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Sonic hedgehog (Shh) is a secreted signaling protein that plays important roles in a variety of developmental processes and also in pathogenesis of some human cancers and congenital diseases. Molecules that function downstream of Shh, however, still remain elusive. Here we searched for Shh-responsive genes by using an in-house cDNA microarray. Two genes were newly identified to be Shh responsive in neuroepithelial cell line MNS-70: the metal-binding protein Ceruloplasmin (Cp) and the serine protease inhibitor inter-α-trypsine inhibitor heavy chain H3 (ITIH3). In MNS-70 cells, expression of ITIH3 was regulated by Gli zinc-finger transcription factors downstream of Shh, whereas Cp appeared to be regulated by Gli-independent pathways. Cp mRNA was detected in the developing mouse brain, where its expression domain was closely adjacent to that of Shh. These results demonstrate that microarray technology provides a useful tool for studying expression of developmentally regulated genes.

We isolated a cDNA clone encoding a novel Src homology (SH)2 domain-containing protein of 47 kDa from a human cDNA library. As its transcript was predominantly expressed in hematopoietic cells, this gene was termed HSH2 for hematopoietic SH2 protein. This protein contains several putative protein-binding motifs, SH3-binding proline-rich regions, and phosphotyrosine sites, but lacks enzymatic motifs. In a yeast two-hybrid screen, we identified a cytokine-regulated tyrosine kinase c-FES and an activated Cdc42-associated tyrosine kinase ACK1 as HSH2 interactors. HSH2 bound c-FES via its C-terminal region as well as its N-terminal region including the SH2 domain, whereas it bound ACK1 via its N-terminal proline-rich region. Furthermore, these two kinases bound and tyrosine-phosphorylated HSH2 in mammalian cells. Hence, we postulate that HSH2 functions as an adapter protein involved in tyrosine kinase signaling, and possibly regulates cytokine signaling and cytoskeletal reorganization, in hematopoietic cells.

Helicobacter pylori infection stimulates several intracellular signaling pathways and is accompanied by increased gene expression in gastric epithelial cells. High-density cDNA microarray was used to characterize the mRNA expression profile of genes in human gastric cancer cells (MKN45, AGS) cocultured with H. pylori. Coculture with cag pathogenicity island (PAI)-positive H. pylori (wild-type) significantly up-regulated mRNA expression in 8 of 2304 genes tested. In 6 (interleukin-8, IκBα, A20, ERF-1, keratin K7, glutathione peroxidase) of the 8 genes, up-regulation was confirmed by RT-PCR. In coculture with isogenic cagE-negative mutant (ΔcagE), which encodes a type IV secretion system with other genes in the cag PAI, no significant up-regulation was found. We further analyzed the role of A20. Transfection of expression vector encoding A20 resulted in an inhibition of H. pylori-mediated NF-κB activation, indicating that H. pylori-mediated A20 expression could be a negative regulator of NF-κB activation. Taken together, these results indicate the importance of microarray technology as a tool for analyzing the complex interplay between H. pylori and the host.

The major issue in the post-genome sequencing era is determination of gene expression patterns in variety of biological systems. A microarray system is a powerful technology for analyzing the expression profile of thousands of genes at one experiment. In this study, we identified highly expressed genes in mouse brain using the cDNA microarray carrying 2304 cDNAs derived from oligo-capped mouse cDNA library. Nine genes were highly expressed in adult mouse brain compared to kidney, liver, and skeletal muscle. Tissue distribution analysis by reverse transcription-coupled polymerase chain reaction (RT-PCR) revealed that consistent with the microarray data, all of the selected 9 genes were predominantly expressed in the brain. A database search showed that 5 of the 9 genes, MBP, SC1, HiAT3, S100 protein-beta, and SNAP25, were previously known to be expressed at high level in the brain and in the nervous system. One gene was highly sequenced similar to rat S-Rex-s/human NSP-C, suggesting that the gene is a mouse homologue. The remaining three genes did not match to known genes in the GenBank/EMBL database, indicating that these are novel genes highly expressed in the brain. Taken together, our cDNA microarray system can be an excellent tool for identifying differentially expressed genes in mouse brain.

