Genomic analysis of the rhg1 locus: candidate genes that underlie soybean resistance to the cyst nematode.

The rhg1 gene or genes lie at a recessive or co-dominant locus, necessary for resistance to all Hg types of the soybean (Glycine max (L.) Merr.) cyst nematode (Heterodera glycines I.). The aim here was to identify nucleotide changes within a candidate gene found at the rhg1 locus that were capable of altering resistance to Hg types 0 (race 3). A 1.5 +/- 0.25 cM region of chromosome 18 (linkage group G) was shown to encompass rhg1 using recombination events from four near isogenic line populations and nine DNA markers. The DNA markers anchored two bacterial artificial chromosome (BAC) clones 21d9 and 73p6. A single receptor like kinase (RLK; leucine rich repeat-transmembrane-protein kinase) candidate resistance gene was amplified from both BACs using redundant primers. The DNA sequence showed nine alleles of the RLK at Rhg1 in the soybean germplasm. Markers designed to detect alleles showed perfect association between allele 1 and resistance to soybean cyst nematode Hg types 0 in three segregating populations, fifteen additional selected recombination events and twenty-two Plant Introductions. A quantitative trait nucleotide (QTN) [corrected] in the RLK at rhg1 was inferred that alters A87 to V87 in the context of H274 rather than N274. [corrected] Contiguous DNA sequence of 315 kbp of chromosome 18 (about 2 cM) contained additional gene candidates that may modulate resistance to other Hg-types including a variant laccase, a hydrogen-sodium ion antiport and two proteins of unknown function. A molecular basis for recessive and co-dominant resistance that involves interactions among paralagous disease-resistance genes was inferred that would improve methods for developing new nematode-resistant soybean cultivars.

Three minimum tile paths from bacterial artificial chromosome libraries of the soybean (Glycine max cv. 'Forrest'): tools for structural and functional genomics.

BACKGROUND: The creation of minimally redundant tile paths (hereafter MTP) from contiguous sets of overlapping clones (hereafter contigs) in physical maps is a critical step for structural and functional genomics. Build 4 of the physical map of soybean (Glycine max L. Merr. cv. 'Forrest') showed the 1 Gbp haploid genome was composed of 0.7 Gbp diploid, 0.1 Gbp tetraploid and 0.2 Gbp octoploid regions. Therefore, the size of the unique genome was about 0.8 Gbp. The aim here was to create MTP sub-libraries from the soybean cv. Forrest physical map builds 2 to 4. RESULTS: The first MTP, named MTP2, was 14,208 clones (of mean insert size 140 kbp) picked from the 5,597 contigs of build 2. MTP2 was constructed from three BAC libraries (BamHI (B), HindIII (H) and EcoRI (E) inserts). MTP2 encompassed the contigs of build 3 that derived from build 2 by a series of contig merges. MTP2 encompassed 2 Gbp compared to the soybean haploid genome of 1 Gbp and does not distinguish regions by ploidy. The second and third MTPs, called MTP4BH and MTP4E, were each based on build 4. Each was semi-automatically selected from 2,854 contigs. MTP4BH was 4,608 B and H insert clones of mean size 173 kbp in the large (27.6 kbp) T-DNA vector pCLD04541. MTP4BH was suitable for plant transformation and functional genomics. MTP4E was 4,608 BAC clones with large inserts (mean 175 kbp) in the small (7.5 kbp) pECBAC1 vector. MTP4E was suitable for DNA sequencing. MTP4BH and MTP4E clones each encompassed about 0.8 Gbp, the 0.7 Gbp diploid regions and 0.05 Gbp each from the tetraploid and octoploid regions. MTP2 and MTP4BH were used for BAC-end sequencing, EST integration, micro-satellite integration into the physical map and high information content fingerprinting. MTP4E will be used for genome sequence by pooled genomic clone index. CONCLUSION: Each MTP and associated BES will be useful to deconvolute and ultimately finish the whole genome shotgun sequence of soybean.

Genomic analysis of a region encompassing QRfs1 and QRfs2: genes that underlie soybean resistance to sudden death syndrome.

