Nucleotide sequence analysis of <Emphasis Type="Italic">S</Emphasis>-locus genes to unify <Emphasis Type="Italic">S</Emphasis> haplotype nomenclature in radish

Radish, belonging to the family Brassicaceae, has a self-incompatibility which is controlled by multiple alleles on the S locus. To employ the self-incompatibility in an F₁ breeding system, identification of S haplotypes is necessary. Since collection of S haplotypes and determination of nucleotide sequences of SLG, SRK, and SCR alleles in cultivated radish have been conducted by different groups independently, the same or similar sequences with different S haplotype names and different sequences with the same S haplotype names have been registered in public databases, resulting in confusion of S haplotype names for researchers and breeders. In the present study, we developed S homozygous lines from radish F₁ hybrid cultivars in Japan and determined the nucleotide sequences of SCR, the S domain and the kinase domain of SRK, and the SLG of a large number of S haplotypes. Comparing these sequences with our previously published sequences, the haplotypes were ordered into 23 different S haplotypes. The sequences of the 23 S haplotypes were compared with S haplotype sequences registered by different groups, and we suggested a unification of these S haplotypes. Furthermore, dot-blot hybridization using SRK allele-specific probes was examined for developing a standard method for S haplotype identification.     

Identification of QTLs Associated with Conversion of Sucrose to Hexose in Mature Fruit of Japanese Pear

Sweetness is the most important trait for fruit breeding and is fundamentally determined by both total and individual sugar contents. We analyzed the contents of sucrose, fructose, glucose, and sorbitol in mature fruit in an F₁ population derived from crossing modern Japanese pear cultivar ‘Akizuki’ and breeding line ‘373-55’. A genetic linkage map was constructed using simple sequence repeats (SSRs) and single-nucleotide polymorphisms (SNP). We identified two regions associated with individual sugar contents on linkage group (LG) 1 and LG 7. The percentages of the variance in sucrose, fructose, and glucose explained by the quantitative trait loci (QTLs) were 26.6, 15.9, and 18.5%, respectively, for the region on LG 1, and 22.2, 20.0, and 9.5%, respectively, for the region on LG 7. In both regions, genotypes associated with increases in sucrose were associated with decreases in both fructose and glucose. The 1.5-LOD support intervals of the QTLs on LGs 1 and 7 include SSRs within the regions flanking acid invertase genes PPAIV3 and PPAIV1, respectively. Because acid invertase is a key enzyme in the conversion of sucrose to hexose, these are promising candidates for genes underlying those QTLs and controlling individual sugar contents. We also found one region on LG 11 that explained 21.4% of the variation in total sugar content but was not significantly associated with variation for individual sugars. The information obtained in this study will accelerate research and breeding programs to improve fruit sweetness.     

Association studies of N-acetyl-amino acid N,N-dimethylamides in carbon tetrachloride

The self-association of N-acetylglycine N,N-dimethylamide, N-acetyl-L -valine N,N-dimethylamide, and N-acetyl-L -phenylalanine N,N-dimethylamide in carbon tetrachloride was investigated by using ir and ¹H-nmr methods. It was concluded from ir measurements that the associated species is the dimer formed as a result of the simultaneous formation of two intermolecular hydrogen bonds. This is supported by the results of ¹H-nmr measurements. Thermodynamic quantities for the association were determined from the temperature and concentration dependence of the NH proton chemical shifts of the sample solutions. Compared with the Gly derivative, L -Val and L -Phe derivatives have larger values of ?ΔH for association, which shows good correlation with Δv_NH values, the difference between the maxima of the monomer and dimer bands, obtained from ir spectra. This is due to the less stable monomer conformation and to the stronger intermolecular hydrogen bonding of the dimers in L -Val and L -Phe derivatives. The line shapes of both methyl proton resonances of L -Val residue and methylene proton resonances of L -Phe residue were found to vary with concentration and temperature of the sample solutions. These data indicate that the rotation about the C^α—C^β bond is restricted by the steric hindrance present in the associated dimers. All these experimental results can be related to the fact that L -Val and L -Phe derivatives have a warped framework because of the bulky side chains, whereas the Gly derivative has a planar framework.     

