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H Ariga

Publications and source records attributed to H Ariga.

At least 91 records · Page 5Linked to original sources
PubMed1994

A novel DNA replication origin identified in the human heat shock protein 70 gene promoter.

A general and sensitive method for the mapping of initiation sites of DNA replication in vivo, developed by Vassilev and Johnson, has revealed replication origins in the region of simian virus 40 ori, in the regions upstream from the human c-myc gene and downstream from the Chinese hamster dihydrofolate reductase gene, and in the enhancer region of the mouse immunoglobulin heavy-chain gene. Here we report that the region containing the promoter of the human heat shock protein 70 (hsp70) gene was identified as a DNA replication origin in HeLa cells by this method. Several segments of the region were cloned into pUC19 and examined for autonomously replicating sequence (ARS) activity. The plasmids carrying the segments replicated episomally and semiconservatively when transfected into HeLa cells. The segments of ARS activity contained the sequences previously identified as binding sequences for a c-myc protein complex (T. Taira, Y. Negishi, F. Kihara, S. M. M. Iguchi-Ariga, and H. Ariga, Biochem. Biophys. Acta 1130:166-174, 1992). Mutations introduced within the c-myc protein complex binding sequences abolished the ARS activity. Moreover, the ARS plasmids stably replicated at episomal state for a long time in established cell lines. The results suggest that the promoter region of the human hsp70 gene plays a role in DNA replication as well as in transcription.

Base Sequence↗
PubMed1994

Identification and cDNA cloning of single-stranded DNA binding proteins that interact with the region upstream of the human c-myc gene.

We have previously reported that a c-myc protein complex binds to the region upstream of the c-myc gene, where exist an origin of cellular DNA replication (ori) and a transcriptional enhancer. Both functions require a 21 bp long sequence, while the c-myc protein complex recognizes a 7 bp consensus therein. It was recently reported that single-stranded DNA binding proteins bound specifically to sequences that play roles in DNA replication or transcription. We examined for proteins binding to the single-stranded DNAs of the 21 bp element (myc(H-P)21). In a band shift assay with HL60 cells nuclear extract, probes of either the plus strand or the minus strand gave rise to specific signals. Mutation introduced within a short consensus (A/TCTA/TA/TT) present in both strands completely abolished binding in either case. Southwestern blotting analysis showed that proteins of molecular weight 105, 80, 50, 45, 40, 39.5 and 14 kDa bound sequence-specifically to either strand and 22 kDa to minus strand to the cognate A/TCTA/TA/TT consensus. These single-stranded DNA binding proteins were named MSSP, c-myc gene single strand binding proteins. We attempted to isolate the cDNAs encoding these proteins by screening a human cDNA library with the plus single-stranded oligonucleotide as a probe. Among several positive clones, we have characterized one, termed MSSP-1. MSSP-1 produced in E. coli as a fusion protein with GST specifically interacted with single-stranded TCTTAT (plus myc(H-P)21) and ACT-ATT (in minus myc(H-P)21), the consensus of which can be referred to as A/TCTA/TA/TT. Sequence analysis of MSSP-1 cDNA revealed that two domains thereof are homologous to the RNA binding motifs common to several ribonucleoproteins. Interestingly, the MSSP-1/GST fusion protein specifically recognized myc(H-P)21 not only in single-stranded but also in double-stranded forms. Binding properties of MSSP-1 imply its functions in DNA replication. Furthermore, when the AT stretch in the SV40 ori core was substituted by TCTTAT, MSSP-1 promoted viral DNA replication depending on the consensus sequences.

Amino Acid Sequence↗
PubMed1993

Transcriptional regulation of the N-myc gene: identification of positive regulatory element and its double- and single-stranded DNA binding proteins.

The N-myc gene is amplified and overexpressed in neuroblastoma, retinoblastoma and small cell lung carcinoma, and is considered to be related to cell proliferation and/or differentiation. The transcriptional regulatory sequences of the c-myc gene have been already identified, while those of N-myc have remained obscure for a long time. In this report, we have identified several positive and negative transcriptional regulatory elements in the upstream region of the mouse N-myc gene. Among them, an activating sequence spanning -860 to -797 bp (63 bp) could be reduced to a functional core of 21 bp from -846 to -826. This sequence, termed N21 box, worked as a positive transcriptional element when linked directly upstream (but not downstream) of the putative N-myc promoter in HeLa, not in IMR32 cells. At least two proteins, of 42 kDa and 100 kDa, bound to the double-stranded N21 box, and were expressed in HeLa as well as in IMR32 cells. Moreover, the plus strand of N21 box could be specifically bound by a species of 42 kDa from either cell type and by a 37 kDa protein found only in HeLa cells. These proteins may be factors binding to positive transcriptional regulatory elements and may have a role in the regulation of N-myc expression.

