<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Massive genetic introgression in threatened northern crested newts (<Emphasis Type="Italic">Triturus cristatus</Emphasis>) by an invasive congener (<Emphasis Type="Italic">T. carnifex</Emphasis>) in Western Switzerland

Genetic pollution through introgressive hybridization of local species by exotic relatives is a major, yet neglected aspect of biological invasions, particularly in amphibians where human introductions are frequent. In Western Switzerland, crested newts make an interesting case: the Italian species Triturus carnifex was introduced at least a century ago within the range of the native and threatened T. cristatus. To understand the genetic consequences of this introduction and inform wildlife management authorities, we conducted a genetic survey on the remaining northern crested newt populations known in the area, using newly-developed species-diagnostic nuclear (microsatellites) and mitochondrial (control region) DNA markers. We documented massive nuclear introgression by the T. carnifex genome, which has completely replaced T. cristatus in most populations, especially in the Geneva area where the introduction was originally reported. However, many of these individuals retained the ancestral T. cristatus mtDNA, which could be explained by asymmetric introgression between the two species, stemming from demographic and/or selective processes. Analyses of genetic diversity support multiple events of T. carnifex releases, most-likely of proximate North Italian origin. We pinpointed the last indigenous populations in the region and recommend to prioritize their protection. Our study demonstrates the invasive potential of introduced taxa through introgressive hybridization, alerts about the underestimated rate of illegal amphibian translocations, and emphasizes the need for genetic analyses to monitor such invasions.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Isolation and characterization of the <Emphasis Type="Italic">Larix gmelinii </Emphasis><Emphasis Type="Italic">ANGUSTIFOLIA</Emphasis> (<Emphasis Type="Italic">LgAN</Emphasis>) gene

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.     

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.     

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.     

Analysis of alien introgression in coffee tree (<Emphasis Type="Italic">Coffea</Emphasis><Emphasis Type="Italic">arabica</Emphasis> L.)

The transfer of desired traits from related wild diploid Coffea species into the cultivated allotetraploid C. arabica is essential in coffee breeding to develop pest/disease-resistant cultivars. The present work is an attempt to gain insights into alien introgression in C. arabica. An F2 population derived from a cross between T5296 and Et6 was analysed with simple sequence repeat (SSR; microsatellite) and amplified fragment length polymorphism (AFLP) molecular markers. The T5296 is a derivative of an interspecific hybrid introgressed by the diploid C. canephora species and Et6 is a wild Ethiopian accession of C. arabica. The origin of the revealed polymorphism was determined by comparisons using representative accessions from C. arabica and its two diploid parental species, C. eugenioides and C. canephora. The number and mode of inheritance of canephora-introgressed segments were investigated, as well as their sub-genome localisation and rate of recombination. The results suggested that the transfer of desirable genes into C. arabica from C. canephora is not limited by the ploidy level differences or the suppression of recombination between the different genomes.     

<Emphasis Type="Italic">Rhodocollybia</Emphasis> in neotropical montane forests

Traditional concepts of Collybia (Agaricales) have included a variety of morphologically disparate taxa. Reapplication of some characters used in circumscribing Collybia s.l. suggests realignment of well-known North American and European species into three genera, viz. Collybia s.s, Rhodocollybia, and Gymnopus. Rhodocollybia is recognized here at generic rank based on morphological characteristics and phylogenetic signaling. Seven new species epithets under Rhodocollybia, and a new combination under Gymnopus, are proposed. A key to species of Rhodocollybia found in neotropical montane forests is provided.     

