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K Ozato

Publications and source records attributed to K Ozato.

At least 91 records · Page 5Linked to original sources
PubMed1995

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.

Amino Acid Sequence↗
PubMed1995

Transcription factor TFIIB and the vitamin D receptor cooperatively activate ligand-dependent transcription.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates gene transcription through binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily. Sequence-specific transcription factors, including nuclear hormone receptors, are thought to interact with the basal transcription complex to regulate transcription. In glutathione S-transferase fusion-based protein-protein binding assays we found that VDR specifically binds to TFIIB, a component of the basal complex, and that the interaction requires select domains of each protein. To assess the functional significance of this interaction, transfection assays were performed with a 1,25(OH)2D3-responsive reporter construct. In P19 embryonal carcinoma cells cotransfection of VDR and TFIIB cooperatively activated reporter transcription, while each factor alone gave very low to no activation. This activation was dependent on 1,25(OH)2D3 and the dose of TFIIB and VDR transfected, demonstrating that a nuclear hormone receptor functionally interacts with TFIIB in vivo. In contrast, transfection of NIH 3T3 cells generated strong reporter activation by 1,25(OH)2D3 in the presence of VDR alone, and cotransfection of TFIIB led to specific dose-dependent repression of reporter activity. Taken together, these results indicate that TFIIB-nuclear hormone receptor interaction plays a critical role in ligand-dependent transcription, which is apparently modulated by a cell-type-specific accessory factor.

Adult↗
PubMed1995

Physical mapping of the retinoid X receptor B gene in mouse and human.

Retinoid X receptors (RXRs) are zinc finger-containing nuclear transcription factors. They belong to the nuclear receptor superfamily that contains retinoid receptors, vitamin D receptors, thyroid hormone receptors, and steroid hormone receptors as well as the so-called orphan receptors. We previously mapped all three RXR genes on mouse chromosomes, using a panel of Mus spretus-Mus musculus interspecific backcross mice: Namely, the RXRA-gene (Rxra) on Chr 2 near the centromere, the RXRB gene (Rxrb) on Chr 17 in the H2 region, and the RXRG gene (Rxrg) on distal Chr 1. Using cosmid clones that cover the major histocompatibility complex (MHC) region, we determined the precise physical map positions of the gene encoding mouse and human RXRB, respectively. The mouse gene (Rxrb) maps between H2-Ke4 and H2-Ke5: namely, immediately telomeric to H2-Ke4 which encodes a histidine-rich transmembrane protein, and 12 kilobases centromeric to H2-Ke5 which is expressed in lymphoid tissues. Rxrb and H2-Ke4 are transcribed into opposite directions from a CpG-rich promoter of about 250 base pairs. This gene organization is well conserved also in the human genome at the HLA-DP subregion of Chr 6p, underscoring the strong conservation of the gene organization in the MHC region between the two mammals.

Animals↗
PubMed1995

NF kappa B and interferon regulatory factor 1 physically interact and synergistically induce major histocompatibility class I gene expression.

Major histocompatibility (MHC) class I gene expression is synergistically induced by the cytokines TNF-alpha and IFN-gamma. However, the mechanism that results in synergistic activation of these genes has remained unclear. We demonstrated here that TNF-alpha induced binding of NF kappa B p50 and p65 to the NF kappa B-like element of the MHC class I promoter termed region I and IFN-gamma induced binding of IRF-1 to the adjacent interferon consensus sequence (ICS). We further demonstrated that NF kappa B and IRF-1 physically interacted with each other and cooperatively induced MHC class I gene expression when cotransfected into CHP-126 neuroblastomas. These results provide a molecular mechanism by which TNF-alpha and IFN-gamma synergistically induce the expression of a variety of genes involved in immune responses, including MHC class I.

Base Sequence↗
PubMed1995

Rapid isolation and characterization of 118 novel C2H2-type zinc finger cDNAs expressed in human brain.

