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K Ozato

Publications and source records attributed to K Ozato.

At least 145 records · Page 8Linked to original sources
PubMed1991

Negative regulation of the major histocompatibility complex class I promoter in embryonal carcinoma cells.

Transcription of major histocompatibility complex (MHC) class I genes is negatively regulated in undifferentiated F9 mouse embryonal carcinoma cells via the conserved upstream regulatory region. This region contains constitutive enhancers and an inducible enhancer, the interferon consensus sequence (ICS), that is responsible for interferon-induced transcription. A series of mutations in the ICS, but not in the enhancer elements, resulted in an increase in expression of the MHC class I promoter in F9 cells. However, these ICS mutants did not increase promoter activity in F9 cells differentiated after retinoic acid treatment. Results of mobility-shift DNA-binding assays and methylation interference experiments showed that undifferentiated F9 cells contained a factor(s) that bound to a sequence within the 5' and central part of the ICS. This binding site, termed the MHC negative regulatory element (NRE), coincided with the site of mutations that increased promoter activity in F9 cells and was distinct from the element to which interferon-response factors bind. The factor(s) that binds to the MHC NRE was not detected in differentiated F9 cells treated with retinoic acid or in other cells expressing MHC class I genes. Finally, introduction of concatenated, double-stranded NRE oligomers, but not oligomers of unrelated sequences, into F9 cells abolished negative regulation of the MHC class I promoter activity, providing evidence that the NRE binding factor is responsible for repression of the MHC class I genes in F9 cells.

Animals↗
PubMed1991

The AP-1 site and the cAMP- and serum response elements of the c-fos gene are constitutively occupied in vivo.

The c-fos proto-oncogene is inducible by cAMP, phorbol esters, serum, and growth factors. The induction by cAMP is mediated by the conserved cAMP response element (CRE), while induction by phorbol esters, serum, and growth factors requires a distal element called the serum response element (SRE). In addition to these elements, a consensus AP-1 transcription factor binding site is located next to SRE. Upstream regions of the mouse and human c-fos genes were footprinted in vivo by the ligation-mediated polymerase chain (PCR). Our results show that all three elements are constitutively protected in mouse liver and lung and in cultured human A431 cells. No major change in the protection profile was detected in A431 cells following stimulation with epidermal growth factor or in mice at birth, when c-fos is known to be induced. These results suggest that the inducible cis elements of the c-fos gene are poised, ready to respond immediately to external signals.

Animals↗
PubMed1990

A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo.

The murine oct-3 gene encodes a transcription factor containing a POU-specific domain and a homeodomain. In marked contrast to other homeodomain-encoding genes, oct-3 is expressed in the totipotent and pluripotent stem cells of the pregastrulation embryo and is down-regulated during differentiation to endoderm and mesoderm, suggesting that it has a role in early development. The oct-3 gene is also expressed in primordial germ cells and in the female germ line.

Amino Acid Sequence↗
PubMed1990

An interferon gamma-regulated protein that binds the interferon-inducible enhancer element of major histocompatibility complex class I genes.

Interferons (IFNs) induce transcription of major histocompatibility complex (MHC) class I genes through the conserved IFN consensus sequence (ICS) that contains an IFN response motif shared by many IFN-regulated genes. By screening mouse lambda ZAP expression libraries with the ICS as a probe, we isolated a cDNA clone encoding a protein that binds the ICS, designated ICSBP. Protein blot analysis with labeled oligonucleotide probes showed that ICSBP binds not only the MHC class I ICS but also IFN response motifs of many IFN-regulated genes, as well as a virus-inducible element of the IFN-beta gene. The ICSBP cDNA encodes 424 amino acids and a long 3' untranslated sequence. The N-terminal 115 amino acids correspond to a putative DNA-binding domain and show significant sequence similarity with other cloned IFN response factors (IRF-1 and IRF-2). Because of the structural similarity and shared binding specificity, we conclude that ICSBP is a third member of the IRF gene family, presumably playing a role in IFN- and virus-mediated regulation of many genes. Although IRF-1 and IRF-2 share some similarity in their C-terminal regions, ICSBP shows no similarity to IRF-1 or IRF-2 in this region, suggesting that it is more distantly related. We show that ICSBP mRNA is expressed predominantly in lymphoid tissues and is inducible preferentially by IFN-gamma. The induction by IFN-gamma appears to be predominant in lymphocytes and macrophages, implying that ICSBP plays a regulatory role in cells of the immune system. The presence of multiple factors that bind common IFN response motifs may partly account for the complexity and diversity of IFN action as well as IFN-regulated gene expression.

