野村 信夫

In human mitochondria, 10 mRNAs species are generated from a long polycistronic precursor that is transcribed from the heavy chain of mitochondrial DNA, in theory yielding equal copy numbers of mRNA molecules. However, the steady-state levels of these mRNAs differ substantially. Through absolute quantification of mRNAs in HeLa cells, we show that the copy numbers of all mitochondrial mRNA species range from 6000 to 51 000 molecules per cell, indicating that mitochondria actively regulate mRNA metabolism. In addition, the copy numbers of mitochondrial mRNAs correlated with their cellular half-life. Previously, mRNAs with longer half-lives were shown to be stabilized by the LRPPRC/SLIRP complex, which we find that cotranscriptionally binds to coding sequences of mRNAs. We observed that the LRPPRC/SLIRP complex suppressed 3' exonucleolytic mRNA degradation mediated by PNPase and SUV3. Moreover, LRPPRC promoted the polyadenylation of mRNAs mediated by mitochondrial poly(A) polymerase (MTPAP) in vitro. These findings provide a framework for understanding the molecular mechanism of mRNA metabolism in human mitochondria.

Koji Ichiyama, Sindhoora Bhargavi Gopala Reddy, Li Feng Zhang, Wei Xin Chin

Sic family kinases (SFKs) are the earliest known family of tyrosine kinases and are widely thought to play essential roles in cellular signal transduction. Although numerous functional analyses have been performed, no study has analyzed the specificity of all SFKs on an equal platform. To gain a better understanding of SFK phosphorylation, we designed a high-throughput in vitro kinase assay on the subproteome scale using surface plasmon resonance. We reacted each of the 11 human SFKs with 519 substrate proteins, and significant phosphorylation was detected in 33.6% (1921) of the total 5709 kinase substrate combinations. A large number of novel phosphorylations were included among them. Many substrates were shown to be phosphorylated by multiple SFKs, which might reflect functional complementarity of SFKs. Clustering analysis of phosphorylation results grouped substrates into 10 categories, while the similarity of SFK catalytic specificity exhibited no significant correlation with that of amino acid sequences. In silico predictions of SRC-specific phosphorylation sites were not consistent with experimental results, implying some unknown SRC recognition modes. In an attempt to find biologically meaningful novel substrates, phosphorylation data were integrated with annotation data. The extensive in vitro data obtained in this study would provide valuable clues for further understanding SFK-mediated signal transduction.

Transcriptomics is an objective index that reflects the overall condition of cells or tissues, and transcriptome technology, such as DNA microarray analysis, is now being introduced for the quality control of medical products. In this study, we applied DNA microarray analysis to evaluate the character of Japanese encephalitis (JE) vaccines. When administered into rat peritoneum, Vero cell-derived and mouse brain-derived JE vaccines induced similar gene expression patterns in liver and brain. Body weights and blood biochemical findings were also similar after administration of the two vaccines. Our results suggest that the two JE vaccines are likely to have equivalent characteristics with regard to reactivity in rats.

We analyzed diversity of mRNA produced as a result of alternative splicing in order to evaluate gene function. First, we predicted the number of human genes transcribed into protein-coding mRNAs by using the sequence information of full-length cDNAs and 5'-ESTs and obtained 23 241 of such human genes. Next, using these genes, we analyzed the mRNA diversity and consequently sequenced and identified 11 769 human full-length cDNAs whose predicted open reading frames were different from other known full-length cDNAs. Especially, 30% of the cDNAs we identified contained variation in the transcription start site (TSS). Our analysis, which particularly focused on multiple variable first exons (FEVs) formed due to the alternative utilization of TSSs, led to the identification of 261 FEVs expressed in the tissue-specific manner. Quantification of the expression profiles of 13 genes by real-time PCR analysis further confirmed the tissue-specific expression of FEVs, e.g. OXR1 had specific TSS in brain and tumor tissues, and so on. Finally, based on the results of our mRNA diversity analysis, we have created the FLJ Human cDNA Database. From our result, it has been understood mechanisms that one gene produces suitable protein-coding transcripts responding to the situation and the environment.