Animals↗
PubMed1993

Membrane cofactor protein (CD46) protects cells predominantly from alternative complement pathway-mediated C3-fragment deposition and cytolysis.

Membrane cofactor protein (MCP) cDNA was transfected into Chinese hamster ovarian tumor (CHO) cells and the functional properties of the expressed protein were studied. Cells adherent to flasks were essential for continuous expression of MCP on CHO cells. If the cells were maintained in noncoated flasks, MCP expression was markedly reduced but upon being transferred to coated flasks reexpressed the protein. MCP expressed on CHO cells had the expected m.w., approximately 50 kDa, and possessed factor I-cofactor activity. By a propidium iodide incorporation assay and by 51Cr release assay, antibody-sensitized CHO cells expressing MCP were protected from C-mediated cytotoxicity. The inhibition of lytic activity correlated with a decrease in C3 deposition. This host cell protective activity was exerted efficiently for the alternative pathway. The classical pathway was not blocked by MCP unless the cells were presensitized with low concentrations of antibody. These results imply that MCP primarily protects host cells from alternative pathway-mediated C3 targeting. In a pathologic state such as autoimmune diseases, the binding of an autoantibody to a target may overcome this protective effect of MCP via the classical pathway.

Animals↗
PubMed1993

Stimulation of SV40 DNA replication and transcription by Alu family sequence.

The sequence motif GGAGGC (Alu core) is present in the Alu family repeats, where it is required for RNA polymerase III promoter function. This motif is also found in the SV40 origin (ori) of replication. Here, an oligonucleotide containing the Alu sequence was inserted into pSV2CAT, a plasmid composed of the SV40 enhancer/promoter/ori linked to the bacterial chloramphenicol acetyltransferase gene (CAT), to see the effect of the Alu sequence on SV40 DNA replication and transcription. Results of transfection experiments in human HeLa cells showed that the Alu sequence stimulated sequence-specifically replication and transcription in the SV40 system. Stimulation effects on DNA replication were observed when the Alu sequence was placed upstream of enhancer/promoter/ori in either orientation, while effects on transcription were detected only when it was inserted in the normal orientation. These effects correlate with sequence-specific binding of two proteins (40 kDa and 120 kDa) to this motif. In fact, binding was abolished by a mutation in the cognate sequence that disrupted stimulation of replication and transcription. Both proteins bind duplex DNA, while the 40 kDa one also binds the minus strand with high affinity.

Base Sequence↗
PubMed1993

Mammalian genomic sequences can substitute for the SV40 AT stretch in sustaining replication of the SV40 origin of replication.

The core of the SV40 origin of replication (ori) contains a stretch of adenine (A) and thymine (T) residues. This region is very conserved among the papova viruses, and is known to be extremely sensitive to mutations. So far, mutations have been found to drastically reduce, and in most cases abolish, replication. The AT stretch has been shown to be the target for several host cellular proteins that belong to the replication machinery. We reasoned that, in this light, there might exist cellular DNA sequences that can substitute for the SV40 AT stretch. To study this possibility, we digested mammalian genomic DNA and inserted the fragments instead of the SV40 AT stretch in a plasmid carrying the SV40 ori core. The resulting pool was analyzed by a 'replication trap' in CosI cells. We present evidence that there are indeed several mammalian sequences that can substitute for the SV40 AT stretch. All of them are rich in adenines and thymines but, surprisingly, these sequences differ from the wild-type SV40 AT stretch to such extent that at first sight they would seem unlikely to replicate. This is all the more impressive if we consider that another AT-rich sequence from the yeast TRP1 gene, which also carries a similar variation, cannot substitute for the SV40 AT stretch.

Adenine↗
PubMed1993

Identification of the initiation region of DNA replication in the murine immunoglobulin heavy chain gene and possible function of the octamer motif as a putative DNA replication origin in mammalian cells.

