Identification and Characterization of a Cryptic Bifunctional Type I Diterpene Synthase Involved in Talaronoid Biosynthesis from a Marine-Derived Fungus.

We report the identification of the tnd biosynthetic cluster from the marine-derived fungus Aspergillus flavipes and the in vivo characterization of a cryptic type I diterpene synthase. The heterologous expression of the bifunctional terpene synthase led to the discovery of a diterpene backbone, talarodiene, harboring a benzo[a]cyclopenta[d]cyclooctane tricyclic fused ring system. The conversion of geranylgeranyl diphosphate to talarodiene was investigated using 13C-labeling studies, and stable isotope tracer experiments showed the biotransformation of talarodiene into talaronoid C.

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha.

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

Mapping a kingdom-specific functional domain of squalene synthase.

Squalene synthase catalyzes the first committed step in sterol biosynthesis and consists of both an amino-terminal catalytic domain and a carboxy-terminal domain tethering the enzyme to the ER membrane. While the overall architecture of this enzyme is identical in eukaryotes, it was previously shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implied a unique component of the fungal carboxy-terminal domain was responsible for the complementation phenotype. To identify this motif, we used Saccharomyces cerevisiae with a squalene synthase knockout mutation, and expressed intact and chimeric squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. However, when highly expressed, non-fungal squalene synthases could not complement the yeast mutation and instead led to the accumulation of a toxic intermediate(s) as defined by mutations of genes downstream in the ergosterol pathway. Restoration of the complete complementation phenotype was mapped to a 26-amino acid hinge region linking the catalytic and membrane-spanning domains specific to fungal squalene synthases. Over-expression of the C-terminal domain containing a hinge domain from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Because this hinge region is unique to and highly conserved within each kingdom of life, the data suggests that the hinge domain plays an essential functional role, such as assembly of ergosterol multi-enzyme complexes in fungi.

Experimental Genome-Wide Determination of RNA Polyadenylation in Chlamydomonas reinhardtii.

The polyadenylation of RNA is a near-universal feature of RNA metabolism in eukaryotes. This process has been studied in the model alga Chlamydomonas reinhardtii using low-throughput (gene-by-gene) and high-throughput (transcriptome sequencing) approaches that recovered poly(A)-containing sequence tags which revealed interesting features of this critical process in Chlamydomonas. In this study, RNA polyadenylation has been studied using the so-called Poly(A) Tag Sequencing (PAT-Seq) approach. Specifically, PAT-Seq was used to study poly(A) site choice in cultures grown in four different media types-Tris-Phosphate (TP), Tris-Phosphate-Acetate (TAP), High-Salt (HS), and High-Salt-Acetate (HAS). The results indicate that: 1. As reported before, the motif UGUAA is the primary, and perhaps sole, cis-element that guides mRNA polyadenylation in the nucleus; 2. The scope of alternative polyadenylation events with the potential to change the coding sequences of mRNAs is limited; 3. Changes in poly(A) site choice in cultures grown in the different media types are very few in number and do not affect protein-coding potential; 4. Organellar polyadenylation is considerable and affects primarily ribosomal RNAs in the chloroplast and mitochondria; and 5. Organellar RNA polyadenylation is a dynamic process that is affected by the different media types used for cell growth.

HIV pre-test information, discussion or counselling? A review of guidance relevant to the WHO European Region.

In the context of a shift from exceptionalism to normalisation, this study examines recommendations/evidence in current pan-European/global guidelines regarding pre-test HIV testing and counselling practices in health care settings. It also reviews new research not yet included in guidelines. There is consensus that verbal informed consent must be gained prior to testing, individually, in private, confidentially, in the presence of a health care provider. All guidelines recommend pre-test information/discussion delivered verbally or via other methods (information sheet). There is agreement about a minimum standard of information to be provided before a test, but guidelines differ regarding discussion about issues encouraging patients to think about implications of the result. There is heavy reliance on expert consultation in guideline development. Referenced scientific evidence is often more than ten years old and based on US/UK research. Eight new papers are reviewed. Current HIV testing and counselling guidelines have inconsistencies regarding the extent and type of information that is recommended during pre-test discussions. The lack of new research underscores a need for new evidence from a range of European settings to support the process of expert consultation in guideline development.