Protein-protein interactions (PPIs) play key roles in all cellular processes and hence are useful as potential targets for new drug development. To facilitate the screening of PPI inhibitors as anticancer drugs, the authors have developed a high-throughput screening (HTS) system using an in vitro protein fragment complementation assay (PCA) with monomeric Kusabira-Green fluorescent protein (mKG). The in vitro PCA system was established by the topological formation of a functional complex between 2 split inactive mKG fragments fused to target proteins, which fluoresces when 2 target proteins interact to allow complementation of the mKG fragments. Using this assay system, the authors screened inhibitors for TCF7/beta-catenin, PAC1/PAC2, and PAC3 homodimer PPIs from 123,599 samples in their natural product library. Compound TB1 was identified as a specific inhibitor for PPI of PAC3 homodimer. TB1 strongly inhibited the PPI of PAC3 homodimer with an IC(50) value of 0.020 mu M and did not inhibit PPI between TCF7/beta-catenin and PAC1/PAC2 even at a concentration of 250 mu M. The authors thus demonstrated that this in vitro PCA system applicable to HTS in a 1536-well format is capable of screening for PPI inhibitors from a huge natural product library. (Journal of Biomolecular Screening 2009:970-979)

Completion of human genome sequencing has greatly accelerated functional genomic research. Full-length cDNA clones are essential experimental tools for functional analysis of human genes. In one of the projects of the New Energy and Industrial Technology Development Organization (NEDO) in Japan, the full-length human cDNA sequencing project (FLJ project), nucleotide sequences of approximately 30 000 human cDNA clones have been analyzed. The Gateway system is a versatile framework to construct a variety of expression clones for various experiments. We have constructed 33 275 human Gateway entry clones from full-length cDNAs, representing to our knowledge the largest collection in the world. Utilizing these clones with a highly efficient cell-free protein synthesis system based on wheat germ extract, we have systematically and comprehensively produced and analyzed human proteins in vitro. Sequence information for both amino acids and nucleotides of open reading frames of cDNAs cloned into Gateway entry clones and in vitro expression data using those clones can be retrieved from the Human Gene and Protein Database (HGPD, http://www.HGPD.jp). HGPD is a unique database that stores the information of a set of human Gateway entry clones and protein expression data and helps the user to search the Gateway entry clones.

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

We propose that cDNA microarray analysis can be used in the quality control of pandemic and endemic influenza vaccine. Based on the expression profiles of 76 genes in the rat lung one day after inoculation of influenza vaccine, we can distinguish whole-virion influenza vaccine (PDv: pandemic influenza vaccine and WPv: whole virion-particle vaccine) and sub-virion vaccine (HA vaccine) from saline. Among these 76 genes, we found genes up-regulated by influenza infection, as well. as genes involved in the immune response, and interferon. Hierarchical clustering of each influenza vaccine by the expression profiles of these 76 genes matched data from current quality control tests in Japan, such as the abnormal toxicity test (ATT) and the leukopenic toxicity test (LTT). Thus, it can be concluded that cDNA microarray technology is an informative, rapid and highly sensitive method with which to evaluate the quality of influenza vaccines. Using DNA microarray system, consistent with the results of the ATT and LTT, it was clarified that there was no difference in vaccine quality between PDv and WPv. (C) 2008 Elsevier Ltd. All rights reserved.

Discoidin domain receptor ( DDR) is a cell- surface receptor tyrosine kinase activated by the binding of its discoidin ( DS) domain to fibrillar collagen. Here, we have determined the NMR structure of the DS domain in DDR2 ( DDR2- DS domain), and identified the binding site to fibrillar collagen by transferred cross- saturation experiments. The DDR2- DS domain structure adopts a distorted jellyroll fold, consisting of eight beta-strands. The collagen-binding site is formed at the interloop trench, consisting of charged residues surrounded by hydrophobic residues. The surface profile of the collagen- binding site suggests that the DDR2- DS domain recognizes specific sites on fibrillar collagen. This study provides a molecular basis for the collagen- binding mode of the DDR2- DS domain.

