[ACF and chromatin reconfiguration].
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Adenosine Triphosphate↗
Biomedical subjects
M Muramatsu
Publications and source records attributed to M Muramatsu.
Molecular cloning, localization, and developmental expression of mouse brain finger protein (Bfp)/ZNF179: distribution of bfp mRNA partially coincides with the affected areas of Smith-Magenis syndrome.
Bfp (brain finger protein) is a member of the RING finger protein family, which is highly expressed in the brain. We have previously shown that one copy of the human bfp gene, mapped at 17p11.2, was actually deleted in six of six Smith-Magenis syndrome (SMS) patients. Now we have isolated the mouse bfp cDNA. Using in situ hybridization and immunohistochemistry, the distribution of mouse bfp mRNA and protein was identified especially in neural cells of the cerebral cortex, hippocampus, lateral amygdaloid nucleus, and ventromedial hypothalamus. In primary culture of the whole brain in a neonatal mouse, the Bfp protein was detected in both neuron and glial cells, and its subcellular localization was predominantly in the nucleus, but some amounts were also found in the cytoplasm. The bfp mRNA was also expressed strongly in the marginal zone of brain vesicles, optic stalk, and cartilage primordium, which are part of the critical tissues frequently involved in SMS patients, and in such tissues as nasal epithelium and primordium of follicles in a 13. 5-dpc embryo. Subsequently, its amount in the developing brain further increased during embryogenesis, reaching the highest level in the adult brain. These findings suggest a possibility that Bfp might be involved in the pathogenesis of Smith-Magenis syndrome as a regulator protein related to neural differentiation and function.
Abnormalities, Multiple↗
NOLP: identification of a novel human nucleolar protein and determination of sequence requirements for its nucleolar localization.
This study reports cDNA isolation and partial characterization of a novel human nucleolar protein isolated by "nuclear transportation trap" described previously. The cDNA encodes a putative polypeptide of 524 amino acids with a short Escherichia coli DNA helicase homologous region, an acid-rich domain, three potential base-rich nuclear localization signals (NLSs), a serine-rich domain, and a deduced coiled-coil domain. The protein has no known prominent similarities with any other protein in the protein databases. Tissue distribution analysis demonstrated a predominant expression in brain and testis. To determine the sequence requirements for nucleolar targeting, a set of deletion constructs with a fluorescent tag were transiently expressed in COS-7 cells. We revealed that a region of 30 amino acids (position 342-371), which overlaps the first and second NLS, is sufficient for nucleolar localization. Furthermore, the adjacent region of 30 amino acids (position 372-401), which contains the third NLS, is sufficient for nuclear localization. These results suggest that this novel nucleolar protein has at least two distinct domains for directing to different subnuclear destinations.
Amino Acid Sequence↗
Identification of stable RNA hairpins causing band compression in transcriptional sequencing and their elimination by use of inosine triphosphate.
To identify stable RNA secondary structure causing band compression, 30 lambda DNA clones and four cDNA clones (about 10 kb in total length) were sequenced using Transcriptional Sequencing, which is based on the phage RNA polymerase chain termination reaction with fluorescent 3' deoxynucleoside triphosphate, using the canonical set of rNTPs for the substrate. Electrophoresis was performed on acrylamide gel containing 7 M urea at 50 degrees C using ABI 377 DNA sequencer. A total of 159 band compressions were identified, and most compression sites seem to be due to hairpin structures. We also found that the presence of rITP in place of rGTP in the sequencing reaction can entirely eliminate all band compressions. The use of rITP gave a better peak uniformity and resolution in the sequencing gel in the case of lambda DNA than with c7rGTP, leading to improved accuracy in the sequence determination. Substitution of the base analog rITP for rGTP should be useful for accurate sequencing determination.
Bacteriophage lambda↗
Cloning of a novel rat gene, DB83, that encodes a putative membrane protein.
Using a partial cDNA sequence and a 5'-RACE technique, we isolated a novel cDNA from rat liver referred to as DB83. DB83 had four hydrophobic trans-membrane domains and one N-myristoylation site as well as multiple possible phosphorylation sites. The db83 gene was highly expressed in the liver and significantly in brain, lungs and kidneys. We suggest that DB83 is a tissue-specific putative membrane protein. p6
Amino Acid Sequence↗
Molecular cloning of a novel RING finger-B box-coiled coil (RBCC) protein, terf, expressed in the testis.
