Cloning of two SOS1 transporters from the seagrass <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>. SOS1 transporters from <Emphasis Type="Italic">Cymodocea</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis> mediate potassium uptake in bacteria

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na⁺-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na⁺, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na⁺ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K⁺-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K⁺. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K⁺ and Rb⁺ transporter. CnSOS1B mediated a transient, extremely rapid K⁺ or Rb⁺ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K⁺ and Rb⁺ uptake characteristics in bacteria.     

Molecular characterization the <Emphasis Type="Italic">YABBY</Emphasis> gene family in <Emphasis Type="Italic">Oryza sativa</Emphasis> and expression analysis of <Emphasis Type="Italic">OsYABBY1</Emphasis>

Members of the YABBY gene family have a general role that promotes abaxial cell fate in a model eudicot, Arabidopsis thaliana. To understand the function of YABBY genes in monocots, we have isolated all YABBY genes in Oryza sativa (rice), and revealed the spatial and temporal expression pattern of one of these genes, OsYABBY1. In rice, eight YABBY genes constitute a small gene family and are classified into four groups according to sequence similarity, exon-intron structure, and organ-specific expression patterns. OsYABBY1 shows unique spatial expression patterns that have not previously been reported for other YABBY genes, so far. OsYABBY1 is expressed in putative precursor cells of both the mestome sheath in the large vascular bundle and the abaxial sclerenchyma in the leaves. In the flower, OsYABBY1 is specifically expressed in the palea and lemma from their inception, and is confined to several cell layers of these organs in the later developmental stages. The OsYABBY1-expressing domains are closely associated with cells that subsequently differentiate into sclerenchymatous cells. These findings suggest that the function of OsYABBY1 is involved in regulating the differentiation of a few specific cell types and is unrelated to polar regulation of lateral organ development.     

Mapping of the Ogura fertility restorer gene <Emphasis Type="Italic">Rfo</Emphasis> and development of <Emphasis Type="Italic">Rfo</Emphasis> allele-specific markers in canola (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Ogura cytoplasmic male sterility (CMS) and its corresponding nuclear fertility restorer gene, Rfo, have been introduced from radish to Brassica species by interspecific crosses. Rfo restores male fertility by altering the translational expression of Orf138, a mitochondrial gene, whose expression results in the male sterile phenotype. This system has been extensively investigated and breeding restorer lines for the Ogura CMS has become a major objective for hybrid seed production in many canola breeding programs. In this study, we have sequenced genomic clones of Rfo amplified from a canola restorer line R2000, licensed from INRA, France, and a Dow AgroSciences non-restorer line Nexera 705 using primers designed from the radish Rfo sequence (GenBank accession AJ550021). Sequence alignment revealed three homologous sequences of Rfo. Two of the sequences were present in both R2000 and Nexera 705 but the third one was present only in R2000. These results suggested that the first two sequences could be the homoeologous sequences of Rfo already existing in the canola genome and the third one could be the radish Rfo introduced into canola. Based on the sequence differences between the restorer and non-restorer lines, Rfo allele-specific PCR markers were developed. We also developed a high throughput, Rfo allele-specific Invader^® assay through Third Wave Technologies. Linkage analysis revealed a co-segregation between the allele-specific marker and the phenotypes for fertility restoration. This allele-specific marker has been mapped in the linkage group N19 and proved to be very useful for direct selection of Rfo alleles for fertility restoration during marker-assisted introgression of the Ogura restorer for hybrid development in canola.     

