Biomedical subjects

Y Sakaki

Publications and source records attributed to Y Sakaki.

At least 91 records · Page 5Linked to original sources

PubMed1997

Screening for imprinted genes by allelic message display: identification of a paternally expressed gene impact on mouse chromosome 18.

A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes. In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal F1 hybrids and pooled backcross progenies. From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified. The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene Impact on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function. In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages. Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.

Alleles↗

PubMed1997

A novel human CC chemokine PARC that is most homologous to macrophage-inflammatory protein-1 alpha/LD78 alpha and chemotactic for T lymphocytes, but not for monocytes.

By searching the expressed sequence tag (EST) database, we identified partial cDNA sequences encoding a polypeptide with significant sequence identity to the human CC chemokine macrophage-inflammatory protein-1 alpha (MIP-1 alpha)/LD78 alpha. We determined the complete cDNA sequence that contained a reading frame of 89 amino acids with 61% identity to human MIP-1 alpha/LD78 alpha. The mRNA was expressed constitutively at high levels in human lung and at low levels in some lymphoid tissues. Furthermore, the mRNA was strongly induced in several human cell lines, including monocytic U937 cells, by PMA. From these results, we designated this novel CC chemokine as PARC from pulmonary and activation-regulated chemokine. In situ hybridization analyses showed that alveolar macrophages, follicular dendritic cells in the germinal centers of regional lymph nodes, and peripheral blood monocytes stimulated with LPS express PARC mRNA. Using the human CC chemokine yeast artificial chromosome contig that we constructed recently, we mapped the PARC gene (SCYA18) within one of the two subregions of the CC chemokine gene cluster at chromosome 17q11.2. To investigate its biologic activity, the PARC protein was expressed in insect cells. PARC was chemotactic for both activated (CD3+) T cells and nonactivated (CD14-) lymphocytes, but not for monocytes or granulocytes. Binding analysis using PARC fused with alkaline phosphatase-(His)6 showed the presence of a single class of receptors for PARC on lymphocytes with a Kd of 1.9 nM and 590 sites/cell. Thus, PARC is a novel CC chemokine with a close phylogenic relationship with MIP-1 alpha/LD78 alpha, but with a highly selective activity on lymphocytes.

Amino Acid Sequence↗

PubMed1997

A novel method for making nested deletions and its application for sequencing of a 300 kb region of human APP locus.

We developed a novel in vitro method for making nested deletions and applied it to a large-scale DNA sequencing. A DNA fragment to be sequenced (up to 15 kb long) was cloned with a new vector possessing two unique Sfi I sites, digested by Sfi I and ligated to generate a large head-to-tail concatemer. The large concatemer was randomly fragmented by sonication and then redigested by Sfi I to separate insert and vector DNAs. The fragments of various length were then cloned into the other vector(s) specifically designed for selective cloning of insert-derived DNA fragments to generate a library of nested deletions. This method allowed a single person to generate >20 nested deletion libraries sufficient to cover 100 kb in a few days. We applied the method for sequencing of P1 clones and successfully determined the complete sequence of approximately 300 kb of the human amyloid precursor protein (APP) locus on chromosome 21 with a redundancy of 3.8, reasonably low cost and very few gaps remaining to be closed. Development of some new instruments and software is also described which makes this method more applicable for large-scale sequencing.

Amyloid beta-Protein Precursor↗

PubMed1997

Identification of critical CpG sites for repression of L1 transcription by DNA methylation.

L1 (LINE-1) is an interspersed non-LTR retrotransposon and several genetic defects caused by L1 transposition have been reported. L1 is thus considered as a potential mutagen. However, this potentially hazardous insertional event seems to be rare in spite of the presence of 3000 or more L1 elements of full or nearly full length in the human genome. Thus there must exist a mechanism(s) for repressing the expression of most, if not all, L1 elements. Some studies suggested that methylation plays a major role in the repression of L1 expression. However, no direct evidence has been presented and further study is required to draw a conclusion. We thus studied the effect of methylation on L1 transcription in vivo and in vitro. Transfection of plasmid which contained a L1 promoter linked to cat gene into HeLa cells showed that methylation did repress the L1 promoter activity. In vitro transcription studies using mutagenized templates indicated that methylation of the first seven CpGs in L1 promoter, particularly four CpGs at +52, +58, +61 and +70 was essential for the inhibition. These results suggest that there exists a mechanism to regulate the L1 transcription through the region-specific methylation.

