Interaction between uranium and the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>3</Subscript> of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis> strain G20

Cytochrome c(3) of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c(3) in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c(3) ( cycA) to lacZ. Instead, cytochrome c(3) protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c(3) with U(IV) was interpreted to be non-specific, since pure cytochrome c(3) adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe(2)O(3)), and commercially available U(IV) oxide.     

Two <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> (<Emphasis Type="Italic">FT</Emphasis>) homologs in <Emphasis Type="Italic">Chenopodium rubrum</Emphasis> differ in expression patterns

FLOWERING LOCUS T (FT) like genes are crucial regulators (both positive and negative) of flowering in angiosperms. We identified two FT homologs in Chenopodium rubrum, a short-day species used as a model plant for the studies of photoperiodic flower induction. We found that CrFTL1 gene was highly inducible by a 12-h dark period, which in turn induced flowering. On the other hand, photoperiodic treatments that did not induce flowering (short dark periods, or a permissive darkness interrupted by a night break) caused only a slight increase in CrFTL1 mRNA level. We demonstrated diurnal oscillation of CrFTL1 expression with peaks in the middle of a light period. The oscillation persisted under constant darkness. Unlike FT homologs in rice and Pharbitis, the CrFTL1 expression under constant darkness was very low. The CrFTL2 gene showed constitutive expression. We suggest that the CrFTL1 gene may play a role as a floral regulator, but the function of CrFTL2 remains unknown.     

Complete hydrolysis of <Emphasis Type="Italic">myo</Emphasis>-inositol hexakisphosphate by a novel phytase from <Emphasis Type="Italic">Debaryomyces castellii</Emphasis> CBS 2923

Debaryomyces castellii phytase was purified to homogeneity in a single step by hydrophobic interaction chromatography. Its molecular mass is 74 kDa with 28.8% glycosylation. Its activity was optimal at 60°C and pH 4.0. The K _m value for sodium phytate was 0.532 mM. The enzyme exhibited a low specificity and hydrolyzed many phosphate esters. The phytase fully hydrolyzed myo-inositol hexakisphosphate (or phytic acid, Ins P₆) to inositol and inorganic phosphate. The sequence of Ins P₆ hydrolysis was determined by combining results from high-performance ionic chromatography and nuclear magnetic resonance. D. castellii phytase is a 3-phytase that sequentially releases phosphate groups through Ins (1,2,4,5,6) P₅, Ins (1,2,5,6) P₄, Ins (1,2,6) P₃, Ins (1,2) P₂, Ins (1 or 2) P₁, and inositol (notation 3/4/5/6/1 or 2).     

<Emphasis Type="Italic">kitb</Emphasis>, a second zebrafish ortholog of mouse <Emphasis Type="Italic">Kit</Emphasis>

The large numbers of duplicated pairs of genes in zebrafish compared to their mammalian counterparts has lead to the notion that expression of zebrafish co-orthologous pairs in some cases can together describe the expression of their mammalian counterpart. Here, we explore this notion by identification and analysis of a second zebrafish ortholog of the mammalian Kit receptor tyrosine kinase (kitb). We show that in embryos, kitb is expressed in a non-overlapping pattern to that of kita, in the anterior ventral mesoderm, Rohon-beardRohon–Beard neurons, the otic vesicle, and trigeminal ganglia. The expression pattern of kita and kitb in zebrafish together approximates that of Kit in mouse, with the exception that neither zebrafish kit gene is expressed in primordial germ cells, a site of kit expression in the mouse embryo. In addition, zebrafish kita is expressed in a site of zebrafish primitive hematopoiesis but not required for blood development, and we fail to detect kitb expression in sites of zebrafish hematopoiesis. Thus, the expression and function of zebrafish kit genes cannot be described as a simple partition of the expression and function of mouse Kit. We discuss the possibility that these unaccounted for expression domains and functions are derived from more ancestral gene duplications and partitioning instead of the relatively recent teleost teleost-specific duplication. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Expression patterns of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">dpp</Emphasis> in the gastropod <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

We isolated the full-length cDNAs of engrailed and dpp-BMP2/4 orthologues from the pond snail Lymnaea stagnalis and examined their expression patterns during development by the whole mount in situ hybridization. At the gastrula and trochophore stages, engrailed is expressed in the peripheral ectoderm of the presumptive and invaginating shell gland, corroborating its role in the shell formation that is widely conserved among molluscs. At the same stages, dpp-BMP2/4 is expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum. Unlike in the gastropod Patella vulgata, our results suggested that dpp-BMP2/4 has a role in the shell formation, rather than in the regional specification and that it could be involved in the specification pathway of the left–right asymmetry of the developing shell in L. stagnalis.     

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.     

Nucleosome transactions on the<Emphasis Type="Italic"> Hypocrea jecorina</Emphasis> (<Emphasis Type="Italic"> Trichoderma reesei</Emphasis>) cellulase promoter<Emphasis Type="Italic"> cbh2</Emphasis> associated with cellulase induction

The 5 regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes –1 and –2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome –1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5 regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing conditions by repositioning nucleosome –1.Communicated by C. A. M. J. J. van den Hondel     

<Emphasis Type="Italic">smyd1</Emphasis> and <Emphasis Type="Italic">smyd2</Emphasis> are expressed in muscle tissue in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Epigenetic modifications of histone play important roles for regulation of cell activity, such as cell division, cell death, and cell differentiation. A SET domain consisting of about 130 amino acids has lysine methyltransferase activity in the presence of the cosubstrate S-adenosyl-methionine. More than 60 SET domain-containing proteins have been predicted in various organisms. One of them, the SMYD family genes which contain a SET domain and a zinc-finger MYND domain are reported to regulate cell cycle and muscle formation. Here we examined the expression and function of smyd1 and 2 in Xenopus. smyd1 and 2 were expressed in various muscle tissues. While smyd1 expression was observed mainly in cardiac muscle and skeletal muscle, smyd2 expression was done abundantly in skeletal muscle and face region. Moreover, by loss-of-function experiments using antisense morpholino oligonucleotides, it was suggested that smyd1 and 2 related to muscle cells differentiation.     

<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.     

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.