C2H2-type zinc finger genes comprise one of the largest gene families in the human genome. These proteins are involved in genetic regulation and development and are quite conserved throughout evolution. The finger domains commonly contain the small linker peptide TGEKP between some finger units. Here, we report the isolation of 133 human zinc finger cDNAs, of which 118 are novel. These clones were isolated from human brain cDNA libraries using oligonucleotide hybridization followed by expressed sequence tag (EST) analysis, sequencing from the conserved linker region using degenerate oligonucleotide primers. This directed partial sequencing approach to cDNA isolation and characterization, signature sequencing, combines the speed of EST automatic sequencing with the focus of specific cDNA family analysis. Signature sequencing minimizes the generation of less informative random EST sequences and provides a unique relative position for sequence comparison. We also show that there is an even distribution of these RNAs from this brain cDNA library, and that these cDNAs contain N-terminal domains found in other zinc finger genes. This rapid focused sequencing approach should be applicable to any family of cDNAs containing short conserved signature peptide sequences.

Amino Acid Sequence↗
PubMed1995

Vesicular stomatitis virus infection induces a nuclear DNA-binding factor specific for the interferon-stimulated response element.

Vesicular stomatitis virus (VSV) has a broad host range. It replicates in the cytoplasm and causes rapid cytopathic effects. We show that following VSV infection, a nuclear factor that binds to a select set of interferon-stimulated responsive elements (ISRE) is induced in many cell types. This factor, tentatively called VSV-induced binding protein (VIBP), was estimated to have an approximate molecular mass of 50 kDa and was distinct from known members of the interferon regulatory factor family, that are known to bind to the ISRE. Induction of VIBP required tyrosine kinase activity but did not require cellular transcription. Treatment of cells with cycloheximide, which inhibits translation, only partially inhibited induction of VIBP. However, type I interferons and staurosporine, both of which inhibit VSV transcription, inhibited VIBP induction. Moreover, a double-stranded RNA analog, poly(I)-poly(C) also induced a DNA-binding activity very similar to that of VIBP. These results indicate that a preexisting cellular protein is activated upon VSV infection and that this activation requires primary viral transcripts. The functional activity of VIBP was analyzed in cells stably transfected with a herpesvirus thymidine kinase-luciferase reporter gene that is under control of the ISRE. While activity of the control promoter without ISRE was strongly inhibited following VSV infection (as a result of virus-mediated transcriptional shutdown of the host cell), the inhibition was reversed by the ISRE-containing promoter, albeit partially, which suggests that VSV infection differentially affects transcription of host genes. Although VIBP was induced in all other cells tested, it was not induced in embryonal carcinoma cells after VSV infection, suggesting developmental regulation of VIBP inducibility.

Amino Acid Sequence↗
PubMed1995

Mouse retinoid X receptor contains a separable ligand-binding and transactivation domain in its E region.

Steroid, thyroid, and retinoid hormones exert their biological functions by interacting with their cognate nuclear receptors. Upon binding receptors, hormones induce a protease-resistant structural change in the receptor ligand-binding domain and subsequently activate the receptors. Utilizing partial proteolysis, we have been able to delineate a region in the mouse retinoid X receptor beta (mRXR beta) required for ligand binding. A separable activation domain within the mRXR beta E region has been identified. The activation domain, which is 21 amino acids in length, is located at the extreme C terminus of mRXR beta. This domain is not required for ligand binding since removal of this sequence neither eliminates the ligand-induced, protease-resistant conformational change nor alters the ligand-enhanced DNA binding. Furthermore, deletion of this activation domain converts the receptor into a transcriptional silencer. Finally, a further truncation of 9 amino acids (for a total of 30 amino acids) from the C terminus results in a mutant which does not undergo the protease-resistant conformational change and cannot bind DNA as a homodimer. Nevertheless, this mutant is still able to form a heterodimer with the thyroid hormone receptor. Therefore, homodimerization and heterodimerization can be distinguished by this nine-amino-acid sequence.

Amino Acid Sequence↗
PubMed1995

Initiation of the transgenic lacZ gene expression in medaka (Oryzias latipes) embryos.

