Genetic mapping of genes for susceptibility to black spot disease in Japanese pears.

Black spot disease, which is caused by the Japanese pear pathotype of Alternaria alternata (Fr.) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We identified the exact positions and linkage groups (LGs) of the genes for susceptibility to black spot in the Japanese pear (Pyrus pyrifolia Nakai) cultivars 'Osa Nijisseiki' (gene Ani) and 'Nansui' (gene Ana). Segregation of susceptibility and resistance fitted the expected ratio of 1:1 in progeny of 'Nansui' but showed a slight distortion in progeny of 'Osa Nijisseiki'. We mapped the genes for susceptibility to black spot in both populations using a genome scanning approach. The simple sequence repeat (SSR) markers CH04h02 and CH03d02 showed tight linkage to Ani and Ana. Although Ani and Ana are derived from different sources, both genes are located at the top region of LG 11. Information about the positions of the susceptibility genes and the molecular markers linked to them will be useful for marker-assisted selection in pear breeding programs.

Genetic mapping of the pear scab resistance gene Vnk of Japanese pear cultivar Kinchaku.

Pear scab (caused by Venturia nashicola) is one of the most harmful diseases of pears, especially Japanese and Chinese pear species. The molecular identification and early selection of resistant plants could greatly improve pear breeding. We have identified the position of the scab resistance gene, designated Vnk in an indigenous Japanese pear cultivar Kinchaku, within the pear genome by using simple sequence repeat (SSR) markers derived from pear and apple. The position of Vnk was identified in the central region of linkage group 1 of Kinchaku. Several amplified fragment length polymorphism (AFLP) markers linked to Vnk were obtained by bulked segregant analysis. Among them, the AFLP marker closest to Vnk was converted into a sequence tagged site (STS) marker. Four random amplified polymorphic DNA (RAPD) markers previously found to be loosely associated with Vnk (Iketani et al. 2001) were successfully converted into STS markers. Six markers (one SSR Hi02c07 and five STSs converted from AFLP and RAPD) showed tight linkages to Vnk, being mapped with distances ranging from 2.4 to 12.4 cM. The SSR CH-Vf2, which was isolated from a BAC clone of the contig containing the apple scab gene Vf, was mapped at the bottom of linkage group 1 in Kinchaku, suggesting that the Vnk and Vf loci are located in different genomic regions of the same homologous linkage group.

Identification of two loci for resistance to clubroot (Plasmodiophora brassicae Woronin) in Brassica rapa L.

In an analysis of 114 F(2) individuals from a cross between clubroot-resistant and susceptible lines of Brassica rapa L., 'G004' and 'Hakusai Chukanbohon Nou 7' (A9709), respectively, we identified two loci, Crr1 and Crr2, for clubroot (caused by Plasmodiophora brassicae Woronin) resistance. Each locus segregated independently among the F(2) population, indicating that the loci reside on a different region of chromosomes or on different chromosomes. Genetic analysis showed that each locus had little effect on clubroot resistance by itself, indicating that these two loci are complementary for clubroot resistance. The resistance to clubroot was much stronger when both loci were homozygous for resistant alleles than when they were heterozygous. These results indicate that clubroot resistance in B. rapa is under oligogenic control and at least two loci are necessary for resistance.

Isolation and characterization of microsatellites in Brassica rapa L.

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.

Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens.

The rice chitinase gene (RCC2), classified as class I chitinase, was introduced into the somatic embryos of grapevine (Vitis vinifera L. cv. Neo Muscut) by Agrobacterium infection. After co-cultivation with Agrobacterium, somatic embryos were transferred onto Murashige and Skoog hormone-free medium supplemented with 50 mg/l kanamycin. Transformed secondary or tertiary embryos were selected, and then more than 20 transgenic plantlets were recovered. Two transformants showed enhanced resistance against powdery mildew caused by Uncinula necator. Few disease symptoms were observed on leaves of these transformants compared with those of the non-transformant, although browning and necrotic symptoms, which seemed to constitute a hypersensitive reaction, were observed. Scanning electron microscopic observation revealed that conidial germination, mycelial growth and conidial formation were suppressed on the leaf surface of the transformant. The transgenic grapevines obtained also exhibited slight resistance against Elisinoe ampelina inducing anthracnose, resulting in a reduction in disease lesions. The relationship between the expression of the foreign chitinase gene and the disease resistance is discussed.

