Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17 , which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries ‫ف‬ 500,000 insertions. We analyzed Ͼ 42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17 , the utility of the mutant lines, and the knockout gene database are discussed.

Detection of cancer at early stages that can be treated through surgery is a difficult task. One methodology for cancer biomarker discovery exploits the fact that glycoproteins produced by cancer cells have altered glycan structures, although the proteins themselves are common, ubiquitous, abundant, and familiar. However, as cancer tissue at the early stage probably constitutes less than 1% of the normal tissue in the relevant organ, only 1% of the relevant glycoproteins in the serum should have altered glycan structures. Here, we describe our strategy to approach the detection of these low‐level glycoproteins: (a) a quantitative real‐time PCR array for glycogenes to predict the glycan structures of secreted glycoproteins; (b) analysis by lectin microarray to select lectins that distinguish cancer‐related glycan structures on secreted glycoproteins; and (c) an isotope‐coded glycosylation site‐specific tagging high‐throughput method to identify carrier proteins with the specific lectin epitope. Using this strategy, we have identified many glycoproteins containing glycan structures that are altered in cancer cells. These candidate glycoproteins were immunoprecipitated from serum using commercially available antibodies, and their glycan alteration was examined by a lectin microarray. Finally, they were analyzed by multistage tandem MS.

No abstract

No abstract

Glycan alterations of proteins, a common feature of cancer cells, are associated with carcinogenesis, invasion and metastasis. Glycomics, the study of glycans and glycan-binding proteins in various biological systems, is an emerging field in the postgenome and postproteomics era. However, systematic and robust strategies for glycomics are still not fully established because the structural analysis of glycans, which comprise different patterns of branching, various possible linkage positions as well as monomer anomericity, is technically difficult. Here, we introduce a new strategy for glyco-alteration analysis of glycoproteins by using multiple glycan profiling tools. To understand glycan alterations of proteins by correlating the glycosyltransferase expression profile with the actual glycan structure, we systematically used three glycan profiling tools: (1) multiplex quantitative PCR (qPCR) array format for profiling the expression pattern of glycogenes, (2) lectin microarray as a multiplex glycan-lectin interaction analysis system for profiling either a pool of cell glycoproteins or a target glycoprotein, and (3) tandem mass spectrometry for identifying the glycan structure connected to a target glycoprotein. Using our system, we successfully identified glycan alterations on alpha-fetoprotein (AFP), including a novel LacdiNAc structure in addition to previously reported alterations such as alpha1,6 fucosylation.