In total, 4165 medaka (Oryzias latipes) oocytes were injected with three DNA constructs separately, and results showed that exogenous lacZ expression was transient and stage-dependent. The initiation of the transgene expression was at the mid-blastula stage for embryos derived from oocytes injected with pmiwZ, containing the long terminal repeat (LTR) of the Rous sarcoma virus, and with pCAGGS-lacZ, containing the enhancer and promoter of the immediate early gene of the human cytomegalovirus, respectively, whereas embryos derived from oocytes injected with pMoZtk, containing the LTR of the Moloney murine leukemia virus, started expression at the late-blastula stage. These reveal that the earliest onset of the exogenous lacZ gene should be by the mid-blastula stage. Therefore the mid-blastula transition phenomenon in embryogenesis known in other animal species exists in medaka embryos.

Animals↗
PubMed1994

Mechanisms for synergistic activation of thyroid hormone receptor and retinoid X receptor on different response elements.

The thyroid hormone receptors (TR) form heterodimers with the retinoid X receptors (RXR) and activate target genes through thyroid-responsive elements (TRE). Heterodimerization elevates the DNA binding efficiency and thus can result in functional synergism between TR and RXR. Here we demonstrate that DNA sequences dictate the cooperative activation between TR and RXR despite the high affinity binding of the heterodimer to those TREs. We provide evidence that the C-terminal activation domain of RXR can modulate the triiodothyronine (T3) responsiveness of TR/RXR heterodimers on reporter genes without altering the DNA binding properties of the heterodimers. The modulation function of this relatively small region is under the control of specific TRE sequences and promoter context. These data indicate that this C-terminal region of RXR is likely involved in receptor-cellular factor(s) interactions. Finally, we propose that the synergistic activation by TR and RXR is achieved through elevated DNA binding and, dependent on the DNA sequence, the interaction of RXR with other transcription factors.

Base Sequence↗
PubMed1994

Characterization of hUCRBP (YY1, NF-E1, delta): a transcription factor that binds the regulatory regions of many viral and cellular genes.

The UCRBP (YY1, delta, NF-E1) protein has been isolated for its ability to bind to the UCR (upstream conserved region) site present in the conserved murine leukemia virus long terminal repeat. UCRBP carries a highly charged N-terminal domain and four C2-H2-type zinc fingers at its C-terminal end. The present study reveals the following results: (i) The UCR site is present in the upstream and/or regulatory regions of numerous mammalian cellular and viral genes to which both recombinant and cellular UCRBP bind. UCR sites are also found in the regulatory regions of repetitive sequences including human LINE-1 elements and mouse intracisternal-A particle sequences. (ii) By immunological and UV cross-linking experiments, we found that two proteins, of approx. 68 kDa and an antigenically related protein of approx. 40 kDa, account for much of the UCR-binding activity in T-lymphocytes. (iii) There is evidence that UCRBP acts as a phosphoprotein. Eight consensus phosphorylation sites are found in the deduced amino-acid sequence of human UCRBP. The cellular UCR-binding activity was abolished by phosphatase treatment, and there is an incremental increase in apparent molecular mass between the cytoplasmic and nuclear forms of the protein, suggesting phosphorylation. (iv) Although UCRBP has been previously shown to act as a transcriptional repressor, we show here that UCRBP can also act as a positive transactivator of a reporter driven by UCR elements when used in co-transfection assays. This transactivation occurred in a dose-restricted manner and was absent at high concentrations of a UCRBP expression plasmid, indicating a complex mode of function.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Induction by concanavalin A of specific mRNAs and cytolytic function in a CD8-positive T cell hybridoma.

