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Biomedical subjects

Sumio Sugano

Publications and source records attributed to Sumio Sugano.

At least 37 records · Page 2Linked to original sources
PubMed2005

A novel method for development of malaria vaccines using full-length cDNA libraries.

We describe a novel method to screen malaria DNA vaccine candidates using a full-length cDNA library and a murine malaria infection model. For the development of effective malaria vaccines, much effort has been made with meager success. The completion of genome sequencing of Plasmodium falciparum has provided invaluable information for achieving this goal. We have been studying full-length cDNA libraries of malaria parasites as a part of genome analysis. Mice vaccinated with a DNA vaccine consisting of 2000 pooled clones showed significantly prolonged survival after challenge infection. In addition, spleen cells of vaccinated mice produced augmented levels of IL-2 and IFN-gamma when incubated with the crude parasite antigens, indicating that cellular immunity plays an important role in the protection. This approach will not only form the basis for development of malaria vaccines but will also be applicable to other parasites and pathogenic microorganisms.

Animals↗
PubMed2005

Substitution rate and structural divergence of 5'UTR evolution: comparative analysis between human and cynomolgus monkey cDNAs.

The substitution rate and structural divergence in the 5'-untranslated region (UTR) were investigated by using human and cynomolgus monkey cDNA sequences. Due to the weaker functional constraint in the UTR than in the coding sequence, the divergence between humans and macaques would provide a good estimate of the nucleotide substitution rate and structural divergence in the 5'UTR. We found that the substitution rate in the 5'UTR (K5UTR) averaged approximately 10%-20% lower than the synonymous substitution rate (Ks). However, both the K5UTR and nonsynonymous substitution rate (Ka) were significantly higher in the testicular cDNAs than in the brain cDNAs, whereas the Ks did not differ. Further, an in silico analysis revealed that 27% (169/622) of macaque testicular cDNAs had an altered exon-intron structure in the 5'UTR compared with the human cDNAs. The fraction of cDNAs with an exon alteration was significantly higher in the testicular cDNAs than in the brain cDNAs. We confirmed by using reverse transcriptase-polymerase chain reaction that about one-third (6/16) of in silico "macaque-specific" exons in the 5'UTR were actually macaque specific in the testis. The results imply that positive selection increased K5UTR and structural alteration rate of a certain fraction of genes as well as Ka. We found that both positive and negative selection can act on the 5'UTR sequences.

Animals↗
PubMed2005

Genome-wide analysis reveals strong correlation between CpG islands with nearby transcription start sites of genes and their tissue specificity.

It has been envisaged that CpG islands are often observed near the transcriptional start sites (TSS) of housekeeping genes. However, neither the precise positions of CpG islands relative to TSS of genes nor the correlation between the presence of the CpG islands and the expression specificity of these genes is well-understood. Using thousands of sequences with known TSS in human and mouse, we found that there is a clear peak in the distribution of CpG islands around TSS in the genes of these two species. Thus, we classified human (mouse) genes into 6600 (2948) CpG+ genes and 2619 (1830) CpG- ones, based on the presence of a CpG island within the -100: +100 region. We estimated the degree of each gene being a housekeeper by the number of cDNA libraries where its ESTs were detected. Then, the tendency that a gene lacking CpG islands around its TSS is expressed with a higher degree of tissue specificity turned out to be evolutionarily conserved. We also confirmed this tendency by analyzing the gene ontology annotation of classified genes. Since no such clear correlation was found in the control data (mRNAs, pre-mRNAs, and chromosome banding pattern), we concluded that the effect of a CpG island near the TSS should be more important than the global GC content of the region where the gene resides.

Animals↗
PubMed2005

5'SAGE: 5'-end Serial Analysis of Gene Expression database.

To comprehensively identify transcription start sites and the frequencies of individual mRNAs in human cell libraries, a method of 5' end Serial Analysis of Gene Expression (SAGE) was developed recently, which makes it possible to collect a large amount of start site information, and subsequently, we have established a related database server called 5'SAGE. This database displays the observed frequencies of individual 5' end SAGE tags and previously unknown transcription start sites in the promoter regions, introns and intergenic regions of known genes. 5'SAGE will be useful for analyzing promoter regions and start site variation in different tissues, and is freely available at http://5sage.gi.k.u-tokyo.ac.jp/.

