Nuclear Localization Signals Enhance Germline Transmission of a Transgene in Zebrafish

We report that cytoplasmic injection into zebrafish eggs of 104 copies of plasmid DNA complexed to nuclear localization signal (NLS) peptides, as compared to 106 copies of naked DNA, increased nuclear uptake of transgene DNA early during embryo development and enhanced transgene integration frequency into the germline of founders. Monitoring the dynamics of nuclear uptake of DNA-NLS complexes by fluorescence in situ hybridization (FISH) of interphase nuclei indicates that NLS enhances both the proportion of nuclei importing DNA during early embryo development, and the amount of DNA imported by individual nuclei. The use of NLS increases the proportion of germline transgenic founders from 14 to 43% (P < 0.01) as assessed by polymerase chain reaction analysis of F1s. From germline transgenic DNA-NLS-injected founders, 47% transgenic F1s are obtained in wild-type crosses, as opposed to 6% from naked DNA-injec...     

Increasing the variability of Lycopersicon peruvianum Mill. by protoplast fusion with Petunia hybrida L.

Somatic hybridization of Lycopersicon peruvianum and Petunia hybrida was carried out to transfer cytoplasmic male sterility from Petunia to Lycopersicon. Cytological, morphological and biochemical analyses were performed to characterize the regenerated plants. Two regenerated plants, R₃ and R₆, were male sterile. R₃ possessed chromosomes morphologically similar to those of both parental types. Leaf morphologies of these two plants and a third plant, R₇, were intermediate between the two parents. The stability of RUBPCase was verified during parental plant development and after in vitro culture. Plant R₇ presented a new form of the large subunit of RUBPCase.     

Ability of the endangered species Papaver fauriei to produce hybrids with a cultivated poppy (Papaver sp.)

Papaver fauriei is an endemic and endangered species that grows only on the gravelly alpine slopes of Mt. Rishiri, Japan. Cultivated poppy (Papaver sp.), the species name of which is unknown, has been introduced to the natural habitats of P. fauriei through human activities. Because the appearance and internal transcribed spacer (ITS) sequences of these two poppies are highly similar, it is of concern that they could produce hybrids in their natural habitats. Thus, first, the ability of these two poppies to produce hybrids was analyzed by artificial fertilization in this study. A large number of seeds were produced by reciprocal crosses between P. fauriei and the cultivated poppy, comparable with the number of seeds obtained by self‐ or cross‐fertilization of P. fauriei or the cultivated poppy. In addition, high germination was observed for seeds obtained from crosses between the two poppies, and deleterious phenotypes, such as albinism and dwarfism, were not detected in the F₁ generation. These results indicate that after pollination, there is no reproductive isolation between the two poppies. Second, we sequenced the internal transcribed spacer (ITS) region of 240 poppy individuals collected from the gravelly alpine slopes of Mt. Rishiri, and 66 showed the sequence of P. fauriei, whereas 174 showed the sequence of the cultivated poppy. However, the ITS sequence that confirms hybridism between the two poppies was not detected in these individuals, indicating that hybridization of P. fauriei and the cultivated poppy rarely occurs under natural conditions. Unknown mechanism(s) appear to prevent cross‐pollination between the two poppies.     

A reassessment of explanations for discordant introgressions of mitochondrial and nuclear genomes

Hybridization is increasingly recognized as a significant evolutionary process, in particular because it can lead to introgression of genes from one species to another. A striking pattern of discordance in the amount of introgression between mitochondrial and nuclear markers exists such that substantial mitochondrial introgression is often found in combination with no or little nuclear introgression. Multiple mechanisms have been proposed to explain this discordance, including positive selection for introgressing mitochondrial variants, several types of sex‐biases, drift, negative selection against introgression in the nuclear genome, and spatial expansion. Most of these hypotheses are verbal, and have not been quantitatively evaluated so far. We use individual‐based, multilocus, computer simulations of secondary contact under a wide range of demographic and genetic scenarios to evaluate the ability of the different mechanisms to produce discordant introgression. Sex‐biases and spatial expansions fail to produce substantial mito‐nuclear discordance. Drift and nuclear selection can produce strong discordance, but only under a limited range of conditions. In contrast, selection on the mitochondrial genome produces strong discordance, particularly when dispersal rates are low. However, commonly used statistical tests have little power to detect this selection. Altogether, these results dismiss several popular hypotheses, and provide support for adaptive mitochondrial introgression.     

In situ hybridization of corticotropin-releasing factor-encoding messenger RNA in the hypothalamus of the white sucker,Catostomus commersoni

Summary In situ hybridization procedure with a ³²P-labelled synthetic oligonucleotide probe was used to detect corticotropin-releasing factor-encoding messenger RNA (CRF mRNA) in the hypothalamus of the white sucker, Catostomus commersoni. Adjacent sections were immunostained by a sucker CRF-specific antiserum. CRF mRNA-containing neurons were identified by autoradiography in the magnocellular and parvocellular subdivisions of the preoptic nucleus (PON). Many of these neurons were also immunostained by sucker antiserum, showing the same distribution patterns. These results confirm the presence of CRF mRNA and CRF peptide in the white sucker hypothalamus and support the view that the magnocellular and parvocellular neurons of the PON may be involved in the control of adrenocorticotropic hormone secretion from the pituitary in the white sucker.     

Mercury-induced genes in Arabidopsis thaliana: identification of induced genes upon long-term mercuric ion exposure

Mercuric‐ion‐induced gene expression was studied in Arabidopsis thaliana Columbia wild type. Rosettes of plants grown for 21 d on agar medium supplemented with 20, 30 and 40 µm HgCl₂ were pooled and used to isolate cDNAs of induced genes by suppression subtractive hybridization. Of the 576 clones isolated initially, 31 turned out to be mercury‐induced by Northern hybridization. However, kinetic studies using cDNA arrays clearly showed that seven genes were exclusively mercuric‐ion‐induced, 14 were induced by mercury but also affected by a diurnal rhythm, and 10 clones were only modulated by the day–night cycle. The expression levels of the metal‐induced genes increased from 1·5‐fold to 10‐fold. Functional classification resulted in genes encoding proteins for the photosynthetic apparatus and for the antioxidative system. In addition, unexpected genes, whose connection to mercury ion stress is not evident, were identified.     

The anammox case-a new experimental manifesto for microbiological eco-physiology

The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional ‘cultivation based techniques’ alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers. This revised version was published online in August 2006 with corrections to the Cover Date.