Integration of linkage and chromosome maps of red clover (Trifolium pratense L.).

Red clover (Trifolium pratense L.) is a forage legume and an allogamous diploid plant (2n = 14; 440 Mb). Here, we examine the 7 prometaphase chromosomes of red clover using fluorescence in situ hybridization (FISH) with ribosomal RNA sequences, pericentromeric and telomeric repeats, as well as bacterial artificial chromosome (BAC) clones. Position of hybridization signals and chromosome condensation patterns were quantified by the help of the chromosome image analysis system ver. 4.0 (CHIAS IV). Fourteen BAC clones belonging to linkage groups (LG) 1-7 hybridized to individual chromosomes 4, 2, 6, 5, 1, 7, and 3, respectively. Quantitative analysis of FISH mapping and chromosome analysis using CHIAS IV allowed us to construct a quantitative idiogram that constitutes the comprehensive chromosome map of red clover. Chromosomal positions of the 26S rDNA locus were detected at a heterozygous locus on chromosome 6 in the variety HR, and polymorphisms of rDNA loci were observed in other varieties, although chromosomal positions of some BAC clones did not vary among HR and other varieties. These results demonstrate chromosomal collinearity among allogamous red clover varieties. This integration of genetic linkage and quantitative chromosome maps should provide valuable insight into allogamous legume genetics.

ARR1, a transcription factor for genes immediately responsive to cytokinins.

Cytokinins are a class of phytohormones involved in various physiological events of plants. The Arabidopsis sensor histidine kinase CRE1 was recently reported to be a cytokinin receptor. We used a steroid-inducible system to show that the transcription factor-type response regulator ARR1 directs transcriptional activation of the ARR6 gene, which responds to cytokinins without de novo protein synthesis. This fact, together with characteristics of ARR1-overexpressing plants and arr1 mutant plants, indicates that the phosphorelay to ARR1, probably from CRE1, constitutes an intracellular signal transduction occurring immediately after cytokinin perception.

Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response.

Chloroplasts relocate their positions in a cell in response to the intensity of incident light, moving to the side wall of the cell to avoid strong light, but gathering at the front face under weak light to maximize light interception. Here, Arabidopsis thaliana mutants defective in the avoidance response were isolated, and the mutated gene was identified as NPL1 (NPH-like 1), a homolog of NPH1 (nonphototropic hypocotyl 1), a blue light receptor used in phototropism. Hence, NPL1 is likely a blue light receptor regulating the avoidance response under strong light.

Collagen content and architecture of the Iliotibialis lateralis muscle in male chicks and broilers with different growth rates fed on different nutritional planes.

1. Varying growth rates in chickens were induced by different nutritional regimes. The collagen content and architecture of iliotibialis lateralis (ITL) muscle were compared among 21-d-old chick types and broiler types at 80 or 95 d of age. 2. Relative size of ITL muscle was greater in the rapid growing (1.16% of live weight) than the slow growing chicks (1.02% of live weight). The 80-d-old broilers with a compensatory growth phase after an earlier slow growth period produced ITL muscle at 1.65-1.69% of live weight. The ITL muscle in 80- and 95-d-old broilers with restricted later growth after an earlier rapid growth period was 1.29 and 1.49% of live weight, respectively. 3. Collagen content of ITL muscle did not differ between chick types and also among the broiler types. However, collagen concentration decreased from 6.00-6.51 mg/g in the chicks to 3.33-4.00 mg/g in the broilers. 4. Thick and thin perimysia and honeycomb endomysia were viewed by scanning electron microscope (SEM) photography. In the perimysia, a central wide layer of longitudinal collagen fibres and peripheral narrow band of transverse fibres were distinguished. Collagen baskets of adipocytes were observed in the perimysia. 5. Perimysial collagen fibres markedly increased in number and formed a larger fibre cluster during growth from chicks to broilers. Endomysia changed from thin to thicker meshwork with growth. However, the collagen architecture of the muscle in broilers did not change under different nutritional regimes. 6. In conclusion, ITL muscle of chicken develops optimally when body growth is enhanced, but the collagen content and architecture in broilers are not affected by different growth processes.