Production, purification, and assay of recombinant proteins for in vitro biochemical analyses of the plant polyadenylation complex.

In the post-genomic era where gene sequences are available for many organisms, attention has shifted from DNA to the workhorses of the cell-RNA and protein. A number of proteins, as recent studies indicate, seem to possess RNA-binding and RNA cleavage activities. In order to understand the events that comprise RNA processing such as splicing, 3' end processing, and even RNA turnover, well established methods are necessary. Bacterial recombinant proteins afford an invaluable opportunity to produce proteins in an economical and reproducible fashion in order to study these activities. This chapter describes various experimental protocols to begin the elucidation of the many events that surround RNA processing at the 3' end of a transcript.

Now playing: farnesol in the biofilm.

The evolutionary pathway of specialized metabolism often takes unexpected, perplexing turns. In this issue of Chemistry & Biology, Feng and coworkers provide evidence for a unique phosphatase whose enzymatic product plays a critical role in biofilm formation in Bacillus subtilis.

Structure-function mapping of key determinants for hydrocarbon biosynthesis by squalene and squalene synthase-like enzymes from the green alga Botryococcus braunii race B.

Squalene and botryococcene are branched-chain, triterpene compounds that arise from the head-to-head condensation of two molecules of farnesyl diphosphate to yield 1'-1 and 1'-3 linkages, respectively. The enzymes that catalyze their formation have attracted considerable interest from the medical field as potential drug targets and the renewable energy sector for metabolic engineering efforts. Recently, the enzymes responsible for botryococcene and squalene biosynthesis in the green alga Botryococcus braunii race B were characterized. To better understand how the specificity for the 1'-1 and 1'-3 linkages was controlled, we attempted to identify the functional residues and/or domains responsible for this step in the catalytic cascade. Existing crystal structures for the mammalian squalene synthase and Staphylococcus dehydrosqualene synthase enzymes were exploited to develop molecular models for the B. braunii botryococcene and squalene synthase enzymes. Residues within the active sites that could mediate catalytic specificity were identified, and reciprocal mutants were created in an attempt to interconvert the reaction product specificity of the enzymes. We report here the identification of several amino acid positions contributing to the rearrangement of the cyclopropyl intermediate to squalene, but these same positions do not appear to be sufficient to account for the cyclopropyl rearrangement to give botryococcene.

'Mobile men with money': HIV prevention and the erasure of difference.

Mobile Men with Money is one of the latest risk categories to enter into HIV prevention discourse. Used in countries in Asia, the Pacific and Africa, it refers to diverse groups of men (e.g. businessmen, miners and itinerant wage labourers) who, in contexts of high population movement and economic disparity, find themselves at heightened risk of HIV as members of a 'most-at-risk population', or render others vulnerable to infection. How adequate is such a description? Does it make sense to develop HIV prevention programmes from such understandings? The history of the epidemic points to major weaknesses in the use of terminologies such as 'sex worker' and 'men who have sex with men' when characterising often diverse populations. Each of these terms carries negative connotations, portraying the individuals concerned as being apart from the 'general population', and posing a threat to it. This paper examines the diversity of men classified as mobile men with money, pointing to significant variations in mobility, wealth and sexual networking conducive to HIV transmission. It highlights the patriarchal, heteronormative and gendered assumptions frequently underpinning use of the category and suggests more useful ways of understanding men, masculinity, population movement, relative wealth in relation to HIV vulnerability and risk.

Functional identification of triterpene methyltransferases from Botryococcus braunii race B.