An origin region of DNA replication in the murine immunoglobulin heavy chain (IgH) gene was identified by BrdU pulse labeling and PCR amplification methods. The origin region spans about 1000 base pairs and contains the region of transcriptional enhancer in which the octamer sequence is present. The octamer sequence, TNATTTGCAT, is a well-conserved promoter/enhancer element responsible for B cell-specific transcription and is also found in the regulatory sequences for histone genes and others. Its activity as an autonomously replicating sequence was further examined. The murine IgH enhancer region containing the octamer motif was cloned in pUC18 and transfected to HeLa cells. After 60-65 h, the low molecular weight DNA was extracted and the degree to which the plasmid DNA had been replicated in the cells was measured by back-transformation of competent bacteria. Five to ten copies of the plasmid were detected per cell. The replicated plasmid-form DNA could be detected by this assay for at least 7 days after transfection. Synthetic oligonucleotides corresponding to the octamer and the Ephrussi box in the IgH enhancer were also cloned into pUC18 and examined for replicating activity. These plasmids replicated provided that the octamer sequence remained intact, irrespective of the Ephrussi box sequence and of the sites of insertion. These results suggest that the octamer transcriptional element may also serve as a putative origin for cellular DNA replication.

Animals↗
PubMed1993

BOX DNA: a novel regulatory element related to embryonal carcinoma cell differentiation.

BOX DNA was previously isolated from the DNA sequence inserted in the enhancer B domain of mutant polyomavirus (fPyF9) DNA. We also reported that BOX DNA functioned negatively on DNA replication and transcription of another polyomavirus mutant (PyhrN2) in F9-28 cells, a subclone of mouse F9 embryonal carcinoma (EC) cells expressing the polyomavirus large T antigen. In this study, we demonstrate that BOX DNA enhances transcription from the thymidine kinase (TK) promoter in various EC cells. One or three copies of BOX DNA, linked to the bacterial chloramphenicol acetyltransferase gene under the control of the herpes simplex virus TK promoter, activated promoter activity in F9, P19, and ECA2 cells. Band shift assays using BOX DNA as a probe revealed that specific binding proteins were present in all EC cells examined; the patterns of BOX DNA-protein complexes were the same among them. A mutation introduced within BOX DNA abolished enhancer activity as well as the formation of specific DNA-protein complexes. In non-EC cells, including L and BALB/3T3 cells, the enhancer activity of BOX DNA on the TK promoter was not observed, although binding proteins specific to the sequence exist. In band shift assays, the patterns of the DNA-protein complexes of either L or BALB/3T3 cells were different from those of EC cells. Furthermore, the enhancer activity of BOX DNA decreased upon differentiation induction in all EC cells examined, of different origins and distinct differentiation ability. In parallel with the loss of enhancer activity, the binding proteins specific for BOX DNA decreased in these cells. Moreover, we cloned a genomic DNA of F9, termed BOXF1, containing BOX DNA sequence approximately 400 bp upstream from the RNA start site of the gene. BOXF1, containing a TATA-like motif and the binding elements for Sp1 and Oct in addition to BOX DNA, possessed promoter activity deduced by a BOXF1-chloramphenicol acetyltransferase construct. Deletion analyses of the construct revealed that the transcription of BOXF1 gene is regulated by BOX DNA, preferentially in undifferentiated EC cells versus differentiated cells. Hence, BOX DNA is probably a novel transcriptional element related to EC cell differentiation.

Animals↗
PubMed1992

The upstream region of the mouse N-myc gene: identification of an enhancer element that functions preferentially in neuroblastoma IMR32 cells.

The various members of the myc gene family, including c-myc and N-myc, are supposed to play a role in the regulation of cell cycle and proliferation. Whereas c-myc is expressed nearly ubiquitously, the N-myc gene product is found mainly in actively proliferating neural tissues such as early development tissues or in retinoblastomas and neuroblastomas. In this report, the upstream region of mouse N-myc gene was ligated to pSVPCAT, which carries the simian virus 40 (SV40) promoter and bacterial chloramphenicol acetyltransferase (CAT) gene, and transcriptional activities were examined by CAT and S1 protection assays after transfection of the DNAs into human cervical carcinoma HeLa or neuroblastoma IMR32 cells. Several regulatory regions were identified: two promoting regions (-980 to -860 and -279 to +108) and an inhibiting one (-860 to -797). The region spanning positions -980 to -860 increased CAT expression independently of orientation and distance to the SV40 promoter, indicating that the element is a typical enhancer. Moreover, the expression levels from this enhancer were higher in IMR32 cells than in HeLa cells, indicating that action has, if not cell-type specificity, cell-type preference. These findings may provide useful bases for the understanding of the cell-type specific regulation of N-myc expression.