Binding Sites↗

PubMed1997

Differentially expressed MHC class II-associated invariant chain in rat stomach pyloric mucosa with N-methyl-N-nitro-nitrosoguanidine exposure.

Administration of N-methyl-N'-nitro-N-nitrosoguanidine, a glandular stomach carcinogen, at the concentration of 100 microg/ml in drinking water for 8 days induced the appearance of a MHC class II-associated invariant chain in the target organ of stomach pyloric mucosa of male Lewis rats. The up-regulation of the MHC class II-associated invariant chain was revealed by fluorescent differential display analysis, reverse transcription-PCR, Northern blot, and histochemical staining. The appearance of MHC class II and MHC class I was also demonstrated by reverse transcription-PCR and Northern blot. The results suggest the involvement of MHC-controlled immune reactions in chemically-induced stomach carcinogenesis.

Animals↗

PubMed1997

The human MCP-2 gene (SCYA8): cloning, sequence analysis, tissue expression, and assignment to the CC chemokine gene contig on chromosome 17q11.2.

Monocyte chemotactic proteins (MCPs) form a subfamily of chemokines that recruit leukocytes to sites of inflammation and that may contribute to tumor-associated leukocyte infiltration and to the antiviral state against HIV infection. With the use of degenerate primers that were based on CC chemokine consensus sequences, the known MIP-1 alpha/LD78 alpha, MCP-1, and MCP-3 genes and the previously unidentified eotaxin and MCP-2 genes were isolated from a YAC contig from human chromosome 17q11.2. The amplified genomic MCP-2 fragment was used to isolate an MCP-2 cosmid from which the gene sequence was determined. The MCP-2 gene shares with the MCP-1 and MCP-3 genes a conserved intron-exon structure and a coding nucleotide sequence homology of 77%. By Northern blot analysis the 1.0-kb MCP-2 mRNA was predominantly detectable in the small intestine, peripheral blood, heart, placenta, lung, skeletal muscle, ovary, colon, spinal cord, pancreas, and thymus. Transcripts of 1.5 and 2.4 kb were found in the testis, the small intestine, and the colon. The isolation of the MCP-2 gene from the chemokine contig localized it on YAC clones of chromosome 17q11.2, which also contain the-eotaxin, MCP-1, MCP-3, and NCC-1/MCP-4 genes. The combination of using degenerate primer PCR and YACs illustrates that novel genes can efficiently be isolated from gene cluster contigs with less redundancy and effort than the isolation of novel ESTs.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of a novel human CC chemokine liver and activation-regulated chemokine (LARC) expressed in liver. Chemotactic activity for lymphocytes and gene localization on chromosome 2.

Partial overlapping cDNA sequences likely to encode a novel human CC chemokine were identified from the GenBank Expressed Sequence Tag data base. Using these sequences, we isolated full-length cDNA encoding a protein of 96 amino acid residues with 20-28% identity to other CC chemokines. By Northern blot, this chemokine was mainly expressed in liver among various tissues and strongly induced in several human cell lines by phorbol myristate acetate. We thus designated this chemokine as LARC from Liver and Activation-Regulated Chemokine. We mapped the LARC gene close to the chromosomal marker D2S159 at chromosome 2q33-q37 by somatic cell and radiation hybrid mappings and isolated two yeast artificial chromosome clones containing the LARC gene from this region. To prepare LARC, we subcloned the cDNA into a baculovirus vector and expressed it in insect cells. The secreted protein started at Ala-27 and was significantly chemotactic for lymphocytes. At a concentration of 1 microg/ml, it also showed a weak chemotactic activity for granulocytes. Unlike other CC chemokines, however, LARC was not chemotactic for monocytic THP-1 cells or blood monocytes. LARC tagged with secreted alkaline phosphatase-(His)6 bound specifically to lymphocytes, the binding being competed only by LARC and not by other CC or CXC chemokines. Scatchard analysis revealed a single class of receptors for LARC on lymphocytes with a Kd of 0.4 nM and 2100 sites/cell. Collectively, LARC is a novel CC chemokine, which may represent a new group of CC chemokines localized on chromosome 2.