Collagen content and architecture of the puboischiofemoralis muscle in male chicks and broilers with different growth rates on various nutritional planes.

1. Various growth rates of chickens were induced with different nutritional regimes, and the collagen content and architecture of the medial part of the puboischiofemoralis muscle were compared among 21-d-old chicks and 80- or 95-d-old broilers. 2. The percentage muscle weight relative to live weight increased from chicks to 80-d-old broilers and the 95-d-old broilers attained the largest percentage. An inter-relationship of the percentage muscle weight and the growth rates of birds could not be determined. 3. Collagen concentration was related to the growth rates for the first 21 d post hatching and maintained the same level during the later stages up to 80 d. The 95-d-old broilers, that were subjected to early rapid growth followed by restricted later growth, had the highest collagen content. 4. On SEM (Scanning Electron Microscopy) photographs, endomysial honeycombs were small and encircled by perimysia of a collagen network with small mesh size. Thin and thick perimysia were distinguished and the expanded portion of thick perimysia was also observed. Generally, the perimysia were made up of rough collagen tissue where fatty tissue developed, especially in the broilers. 5. Perimysial collagen fibres with mainly transverse striation were divided into two fundamental types, wide collagen platelets and narrow cords. With growth from the chick to broiler stage, features of the collagen fibres did not change regardless of expansion of the thick perimysia. Endomysia increased slightly from thin to thick meshwork as growth progressed. However, the collagen architecture of the muscle in broilers did not change under different nutritional regimes. 6. In conclusion, the puboischiofemoralis muscle of chickens develops relative to live weight when later growth is limited in broilers, but the collagen architecture is not affected by the different growth rates.

Principal component analysis uncovers cytomegalovirus-associated NK cell activation in Ph+ leukemia patients treated with dasatinib.

Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) in a proportion of Ph+ leukemia patients, which associates with a better prognosis. The lymphocytosis is predominantly observed in cytomegalovirus (CMV)-seropositive patients, yet detectable CMV reactivation exists only in a small fraction of patients. Thus, etiology of the lymphocytosis still remains unclear. Here, we identified NK cells as the dominant LGLs expanding in dasatinib-treated patients, and applied principal component analysis (PCA) to an extensive panel of NK cell markers to explore underlying factors in NK cell activation. PCA displayed phenotypic divergence of NK cells that reflects CMV-associated differentiation and genetic differences, and the divergence was markedly augmented in CMV-seropositive dasatinib-treated patients. Notably, the CMV-associated highly differentiated status of NK cells was already observed at leukemia diagnosis, and was further enhanced after starting dasatinib in virtually all CMV-seropositive patients. Thus, the extensive characterization of NK cells by PCA strongly suggests that CMV is an essential factor in the NK cell activation, which progresses stepwise during leukemia and subsequent dasatinib treatment most likely by subclinical CMV reactivation. This study provides a rationale for the exploitation of CMV-associated NK cell activation for treatment of leukemias.

Identification of human DAN gene, mapping to the putative neuroblastoma tumor suppressor locus.

The expression of DAN gene (previously designated as N03 gene) is significantly reduced in a variety of transformed rat fibroblasts, including v-src- (SR-3Y1), SV40- and v-mos-transformed 3Y1 cells, compared with that in parental 3Y1 cells. Recently, DAN gene has been shown to possess a tumor suppressive activity when it is overexpressed in SR-3Y1 cells (Ozaki & Sakiyama, 1994). To assess the involvement of DAN gene with human neoplasms, we have isolated human DAN counterpart from a normal lung cDNA library by using rat DAN cDNA as a probe, and determined its chromosomal location. Human DAN gene mapped to chromosome 1p36.11-p36.13, which is well known to show highly significant linkage with the genesis and/or progression of human neuroblastoma. Southern blot analysis on tumor DNA from 26 patients with neuroblastoma has detected three patients showing genomic rearrangement or deletion within or closely linked to the DAN gene locus. Collectively, we propose that human DAN gene is a possible candidate for a tumor suppressor gene of human neuroblastoma.

