Rapid detection of S-glycoproteins of self-incompatible crucifers using Con-A reaction

Summary As S-specific glycoproteins react with concanavalin A (ConA), they could be stained sensitively with ConA-FITC after cellulose acetate isoelectric focusing of stigma proteins in self-incompatible crucifers. By this method the S genotype of an individual plant could be inferred within 4 h using 4 flowers. This simple and rapid method may be applicable for describing S genotypes in practical F₁ breeding.     

Distribution of S haplotypes in commercial cultivars of Brassica rapa

Self‐incompatibility in Brassicaceae plants is sporophytically controlled by a single multi‐allelic locus (S locus), which contains at least three highly polymorphic genes expressed in the stigma (SLG and SRK) and in the pollen (SCR/SP11). Using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) analysis with SXG‐specific primer pairs, the S haplotypes of F₁ hybrid and open‐pollinated commercial cultivars of Brassica rapa were identified. The number of S haplotypes detected in the F₁ hybrid cultivars of Chinese cabbage, komatsuna, pak‐choi, turnip, open‐pollinated cultivars of Chinese cabbage and turnip were 9, 9, 4, 11, 13 and 12, respectively. Nine of them had different PCR‐RFLP profiles from those of the S‐tester lines that determined the SLG sequences. Four SLG sequences in the F₁ hybrid cultivars were determined and named S⁵³, S⁵⁴, S⁵⁵ and S⁵⁶, respectively. It is demonstrated that the PCR‐RFLP analysis using specific primer pairs of SLG and SRK is useful for identification of the S haplotypes, in both, S homozygous and S heterozygous plants of B. rapa. The possibility of using this method routinely in breeding programmes, and in the evaluation of F₁ hybrid seed purity, is discussed.     

Mineral composition of peach leaves affected by iron chlorosis

Abstract

The effect of Fe chlorosis on the mineral composition of field grown peach tree leaves was studied in two different areas. No significant differences in total Fe content were found, whereas 2,2’ bipyridyl extractable Fe, K and the K/Ca ratio were significantly affected in both experiments. Phosphorus and the P/Fe ratio were significantly affected only in one experiment.     

Haplotype C of growth hormone (GH) gene in Japanese Black cattle: Structure of GH protein and a novel method for detection of the gene

From a series of studies on single nucleotide polymorphisms (SNPs) in the bovine growth hormone (GH) gene of Japanese Black cattle, the type‐C (127^Val and 172^Met) that is specific for this breed has been intensively focused upon because of the economic importance for carcass traits, such as intramuscular oleic acid contents. In the present study, we intended to analyze the 3‐D structure of GH of haplotype C, and developed a novel method to detect the type C gene. Three‐D analysis of the type C protein showed that the amino acid residues (127^Val and 172^Met), which are present in the third and fourth helixes, respectively, and are important for binding with GH receptors, are shifted to deeper positions in the molecule compared with that for type A (127^Leu and 172^Thr), implying the alteration of binding interaction with receptors. A novel, efficient and cost‐effective method (Dot‐blot‐SNP technique) for type C genotyping was successfully established, of which the basal method was a reported genotyping of SNPs for a large number of plants, reducing the cost to 10% or less of direct sequencing.     

Mediators of nonadrenergic, noncholinergic relaxation in Sprague Dawley rat intestine: comparison with the mediators of other strains

Participation of nitric oxide, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) in nonadrenergic, noncholinergic (NANC) relaxation of longitudinal muscle of various intestinal regions in Sprague Dawley rats (8-week-old) was studied in vitro. Nitric oxide was suggested to participate in NANC relaxation of every intestinal region studied. But the participation was partial and its extent varied among the regions: significant in the proximal colon and rectum, and moderate in the jejunum, ileum and distal colon. Participation of PACAP in NANC relaxation was suggested only in the distal colon, while that of VIP was not detected in any of regions. Results obtained in the present study indicate that extent of participation of nitric oxide in NANC relaxation in Sprague Dawley rat intestine is more significant than those of other strains, Wistar and Wistar-ST.     

