Toward unraveling the structure of Brassica rapa genome

Genomic research in any organism encompasses understanding structure of the target genome and genes, their function, and evolution. Brassica rapa , which is phylogenetically related to Arabidopsis thaliana , is an important species with respect to its uses as vegetable, oil, and fodder. The availability of suitable genetic and genomic resources is a prerequisite to undertake genomic research in B. rapa . We have developed reference mapping populations of Chinese cabbage ( B. rapa ssp. pekinensis ) comprising 78 doubled haploid lines and over 250 recombinant inbred lines. Two Bacterial Artificial Chromosome (BAC) libraries, generated by restriction enzymes Hin dIII (KBrH) and Bam HI (KBrB), comprise 56 592 and 50 688 clones, respectively. We have also constructed 22 cDNA libraries from different plant tissues consisting of 104 914 clones with an average length of 575 bp. Initial BAC-end sequence analysis of 1473 clones of the KBrH library led us to understand the structure of B. rapa genome with respect to extent of genic sequences and their annotation, and relative abundance of different types of repetitive DNAs. Full-length sequence analysis of BAC clones revealed extensive triplication of B. rapa DNA segments coupled with variable gene losses within the segments. The formulation of the 'Multinational Brassica Genome Project' has laid the foundation to sequence the complete genome of B. rapa ssp. pekinensis by the international Brassica research community. It has been proposed to undertake BAC-to-BAC sequencing of genetically mapped seed BACs. In recent years, development of bioinformatics tools in Brassica has given a boost to structural genomics research in Brassica species. The research undertaken with the availability of various genomic resources in the public domain has added to our understanding of the structure of B. rapa .     

Mapping quantitative trait loci for yield components and morphological traits in an advanced backcross population between Oryza grandiglumis and the O. sativa japonica cultivar Hwaseongbyeo

Introgression has been achieved from wild species Oryza grandiglumis (2n=48, CCDD, Acc. No. 101154) into O. sativa subsp. japonica cv. Hwaseongbyeo as a recurrent parent. An advanced introgression (backcross) line, HG101, produced from a single plant from BC₅F₃ families resembled Hwaseongbyeo, but it showed differences from Hwaseongbyeo in several traits, including days to heading and culm length. To detect the introgressions, 450 microsatellite markers of known chromosomal position were used for the parental survey. Of the 450 markers, 51 (11.3%) detected O. grandiglumis segments in HG101. To characterize the effects of alien genes introgressed into HG101, an F_2:3 population (150 families) from the cross Hwaseongbyeo/HG101 was developed and evaluated for 13 agronomic traits. Several lines outperformed Hwaseongbyeo in several traits, including days to heading. Genotypes were determined for 150 F₂ plants using simple sequence repeat markers. Qualitative trait locus (QTL) analysis was carried out to determine the relationship between marker genotype and the traits evaluated. A total of 39 QTL and 1 gene conferring resistance to blast isolate were identified using single-point analysis. Phenotypic variation associated with each QTL ranged from 4.2 to 30.5%. For 18 (46.2%) of the QTL identified in this study, the O. grandiglumis-derived alleles contributed a desirable agronomic effect despite the overall undesirable characteristics of the wild phenotype. Favorable wild alleles were detected for days to heading, spikelets per panicle, and grain shape traits. Grain shape QTL for grain weight, thickness, and width identified in the F_2:3 lines were further confirmed based on the F₄ progeny test. The confirmed locus, tgw2 for grain weight is of particular interest because of its independence from undesirable height and maturity. Several QTL controlling amylose content and grain traits have not been detected in the previous QTL studies between Oryza cultivars, indicating potentially novel alleles from O. grandiglumis. The QTL detected in this study could be a rich source of natural genetic variation underlying the evolution and breeding of rice.     

Middle Pleistocene handaxes from the Korean Peninsula

We present four biface assemblages from an archaeologically poorly known region of the Old World: Middle Pleistocene Korea. The handaxes are derived from a series of Middle Pleistocene localities in the Imjin/Hantan River Basins (IHRB) in Korea. The best known of these localities is Chongokni, although a number of equally important sites in the IHRB have been discovered and excavated over the course of the past two decades (e.g., Kumpari, Chuwoli, and Kawoli). Reanalysis of the age of the Chongokni deposits suggests a hominin occupation between 350-300 ka. Comparative study of the IHRB handaxes with the well-known bifacial implements from Olorgesailie (Kenya) and Hunsgi-Baichbal (India) indicates that the often-noted "thick" trait of the East Asian handaxes differs at a statistical level across the various regions of the Old World. The finds from the IHRB sites, and the Chinese sites of Bose and Dingcun that contain handaxes-like implement, question the validity of the Movius Line sensu stricto. However, why East Asian Middle Pleistocene hominins did not consistently produce more refined bifaces across broader regional and/or temporal facies, remains open to question. Thus, the absence of similar sites in wider areas of Early and Middle Pleistocene East Asia suggests that the Movius Line sensu lato is still supportable and warrants additional detailed cross comparative studies of the stone toolkits east and west of the line.     

Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases

We cloned lipG, which encoded a lipolytic enzyme, from a Korean tidal flat metagenomic library. LipG was related to six putative lipases previously identified only in bacterial genome sequences. These enzymes comprise a new family. We partially characterized LipG, providing the first experimental data for a member of this family.     

Immobilization of Horseradish Peroxidase on Multi-Wall Carbon Nanotubes and its Electrochemical Properties

Horseradish peroxidase (HRP) was immobilized on carboxylated multi-wall carbon nanotubes in the presence of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The immobilized HRP maintained its oxidative activity for guaiacol over a broad range of pH values (4–9). An electrode of graphite rod, 6 mm diam. was fabricated using the immobilized HRP. Cyclic voltammetry of the enzyme electrode confirmed electron transfer between the immobilized HRP and the electrode in the presence of H₂O₂ but without an added mediator or a reducing substrate.     

Optimal Production of Poly-γ-glutamic Acid by Metabolically Engineered Escherichia coli

Metabolically-engineered Escherichia coli strains were developed by cloning poly-γ-glutamic acid (γ-PGA) biosynthesis genes, consisting of pgsB, pgsC and pgsA, from Bacillus subtilis The metabolic and regulatory pathways of γ-PGA biosynthesis in E. coli were analyzed by DNA microarray. The inducible trc promoter and a constitutive promoter (P_HCE) derived from the d-amino acid aminotransferase (D-AAT) gene of Geobacillus toebii were employed. The constitutive HCE promoter was more efficient than inducible trc promoter for the expression of γ-PGA biosynthesis genes. DNA microarray analysis showed that the expression levels of several NtrC family genes, glnA, glnK, glnG, yhdX, yhdY, yhdZ, amtB, nac, argT and cbl were up-regulated and sucA, B, C, D genes were down-regulated. When (NH₄)₂SO₄ was added at 40 g/l into the feeding solution, the final γ-PGA concentration reached 3.7 g/l in the fed-batch culture of recombinant E. coli/pCOpgs.     

Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, by Candida sp. SY16 using fed-batch fermentation

Candida sp. strain SY16 produces a glycolipid-type biosurfactant, mannosylerythritol lipid (MEL-SY16), which can reduce the surface tension of a culture broth from 72 to 30 dyne cm⁻¹ and highly emulsify hydrocarbons when cultured in soybean-oil-containing media. As such, laboratory-scale fermentation for MEL-SY16 production was performed using optimized conditions. In batch fermentation, MEL-SY16 was mainly produced during the stationary phase of growth, and the concentration of MEL-SY16 reached 37 g l⁻¹ after 200 h. The effect of pH control on the production of MEL-SY16 was also examined in batch fermentation. The highest production yield of MEL-SY16 was when the pH was controlled at 4.0, and the production was significantly improved compared to batch fermentation without pH control. In fed-batch fermentation, glucose and soybean oil (1:1, w/w) were used in combination as the initial carbon sources for cell growth, and soybean oil was used as the feeding carbon source during the MEL production phase. The feeding of soybean oil resulted in the disappearance of any foam and a sharp increase in the MEL production until 200 h, at which point the concentration of MEL-SY16 was 95 g l⁻¹. Among the investigated culture systems, the highest MEL-SY16 production and volumetric production rate were achieved with fed-batch fermentation.     

A membrane-associated metalloprotease of Taenia solium metacestode structurally related to the FACE-1/Ste24p protease family

The CaaX proteases are intimately involved in the post-translational modification of prenylated proteins and play a critical role in the activation/stabilization of membrane-bound or secreted molecules constituting the CAAX protein family. In this study, we have isolated a full-length cDNA putatively encoding a type I CaaX protease of the Taenia solium metacestode (TsM), which an agent causative of human neurocysticercosis. The cDNA, designated TsSte24p, comprised 1,505 bp and coded for an open reading frame of 472 amino acids with predicted Mr 54.5 kDa. This monoexonic TsSte24p gene existed as a single copy within the TsM genome and constantly expressed in the parasite from metacestode to adult stages. The TsSte24p exhibited the typical CaaX protease topology, including seven transmembrane domains and a metalloprotease segment with a zinc-binding motif. It shared a significant degree of sequence identity with the type I CaaX proteases such as Saccharomyces cerevisiae Ste24p and Caenorhabditis elegans CeFACE-1. A comparative phylogenetic analysis demonstrated that this protein family is tightly conserved across taxa, from bacteria to mammals. The bacterially expressed recombinant TsSte24p showed proteolytic activity, with an optimal pH of 7.5. The enzyme activity was significantly inhibited by EDTA. Its activity was increased in the presence of low concentrations of the Zn2+(0.001-0.01 mM); but was reversibly down-regulated at high doses (over 0.1 mM). The native TsSte24p appeared to function as a homodimer, the subunits of which were linked to each other via covalent disulfide bond. The protein was localized in the bladder wall and scolex with differential patterns of distribution. Our results indicated that TsSte24p is a zinc-dependent metalloprotease, which belongs to the FACE-1/Ste24p protease family.