Occurrence of Potential Brassinosteroid Precursor Steroids in Seeds of Wheat and Foxtail Millet

Triticum aestivum L.) and foxtail millet (Setaria italica Beauv.) were found by GC-MS to contain, in addition to bulk sterols, 4-en-3-one steroids including 24-ethylcholesta-4,24(28)Z- dien-3-one (a new steroid), 24-methylcholest-4-en-3-one, 24-ethylcholesta-4,22E-dien-3-one and 24-ethylcholest-4-en-3-one, as well as 5α-steroidal 3-one compounds including 24-methyl-5α-cholestan-3-one, 24-ethyl-5α-cholestan-3-one and 24-ethyl 5α-cholest-22E-en-3-one (in S. italica only). Analysis of free sterol and steryl ester fractions indicated that campestanol and sitostanol were present at high levels in both seeds. These results suggest that the seeds of T. aestivum and S. italica synthesize campestanol from campesterol via 24-methylcholest-4-en-3-one and 24-methyl-5α-cholestan-3-one as has already been demonstrated in Arabidopsis thaliana L., and also produce sitostanol from sitosterol via 24-ethylcholest-4-en-3-one and 24-ethyl-5α-chotestan-3-one. Biosynthetic relationships of campestanol and sitostanol with C₂₈ and C₂₉ brassinosteroids are discussed. Received 4 September 1998/ Accepted in revised form 26 November 1998     

Isolation and characterization of sex chromosome rearrangements generating male muscle dystrophy and female abnormal oogenesis in the silkworm, Bombyx mori

In deletion-mapping of W-specific RAPD (W-RAPD) markers and putative female determinant gene (Fem), we used X-ray irradiation to break the translocation-carrying W chromosome (W ^Ze ). We succeeded in obtaining a fragment of the W ^Ze chromosome designated as Ze ^W, having 3 of 12 W-RAPD markers (W-Bonsai, W-Yukemuri-S, W-Yukemuri-L). Inheritance of the Ze ^W fragment by males indicates that it does not include the Fem gene. On the basis of these results, we determined the relative positions of W-Yukemuri-S and W-Yukemuri-L, and we narrowed down the region where Fem gene is located. In addition to the Ze ^W fragment, the Z chromosome was also broken into a large fragment (Z₁) having the + ^sch (1-21.5) and a small fragment (Z₂) having the + ^od (1-49.6). Moreover, a new chromosomal fragment (Ze ^WZ₂) was generated by a fusion event between the Ze ^W and the Z₂ fragments. We analyzed the genetic behavior of the Z₁ fragment and the Ze ^WZ₂ fragment during male (Z/Z₁ Ze ^WZ₂) and female (Z₁ Ze ^WZ₂/W) meiosis using phenotypic markers. It was observed that the Z₁ fragment and the Z or the W chromosomes separate without fail. On the other hand, non-disjunction between the Ze ^WZ₂ fragment and the Z chromosome and also between the Ze ^WZ₂ fragment and the W chromosome occurred. Furthermore, the females (2A: Z/Ze ^WZ₂/W) and males (2A: Z/Z₁) resulting from non-disjunction between the Ze ^WZ₂ fragment and the W chromosome had phenotypic defects: namely, females exhibited abnormal oogenesis and males were flapless due to abnormal indirect flight muscle structure. These results suggest that Z₂ region of the Z chromosome contains dose-sensitive gene(s), which are involved in oogenesis and indirect flight muscle development.     

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.     

Stable nuclear transformation of the Closterium peracerosum-strigosum-littorale complex

Although charophycean algae form a relevant monophyly with embryophytes and hence occupy a fundamental place in the development of Streptophyta, no tools for genetic transformation in these organisms have been developed. Here we present the first stable nuclear transformation system for the unicellular Zygnematales, the Closterium peracerosum-strigosum-littorale complex (C. psl complex), which is one of the most useful organisms for experimental research on charophycean algae. When a vector, pSA106, containing the dominant selectable marker ble (phleomycin-resistant) gene and a reporter cgfp (Chlamydomonas-adapted green fluorescent protein) gene was introduced into cells via particle bombardment, a total of 19 phleomycin-resistant cells were obtained in the presence of a low concentration of phleomycin. Six isogenic strains isolated using conditioned medium showed consecutive cgfp expression and long-term stability for phleomycin resistance. DNA analyses verified single or tandem/redundant integration of ~10 copies of pSA106 into the C. psl complex genome. We also constructed an overexpression vector, pSA1102, and then integrated a CpPI gene encoding minus-specific sex pheromone into pSA1102. Ectopic overexpression of CpPI and the pheromonal function were confirmed when the vector pSA1102_CpPI was introduced into mt(+) cells. The present efficient transformation system for the C. psl complex should provide not only a basis for molecular investigation of Closterium but also an insight into important processes in early development and evolution of Streptophyta.     