Candidate genes were identified for two loci, QRfs2 providing resistance to the leaf scorch called soybean (Glycine max (L.) Merr.) sudden death syndrome (SDS) and QRfs1 providing resistance to root infection by the causal pathogen Fusarium solani f.sp. glycines. The 7.5 +/- 0.5 cM region of chromosome 18 (linkage group G) was shown to encompass a cluster of resistance loci using recombination events from 4 near-isogenic line populations and 9 DNA markers. The DNA markers anchored 9 physical map contigs (7 are shown on the soybean Gbrowse, 2 are unpublished), 45 BAC end sequences (41 in Gbrowse), and contiguous DNA sequences of 315, 127, and 110 kbp. Gene density was high at 1 gene per 7 kbp only around the already sequenced regions. Three to 4 gene-rich islands were inferred to be distributed across the entire 7.5 cM or 3.5 Mbp showing that genes are clustered in the soybean genome. Candidate resistance genes were identified and a molecular basis for interactions among the disease resistance genes in the cluster inferred.

[Thoracoscopic surgery using a new bronchial blocker].

We report use of a new bronchial blocker through a single-lumen endotracheal tube to achieve one-lung ventilation to perform thoracoscopic operation in patients in whom placement of the double-lumen tube failed and difficult intubation is predicted. The bronchial blocker tube was placed into the aimed bronchus under the bronchoscopic vision and the cuff of the blocker was inflated to achieve one-lung ventilation. In all of the 4 patients, the bronchial blocker could be inserted and placed safely, quickly, and exactly under the fiberoptic flexible bronchoscopic vision to perform thoracoscopic operation without any complications. The new bronchial blocker tube through the indwelling endotracheal tube may have advantages in situations where placement of double-lumen endotracheal tubes is technically impossible or inappropriate. The use of the new bronchial blocker tube will, however, require careful evaluation in larger series.

[Thoracoscopic pericardial resections for malignant pericardial effusions].

We have performed 5 thoracoscopic pericardial resections for malignant pericardial effusions. An initial trocar was placed in the seventh or eighth intercostal space posterior to the midaxillary line. Two additional trocars were placed, usually in the sixth intercostals spaces in the anterior axillary and posterior axillary lines. Using an endoscopic grasping instrument and scissors through the working ports, a pericardial resection was performed. All patients were successfully managed by thoracoscopic pericardial resections. Two of the 5 patients had associated malignant pleural effusions that were able to be managed at the same time by thoracoscopy. The average chest tube duration was 1.8 days. There has been no reaccumulation of pericardial effusions in all patients at an average follow-up of 5 months. The thoracoscopic approach could be minimally invasive and the procedure of choice in performing pericardial resections in selected patients with malignant pericardial effusions who are expected to have a reasonable life expectancy.

[A thoracoscopic technique with fibrin glue and polyglycolic acid mesh for the injured lung during thoracoscopic operation].

The injury of the lung occurred during thoracoscopic operation, especially, of the fragile, severely emphysematous lung is often difficult to treat. A fibrin glue spraying device in which an air compressor was connected to an intravenous catheter was constructed, and connected to a Dupuloject syringe. Fibrin glue was administered by spraying on the target lesion under a thoracoscopic vision. The procedure was done while the tip of the intravenous catheter was held and manipulated with forceps through a thoracoport. And then, the polyglycolic acid (PGA) mesh that had been cut into small pieces was put on the target lesion with forceps. Fibrin glue was sprayed on the lesion again in the above-mentioned manner. When necessary, the procedure was repeated. Our technique during thoracoscopic operation may be easy to manipulate and be able to apply the target lesion uniformly within a short time with a successful repair.

[Thoracoscopic surgery for metastatic lung tumors: computed tomography-guided localization with use of a needle with a suture].

When a metastatic lung tumor is found to be deep to the visceral surface of the pleura, or when it is found to be a small lesion, it may important to help identify the lesion by preoperative methods in order to localize it at the time of thoracoscopic operation. We performed computed tomography-guided localization of metastatic lung tumors with use of a needle with a suture in 11 cases prior to thoracoscopic resection. Placement of the needle, immediately before moving to the operation room were successfully performed in all patients. It took from 16 to 25 minutes (20.3 minutes on average). Complications included minimal pneumothorax in 8 patients, mild airway bleeding in 2, and penetration through the middle lobe to the lower lobe of the right lung in 1. However, treatment was not necessary in any of the patients. Thoracoscopic resections were successfully achieved in all patients. Our technique may be a simpler technique and advantageous for identification of small lesions and lesions deep to the visceral surface of the pleura during the thoracoscopic operation for metastatic lung tumors.