RING finger is a variant zinc finger motif present in a new family of proteins including transcription regulators. Here, utilizing the polymerase chain reaction with degenerate primers, we isolated a genomic DNA fragment containing the RING finger motif. Using this fragment as a probe, we have identified a novel cDNA from rat testis library. Then, the human homologue of the terf cDNA was also isolated from a testis library. This gene was designated testis RING finger protein (terf) because the corresponding transcripts were detected almost exclusively in the testis by Northern blot analysis. Both cDNAs encode an open reading frame of 477 amino acids sharing high homology (74% identity at the protein level) between two species. The terf contains an N-terminal RING finger domain, one B-box domain, middle coiled-coil domain, and a C-terminal domain, belonging to the RING finger-B box-coiled coil (RBCC) family. Several RBCC proteins, such as PML, TIF1alpha and RFP, have transformation capabilities when found in chromosomal translocations. Among the members of the RBCC family, the terf shares highest homology (40% identity at the protein level) with RFP that is expressed only in the testis in normal tissues. Structural similarity raises the possibilities that the terf gene might be also involved in carcinogenesis or cell transformation.
Amino Acid Sequence↗
Characterization of functional domains of an embryonic stem cell coactivator UTF1 which are conserved and essential for potentiation of ATF-2 activity.
We have recently cloned a cDNA encoding an embryonic stem cell transcriptional coactivator termed UTF1 from the mouse F9 teratocarcinoma cell line (Okuda, A., Fukushima, A., Nishimoto, M., Orimo, A., Yamagishi, T., Nabeshima, Y., Kuro-o, M., Nabeshima, Y., Boon, K., Keaveney, M., Stunnenberg, H.G., and Muramatsu, M. (1998) EMBO J. 17, 2019-2032). Here we have cloned a cDNA for human UTF1 and identified two highly conserved domains termed conserved domain (CD)1 and CD2. Human UTF1, like that of mouse, binds to ATF-2 and the mutagenesis analyses reveal that the leucine zipper motif within the CD2 of the UTF1 and metal binding motif of ATF-2 are involved in this interaction. The factor also binds to TATA-binding protein containing complex. By means of immunoprecipitation analysis, we mapped two domains which are independently able to bind to the complex. Importantly, both domains are located within the conserved domains (one in CD1 and the other in CD2). Furthermore, transient transfection analyses point out the importance of these domains for activating ATF-2. Thus, these results suggest that these two conserved domains identified here play important roles in activating specific transcription at least in part by supporting physical interaction between the upstream factor, ATF-2, and basal transcription machinery.
Activating Transcription Factor 2↗
Molecular features of a new human lymphoma cell line carrying both BCL2 and BCL6 gene rearrangements.
Chromosomal translocations and/or their molecular equivalents involving the BCL6 gene on 3q27 band have been suggested to be involved in the development of non-Hodgkin's lymphoma of B-cell type (B-NHL). The rearrangement of BCL6 sometimes coexists with other translocations specific to B-NHL. Here, we report a novel B-cell lymphoma cell line, YM, established from a patient with diffuse large cell lymphoma. The YM cells expressed B-cell-associated antigens in addition to mu delta/kappa monoclonal immunoglobulin. Southern blot analysis of DNA from YM cells demonstrated rearrangement of the BCL2 gene within the 5' flanking region (5'-BCL2). Polymerase chain reaction (PCR) using primer pairs for the BCL2 exons 1 and 2, and for the constant region of the immunoglobulin kappa light chain gene (IGkappa) revealed PCR products encompassing the 5'-BCL2/IGkappa fusion, indicating that the YM cells had a t(2;18)(p11;q21) translocation. The BCL6 gene was rearranged at a point within the first intron, and cloning of the rearranged BCL6 revealed unidentified sequences juxtaposed to the 5' side of the gene. The isolated clones were mapped to 16p11.2 by high resolution fluorescence in situ chromosomal hybridization. Thus, the YM cells carried a 3q27 translocation involving 16p11.2 as a partner. Chromosome painting of metaphase spreads confirmed that the YM cells had both t(2;18) and t(3;16). Northern blot analysis using a fragment immediately adjacent to the breakpoint on 16p11.2 revealed transcriptional activity within this locus. The YM cells expressed abundant transcripts with aberrant sizes from BCL2 and BCL6, indicating deregulated overexpression of the two genes resulting from the t(2;18) and t(3;16). The YM cell line will therefore be useful to study whether BCL2 and BCL6 genes collaborate in the pathogenesis of B-NHL.