A previous report from this laboratory described the production of CD8+, class I-specific T cell hybridomas which developed specific cytolytic activity and the ability to secrete IL-2 upon Con A or specific Ag stimulation. Unlike normal lymphocytes or long-term CTL lines for which exposure to Ag triggers both differentiation and proliferation, T cell hybridoma lines can be activated functionally against a background of continuous proliferation. They therefore provide a unique system with which to study the molecular events involved in the induction of cytolytic function. The expression of mRNA from a series of genes was evaluated by Northern hybridization at various times after Con A stimulation of the H-2Ld-specific CD8+ 3D9 hybridoma. Induction of the c-fos proto-oncogene by 45 min poststimulation was followed shortly by c-myc induction. Perforin mRNA was expressed at a low level in the unstimulated hybridomas, but was down-regulated upon Con A stimulation to levels undetectable by PCR. Interestingly, production of granzyme A mRNA was strongly induced by 45 min after Con A stimulation. In the CD8+ RT-1.3G3 hybridoma, which is nonlytic and specific for the HIV-1 envelope glycoprotein, c-fos but not granzyme A mRNA was induced by 45 min poststimulation, and no granzyme A mRNA was detectable at any time. Thus, a significant role for granzyme A in the induction of cytolytic activity is suggested. Cytolysis by the 3D9 hybridoma involved both target cell membrane damage and DNA fragmentation, and both Ca(2+)-dependent and Ca(2+)-independent cytolysis were observed. Although TNF-alpha mRNA was induced by 4 h poststimulation, Ab to TNF-alpha failed to inhibit the Ca(2+)-independent lysis observed, leaving the basis for the observed Ca(2+)-independent lysis unexplained.

Animals↗
PubMed1994

The mouse Rxrb gene encoding RXR beta: genomic organization and two mRNA isoforms generated by alternative splicing of transcripts initiated from CpG island promoters.

Two major isoforms of retinoid X receptor beta (RXR beta; H-2RIIBP), encoded by the Rxrb gene, have been identified in the mouse. Northern analysis of Rxrb mRNA showed two close bands of 2.8 and 2.6 kb in many tissues and cell lines. They are designated as mRxr beta 1 and mRxr beta 2, respectively. Some rapidly growing cell lines and spleen tissue had about twofold more Rxr beta 1 mRNA than Rxr beta 2 whereas most adult tissues had similar amounts of both beta 1 and beta 2. Amino acid (aa) sequences deduced from cDNAs show an extra N-terminal domain of 72 aa for RXR beta 1 that is well conserved between mouse and human, but not found in RXR beta 2. These isoforms are generated from separate exons transcribed from different CpG island promoters and spliced into the common acceptor site in the transactivation domain by an alternative splicing. The Rxrb gene contains an intron in the midst of the first zinc-finger coding region. This is different from the retinoic acid receptor (RAR) and other nuclear receptor superfamily genes that contain an intron between the first and the second zinc-finger coding regions. These results, together with their unique ability to form heterodimers with other members of the superfamily, suggest a distinct phylogenic position for the Rxr genes.

Alternative Splicing↗
PubMed1994

Recombinant thyroid hormone receptor and retinoid X receptor stimulate ligand-dependent transcription in vitro.

The thyroid hormone and retinoid X receptors form a heterodimer with each other and mediate thyroid hormone (T3)-dependent transcription. Retinoid X receptor, in addition, forms a homodimer and mediates 9-cis-retinoic acid-dependent transcription. Here, recombinant thyroid hormone receptor and recombinant retinoid X receptor beta expressed from baculovirus vectors have been studied for ligand-mediated activation of transcription in vitro. We show that the two recombinant receptors, most likely as a heterodimer, cooperatively enhance transcription in vitro from a template containing functional T3 responsive elements. The enhancement was specific for the T3 responsive element and was greatest when T3 was added to the reaction (approximately 14-fold increase). Albeit to a lesser degree, the two receptors also directed transcription in the absence of T3. Template competition experiments suggest that the two receptors enhance formation of the preinitiation complex and that activation by T3 occurs when the ligand binds the receptor prior to (or during), but not after, the formation of the preinitiation complex. Although 9-cis-retinoic acid had no effect on the T3-dependent transcription, this ligand activated transcription in vitro directed by recombinant retinoic X receptor beta, most likely as a homodimer. This activation was observed when using nuclear extracts from embryonal carcinoma cells as a source of basal transcription factors, but not those from B lymphocytes. These results demonstrate that transcriptional activation mediated by T3 and 9-cis-retinoic acid can be reconstituted in vitro with the respective recombinant receptors.

Animals↗
PubMed1994

Regulation of IFN-gamma-induced nuclear expression of IFN consensus sequence binding protein in murine peritoneal macrophages.

