapping of the genes regulated by all transcriptionally acting response regulators in a single bacterium. Our results enabled functional predictions for several response regulators and include key processes of carbon, nitrogen and energy metabolism, cell motility and biofilm formation, and responses to stresses such as nitrite, low potassium and phosphate starvation. Our study also led to the prediction of new genes and regulatory networks, which found corroboration in a compendium of transcriptome data available for D. vulgaris. For several regulators we predicted and experimentally verified the binding site motifs, most of which were discovered as part of this study.ConclusionsThe gene targets identified for the response regulators allowed strong functional predictions to be made for the corresponding two component systems. By tracking the D. vulgaris regulators and their motifs outside the Desulfovibrio spp. we provide testable hypotheses regarding the functions of orthologous regulators in other organisms. The in vitro array based method optimized here is generally applicable for the study of such systems in all organisms