MicroRNAs (miRNAs), which are non-coding RNAs 18–25 nt in length, regulate a variety of biological processes, including vertebrate development. To identify new species of miRNA and to simultaneously obtain a comprehensive quantitative profile of small RNA expression in mouse embryos, we used the massively parallel signature sequencing technology that potentially identifies virtually all of the small RNAs in a sample. This approach allowed us to detect a total of 390 miRNAs, including 195 known miRNAs covering ∼80% of previously registered mouse miRNAs as well as 195 new miRNAs, which are so far unknown in mouse. Some of these miRNAs showed temporal expression profiles during prenatal development (E9.5, E10.5 and E11.5). Several miRNAs were positioned in polycistron clusters, including one particular large transcription unit consisting of 16 known and 23 new miRNAs. Our results indicate existence of a significant number of new miRNAs expressed at specific stages of mammalian embryonic development and which were not detected by earlier methods.

Using a differential display technique, we identified two genes that are down-regulated in human gastric cancer tissue as compared to normal gastric mucosa. The down-regulated expression of these genes in gastric cancer tissue was confirmed by northern blotting analysis and RT-PCR. One, CA11, was a novel gene expressed predominantly in the stomach and was depleted in all of the gastric cancer cell lines examined. The other gene, GC36, was homologous to the digestive tract-specific calpain gene, nCL-4. The expression of both GC36 and nCL-4 was suppressed or depleted in gastric cancer cell lines of differentiated and poorly differentiated types. This is the first report of genes, the expression of which is down-regulated with considerable frequency in gastric cancer.Key words: Differential display -Down-regulated expression -Gastric cancer -Digestive tractspecific calpain nCL-4Despite a decrease in incidence, gastric cancer is still one of the most common malignancies in the world. The genetic basis of this disease is not well understood. Amplification of c-met, c-erbB-2 and K-sam genes, or point mutations of K-ras, p53 and APC are known to occur in gastric cancer.1, 2) Recently, it was reported that germline mutation of E-cadherin caused a reduction in the amount of functional protein and led to a high rate of gastric cancer in a Maori family.3) However, the frequencies of these genetic alterations are not high.Differential display (DD) 4) is a rapid and effective method to identify differences in gene expression between related cells. Using DD, genes that are differentially expressed between normal gastric mucosa and gastric cancer tissue were investigated. The results are illustrated in Fig. 1. The bands of about 760 bp and 190 bp derived from normal tissue, the intensities of which were higher than those of the corresponding bands from the tumor tissue, were cut out from the polyacrylamide gel. We isolated the cDNA fragments of interest in the excised bands from the polyacrylamide gels with a rapid selection system using agarose gel electrophoresis containing a base-specific DNA ligand without cloning, as described previously.5) We also cut out the corresponding regions showing low or no expression in the tumor lane. After reamplification of these fragments from the normal and tumor lanes, the cDNA fragments of interest were selected by side-byside comparison between normal and tumor lanes on agarose gels containing the base-specific DNA ligand bisbenzimide-polyethylene glycol polymer.6) The selected cDNA fragments were again purified by preparative electrophoresis on agarose gels containing phenyl neutral red-polyethylene glycol polymer. A cDNA library of mRNA extracted from normal gastric mucosa was constructed in the Uni-ZAP XR vector (Stratagene, La Jolla, CA) and screened using 32 P-labeled reamplified fragments as probes. We isolated two novel genes, CA11 and GC36, showing tissuespecific expression and down-regulated expression in gastric cancer tissue.The size of the largest clone of CA11 isolated from ...

The high molecular weight cldemon (h-

We cloned the gene encoding the thermostable DNA polymerase from the archaeon Pyrococcus furiosus. The DNA fragment of 2785 base pair (bp) containing the structural gene for DNA polymerase was sequenced. DNA polymerase (Pfu polymerase), as deduced from the DNA sequence, consisted of 775 amino acids, had a molecular weight of 90, 109, and was structurally homologous to the alpha-like DNA polymerases (family B) represented by human DNA polymerase alpha and Escherichia coli DNA polymerase II. An unrooted phylogenetic tree of the alpha-like DNA polymerases based on the amino acid sequence alignment was constructed. Pfu polymerase, with two other archaeon polymerases, constitutes a group with some animal viruses. The transcription initiation sites of the pol gene were identified by analysis of in vivo transcripts of both from P. furiosus and E. coli, and the promoters were assigned upstream of the pol coding region. A typical promoter sequence for the archaeon was found at a reasonable distance from the transcription initiation site in P. furiosus.