Amino Acid Sequence↗
PubMed1990

A constitutive damage-specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells.

Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts.

Animals↗
PubMed1990

Regulation of the mouse alpha A-crystallin gene: isolation of a cDNA encoding a protein that binds to a cis sequence motif shared with the major histocompatibility complex class I gene and other genes.

We have shown by site-directed mutagenesis that the sequence between positions -69 and -40 of the mouse alpha A-crystallin gene is crucial for tissue-specific gene expression in a transfected mouse lens epithelial cell line transformed with the early region of simian virus 40. Gel retardation experiments with synthetic oligodeoxynucleotides revealed a mouse lens nuclear protein which bound specifically to the palindromic sequence 5'-GGGAAATCCC-3' at positions -66 to -57 in the alpha A-crystallin promoter. By screening a bacteriophage lambda gt11 expression library of the transformed lens cells, we isolated a 2.5-kilobase-pair cDNA encoding a fusion protein which bound to this sequence and to the regulatory element of the major histocompatibility complex (MHC) class I gene. This cDNA hybridized to a 10-kilobase-pair polyadenylated RNA present in many different tissues, including lens. It encoded a protein, tentatively called alpha A-CRYBP1, containing at least two zinc fingers. alpha A-CRYBP1 is either homologous or very similar to the human nuclear proteins MBP-1 (Baldwin et al., Mol. Cell. Biol. 10:1406-1414, 1990), PRDII-BFI (Fan and Maniatis, Genes Dev. 4:29-42, 1990), and HIV-EP1 (Maekawa et al., J. Biol. Chem. 264:14591-14593, 1989), which bind to regulatory elements of the MHC class I, beta interferon, and human immunodeficiency virus genes, respectively. Our results suggest that the lens-specific alpha A-crystallin, MHC class I, beta interferon and other genes have a similar cis-acting DNA regulatory motif that shares alpha A-CRYBPI, MBP-1, PRDII-BF1, HIV-EP1, or other closely related proteins as trans-acting factors.

Amino Acid Sequence↗
PubMed1989

Expression of mutant H-2d proteins encoded by class I genes which alternatively process the 5' end of their transcripts.

Mutation of the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes generated alternatively spliced transcripts when the constructs were transfected into L cells (J. Immunol. 143:1018). The H-2Dd transcripts contained an additional 84 nucleotides derived from the first intervening sequence, whereas 60 extra bases were included in the H-2Kd mRNA. Proteins derived from these transcripts were recognized by mAb. Moreover, both Ag served as recognition elements for CTL, and the mutant H-2Kd molecule functioned as a restricting element for an Ag peptide. As a result of alternative splicing, the mutant proteins should have additional residues at their NH2 termini to increase their lengths by 28 (Dd) or 20 (Kd) amino acids. Immunoprecipitation and analysis on SDS-PAGE demonstrated that the mutant H-2Kd molecule was indeed larger than the normal H-2Kd protein, but the mutant and wild-type H-2Dd Ag were the same size. In addition, treatment of H-2Dd mutant and normal Ag with N-glycanase produced molecules of equal size, demonstrating that the mutant protein was completely glycosylated. Limited amino acid sequencing of this Ag indicated that it was normal H-2Dd. Therefore, before its transfer to the cell surface, post-translational modifications remove the additional NH2-terminal residues of the mutant Dd but not Kd protein.

Amino Acid Sequence↗
PubMed1989

Developmental and tissue-specific expression of nuclear proteins that bind the regulatory element of the major histocompatibility complex class I gene.