Ca2+/calmodulin-dependent protein kinase kinase (CaM-KK) is a novel member of the CaM kinase family, which specifically phosphorylates and activates CaM kinase I and IV. In this study, we characterized the CaM-binding peptide of αCaM-KK (residues 438–463), which suppressed the activity of constitutively active CaM-KK (84–434) in the absence of Ca2+/CaM but competitively with ATP. Truncation and site-directed mutagenesis of the CaM-binding region in CaM-KK reveal that Ile441is essential for autoinhibition of CaM-KK. Furthermore, CaM-KK chimera mutants containing the CaM-binding sequence of either myosin light chain kinases or CaM kinase II located C-terminal of Leu440, exhibited enhanced Ca2+/CaM-independent activity (60% of total activity). Although the CaM-binding domains of myosin light chain kinases and CaM kinase II bind to the N- and C-terminal domains of CaM in the opposite orientation to CaM-KK (Osawa, M., Tokumitsu, H., Swindells, M. B., Kurihara, H., Orita, M., Shibanuma, T., Furuya, T., and Ikura, M. (1999) Nat. Struct. Biol.6, 819–824), the chimeric CaM-KKs containing Ile441remained Ca2+/CaM-dependent. This result demonstrates that the orientation of the CaM binding is not critical for relief of CaM-KK autoinhibition. However, the requirement of Ile441for autoinhibition, which is located at the −3 position from the N-terminal anchoring residue (Trp444) to CaM, accounts for the opposite orientation of CaM binding of CaM-KK compared with other CaM kinases.

A human cDNA encoding a novel zinc-finger protein, ZNF274, was identified by the “nuclear transportation trap” method (Ueki, N., Oda, T., Kondo, M., Yano, K., Noguchi, T., and Muramatsu, M., 1998, Nat. Biotechnol.16: 1338–1342). Based on sequence analysis of the full-length cDNA, this novel gene has two alternative splicing forms, ZNF274a and ZNF274b, which encode putative proteins of 621 and 584 amino acids, respectively. ZNF274a contains five C2H2-type zinc-finger motifs, two KRAB-A (Kruppel-associated box) domains, and one leucine-rich domain. ZNF274b lacks the first KRAB-A domain at the N-terminus. ZNF274 mRNA is detected in various human tissues by Northern analysis. The ZNF274 gene is mapped distal to marker RPS281 in the human chromosome 19qter region, by RH mapping. The KRAB domains of ZNF274 exhibited transcription repressor activity when tested in GAL4 fusion protein assays. EGFP–ZNF274 fusion protein expressed in COS7 cells predominantly localized to the nucleoli. A series of deletion constructs revealed that a minimal domain consisting of the third and fourth zinc-fingers possesses nucleolar targeting ability. These results suggest that ZNF274 is a ubiquitous transcription repressor that plays a role in the nucleoli.

ABSTRACTIdentification of a novel mouse nuclear protein termed activator of basal transcription 1 (mABT1) that associates with the TATA-binding protein (TBP) and enhances basal transcription activity of class II promoters is described. We also identify mABT1 homologous counterparts in Caenorhabditis elegansand Saccharomyces cerevisiaeand show the homologous yeast gene to be essential for growth. The mABT1 associated with TBP in HeLa nuclear extracts and with purified mouse TBP in vitro. In addition, ectopically expressed mABT1 was coimmunoprecipitated with endogenous TBP in transfected cells. More importantly, mABT1 significantly enhanced transcription from an adenovirus major late promoter in a reconstituted cell-free system. We furthermore demonstrate that mABT1 consistently enhanced transcription from a reporter gene with a minimal core promoter as well as from reporter genes with various enhancer elements in a cotransfection assay. Taken together, these results suggest that mABT1 is a novel TBP-binding protein which can function as a basal transcription activator.

Identification of a novel mouse nuclear protein termed activator of basal transcription 1 (mABT1) that associates with the TATA-binding protein (TBP) and enhances basal transcription activity of class II promoters is described. We also identify mABT1 homologous counterparts in Caenorhabditis elegansand Saccharomyces cerevisiaeand show the homologous yeast gene to be essential for growth. The mABT1 associated with TBP in HeLa nuclear extracts and with purified mouse TBP in vitro. In addition, ectopically expressed mABT1 was coimmunoprecipitated with endogenous TBP in transfected cells. More importantly, mABT1 significantly enhanced transcription from an adenovirus major late promoter in a reconstituted cell-free system. We furthermore demonstrate that mABT1 consistently enhanced transcription from a reporter gene with a minimal core promoter as well as from reporter genes with various enhancer elements in a cotransfection assay. Taken together, these results suggest that mABT1 is a novel TBP-binding protein which can function as a basal transcription activator.

