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101.
Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) are increasingly used for cultivar identification, construction of genetic maps, genetic diversity assessment, association mapping and marker-assisted breeding. Although there are several highly sensitive methods for the detection of polymorphisms, most of them are often beyond the budget of medium-throughput academic laboratories or seed companies. Heteroduplex analysis by enzymatic cleavage (CEL1CH) or denaturing high-performance liquid chromatography (dHPLC) has been successfully used to examine genetic variation in several plant and animal species. In this work, we assess and compare the performance of both methods in sunflower by genotyping SNPs from a set of 24 selected polymorphic candidate genes. The CEL1CH method allowed us to accurately detect allele differences in 10 out of 24 regions using an in-house prepared CEL1 enzyme (celery single strand endonuclease 1, Apium graveolens L.). Similarly, a total of 11 regions were successfully optimized for dHPLC analysis. As a scaling-up approach, both strategies were tested to genotype either 42 SNPs/indels in 22 sunflower accessions from the local germplasm bank or 33 SNPs/indels in 90 recombinant inbred lines (RILs) for genetic mapping purposes. Summarizing, a total of 601 genotypes were efficiently analyzed either with CEL1CH (110) or dHPCL (491). In conclusion, CEL1CH and dHPLC proved to be robust, complementary methods, allowing medium-scale laboratories to scale up the number of both SNPs and individuals to be included in genetic studies and targeted germplasm diversity characterization (EcoTILLING).  相似文献   
102.
Proteomics is increasingly being used to understand enzyme expression and regulatory mechanisms involved in the accumulation of storage reserves in crops with sequenced genomes. During the past six years, considerable progress has been made to characterize proteomes of both mature and developing seeds, particularly oilseeds - plants which accumulate principally oil and protein as storage reserves. This review summarizes the emerging proteomics data, with emphasis on seed filling in soy, rapeseed, castor and Arabidopsis as each of these oilseeds were analyzed using very similar proteomic strategies. These parallel studies provide a comprehensive view of source-sink relationships, specifically sucrose assimilation into organic acid intermediates for de novo amino acid and fatty acid synthesis. We map these biochemical processes for seed maturation and illustrate the differences and similarities among the four oilseeds. For example, while the four oilseeds appear capable of producing cytosolic phosphoenolpyruvate as the principal carbon intermediate, soybean and castor also express malic enzymes and malate dehydrogenase, together capable of producing malate that has been previously proposed to be the major intermediate for fatty acid synthesis in castor. We discuss these and other differences in the context of intermediary metabolism for developing oilseeds.  相似文献   
103.
A new technology has been developed by immunologix that allows human antibodies to be quickly generated against virtually any antigen. Using a novel process, naïve human B cells are isolated from tonsil tissue and transformed with efficiency up to 85%, thus utilizing a large portion of the human VDJ/VJ repertoire. Through ex vivo stimulation, the B cells class switch and may undergo somatic hypermutation, thus producing a human “library” of different IgG antibodies that can then be screened against any antigen. Since diversity is generated ex vivo, sampling immunized or previously exposed individuals is not necessary. Cells producing the antibody of interest can be isolated through limiting dilution cloning and the human antibody from the cells can be tested for biological activity. No humanization is necessary because the antibodies are produced from human B cells. By eliminating immunization and humanization steps and screening a broadly diverse library, this platform should reduce both the cost and time involved in producing therapeutic monoclonal antibody candidates.Key words: human, antibody, monoclonal, novel platform, naïve, B cell, therapeutic  相似文献   
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105.
Splenic marginal zone lymphomas (SMZL) are an uncommon type of B-cell non-Hodgkin's lymphoma (NHL-B) in which no specific chromosomal translocations have been described. In contrast, the most frequent cytogenetic abnormality is the loss of the long arm of chromosome 7 (7q). Previous reports have located this loss in the 7q32 region. In order to better characterize the genomic imbalances in SMZL, molecular studies were carried out in 73 patients with SMZL. To gain insight into the mapping at 7q a tiling array was also used. The results confirmed the loss of 7q as the most frequent change. In addition, several abnormalities, including 4q22.1, 1q21.3-q22, 6q25.3, 20q13.33, 3q28, 2q23.3-q24.1 and 17p13, were also present. A loss of 7q22.1 at 99925039-101348479 bp was observed in half of the cases. The region of 7q22.1 has not previously been characterised in SMZL. Our results confirmed the presence of a new region of loss on chromosome 7 in these NHL.  相似文献   
106.
We have recently identified an intronic polymorphic CA-repeat region in the human endothelial nitric oxide synthase (eNOS) gene as an important determinant of the splicing efficiency, requiring specific binding of hnRNP L. Here, we analyzed the position requirements of this CA-repeat element, which revealed its potential role in alternative splicing. In addition, we defined the RNA binding specificity of hnRNP L by SELEX: not only regular CA repeats are recognized with high affinity but also certain CA-rich clusters. Therefore, we have systematically searched the human genome databases for CA-repeat and CA-rich elements associated with alternative 5' splice sites (5'ss), followed by minigene transfection assays. Surprisingly, in several specific human genes that we tested, intronic CA RNA elements could function either as splicing enhancers or silencers, depending on their proximity to the alternative 5'ss. HnRNP L was detected specifically bound to these diverse CA elements. These data demonstrated that intronic CA sequences constitute novel and widespread regulatory elements of alternative splicing.  相似文献   
107.
108.
There is evidence that severe dengue disease is associated with alterations of the microvascular endothelium. We examined the hypothesis that activation and damage of microvascular endothelial cells (EC) could be induced by inflammatory mediators present in dengue patient's sera. We cultured human microvascular EC (HMEC-1) in vitro with sera from patients with acute dengue infection. Sera from patients with acute dengue induced an increase in ICAM-1 expression on HMEC-1. This effect was greater with samples from the acute febrile phase than with samples from the convalescent phase of the disease. Acute dengue sera had elevated levels of TNF-alpha and the endothelial activating effect of acute dengue sera was inhibited up to 80% by pre-treatment with monoclonal antibodies against TNF-alpha. Furthermore, acute dengue sera induced apoptosis in HMEC-1. These findings support the pathophysiologic significance of microvascular EC and serum inflammatory mediators in dengue.  相似文献   
109.
The role of host factors in regulating bacterial transposition has never been comprehensively addressed, despite the potential consequences of transposition. Here, we describe a screen for host factors that influence transposition of IS903, and the effect of these mutations on two additional transposons, Tn10 and Tn552. Over 20,000 independent insertion mutants were screened in two strains of Escherichia coli; from these we isolated over 100 mutants that altered IS903 transposition. These included mutations that increased or decreased the extent of transposition and also altered the timing of transposition during colony growth. The large number of gene products affecting transposition, and their diverse functions, indicate that the overall process of transposition is modulated at many different steps and by a range of processes. Previous work has suggested that transposition is triggered by cellular stress. We describe two independent mutations that are in a gene required for fermentative metabolism during anaerobic growth, and that cause transposition to occur earlier than normal during colony development. The ability to suppress this phenotype by the addition of fumarate therefore provides direct evidence that transposition occurs in response to nutritional stress. Other mutations that altered transposition disrupted genes normally associated with DNA metabolism, intermediary metabolism, transport, cellular redox, protein folding and proteolysis and together these define a network of host proteins that could potentially allow readout of the cell's environmental and nutritional status. In summary, this work identifies a collection of proteins that allow the host to modulate transposition in response to cell stress.  相似文献   
110.
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