Functional differentiation in the anterior gills of the aquatic air-breathing fish, <Emphasis Type="Italic">Trichogaster leeri</Emphasis>

The fish gill is a multifunctional organ responsible for gas exchange and ionic regulation. It is hypothesized that both morphological and functional differentiation can be found in the gills of the aquatic air-breathing fish, Trichogaster leeri. To test this, we used the air-breathing fish, Trichogaster leeri, to investigate various morphological/functional parameters. First, we evaluated the importance of performing the aquatic surface respiration behavior in T. leeri. A reduced survival rate was observed when fish were kept in the restrained cages in hypoxic conditions. On the gross anatomy of gills, we found evidence of both morphological and functional modification in the first and the second gills and are responsible for ionic regulation. There were large-bore arterioarterial shunts in the fourth gill arch. It is specialized for the transport of oxygenated blood and is less responsive to environmental stress. In addition, the anterior and the posterior gills differed in the Na⁺, K⁺-ATPase activity upon ionic stresses. That is, only the Na⁺, K⁺-ATPase activity of the anterior two gills was up-regulated significantly in the deionized water. Lastly, we found that the number of mitochondria-rich cells in the first and the second gills increased following ionic stress and no difference was found in the third and the fourth gills following such an exposure. These results supported the hypothesis that there are morphological and functional differences between anterior and posterior gill arches within the air-breathing Trichogaster leeri. In contrast, no significant difference was found among gills in gross anatomy, filament density and Na⁺, K⁺-ATPase activity in the non-air-breather, Barbodes schwanenfeldi.     

A<Emphasis Type="Italic"> Dickkopf</Emphasis>-<Emphasis Type="Italic">3</Emphasis>-related gene is expressed in differentiating nematocytes in the basal metazoan<Emphasis Type="Italic"> Hydra</Emphasis>

In vertebrate development the Dickkopf protein family carries out multiple functions and is represented by at least four different genes with distinct biological activities. In invertebrates such as Drosophila and Caenorhabditis, Dickkopf genes have so far not been identified. Here we describe the identification and characterization of a Dickkopf gene with a deduced amino acid sequence closely related to that of chicken Dkk-3 in the basal metazoan Hydra. HyDkk-3 appears to be the only Dickkopf gene in Hydra. The gene is expressed in the gastric region in nematocytes at a late differentiation stage. In silico searches of EST and genome databases indicated the absence of Dkk genes from the protostomes Drosophila and Caenorhabditis, whereas within the deuterostomes, a Dkk-3 gene could be identified in the genome of the urochordate Ciona intestinalis. The results indicate that at an early stage of evolution of multicellularity Dickkopf proteins have already played important roles as developmental signals. They also suggest that vertebrate Dkk-1, 2 and 4 may have originated from a common ancestor gene of Dkk-3.H. Fedders and R. Augustin contributed equally to this workEdited by D. Tautz     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

Oxaloacetate decarboxylase of <Emphasis Type="Italic">Vibrio cholerae</Emphasis>: purification,characterization, and expression of the genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The oxaloacetate decarboxylase (OAD) Na⁺ pump consists of subunits , , and , which are expressed from an oadGAB gene cluster present in various anaerobic bacteria. Vibrio cholerae has two copies of oad genes, which are termed oad-1 and oad-2. The oad-2 genes are part of the citrate fermentation operon, while the oad-1 genes are flanked by genes encoding products not involved in a catabolic pathway. The gene sequences of oad-1 and oad-2 of V. cholerae strain O395-N1 were determined. The apparent frameshift in the published sequence of the oadA-2 gene from V. cholerae El Tor N16961 was not present in strain O395-N1. Upon anaerobic growth of V. cholerae on citrate, exclusively the oad-2 genes are expressed. OAD was isolated from these cells by monomeric avidin–Sepharose affinity chromatography. The enzyme was of higher specific activity than that from Klebsiella pneumoniae and was significantly more stable. Decarboxylase activity was Na⁺ dependent, and the activation profile showed strong cooperativity with a Hill coefficient n_H=1.8. Oxalate and oxomalonate inhibited the enzyme with half-maximal concentrations of 10 M and 200 M, respectively. After reconstitution into proteoliposomes, the enzyme acted as a Na⁺ pump. With size-exclusion chromatography, the enzyme eluted in a symmetrical peak at a retention volume corresponding to an apparent molecular mass of approximately 570 kDa, suggesting a tetrameric structure for OAD-2. The two oad gene clusters were heterologously expressed in Escherichia coli, and the decarboxylases were isolated from the host cells.     