The <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>/<Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic"> cjaA</Emphasis> (<Emphasis Type="Italic">cj0982c</Emphasis>) Gene Encodes an N-Glycosylated Lipoprotein Localized in the Inner Membrane

The Campylobacter coli 72Dz/92 cjaA gene (orthologue of cj0982c of C. jejuni NCTC 11168) product is a highly immunogenic, amino acid-binding protein. CjaA was palmitic acid-modified when processed in E. coli. In addition, site-directed mutagenesis of the Cys residue of the LAAC motif of its signal sequence confirmed that CjaA is a lipoprotein when processed in Campylobacter. Localization of the protein appeared to be host dependent. In Campylobacter, CjaA was recovered mainly as an inner-membrane protein, whereas in E. coli most of the protein was present in the periplasmic space. Interestingly, antiserum raised against Campylobacter glycine-extracted material also recognized CjaA produced by Campylobacter and Escherichia coli, indicating that at least part of the protein may be surface exposed. Site-directed mutagenesis of the Asn residues of two putative N-linked glycosylation sites (NIS and NFT) showed that CjaA is glycosylated and that only the first N-X-S/T sequeon serves as a glycan acceptor.     

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.     

Production of transgenic <Emphasis Type="Italic">Pinus armandii</Emphasis> plants harbouring <Emphasis Type="Italic">btCry</Emphasis>III(<Emphasis Type="Italic">A</Emphasis>) gene

A synthetic chimeric gene SbtCryIII(A) encoding the insecticidal protein btCryIII(A), was transformed into Pinus armandii embryos and embryogenic calli using Agrobacterium tumefaciens. Polymerase chain reaction and genomic DNA Southern blot analysis showed that the SbtCryIII(A) gene was integrated into the genome of transgenic Pinus armandii plants, and Northern blot analysis indicated that the SbtCryIII(A) gene was transcribed.     

Three new species of <Emphasis Type="Italic">Stigmidium</Emphasis> and <Emphasis Type="Italic">Sphaerellothecium</Emphasis> (lichenicolous ascomycetes) on <Emphasis Type="Italic">Stereocaulon</Emphasis>

Sphaerellothecium stereocaulorum sp. nov., Stigmidium beringicum sp. nov., Stigmidium stereocaulorum sp. nov. and goniocysts are described on Stereocaulon species from the northern Holarctic. Endococcus nanellus is reported new to Alaska and Mongolia. Taxonomical novelties Sphaerellothecium stereocaulorum Zhurb. & Triebel, Stigmidium beringicum Zhurb. & Triebel, Stigmidium stereocaulorum Zhurb. & Triebel.     

<Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> an environmental host for <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> and <Emphasis Type="Italic">Shigella sonnei</Emphasis>

The interaction between Shigella dysenteriae or Shigella sonnei and Acanthamoeba castellanii was studied by viable counts, gentamicin assay and electron microscopy. The result showed that Shigella dysenteriae or Shigella sonnei grew and survived in the presence of amoebae for more than 3 weeks. Gentamicin assay showed that the Shigella were viable inside the Acanthamoeba castellanii which was confirmed by electron microscopy that showed the Shigella localized in the cytoplasm of the Acanthamoeba castellanii. In conclusion, the relationship between Shigella dysenteriae and Shigella sonnei with Acanthamoeba castellanii is symbiotic, and accordingly free-living amoebae may serve as a transmission reservoir for Shigella in water.     

Linkage of <Emphasis Type="Italic">Patr-AL</Emphasis> to <Emphasis Type="Italic">Patr-A</Emphasis> and-<Emphasis Type="Italic">B</Emphasis> in the major histocompatibility complex of the common chimpanzee (<Emphasis Type="Italic">Pan troglodytes</Emphasis>)

Patr-AL is a recently described gene found only in the common chimpanzee, but closely related in structure to the highly polymorphic Patr-A and HLA-A genes of the chimpanzee and human MHCs, respectively. Unlike Patr-A and HLA-A, the Patr-AL gene has little polymorphism and is not fixed in the chimpanzee genome. To determine whether Patr-AL is located in the MHC or elsewhere, we compared segregation of the Patr-AL gene with segregation of Patr-A and - B alleles in chimpanzee families. The results demonstrate that Patr-AL is an MHC class I gene present on different MHC haplotypes as defined by their combination of Patr-A and B alleles.