Molecular mapping of <Emphasis Type="Italic">Rym17</Emphasis>, a dominant and <Emphasis Type="Italic">rym18</Emphasis> a recessive barley yellow mosaic virus (BaYMV) resistance genes derived from <Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.

PK23-2, a line of six-rowed barley (Hordeum vulgare L.) originating from Pakistan, has resistance to Japanese strains I and III of the barley yellow mosaic virus (BaYMV). To identify the source of resistance in this line, reciprocal crosses were made between the susceptible cultivar Daisen-gold and PK23-2. Genetic analyses in the F₁ generation, F₂ generation, and a doubled haploid population (DH45) derived from the F₁ revealed that PK23-2 harbors one dominant and one recessive resistance genes. A linkage map was constructed using 61 lines of DH45 and 127 DNA markers; this map covered 1268.8 cM in 10 linkage groups. One QTL having a LOD score of 4.07 and explaining 26.8% of the phenotypic variance explained (PVE) for resistance to BaYMV was detected at DNA marker ABG070 on chromosome 3H. Another QTL having a LOD score of 3.53 and PVE of 27.2% was located at marker Bmag0490 on chromosome 4H. The resistance gene on chromosome 3H, here named Rym17, showed dominant inheritance, whereas the gene on chromosome 4H, here named rym18, showed recessive inheritance in F₁ populations derived from crosses between several resistant lines of DH45 and Daisen-gold. The BaYMV recessive resistance genes rym1, rym3, and rym5, found in Japanese barley germplasm, were not allelic to rym18. These results revealed that PK23-2 harbors two previously unidentified resistance genes, Rym17 on 3H and rym18 on 4H; Rym17 is the first dominant BaYMV resistance gene to be identified in primary gene pool. These new genes, particularly dominant Rym17, represent a potentially valuable genetic resource against BaYMV disease.     

Characterization of suppressor T cell clones derived from a mouse tolerized with conjugates of ovalbumin and monomethoxypolyethylene glycol.

The induction of antigen-specific tolerance in mice by conjugates of ovalbumin (OVA) and monomethoxypolyethylene glycol (mPEG) previously had been shown to be associated with the generation of antigen-specific suppressor T (Ts) cells. For the elucidation of the nature of these Ts cells, five nonhybridized OVA-specific Ts cell clones were generated from the spleen cells of a BDF1 mouse which had been immunosuppressed by the tolerogenic conjugate, OVA(mPEG)12. The cloned Ts cells were maintained in vitro by periodic stimulation with OVA and feeder cells and were able to suppress the in vitro antibody production in an OVA-specific and MHC class I (H-2Kd or H-2Dd)-restricted manner. All these Ts cell clones were shown to be Thy1.2+, CD4-, CD5-, CD8+, and to express CD3 and the alpha beta heterodimer of the T cell receptor. The cell-free extracts of these cells contained soluble suppressor factors which could mimic in vitro the suppressive activity of the intact cells. In contrast to cytotoxic T lymphocytes (CTL), none of the cloned Ts cells were endowed with cytolytic activity as revealed in the perforin-mediated microhemolysis and in the 18-hr51Cr release assays. These results demonstrate that (i) OVA-specific Ts cell clones can be generated from mice pretreated with OVA(mPEG)12 by employing conventional T cell culture techniques, and (ii) these Ts cells are functionally different from conventional CD8+ CTL.     

Phylogeography of lateral plate dimorphism in the freshwater type of ninespine sticklebacks, genus Pungitius