Animals↗
PubMed1992

Stimulation of SV40 DNA replication by the human c-myc enhancer.

In earlier studies we had shown that a transcriptional enhancer sequence exists about 2 kb upstream of the human c-myc gene. The core sequence necessary for enhancer activity was defined therein as a 21 bp nucleotide element, which also showed autonomous replicating activity [EMBO J. (1988) 7, 3135-3142; EMBO J. (1989) 8, 4273-4279]. Recently, several reports have substantiated the notion that transcription and replication can be concertedly regulated in a larger number of cases than expected. In this report, we took the simian virus 40 (SV 40) ori/promoter as a model system. The SV40 enhancer is known to enhance transcription from its ori/promoter, but to reduce its replication (probably due to a negative feedback). The SV40 enhancer was replaced by the c-myc enhancer core in order to see its effect upon SV40 DNA replication and transcription. The results showed that besides stimulating transcription, the c-myc enhancer promoted SV40 DNA replication in monkey CosI cells. Stimulation was only observed when the c-myc enhancer was inserted in the 'up-to-down' orientation to the SV40 promoter. The promoting function of the c-myc enhancer on DNA replication correlated with the transcriptional activation function, as determined by systematic point mutations introduced within the 21 bp core sequence.

Animals↗
PubMed1992

The AT-rich tract of the SV40 ori core: negative synergism and specific recognition by single stranded and duplex DNA binding proteins.

The SV40 origin of replication comprises a run of thymine and adenine residues. Integrity of this AT-rich sequence is known to be essential for replication. We set out to study whether or not these elements can work synergistically to sustain replication. Quite surprisingly, additional copies of the AT stretch linked to a functional SV40 ori core dramatically reduce its replication in Cosl cells, probably by creating some physical block. Interestingly, the same inhibiting effect can be observed with the addition in cis of the yeast ARS consensus, which is homologous to the SV40 AT stretch. This modulation is possibly due to the action of cellular factors that recognize either of the two sequences. In fact, we demonstrate the existence of factor(s) in Cosl crude nuclear extracts that in vitro can specifically bind to either of them. Moreover, we show that these sequence-specific factor(s) (MW about 50 kDa), named SOAP, recognize both single (T-rich strand) and double stranded forms of the AT tracts. Binding to single stranded AT stretches can be specifically inhibited by the corresponding duplex form, but not vice versa.

Base Sequence↗
PubMed1992

c-myc protein complex binds to two sites in human hsp70 promoter region.

We investigated the binding of partially purified (enriched) c-myc protein to the human hsp70 promoter region by band shift and ultraviolet crosslinking assays. In the hsp70 promoter region, two sites were found to be homologous to the c-myc protein complex binding sequence in the c-myc gene. These sites are located at positions -230 and -160 bases relative to the transcription initiation site, overlapping with the region reported for the regulation of hsp70 gene expression by c-myc, and upstream of other regulatory sequences including the heat shock element and the serum responsive element. The results shown here suggest that the c-myc protein complex from human HL-60 cells binds to the two sites of the region directly and sequence specifically. It is therefore suggested that the c-myc protein or protein complex contribute to the regulation of hsp70 gene expression by binding directly to the promoter region.

Autoradiography↗
PubMed1992

Protein complexes bearing myc-like antigenicity recognize two distinct DNA sequences.

The products of the myc proto-oncogene family have been suggested to carry functions important for cell proliferation, differentiation and neoplasia, but the molecular mechanisms of such biological effects have not yet been clarified. We have previously reported that the c-myc protein or protein(s) complexed with the c-myc protein bind to a specific sequence in the region upstream of the c-myc gene, where there exist an origin of cellular DNA replication (ori) and also a transcriptional enhancer. It was recently reported that the c-myc protein forms complexes with a novel helix-loop-helix leucine zipper protein (Max), and that the Myc-Max complex specifically recognizes a DNA sequence different from the sequence we have defined in the c-myc gene. In this report we examine the nuclear extract prepared from human Raji cells for binding to the two different sequences which have been reported as specific binding sequences for c-myc protein or c-myc protein complexes. The binding to one sequence was inhibited in the presence of excess amount of the other sequence, suggesting that the same protein(s) may be necessary for binding to either of the two sequences. Since binding to both sequences was cancelled by pretreatment of the Raji extract with an anti-c-myc protein antibody, it is suggested that proteins carrying myc-like antigenicity are necessary for efficient binding of the nucleoprotein complexes to the two distinct DNA sequences.