Base Sequence↗
Promoter analysis of mouse estrogen-responsive finger protein (efp) gene: mouse efp promoter contains an E-box that is also conserved in human.
The estrogen-responsive finger protein (efp) containing a RING finger motif has been identified as an estrogen-responsive gene in human and mouse. Here, we have characterized the basal promoter region of the mouse efp gene. The promoter lacks the TATA motif, and transcription initiation sites are found at positions -38T, -64A and -73C from the translation initiation site. Deletion analysis of the 5'-flanking region using Jyg-Mc(B) mouse breast cancer cells indicates that the sequence encompassing from -139 to -1 has a basal transcription activity. This region is GC-rich in both mouse and human promoters, and the E-box is precisely matched on the sequence alignment. A mutation experiment with E-box shows that the E-box is functionally active. An electrophoretic mobility shift assay using Jyg-Mc(B) nuclear extracts shows that a transcription factor, USF-1 binds to the E-box in the mouse efp promoter. It has been shown that the E-box in the human efp promoter is indispensable for basal transcriptional activity and binds USF-1. These findings suggest that the mouse efp promoter is regulated by a similar mechanism to that of the human. In mouse, however, we have not found a negative regulatory region that is present in human promoter.
Animals↗
Molecular cloning and characterization of human estrogen receptor betacx: a potential inhibitor ofestrogen action in human.
We have identified and characterized a novel human estrogen receptor (ER) beta isoform, ERbetacx, which is truncated at the C-terminal region but has an extra 26 amino acids due to alternative splicing. The ERbetacx transcript is expressed in testis, ovary, thymus and prostate as well as in human cultured cell lines such as HEC-1, HOS-TE85 and Saos-2 cells. ERbetacx protein is also immunodetectable in these human cells. Biochemical analysis reveals that the average dissociation constants ( K d) of ERalpha and ERbeta for 17beta-estradiol (E2) are 0.2 and 0.6 nM respectively, but ERbetacx has no ligand binding ability. ERalpha and ERbeta proteins bind to the estrogen response element, whereas ERbetacx does not form any shifted complex in gel shift assays. In a transient expression assay, ERbetacx shows no ligand-dependent transactivation ability of a basal promoter and also cannot interact with a cofactor, TIF1alpha, in the presence or absence of E2. ERbetacx preferentially forms a heterodimer with ERalpha rather than that with ERbeta, inhibiting DNA binding by ERalpha. Interestingly, however, it shows a significant dominant negative activity only against ERalpha transactivation. Thus, this study indicates that ERbetacx potentially inhibits ERalpha-mediated estrogen action and that alternative splicing of the C-terminal region and its inhibitory properties are characteristic of several members of nuclear receptor isoforms.
Amino Acid Sequence↗
Human ULK1, a novel serine/threonine kinase related to UNC-51 kinase of Caenorhabditis elegans: cDNA cloning, expression, and chromosomal assignment.
The unc-51 gene, isolated from mutants of Caenorhabditis elegans exhibiting abnormal axonal extension and growth, encodes a novel serine/threonine kinase (K. Ogura, et al., 1994, Genes Dev. 8: 2389-2400). Here we report the molecular cloning and characterization of the human homologue of UNC-51, designated ULK1, for UNC-51 (C. elegans)-like kinase 1. Sequence analysis of the human ULK1 cDNA showed that an open reading frame is composed of 1050 amino acids with a calculated MW of 112.6 kDa and a pI of 8.80. Homology search analysis showed that ULK1 has 41% overall similarity to UNC-51 and 29% similarity to Apg1p of Saccharomyces cerevisiae. Phylogenetic analysis of ULK1, UNC-51, and Apglp suggested that they constitute a novel subfamily of serine/threonine kinases. Southern blot analyses suggested that the ULK1 gene spans 30-40 kb in the human genome as a single-copy gene. Zoo blot analysis indicated that ULK1 kinase is conserved among vertebrates including mammals, birds, reptiles, amphibians, and fish. Northern blot analysis revealed that ULK1 is ubiquitously expressed in adult human tissues such as skeletal muscle, heart, pancreas, brain, placenta, liver, kidney, and lung, whereas UNC-51 is specifically detected in the nervous system of C. elegans. Both FISH and RH mapping confirmed the regional localization of ULK1 to human chromosome 12q24.3.