The freshwater type of ninespine sticklebacks, genus Pungitius, is widely distributed in northern Japan and reproductively isolated from other genetically divergent types endemic to small regions in Japan. This type expresses dimorphism in its lateral plate morphology: complete and partial row morphs. The two morphs show a parapatric distribution in Japan. To clarify the process involving the distribution of these two morphs, we examined their phylogeography based on restriction fragment length polymorphism of an entire mitochondrial DNA (mtDNA). The survey was carried out with seven restriction enzymes on the populations of the freshwater type collected from 41 localities across the distribution range in Japan, and 6 further Pungitius populations from the Okhotsk Sea coast of Russia were appended. An unweighted pair-group method with arithmetic averages (UPGMA) tree among 54 mtDNA haplotypes resolved eight clustering groups that differed in sequence divergence by approximately 1.3%–2.1%. Two of the eight groups were found only in Russia. mtDNA phylogenies constructed by neighbor-joining and Wagner parsimony methods suggested that the haplotypes of each plate morph were polyphyletic. The geographic distribution pattern of these groups suggests that they should be classified into two broad categories, one with extensive distribution and the other with localized distribution of the constituent haplotypes within a group. The former groups were found mainly in the populations with the completely plated morph and the latter groups with the partially plated morph. It is supposed that twice dispersals of dimorphic or complete plated ancestors and genetic differentiation during the interglacial played an important role in the formation of the present distribution of the two morphs in Japan. Received: March 28, 2000 / Revised: November 3, 2000 / Accepted: January 16, 2001     

In-depth proteomic profiling of the normal human kidney glomerulus using two-dimensional protein prefractionation in combination with liquid chromatography-tandem mass spectrometry

The kidney glomerulus plays a pivotal role in ultrafiltration of plasma into urine and also is the locus of kidney disease progressing to chronic renal failure. We have focused proteomic analysis on the glomerulus that is most proximal to the disease locus. In the present study, we aimed to provide a confident, in-depth profiling of the glomerulus proteome. The glomeruli were highly purified from the kidney cortex from a male, 68-year-old patient who underwent nephroureterectomy due to ureter carcinoma. The patient was normal in clinical examinations including serum creatinine and urea levels and liver function, and did not receive any chemotherapy and radiotherapy. The cortical tissue was histologically normal, and no significant deposition of immunoglobulins and complement C3 was observed. We employed a novel strategy of protein separation using 1D (SDS-PAGE) and 2D (solution-phase IEF in combination with SDS-PAGE) prefractionation prior to the shotgun analysis with LC-MS/MS. The protein prefractionation produced 90 fractions, and eventually provided a confident set of identified proteins consisting of 6686 unique proteins (3679 proteins with two or more peptide matches and 3007 proteins with one peptide match), representing 2966 distinct genes. All the identified proteins were annotated and classified in terms of molecular function and biological process, compiled into 1D and 2D protein arrays, consisting of 15 and 75 sections, corresponding to the protein fractions which were defined by MW and pI range, and deposited on a Web-based database (http://www.hkupp.org). The most remarkable feature of the glomerulus proteome was a high incidence of identification of cytoskeleton-related proteins, presumably reflecting the well-developed, cytoskeletal organization of glomerular cells related to their physiological functions.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

Multiple structural transitions of the GroEL subunit are sensitive to intermolecular interactions with cochaperonin and refolding polypeptide

In this study we attempted to determine the specific roles of the numerous conformational changes that are observed in the bacterial chaperonin GroEL, by performing stopped-flow experiments on GroEL R231W in the presence of a refolding substrate protein. The apparent rate of one kinetic phase was decreased by approximately 25% in the presence of prebound unfolded malate dehydrogenase while another phase was suppressed completely under the same conditions, reflecting different effects of the unfolded protein on multiple structural transitions within GroEL. The addition of cochaperonin GroES counteracts the effect of the bound substrate protein in the former case, but had no effect on the latter, more extensive suppression. Using a chemically modified form of GroEL R231W which is incapable of releasing substrate proteins at low temperatures, we identified a conformational transition that is implicated in the release of substrate proteins. Parts of the actual process of substrate protein release were also observed through fluorescence resonance energy transfer experiments involving GroEL and labeled substrate protein. Analysis of the energy transfer data revealed an interesting relationship between substrate protein displacement and a specific structural transition in the GroEL apical domain.     

Synthesis and structure of poly(binaphthyl salen manganese complex) and its application to asymmetric epoxidation

Poly(binaphthyl salen manganese complex)es 3-Mn were synthesized from a 3,3'-diformylbinaphthol derivative, alpha,omega-diamines, and Mn(OAc)2. Their helical structures were well-supported by their IR, UV, and CD spectra. The catalysis of 3-Mn in an asymmetric epoxidation was investigated.