PubMed · 11598625
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M Emi. 2001. [cDNA microarray and SNP analysis].. https://doi.org/10.1272/jnms.68.411
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Thymoma is one of the most common solid tumors in the mediastinum. The recent development of high-density oligonucleotide arrays provides a unique opportunity to generate gene expression profiles of cells from various stages of tumor progression as it occurs in actual neoplastic tissues. We used oligonucleotide arrays to monitor in vivo gene expression levels in early- (stage I or II) and late- (stage IVa) stage thymoma tissues in 36 patients. These in vivo gene expression profiles were verified by real-time quantitative RT-PCR using LightCycler. Using both methods, 2 candidate genes were identified that were more highly expressed in advanced-stage thymomas. One was a well-known gene, c-JUN, and another was an unknown gene, AL050002. AL050002 expression, but not c-JUN expression, was also correlated with the WHO classification (type B3 vs. type B1, B2 or AB). The combined use of oligonucleotide microarray and real-time RT-PCR analyses provides a powerful new approach to elucidate the in vivo molecular events correlated with tumor stage of thymoma.
Specialized cDNA-based microarrays (IronChips) were developed to investigate complex physiological gene-regulatory patterns in iron metabolism. Approximately 115 human cDNAs were strategically selected to represent genes involved either in iron metabolism or in interlinked pathways (eg, oxidative stress, nitric oxide [NO] metabolism, or copper metabolism), and were immobilized on glass slides. HeLa cells were treated with iron donors or iron chelators, or were subjected to oxidative stress (H(2)O(2)) or NO (sodium nitroprusside). In addition, we generated a stable transgenic HeLa cell line expressing the HFE gene under an inducible promoter. Gene-response patterns were recorded for all of these interrelated experimental stimuli, and analyzed for common and distinct responses that define signal-specific regulatory patterns. The resulting regulatory patterns reveal and define degrees of relationship between distinct signals. Remarkably, the gene responses elicited by the altered expression of the hemochromatosis protein HFE and by pharmacological iron chelation exhibit the highest degree of relatedness, both for iron-regulatory protein (IRP) and non-IRP target genes. This finding suggests that HFE expression directly affects the intracellular chelatable iron pool in the transgenic cell line. Furthermore, cells treated with the iron donors hemin or ferric ammonium citrate display response patterns that permit the identification of the iron-loaded state in both cases, and the discrimination between the sources of iron loading. These findings also demonstrate the broad utility of gene-expression profiling with the IronChip to study iron metabolism and related human diseases.
LTR-retrotransposons are abundant components of all eukaryotic genomes and appear to be key players in their evolution. They share with retroviruses a reverse transcription step during their replication cycle. To better understand the replication of retrotransposons as well as their similarities to and differences from retroviruses, we set up an in vitro model system to examine minus-strand cDNA synthesis of the yeast Ty1 LTR-retrotransposon. Results show that the 5' and 3' ends of Ty1 genomic RNA interact through 14 nucleotide 5'-3' complementary sequences (CYC sequences). This 5'-3' base pairing results in an efficient initiation of reverse transcription in vitro. Transposition of a marked Ty1 element and Ty1 cDNA synthesis in yeast rely on the ability of the CYC sequences to base pair. This 5'-3' interaction is also supported by phylogenic analysis of all full-length Ty1 and Ty2 elements present in the Saccharomyces cerevisiae genome. These novel findings lead us to propose that circularization of the Ty1 genomic RNA controls initiation of reverse transcription and may limit reverse transcription of defective retroelements.