Evolution of <Emphasis Type="Italic">Pleopsidium</Emphasis> (Lichenized Ascomycota) S943 Group I Introns and the Phylogeography of an Intron-Encoded Putative Homing Endonuclease

The sporadic distribution of nuclear group I introns among different fungal lineages can be explained by vertical inheritance of the introns followed by successive losses, or horizontal transfers from one lineage to another through intron homing or reverse splicing. Homing is mediated by an intron-encoded homing endonuclease (HE) and recent studies suggest that the introns and their associated HE gene (HEG) follow a recurrent cyclical model of invasion, degeneration, loss, and reinvasion. The purpose of this study was to compare this model to the evolution of HEGs found in the group I intron at position S943 of the nuclear ribosomal DNA of the lichen-forming fungus Pleopsidium. Forty-eight S943 introns were found in the 64 Pleopsidium samples from a worldwide screen, 22 of which contained a full-length HEG that encodes a putative 256-amino acid HE, and 2 contained HE pseudogenes. The HEGs are divided into two closely related types (as are the introns that encode them) that differ by 22.6% in their nucleotide sequences. The evolution of the Pleopsidium intron-HEG element shows strong evidence for a cyclical model of evolution. The intron was likely acquired twice in the genus and then transmitted via two or three interspecific horizontal transfers. Close geographical proximity plays an important role in intron-HEG horizontal transfer because most of these mobile elements were found in Europe. Once acquired in a lineage, the intron-HEG element was also vertically transmitted, and occasionally degenerated or was lost. [Reviewing Editor: Dr. Manyuan Long]     

Characterisation of the novel restriction endonuclease <Emphasis Type="Italic">Sui</Emphasis>I from <Emphasis Type="Italic">Sulfolobus islandicus</Emphasis>

A restriction endonuclease activity from Sulfolobus islandicus REN2H1 was purified by phosphocellulose and cation exchange chromatography. The enzyme cuts DNA at the recognition site GCwGC as could be shown by restriction analysis of plasmids and short synthetic duplex DNA. The cleavage occurs after the first guanosine base and is inhibited by 5-methyl-cytosine methylation. The restriction activity is salt-sensitive and has an optimal activity around 70°C.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

<Emphasis Type="Italic">Anthocyaninless1</Emphasis> gene of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a UDP-glucose:flavonoid-3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.     

Cloning and Preliminary Characterization of <Emphasis Type="Italic">lh3</Emphasis> Gene Encoding a Putative Acetyltransferase from a Rifamycin SV-Producing Strain <Emphasis Type="Italic">Amycolatopsis mediterranei</Emphasis>

An ORF located immediately downstream of glnR gene was cloned from Amycolatopsis mediterranei U32 and was named lh3. Sequence analysis revealed that lh3 encodes a putative acetyltransferase, which shows high amino acid sequence similarities to the mycothiol synthase (MshD) from other actinomycetes. For functional analysis, mutation in lh3 gene was generated by gene replacement with an apramycin resistance gene through homologous recombination. Compared with the wild type strain, the resulting mutant was more sensitive to H₂O₂, apramycin and erythromycin by two- to three-fold. These results suggest that the lh3 gene plays an important role in the course of detoxification in A. mediterranei U32.     

Analysis of <Emphasis Type="Italic">Acropora muricata</Emphasis> Calmodulin (<Emphasis Type="Italic">CaM</Emphasis>) Indicates That Scleractinian Corals Possess the Ancestral Exon/Intron Organization of the Eumetazoan <Emphasis Type="Italic">CaM</Emphasis> Gene

Calmodulin (CaM), belonging to the tropinin C (TnC) superfamily, is one of the calcium-binding proteins that are highly conserved in their protein and gene structure. Based on the structure comparison among published vertebrate and invertebrate CaM, it is proposed that the ancestral form of eumetazoan CaM genes should have five exons and four introns (four-intron hypothesis). In this study, we determined the gene structure of CaM in the coral Acropora muricata, an anthozoan cnidarian representing the basal position in animal evolution. A CaM clone was isolated from a cDNA library constructed from the spawned eggs of A. muricata. This clone was composed of 908 nucleotides, including 162 base pairs (bp) of 5′-untranslated region (UTR), 296 bp of 3′-UTR, and an open reading frame 450 bp in length. The deduced amino acid indicated that the Acropora CaM protein is identical to that of the actiniarian, Metridinium senile, and has four putative calcium-binding domains highly similar to those of other vertebrate or invertebrate CaMs. Southern blot analysis revealed that Acropora CaM is a putative single-copy gene in the nuclear genome. Genomic sequencing showed that Acropora CaM was composed of five exons and four introns, with intron II not corresponding to any region in the actiniarian CaM gene, which possesses only four exons and three introns. Our results highlight that the coral CaM gene isolated from A. muricata has four introns at the predicted positions of the early metazoan CaM gene organization, providing the first evidence from the basal eumetazoan phylum to support the four-intron hypothesis.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

<Emphasis Type="Italic">Funastrum rupicola</Emphasis> (<Emphasis Type="Italic">Apocynaceae:</Emphasis><Emphasis Type="Italic">Asclepiadoideae</Emphasis>), a new species from Bolivia

Summary   Funastrum rupicola Goyder, a new species of Apocynaceae: Asclepiadoideae from Bolivia, is described and illustrated. The conservation status of this species is assessed.     

Impact of <Emphasis Type="Italic">cry1AC</Emphasis>-carrying <Emphasis Type="Italic">Brassica rapa</Emphasis> subsp. <Emphasis Type="Italic">pekinensis</Emphasis> on leaf bacterial community

The effects of Chinese cabbage (Brassica rapa subsp. pekinensis) carrying cry1AC derived from Bacillus thuringiensis (Bt) on leaf bacterial community were examined by analyzing the horizontal transfer of trans-gene fragments from plants to bacteria. The effect of plant pathogenic bacteria on the gene transfer was also examined using Pseudomonas syringae pathovar. maculicola. The frequency of hygromycin-resistant bacteria did not alter in Bt leaves, though slight increase was observed in Pseudomonas-infected Bt leaves with no statistical significance. The analysis of bacterial community profiles using the denaturing gradient gel electrophoresis (DGGE) fingerprinting indicated that there were slight differences between Bt and control Chinese cabbage, and also that infected tissues were dominated by P. syringae pv. maculicola. However, the cultured bacterial pools were not found to contain any transgene fragments. Thus, no direct evidence of immediate gene transfer from plant to bacteria or acquisition of hygromycin resistance could be observed. Still, long-term monitoring on the possibility of gene transfer is necessary to correctly assess the environmental effects of the Bt crop on bacteria.