Selective recruitment of <Emphasis Type="Italic">Adh</Emphasis> genes for distinct enzymatic functions in <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alcohol dehydrogenase (ADH) activity in plants is generally associated with glycolytic fermentation, which facilitates cell survival during episodes of low-oxygen stress in water-logged roots as well as chronically hypoxic regions surrounding the vascular core. Work with tobacco and potato has implicated ADH activity in additional metabolic roles, including aerobic fermentation, acetaldehyde detoxification and carbon reutilization. Here a combination of approaches has been used to examine tissue-specific patterns of Adh gene expression in order to provide insight into the potential roles of alcohol dehydrogenases, using Petunia hybrida, a solanaceous species with well-characterized genetics. A reporter-gene study, relying on the promoters of Adh1 and Adh2 to drive expression of the gene for a green fluorescent protein derivative, mgfp5, revealed unexpectedly complex patterns of GFP fluorescence in floral tissues, particularly the stigma, style and nectary. Results of GC-MS analysis suggest the association of ADH with production of aromatic compounds in the nectary. Overall the results demonstrate selective recruitment of Adh gene family members in tissues and organs associated with diverse ADH functions.     

Expression analysis of <Emphasis Type="Italic">Clavata1</Emphasis>-like and <Emphasis Type="Italic">Nodulin21</Emphasis>-like genes from <Emphasis Type="Italic">Pinus sylvestris</Emphasis> during ectomycorrhiza formation

The ecology and physiology of ectomycorrhizal (EcM) symbiosis with conifer trees are well documented. In comparison, however, very little is known about the molecular regulation of these associations. In an earlier study, we identified three EcM-regulated Pinus expressed sequence tags (EST), two of which were identified as homologous to the Medicago truncatula nodulin MtN21. The third EST was a homologue to the receptor-like kinase Clavata1. We have characterized the expression patterns of these genes and of auxin- and mycorrhiza-regulated genes after induction with indole-3-butyric acid in Pinus sylvestris and in a time course experiment during ectomycorrhizal initiation with the co-inoculation of 2,3,5-triiodobenzoic acid, an auxin transport inhibitor. Our results suggest that different P. sylvestris nodulin homologues are associated with diverse processes in the root. The results also suggest a potential role of the Clv1-like gene in lateral root initiation by the ectomycorrhizal fungus.     

<Emphasis Type="Italic">R-spondin1</Emphasis> and <Emphasis Type="Italic">FOXL2</Emphasis>act into two distinct cellular types during goat ovarian differentiation

Background  

Up to now, two loci have been involved in XX sex-reversal in mammals following loss-of-function mutations, PIS (Polled Intersex Syndrome) in goats and R-spondin1 (RSPO1) in humans. Here, we analyze the possible interaction between these two factors during goat gonad development. Furthermore, since functional redundancy between different R-spondins may influence gonad development, we also studied the expression patterns of RSPO2, 3 and 4.     

<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.     

BDNF promotes target innervation of <Emphasis Type="Italic">Xenopus</Emphasis> mandibular trigeminal axons <Emphasis Type="Italic">in vivo</Emphasis>

Background  

Trigeminal nerves consist of ophthalmic, maxillary, and mandibular branches that project to distinct regions of the facial epidermis. In Xenopus embryos, the mandibular branch of the trigeminal nerve extends toward and innervates the cement gland in the anterior facial epithelium. The cement gland has previously been proposed to provide a short-range chemoattractive signal to promote target innervation by mandibular trigeminal axons. Brain derived neurotrophic factor, BDNF is known to stimulate axon outgrowth and branching. The goal of this study is to determine whether BDNF functions as the proposed target recognition signal in the Xenopus cement gland.     

Differential regulation of <Emphasis Type="Italic">chlorophyll a oxygenase</Emphasis> genes in rice

Chlorophyll b is synthesized from chlorophyll a by chlorophyll a oxygenase. We have identified two genes (OsCAO1 and OsCAO2) from the rice genome that are highly homologous to previously studied chlorophyll a oxygenase (CAO) genes. They are positioned in tandem, probably resulting from recent gene duplications. The proteins they encode contain two conserved functional motifs – the Rieske Fe–sulfur coordinating center and a non-heme mononuclear Fe-binding site. OsCAO1 is induced by light and is preferentially expressed in photosynthetic tissues. Its mRNA level decreases when plants are grown in the dark. In contrast, OsCAO2 mRNA levels are higher under dark conditions, and its expression is down-regulated by exposure to light. To elucidate the physiological function of the CAO genes, we have isolated knockout mutant lines tagged by T-DNA or Tos17. Mutant plants containing a T-DNA insertion in the first intron of the OsCAO1 gene have pale green leaves, indicating chlorophyll b deficiency. We have also isolated a pale green mutant with a Tos17 insertion in that OsCAO1 gene. In contrast, OsCAO2 knockout mutant leaves do not differ significantly from the wild type. These results suggest that OsCAO1 plays a major role in chlorophyll b biosynthesis, and that OsCAO2 may function in the dark.     

Cloning of two cellobiohydrolase genes from <Emphasis Type="Italic">Trichoderma</Emphasis><Emphasis Type="Italic">viride</Emphasis> and heterogenous expression in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa. The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml⁻¹ under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70°C, respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively.     

Surface microdeformations and regulation of cell movements in <Emphasis Type="Italic">Xenopus</Emphasis> development

The velocities and directions of movements of individual outer ectodermal cells of Xenopus embryos in the course of normal development from the blastula to the early tail-bud stage, as well as after mechanical relaxation in the early gastrula, were measured. An alternation of the periods of directed movements of large cell masses and local cell wanderings was detected. In both cases, the trajectories of individual cells consisted primarily of orthogonal segments. Cell movements were measured on two scales. At a smallscale consideration (time intervals of the order of several hours and distances of the order of tens of microns), fairly slight linear stretching and compressive deformations were detected, which looked like gentle smooth gradients along which the upward morphogenetic movements of cells were directed. At a large-scale consideration (time intervals of the order of tens of minutes and distances of the order of microns), quasi-periodic fluctuations of velocities of individual cells partly correlated in time were found. The differences between these velocities generated microdeformations, which reached several tens of percent and developed within time intervals not more than 10 min. Measurements of relative magnitudes of mechanical forces influencing the cell walls suggests that microdeformations generate local stretching and compressive deformations modulating smoother tension gradients.     

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.     

<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.     

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.