Molecular linkage mapping and phylogeny of the chalcone synthase multigene family in soybean.

Chalcone synthase (CHS), the key enzyme in the flavonoid biosynthesis pathway, is encoded by a multigene family, CHS1-CHS8 and dCHS1 in soybean. A tandem repeat of CHS1, CHS3 and CHS4, and dCHS1 that is believed to be located in the vicinity comprises the I locus that suppresses coloration of the seed coat. This study was conducted to determine the location of all CHS members by using PCR-based DNA markers. Primers were constructed based on varietal differences in either the nucleotide sequence of the 5'-upstream region or the first intron of two cultivars, Misuzudaizu, with a yellow seed coat (II), and Moshidou Gong 503, with a brown seed coat (ii). One hundred and fifty recombinant inbred lines that originated from a cross between these two cultivars were used for linkage mapping together with 360 markers. Linkage mapping confirmed that CHS1, CHS3, CHS4, dCHS1, and the I locus are located at the same position in molecular linkage group (MLG) A2. CHS5 was mapped at a distance of 0.3 cM from the gene cluster. CHS2 and CHS6 were located in the middle region of MLGs A1 and K, respectively, while CHS7 and CHS8 were found at the distal end of MLGs D1a and B1, respectively. Phylogenetic analysis indicated that CHS1, CHS3, CHS4, and CHS5 are closely related, suggesting that gene duplication may have occurred repeatedly to form the I locus. In addition, CHS7 and CHS8 located at the distal end and CHS2, CHS6, and CHS members around the I locus located around the middle of the MLG are also related. Ancient tetraploidization and repeated duplication may be responsible for the evolution of the complex genetic loci of the CHS multigene family in soybean.

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice.

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion-exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SWXL and G3000SWXL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SWXL and G4000SWXL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 degrees C, respectively. The enzyme activity was completely inhibited by HgCl2 and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995. Plant Cell 7: 417-429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al. 1988, J Biol Chem 263: 9271-9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type.

Nucleotide sequence analysis of the 3' terminal region of a wasabi strain of crucifer tobamovirus genomic RNA: subgrouping of crucifer tobamoviruses.

The 3' terminal 2378 nucleotides of a wasabi strain of crucifer tobamovirus (CTMV-W) infectious to crucifer plants was determined. This includes the 3' non-coding region of 235 nucleotides, coat protein (CP) gene (468 nucleotides), movement protein (MP) gene (798 nucleotides) and C-terminal partial readthrough portion of 180 K protein gene (940 nucleotides). Comparison of the sequence with homologous regions of thirteen other tobamovirus genomes showed that it had much higher identity to those of four other crucifer tobamoviruses, 85.2% to cr-TMV and turnip vein-clearing virus (TVCV), 87.4% to oilseed rape mosaic virus (ORMV) and 87.1% to TMV-Cg, than to those of other tobamoviruses. Thus CTMV-W was most similar to ORMV and TMV-Cg in sequence, but only marginally so, whereas the location and size of its MP gene was the same as cr-TMV amd TVCV. These results, together with other analyses, show that CTMV-W is a new crucifer tobamovirus, that the five crucifer tobamoviruses can be classified into two subgroups based on MP gene organization, and that the rate of sequence change is not the same in all lineages.

The complete nucleotide sequence of the rice grassy stunt virus genome and genomic comparisons with viruses of the genus Tenuivirus.

Rice grassy stunt virus (RGSV, IRRI isolate) has six genomic RNA segments. The nucleotide (nt) sequences of RNAs 1-4 were determined. The cumulative length of the RGSV genome, including RNAs 5 and 6, was 25142 nt. All six RNA segments had an ambisense coding strategy and almost identical terminal sequences over 17 nt. The virus complementary (vc) sequence of the largest segment, RNA1, had an open reading frame encoding a protein of Mr 339133 (the 339.1K protein), while the virus sense (v) sequence encoded a protein of Mr 18910 in the 5'-proximal region. The predicted 339.1K protein contained the highly conserved motifs of the RNA-dependent RNA polymerase and a short but distinct Arg/Gly-rich stretch at the C terminus. The putative RNA polymerase showed strong similarity with that of rice stripe tenuivirus (RSV); they shared 37.9% amino acid identity over 2140 residues. The predicted proteins of Mr 23280 on vRNA2 and 93 879 on vcRNA2 were only slightly similar in sequence to the proteins encoded by vRNA2 and vcRNA2 of other tenuiviruses. The predicted proteins encoded by RNA3 and RNA4 did not show significant similarity to any database proteins. Only the putative RNA polymerase encoded on RNA1 was well-conserved between RGSV and RSV. The low sequence similarities in proteins encoded by RNAs 2, 5 and 6, together with the unique RNA segments 3 and 4, indicate that RGSV may be distinct from other tenuiviruses.

Isoprenoid synthesis gene for geranylgeranyl diphosphate synthase from a hyperthermophile, Pyrococcus sp. strain OT3.

Pyrococcus sp. strain OT3, a hyperthermophilic archaeon that was isolated by the authors was found to contain tetraether lipid mainly in the membrane lipid, which was quite different from the other hyperthermophiles (Masuchi et al. 1997). Those isoprenoids are synthesized by a family of isoprenyl diphosphate synthases from isopentenyl diphosphate to allylic diphosphates. The gene that encodes one of these families, geranylgeranyl diphosphate synthase (GGPPSase), from this strain was cloned and sequenced. This coding gene has a 960-bp (320aa) sequence. The putative Shine-Dalgarno sequence was six bases upper of start codon, exactly the same as Methanobacterium thermoautotrophicum, a methnogenic thermophile. Comparison of the amino acid sequence of 13 organisms including Eukarya, Bacteria, and Archaea showed that Archaea strains including Pyrococcus sp. strain OT3 consisted of a separate group from the others, but five conservative regions are very homologous.