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature396, 584–587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK α and β shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathioneS-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e.autophosphorylation) and PKB activation.

Using digoxigenin (DIG)-based differential hybridization, a series of immediate early genes (IEG) was identified following the adipogenic stimulation in 3T3-L1 preadipocyte cells. Most of the known IEGs were identified as well as new members such as zf9 and Stra13. To delineate possible signaling pathways accounting for these gene expression, a subset of specific kinase inhibitors, SB203580, PD98059, rapamycin, LY294002, and Ro-32-0432, which inhibit p38 (HOG), MEK (MAPKK), S6 kinase, PI3 kinase, and protein kinase C (PKC), respectively, were employed. The IEGs were classified into three categories according to their susceptibility to the inhibitors. Expression of the first group (c-fos, jun-B, egr-1, tis11, tis21, thrombospondin-1, erp, thyroid hormone receptor [N-10], cyr61, and zf9) was mainly dependent on PKC and MEK pathways, while that of the second class (gene33 and tis10) exhibited an additional dependence on PI3 kinase pathways. The third one (Id-3, gly96, and Stra13) was characterized in that none of these inhibitors interfered with gene expression. Our results suggest that the induction of IEGs by the adipogenic stimuli is mediated by common as well as distinct signaling pathways.

In a previous study we found that HTLV‐I encoded Tax transactivator, which binds to NF‐κ‐B1 p105, suppresses p105‐mediated Iκ‐B activity, thereby allowing entry of NF‐κB p65 (Re1A) and p50 into the nucleus. In the present report, we compared the effect of Tax on NF‐κB2 p100, which also binds to Tax, with that of p105 in transfected COS7 cells. While p 105 is processed to the DNA binding form, p50, processing of p100 was much less efficient both in the presence and absence of Tax. Both p105 and p100 showed IfκB activity in sequestering NF‐κB p65 into the cytoplasm. However, only p105‐mediated IκB activity, and not that of p100, was inhibited by Tax. Chimeric molecules between p100 and p105 suggested that inefficient processing of p100 can be attributed to the Rel homologous domain, rather than to the ankyrin repeat domain. p100, but not p105, potently suppressed Tax‐ and p65‐induced transactivation of a GM‐CSF promoter in Jurkat cells. Taken together, these results suggest that p105 and p100 may have distinct effects on Tax‐induced transactivation events.

The promoter of TGF-β type II receptor lacks TATA box or CAAT box. In order to define the exact transcriptional start site(s), we used “oligo-capping” method developed by ourselves. The major transcripts were started from GAA located between −35 and −33 relative to 5′ end of cDNA. Other minor transcripts started from −4, +11 and +17. Deletion analysis of this promoter region revealed that it contained two adjacently located promoters (P1 and P2) capable of acting by itself. The P1 is located between −22 and +55 which covers the minor transcriptional start sites described above. The P2 is located just upstream of the P1 between −137 and −22. The results showed all of the transcripts were started either from P1 or P2.

To define the role of the auto‐inhibitory region of protein kinase C (PKC), 22‐23‐Gly24‐Ala25‐Leu26‐Arg27, site‐directed mutations were introduced into the basic residues. Three mutants, PKCAla22,23, PKCAla27and PKCAla22,23,27, apparently fell into two distinct types with regard to their biochemical properties and biological activities, as judged by the enhancement of a c‐fospromoter in Jurkat cells and by the initiation of germinal vesicle breakdown (GVBD) in Xenopus laevisoocytes. (i) PKCAlu22,23and PKCAla27had activators independent in vitro kinase activity, high phosphorylation levels in vivo, and localized in both cytosolic and particulate fractions. These mutants were not fully biologically active. (ii) PKCAla22,23,27had a low phosphorylation level in vivo, was found predominantly in the particulate fraction and was the most biologically active. These results suggest that basic residues in the auto‐inhibitory domain account for the regulation of kinase activity and the cytosolic retention of PKC. The particulate association or the cytosolic clearance of PKC may facilitate signal transduction in the cell.