Breast cancer is the most common cancer in women worldwide. To identify novel amplicons involved in the mammary carcinogenesis, we constructed gene expression maps of chromosomes in 35 human breast cancer cell lines and extracted six candidate amplicons containing highly expressed gene clusters on chromosomes 8, 17, and X. We also confirmed the presence of the identified amplicons in clinical specimens by Southern blot analysis. Highly expressed genes identified in the amplicons will contribute to the characterization of breast cancer phenotypes, thereby providing novel targets for anticancer therapies. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A new member of the piericidin family, JBIR-02, was isolated from mycelium of Streptomyces sp. ML55 together with two known piericidin derivatives, piericidin A(1) and IT-143-B. The structure was determined on the basis of spectroscopic data. JBIR-02 inhibited nuclear export of beta-arrestin in HeLa cells at the concentration of 20 mu M.

Endothelial cells play an important role in terms of biological functions by responding to a variety of stimuli in the blood. However, little is known about the molecular mechanism involved in rendering the variety in the cellular response. To investigate the variety of the cellular responses against exogenous stimuli at the gene expression level, we attempted to describe the cellular responses with comprehensive gene expression profiles, dissect them into multiple response patterns, and characterize the response patterns according to the information accumulated so far on the genes included in the patterns. We comparatively analyzed in parallel the gene expression profiles obtained with DNA microarrays from normal human coronary artery endothelial cells (HCAECs) stimulated with multiple cytokines, interleukin-1 beta, tumor necrosis factor-alpha, interferon-beta, interferon-gamma, and oncostatin M, which are profoundly involved in various functional responses of endothelial cells. These analyses revealed that the cellular responses of HCAECs against these cytokines included at least 15 response patterns specific to a single cytokine or common to multiple cytokines. Moreover, we statistically extracted genes contained within the individual response patterns and characterized the response patterns with the genes referring to the previously accumulated findings including the biological process defined by the Gene Ontology Consortium (GO). Out of the 15 response patterns in which at least one gene was successfully extracted through the statistical approach, 11 response patterns were differentially characterized by representing the number of genes contained in individual criteria of the biological process in the GO only. The approach to dissect cellular responses into response patterns and to characterize the pattern at the gene expression level may contribute to the gaining of insight for untangling the diversity of cellular functions. (c) 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

We have developed a device for pinpoint delivery of chemicals, proteins, and nucleic acids into cultured cells. The principle underlying the technique is the flow of molecules from the culture medium into cells through a rupture in the plasma membrane made by a needle puncture. DNA transfection is achieved by stabbing the needle tip into the nucleus. The CellBee device can be attached to any inverted microscope, and molecular delivery can be coupled with conventional live cell imaging. Because the position of the needle relative to the targeted cultured cells is computer-controlled, efficient delivery of molecules such as rhodamine into as many as 100 HeLa cells can be completed in 10 min. Moreover, specific target cells within a single dish can be transfected with multiple DNA constructs by simple changes of culture medium containing different plasmids. In addition, the nanosized needle tip enables gentle molecular delivery, minimizing cell damage. This method permits DNA transfection into specific hippocampal neurons without disturbing neuronal circuitry established in culture.

We report a novel in vitro high-throughput (HTP) kinase assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as kinase activity screening, kinase substrate profiling, and kinase HTP screening of kinase inhibitors. (c) 2006 Elsevier Inc. All rights reserved.

The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.

Cypoviruses are insect viruses that produce a cytoplasmic crystalline particle called the polyhedron in which progeny virions are occluded. The virion structural protein, VP3, is implicated in the occlusion of viral particles into polyhedra. In this study, we determined the amino acid sequence of VP3 required for occlusion of viral particles into polyhedra and proposed that this sequence could be used as an immobilization signal to direct the stable incorporation of foreign proteins into polyhedra. A large-scale survey revealed that the immobilization signal could, in fact, direct the incorporation of a variety of human proteins into polyhedra. Immune reactivity and protein-protein interactions were detected on the surface of polyhedra containing immobilized foreign proteins, and these particles were shown to be highly stabilized against dehydration. We showed that these particles could be arrayed onto a glass slide by standard spotting and laser manipulation methods. Thus, this approach is well suited for protein expression, purification, and the development of protein microarrays.