Expression of MHC class I genes varies according to developmental stage and type of tissues. To study the basis of class I gene regulation in tissues in vivo, we examined binding of nuclear proteins to the conserved cis sequence of the murine H-2 gene, class I regulatory element (CRE), which contains two independent factor-binding sites, region I and region II. In gel mobility shift analyses we found that extracts from adult tissues that express class I genes, such as spleen and liver, had binding activity to region I. In contrast, extracts from brain, which does not express class I genes, did not show region I binding activity. In addition, fetal tissues that express class I gene at very low levels, also did not reveal region I binding activity. Binding activity to region I became detectable during the neonatal period when class I gene expression sharply increases. Most of these tissues showed binding activity to region II, irrespective of class I gene expression. Although region II contained a sequence similar to the AP-1 recognition site, AP-1 was not responsible for the region II binding activity detected in this work. These results illustrate a correlation between region I binding activity and developmental and tissue-specific expression of MHC class I genes. The CRE exerts an enhancer-like activity in cultured fibroblasts. We evaluated the significance of each factor binding to CRE. Single 2-bp mutations were introduced into the CRE by site-directed mutagenesis and the ability of each mutant to elicit the enhancer activity was tested in transient CAT assays. A mutation that eliminated region I protein binding greatly impaired enhancer activity. A mutation that eliminated region II binding also caused a lesser but measurable effect. We conclude that region I and region II are both capable of enhancing transcription of the class I gene. These results indicate that in vivo regulation of MHC class I gene expression is mediated by binding of trans-acting factors to the CRE.

Animals↗
PubMed1989

Site-specific mutagenesis of the class I regulatory element the Q10 gene allows expression in non-liver tissues.

Classical transplantation Ag are found on nearly all cells, whereas Ag encoded by the genes of the Qa/Tla region have a restricted tissue distribution. To investigate the cause of these different patterns of expression, we have compared the regulatory regions of Q10, a Qa region gene that is expressed only in liver, with those of H-2Ld. Gel retardation analysis shows that the nuclear factor (rI) that binds to the inverted repeat (TGGGGATTCCCCA) of the class I regulatory element (CRE) present in H-2Ld and other classical class I genes does not bind to the equivalent region of the Q10 gene. However, the Q10 CRE binds another nuclear factor (rII) that binds to the H-2Ld CRE. The sequence of the Q10 CRE differs from the sequence present in classical class I genes by three nucleotides, two of these changes are within the inverted repeat sequence (TGaGGAcTCCCCA) and disrupt the region of dyad symmetry. We have used site-specific in vitro mutagenesis to individually change these two nucleotides and have investigated the ability of this region to promote transcription with and without these substitutions. A change of either base restores transcriptional activity in chloramphenicol acetyl transferase assays and allows for binding of the rI nuclear factor. These results suggest that the failure of the Q10 CRE to bind the rI nuclear factor may play a role in preventing Q10 expression in tissues other than the liver and fetal yolk sac. Thus, the dichotomy between the widespread tissue expression of classical class I genes and the restricted tissue expression of class I genes of the Qa/Tla region may be due in part to differences in the cis acting regulatory sequences that interact with trans-acting nuclear factors to direct transcription.

Animals↗
PubMed1989

Expression of ets genes in mouse thymocyte subsets and T cells.

The cellular ets genes (ets-1, ets-2, and erg) have been identified by their sequence similarity with the v-ets oncogene of the avian erythroblastosis virus, E26. Products of the ets-2 gene have been detected in a wide range of normal mouse tissues and their expression appears to be associated with cell proliferation in regenerating liver. In contrast, the ets-1 gene was previously shown to be more highly expressed in the mouse thymus than in other tissues. Because the thymic tissue contains various subsets of cells in different stages of proliferation and maturation, we have examined ets gene expression in fetal thymocytes from different stages of development, in isolated subsets of adult thymocytes, and in peripheral T lymphocytes. Expression of the ets-1 gene was first detected at day 18 in fetal thymocytes, corresponding to the first appearance of CD4+ (CD4+, CD8-) thymocytes, and reaches maximal/plateau levels of expression in the thymus at 1 to 2 days after birth. The ets-2 gene expression is detected at least 1 day earlier, coinciding with the presence of both double-positive (CD4+, CD8+) and double-negative (CD4-, CD8-) blast thymocytes and reaches maximal/plateau levels 1 day before birth. In the adult thymus, ets-1 and ets-2 mRNA expression is 10- to 8-fold higher respectively in the CD4+ subset than in the other subsets examined. Higher levels of p55 ets-1 protein were also shown to exist in the CD4+ subset. Because the CD4+ thymic subset is the pool from which the CD4+ peripheral, helper/inducer T cells are derived, the ets gene expression was examined in lymph node T cells. Both the CD4+ and the CD8+ T cells subsets had lower ets RNA levels than the CD4+ thymocytes. These results suggest that ets-2 and more particularly ets-1 gene products play an important role in T cell development and differentiation and are not simply associated with proliferating cells, which are observed at a higher frequency in fetal thymocytes, or dull Ly-1 (low CD5+), and double-negative (CD4-, CD8-) adult thymocytes. Selectively enhanced expression of ets-1 gene may be observed in thymic CD4+ thymocytes because these cells have uniquely encountered MHC class II or other Ag in the thymic environment. These cells may have been subsequently stimulated to activate the ets genes in conjunction with their differentiation of helper/inducer function(s) and expression of mature TCR.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1989