Purification and characterization of alpha-glucosidases produced by Saccharomyces in response to three distinct maltose genes.

alpha-Glucosidases or maltases (EC 3.2.1.20) were purified to electrophoretic homogeneity from a respective strain of Saccharomyces cerevisiae which carries a single MAL gene, either MAL alpha, MAL beta, or MAL gamma, using gluconate-Sepharose affinity chromatography and isoelectrofocusing. Of these maltases, two types of maltase were obtained from the MAL gamma strain, the pI values of which were 5.6 and 5.9. From the MAL alpha and MAL beta strain was obtained only one type of maltase with the pI at 5.6 which was identical to one of the maltases from the MAL gamma strain. These four maltases possessed the same properties, except for pI. They were monomers with molecular weights of between 66 000 and 67 000. With regard to the substrate specificity, they hydrolyzed maltose and sucrose exclusively but not alpha-methylglucoside nor maltooligosaccharide. They did not differ in immunological properties.

Primary structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata and its cDNA: structural similarity to the B-chain from plant lectin, ricin.

CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc) specific lectin purified from a marine invertebrate Cucumaria echinata has a strong hemolytic activity especially toward human and rabbit erythrocytes. We determined the primary structure of the CEL-III by examining the amino acid sequences of the protein and the nucleotide sequence of the cDNA. The cDNA encoding CEL-III has 1823 nucleotides and an open reading frame of 1296 nucleotides. CEL-III is composed of 432 amino acid residues with a M(r) of 47¿ omitted¿457 and has six internal tandem repeats, each with of 40-50 amino acids, comprising the N-terminal two-thirds of the molecule. Similar repeats are found in the B-chains of cytotoxic plant lectins, such as ricin and abrin, where six repetitive sequences extend throughout the molecules. A hydropathy plot predicts hydrophobic segments in the C-terminal region of CEL-III. These findings suggest that the N-terminal region of CEL-III plays an important role in binding to carbohydrate receptors on the target cell membranes, an event which triggers an intermolecular hydrophobic interaction of the C-terminal region, the result being oligomerization of CEL-III to lead to pore-formation in erythrocyte membrane.

[Intraoperative frozen section diiagnosis of small peripheral lung adenocarcinoma for limited operation].

Sixty-two cases of peripheral lung adenocarcinoma 2 cm or less in diameter were diagnosed as type A, B, or C according to Noguchi classifications by intraoperative frozen section diagnosis. Four of 19 type A were changed finally to type B by postoperative pathologic examination. Likewise 10 of 34 type B were changed finally to 1 type A, 8 type C, and 1 type D. Three of 9 type C were changed finally to 2 type B and 1 type D. Frozen section diagnosis for small peripheral lung adenocarcinoma has some special problems and limitations. Only cases of type A diagnosed by frozen section diagnosis should be permitted to undergo limited surgical resection.

[Clinical experience with aortic connector system].

From April to December 2002, 40 patients underwent coronary artery bypass grafting (CABG) using the St. Jude Medical (Minneapolis) Symmetry bypass system (aortic connector system: ACS). 59 proximal anastomoses (51 saphenous vein grafts, 8 radial artery grafts) were performed with the ACS. One saphenous vein graft occluded during operation. Postoperative evaluation of the anastomotic patency was carried out by angiography in 45 grafts. Five of the saphenous vein grafts were occluded (5/38). One patient who was shock state before operation presented with postoperative unconsciousness. Another patient died at 8th postoperative day caused by ventricular fibrillation. We conclude that the ACS produces a simple, quick way of performing the proximal anastomosis without the need for clamping the aorta, allows reducing risk of embolization by aortic manipulation. However, it is necessary to discuss sufficiently using the ACS, because the graft patency with the ACS is lower than with standard suturing technique.