We report the first genome-wide identification and characterization of alternative splicing in human gene transcripts based on analysis of the full-length cDNAs. Applying both manual and computational analyses for 56 419 completely sequenced and precisely annotated full-length cDNAs selected for the H-Invitational human transcriptome annotation meetings, we identified 6877 alternative splicing genes with 18 297 different alternative splicing variants. A total of 37 670 exons were involved in these alternative splicing events. The encoded protein sequences were affected in 6005 of the 6877 genes. Notably, alternative splicing affected protein motifs in 3015 genes, subcellular localizations in 2982 genes and transmembrane domains in 1348 genes. We also identified interesting patterns of alternative splicing, in which two distinct genes seemed to be bridged, nested or having overlapping protein coding sequences (CDSs) of different reading frames (multiple CDS). In these cases, completely unrelated proteins are encoded by a single locus. Genome-wide annotations of alternative splicing, relying on full-length cDNAs, should lay firm groundwork for exploring in detail the diversification of protein function, which is mediated by the fast expanding universe of alternative splicing variants.

H-Invitational Database (H-InvDB; http://www.h-invitational.jp/) is a human transcriptome database, containing integrative annotation of 41,118 full-length cDNA clones originated from 21,037 loci. H-InvDB is a product of the H-Invitational project, an international collaboration to systematically and functionally validate human genes by analysis of a unique set of high quality full-length cDNA clones using automatic annotation and human curation under unified criteria. Here, 19,574 proteins encoded by these cDNAs were classified into 11,709 function-known and 7865 function-unknown hypothetical proteins by similarity with protein databases and motif prediction (InterProScan). The proportion of "hypothetical proteins" in H-InvDB was as high as 40.4%. In this study, we thus conducted data-mining in H-InvDB with the aim of assigning advanced functional annotations to those hypothetical proteins. First, by data-mining in the H-InvDB version of GTOP. we identified 337 SCOP domains within 7865 H-Inv hypothetical proteins. Second, by data-mining of predicted subcellular localization by SOSUI and TMHMM in H-InvDB, we found 1032 transmembrane proteins within H-Inv hypothetical proteins. These results clearly demonstrate that structural prediction is effective for functional annotation of proteins with unknown functions. All the data in H-InvDB are shown in two main views, the cDNA view and the Locus view, and five auxiliary databases with web-based viewers; DiseaseInfo Viewer, H-ANGEL, Clustering Viewer, G-integra and TOPO Viewer; the data also are provided as flat files and XML files. The data consists of descriptions of their gene structures, novel alternative splicing isoforms, functional RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs in relation with orphan diseases, gene expression profiling, and comparisons with mouse full-length cDNAs in the context of molecular evolution. This unique integrative platform for conducting in silico data-mining represents a substantial contribution to resources required for the exploration of human biology and pathology. (C) 2005 Elsevier B.V. All rights reserved.

We have developed a system to classify cellular forms of fusion proteins with an enhanced yellow fluorescent protein (EYFP) into subcellular compartments using images by a fluorescent microscope. The system aims at image classification to accommodate the multiplicity of cellular forms. The cellular forms automatically searched for in the images are classified with excellent reliability using statistical pattern recognition. The patterns are extended models of the cellular forms which are the result of protein localization as seen in seven standard subcellular compartments, including in the apoptosis and the overexpression. The image itself is classified by the majority of cellular forms it contains so as to reflect the characteristics of cell population, rejecting cellular forms with low reliability and in the apoptosis. We have found that our system is 97.9% accurate in classifying cellular forms into subcellular compartments. Copyright 2004 Chem-Bio Informatics Society.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at similar to58% compared with a peak at similar to42% for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at similar to42%, relatively low compared with that of protein-coding cDNAs.