Amino Acid Sequence↗
Rapid fragmentation of vimentin in human skin fibroblasts exposed to tamoxifen: a possible involvement of caspase-3.
Tamoxifen (TAM), an anti-estrogen compound, is widely used for chemotherapy of breast cancer, although the molecular mechanisms underlying TAM cytotoxicity are obscure. Here, we show that TAM dramatically caused degradation of vimentin (VIM) in human skin fibroblasts, in a time and dose dependent manner. Addition of caspase-3 inhibitor, Z-DEVD-FMK, inhibited formation of some fragments of VIM, and caspase-3 was proteolytically activated by TAM treatment. Expression of functional estrogen receptors were negative in these cells, and neither transcription nor protein synthesis was required for TAM-induced degradation of VIM. Moreover, quinestrol, an ethinyl estradiol derivative, weakly degraded VIM, whereas neither estradiols nor estriol had any effects. Taken together, TAM may induce fragmentation of VIM associated with an activation of caspase-3, which may be attributed to non-genomic actions of TAM.
Antineoplastic Agents, Hormonal↗
GPIIb/IIIa integrin antagonists with the new conformational restriction unit, trisubstituted beta-amino acid derivatives, and a substituted benzamidine structure.
Ethyl N-[3-(2-fluoro-4-(thiazolidin-3-yl(imino)methyl)benzoyl)amino-2, 2-dimethylpentanoyl]piperidine-4-acetate 40 (NSL-96184) is a highly potent and orally active fibrinogen receptor antagonist, which is characterized by the presence of the trisubstituted beta-amino acid residue, 3-ethyl-2,2-dimethyl-beta-alanine. This compound was developed on the basis of the SAR study of N-[3-(N-4-amidinobenzoyl)amino-2, 2-dimethyl-3-phenylpropionyl]piperidine-4-acetic acid 1(NSL-95301) with the derivatization focused on the central trisubstituted beta-amino acid unit as well as the basic amidinobenzoyl unit, and the esterification of the carboxyl group for prodrug composition. Compound 1, which was reported in our previous study, was discovered by the application of combinatorial chemistry. The molecular modeling study suggests that the trisubstituted beta-amino acid unit is responsible for fixing the molecule to its active conformation. Compound 40 showed an excellent profile in the in vitro and in vivo studies for its human platelet aggregation inhibitory activity and oral availability in guinea pigs. This oral availability largely depends on the modification of the amidino group with a cyclic secondary amine, i.e., thiazolidine in 40. In in vivo studies, the onset of the antiplatelet action of 40 is very fast after oral administration, whereas its duration of action is relatively short. These results suggest that 40 has an excellent therapeutic potential, especially for antithrombotic treatment in the acute phase. 3-Substituted-2,2-dimethyl-beta-amino acid residues would serve as new and useful linear templates to restrict the conformational flexibility of peptidomimetics.
Administration, Oral↗
Recognition sites of 3'-OH group by T7 RNA polymerase and its application to transcriptional sequencing.
When analyzing the elongation mechanisms in T7 RNA polymerase (T7 RNAP)by using site-directed mutagenesis and a protein expression system, we identified the recognition sites of the rNTP 3'-OH group in T7 RNAP. On the basis of three-dimensional crystal structure analysis, we selected and analyzed six candidate sites interacting with the 3'-OH group of rNTP in T7 RNAP. We found that the Phe-644 and Phe-667 sites are responsible for the high selectivity of T7 RNAP for rNTPs. Also, we constructed the protein mutations of these residues, F644Y and F667Y, which display a >200-fold higher affinity than the wild type for 3'-dNTPs. These findings indicate that the phenylalanine residues of 644 and 667 specifically interact with the 3'-OH group. Thus, these mutants, F644Y and F667Y, with incorporation of 3'-dNTP terminators, which is similar to native rNTPs, can offer low backgrounds and equal intensities of the sequencing ladders in our method, called "transcriptional sequencing. "
Amino Acid Sequence↗
Identification of mouse ULK1, a novel protein kinase structurally related to C. elegans UNC-51.
