Identification and Cloning of waaF (rfaF) from Bordetella pertussis and Use To Generate Mutants of Bordetella spp. with Deep Rough Lipopolysaccharide

A DNA locus from Bordetella pertussis capable of reconstituting lipopolysaccharide (LPS) O-antigen biosynthesis in Salmonella typhimurium SL3789 (rfaF511) has been isolated, by using selection with the antibiotic novobiocin. DNA within the locus encodes a protein with amino acid sequence similarity to heptosyltransferase II, encoded by waaF (previously rfaF) in other gram-negative bacteria. Mutation of this gene in B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica by allelic exchange generated bacteria with deep rough LPS phenotypes consistent with the proposed function of the gene as an inner core heptosyltransferase. These are the first LPS mutants generated in B. parapertussis and B. bronchiseptica and the first deep rough mutants of any of the bordetellae.     

In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase.

Controlled expression of cellular and viral genes through alternative precursor messenger RNA (pre-mRNA) splicing requires serine/arginine-rich (SR) proteins. The Clk1 kinase, which phosphorylates SR proteins, is regulated through alternative splicing of the Clk1 pre-mRNA, yielding mRNAs encoding catalytically active and truncated inactive polypeptides (Clk1 and Clk1T, respectively). We present evidence that Clk1 and Clk1T proteins regulate the splicing of Clk1 and adenovirus pre-mRNAs in vivo. The peptide domain encoded by the alternatively spliced exon of Clk1 is essential for the regulatory activity of the Clk1 kinase. This is the first direct demonstration of an in vivo link between alternative splicing and protein kinase activity.     

Root-zone constraints and plant-based solutions for dryland salinity

Limitations to agricultural productivity imposed by the root-zone constraints in Australian dryland soils are severe and need redemption to improve the yields of grain crops and thereby meet world demand. Physical, chemical and biological constraints in soil horizons impose a stress on the plant and restrict plant growth and development. Hardsetting, crusting, compaction, salinity, sodicity, acidity, alkalinity, nutrient deficiencies and toxicities due to boron, carbonates and aluminium are the major factors that cause these constraints. Further, subsoils in agricultural regions in Australia have very low organic matter and biological activity. Dryland salinity is currently given wide attention in the public debate and government policies in Australia, but they only focus on salinity induced by shallow groundwater. However, the occurrence of transient salinity in root-zone layers in the regions where water tables are deep is an important issue with potential for larger economic loss than water table-induced seepage salinity. Root-zone constraints pose a challenge for salinity mitigation in recharge as well as discharge zones. In recharge zones, reduced water movement in sodic horizons results in salt accumulation in the root zone resulting in chemical and physical constraints that reduce transpiration that, in turn, upsets salt balance and plant growth. High salinity in soil and groundwater restricts the ability of plants to reduce water table in discharge zones. Thus plant-based strategies must address different kinds of limitations in soil profiles, both in recharge and discharge zones. In this paper we give an overview of plant response to root-zone constraints but with an emphasis on the processes of salt accumulation in the root-zone of soils. We also examine physical and chemical methods to overcome subsoil limitations, the ability of plants to adapt to and ameliorate these constraints, soil modification by management of agricultural and forestry ecosystems, the use of biological activity, and plant breeding for resistance to the soil constraints. We emphasise that soil scientists in cooperation with agronomists and plant breeders should design site-specific strategies to overcome multiple soil constraints, with vertical and lateral variations, and to develop plant-based solutions for dryland salinity.     

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

The renin-angiotensin and the kallikrein-kinin systems

The renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS) each encompasses a large number of molecules, with several participating in both systems. The RAS generates a family of bioactive angiotensin peptides with varying biological activities. These include angiotensin-(1-8) (Ang II), angiotensin-(2-8) (Ang III), angiotensin-(3-8) (Ang IV), and angiotensin-(1-7) [Ang-(1-7)]. Ang II and Ang III act on type 1 (AT(1)) and type 2 (AT(2)) angiotensin receptors, whereas, Ang IV and Ang-(1-7) act on their own receptors. The KKS also generates a family of bioactive peptides with varying biological activities. These include hydroxylated and non-hydroxylated bradykinin and kallidin peptides and their carboxypeptidase metabolites des-Arg(9)-bradykinin and des-Arg(10)-kallidin. Whereas bradykinin and kallidin act mainly via the type 2 bradykinin (B(2)) receptor, des-Arg(9)-bradykinin and des-Arg(10)-kallidin act mainly via the type 1 bradykinin (B(1)) receptor. The AT(1) receptor forms heterodimers with the AT(2) and B(2) receptors and there is cross talk between the AT(1) and epidermal growth factor receptors. The B(2) receptor also interacts with angiotensin converting enzyme and nitric oxide synthase. Both angiotensin and kinin peptides are metabolised by many different peptidases that are important determinants of the activities of the RAS and KKS, and several of which participate in both systems.     

Cation loss during accumulation of heavy metal cations by Saccharomyces cerevisiae

Summary Cu²⁺ accumulation byS. cerevisiae resulted in rapid release of 70% of cellular K⁺, followed by a slower release of approximately 60% of cellular Mg²⁺, but little loss of Ca²⁺. Co²⁺ was accumulated in smaller quantities and caused a smaller loss of physiological cations than either Cu²⁺ or Cd²⁺. Mg²⁺ release during copper accumulation was maximal at pH 6. Mg²⁺ release during Cu²⁺ accumulation increased with temperature and salinity of the suspension.     

Primary reactions of the LOV2 domain of phototropin,a plant blue-light photoreceptor

The phototropins constitute an important class of plant photoreceptor kinases that control a range of physiological responses, including phototropism, light-directed chloroplast movement, and light-induced stomatal opening. The LOV2 domain of phototropin binds a molecule of flavin mononucleotide (FMN) and undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine residue and the C(4a) atom of FMN. This product state promotes C-terminal kinase activation and downstream signal transduction. Here, we report the primary photophysics and photochemistry of LOV2 domains of phototropin 1 of Avena sativa (oat) and of the phy3 photoreceptor of Adiantum capillus-veneris (maidenhair fern). In agreement with earlier reports [Swartz, T. E., et al. (2001) J. Biol. Chem. 276, 36493-36500], we find that the FMN triplet state is the reactive species from which the photoreaction occurs. We demonstrate that the triplet state is the primary photoproduct in the LOV2 photocycle, generated at 60% efficiency. No spectroscopically distinguishable intermediates precede the FMN triplet on the femtosecond to nanosecond time scale, indicating that it is formed directly via intersystem crossing (ISC) from the singlet state. Our results indicate that the majority of the FMN triplets in the LOV2 domain exist in the protonated form. We propose a reaction mechanism that involves excited-state proton transfer, on the nanosecond time scale or faster, from the sulfhydryl group of the conserved cysteine to the N5 atom of FMN. This event promotes adduct formation by increasing the electrophilicity of C(4a) and subsequent nucleophilic attack by the cysteine's thiolate anion. Comparison to free FMN in solution shows that the protein environment of LOV2 increases the ISC rate of FMN by a factor of 2.4, thus improving the yield of the cysteinyl-flavin adduct and the efficiency of phototropin-mediated signaling processes.     

A SAS-6-Like Protein Suggests that the Toxoplasma Conoid Complex Evolved from Flagellar Components

SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.