The sequence of the human genome has been determined. The next task is to determine the function of the genes. Classifying cellular forms of proteins encoded by human cDNA clones is a primary step toward understanding the biological role of proteins and their coding genes. We report here our ongoing work on an automatic system to facilitate this classification. Our system handles the transfection, incubation, acquisition of microscopic images of the cells, and the classification of forms there appearing in the images. Our system correctly classified proteins by their forms at a rate of 90% in feasibility studies. (C) 2003 Academie des sciences. Published by Elsevier SAS. All rights reserved.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively, From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.

The introduction of a human chromosome 1 via microcell-mediated chromosome transfer (MMCT) induces the cellular senescence in mouse melanoma B16-F10 cells. The senescent cells maintained still the telomerase activity, which is frequently associated with immortal growth of human cells, suggesting that a telomerase-independent mechanism is involved in the senescence observed in this mouse cell line. To map the senescence-inducing gene to a specific chromosomal region, we took two experimental approaches: identification of a minimal region with the senescence-inducing activity via MMCT of a series of subchromosomal transferrable fragments (STFs), each consisting of a different profile of human chromosome 1-derived regions, and identification of a region commonly deleted from the transferred chromosome 1 in the revertant clones that escaped cellular senescence. These approaches identified a 2.7-3.0 Mb of senescence-inducing region shared among the active STFs and a 2.4-3.0 Mb of commonly deleted region in the revertant clones. These two regions overlapped each other to map the responsible gene at the 450 to 600-kb interval between UniSTS93710 and D1S3542 on chromosome 1q42.3. This study provides essential information and materials for cloning and characterization of a novel senescence-inducing gene that functions in a telomerase-independent pathway, which is likely to be conserved between mice and humans.

Cell cycle defects have been associated with the process of carcinogenesis in many studies. Here we report the cloning and analysis of the novel gene KIAA0008 (GenBank acc. no. D13633). Chromosomal localization experiments assigned the gene to chromosome 14q22-q23. The mRNA transcript was found to be cell cycle regulated, expressed at S-phase, and maintained at both G2- and M-phases. In sit-it hybridization showed expression in proliferating colon and breast (tumor) tissues. Structurally, KIAA0008 shares homology with the Drosophila melanogaster discs large-1 (dlg1) tumor suppressor gene and membrane-associated guanylate kinase protein family members. The potential involvement of KIAA0008 in cell proliferation is discussed, along with its sequence identity and tissue distribution.

Amplification of the 3q26 region appears to occur frequently among esophageal squamous cell carcinomas (ESCs). We examined ESC cell lines for amplification and expression levels of four genes in this region: SNO and EVI1, which encode proteins antagonizing transforming growth factor-beta signaling, and two other putative target genes, TERC and PIK3CA Amplification of SNO was accompanied by significant increases in its expression level, suggesting that this gene is activated in an amplification-dependent manner. SNO was also amplified in 5 of 44 primary ESCs (11.4%). However, expression levels of EVI1, TERC, and PIK3CA did not correlate with their copy-numbers, even though EVI1 and TERC showed the same amplification pattern as SNO. Taken together, the data suggest that of the four candidates, SNO is the most probable target in the 3q26 amplicon for involvement in the progression of ESC. (C) 2001 Academic Press.

