Analysis of expressed sequence tags from Brassica rapa L. ssp. pekinensis

Non-redundant expressed sequence tags (ESTs) were generated from six different organs at various developmental stages of Chinese cabbage, Brassica rapa L. ssp. pekinensis. Of the 1,295 ESTs, 915 (71%) showed significantly high homology in nucleotide or deduced amino acid sequences with other sequences deposited in databases, while 380 did not show similarity to any sequences. Briefly, 598 ESTs matched with proteins of identified biological function, 177 with hypothetical proteins or non-annotated Arabidopsis genome sequences, and 140 with other ESTs. About 82% of the top-scored matching sequences were from Arabidopsis or Brassica, but overall 558 (43%) ESTs matched with Arabidopsis ESTs at the nucleotide sequence level. This observation strongly supports the idea that gene-expression profiles of Chinese cabbage differ from that of Arabidopsis, despite their genome structures being similar to each other. Moreover, sequence analyses of 21 Brassica ESTs revealed that their primary structure is different from those of corresponding annotated sequences of Arabidopsis genes. Our data suggest that direct prediction of Brassica gene expression pattern based on the information from Arabidopsis genome research has some limitations. Thus, information obtained from the Brassica EST study is useful not only for understanding of unique developmental processes of the plant, but also for the study of Arabidopsis genome structure.     

Development of a molecular marker specific to a novel CMS line in radish (Raphanus sativus L.)

In this study, we have investigated the cytoplasmic male sterility (CMS) of a novel male sterile radish line, designated NWB CMS. The NWB CMS was crossed with 16 fertile breeding lines, and all the progenies were completely male sterile. The degree of male sterility exhibited by NWB CMS is more than Ogura CMS from the Cruciferae family. The NWB CMS was found to induce 100% male sterility when crossed with all the tested breeding lines, whereas the Ogura CMS did not induce male sterility with any of the breeding lines. PCR analysis revealed that the molecular factor that influenced Ogura CMS, the orf138 gene, was absent in the NWB CMS line, and that the orf138 gene was not also expressed in this CMS line. In order to identify the cytoplasmic factors that confer male sterility in the NWB CMS line, we carried out RFLP analyses with 32 mitochondrial genes, all of which were used as probes. Fourteen genes exhibited polymorphisms between the NWB CMS line and other radish cultivars. Based on these RFLP data, intergenic primers were developed in order to amplify the intergenic regions between the polymorphic genes. Among these, a primer pair at the 3′ region of the atp6 gene (5′-cgcttggactatgctatgtatga-3′) and the 5′ region of the nad3 gene (5′-tcatagagaaatccaatcgtcaa-3′) produced a 2 kbp DNA fragment as a result of PCR. This DNA fragment was found to be specific to NWB CMS and was not present in other CMS types. It appears that this fragment could be used as a DNA marker to select NWB CMS line in a radish-breeding program.     

Molecular and biochemical analyses of OsRab7, a rice Rab7 homolog

Rab7 is a small GTP-binding protein important in early to late endosome/lysosome vesicular transport in mammalian cells. We have isolated a Rab7 cDNA clone, OsRab7, from a cold-treated rice cDNA library by the subtraction screening method. The cDNA encodes a polypeptide of 206 amino acids with a calculated molecular mass of about 23 kDa. Its predicted amino acid sequence shows significantly high identity with the sequences of other Rab7 proteins. His-tagged OsRab7 bound to radiolabeled GTPgammaS in a specific and stoichiometric manner. Biochemical and structural properties of the Rab7 wild type (WT) protein were compared to those of Q67L and T22N mutants. The detergent 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS) increased the guanine nucleotide binding and hydrolysis activities of Rab7WT. The OsRab7Q67L mutant showed much lower GTPase activity compared to the WT protein untreated with CHAPS, and the T22N mutant showed no GTP binding activity at all. The OsRab7Q67L mutant was constitutively active for guanine nucleotide binding while the T22N mutant (dominant negative) showed no guanine nucleotide binding activity. When bound to GTP, the Rab7WT and the Q67L mutants were protected from tryptic proteolysis. The cleavage pattern of the Rab7T22N mutant, however, was not affected by GTP addition. Northern and Western blot analyses suggested that OsRab7 is distributed in various tissues of rice. Furthermore, expression of a rice Rab7 gene was differentially regulated by various environmental stimuli such as cold, NaCl, dehydration, and ABA. In addition, subcellular localization of OsRab7 was investigated in the Arabidopsis protoplasts by a double-labeling experiment using GFP-fused OsRab7 and FM4-64. GFP-OsRab7 is localized to the vacuolar membrane, suggesting that OsRab7 is implicated in a vesicular transport to the vacuole in plant cells.     