Allelic loss of chromosome 16q in endometrial cancer: correlation with poor prognosis of patients and less differentiated histology.

Deletion of certain chromosomal regions can be demonstrated in malignant cells. Chromosome 16q is one of the regions where allelic loss is frequently detected in carcinoma of the breast and many other tumors, suggesting that gene(s) which retard tumor growth may exist here. To elucidate the clinicopathological significance of chromosome 16q, loss of heterozygosity (LOH) was investigated using microsatellite polymorphism analysis in 58 patients with endometrial lesions (50 with endometrial carcinoma and 8 who had hyperplasia with or without atypia). When 11 regions of chromosome 16q were examined, LOH was found in 20 patients with carcinoma (40%) and none of the patients with hyperplasia. The tumors of 9 of the 20 patients (45%) showed total loss of 16q, while the others (55%) showed partial deletion. Tumors with LOH were histologically less differentiated than those without LOH (P = 0.038, chi2 test). Patients with tumors showing LOH of 16q had a worse prognosis than those without LOH according to Kaplan-Meier survival analysis (P=0.0158, log-rank test). In addition, LOH of 16q showed a significant relationship to prognosis by Cox regression analysis. Deletion mapping of 16q demonstrated that two regions (16q22.1 and 16q22.2-23.1) were frequently involved. Patients with 16q22.1 LOH had a poorer prognosis than those with intact 16q22.1 (P=0.0003, log-rank test). These findings suggest that gene(s) of which defect is possibly related to the aggressiveness of endometrial cancer are localized on a limited region of 16q that includes 16q22.1.

Mutation and allelic loss of the p53 gene in endometrial carcinoma. Incidence and outcome in 92 surgical patients.

BACKGROUND: Alterations of the p53 gene are involved in the development of diverse human malignancies, but their incidence and clinicopathologic features are still not well characterized for endometrial carcinoma. METHODS: To investigate the clinicopathologic significance of p53, mutations and loss of heterozygosity (LOH) in endometrial carcinoma in 92 patients with this disease were examined. RESULTS: Mutations of p53 were detected in 20 (22%) of the 92 patients with carcinoma, and LOH was detected in 23 (32%) of the 72 patients in whom heterozygosity of the gene was available. There was a significant correlation between the occurrence of mutation and LOH. Mutations and LOH were more frequent in patients with Grade 3 tumors than in those with Grades 1 and 2 tumors (P = 0.0498, P = 0.0051, respectively). Patients with LOH had a poorer postoperative survival than those without LOH (P = 0.0022, log-rank test), and patients with both LOH and mutation showed the worst prognosis (P < 0.0001, log rank test). Loss of heterozygosity of the p53 gene showed a significant relation to prognosis that was independent of tumor stage, histologic grade, and muscular invasion. CONCLUSIONS: Mutation and LOH of the p53 gene are prognostic indicators in patients with endometrial carcinoma, suggesting that alterations of p53 may play an important role in the development of this cancer.

Efficacy of lateral retinacular release for painful bipartite patella.

We treated painful bipartite patella with a modified lateral retinacular release technique in 15 patients (16 knees). Bony union of the separated fragment and the patella was obtained in 15 of 16 knees within 8 months of surgery. Sustained traction acting on the patella laterally and proximally is presumed to cause the pain. The surgical technique to reduce this force proved effective not only in relieving the pain but also in achieving bony union. Painful bipartite patella can lead to excessive lateral pressure syndrome or patellar compression syndrome, these complications can be effectively treated by this surgical technique. In contrast to conventional treatments, such as excision of smaller fragments or osteosynthesis to achieve bony union, the modified lateral retinacular release technique is easy to perform and provides an effective means for relieving patellofemoral pain and achieving bony union.

Patterns of failure and survival in locally advanced carcinoma of the uterine cervix treated with high dose-rate intracavitary irradiation system.