Loss of paternal gene expression at the imprinted domain on proximal human chromosome 15 causes Prader-Willi syndrome (PWS), a complex multiple-anomaly disorder involving variable mental retardation, hyperphasia leading to obesity and infantile hypotonia with failure to thrive. Although numerous paternally expressed transcripts have been identified that reside in the candidate region, the individual contributions to the development of PWS have not been firmly established. Recent studies of mouse models carrying a cytogenetic deletion suggest that paternal deficiency of the SNRPN-IPW interval is critical for perinatal lethality of potential relevance to PWS, Here we determined the allelic expression profiles of a total of 118 cDNA clones using monochromosomal hybrids retaining either a paternal or maternal human chromosome 15, Our results demonstrated a preponderance of unusual transcripts lacking protein-coding potential that were expressed exclusively from the paternal copy of the critical interval. This interval was also found to encompass a large direct repeat (DR) cluster displaying a potentially active chromatin conformation of paternal origin, as suggested by enhanced sensitivity to nuclease digestion. Database searches revealed an unexpected organization of tandemly repeated consensus elements, all of which possessed well-defined box C and D sequences characteristic of small nucleolar RNAs (snoRNAs), Southern blot analysis further demonstrated a considerable degree of phylogenetic conservation of the DR locus in the genomes of all mammalian species tested, but not in chicken, Xenopus and Drosophila, These findings imply a potential direct contribution of the DR locus, representing a cluster of multiple snoRNA genes, to certain phenotypic features of PWS.

Forty-one cDNA clones of human functional genes were newly mapped to chromosomes of the musk shrew (Suncus murinus, Insectivora) by fluorescence in situ hybridization, and a comparative cytogenetic map of 51 genes, including 10 genes reported in our previous study, was constructed between human (HSA) and musk shrew (SMU) chromosomes. In this comparative map, the 51 genes localized to human autosomes, except HSA 8, 16, and 20, were mapped to 15 shrew autosomes, except SMU 4, 16, 17 and 18. Twelve conserved segments were identified between human and shrew chromosomes, and six segments among the musk shrew, human, and mouse. Our results defined the presence of at least one inversion and several interchromosomal rearrangements that occurred during evolution after the two species diverged from a common ancestor. Localization of three major histocompatibility complex (MHC) genes to shrew chromosome 3 suggested that the MHC genes of the musk shrew are located in a cluster on chromosome 3. The cytogenetic map constructed in this study is the first cytogenetic map with many functional genes in insectivore species. This approach provides clues for clarifying the chromosomal evolution in this order. Copyright (C) 2001 S. Karger AG, Basel.

The BRCT (<(BR)under bar>ECA1 (C) under bar-(t) under bar erminus) superfamily includes a large number of nuclear proteins closely involved in DNA repair, recombination, and cell-cycle control. The human cDNA clone NFBD1 (previously designated KIAA0170) encodes a novel protein (2089 amino acids in length; calculated molecular mass 226,440 D) with possible BRCT domains at its carboxy terminus (amino acid residues 1894-2089), This gene product has been described as one of the BRCT superfamily proteins. However, its biological significance has been unclarified, Expression of green fluorescent protein (GFP)-tagged full-length NFBD1 or a series of deletion mutants indicated that NFBD1 was localized to the nucleus in various mammalian cells, and a 197-amino acid segment near the amino terminus (amino acid residues 142-338) contained a nuclear targeting signal. In vitro DNA-binding experiments showed that the highly basic region of NFBD1 (amino acid residues 1841-1893) possessed DNA-binding activity. The region encoding amino acids 508-995 of NFBD1 fused inframe with GAL4 DNA-binding domain activated transcription in both yeast and mammalian cells, while the possible BRCT domains of NFBD1 failed to induce transcription in mammalian cells. Overexpression of antisense NFBD1 RNA in a p53-deficient human osteogenic sarcoma cell line (SAOS-2) resulted in remarkable suppression of SAOS-2 colony formation. These results suggest that NFBD1 is a nuclear transcriptional transactivator with possible BRCT domains and may contribute to cell growth control.