Cryptic assemblages in coral-rubble interstices along a terrestrial-sediment gradient

The assemblage composition of cryptic mobile animals inhabiting coral rubble was sampled using mesh traps containing clean coral rubble, and used as indicators of land-based pollution at 14 sites in three coral lagoons at Ishigaki Island, southern Japan. Cluster analyses identified three groups of large mobile animal assemblages (molluscs, echinoderms, fishes, decapod and stomatopod crustaceans). Using a distance-based redundancy analysis (db-RDA) there was a significant relationship between the assemblage composition and environmental variables. The 1st axis of the db-RDA ordination was regarded as the land-based pollution gradient because of the strong relationship with silicate sediment, turbidity, and salinity, indicating effects of terrestrial-sediment runoff. Species response curves were derived from a plot of the number of individuals against the 1st axis of the db-RDA sites sample score. The response curves of Galathea mauritiana, an indicator species for the intermediate sites, were unimodal along the land-based pollution gradient. This study demonstrates the use of traps containing clean coral rubble for nondestructive quantitative sampling and environmental monitoring in coral lagoons, and their potential for monitoring changes in the reef environment. Communicated by Environmental Editor Prof. Rob van Woesik.     

A novel phosphatidylcholine which contains pentadecanoic acid at sn-1 and docosahexaenoic acid at sn-2 in Schizochytrium sp. F26-b

Docosahexaenoic acid (DHA, 22:6n-3)-containing phospholipids are a ubiquitous component of the central nervous system and retina, however their physiological and pharmacological functions have not been fully elucidated. Here, we report a novel DHA-containing phosphatidylcholine (PC) in a marine single cell eukaryote, Schizochytrium sp. F26-b. Interestingly, 31.8% of all the fatty acid in F26-b is DHA, which is incorporated into triacylglycerols and various phospholipids. In phospholipids, DHA was found to make up about 50% of total fatty acid. To identify phospholipid species containing DHA, the fraction of phospholipids from strain F26-b was subjected to normal phase high-performance liquid chromatography (HPLC). It was found that DHA was incorporated into PC, lyso-PC, phosphatidylethanolamine, and phosphatidylinositol. The major DHA-containing phospholipid was PC in which 32.5% of the fatty acid was DHA. The structure of PC was analyzed further by phospholipase A2 treatment, fast atom bombardment mass spectrometry, and 1H- and 13C-NMR after purification of the PC with reverse phase HPLC. Collectively, it was clarified that the major PC contains pentadecanoic acid (C15:0) at sn-1 and DHA at sn-2; the systematic name of this novel PC is therefore "1-pentadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine."     

Escape of leukemia blasts from HLA-specific CTL pressure in a recipient of HLA one locus-mismatched bone marrow transplantation

A case of leukemia escape from an HLA-specific cytotoxic T lymphocyte (CTL) response in a recipient of bone marrow transplantation is presented. Only the expression of HLA-B51, which was a mismatched HLA locus in the graft-versus-host direction, was down-regulated in post-transplant leukemia blasts compared with that in pre-transplant blasts. All CTL clones, that were isolated from the recipient's blood when acute graft-versus-host disease developed, recognized the mismatched B(?)51:01 molecule in a peptide-dependent manner. The pre-transplant leukemia blasts were lysed by CTL clones, whereas the post-transplant leukemia blasts were not lysed by any CTL clones. The IFN-γ ELISPOT assay revealed that B(?)51:01-reactive T lymphocytes accounted for the majority of the total alloreactive T lymphocytes in the blood just before leukemia relapse. These data suggest that immune escape of leukemia blasts from CTL pressure toward a certain HLA molecule can lead to clinical relapse after bone marrow transplantation.     

Aligned genetic linkage maps of apple rootstock cultivar ‘JM7’ and Malus sieboldii ‘Sanashi 63’ constructed with novel EST-SSRs

Identification of markers associated with genes of interest and quantitative trait loci (QTLs), combined with high-density genetic linkage maps, can help reduce labor and costs by enabling marker-assisted selection (MAS). In this study, a dwarfing apple rootstock cultivar ??JM7?? (Malus prunifolia × Malus pumila ??Malling 9??) and wild apple Malus sieboldii ??Sanashi 63?? (section Sorbomalus) were used for constructing genetic linkage maps. Here, a species from section Sorbomalus was used for the first time as a target species in a genome-wide mapping study. We also developed and mapped 137 novel-expressed sequence tag-simple sequence repeat (EST-SSR) markers. The genetic linkage maps of ??JM7?? and ??Sanashi 63?? consisted of 415 and 310 loci and spanned 998.0 and 981.8?cM, respectively, comparable to the reference map of Malus × domestica ??Discovery??. A BLASTN search revealed that all of the EST-SSR sequences used in this study exhibited very high homology to one or more previously characterized apple genome contigs. Although the most homologous contigs of 89 EST-SSRs were located within the same linkage groups (LGs) identified by mapping analysis, the other 48 EST-SSRs were aligned into contigs positioned in different LGs than those identified by mapping. When search criteria were expanded to include the five most homologous contigs of each EST-SSR, at least one of the top five contigs for 15 of these 48 EST-SSRs corresponded to the LG obtained by mapping. The maps of ??JM7?? and ??Sanashi 63?? may be useful for analyzing important rootstock characteristics and identifying markers for MAS.