Botryococcus braunii race B is a colony-forming, green algae that accumulates triterpene oils in excess of 30% of its dry weight. The composition of the triterpene oils is dominated by dimethylated to tetramethylated forms of botryococcene and squalene. Although unusual mechanisms for the biosynthesis of botryococcene and squalene were recently described, the enzyme(s) responsible for decorating these triterpene scaffolds with methyl substituents were unknown. A transcriptome of B. braunii was screened computationally assuming that the triterpene methyltransferases (TMTs) might resemble the S-adenosyl methionine-dependent enzymes described for methylating the side chain of sterols. Six sterol methyltransferase-like genes were isolated and functionally characterized. Three of these genes when co-expressed in yeast with complementary squalene synthase or botryococcene synthase expression cassettes resulted in the accumulation of mono- and dimethylated forms of both triterpene scaffolds. Surprisingly, TMT-1 and TMT-2 exhibited preference for squalene as the methyl acceptor substrate, whereas TMT-3 showed a striking preference for botryococcene as its methyl acceptor substrate. These in vivo preferences were confirmed with in vitro assays utilizing microsomal preparations from yeast overexpressing the respective genes, which encode for membrane-associated enzymes. Structural examination of the in vivo yeast generated mono- and dimethylated products by NMR identified terminal carbons, C-3 and C-22/C-20, as the atomic acceptor sites for the methyl additions to squalene and botryococcene, respectively. These sites are identical to those previously reported for the triterpenes extracted from the algae. The availability of closely related triterpene methyltransferases exhibiting distinct substrate selectivity and successive catalytic activities provides important tools for investigating the molecular mechanisms responsible for the specificities exhibited by these unique enzymes.

Young people and sexual agency in rural Uganda.

This paper offers an analysis of young people's sexual agency in rural Uganda. Drawing on definitions of agency from within the international development literature, it focuses on: decision-making processes leading to young people's involvement in relationships; actions undertaken to maintain 'secret' relationships in contexts where young people's sexual agency is generally prohibited; transactional and gendered negotiations between young people involved within a relationship; and a range of outcomes arising from young people's sexual activity. An understanding of the dynamics and temporal nature of young people's sexual agency, and the consequences that follow from it, challenges the widely held view that young people do not know what they are doing in relation to their sexual health. This should enable practitioners to identify avenues for developing HIV prevention and sexual health programmes that are more fully based in, and driven by, the realities of young people's sexual lives.

The Arabidopsis ortholog of the 77 kDa subunit of the cleavage stimulatory factor (AtCstF-77) involved in mRNA polyadenylation is an RNA-binding protein.

The 77 kDa subunit of the polyadenylation cleavage stimulation factor (CstF77) is important in messenger RNA 3' end processing. Previously, we demonstrated that AtCstF77 interacts with AtCPSF30, the Arabidopsis ortholog of the 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor. In further dissecting this interaction, it was found that the C-terminus of AtCstF77 interacts with AtCPSF30. Remarkably, we also found that the C-terminal domain of AtCstF77 possesses RNA-binding ability. These studies therefore reveal AtCstF77 to be an RNA-binding protein, adding yet another RNA-binding activity to the plant polyadenylation complex. This raises interesting questions as to the means by which RNAs are recognized during mRNA 3' end formation in plants.

Synthesis and characterization of covalently linked single-stranded DNA oligonucleotide-dendron conjugates.

A solution-phase synthesis and characterization of covalent DNA-dendron conjugates is presented. Thiol-terminated 12-base oligonucleotides were added to second- and third-generation triazine-based dendrons via thiol/disulfide exchange chemistry. Single-stranded DNA oligonucleotides were successfully attached to dendrons at the core, the periphery, and both. Proof of structure for these architectures is derived primarily from mass spectrometry and polyacrylamide gel electrophoresis and complemented by labeling analysis using Ellman's reagent and degradation analysis using a reducing agent.

Catalytic double-bond metathesis without the transition metal.

Iminophosphoranes of the type X(3)P=NR (X = Cl, pyrrolyl; R = alkyl, aryl) catalytically metathesize C=N bonds of carbodiimides via an addition/elimination mechanism that, despite the lack of d orbital participation in P-N bonding, conserves the key features of metal-catalyzed olefin metathesis. Diazaphosphetidine intermediates, produced by the formal [2 + 2] addition of carbodiimides to the P=N bond, have been isolated and characterized. All phosphorus-containing species in the complex catalytic reaction mixtures have been identified and their origins explained. The kinetics of addition of diisopropylcarbodiimide to Cl(3)P=NPr(i)() and subsequent elimination were studied, and rate constants were determined: k(add) = 1.7 x 10(-3) (+/-0.1 x 10(-3)) M s(-1) and k(elim) = 4.0 x 10(-4) (+/-0.3 x 10(-4)) s(-1). The rate of these reactions corresponds well with the observed catalytic TOF of 1.44 TO/P/h.