The distal region of a short arm of chromosome Ip is frequently deleted in many human cancers including neuroblastoma (NBL), in which it has been narrowed down to the smallest region of overlap between D1S244 and D1S214 (similar to 7 cM). During the search for the candidate tumor suppressor genes mapped within the region, we found the KIAA0591 gene which encoded a new human kinesin-related protein with a homology to human axonal transporter of synaptic vesicles (ATSV). The kinesin is an intracellular motor protein and often associated with neuronal differentiation and survival. Here we identified a complete open reading frame of the KIAA0591 gene by screening a cDNA library derived from human substantia nigra. The KIAA0591 protein contains a possible pleckstrin homology (PH) domain at its carboxy-terminus. However, it did not possess a force-generating motor domain which is well conserved among kinesin superfamily members (KIFs). Northern blot analysis demonstrated that KIAA0591 mRNA was preferentially expressed in both adult and fetal brains, kidney, skeletal muscle and pancreas. KIAA0591 was expressed in favorable NBLs at higher levels than in unfavorable NBLs, although RT-PCR SSCP analysis showed no mutation within the coding region of the KIAA0591 gene, when 8 neuroblastoma tissues and 15 neuroblastoma-derived cell lines were examined. Thus, the full-length KIAA0591 gene may be a novel member of human KIF superfamily which lacks motor domain and might function as a tumor suppressor in an epigenetic but not a classic Knudson's manner.

Histone deacetylases are the catalytic subunits of multiprotein complexes that are targeted to specific pro meters through their interaction with sequence-specific DNA-binding factors. We have cloned and characterized a new human cDNA, HDAC-A, with homology to the yeast HDA1 family of histone deacetylases. Analysis of the predicted amino acid sequence of HDAC-A revealed an open reading frame of 967 amino acids containing two domains: a NH2-terminal domain with no homology to known proteins and a COOH-terminal domain with homology to known histone deacetylases (42% similarity to RPD3, 60% similarity to HDA1), Three additional human cDNAs with high homology to HDAC-A were identified in sequence data bases, indicating that HDAC-A itself is a member of a new family of human histone deacetylases. The mRNA encoding HDAC-A was differentially expressed in a variety of human tissues. The expressed protein, HDAC-Ap, exhibited histone deacetylase activity and this activity mapped to the COOH-terminal region (amino acids 495-967) with homology to HDA1p, in immunoprecipitation experiments, HDAC-A interacted specifically with several cellular proteins, indicating that it might be part of a larger multiprotein complex.

Poly(ADP-ribose)polymerase is a nuclear NAD-dependent enzyme and an essential nick sensor involved in cellular processes where nicking and rejoining of DNA strands are required. The inter-alpha-inhibitor family is comprized of several plasma proteins that all harbor one or more so-called heavy chains designated H1-H4. The latter originate from precursor polypeptides H1P-H4P whose upper two thirds are highly homologous. We now describe a novel protein that includes (i) a so-called BRCT domain found in many proteins involved in DNA repair, (ii) an area that is homologous to the NAD-dependent catalytic domain of poly(ADP-ribose)polymerase, (iii) an area that is homologous to the upper two thirds of precursor polypeptides H1P-H4P and (iv) a proline-rich region with a potential nuclear localization signal. This protein now designated PH5P points to as yet unsuspected links between poly(ADP-ribose)polymerase and the inter-alpha-inhibitor family and is likely to be involved in DNA repair. (C) 1999 Federation of European Biochemical Societies.

Expression profiles of 5058 human gene transcripts represented by an array of 7451 clones from the First IMAGE Consortium cDNA library from infant brain have been collected by semiquantitative hybridization of the array with complex probes derived by reverse transcription of mRNA from brain and five other human tissues. Twenty-one percent of the clones corresponded to transcripts that could be classified in general categories of low, moderate, or high abundance. These expression profiles were integrated with cDNA clone and sequence clustering and gene mapping information From an upgraded version of the Genexpress Index. For seven gene transcripts found to be transcribed preferentially or specifically in brain, the expression profiles were confirmed by Northern blot analyses of mRNA from eight adult and four fetal tissues, and 15 distinct regions of brain. In four instances, further documentation of the sites of expression was obtained by in situ hybridization of rat-brain tissue sections. A systematic effort was undertaken to Further integrate available cytogenetic, genetic, physical, and genic map informations through radiation-hybrid mapping to provide a unique validated map location For each of these genes in relation to the disease map. The resulting Genexpress IMAGE Knowledge Base is illustrated by five examples presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/EXPR/welcome.html.