Characterization of S tester lines in Brassica oleracea: polymorphism of restriction fragment length of SLG homologues and isoelectric points of S-locus glycoproteins

 Forty three S tester lines of Brassica oleracea were characterized using DNA and protein gel-blotting analyses. DNA gel-blot analysis of HindIII-digested genomic DNA with class-I and class-II SLG probes revealed that 40 lines could be classified as class-I S haplotypes while three lines could be classified as class-II S haplotypes. The band patterns in the S tester lines were highly polymorphic. Although the S tester lines typically showed two bands corresponding to SLG and SRK in the analysis with the class-I SLG probe, only one band was observed in the S ²⁴ homozygote. This band was identified as SRK, suggesting that this haplotype has no class-I SLG band. In the analysis using the class-II SLG probe, one plant yielded a different band pattern from the known class-II haplotypes, S ² , S ⁵ and S ¹⁵ . Unexpectedly, this plant was reciprocally cross-incompatible with the S ² haplotype. Therefore, it was designated as S ^2-b . We found an S ¹³ haplotype having a restriction fragment length polymorphism different from that of the S ¹³ homozygotes of the S tester line. These findings indicate that S homozygous lines with the same S specificity do not necessarily show the same band pattern in the DNA gel-blot analysis. Soluble stigma proteins of 32 S homozygotes were separated by isoelectric focusing and detected using anti-S ²² SLG antiserum. S haplotype-specific bands were detected in 27 S homozygotes but not in five S homozygotes, including the S ²⁴ homozygote. This is consistent with the observation that the S ²⁴ haplotype had no SLG band. Received: 13 July 1998 / Accepted: 29 September 1998     

Species-Independent Inhibition of Abnormal Prion Protein (PrP) Formation by a Peptide Containing a Conserved PrP Sequence

Conversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions. Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.     

Synthesis, Solution Structure, and Phylum Selectivity of a Spider ��-Toxin That Slows Inactivation of Specific Voltage-gated Sodium Channel Subtypes