Osteogenesis associated with bone gla protein gene expression in diffusion chambers by bone marrow cells with demineralized bone matrix.

Diffusion chambers with rat bone marrow cells and demineralized bone matrix (DBM) were implanted subcutaneously to syngeneic 8-week-old rats and were harvested every week 3-7 weeks after implantation, and histochemical examination, determination of alkaline phosphatase activity, total calcium and phosphorus, the bone-specific vitamin K-dependent gla-containing protein (BGP) content, and detection of BGP mRNA relative to mineralization were performed. Alkaline phosphatase in diffusion chamber implants reached the highest activity at 4 weeks and then decreased. Calcium and phosphorus deposits occurred at 4 weeks after implantation and were followed by marked increases until 7 weeks, which was comparable to the accumulation of BGP. The BGP gene within the diffusion chambers began to be expressed at 5 weeks, and its expression increased markedly at 7 weeks after implantation. At 4-5 weeks after implantation, new bone adjacent to the membrane filters and cartilage toward the center of the diffusion chamber were observed histochemically. Light microscopic and immunohistologic examinations of chambers with marrow cells and DBM revealed production of mineralized matrices, typical of bone characterized by the appearance of BGP and mineralized nodules. In contrast, bone marrow cells alone did not show extensive bone formation and yielded very low values for these biochemical parameters. The present experiments demonstrate the potential of bone marrow cells and DBM to produce not only cartilage formation but also membranous bone formation associated with increasing expression of BGP mRNA during the later stages of bone formation, as well as a marked accumulation of BGP.

A novel polymorphism in the promoter region for the human osteocalcin gene: the possibility of a correlation with bone mineral density in postmenopausal Japanese women.

We present a polymorphism of the human osteocalcin gene (also known as BGP, for bone Gla protein) due to a 1 base pair (bp) substitution from cytosine to thymine at position 298 nucleotides (nt), which is at position 198 nt upstream from the BGP exon 1. This mutation was detected by single-strand conformation polymorphism analysis after polymerase chain reaction for the osteocalcin gene fragment (326 bp) and sequencing analysis. The cytosine/thymine polymorphism can be defined by restriction fragment length polymorphism analysis using a modified primer pair and the restriction endonuclease HindIII. The osteocalcin genotype was determined in 160 postmenopausal Japanese women (age 48-80 years). Osteocalcin alleles were designated according to the absence (H) or presence (h) of the HindIII restriction site. There were 12 HH, 49 Hh, and 99 hh individuals, and the allele frequencies were 22.8% for H and 77.2% for h. To determine if genetic variation influences bone mineral density (BMD) and thus can be a determinant of susceptibility to osteoporosis in older women, we examined the association of BMD with the osteocalcin genotypes found in the present study. The subjects with genotype HH had the smallest BMD and those with hh had the greatest BMD among subjects, but these differences did not reach statistical significance. The HindIII genotype showed a significant effect on the prevalence of osteopenia in the subjects, that is, women with genotype HH had a 5.74 times greater risk for osteopenia (p < 0.05) and those with genotype Hh had a 1.59 times greater risk than women with genotype hh. We identified the osteocalcin gene polymorphism, detected with the HindIII genotype, which was suggested to influence bone density and is a possible genetic marker for bone metabolism.

Osteogenic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics.

Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone-forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.

Compilation of all genes encoding bacterial two-component signal transducers in the genome of the cyanobacterium, Synechocystis sp. strain PCC 6803.

Bacteria have devised sophisticated signaling systems for eliciting a variety of adaptive responses to their environment, which are generally referred to as the "two-component regulatory system." The widespread occurrence of the two-component systems in both prokaryotes and eukaryotes implies that it is a powerful device for a wide variety of adaptive responses of cells to their environment. The two-component signal transducers contain one or more of three conserved and characteristic phosphotransfer signaling domains, named the "transmitter, receiver, and alternative transmitter." The recently determined entire genomic sequence of Synechocystis sp. strain PCC 6803 allowed us to compile systematically a complete list of genes encoding such two-component signal transduction proteins. The results of such an effort, made in this study, revealed that at least 80 ORFs were identified as members of the two-component signal transducers in this single species of cyanobacteria.