A novel protein kinase related to the C. elegans serine/threonine kinase UNC-51 was cloned from mouse. The UNC-51-Like Kinase (ULK)1 is encoded by a cDNA of 1051 amino acids with calculated MW of 113 kDa. Comparison of the ULK1 and UNC-51 shows the highest conservation in the amino-terminal kinase domain, which is followed by a proline/serine-rich (PS) domain and a conserved carboxyl-terminal (C) domain. ULK1 mRNA is expressed in various tissues, and is mapped to mouse chromosome 5F and rat chromosome 12q16.3, by fluorescent in situ hybridization. HA-tagged ULK1 is expressed as a protein of approximately 150 kDa in COS7 cells and is auto-phosphorylated in vitro in its PS domain. We propose that ULK1, UNC-51 and a yeast protein kinase Apg1p comprise a novel subfamily of protein kinase, which is structurally conserved among eukaryotes.
Amino Acid Sequence↗
Characterization of gene expression in mouse blastocyst using single-pass sequencing of 3995 clones.
To study the gene expression profile in the mouse blastocyst and to identify embryonic stage-specific genes, we randomly selected cDNAs derived from mouse blastocysts and sequenced a total of 3995 clones from one or both ends. Excluding the uninformative clones, 3395 clones were grouped as 937 different kinds of genes. Among these, 465 and 406 species showed similarity to known genes and expressed sequence tags (ESTs), respectively, whereas 66 species showed no significant similarity to any genes in known databases. Analysis of these cDNAs revealed that this library contained a variety of functional genes as well as genes that have not been detected in the human EST database; it should provide us with a useful resource for molecular analysis of developmental mechanisms. Although the human EST project is considered to represent roughly half of all genes, our findings indicate that many early stage developmental genes remain to be identified.
Animals↗
A methylation imprint mark in the mouse imprinted gene Grf1/Cdc25Mm locus shares a common feature with the U2afbp-rs gene: an association with a short tandem repeat and a hypermethylated region.
We identified a sperm-specific methylation imprint mark (Site II) associated with a short tandem repeat sequence and a site/region methylated in both gametes (Site I) in the Grf1 locus on mouse chromosome 9, which shared a common feature with the U2afbp-rs gene. Sites or regions of gamete-specific methylation in imprinted genes are strong candidates for carrying information regarding the parental origin of alleles. The gamete-specific methylation pattern of Sites I and II was conserved after fertilization, but attained the somatic cell pattern by the blastocyst stage. In primordial germ cells, Site I was methylated, but Site II was unmethylated in both male and female embryos, suggesting that the sperm-specific methylation imprint mark in Site II was established during spermatogenesis. These common features in methylation imprint regions may be a clue to identifying regions carrying primary information for the imprinting regulation.
Animals↗
UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells.
We have obtained a novel transcriptional cofactor, termed undifferentiated embryonic cell transcription factor 1 (UTF1), from F9 embryonic carcinoma (EC) cells. This protein is expressed in EC and embryonic stem cells, as well as in germ line tissues, but could not be detected in any of the other adult mouse tissues tested. Furthermore, when EC cells are induced to differentiate, UTF1 expression is rapidly extinguished. In normal mouse embryos, UTF1 mRNA is present in the inner cell mass, the primitive ectoderm and the extra-embryonic tissues. During the primitive streak stage, the induction of mesodermal cells is accompanied by the down-regulation of UTF1 in the primitive ectoderm. However, its expression is maintained for up to 13.5 days post-coitum in the extra-embryonic tissue. Functionally, UTF1 boosts the level of transcription of the adenovirus E2A promoter. However, unlike the pluripotent cell-specific E1A-like activity, which requires the E2F sites of the E2A promoter for increased transcriptional activation, UTF1-mediated activation is dependent on the upstream ATF site of this promoter. This result indicates that UTF1 is not a major component of the E1A-like activity present in pluripotent embryonic cells. Further analyses revealed that UTF1 interacts not only with the activation domain of ATF-2, but also with the TFIID complex in vivo. Thus, UTF1 displays many of the hallmark characteristics expected for a tissue-specific transcriptional coactivator that works in early embryogenesis.
Activating Transcription Factor 2↗