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The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

To clarify the upstream regulatory mechanism of mitogen-activated protein kinase (MAPK), we performed the reverse transcriptase-based polymerase chain reaction (RT ± PCR) with degenerate primers synthesized based on sequences conserved among the kinase domains of yeast MAPK kinase kinases (MAPKKKs), Ste11, Bck1, and Byr2. We isolated several mammalian cDNA fragments that encode kinase subdomains sharing signi®cant sequence homology with yeast MAPKKKs. Subsequent screening of a HeLa cell cDNA library using one of these cDNA fragments as a probe resulted in the isolation of a full-length cDNA that encodes a novel protein kinase. The catalytic domain sequence of this gene product is closely related to those of budding yeast Sps1 and Ste20 protein kinases. Thus, we call this protein YSK1 (Yeast Sps1/Ste20-related Kinase 1). The transcript of YSK1 was detected in a wide range of tissues and cells. Immunoprecipitated YSK1 shows protein kinase activity. Although YSK1 is signi®cantly similar in its kinase domain to kinases of the yeast and mammalian MAPK pathways, the overexpression of YSK1 did not lead to the activation of the ERK (extracellular signal-regulated kinase) pathway, JNK (c-Jun NH 2 -terminal kinase)/SAPK (stress-activated protein kinase) pathway, or p38/Mpk2 pathway. These results suggest that YSK1 may be involved in the regulation of a novel intracellular signaling pathway.

We have developed a sequencing method based on the RNA polymerase chain termination reaction with rhodamine dye attached to 3-deoxynucleoside triphosphate (3-dNTP). This method enables us to conduct a rapid isothermal sequencing reaction in <30 min, to reduce the amount of template required, and to do PCR direct sequencing without the elimination of primers and 2-dNTP, which disturbs the Sanger sequencing reaction. An accurate and longer read length was made possible by newly designed four-color dye-3-dNTPs and mutated RNA polymerase with an improved incorporation rate of 3-dNTP. This method should be useful for large-scale sequencing in genome projects and clinical diagnosis.High throughput DNA sequencing is essential technology for genome projects and clinical diagnosis (1). Recent developments for high throughput DNA sequencing include multiple capillary array sequencer, enzyme and fluorescent primer or fluorescent dideoxynucleotide (ddNTP) for sequencing reactions (2-5). The ideal sequencing reaction should be accurate and quick and easy to perform, enabling automation of a large number of reactions.Currently, cycle sequencing chemistry, employing dye primers and dye terminators, is widely used. Dye-primer chemistry is useful for long-read sequencing due to the uniform incorporation of four types of ddNTP, resulting in an even peak height for each signal. However, this requires four independent reactions and the sequencing pattern is sometimes flawed by false stops at some sites with no incorporation of ddNTPs. On the other hand, dye-terminator chemistry was developed as a one-tube reaction without false stops, but shows various incorporation rates for the four color terminators resulting in failure of long-read sequencing. Recently, ''ThermoSequenase'', a newly developed enzyme mutated to make the incorporation uniform, allowed the improvement of long read sequencing (5). However, cycle sequencing has the drawback of a long reaction time (2-to 3-hr reaction) because of its requirement of temperature cycling.At present, two independent methods for preparing DNA templates are available. One is DNA cloning using a plasmid vector and the other is PCR. Although PCR has limitations on the size or sequence to be amplified, it is very convenient to prepare template(s) directly from plasmids in Escherichia coli and cells from tissues without cloning and library construction (6). From this aspect, PCR allows automation of template preparation because it can amplify very rapidly DNA fragment(s) from a large number of samples. However, in the direct sequencing of PCR products by using Dye-terminator chemistry, unreacted 2Ј-dNTP and primers must be eliminated to avoid interference with the subsequent sequencing reaction. Although efforts have been made to quickly purify the PCR product such as enzymatic degradation using exonuclease I and shrimp alkaline phosphatase, most protocols are timeconsuming, laborious, and expensive (7,8).To overcome the above problems, we pursued a completely different approach. Based ...

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