Amino Acid Sequence↗

PubMed1997

Fluorescent differential display analysis of gene expression in differentiating neuroblastoma cells.

Identification of differentially-expressed genes provides an important step toward the elucidation of molecular mechanisms underlying a variety of biological processes. A novel PCR-based approach to detect and clone such transcripts is the so-called differential display (DD). We established an improved DD protocol that can be performed on an automated fluorescent DNA sequencer to ensure high throughput as well as operational safety. Using this fluorescent DD (FDD) technique, we analyzed the gene expression profile in the retinoic acid-induced differentiation of a human neuroblastoma cell line SH-SY5Y. Screening with 102 primer combinations at eight different time points revealed 66 cDNA bands with variously different behaviors out of approximately 6000 bands displayed. Subsequent analyses with 15 cloned species confirmed the differential expression of corresponding transcripts in all the cases, thereby demonstrating the high reliability of FDD analysis. These clones were composed of seven novel and eight known genes, the latter of which included those that had never been described in the context of neuronal differentiation. These results indicate that FDD is an effective approach to obtain not only novel genes but also clues to possible novel functions of known genes involved in various biological phenomena.

Amino Acid Sequence↗

PubMed1997

Capacitative Ca2+ influx in the neural retina of chick embryo.

Depletion of intracellular Ca2+ stores induces a capacitative Ca2+ influx in non-neural cells. It has been unknown whether the capacitative Ca2+ influx occurs in the cells of nervous systems. We found the capacitative Ca2+ influx in the neural retina of early embryonic chick with Fura-2 fluorescence measurements. A Ca(2+)-free medium containing thapsigargin (500 nM), an inhibitor of Ca(2+)-ATPase of intracellular Ca2+ stores, was applied to the neural retina of embryonic day 3 (E3) chick. A rise in intracellular Ca2+ concentration was evoked after the reintroduction of extracellular Ca2+, and this Ca2+ rise was suppressed by Zn2+ (1 mM) and Ni2+ (5 mM). The developmental changes in the Ca2+ rise induced by thapsigargin (250 nM) were studied from E3 to E13. The thapsigargin-induced Ca2+ rise was largest at E3, declined rapidly toward E6, and then decreased gradually until E13, when the Ca2+ rise almost disappeared. This developmental profile correlated with the decline in the mitotic activities of the retinal cells studied by Prada et al. The fluorescence imaging with the vertical slice of the E9 retina showed that the site at which the thapsigargin-induced Ca2+ rise was largest was the most outer layer of the retina, where proliferating cells are located. This spatial distribution and the above developmental profile may suggest that the capacitative Ca2+ influx occurs at the early period of neurogenesis when the cells have mitotic activities.

Animals↗

PubMed1997

Comparison of amyloid deposition in two lines of transgenic mouse that model familial amyloidotic polyneuropathy, type I.

We previously produced a transgenic mouse line designated MT-hMet30 by introducing the human mutant transthyretin (TTR) gene carrying the mouse metallothionein promoter, and showed that the presence of human variant TTR is sufficient for amyloid deposition in various tissues of these transgenic mice. However, the expression pattern of human mutant transthyretin gene in the mouse was different from that in man. To analyse pathologic processes, it is essential to establish a transgenic mouse line in which the development and tissue-specific expression of the human mutant TTR gene is the same as in man. Thus, we produced two additional transgenic mouse lines carrying the human mutant TTR gene containing either 0.6 kb (0.6-hMet30) or 6.0 kb (6.0- hMet30) of the upstream region. The expression levels of 6.0-hMet 30 gene in the liver and serum were the same as in man and about 10 times higher than those of 0.6-hMet30 gene in the liver and serum were the same as similar tissues to human patients except for the peripheral and autonomic nervous tissues. The amyloid deposition started earlier and was more extensive in 6.0-hMet30 than 0.6-hMet30 mice, suggesting that the serum levels of human mutant TTR are correlated with the occurrence and degree of amyloid deposition, to some extent. Neither amyloid deposition nor degenerative changes were observed in the peripheral and autonomic nervous systems despite the transgene expression in the choroid plexus of the 6.0-hMet30 mice. In the 6.0-hMet30 mice, amyloid deposition started at 9 months of age, although the serum level of human mutant TTR reached the adult level at 1 month. These results suggest that intrinsic environmental factors other than the mutant gene are involved in the late-onset deposition of amyloid fibrils. Transgenic mice described here should be useful for analysing such factors.

