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Biomedical subjects

Y Sakaki

Publications and source records attributed to Y Sakaki.

At least 127 records · Page 7Linked to original sources

RNA polymerase III dependence of the human L1 promoter and possible participation of the RNA polymerase II factor YY1 in the RNA polymerase III transcription system.

From the general views of the eukaryotic transcription systems, L1 (or L1-like) retrotransposons that encode some proteins are unusual. L1, unlike other protein-coding elements, is transcribed through an internal promoter. And the L1 internal promoter, unlike other internal promoters, is thought to be RNA polymerase II (pol II) dependent, because the L1 transcript has a large size (approximately 6 kb), protein coding capacity and a 3' terminal polyadenylation signal followed by a poly(A) tail, and also because transcription from the promoter of Drosophila L1-like element jockey was highly sensitive to alpha-amanitin. However, our in vitro transcription study reveals that transcription from the human L1 promoter is highly sensitive to tagetitoxin, a selective inhibitor of RNA polymerase III (pol III), but insensitive to 1 micrograms/ml of alpha-amanitin, indicating that the human L1 promoter is pol III-dependent. The pol III dependence is further supported by our observation that L1 and pol III-dependent tRNA gene promoters share a common nuclear factor YY1. There is evidence that YY1 is also a pol II transcription factor. We thus propose that YY1 is a possible member of the pol III transcription system.

Base Sequence↗

Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (KN) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The KN values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (KA) were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

Amino Acid Sequence↗

Differential display analysis of gene expression in developing embryos of Xenopus laevis.

Differential display (DD), an arbitrarily primed RT-PCR fingerprinting technique, is a novel approach for the search of differentially expressed transcripts. Using our improved DD protocol, reproducible cDNA fingerprints were successfully obtained from RNAs of Xenopus laevis embryos at six representative stages. Parallel comparison among the fingerprints revealed a number of bands with differential expression patterns. Analysis with clones of three randomly chosen bands confirmed that their expression patterns were faithfully reflected on fingerprints, thereby proving the reliability and validity of the approach. Nucleotide sequencing of these clones revealed that one is identical with a known transcript (cardiac actin), the second is a novel developmentally regulated gene showing no significant homology with those reported previously, and the last is a close but unique relative of XK endo B gene showing somewhat different spatial expression pattern. These results indicated that the DD analysis provides a rapid and reliable way for the identification of novel differentially expressed genes as well as a unique 'scope' for the survey of the changes in overall gene expression profiles occurring in the early embryonic development of Xenopus as well as of other organisms.

Amino Acid Sequence↗

High-density cDNA filter analysis: a novel approach for large-scale, quantitative analysis of gene expression.

In order to analyze the expression profiles of a large number of genes in the tissues (or cells) of interest, and to identify the genes preferentially expressed in the tissues, we have developed a large-scale gene expression analysis system. It is based on the hybridization of the mRNAs from the tissues with a high-density cDNA filter followed by the quantitative measurement of the amount of the hybridized mRNA on each cDNA spot. By employing a high-performance bioimaging analyzer, the system allowed us to compare the expression profiles of thousands of genes (cDNAs) simultaneously with a sensitivity comparable to conventional Northern blotting analysis. By this system (called high-density cDNA filter analysis or HDCFA), the expression profiles of 2505 cloned human brain cDNAs (genes) were monitored. Through the comparison of the expression profiles of these cDNAs in the adult brain, fetal brain and adult liver, about one half of these brain cDNAs (1239 clones) were identified as the candidates which were expressed preferentially in the brain. Among these, 408 and 288 clones were found to be preferentially expressed in the adult and fetal brain, respectively. The results have shown that the system may be widely applicable for analysis of the gene expression profiles of various tissues on a large scale.

Base Sequence↗

Structures of genes encoding TATA box-binding proteins from Trimeresurus gramineus and T. flavoviridis snakes.

A cDNA encoding the Trimeresurus gramineus (Tg; green habu snake) TATA-box-binding protein (TgTBP) was cloned and sequenced. The cDNA encodes a 33-kDa protein with an extensive sequence similarity to those derived from other organisms, except for the N-terminal domain. Genes encoding TgTBP and Trimeresurus flavoviridis (Tf; habu snake) TBP (TfTBP) were isolated using a TgTBP cDNA and their nt sequences were determined. They are the first TBP genes entirely sequenced in higher animals. Both genes span over 15 kb and are constructed from eight exons and seven introns. Comparison of the loci of introns on the aligned amino-acid sequences of TBP from six organisms (Tg, Tf, mouse, Arabidopsis thaliana, Schizosaccharomyces pombe and Acanthamoeba castellanii) indicated that there are three highly conserved loci in the C-terminal domain.

Amino Acid Sequence↗

Molecular evolution of group II phospholipases A2.