5' Flanking Region↗
PubMed2005

dbQSNP: a database of SNPs in human promoter regions with allele frequency information determined by single-strand conformation polymorphism-based methods.

We present a database, dbQSNP (http://qsnp.gen.kyushu-u.ac.jp/), that provides sequence and allele frequency information for single-nucleotide polymorphisms (SNPs) located in the promoter regions of human genes, which were defined by the 5' ends of full-length cDNA clones. We searched for the SNPs in these regions by sequencing or single-strand conformation polymorphism (SSCP) analysis. The allele frequencies of the identified SNPs in two ethnic groups were quantified by SSCP analyses of pooled DNA samples. The accuracy of our estimation is supported by strong correlations between the frequencies in our data and those in other databases for the same ethnic groups. The frequencies vary considerably between the two ethnic groups studied, suggesting the need for population-based collections and allele frequency determination of SNPs, in, e.g., association studies of diseases. We show profiles of SNP densities that are characteristic of transcription start site regions. A fraction of the SNPs revealed a significantly different allele frequency between the groups, suggesting differential selection of the genes involved.

Alleles↗
PubMed2005

Identification and characterization of a canine highly similar to retinoic acid receptor alpha.

A canine highly similar to retinoic acid receptor alpha (canine HS-RARa) cDNA was isolated from the spleen tissue. A database search and the alignment revealed that the canine cDNA was most similar to highly similar type of human RARa and was named canine HS-RARa. The expression of the genes encoding RARa in the dog was the highest in the testis and moderate in the blood, lymph node, mammary gland, pancreas, salivary gland, spleen, thyroid gland, tonsil and uterus. The nucleotide sequence encoded the 462-amino acid containing the conserved sequence motif of RARa. Though the amino acid sequences were well-conserved among species, some unique arrangements were observed within each class. In the phylogenetic analysis, each species separated according to their class. In the branch of mammals, the dog is in the cluster of humans, mice and western wild mice. However, hamsters and rats formed another branch.

Amino Acid Sequence↗
PubMed2005

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

5' Flanking Region↗
PubMed2005

Growth characteristics of canine distemper virus in a new cell line CCT cells originated from canine malignant histiocytosis.

Canine distemper virus (CDV) growth and the morphological characterization were examined in a cell line established from a canine malignant histiocytosis (CCT cell line). The susceptibility of the CCT cells to 3 CDV strains, FXNO, YSA-TC and MD-77 was shown by detection of the antigen in the indirect fluorescent assay. After passaging 4 and 9 times through the CCT cells, only FXNO strain could produce the syncytia where demonstrated the antigens. Titers of 9 passaged viruses through the CCT cells showed slightly higher in the CCT cells than those in Vero cells. Morphological characterization of karyorrhexis and specific DNA ladder by extracted DNA electrophoresis indicated apoptosis in the CDV infected CCT cells.

Animals↗
PubMed2004

Role of a BCL9-related beta-catenin-binding protein, B9L, in tumorigenesis induced by aberrant activation of Wnt signaling.

Wnt signaling plays a crucial role in a number of developmental processes and in tumorigenesis. beta-Catenin is stabilized by Wnt signaling and associates with the TCF/LEF family of transcription factors, thereby activating transcription of Wnt target genes. Constitutive activation of beta-catenin-TCF-mediated transcription resulting from mutations in adenomatous polyposis coli (APC), beta-catenin, or Axin is believed to be a critical step in tumorigenesis among divergent types of cancers. Here we show that the transactivation potential of the beta-catenin-TCF complex is enhanced by its interaction with a BCL9-like protein, B9L, in addition to BCL9. We found that B9L is required for enhanced beta-catenin-TCF-mediated transcription in colorectal tumor cells and for beta-catenin-induced transformation of RK3E cells. Furthermore, expression of B9L was aberrantly elevated in about 43% of colorectal tumors, relative to the corresponding noncancerous tissues. These results suggest that B9L plays an important role in tumorigenesis induced by aberrant activation of Wnt signaling.