Magi 4, now renamed δ-hexatoxin-Mg1a, is a 43-residue neurotoxic peptide from the venom of the hexathelid Japanese funnel-web spider (Macrothele gigas) with homology to δ-hexatoxins from Australian funnel-web spiders. It binds with high affinity to receptor site 3 on insect voltage-gated sodium (Na_V) channels but, unlike δ-hexatoxins, does not compete for the related site 3 in rat brain despite being previously shown to be lethal by intracranial injection. To elucidate differences in Na_V channel selectivity, we have undertaken the first characterization of a peptide toxin on a broad range of mammalian and insect Na_V channel subtypes showing that δ-hexatoxin-Mg1a selectively slows channel inactivation of mammalian Na_V1.1, Na_V1.3, and Na_V1.6 but more importantly shows higher affinity for insect Na_V1 (para) channels. Consequently, δ-hexatoxin-Mg1a induces tonic repetitive firing of nerve impulses in insect neurons accompanied by plateau potentials. In addition, we have chemically synthesized and folded δ-hexatoxin-Mg1a, ascertained the bonding pattern of the four disulfides, and determined its three-dimensional solution structure using NMR spectroscopy. Despite modest sequence homology, we show that key residues important for the activity of scorpion α-toxins and δ-hexatoxins are distributed in a topologically similar manner in δ-hexatoxin-Mg1a. However, subtle differences in the toxin surfaces are important for the novel selectivity of δ-hexatoxin-Mg1a for certain mammalian and insect Na_V channel subtypes. As such, δ-hexatoxin-Mg1a provides us with a specific tool with which to study channel structure and function and determinants for phylum- and tissue-specific activity.Voltage-gated sodium (Na_V)⁴ channels are responsible for the generation and propagation of electrical signals in excitable cells. At least nine different genes encoding distinct Na_V channels isoforms have been identified, and functionally expressed, in mammals (1). They are characterized by their sensitivity to TTX, with Na_V1.5, Na_V1.8, and Na_V1.9 being TTX-insensitive or TTX-resistant, and the remaining subtypes being sensitive to nanomolar concentrations of TTX. In addition, localization of the subtypes also varies, with Na_V1.1–1.3 mostly distributed in the central nervous system, Na_V1.6–1.9 principally located in the peripheral nervous system, and Na_V1.4 and Na_V1.5 found in skeletal and cardiac muscle, respectively. The structural diversity of Na_V channels also coincides with variations in physiological and pharmacological properties (2). In contrast, insects express only one gene (para) that undergoes extensive alternative splicing and RNA editing (3). The para-encoded Na_V channel is exceptionally well conserved across diverse orders of insects, with the level of identity ranging from 87 to 98% (3). This is one reason why insecticides that target insect Na_V channels have broad activity across many insect orders. In contrast, para-type Na_V channels have significantly lower levels of identity with the various types of mammalian Na_V channels with the level of identity typically around 50–60% (3). This explains why a high degree of phylogenetic specificity can be achieved with both Na_V channel toxins and insecticides that target the Na_V channel.At least seven distinct toxin-binding sites have been identified by radioligand binding and electrophysiological studies on vertebrate and insect Na_V channels (4, 5). Toxins interacting with these neurotoxin receptor sites have been instrumental in the study of Na_V channel topology, function, and pharmacology (6). In particular, a wide range of scorpion α-toxins, sea anemone toxins, and spider δ-hexatoxins (formerly δ-atracotoxins (7)) compete for binding to receptor site-3 on the extracellular surface of Na_V channels. These polypeptide toxins all inhibit the fast inactivation of Na_V channels to prolong Na⁺ currents (I_Na), despite huge diversity in primary and tertiary structures (8, 9). Nevertheless, receptor site-3 has not yet been fully characterized but is believed to involve domains DI/S5-S6, DIV/S5-S6, as well as DIV/S3-S4 (9). Most importantly, however, toxin characterization is often limited to studies using whole-cell I_Na or binding studies on neuronal membranes where there are mixed populations of Na_V channel subtypes. For all of these toxins, the precise pattern of Na_V channel subtype selectivity is either unknown or at best is incomplete.Recently, it was found that receptor site-3 was also recognized by a 43-residue spider toxin, originally named Magi 4, from the hexathelid spider Macrothele gigas (Iriomote, Japan). It binds with high affinity to insect Na_V channels but, similar to scorpion α-like toxins, does not compete for the related site-3 in rat brain synaptosomes, despite being lethal by intracranial injection (10). Magi 4 shares significant homology to four δ-hexatoxin (HXTX)-1 family peptides and δ-actinopoditoxin-Mb1a (formerly δ-missulenatoxin-Mb1a; Fig. 1) but no sequence homology to scorpion α-toxins. Neurochemical studies have shown that δ-HXTX-1 toxins compete at nanomolar concentrations with both anti-mammalian (e.g. Aah2 and Lqh2) and anti-insect (e.g. LqhαIT) scorpion toxins for site-3 (11–13). The three-dimensional structures of δ-HXTX-Ar1a and δ-HXTX-Hv1a peptides have been determined (14, 15) and possess core β regions stabilized by four disulfide bonds, placing them in the inhibitory cystine knot (ICK) structural family (16).Open in a separate windowFIGURE 1.Primary and secondary structure of δ-HXTX-Mg1a. A, comparison of the primary sequence of δ-HXTX-Mg1a and δ-HXTX-Mg1b (formerly Magi 14) with currently known members of the δ-HXTX-1 family and δ-AOTX-Mb1a (δ-actinopoditoxin-Mb1a, formerly δ-missulenatoxin-Mb1a). Homologies are shown relative to δ-HXTX-Mg1a; identities are boxed in gray, and conservative substitutions are in gray italic text. Gaps (dashes) have been inserted to maximize alignment. The disulfide bonding pattern for the strictly conserved cysteine residues determined for δ-HXTX-Mg1a (this study), δ-HXTX-Ar1a (55), and δ-HXTX-Hv1a (15) is indicated above the sequences; it is assumed that δ-AOTX-Mb1a (36), δ-HXTX-Hs20.1a (8), and δ-HXTX-Hv1b (56) have the same disulfide bonding pattern. The percentage identity and homology with δ-HXTX-Mg1a is shown to the right of the sequences. B, summary of δ-HXTX-Mg1a NMR data. Sequential NOEs, classified as very weak, weak, medium, and strong, are represented by the thickness of bars. Filled diamonds indicate backbone amide protons that form hydrogen bonds. ³J_NHCα coupling constants are indicated by ↑ (>8 Hz) and ↓ (<5.5 Hz). Secondary structure is shown at the bottom of the figure where rectangles represent β-turns (the type of turn is indicated in the rectangle) and arrows represent β-sheets.The aim of this study was to first determine the solution structure of Magi 4 and second to investigate the ability of Magi 4 to discriminate between different Na_V channels subtypes. Here we report the tertiary structure of Magi 4 by ¹H NMR and show its disulfide bonding pattern and three-dimensional structure are homologous to δ-HXTX-1 toxins. We highlight the key residues in Magi 4 that appear to be topologically similar to those residues known to be part of the pharmacophore for site-3 scorpion α-toxins, despite Magi 4 having a different overall structure to scorpion α-toxins (11). In addition, we provide a detailed characterization of the selectivity and mode of action of Magi 4 on nine cloned mammalian and insect Na_V channel subtypes, including a detailed characterization on insect neurotransmission. Given that the toxin potently slows the inactivation of Na_V channels, it should be renamed δ-hexatoxin-Mg1a (δ-HXTX-Mg1a) in accordance with the rational nomenclature recently proposed for naming spider peptide toxins (7) (see ArachnoServer spider toxin data base).     

