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TGICL is a pipeline for analysis of large Expressed Sequence Tags (EST) and mRNA databases in which the sequences are first clustered based on pairwise sequence similarity, and then assembled by individual clusters (optionally with quality values) to produce longer, more complete consensus sequences. The system can run on multi-CPU architectures including SMP and PVM.

R e s e a R c h a R t i c l e1 8 1 6jci.org Volume 126Number 5 May 2016 26, 54, 55). We assayed the level of GTP-Rab5 in brains of 12-monthold Ts65Dn and 2N mice following a published protocol (56). As previously reported (28), the level of full-length APP in Ts65Dn was approximately 1.5-fold than in 2N samples ( We next tested whether the increase in App gene dose in Ts65Dn BFCNs was responsible for enlargement of Rab5 + endosomes (Figure 1). By immunoblotting, the APP siRNA caused an approximately 30% reduction in the level of full-length APP, as compared with control siRNA ( Figure 2E). Rab5 + puncta in BFCNs treated with either the APP siRNA or control siRNA were analyzed (Figure 2, F and G) as in Figure 1. Large, sometimes lobulated Rab5 + puncta were seen in cultures treated with the control siRNA, whereas these structures were typically smaller and rounded in cultures treated with the APP siRNA ( Figure 2G). Treatment with the APP siRNA significantly reduced the size of Rab5 + puncta in Ts65Dn neurons to a value equivalent to that in 2N neurons ( Figure 2F). Thus, increased App gene dose is necessary for increased Rab5 activation and for early endosome enlargement in Ts65Dn neurons.Full-length APP and β-CTF caused enlargement of early endosomes in PC12 cells. To determine how increased APP expression caused an increase in Rab5 activation, we asked which APP product(s) were responsible (Supplemental Figure 1A). We transfected PC12M cells with full-length APP-GFP, C99-GFP (β-CTF), C83-GFP (α-CTF), or AICD-GFP and examined endosomes by live cell imaging (Supplemental Figure 1B). Bright foci of GFP + intracellular structures were present in PC12M cells that overexpressed APP-GFP or C99-GFP. In contrast, cells expressing C83-GFP or AICD-GFP showed diffuse, hazy signals for GFP, with occasional foci in C83-GFP cells. In APP-GFP and C99-GFP cells, the GFP + intracellular structures were, on average, approximately 2 μm 2 (Supplemental Figure 1E). GFP signals in C83-GFP or AICD-GFP cells contained speckled small puncta within the haze, as well as a small number of larger bright puncta, as seen in cells expressing C99-GFP and APP-GFP (Supplemental Figure 1B). However, the average puncta size in C83-GFP and AICD-GFP cells was approximately 1.2 and 1.3 μm 2 , respectively. Thus, overexpressing APP and β-CTF, but not α-CTF or AICD, routinely induced formation of enlarged, bright intracellular structures. We also tested two APP mutants: APP M596V and APP SWE . APP M596V , which abolishes β-secretase cleavage, prevents production of β-CTF (57); APP SWE enhances β-secretase cleavage to increase the level of β-CTF (57). Both induced the formation of enlarged intracellular structures (Supplemental Figure 1C).We examined colocalization of APP or C99 with Rab5 in cotransfection experiments; APP-mCherry with GFP-Rab5 WT ( Figure 3B); C99-GFP with mCherry-Rab5 WT ( Figure 3C); and Rab5 + endosomes (26, 28, 37) was correlated with reduced endosomal trafficking and signaling of nerve growth factor (NGF), leading to degeneration...

Large-scale single-pass sequencing of cDNAs from different plants has provided an extensive reservoir for the cloning of genes, the evaluation of tissue-specific gene expression, markers for map-based cloning, and the annotation of genomic sequences. Although as of January 2000 GenBank contained over 220,000 entries of expressed sequence tags (ESTs) from plants, most publicly available plant ESTs are derived from vegetative tissues and relatively few ESTs are specifically derived from developing seeds. However, important morphogenetic processes are exclusively associated with seed and embryo development and the metabolism of seeds is tailored toward the accumulation of economically valuable storage compounds such as oil. Here we describe a new set of ESTs from Arabidopsis, which has been derived from 5- to 13-d-old immature seeds. Close to 28,000 cDNAs have been screened by DNA/DNA hybridization and approximately 10,500 new Arabidopsis ESTs have been generated and analyzed using different bioinformatics tools. Approximately 40% of the ESTs currently have no match in dbEST, suggesting many represent mRNAs derived from genes that are specifically expressed in seeds. Although these data can be mined with many different biological questions in mind, this study emphasizes the import of photosynthate into developing embryos, its conversion into seed oil, and the regulation of this pathway.