Gene Function Analysis

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Samples used in this study were approved by local ethics committees, and informed consent was obtained from each patient with available follow-up information. Three independent experiments were performed to analyze relative gene expression and each sample was tested in triplicate.

IHC was performed according to standard protocols. The final immunoreactivity score of each sample was acquired by multiplying the intensity and density scores. In summary, our results provide a comprehensive bioinformatics analysis of genes and pathways which may be involved in the progression of EC. GO and KEGG pathway enrichment analysis showed that DEGs mainly participated in the process of cell adhesion, cell proliferation, survival, invasion, metastasis and angiogenesis.

The central pathways and significant genes identified by us can be used to distinguish EC samples from normal specimens. Our discovery may be of vital importance for investigating the complex interacting mechanisms underlying EC carcinogenesis and designing specific treatments for patients with EC, particularly the immunotherapy. Co Shanghai, P. China for their technical assistance with bioinformatics analysis.

Large-scale gene function analysis with the PANTHER classification system

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Background

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Background

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Gene expression analysis

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Code availability

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Mutat Res. Hu D, Jiang Z. Phospholipase C delta 1 is a novel 3p Genomic and molecular characterization of esophageal squamous cell carcinoma. Since considerable efforts are needed for the propagation of mutant lines to the second generation, a phenotypic marker of mutation frequency in the early stages of population development is often desirable. Thereby, seven phenotypic descriptors among those listed and promoted by the International Board for Plant Genetic Resources [ 13 ] were chosen for the phenotypic screening of our population.

Moreover, because of the out-crossing nature of sunflower and the need to bag the inflorescences for self-pollination, the phenotypic scoring for flowering or flower morphology could not be performed. Some of the mutant lines 37 showed a single altered trait, while lines displayed multiple mutant traits.

Moreover, future screenings of the M 3 generation will clarify the nature of the observed sunflower mutant lines by comparison of the phenotypic percentages recorded in two generations, as reported for indica rice IR64 [ 12 ] and tomato [ 51 ], where not all the identified M 2 mutant lines were confirmed in the next generation.

Information about the genetic uniformity of both the untreated and mutagenized plant material and about the maintenance of seed stocks have been provided. This molecular marker analysis allowed us to detect the existence of any contamination of the population. The original seed stock represented an inbred line F 8 , and two more selfing generations were performed to obtain the M 2 progeny; thereby a high level of genetic uniformity and homozygosity was expected.

However, out-crossing events with unwanted pollen may occur in field conditions, when an open pollinated species is analyzed and obliged to self-fertilize by bagging its inflorescences. Wu et al. Moreover, SSR analysis allowed Xin et al. Several enzymes have been used for mismatching specific cleavage, but Cel I is the most common one in TILLING projects [ 11 , 45 ] for genotyping applications [ 53 ] and detection of heterozygous polymorphism [ 54 ].

The Cel I-mismatch cleavage assay allowed us to establish the best experimental conditions by the background reduction from gel images and the increase in SNP resolution power. The results obtained in our test provided a further advantage, since the use of minimal enzyme concentrations translated into a considerable economic saving. Since DNA sample quality, normalization and pooling level can directly affect the efficiency and cost of mutation discovery [ 9 , 11 ], the main goal of our DNA pooling test was to maximize the throughput by increasing DNA pooling level while still clearly detecting the expected polymorphism.

Although the simulation of a fold pool of individual samples resulted in a successful detection of mutations, the 8-fold pool and the two-dimensional format were chosen as the pooling strategy for our sunflower TILLING population. Our interest was first focused on some key enzymes of the fatty acid pathway, because of the interest in increasing the nutritional value of sunflower oil by the reduction of the ratio of saturated to unsaturated fatty acids.

Moreover, P. Therefore the availability of a stable and effective system, as genetic resistance, for the pest-control results of prime importance. Since few genomic sequences are publicly available for sunflower, the reverse genetic screening was preceded by an accurate reconstruction of candidate gene models, by the amplification and the subsequent sequencing of short overlapping fragments.

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Functional Analysis Program | National Human Genome Research Institute

In this way, new primer pairs flanking this region could be targeted to the intronic sequences, with the aim of improving the screening efficiency on the coding regions in the pilot assay. In total, nine mutant lines have been identified. Each has been confirmed by sequencing and genotyped by microsatellite markers to exclude any individuals originating from cross-pollination events. The development of TILLING technology can lead to the identification of new alleles that may be directly of value for crop improvement.

Furthermore, intensive investigation of the role of key genes, becomes a feasible goal, especially important where genomic information is lacking, as it is in sunflower. Moreover, TILLING methodology makes it possible to focus on specific genes or genomic regions, bypassing problems with other functional genomic tools such as T-DNA knock-outs or RNAi-based gene silencing that require the generation of transgenic plants.

Thus we have developed and established an exciting tool for forward and reverse genetics in sunflower, one that is available for scientific collaborations and that aims to contribute to a global understanding of sunflower gene organization and regulation. Genetic resources, chromosome engineering and crop improvement. Edited by: Singh RJ. Chapter 5. Seiler GJ: Utilization of wild sunflower species for the improvement of cultivated sunflower. Field Crops Research. Department of Agriculture.

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