Structural analysis of a recA-like gene in the genome of Arabidopsis thaliana.

A recA-like gene was identified in the genome of Arabidopsis thaliana by means of PCR using primers designed on the basis of previously reported amino acid sequences of eukaryotic RecA-like proteins. The structure of the gene, termed ArLIM15, was investigated by comparing the primary structure of the genomic DNA with that of the corresponding cDNA. The open reading frame, which was split into 15 exons, was established to have the capacity for encoding a 37.3-kDa polypeptide. The amino acid sequence of the putative product of ArLIM15 showed a high degree of similarity to that of LIM15 in the monocotyledonous plant Lilium, including a 93% identity, and to those of other recA-like genes in yeasts and vertebrates with identities of 69-71%. Phylogenetic analysis indicated ArLIM15 to be much closer to meiosis-specific LIM15 and DMC1 in Saccharomyces cerevisiae than to RAD51 in S. cerevisiae and its homologues on an evolutionary scale.

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.

Generation of 7137 non-redundant expressed sequence tags from a legume, Lotus japonicus.

For comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 22,983 5' end expressed sequence tags (ESTs) were accumulated from normalized and size-selected cDNA libraries constructed from young (2 weeks old) plants. The EST sequences were clustered into 7137 non-redundant groups. Similarity search against public non-redundant protein database indicated that 3302 groups showed similarity to genes of known function, 1143 groups to hypothetical genes, and 2692 were novel sequences. Homologues of 5 nodule-specific genes which have been reported in other legume species were contained in the collected ESTs, suggesting that the EST source generated in this study will become a useful tool for identification of genes related to legume-specific biological processes. The sequence data of individual ESTs are available at the web site: http://www.kazusa.or.jp/en/plant/lotus/EST/.

Expression profiles of a human gene identified as a structural homologue of meiosis-specific recA-like genes.

Using the cDNA clone from mouse testis which carries the conserved sequences among meiosis-specific recA-like genes, a highly homologous cDNA clone was isolated from a cDNA library of human testis. The clone had the coding capacity of a protein consisting of 340 amino acid residues, which coincides with the average size of putative eukaryotic recA-like proteins. When expression of the corresponding gene, named HsLIM15, in various tissues was examined by reverse transcription-PCR, products of two different sizes were detected in testis: While the longer was predominantly seen in the testis, the shorter was commonly to all the tissues including the testis. Analysis of the sequences indicated that the longer product corresponded to the above cDNA clone, and the shorter one was its deletion product missing an internal 165 bp portion. The result strongly suggests that the mRNA species coding for the putative meiosis-specific RecA-like protein in human is predominantly expressed in testis possibly as an alternative splicing product of a ubiquitously expressed gene.

Characterization of a mouse recA-like gene specifically expressed in testis.

Using the sequence information on a class of recA-like genes, which have been isolated from meiosis-specific cDNA libraries of Lilium and budding yeast and identified in various plant cells, a new gene was screened from a mouse testis cDNA library. The putative product of the gene, termed MmLim15, was 37.8 kDa consisting of 340 amino acid residues. The predicted amino acid sequence showed 72-78% similarity to those of Lim15 from Lilium and other meiosis-specific RecA-like proteins. Similarity was also found to another class of recA-like proteins, Rad51 and its homologues, which have been detected in both budding and fission yeasts and various animal cells, but to a lesser extent and the similarity patterns were somewhat different from those for Lim15 type proteins, revealing that the two classes of recA-like genes are phylogenetically separate. Reverse transcription-PCR using poly(A)+ RNAs from various tissues as templates indicated that transcription of MmLim15 occurred specifically in the testis, suggesting that the MmLim15 product participates in meiotic recombination.