Accounting for numbers of expressions of specific genotypes using a modified gene-flow method

The objective of this study was to develop a modified discounted gene-flow method and calculate numbers of cumulative discounted expressions (CDE) and value of using a Japanese Black cattle bull carrying specific alleles of interest. The discounted gene-flow method was modified to consider not only parent's genotypes but also the allele frequency in the herd. Input parameters representing a typical situation in a Japanese Black cattle herd were used to calculate the CDE and the value of using sires genotyped for recessive genes assuming biallelic loci (A and a) and a herd at Hardy–Weinberg equilibrium. The quantitative trait assumed was marbling and the effect of genotype aa was 1 Beef Marbling Standards (BMS) above that of an animal with either genotype Aa or AA. The effects of gene frequency and discount and survival rates on the CDE were determined for different genotypes of sires. Benefits from using either aa sires or Aa sires above that of using unknown sires at various gene frequencies were also determined. The CDE of aa sires were larger than those of Aa sires under all gene frequencies. The differences in the CDE between aa and Aa sires ranged from 34% at gene frequency of 0.7 to 71% at gene frequency of 0.01. An increase in the discount rates and a decrease in survival rates were associated with a decrease in expressions in cases using aa and Aa sires. The benefits of using aa sires were high when the gene frequency in the herd was 0.5. The benefit of aa sires were approximately 3023 yen more than that for unknown sires in the population when the gene frequency was 0.5. At gene frequencies higher than 0.5, use of Aa sires was not beneficial. This study has demonstrated how the gene frequencies in the herd and the genotypes of sires are critical in determining the benefits of using single recessive gene in the Japanese Black cattle.     

Structural differences between myofibrillar protein,paratropomyosin, and tropomyosin as revealed by high‐performance liquid chromatography

Paratropomyosin (PTM) composes myofibril functions to weaken the rigor linkages formed between actin and myosin during postmortem aging of muscles. PTM has the similar physico‐chemical properties as tropomyosin (TM) that is a regulatory protein of myofibrils. So far, it is unclear whether PTM is definitely different from TM, because the primary structure of PTM has not been determined yet. The aim of this study was to clarify structural difference of PTM from TM. PTM was prepared by column chromatography immediately after slaughter from broiler breast muscle, and purified by high‐performance liquid chromatography (HPLC). Purified PTM was successfully separated from TM, and the recovered PTM molecule was reduced with dithiothreitol to separate again by HPLC. Two subunits were obtained and peptides from each digested subunit by V8 protease were recovered by HPLC, and then amino acid sequences of the peptides were analyzed by protein sequencing. As a result, some amino acid residues were replaced from that of TMα1 isoform which is the major isoform of TM, and also was different between the two subunits. Therefore, it is concluded that PTM clearly differs from TM and it is suggested that functional difference in PTM from TM is attributed to amino acid replacements in subunits composing PTM.     

Cloning and characterization of the glutamate dehydrogenase gene in Streptococcus bovis

Streptococcus bovis, an etiologic agent of rumen acidosis in cattle, is a rumen bacterium that can grow in a chemically defined medium containing ammonia as a sole source of nitrogen. To understand its ability to assimilate inorganic ammonia, we focused on the function of glutamate dehydrogenase. In order to identify the gene encoding this enzyme, we first amplified an internal region of the gene by using degenerate primers corresponding to hexameric family I and NAD(P)⁺ binding motifs. Subsequently, inverse PCR was used to identify the whole gene, comprising an open reading frame of 1350 bp that encodes 449 amino acid residues that appear to have the substrate binding site of glutamate dehydrogenase observed in other organisms. Upon introduction of a recombinant plasmid harboring the gene into an Escherichia coli glutamate auxotroph lacking glutamate dehydrogenase and glutamate synthase, the transformants gained the ability to grow on minimal medium without glutamate supplementation. When cell extracts of the transformant were resolved by blue native polyacrylamide gel electrophoresis followed by activity staining, a single protein band appeared that corresponded to the size of S. bovis glutamate dehydrogenase. Based on these results, we concluded that the gene obtained encodes glutamate dehydrogenase in S. bovis.