The nucleotide sequences of 13 cDNAs encoding group II phospholipases A2 (PLA2s), which are from viperidae snake venoms and from mammalian sources, were aligned and analyzed by phylogenetic trees constructed using various components of the sequences. The evolutionary trees derived from the combined sequences of the untranslated (5' and 3') region and the signal peptide region of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. These observations indicated that the mature protein-coding region has evolved through a process differently from the untranslated and signal peptide regions. The trees built from the nucleotide differences at each of three positions of codons in the mature protein-coding region suggested that snake-venom-gland PLA2 genes have evolved via a process different from mammalian PLA2 genes. The occurrence of accelerated evolution has been recently discovered in Trimeresurus flavoviridis venom-gland group II PLA2 isozyme genes (Nakashima et al. 1993, Proc Natl Acad Sci USA 90:5964-5968), so the present phylogenetic analysis together with the estimation of nucleotide divergence of cDNAs provides further evidence that snake-venom-group II PLA2 isozyme genes have evolved by accelerated evolution to gain diverse physiological activities.

Amino Acid Sequence↗

Haplotype analysis of common transthyretin mutations.

The most frequent transthyretin (TTR) variant associated with hereditary amyloidosis is TTR Met 30, which has its major focus in Portugal, although it also occurs in many other countries. The distribution of the mutation and its occurrence in a CpG dinucleotide lead us to question the origin of the mutation and the possibility of its having originated in Portugal. In order to investigate these questions, we studied the distribution of haplotypes associated with the Met 30 mutation in families from different European countries. All the analysed Portuguese families presented the same haplotype associated with the Met 30 mutation (haplotype I). The same was found for the Swedish and Spanish families studied. However, a distinct haplotype (haplotype III) was found in three families, one Italian, one English and one Turkish. These results suggest that, although the Portuguese Met 30 carriers might have one founder, the mutation probably recurred in populations in Europe in a similar manner to that reported in Japan. In this study, we have also analysed the haplotypes associated with other TTR variants frequent in the Portuguese population.

Amyloidosis↗

Study of the regenerating newt retina by electrophysiology and immunohistochemistry (bipolar- and cone-specific antigen localization).

Immunohistochemical and electrophysiological examinations were carried out to investigate the sequence of appearance of the retinal neurons during regeneration after a complete surgical removal of the original retina of the newt. We produced a monoclonal antibody, RB-1, specific for cone photoreceptors and a subtype of bipolar cells in adult newt retina. This antibody was used as a major tool for this analysis. Appearance of spiking activity as a possible marker of ganglion cell differentiation was examined with whole-cell patch-clamp techniques. Spiking cells, which possessed voltage-dependent Na+, K+, and Ca2+ channels similar to those of mature ganglion cells, appeared in the regenerating retina by 24 days before cone photoreceptors had been labeled by the RB-1 antibody. Cones and ganglion cells differentiated before the retina had been segregated into distinct synaptic layers. The RB-1-labeled bipolar cells as well as PKC-immunoreactive bipolar cells appeared in the regenerating retina after the segregation of the synaptic layers. Their appearance seemed to coincide with the appearance of immunoreactive amacrine cells described previously (Negishi et al. [1992] Dev. Brain Res. 68:255-264). During embryonic development of the newt retina, cone photoreceptors appeared prior to bipolar cells. Thus the process of reformation of a functional retina seems to follow the same steps as differentiation of retina during development.

Animals↗

Genomic analysis of a NF1-related pseudogene on human chromosome 21.

A neurofibromatosis type-1 (NF1)-related locus on human chromosome 21 has been characterized. A detailed genomic mapping performed by yeast artificial chromosome (YAC) dot-hybridization revealed that the NF1-related locus is close to the sequence-tagged site (STS), D21S329 (G52E12), which is located on the proximal region of 21q11.2. Sequence analysis showed that this locus seemed to be conserved to the NF1 gene only in several partial regions. Two exon-like segments corresponding to exons 8 and 9 of NF1 were found, in addition to two previously found fragments corresponding to exons 7 and 11. Other exon-like segments were not found in the region so far sequenced. Comparing these homologous segments with the NF1 cDNA, a 2-bp deletion appeared in exon 8 of the locus, resulting in the existence of stop codons in all reading frames. In addition, there were no amplified fragments derived from the related locus by reverse transcription-PCR. Thus, our results suggest that the NF1-related locus on chromosome 21 is a nonprocessed pseudogene.

Animals↗

Fluorescent differential display: arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer.

We established robust, reliable protocols for 'Differential Display (DD),' an RNA fingerprinting method originally developed by Liang and Pardee [(1992) Science 257, 967-971] using RT-PCR with arbitrary primers. Our protocols are optimized so that reliable DD analysis can be performed on a fluorescent DNA sequencer to ensure high throughput as well as improved operational safety, compared with the original one using radioactive compounds. Such 'Fluorescent Differential Display (FDD)' techniques will accelerate the identification of differentially expressed as well as polymorphic transcripts to address various biological questions.