IFNs are well characterized macrophage-activating agents. Their varied effects are largely mediated via the induction of many genes, whose products act in concert to induce macrophage differentiation. Homologous DNA sequences have been found upstream of the promoter in many of these IFN-inducible genes and bind a family of trans-acting proteins. Interferon consensus sequence binding protein (ICSBP) is one member of this family of interferon regulatory factors (IRF) and is structurally related within the DNA-binding domain to the other members, IRF-1, IRF-2, and ISGF3 gamma. ISCBP mRNA levels become elevated in response to IFN-gamma; however, little is known about the regulation of ICSBP expression at the protein level. In this study, anti-ICSBP peptide Abs were used to quantify and localize ICSBP in murine peritoneal exudate macrophages. Western blot analysis of cytoplasmic and nuclear extracts from treated and control cells revealed ICSBP to be induced by IFN-gamma and not by IFN-alpha and to exist primarily in the nucleus. The regulation of ICSBP induction by IFN-gamma was consistent with the characteristics found at the mRNA level; inhibition by IFN-alpha or glucocorticoids and the requirement for protein kinase C (as determined pharmacologically). The time course of IFN-gamma-induced ICSBP showed an induction of protein that required approximately 12 h to reach maximal levels. Induced ICSBP was relatively stable, exhibiting a half-life of approximately 48 h. Indirect immunofluorescence also demonstrated ICSBP to be an IFN-gamma-inducible protein that is strongly localized to the nucleus.

Amino Acid Sequence↗
PubMed1994

Transcriptional induction of genes by IFN-beta in mouse cells is regulated by a transcription factor similar to human ISGF-3.

Previous studies of IFN-stimulated transcription factors in murine cells have identified a variety of trans-acting factors that bind to the IFN-stimulated response element (ISRE) whose role in gene expression remain unclear. The present investigation was undertaken to delineate the signal transduction pathway as well as to identify the transcription factors regulated by murine IFN-beta in L929 cells. Tyrosine kinase inhibitor, Genistein, abrogated gene induction and activation of transcription factors by IFN-beta. As early as 5 min after IFN-beta treatment, a transcription factor was activated in the cytoplasm which subsequently migrated into the nucleus. Anti-phosphotyrosine antibodies detected a specific transcription factor induced by mIFN-beta. Antibodies raised against human ISGF-3 subunit proteins p48, p84, p91 and p113 recognized this factor in the cytoplasm as well as in the nucleus of IFN-beta-treated L929 cells. An antibody raised against an oligopeptide of human p113 (residues 435-450) recognized the ISGF-3 complexes both in human and murine cells. However, a different antibody against the C-terminus of human p113 (residues 671-806) did not recognize the ISGF-3 like complex in mouse cells, indicating differences in the primary sequence of these proteins.

Animals↗
PubMed1994

Quantitative increases in DNA binding affinity and positional effects determine 9-cis retinoic acid induced activation of the retinoid X receptor beta homodimer.

Retinoid X receptors (RXRs) exert transcriptional activities through heterodimerization with members of the nuclear hormone receptor superfamily. RXRs also act as homodimers and stimulate transcription from an RXR responsive element (RXRE) when bound to 9-cis-retinoic acid (9cRA). Here direct effects of 9cRA have been examined on biochemical and mechanistic parameters of RXR beta. It is shown that 9cRA significantly increases RXR beta homodimer binding affinity to an RXRE (Kd without ligand = 18 nM, Kd with ligand = 6 nM), while decreasing significantly the affinity of RXR beta/thyroid hormone receptor (T3R alpha) heterodimer binding to the same element. Effects on other response elements are also examined. The RXR beta homodimer was found to contact both halves of the RXRE direct repeat, irrespective of the effect of added ligand, while the RXR beta/T3R alpha heterodimer contacted the element only through a specific half-site. Binding of the homodimer to the element functionally activates RXR beta, since RXR beta enhanced transcription in vitro from a specific template in a ligand-dependent fashion. In agreement, transfection of RXR beta alone (but not RXR beta/T3R alpha) led to ligand-dependent activation of a reporter containing the RXRE. Taken together, 9cRA facilitates functional activation of the RXR beta homodimer in an element-dependent manner.

Animals↗