We retrospectively analyzed 71 patients with locally advanced carcinoma of the uterine cervix treated by irradiation using high dose-rate intracavitary brachytherapy between 1978 and 1985. Seven patients were Stage IIIa, 46 Stage IIIb, and 18 Stage IVa. Five-year survivals for Stage IIIa, IIIb, and IVa were 71.4, 60.9, and 16.7%, respectively. An analysis of patterns of failure demonstrated that loco-regional recurrences were observed in 1 (14.3%) for Stage IIIa, 6 (13.0%) for Stage IIIb, and 9 (50.0%) for Stage IVa. The incidence of recurrence outside the pelvis observed in Stage IIIb patients (7 para-aortic nodes, 5 distant metastases) was much higher than that of local recurrence. Five patients (7.0% of the total: 1 with Stage IIIa, 3 with Stage IIIb, 1 with Stage IVa) required surgery to manage the complications. These data suggest that a high dose-rate intracavitary irradiation system is an effective tool for the treatment of cervical cancer. Further efforts to control metastatic lesions outside the pelvis are required for patients with Stage IIIb. To increase a loco-regional control rate for patients with Stage IVa disease, it is important to give additional treatment such as chemotherapy in conjunction with radiation therapy.

[Clinico-pathological study and appraisal of treatment for endometrial cancer at the Ehime University Hospital].

Clinical and pathological studies were performed on 51 cases with endometrial cancer. The results are as follows: The average age was 58.9 years and the range 41 to 80 years. Forty-one (80.4%) patients were postmenopausal and the average menopausal age was 49.1 years. All cases were symptomatic and in 35 (76.5%) cases the postmenopausal bleeding was noted as a chief complaint. The chief complications were obesity (37.5%), hypertension (25.0%), infertility (13.7%) and diabetes mellitus (9.4%). Of 51 cases, 36(70.6%) were in Stage I, 7(13.7%) in Stage II, 6(11.8%) in Stage III and 2(3.9%) in Stage IV. When the depth of the invasion was classified into 3 grades, less than 1/3 of the muscular layer, between 1/3 and 2/3, and over 2/3 in 48 cases examined, they were observed in 20(41.7%), 10(20.8%) and 18 cases (37.5%) respectively. There were 6 cases (15%) with lymph node involvement in 40 cases examined. Those in which the depth of invasion was over 2/3, had a significantly higher incidence of lymph node involvement. The five year cumulative survival rate was 66.7%(14/21) for all cases. The clinical stage and age of the patients had a significant correlation with the prognosis of endometrial cancer, but the histological grades, the depth of invasion, and lymph node involvement did not demonstrate the a significant correlation in prognosis in this study. The five year survival rate for the group treated by modified pan-hysterectomy with pelvic lymphadenectomy was 92.3% which was significantly higher than the 25% of the group treated by simple hysterectomy or than the 0% of non-surgical group.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino acid replacement in a mutant lipoprotein of the Escherichia coli outer membrane.

The primary structure of a mutant lipoprotein of the outer membrane of Escherichia coli was investigated. This mutant was previously described as a mutant that forms a dimer of the lipoprotein by an S-S bridge (H. Suzuki et al., J. Bacteriol. 127:1494-1501, 1976). The amino acid analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of tryptic peptides, it was found that the arginine residue at position 57 was replaced with a cysteine residue. The amino terminal structure of the mutant lipoprotein was found to be glycerylcysteine, as in the case of the wild-type lipoprotein. The present results show that the mutation that was previously determined to map at 36.5 min on the E. coli chromosome occurred in the structure gene (lpp) for the lipoprotein. This was further confirmed by the fact that a merodiploid carrying both lpp+ and lpp produces not only the wild-type lipoprotein but also the mutant lipoprotein.

Novel mutation that causes a structural change in a lipoprotein in the outer membrane of Escherichia coli.

A novel mutation which caused a structural change in a lipoprotein in the outer-membrane has been found in Escherichia coli K-12. The lipoprotein of the wild-type strain is known to have a peculiar amino terminal structure: glycerylcysteine with two fatty acids attached by ester linkages and one fatty acid by an amide linkage. In contrast to the wild-type lipoprotein, the mutant lipoproteins is isolated from the E. coli envelope as a dimer of molecular weight of about 15,000. The dimer can be reduced by mercaptoethanol to the lipoprotein monomer of molecular weight of about 7,500. The monomer has a free thiol group which is susceptible to monoiodacetie mutant lipoprotein is extremely low in comparison with that into the wild-type lipoprotein. These results suggest that the mutant is defective in transferring a glycerol group to the thiol group of the amino terminal cysteine residue of the lipoprotein. The gene responsible for this modification reaction has been located at 36.5 min on the E. coli chromosome.