T cell activation: independent induction of killing activity and interleukin 2 secretion in cytolytic hybridomas.

Memory-like cytotoxic T lymphocytes (CTL) hybridomas exhibiting inducible killing activity and IL2 production were used to analyze the anamnestic response of CTL. Four activating agents were examined; anti-Thy-1 monoclonal antibody G7, staphylococcal enterotoxin B, interferon (IFN)-alpha/beta and IFN-gamma. These agents seemed to affect CTL activities in three distinct ways. Anti-Thy-1 monoclonal antibody, like specific antigen, was found to be a potent inducer of specific killing and IL2 production, whereas staphylococcal enterotoxin B induced IL2 production, but not cytolytic activity. On the other hand, IFN-alpha/beta and IFN-gamma effectively stimulated cytotoxicity without inducing IL2 production. The independent triggering of specific killing and IL2 secretion in the monoclonal cytolytic hybridomas suggests that in CTL distinct signals stimulate killing activity and IL2 production. The results also suggest that IFN-alpha/beta and IFN-gamma trigger the cytolytic program through an alternative activation pathway which does not involve the T cell receptor.

Animals↗
PubMed1989

K-252a inhibits the increase in c-fos transcription and the increase in intracellular calcium produced by nerve growth factor in PC12 cells.

K-252a, a kinase inhibitor isolated from the culture broth of Nocardiopsis sp., selectively inhibits, in a dose- and time-dependent fashion, the increased transcription of the protooncogene c-fos induced by nerve growth factor in PC12 cells. Induction of c-fos by epidermal growth factor, A23187, dBcAMP, or TPA in the same cells is not affected. Pretreatment with K-252a for 30 min results in a complete inhibition of the nerve growth factor-induced increase in intracellular calcium. Increases in intracellular calcium induced by carbachol or by high K+ are not altered. K-252a derivatives selective for the inhibition of various known kinases were used to inhibit the nerve growth factor-dependent induction of c-fos mRNA, the nerve growth factor-dependent increase in intracellular calcium levels, and the nerve growth factor-dependent outgrowth of neurites. K-252a is the most effective inhibitor of all three of these actions of nerve growth factor. The possible mechanisms by which K-252a acts on PC12 cells are considered in the light of the characteristics of the inhibitions seen here.

Adrenal Gland Neoplasms↗
PubMed1989

Genetic regulation of class I major histocompatibility complex (MHC) antigen induction on astrocytes.

Neural cells, including astrocytes, normally do not express detectable levels of class I major histocompatibility complex (MHC) molecules, unlike cells of most tissues. However, upon cultivation in vitro, astrocytes begin to express class I molecules, increasing with time after plating. This spontaneous expression was examined in the present study to characterize inducible expression on astrocytes among various strains of mice. Inducible expression, either as a consequence of cultivation or standard gamma-interferon treatment, differed markedly among the strains examined. Analysis of congenic strains on a C57BL/10 (B10) background showed that expression was controlled by genes within the MHC locus. Examination of additional congeneic animals with various recombinations within the MHC showed that high or low expression of MHC molecules correlates with the presence of particular MHC class I genes. In general, H-2a and H-2d class I products are expressed much higher on astrocytes than H-2b and H-2s products. This difference in expression is not seen on spleen cells indicating tissue specificity. Moreover, levels of expression at the cell surface are reflected by the steady-state level of RNA message within astrocytes of the different strains.

