Do phytogeographic patterns reveal biomes or biotic regions?

We present the largest comparative biogeographical analysis that has complete coverage of Australia's geography (20 phytogeographical subregions), using the most complete published molecular phylogenies to date of large Australian plant clades (Acacia, Banksia and the eucalypts). Two distinct sets of areas within the Australian flora were recovered, using distributional data from the Australasian Virtual Herbarium (AVH) and the Atlas of Living Australia (ALA): younger Temperate, Eremaean and Monsoonal biomes, and older southwest + west, southeast and northern historical biogeographical regions. The analyses showed that by partitioning the data into two sets, using either a Majority or a Frequency method to select taxon distributions, two equally valid results were found. The dataset that used a Frequency method discovered general area cladograms that resolved patterns of the Australian biomes, whereas if widespread taxa (Majority method, with >50% of occurrences outside a single subregion) were removed the analysis then recovered historical biogeographical regions. The study highlights the need for caution when processing taxon distributions prior to analysis as, in the case of the history of Australian phytogeography, the validity of both biomes and historical areas have been called into question.     

Multiple virus resistance in transgenic plants conferred by the human dsRNA-dependent protein kinase

We have developed a new strategy for engineering resistance to multipleviruses in plants. The strategy exploits the human double stranded (ds)RNA-dependent protein kinase (PKR). PKR is one of theinterferon-induced enzymes. It confers viral resistance in mammals byinhibitingviral replication through the inactivation of the translational initiationfactor, eIF-2, upon activation by dsRNA. The humanPKR gene was fused to the promoter of theArabidopsis blue copper binding protein gene(BCB) that is induced rapidly in response to wounding. Thechimeric gene cassette was introduced into tobacco plants. Expression of thePKR gene in transgenic tobacco plants was demonstrated byRNA gel blot analysis and autophosphorylation assay of anM _r 68,000 protein. The transgenic plantsexpressing the PKR gene showed significantly reduced viralsymptoms or no viral symptoms at all, when challenged by different plant RNAviruses, such as Cucumber mosaic virus, Tobaccoetch virus, or Potato virus Y. Thus, expressionof a single component in the human interferon pathway, thePKR gene, can effectively confer resistance to multipleviruses in transgenic plants.     

Diversity and varietal classification of Hibiscus syriacus L. with the heterogeneity within retrotransposon-like elements

Retrotransposons are present in multi-copy numbers that are integrated into plant genomes with considerable heterogeneous sequences within a single plant and between plant species, which allows the use of retrotransposons as additional sources of DNA polymorphism. A primer design for the sequence-tagged specific site and cleaved amplified polymorphic sequences (STS-CAPs) that are derived from retrotransposon-like sequences was developed for the molecular marker analysis in Hibiscus syriacus. This method was applied for the detection of sequence variations of intact retrotransposons that exist in plant genomes, which resulted in higher polymorphisms than in the amplified fragment length polymorphism (AFLP). Through STS-CAPs, specific fingerprinting data among H. syriacus varieties can be easily distinguished and generated with reproducible results. It could also be adapted to any species that possess multi-copy retrotransposons for varietal identification as well as the assessment of genetic relationships.     

Cloning and Bacterial Expression of Sesquiterpene Cyclase, a Key Branch Point Enzyme for the Synthesis of Sesquiterpenoid Phytoalexin Capsidiol in UV-Challenged Leaves of Capsicum annuum

Sesquiterpene cyclase, a branch point enzyme in the generalisoprenoid pathway for the synthesis of phytoalexin capsidiol,was induced in detached leaves of Capsicum annuum (pepper) byUV treatment. The inducibility of cyclase enzyme activitiesparalleled the absolute amount of cyclase protein(s) of pepperimmunodetected by monoclonal antibodies raised against tobaccosesquiterpene cyclase. A cDNA library was constructed with poly(A)⁺RNA isolated from 24 h UV-challenged leaves of pepper. A cDNAclone for sesquiterpene cyclase in pepper was isolated by usinga tobacco 5-epi aristolochene synthase gene as a hetero-logousprobe. The predicted protein encoded by this cDNA was comprisedof 559 amino acids and had a relative molecular mass of 65,095.The primary structural information from the cDNA clone revealedthat it shared 77%, 72% and 49% identity with 5-epi aristolochene,vetispiradiene, and cadinene synthase, respectively. The enzymaticproduct catalyzed by the cDNA clone in bacteria was identifiedas 5-epi aristolochene, as judged by argentation TLC. RNA blothybridization demonstrated the induction of an mRNA consistentwith the induction of cyclase enzyme activity in UV-treatedpepper. (Received March 2, 1998; Accepted June 15, 1998)     

A Rapid DNA Extraction Method for RFLP and PCR Analysis from a Single Dry Seed

A single-seed DNA extraction method was developed for rapid identification of plant genotype. The method was applied to 12 plant species, including the oil seeds sesame and soybean. The results were comparable to those obtained for oil-less seeds such as rice. This method will be useful for genotypic selection which requires rapid screening of large populations. It can also be used to identify varietal purity of seed stocks by PCR and RFLP analysis. The method includes two major steps, (i) treatment by proteinase K in an SDS extraction buffer, and (ii) grinding of a single half seed in the buffer after incubation. About 1.5–2 µg of DNA per half seed (the endosperm part) of rice was obtained and more than 200 half seed samples could be handled by one person in a day. The DNA could be used for fingerprinting and detection of target genes in a transgenic plant by PCR. The amplified PCR products from the half seed DNA exhibited the same banding patterns as those from leaf DNA. Yield and quality of DNA extracted from half seeds of rice was also sufficient for RFLP analysis. The remnant half seeds containing the embryo can be maintained for later germination of selected genotypes.     

Analytical estimation of branchwood volume in sugar maple, linked to branchiness

 An analytical link is proposed between branchwood volume and branchiness. A segmented linear model with one parameter is used to describe the branch basal area density along the tree bole and integrated to find a function describing the cumulative branch basal area. It appears that the bases of insertion of the branches defining the base of the light crown correspond to the maximum branch basal area density along the bole. This function is then used together with an individual branch volume equation to find a model that estimates branchwood volume. This model is calibrated with data gathered in 15 stands dominated by sugar maple (Acer saccharum Marsh.) in southern Quebec. A comparison is made with other models of branchwood volume found in the literature. Received: 22 August 1997 / Accepted: 9 February 1998     

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the complex between full-length proteins.     

Tricyclic dihydroquinazolinones as novel 5-HT2C selective and orally efficacious anti-obesity agents

Agonists of the 5-HT_2C receptor have been shown to suppress appetite and reduce body weight in animal models as well as in humans. However, agonism of the related 5-HT_2B receptor has been associated with valvular heart disease. Synthesis and biological evaluation of a series of novel and highly selective dihydroquinazolinone-derived 5-HT_2C agonists with no detectable agonism of the 5-HT_2B receptor is described. Among these, compounds (+)-2a and (+)-3c were identified as potent and highly selective agonists which exhibited weight loss in a rat model upon oral dosing.