Virginia mallow (Sida hermaphrodita (L.) Rusby) as perennial multipurpose crop: biomass yields,energetic valorization,utilization potentials,and management perspectives

This article reviews the scholarly literature dealing with the perennial multipurpose crop Virginia mallow (Sida hermaphrodita (L.) Rusby; Sida in the following). In regions dominated by intensive agricultural management practices, growing Sida holds the potential to combine ecosystem services such as decreasing soil erosion, reducing nitrate leaching as well as enhancing biodiversity, with economic profit for the farmer. After promising biomass yields of Sida were reported from studies performed in Poland about 15 years ago, the interest in this plant species has continuously increased, and different utilization pathways were examined, predominantly by researchers in Poland and Germany. At present, however, a comprehensive overview that summarizes the different lines of research performed regarding the use of Sida is lacking. This review aims at closing this gap. After providing background information on Sida, we summarize the main results obtained from investigations concerning biomass yields, fertilization effects, key findings concerning direct combustion, biogas production, steam gasification, phytoremediation, and alternative utilization pathways. Thereafter, we highlight important aspects of Virginia mallow cultivation practices, including first estimates regarding the costs involved. Finally, we point to existing research gaps. Summarizing the available literature on Sida, we aim at raising the interest of scientists and farmers in this plant species further and to show where future research might tie in with, as the successful cultivation of Sida might represent a worthwhile strategy to transform current agricultural practices in Central Europe into approaches that are more sustainable and resilient against future challenges.     

A reevaluation of the amino acid sequence of human follitropin β-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin β-subunit sequence (hFSHβ), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSHβ with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type B polysaccharide. Demonstration of three types of V regions and their association with H and L chain isotypes

The antibody response to Haemophilus influenzae type b polysaccharide (Hib-PS) is pauciclonal but can vary between different individuals. To estimate the size of this antibody repertoire we examined the constant and V regions of human IgG anti-Hib antibodies from 14 individuals at the clonal level using various serologic and IEF methods. Examination of H chains showed that 11 of 14 individuals produced both IgG1 and IgG2 antibodies, two individuals produced only IgG2 and one individual produced only IgG1 antibody. All 14 individuals produced kappa-containing antibody clones and three persons also produced significant lambda-containing antibody clones. V region heterogeneity was examined by comparing cross-reactivity of anti-Hib-PS antibodies to the capsular polysaccharide of Escherichia coli K100 carbohydrate (K100 CHO). These studies showed that clones of IgG anti-Hib-PS antibodies cross-reactive with K100 CHO were present in 5 of 14 (36%) individuals and also revealed at least three types of V regions among these antibodies. The first type has no cross-reaction with K100 CHO and was found in 13 of the 14 individuals. The second type, found in three of 14 individuals, cross-reacts with K100 CHO and uses a lambda L chain V region. The third type, found in 2 of 14 individuals, cross-reacts with K100 CHO and uses a kappa L chain V region. Although the lambda type V region was found only in association with IgG2, the other two V region types associate with both IgG1 and IgG2. Thus, five IgG antibody clones are serologically discernable. An individual generally responds to Hib-PS by expressing several clones selected from these discernable antibody clones. Indeed, we can observe six individual response patterns among these 14 individuals and conclude that considerable variability in individual responses to Hib-PS can be achieved with very few V regions.     

Feed formulations to reduce N excretion and ammonia emission from poultry manure

This summary focuses on reducing nitrogen (N) and ammonia emissions from poultry manure through the use of improved amino acid digestibilities and enzyme supplementation. Proper feed processing techniques, phase feeding, and the minimization of feed and water waste can contribute to additional minor reductions in these emissions. Reductions in environmental pollution can be achieved through improved diet formulation based on available nutrients in the ingredients, reducing crude protein (CP) levels and adding synthetic amino acids. Use of amino acid and CP digestibilities can reduce N excretion up to 40% and a 25% increase in N digestibility can be achieved with enzyme supplementation in broiler diets. Digestibilities can be measured by two methods: the excreta and ileal amino acid digestibilities. Both methods allow amino acid levels to be reduced by 10% or more. Enzyme supplementation decreases intestinal viscosity, improves metabolizable energy levels, and increases amino acid digestibilities. Many feed manufacturers still use total amino acid content to formulate feeds. To meet amino acid requirements, crystalline amino acids are needed. The use of feather, meat and bone meal must not be overestimated or underestimated and the limiting amino acids such as cystine, tryptophan, and threonine must be carefully analyzed.     

A candidate gene approach identified phytoene synthase as the locus for mature fruit color in red pepper (Capsicum spp.)

Abstrat  The color of mature pepper fruit is determined by the composition of carotenoids. The fruit color of red pepper is genetically determined by three loci, y, c1, and c2. We have been developing a genetic map of hot pepper using RFLP and AFLP markers in the F₂ population of an interspecific cross between Capsicum annuum cv TF68 and Capsicum chinense cv Habanero. The color of the ripe fruit of TF68 is red and Habanero is orange. The red color is dominant over orange in the F₁ and the locus controlling this character has been marked in our SNU Linkage Group 7. To identify the gene or markers tightly linked to the red/orange locus, several candidate genes involved in the carotenoid biosynthesis pathway, namely FPS, GGPS, PSY, PDS, LCY and CCS, were examined. One of the candidate genes, phytoene synthase, cosegregated completely with fruit color in the F₂ population. QTL analysis of the pigment content of F₂ individuals quantified by HPLC also indicated that phytoene synthase is the locus responsible for the development of fruit color. The color, pigment content and genetic behavior of Habanero also suggest that phytoene synthase may be responsible for the c2 gene discriminating between red and orange cultivars. Received: 15 March 2000 / Accepted: 16 August 2000