Animals↗
PubMed1989

Stage-dependent expression of the chicken delta-crystallin gene in transgenic fish embryos.

To study the regulation of gene expression of vertebrate crystallin genes, the chicken delta-crystallin gene was introduced into a small freshwater fish, medaka (Oryzias latipes), which lacks this gene, and its expression was examined immunohistologically at several developmental stages before hatching. The gene expression was detected in the central fiber cells of the lens at an early stage, showing a stage-dependent expression. In non-lens tissues, the expression was barely detectable before tissue differentiation. It first became substantial mainly in mesodermal tissues and then later in a greater variety of tissues, including ectodermal and endodermal ones. Thus, the non-lens expression of delta-crystallin was also stage-dependent, with the stage being dependent on the tissue type. These results from lens and non-lens tissues are discussed in relation to tissue differentiation and two categories of delta-crystallin expression.

Animals↗
PubMed1989

H-2RIIBP, a member of the nuclear hormone receptor superfamily that binds to both the regulatory element of major histocompatibility class I genes and the estrogen response element.

Transcription of major histocompatibility complex (MHC) class I genes is regulated by the conserved MHC class I regulatory element (CRE). The CRE has two factor-binding sites, region I and region II, both of which elicit enhancer function. By screening a mouse lambda gt 11 library with the CRE as a probe, we isolated a cDNA clone that encodes a protein capable of binding to region II of the CRE. This protein, H-2RIIBP (H-2 region II binding protein), bound to the native region II sequence, but not to other MHC cis-acting sequences or to mutant region II sequences, similar to the naturally occurring region II factor in mouse cells. The deduced amino acid sequence of H-2RIIBP revealed two putative zinc fingers homologous to the DNA-binding domain of steroid/thyroid hormone receptors. Although sequence similarity in other regions was minimal, H-2RIIBP has apparent modular domains characteristic of the nuclear hormone receptors. Further analyses showed that both H-2RIIBP and the natural region II factor bind to the estrogen response element (ERE) of the vitellogenin A2 gene. The ERE is composed of a palindrome, and half of this palindrome resembles the region II binding site of the MHC CRE. These results indicate that H-2RIIBP (i) is a member of the superfamily of nuclear hormone receptors and (ii) may regulate not only MHC class I genes but also genes containing the ERE and related sequences. Sequences homologous to the H-2RIIBP gene are widely conserved in the animal kingdom. H-2RIIBP mRNA is expressed in many mouse tissues, in agreement with the distribution of the natural region II factor.

Amino Acid Sequence↗
PubMed1989

Neonatal induction of a nuclear protein that binds to the c-fos enhancer.

The expression of the c-fos gene is transiently induced at birth in most organs in the mouse. To study the basis of this induction we searched for a nuclear factor that binds to the 5' regulatory region of the c-fos gene. Gel mobility shift assays with tissue extracts revealed fast (band I) and slow (band III) migrating bands, which represent factor binding to the c-fos enhancer, termed the serum response element (SRE). Neonatal extracts preferentially elicited band I, with low or undetectable levels of band III, whereas fetal and adult extracts generated predominantly band III, with reduced levels of band I. These results indicate that the SRE-binding activity changes during perinatal development and that the appearance of band I, which coincides with diminution of band III, correlates with neonatal c-fos induction. Methylation interference and competition analyses showed that the neonatal factor (band I) binds to the SRE at a site different from the adult factor (band III). DNA-binding activity of the adult factor, but not the neonatal factor, was sensitive to phosphatase treatment. Furthermore, the adult factor, but not the neonatal factor, shared antigenic specificity with the human serum response factor (SRF) that is expressed in cultured cells irrespective of c-fos gene induction. We conclude that band I in neonates represents a SRE-binding factor that is distinct from the SRF, which may be responsible for the neonatal induction of the c-fos gene. The band III factor was indistinguishable from the SRF in all criteria tested.

Aging↗