Genetic diagnosis of band 3 deficiency and sexing in bovine preimplantation embryos

Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3(Bov.Yamagata), is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3(Bov.Yamagata). These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals.     

Expression of tumor necrosis factor-alpha in IgM+ B-cells from bovine leukemia virus-infected lymphocytotic sheep

Tumor necrosis factor (TNF)-alpha is thought to be one of the cytokines that account for bovine leukemia virus (BLV)-induced B-cell lymphoproliferative disorder, however, information on TNF-alpha expression in B-cells is limited. In this study, the expression of TNF-alpha in IgM(+) B-cells from BLV-infected sheep with or without lymphocytosis was determined. Freshly isolated IgM(+) B-cells from three sheep with lymphocytosis constitutively transcribed TNF-alpha mRNA. Although TNF-alpha mRNA expression in IgM(+) B-cells was transiently up-regulated after cell culture, TNF-alpha mRNA expression was markedly higher in lymphocytotic sheep when compared to that of non-lymphocytotic sheep or uninfected sheep. Expression of membrane-bound TNF-alpha on IgM(+) B-cells was also augmented in lymphocytotic sheep. TNF-alpha expression in lymphocytotic sheep may support the proliferation of B-cells.     

Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.)

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) genes Crr1 and Crr2 are effective against the mild Plasmodiophora brassicae isolate Ano-01 and the more virulent isolate Wakayama-01, but not against isolate No. 14, classified into pathotype group 3. ‘Akiriso’, a clubroot-resistant F₁ cultivar, showed resistance to isolate No. 14. To increase the durability of resistance, we attempted to identify the CR locus in ‘Akiriso’. CR in ‘Akiriso’ segregated as a single dominant gene and was linked to several molecular markers that were also linked to CRb, a CR locus from cultivar ‘CR Shinki’. We developed additional markers around CRb and constructed partial genetic maps of this region in ‘Akiriso’ and ‘CR Shinki’. The positions and order of markers in the genetic maps of the two cultivars were very similar. The segregation ratios for resistance to isolate No. 14 in F₂ populations derived from each of the two cultivars were also very similar. These results suggest that the CR locus in ‘Akiriso’ is CRb or a tightly linked locus. The newly developed markers in this study were more closely linked to CRb than previously reported markers and will be useful for marker-assisted selection of CRb in Chinese cabbage breeding.     

Furan-2,5-dimethylene-tethered bis-imidacloprid insecticide conferring high potency

Bis-imidacloprid (bis-IMI) analogues with suitable alkylene spacers have plant-systemic insecticidal properties. The alkylene-tethered bis-IMI binds in a unique mode to the insect nicotinic acetylcholine receptor (nAChR) wherein the chloropyridine moieties are embraced by two distinct and distant domains. The heptamethylene spacer optimally bridges these two subsites, yet the linker itself binds in a relatively nonspecific manner. This investigation examines the hypothesis that a bis-IMI analogue with a heteroaromatic tether, which undergoes specific interaction(s) with the newly recognized receptor cavity, may enhance the potency relative to those of the alkylene-tethered derivatives. Remarkably, a novel bis-IMI with a furan-2,5-dimethylene fulcrum showed highest receptor potency and insecticidal activity among the analogues with various chemotype spacers. The nAChR structural model, simulating the binding site interactions of the furan-2,5-dimethylene-tethered bis-IMI, reveals that the furan ring is nestled in a hydrophobic pocket, consisting of three aromatic amino acids, and is stabilized via hydrogen bonding.     

RAPD markers linked to microspore embryogenic ability in Brassica crops

In order to identify the markers linked to microspore embryogenic ability in Brassica crops, RAPD segregation analyses were performed in a microspore-derived (MD) population and a F₂ population derived from F₁between ‘Ho Mei’ (high responsive parent in microspore embryogenesis) and ‘269’ (low responsive parent) in Chinese cabbage, and between ‘Lisandra’ (high responsive parent) and ‘Kamikita’ (low responsive parent) in oil seed rape. After 230 and 143 primers were screened, a total of 148 and 52markers were detected to be polymorphic between the parents in Chinese cabbage and oilseed rape, respectively. Twenty-seven percent of the markers in the MD population showed a significant segregation distortion in both crops. Of the markers showing segregation distortion in the MD population, 71–75% of the markers followed the expected Mendelian segregation ratio in the F₂ population. When the relationships between such distorted markers and microspore embryogenesis of the F₂ population were examined, 7 and 3 markers were identified to be associated with embryogenic ability in Chinese cabbage and oilseed rape, respectively. These markers showed additive effects on embryo yields, and the plants having more alleles of the high responsive parent produced higher embryo yields. These markers maybe useful in marker-assisted selection for improving microspore responsiveness straits in Brassica crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genotypic Variation in Shoot Cadmium Concentration in Rice and Soybean in Soils with Different Levels of Cadmium Contamination

A pot experiment was conducted to investigate whether the shoot cadmium (Cd) concentration in 11 rice and 10 soybean cultivars varied among 4 soils with different levels of Cd contamination. Significant differences in shoot Cd concentration were found among rice or soybean cultivars grown in the 4 soils. The ranking of the rice cultivars for the shoot Cd concentration varied considerably among the soils. On the other hand, the soybean cultivars were ranked similarly in terms of shoot Cd concentration in the 4 soils. Significant and positive correlations were found between the Cd and Zn concentrations and between the Cd and Mn concentrations in the shoot of rice cultivars, when they were grown in 2 soils with relatively moderate levels of Cd contamination. The shoot Cd concentration in the soybean cultivars, however, was not correlated with the concentrations determined for any of the metals (Zn, Mn, Cu, and Fe) across the 4 soils. Significant and positive correlations between the concentrations of Cd in younger shoots and mature seeds were detected among the soybean cultivars in 2 soils used, unlike among the rice cultivars, indicating that it may be difficult to evaluate the genotypic variation in seed Cd concentration using relatively younger shoots in the case of rice. These results revealed that genotypic differences in shoot Cd concentration in rice or soybean are variable or invariable among soils, respectively.     

Stimulatory effect of cyanidin 3-glycosides on the regeneration of rhodopsin

Anthocyanins have been suggested to improve visual functions. This study examined the effect of four anthocyanins in black currant fruits on the regeneration of rhodopsin using frog rod outer segment (ROS) membranes. Cyanidin 3-glycosides, glucoside and rutinoside, stimulated the regeneration, but the corresponding delphinidins showed no significant effect. The formation of a regeneration intermediate was suggested to be accelerated by cyanidin 3-rutinoside. Their effects on the cGMP-phosphodiesterase activity in the ROS membranes were also investigated but found to be negligible. It was concluded that the major effect of anthocyanins in rod photoreceptors is on the regeneration of rhodopsin.