Amino Acid Sequence↗
PubMed2004

Serial analysis of gene expression in sinusoidal endothelial cells from normal and injured mouse liver.

Here we describe gene expression profiles of mouse liver sinusoidal endothelial cells (LSECs) revealed by serial analysis of gene expression (SAGE). We prepared SAGE libraries of LSECs from normal and injured liver by CCl(4) administration, and we obtained 32,867 tags from normal and 37,493 tags from injured liver, representing 6011 unique transcripts. CCl(4) administration upregulated several genes related to cell growth and differentiation (Cdkn1a, Irf1, Il4ra, etc.), whereas it downregulated genes related to cell growth or protein transport (Kdr, Igfbp4, Ap1b1, etc.). To identify genes preferentially expressed in LSEC, we compared our SAGE libraries with 77 publicly available libraries generated from various mouse tissues and cell lines. We identified 23 genes, including Stab2 and uncharacterized genes, as possible markers for LSEC, which will be useful to analyze the specific role for LSECs in normal as well as regenerating liver.

Animals↗
PubMed2004

DKK1, a negative regulator of Wnt signaling, is a target of the beta-catenin/TCF pathway.

Wnt signaling plays an important role in embryonic development and tumorigenesis. These biological effects are exerted by activation of the beta-catenin/TCF transcription complex and consequent regulation of a set of downstream genes. TCF-binding elements have been found in the promoter regions of many TCF target genes and characterized by a highly conserved consensus sequence. Utilizing this consensus sequence, we performed an in silico screening for new TCF target genes. Through computational screening and subsequent experimental analysis, we identified a novel TCF target gene, DKK1, which has been shown to be a potent inhibitor of Wnt signaling. Our finding suggests the existence of a novel feedback loop in Wnt signaling.

Animals↗
PubMed2004

5'-end SAGE for the analysis of transcriptional start sites.

Identification of the mRNA start site is essential in establishing the full-length cDNA sequence of a gene and analyzing its promoter region, which regulates gene expression. Here we describe the development of a 5'-end serial analysis of gene expression (5' SAGE) that can be used to globally identify transcriptional start sites and the frequency of individual mRNAs. Of the 25,684 5' SAGE tags in the HEK293 human cell library, 19,893 matched to the human genome. Among 15,448 tags in one locus of the genome, 85.8%-96.1% of the 5' SAGE tags were assigned within -500 to +200 nt of mRNA start sites using the RefSeq, UniGene and DBTSS databases. This technique should facilitate 5'-end transcriptome analysis in a variety of cells and tissues.

5' Flanking Region↗
PubMed2004

Expression profiling and differential screening between hepatoblastomas and the corresponding normal livers: identification of high expression of the PLK1 oncogene as a poor-prognostic indicator of hepatoblastomas.

Hepatoblastoma is one of the most common malignant liver tumors in young children. Recent evidences have suggested that the abnormalities in Wnt signaling pathway, as seen in frequent mutation of the beta-catenin gene, may play a role in the genesis of hepatoblastoma. However, the precise mechanism to cause the tumor has been elusive. To identify novel hepatoblastoma-related genes for unveiling the molecular mechanism of the tumorigenesis, a large-scale cloning of cDNAs and differential screening of their expression between hepatoblastomas and the corresponding normal livers were performed. We constructed four full-length-enriched cDNA libraries using an oligo-capping method from the primary tissues which included two hepatoblastomas with high levels of alpha-fetoprotein (AFP), a hepatoblastoma without production of AFP, and a normal liver tissue corresponded to the tumor. Among the 10,431 cDNAs randomly picked up and successfully sequenced, 847 (8.1%) were the genes with unknown function. Of interest, the expression profile among the two subsets of hepatoblastoma and a normal liver was extremely different. A semiquantitative RT-PCR analysis showed that 86 out of 1188 genes tested were differentially expressed between hepatoblastomas and the corresponding normal livers, but that only 11 of those were expressed at high levels in the tumors. Notably, PLK1 oncogene was expressed at very high levels in hepatoblastomas as compared to the normal infant's livers. Quantitative real-time RT-PCR analysis for the PLK1 mRNA levels in 74 primary hepatoblastomas and 29 corresponding nontumorous livers indicated that the patients with hepatoblastoma with high expression of PLK1 represented significantly poorer outcome than those with its low expression (5-year survival rate: 55.9 vs 87.0%, respectively, p=0.042), suggesting that the level of PLK1 expression is a novel marker to predict the prognosis of hepatoblastoma. Thus, the differentially expressed genes we have identified may become a useful tool to develop new diagnostic as well as therapeutic strategies of hepatoblastoma.