Seasonal variation of phlorotannin in sargassacean species from the coast of the Sea of Japan

Variations in phlorotannin concentrations among the developmental stages of brown algae have been reported; however, the phlorotannin concentration plasticity associated with fluctuations in environmental factors make it difficult to determine the essential ontogenetic variation. The phlorotannin concentrations in five perennial sargassacean species where newly sprouted branches appear in summer and become fertile the following spring were examined every month during a year; and correlation with the developmental or seasonal environmental factors was determined. Although the phlorotannin fluctuated greatly throughout the year, the fluctuation patterns were relatively similar among the five species: phlorotannin showed a peak during July and August; gradually decreased in the winter; and increased in April. Performing a multiple regression analysis, the phlorotannin concentration did not correlate with thallus size in all species; and phlorotannin amounts were significantly affected by ambient abiotic factors in some species. The phlorotannin contents in newly sprouted branches were always higher than those in the long main branches during all seasons. When the phlorotannin contents were determined monthly for S. fulvellum (Turner) C. Agardh where the thalli were cultured from embryos in outdoor tanks, the phlorotannin concentrations were 3–4% of the dry matter (DM) in the juveniles and decreased to less than 1% of the DM in thalli >7.5 cm in length. However, the phlorotannin in these cultured thalli suddenly increased to 5.3% DM after being transplanted to the inshore coast; and then the concentration gradually decreased. The data show higher phlorotannin concentrations in younger sargassacean algae thalli and fluctuation of the phlorotannin amounts with extrinsic environmental factors.     

Production of TMC-151, TMC-154 and TMC-171, a new class of antibiotics, is specific to ‘Gliocladium roseum’ group

A novel class of fungal metabolites, TMC-151, TMC-154, and TMC-171 series compounds, was found exclusively inGliocladium catenulatum, Clonostachys rosea and closely related strains. These compounds were not detected in any other fungi examined. The production spectrum of each component was correlated to the morphology of the secondary conidiophores and the conidia. TMC-151 was limited toClonostachys rosea (formerlyG. roseum) forming navicular or reniform conidia orG. catenulatum with gray-green conidial masses, whereas TMC-154 and 171 were limited to the strains closely related toGliocladium roseum, which grew more slowly and formed more symmetrical conidia.