Base Sequence↗

Cloning and sequence analysis of the human SNAP25 cDNA.

During the screening of human brain-specific cDNAs by a modified differential hybridization analysis, we found a clone which was highly and specifically expressed in the (adult) brain. This clone was subjected to nucleotide sequence analysis and proven to be a nearly full-length cDNA of a human homologue of the previously reported mouse synaptosomal-associated protein 25 (mSNAP25). The human SNAP25 (hSNAP25) and mSNAP25 showed perfect amino-acid sequence conservation.

Base Sequence↗

Expression of the alpha 2-macroglobulin-encoding gene in rat brain and cultured astrocytes.

The alpha 2-macroglobulin (alpha 2M), a protease inhibitor, is a major acute-phase protein in rats, and is produced in the liver during acute inflammation. Recently, it has been demonstrated that alpha 2M is also produced by cultured astrocytes from newborn rat brain and has neurite-promoting activity. Here, we found that the expression of the alpha 2M gene was significantly enhanced in the brain following intraperitoneal injection of the neurotoxicant, kainic acid (KA), suggesting that alpha 2M acts as an acute-phase protein in the brain, as in the case of the liver, and may be involved in neural repair processes. Expression of alpha 2M in cultured astrocytes was shown to be stimulated by interleukin-6 (IL-6) and/or leukemia inhibitory factor (LIF) in the presence of glucocorticoid. The amount of mRNAs for IL-6 and LIF increased in the brain of KA-injected rats prior to alpha 2M induction. These results strongly suggested that IL-6 and LIF are involved in alpha 2M induction in the brain, as in the case of the liver. Analysis of the cis-acting element(s) and the trans-acting factor(s) suggested that the regulatory mechanism for alpha 2M expression in astrocytes was similar to that in inflamed liver.

Animals↗

Sixty new STSs (sequence-tagged sites) of human chromosome 21.

From human chromosome 21-specific libraries, 22 SfiI linking clones and 38 P1 clones were isolated and regionally mapped on the chromosome. The terminal sequences of these clones were determined and pairs of PCR primers were generated which could specifically amplify the sequenced regions. These sequence-tagged sites (STSs) should be useful for constructing a high resolution map of human chromosome 21.

Base Sequence↗

Polymorphisms of Trimeresurus flavoviridis venom gland phospholipase A2 isozyme genes.

Trimeresurus flavoviridis venom gland phospholipase A2 (PLA2) genes pgPLA 1a and pgPLA 2a encode Asp-49-PLA2 and genes pgPLA 1b and pgPLA 2b encode an isozyme of Asp-49-PLA2. Polymorphisms were found in pairs of pgPLA 1a and pgPLA 2a and of pgPLA 1b and pgPLA 2b for individuals of T. flavoviridis. The occurrence of both homozygotes and heterozygotes was demonstrated.

Animals↗

Absence of linkage disequilibrium at amyloid precursor protein gene locus in Japanese familial Alzheimer's disease with 717Val-->Ile mutation.

To date, eleven independent FAD pedigrees with the 717Val-->Ile mutation have been identified. Interestingly, five pedigrees were of Japanese origin and four were of British origin. The apparent ethnic prediction of this mutation raises the possibility that there is a founder effect in these two island nations. We did not observe any significant linkage disequilibrium in any locus of APP and GT12 loci in the five Japanese FAD pedigrees with the 717Val-->Ile mutation. A founder effect would probably not be present in Japanese FAD pedgrees with the 717Val-->Ile mutation.

Alleles↗

Neuron-specific splicing of the Alzheimer amyloid precursor protein gene in a mini-gene system.

Several forms of Alzheimer amyloid precursor protein (APP) mRNA are generated by alternative splicing. Among them, the APP695 mRNA skipping the exon 7 and 8 is expressed specifically in neurons, suggesting that this alternative splicing is regulated in a neuron-specific manner. As the first step for investigating the mechanism of the neuron-specific splicing, a mini-gene system was developed, in which mini-APP genes consisting of the exon 6, 7, 8, 9 and their flanking regions were introduced into neuronal and nonneuronal cultured cell lines to see their expression profiles. In the system the exon 7 and 8 of the mini-gene were significantly skipped in the neuronal cell, and the deletion study indicated that cis-acting elements for skipping the exons existed in the corresponding skipped-exon and its flanking regions. A small deletion upstream of the exon 8 suppressed the skipping of the exon 8 in the neuronal cell, suggesting that one of the regulatory sequence(s) for the exon skipping exists in a small region upstream of the skipped exon.

Alternative Splicing↗