As a part of our cDNA project for deducing the coding sequence of unidentified human genes, we newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0919 to KIAA1018. The sequencing of these clones revealed that the average sizes of the inserts and corresponding open reading frames were 4.9 kb and 2.6 kb (882 amino acid residues), respectively. A computer search of the sequences against the public databases indicated that predicted coding sequences of 87 genes contained sequences similar to known genes, 53% of which (46 genes) were categorized as proteins relating to cell signaling/communication, cell structure/motility and nucleic acid management. The chromosomal locations of the genes were determined by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of all the genes among 10 human tissues, 8 brain regions (amygdala, corpus callosum, cerebellum, caudate nucleus, hippocampus, substania nigra, subthalamic nucleus, and thalamus), spinal cord, fetal brain and fetal liver were also examined by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

A transgenic pig carrying MMTV/v-Ha-ras was produced by microinjection of DNA into pronuclear stage embryos. Reproductive technologies, such as superovulation, insemination methods and synchronization of recipients were also investigated to establish the efficient systems to obtain pronuclear stage embryos and transgenic pigs aiming at improving efficiency of the protocol for producing transgenic pigs. Integration of the transgene was detected by Southern hybridization analysis in one female among 29 piglets which had been born after transfer of 195 DNA-injected embryos to 8 recipients. Expression of the transgene was detected by RT-PCR in 8 of 17 organs of the founder transgenic pig examined. The transgenic pig transmitted the transgene to 42.3% of 26 offspring. However, development of tumour was not found either in the founder or its progeny during the observation period for 30 and 1-8 months, respectively. The average number of ova collected from donors superovulated with 1500 IU eCG was significantly higher than the number collected from gilts which were superovulated with 1000 IU (26.3 vs 6.8, P&lt 0.05). Donors served by artificial insemination yielded nearly the same number of embryos per head as those mated naturally (14.8 vs 16.1). Recipients of which the estrus was synchronized through administration of either 1500 or 750 IU eCG gave rise to pregnancies (5/5 vs 3/4). These data indicate that transgenic pigs carrying activated oncogene can be produced, though induction of the developmental disorders is yet to be investigated.

We have conducted a sequencing project of human cDNAs which encode large proteins in brain. For selection of cDNA clones to be sequenced in this project, cDNA clones have been experimentally examined by in vitro transcription/translation prior to sequencing. In this study, we tested an alternative approach for picking up cDNA clones having a high probability of carrying protein coding region. This approach exploited 5′-end single-pass sequence data and the GeneMrak program for assessing protein-coding potential, and allowed us to select 74 clones out of 14,804 redundant cDNA clones. The complete sequence data of these 74 clones revealed that 45% of them encoded proteins consisting of more than 500 amino acid residues while all the clones thus selected carried possible protein coding sequences as expected. The results indicated that the GeneMark analysis of 5′-end sequences of cDNAs offered us a simple and effective means to select cDNA clones with protein-coding potential although the sizes of the encoded proteins could not be predicted.

In order to obtain information on the coding sequences of unidentified human genes, we newly determined the sequences of 100 cDNA clones of unknown human genes, which we named KIAA1193 to KIAA1292, from two sets of size-fractionated human adult and fetal brain cDNA libraries. The results of our particular strategy to select cDNA clones which have the potentiality of coding for large proteins in vitro revealed that the average sizes of the inserts and the corresponding open reading frames reached 5.2 kb and 2.8 kb (933 amino acid residues), respectively. By the computational analysis of the predicted amino acid sequences against the OWL and Pfam databases, 58 predicted gene products were classified into the following five functional categories: cell signaling/communication, cell structure/motility, nucleic acid management, protein management and metabolism. It was also found that 30 gene products had homologues in the public databases which were similar in sequence throughout almost their entire regions to the newly identified genes. The chromosomal loci of the genes were assigned by using human-rodent hybrid panels unless their mapping data were already available in the public databases. The expression profiles of the genes were studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.