Disruption of a <Emphasis Type="Italic">fur</Emphasis>-like gene inhibits magnetosome formation in <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis> MSR-1

In this study, a genomic library of Magnetospirillum gryphiswaldense MSR-1 strain was constructed and a fur-like gene (encoding Fur protein, ferric uptake regulator) was isolated and sequenced. This gene consisted of 420 bp and encoded 139 amino acid residues. To investigate the function of this gene in MSR-1, a fur mutant was generated by double crossover with a kanamycin cassette inserted into its coding region. Iron uptake and magnetosome formation were dramatically inhibited by disruption of fur. Iron content analysis of the fur mutant indicated that it contained approximately 0.037% by dry weight, which was at least 10-fold less than that observed in the wild type. Electron microscopy revealed the absence of a magnetosome in the fur mutant, although it was able to tolerate 1 mM H₂O₂ at 10-fold higher level than wild-type. These data suggest that Fur protein may possess a novel function in magnetic bacteria. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 11, pp. 1532–1539.     

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.     

Stereoselective oxidation of racemic 1-arylethanols by basil cultured cells of <Emphasis Type="Italic">Ocimum </Emphasis><Emphasis Type="Italic">basilicum</Emphasis> cv. <Emphasis Type="Italic">Purpurascens</Emphasis>

The biotransformation of racemic 1-phenylethanol (30 mg) with plant cultured cells of basil (Ocimum basilicum cv. Purpurascens, 5 g wet wt) by shaking 120 rpm at 25°C for 7 days in the dark gave (R)-(+)-1-phenylethanol and acetophenone in 34 and 24% yields, respectively. The biotransformation can be applied to other 1-arylethanols and basil cells oxidized the (S)-alcohols to the corresponding ketones remaining the (R)-alcohols in excellent ee.     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

Calcium Homeostasis Change in CD4<Superscript>+</Superscript> T Lymphocytes from Human Peripheral Blood during Differentiation <Emphasis Type="Italic">in vivo</Emphasis>

Resting naive CD4⁺CD45R0^?CD45RA⁺ T cells are sensitive to ionomycin. In contrast, resting CD4⁺CD45RA^?CD45R0⁺ memory T cells show resistance to this Ca²⁺ ionophore. In the present study, the ability of activated T lymphocytes to respond to ionomycin during the transition from naive precursors into memory T cells has been analyzed. Activated CD4⁺CD45RA⁺CD45R0⁺ T cells are always present both in human peripheral blood (HPB) and in the ionomycin-resistant (IR) fraction. Therefore, some activated T cells are resistant toward the Ca²⁺ ionophore. CD69 molecules are markers of the very early stage of T cell activation. However, CD4⁺CD69⁺ T cells have never been found in the IR fraction. Thus, the majority of CD4⁺ T lymphocytes at the early stage of activation are ionomycin-sensitive cells. The proportion of CD4⁺CD25⁺ T cells did not differ significantly in HPB and in the IR fraction. The presence of CD4⁺CD25⁺ T lymphocytes in the IR fraction reflects changes in the Ca²⁺-signaling pathway at this differentiation step of activated cells. Depending on the expression level of CD25 molecules, the population of CD4⁺CD25⁺ cells is divided in T-regulatory (CD25^high) and proliferating (CD25^low) subpopulations. The action of ionomycin results in a decrease in the portion of the CD4⁺CD25^low T-cells, but it leads to an increase in the proportion of the CD4⁺CD25^high T lymphocytes. Consequently, greater portion of CD4⁺CD25^high T lymphocytes and smaller portion of CD4⁺CD25^low T cells are IR cells. Expression of HLA-DR molecules can be used as the marker for the late activation step. The IR fraction is significantly rich in CD4⁺HLA-DR⁺ T lymphocytes in comparison to the blood of the same donor. The link between different differentiation steps of CD4⁺ T-lymphocytes and alterations in calcium ion homeostasis is discussed.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.