Cell Cycle Proteins↗
PubMed2004

GATA-1 and NF-Y cooperate to mediate erythroid-specific transcription of Gfi-1B gene.

Expression of Gfi (growth factor-independence)-1B, a Gfi-1-related transcriptional repressor, is restricted to erythroid lineage cells and is essential for erythropoiesis. We have determined the transcription start site of the human Gfi-1B gene and located its first non-coding exon approximately 7.82 kb upstream of the first coding exon. The genomic sequence preceding this first non-coding exon has been identified to be its erythroid-specific promoter region in K562 cells. Using gel-shift and chromatin immunoprecipitation (ChIP) assays, we have demonstrated that NF-Y and GATA-1 directly participate in transcriptional activation of the Gfi-1B gene in K562 cells. Ectopic expression of GATA-1 markedly stimulates the activity of the Gfi-1B promoter in a non-erythroid cell line U937. Interestingly, our results have indicated that this GATA-1-mediated trans-activation is dependent on NF-Y binding to the CCAAT site. Here we conclude that functional cooperation between GATA-1 and NF-Y contributes to erythroid-specific transcriptional activation of Gfi-1B promoter.

5' Untranslated Regions↗
PubMed2004

Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D.

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

Actins↗
PubMed2004

Characterization of genomic organization of the adenosine A2A receptor gene by molecular and bioinformatics analyses.

The adenosine A(2A) receptor (A(2A)R) is abundantly expressed in brain and emerging as an important therapeutic target for Parkinson's disease and potentially other neuropsychiatric disorders. To understand the molecular mechanisms of A(2A)R gene expression, we have characterized the genomic organization of the mouse and human A(2A)R genes by molecular and bioinformatic analyses. Three new exons (m1A, m1B and m1C) encoding the 5' untranslated regions (5'-UTRs) of mouse A(2A)R mRNA were identified by rapid amplification of 5' cDNA end (5' RACE), RT-PCR analysis and genome sequence analyses. Similar bioinformatics analysis also suggested six variants of the non-coding "exon 1" (h1A, h1B, h1C, h1D, h1E and h1F) in the human A(2A)R gene, which were confirmed by RT-PCR analysis, while three of the human exon 1 variants (h1D, h1E and h1F) were likewise verified by 5' oligonucleotide capping analysis suggesting multiple transcription start sites. Importantly, RT-PCR and quantitative PCR analysis demonstrated that the A(2A)R transcripts with different exon 1 variants displayed tissue-specific expression patterns. For instance, the mouse exon m1A mRNA was detected only in brain (specifically striatum) and the human exon h1D mRNA in lymphoreticular system. Furthermore, the determination of the three new transcription start sites of human A(2A)R gene by 5' oligonucleotide capping and bioinformatics analyses led to the identification of three corresponding promoter regions which contain several important cis elements, providing additional target for further molecular dissection of A(2A)R gene expression. Finally, our analysis indicates that A(2A)R mRNA and a novel transcript partially overlapping with the 3' exon h3, but in opposite orientation to the A(2A)R gene, could conceivably form duplexes to mutually regulate transcript expression. Thus, combined molecular and bioinformatics analyses revealed a new A(2A)R genomic structure, with conserved coding exons 2 and 3 and divergent, tissue-specific exon 1 variants encoding for 5'-UTR. This raises the possibility of generating multiple tissue-specific A(2A)R mRNA species by alternative promoters with varying regulatory susceptibility.

Animals↗