Resistance locus pyramids alter transcript abundance in soybean roots inoculated with Fusarium solani f.sp. glycines.

Soybean Sudden Death Syndrome (SDS) is caused by Fusarium solani f.sp. glycines (Fsg). Six quantitative trait loci (QTLs), each conferring partial resistance to SDS, have been discovered in an Essex x Forrest recombinant inbred line (RIL) population, but their mode of action is not clear. This study aimed to identify genes (ESTs) whose mRNA transcripts were altered in abundance in soybean roots following inoculation of Fsg. Roots of the soybean variety Forrest (four resistance alleles) were inoculated with Fsg, and 14 days later RNA sequences that were differentially expressed relative to uninoculated roots were enriched using suppression subtraction and differential display. The abundance of these RNAs was quantified in inoculated and non-inoculated roots by macroarray hybridizations. A unigene set of 135 ESTs was identified and used in a further macroarray analysis. The abundance of 28 cDNA fragments was increased more than two-fold in inoculated compared to uninoculated roots of RIL 23 (six resistance alleles). In Forrest and Essex (two resistance alleles), the level of only one mRNA was increased two-fold in inoculated roots compared to the uninoculated roots. In Essex most of the mRNAs analyzed decreased in abundance (61/135 showed a two-fold decrease), while in Forrest most mRNA abundances did not change. Among the 28 cDNAs that revealed a two-fold or higher increase in mRNA abundance in RIL 23, 14% code for proteins known to be involved in plant defense, 21% in metabolism, 14% in cell structure and 4% in transport. Unannotated ESTs accounted for 43% of the genes, and 4% of the sequences were previously unknown. The plant defense-related genes that showed a differential response to Fsg inoculation suggested a role for the phenylproponoid pathway in soybean defense against Fsg. In Essex, genes involved in plant defense, cell wall synthesis, ethylene synthesis and metabolism were expressed at lower levels in inoculated roots. The difference in response between the 2-, 4- and 6-gene pyramids suggests that QTLs for SDS resistance serve to delay symptoms or confer resistance by maintaining or increasing the expression of specific genes after inoculation/infection.

Differentially abundant mRNAs in rat liver in response to diets containing soy protein isolate.

Soy diets influence cell growth, regulate lipid metabolism to lower blood cholesterol, and prevent bone losses. These biological effects are most likely due to effects of soy phytochemicals on the expression of genes. In this study, we fed 12 female obese Zucker rats (fa/fa) with a low- or a high-isoflavone soy protein diet and compared the gene expression with animals on a casein diet. Rat livers were compared by differential display of mRNA, and 62 unique sequences were identified. The change in mRNA abundance of these sequences was quantified by cDNA macroarray analysis. Thirty-three mRNAs showed more than twofold increase in abundance on soy diets compared with the control. The corresponding genes include carnitine palmitoyltransferase I, stromal cell-derived factor 1, a protein associated with MYC mRNA, basic transcription element binding protein, and expressed sequence tags (ESTs) of unknown function. Twenty-nine mRNAs showed a less than twofold change in abundance in the two diet treatments. For majority of the genes identified, there was not significant difference between the low- and high-isoflavone diet treatments. Therefore, the contrast between soy protein and casein caused the changes observed in mRNA abundance.

[Initial use of the newly developed voice-controlled robot system for a solitary pulmonary arterio-venous malformation].