Age Factors↗

PubMed1997

Isolation of a novel human gene from the Down syndrome critical region of chromosome 21q22.2.

Down syndrome is the most common birth defect, and is caused by trisomy 21. We identified a novel gene in the so-called Down syndrome critical region by means of computer-aided exon prediction and subsequent cDNA cloning. The gene, designated as DCRA (Down syndrome Critical Region gene A), consists of eight exons of 3,252 bp in total and encodes a large open reading frame of 297 amino acid residues. The open reading frame shows significant homology to Hbeta58, a mouse gene essential for embryogenesis, PEP8, a yeast homologue of Hbeta58, and an expressed sequence tag of Arabidopsis thariana, suggesting that DCRA has some important function that has been conserved during the course of evolution. DCRA is expressed in most tissues examined, including fetal and adult brain, heart, lung, liver, and kidney. The cDNA of the DCRA mouse homologue, Dcra, was also cloned. It is 2,157 bp long and has an open reading frame of 297 amino acid residues, which shows 92% identity to human DCRA. Dcra is expressed in all the embryo and adult tissues examined.

Adult↗

PubMed1997

Gene identification in 1.6-Mb region of the Down syndrome region on chromosome 21.

The Down syndrome (DS) region has been defined by analyses of partial trisomy 21. The 2.5-Mb region between D21S17 and ERG is reportedly responsible for the main features of DS. Within this 2.5-Mb region, we focused previously on a distal 1.6-Mb region from an analysis of Japanese DS patients with partial trisomy 21. Previously we also performed exon-trapping and direct cDNA library screening of a fetal brain cDNA library and identified a novel gene TPRD. Further screening of a fetal heart cDNA library was performed and a total of 44 possible exons and 97 cDNA clones were obtained and mapped on a BamH1 map. By rescreening other cDNA libraries and a RACE reaction, we isolated nearly full-length cDNAs of three additional genes [holocarboxylase synthetase (HCS), G protein-coupled inward rectifier potassium channel 2 (GIRK2), and a human homolog of Drosophila minibrain gene (MNB)] and a coding sequence of a novel inward rectifier potassium channel-like gene (IRKK). The gene distribution and direction of transcription were determined by mapping both ends of the cDNA sequences. We found that these genes, except IRKK, are expressed ubiquitously and are relatively large, extending from 100 kb to 300 kb on the genome. These nearly full-length cDNA sequences should facilitate understanding of the detailed genome structure of the DS region and help to elucidate their role in the etiology of DS.

Amino Acid Sequence↗

PubMed1997

Rare occurrence of ras and p53 gene mutations in mouse stomach tumors induced by N-methyl-N-nitrosourea.

The incidence of point mutations of H-, K- and N-ras and p53 oncogenes in male BALB/c mouse stomach tumors induced with N-methyl-N-nitrosourea (MNU) was examined by direct sequencing and PCR single-strand conformation polymorphism (PCR-SSCP). A mutation of GGT to AGT at K-ras codon 12 was found by SSCP in one adenocarcinoma from a total of 19 specimens including 5 adenocarcinomas, 9 adenomatous hyperplastic regions, 1 squamous cell carcinoma and 4 normal-like stomach regions from 4 mice. No mutations were detected by direct sequencing of H-, K- and N-ras oncogenes at exons 1 (codons 12 and 13) and 2 (codon 61) in a total of 26 specimens comprising 10 adenocarcinomas, 10 adenomatous hyperplastic regions, 2 squamous cell carcinomas and 4 normal-like stomach regions from 6 mice. No mutations were detected by direct sequencing of p53 oncogene at exons 5, 6, 7 and 8 in a total of 30 specimens including 13 adenocarcinomas, 8 adenomatous hyperplastic regions, 2 squamous cell carcinomas, 1 papilloma and 6 normal-like stomach regions from 7 mice. These results suggest that ras and p53 oncogenes do not play a role in mouse stomach carcinogenesis induced by MNU.

Adenoma↗

PubMed1997