Transforming growth factor-β (TGF-β) is a multifunctional polypeptide which plays a crucial role in the regulation of cell proliferation, differentiation, and organogenesis. In the present study, we investigated the expression of signaling receptors for TGF-β in developing mice byin situhybridization, revealing a significant difference in the expression of TGF-β type I and type II receptors. Unexpectedly, the TGF-β type I receptors were exclusively expressed without any detectable expression of the TGF-β type II receptors in developing cerebral cortices. In primary cortical neurons, a neutralizing antibody for TGF-β significantly reduced the expression ofbcl-2and subsequently induced neuronal cell death, indicating that TGF-β functions as a survival factor for cortical neuronsin vitro.Consistent with the result ofin situhybridization, the TGF-β type I but not type II receptors were detected in primary cortical neurons by affinity crosslink and RT–PCR analyses. The concomitant expression of TGF-β2 and the TGF-β type I receptors in developing cerebral cortices suggests that the TGF-β signaling system plays a pivotal role in neuronal differentiation and that unidentified components may be involved in TGF-β signaling in the development of the central nervous system.

A novel protein kinase related to theC. elegansserine/threonine kinase UNC-51 was cloned from mouse. The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa. Comparison of the ULK1 and UNC-51 shows the highest conservation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain. ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescentin situhybridization. HA-tagged ULK1 is expressed as a protein of ∼150 kDa in COS7 cells and is auto-phosphorylatedin vitroin its PS domain. We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes.

Theunc-51gene, isolated from mutants ofCaenorhabditis elegansexhibiting abnormal axonal extension and growth, encodes a novel serine/threonine kinase (K. Ogura,et al.,1994,Genes Dev.8: 2389–2400). Here we report the molecular cloning and characterization of the human homologue of UNC-51, designated ULK1, for UNC-51 (C. elegans)-like kinase 1. Sequence analysis of the human ULK1 cDNA showed that an open reading frame is composed of 1050 amino acids with a calculated MW of 112.6 kDa and a pIof 8.80. Homology search analysis showed that ULK1 has 41% overall similarity to UNC-51 and 29% similarity to Apg1p ofSaccharomyces cerevisiae.Phylogenetic analysis of ULK1, UNC-51, and Apglp suggested that they constitute a novel subfamily of serine/threonine kinases. Southern blot analyses suggested that theULK1gene spans 30–40 kb in the human genome as a single-copy gene. Zoo blot analysis indicated that ULK1 kinase is conserved among vertebrates including mammals, birds, reptiles, amphibians, and fish. Northern blot analysis revealed that ULK1 is ubiquitously expressed in adult human tissues such as skeletal muscle, heart, pancreas, brain, placenta, liver, kidney, and lung, whereas UNC-51 is specifically detected in the nervous system ofC. elegans.Both FISH and RH mapping confirmed the regional localization ofULK1to human chromosome 12q24.3.

This study reports cDNA isolation and partial characterization of a novel human nucleolar protein isolated by “nuclear transportation trap” described previously. The cDNA encodes a putative polypeptide of 524 amino acids with a shortEscherichia coliDNA helicase homologous region, an acid-rich domain, three potential base-rich nuclear localization signals (NLSs), a serine-rich domain, and a deduced coiled-coil domain. The protein has no known prominent similarities with any other protein in the protein databases. Tissue distribution analysis demonstrated a predominant expression in brain and testis. To determine the sequence requirements for nucleolar targeting, a set of deletion constructs with a fluorescent tag were transiently expressed in COS-7 cells. We revealed that a region of 30 amino acids (position 342–371), which overlaps the first and second NLS, is sufficient for nucleolar localization. Furthermore, the adjacent region of 30 amino acids (position 372–401), which contains the third NLS, is sufficient for nuclear localization. These results suggest that this novel nucleolar protein has at least two distinct domains for directing to different subnuclear destinations.

Motivation: A methodology to search for genes associated with multifactorial diseases by integrating the large amount of accumulated knowledge is seriously needed. A comprehensive understanding derived from a holistic view of gene relationship structures can be gained from our proposed analysis called the cross-subspace analysis (CSA). In this analysis, gene objects are generated by machine learning using their term occurrence patterns in MEDLINE abstracts and the degree of relationship between gene objects is quantified by matching these patterns. Results: Structuralization of relationships of a set of genes was performed using CSA, which were retrieved using the terms, ‘obesity’, ‘diabetes’, ‘hypertriglyceridemia’ and ‘hypertension’ that refer to diseases comprising metabolic syndrome, on a 2D plane inferring important biomedical concepts from the gene distribution. Then, we prioritized the significance of 6131 well-annotated human genes in terms of the distance on the plane from the centroid of ‘metabolic syndrome’-related genes distribution. The validity was confirmed by comparing the knowledge extracted by the ordering with existing medical knowledge. Contact: matsunagat@nttdata.co.jp