We herein report an initial experience of thoracoscopic surgery for a solitary arterior-venous malformation (PAVM) with the AESOP 3000 HR voice-controlled robot to hold a thoracoscope. A 52-year-old woman was hospitalized due to a transient loss of consciousness. A brain magnetic resonance image and electroencephalogram (EEG) were normal. A chest computed tomogram indicated a suspicion of PAVM. The definite diagnosis of the PAVM was made by the 3-dimensional computed tomogram and pulmonary angiography. Thoracoscopic operation with the voice-controlled robot (AESOP 3000 HR) was conducted. The operating staffs were able to assemble the AESOP 3000 HR robotic system safely and quickly without difficulty. The robot functioned without problems throughout the procedure. No complications or events related or unrelated to the maneuvers of the robot during the operation were noted. The procedure of the pulmonary resection with the voice-controlled robot was successfully preformed by a single surgeon. The operating time was 110 minutes, and the volume of the intraoperative bleeding was 10 g. The postoperative course was uneventful. The use of the AESOP 3000 HR robot may be more convenient and friendly in thoracoscopic procedure.

A pyramid of loci for partial resistance to Fusarium solani f. sp. glycines maintains Myo-inositol-1-phosphate synthase expression in soybean roots.

Myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) converts glucose 6-phosphate to myo-inositol 1-phosphate in the presence of NAD(+). It catalyzes the first step in the synthesis of myo-inositol and pinitol, and is a rate limiting step in the de novo biosynthesis of inositol in eukaryotes. Therefore, MIPS is involved in biotic and abiotic stress via Ca(2+) signalling. Seedlings of four soybean genotypes were inoculated with Fusarium solani f. sp. glycines, the causative agent of sudden death syndrome (SDS), and differentially abundant mRNAs were identified by differential display. The genotypes carried either zero, two, four or six alleles of the quantitative trait loci (QTLs) that control resistance to SDS in an additive manner. The mRNA abundance of MIPS did not decrease following inoculation in a recombinant inbred line (RIL 23) containing all six resistance alleles of the QTLs conferring resistance to SDS of soybean. However, the abundance of MIPS mRNA was decreased in genotypes containing four, two or no resistance alleles. The specific activity of the MIPS enzyme in vitro followed the same pattern across genotypes. The IP(3) content in the inoculated roots of genotypes with two, four or six resistance alleles were higher compared to the non-inoculated root. The results suggests that a non-additive effect on transcription and translation of MIPS is established in RIL 23 roots by pyramiding six QTLs for resistance to SDS. A role of MIPS in the partial resistance or response of soybean roots to F. solani infection is suggested.

Low incidence of a nucleotide sequence alteration of the neurofibromatosis 2 gene in human breast cancers.

We investigated aberrations of the neurofibromatosis type 2 (NF2) gene in breast tumors of 60 patients by single-strand conformation polymorphism analysis of polymerase chain reaction products followed by nucleotide sequencing. We detected a tumor-specific single-base substitution in codon 398 in exon 12 of the gene, resulting in an alteration of a single amino acid, in DNA from a breast cancer sample. The results indicated possible but infrequent involvement of mutations of the tumor suppressor NF2 gene in human breast cancers.

Association of p53 gene mutations with short survival in pancreatic adenocarcinoma.

Mutations of the p53 gene have been found in a variety of human cancers and are implicated in the biologic functions of cancer. To investigate the clinical implications of p53 mutations in pancreatic adenocarcinoma, we examined the association of mutations of the p53 gene with patients' prognosis. Single-strand conformational polymorphism analysis and direct DNA sequencing were used to detect p53 gene mutations in 37 pancreatic adenocarcinomas. p53 gene mutations were detected in 16 (43%) of the 37 pancreatic adenocarcinomas. Direct sequencing did not reveal preferential clustering at any specific codon. There was no significant association of the presence of p53 gene mutations with histologic types, extent of tumor invasion, the presence of lymph node metastasis, or tumor stage. Univariate analysis showed that survival of patients with p53-gene-mutated tumors was significantly poorer than that of patients with p53-gene-nonmutated tumors (P = 0.02). Cox's multivariate analysis of ten clinicopathologic features including p53 gene mutations revealed that presence of p53 gene mutations (P = 0.026) and curativity of operation (P = 0.014) were independent predictors of survival. Furthermore, the survival of patients with p53-gene-mutated tumor was significantly poorer than that of patients with p53-gene-nonmutated tumors, both in patients who underwent curative operation (P = 0.04) and in patients who underwent non-curative operation (P = 0.01). These results suggested that mutations of the p53 gene might play an important role in cancer aggressiveness and could be a clinically useful predictor of prognosis in patients with pancreatic adenocarcinoma.