Structure of cDNA clones for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from a fern (Asplenium cataractarum rosenstock,aspleniaceae), and its comparison with those of various seed plants

We isolated the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO SSu) from a fern,Asplenium cataractarum and determined its 34 N-terminal amino acid sequence. We obtained a cDNA clone that contains the entire coding region of the SSu from the same fern species, using synthetic oligonucleotide probes derived from the above amino acid sequence. It contains a 525 bp open reading frame capable of coding for a polypeptide with 174 amino acids, 31 bp 5′-and 206 bp 3′-noncoding regions. It was also elucidated that the precursor to the SSu contains a transit peptide of 53 amino acid residues and a mature protein of 121 residues. We compared the deduced amino acid sequence of the fern SSu with those of 11 other vascular plant species (including gymnosperms, monocots and dicots). As low as 55% homology was observed between those of a fern and seed plants. Constancy of the amino acid substitution rate in RuBisCO SSu was supported by our relative rate test. Amino acid substitution rate per year per site for RuBisCO SSu was calculated to be 0.81×10⁻⁹ assuming that the separation between pteridophytes and seed plants arose 380 million years ago.     

High-performance liquid chromatographic determination of short-chain aliphatic aldehydes using 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole as a fluorescence reagent

High-performance liquid chromatographic determination of four short-chain aliphatic aldehydes using fluorescence detection was carried out with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). DBD-H derivatives with three aliphatic aldehydes — formaldehyde, acetaldehyde and propionaldehyde — were synthesized and their fluorescence properties were examined. Relative fluorescence intensities of these compounds in acetonitrile were ca. ten-fold larger than those in aqueous acetonitrile. DBD-hydrazones could be separated by reversed-phase chromatography using aqueous acetonitrile as eluent and detection at 560 nm with excitation at 445 nm. Submicromolar levels of formaldehyde, acetaldehyde, propionaldehyde and butylaldehyde could be determined. The HPLC procedure using propionaldehyde as internal standard was applied to the measurement of acetaldehyde levels in normal human plasma before and 30 min after ingestion of ethanol.     

Chemotaxis away from thiocyanic and isothiocyanic esters in Pseudomonas aeruginosa

Abstract A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae.     

Cloning and nucleotide sequence of fosfomycin biosynthetic genes of Streptomyces wedmorensis

The biosynthetic pathway for production of the antibiotic fosfomycin by Streptomyces wedmorensis consists of four steps including the formation of a C-P bond and an epoxide. Fosfomycin production genes were cloned from genomic DNA using S. wedmorensis mutants blocked at different steps of the biosynthetic pathway. Four genes corresponding to each of the biosynthetic steps were found to be clustered in a DNA fragment of about 5 kb. Nucleotide sequencing of a large fragment revealed the presence of ten open reading frames, including the four biosynthetic genes and six genes with unknown functions.     

Gravitropic reaction of primary seminal roots ofZea mays L. influenced by temperature and soil water potential

The growth of the primary seminal root of maize (Zea mays L.) is characterized by an initial negative gravitropic reaction and a later positive one that attains a plagiotropic liminal angle. The effects of temperature and water potential of the surrounding soil on these gravitropic reactions were studied. Temperatures of 32, 25, and 18C and soil water potentials of −5,−38, and −67 kPa were imposed and the direction of growth was measured for every 1 cm length of the root. The initial negative gravitropic reaction extended to a distance of about 10cm from the graln. Higher temperatures reduced the initial negative gravitropic reaction. Lower soil water potential induced a downward growth at root emergence. A mathematical model, in which it was assumed that the rate of the directional change of root growth was a sum of a time-dependent negative gravitropic reaction and an establishment of the liminal angle, adequately fitted the distance-angle relations. It was suggested that higher temperatures and/or a lower water potential accelerated the diminution of the intitial negative gravitropic reaction.     

Development and characterization of 21 polymorphic microsatellite loci in the aphidophagous gall midge, <Emphasis Type="Italic">Aphidoletes aphidimyza</Emphasis> (Diptera: Cecidomyiidae)

We have developed and characterized 21 microsatellite markers in the aphidophagous gall midge Aphidoletes aphidimyza (Rondani) (Diptera: Cecidomyiidae). All 21 loci tested were polymorphic: the number of alleles ranged from 2 to 17. Allelic richness and observed heterozygosities were higher in females than in males. Several loci had no heterozygosity in males, suggesting that the loci were located on sex chromosomes or E-chromosomes, common to cecidomyiids. The high polymorphism detected in this study suggests the markers will be of value in analyzing genetic structure of field populations.     

Hydrophobic Components in Delipidated Granule of Egg Yolk

Dimethyl sulfide (DMS) is volatile compound important as one of the characteristic flavor compounds of marine food products. The precursor of DMS in marine products is dimethyl-β- propiothetin (DMPT), which is abundunt in green algae. DMPT was effectively extracted by the use of hydrophilic solvents from dried hitoegusa (Monostroma nitidum), a green alga. At around pH 4 and at over pH 9, the extracted DMPT was rapidly degraded to DMS; at around pH 7.5, this degradation was much slower. The DMS obtained volatilized immediately from aqueous solution. However, when the DMPT was formed into a powder with dextrin and heated to release the DMS, 40 — 60% of the DMS remained in the powder. The amount of DMS remaining was 80 % when cyclodextrin was used to form the powder.     

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio‐lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1‐1 and 1‐2). Both PpAN1 genes complemented the A. thaliana an‐1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1‐promoter– uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1‐1 and PpAN1‐2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip‐growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α‐tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1‐1/1‐2 double‐knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.     

Direct and Indirect Control of the Initiation of Meiotic Recombination by DNA Damage Checkpoint Mechanisms in Budding Yeast

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.