Base Sequence↗
PubMed1992

Impact of androgen therapy in Sjögren's syndrome: hormonal influence on lymphocyte populations and Ia expression in lacrimal glands of MRL/Mp-lpr/lpr mice.

Recent research has demonstrated that androgen treatment dramatically curtails lymphocyte infiltration in the lacrimal glands of a mouse model of Sjögren's syndrome. The purpose of the current study was to determine whether this androgen action involves the selective suppression of specific lymphocyte populations or Ia expression in lacrimal tissue. Autoimmune female MRL/Mp-lpr/lpr mice were administered placebo- or testosterone-containing compounds for 0, 17, or 34 d. Then lacrimal glands were obtained and processed for immunohistochemical evaluation. Results demonstrated that in pretreatment mice, lacrimal lymphoid foci were composed predominantly of Thy 1.2+ cells, bearing L3T4 (helper T cell) or B220 surface antigens. In contrast, suppressor T cells (Lyt 2+) and surface IgM-bearing B cells represented minority populations in the immune infiltrates. Class II antigen (Ia) expression was observed on over 40% of the infiltrate lymphocytes and occasionally on epithelial cells close to the lymphoid focus. During the experimental time course, the extent of lymphocyte infiltration increased in glands of placebo-treated mice. This cellular accumulation was associated with an elevation in the frequency of B220+ cells, but not that of other lymphocyte subclasses. Testosterone administration induced a striking diminution in the area encompassed by all immune cell populations. Moreover, hormone therapy significantly reduced the frequency of B220+ cells in focal infiltrates. Overall, these findings demonstrate that androgen exposure stimulates a decrease in the quantity, but not necessarily the entire lymphocyte composition, of lymphoid aggregates in lacrimal glands of MRL/lpr mice.

Animals↗
PubMed1991

Activation of c-myc promoter by c-myc protein in serum starved cells.

The function of the c-myc protein, the product of a proto oncogene, is not clearly understood although many reports, including ours, suggest that the c-myc protein plays several roles in the regulation of transcription and DNA replication. Here we examined the effects of c-myc protein on transcription from the c-myc promoter, and by inference its role in auto-regulation, after introducing into cultured cells a c-myc expression vector and a CAT reporter gene linked to the promoter and upstream region of the human c-myc gene. To minimize the effects of the endogenous c-myc protein on the exogenously added CAT reporter gene, the transfected cells were treated under serum-free conditions. The results show that CAT expression from the myc promoter increased in a dose-dependent manner after addition of the c-myc expression vector, and that it also required the presence of a c-myc binding sequence previously identified 2 kb upstream from c-myc's first exon. Moreover, the domains of the c-myc protein important for transactivation were determined by use of various deletions mutants of c-myc cDNA. The results showed that the N-terminal portion in the c-myc protein was necessary for transactivation beside the C-terminal portion containing basic region, helix-loop-helix, and leucine zipper.

3T3 Cells↗
PubMed1991

A eukaryotic nuclear protein of 130 kDa binds to a bacterial cAMP responsive element.

It has been known that one of the signal transduction mechanisms in Escherichia coli is mediated by cAMP which binds to the receptor protein (CAP), and that CAP complexed with cAMP facilitates gene expression by binding to the specific sequences. To identify a molecular mechanism in eukaryotes similar to a cAMP-mediated pathway in E. coli, the function of the CAP binding site of lac gene in E. coli and the protein(s) interacting with it were examined in a mammalian system. From transient expression studies of the fusion gene between the chloramphenicol acetyltransferase and lac genes, it was found that the lacCAP binding site could act as an enhancer activity on the SV40 promoter, and also as an additive enhancer activity to the SV40 enhancer in HeLa cells. However, the activity was not stimulated by cpt-cAMP (a highly stable analogue of cAMP) in HeLa cells, although it was induced in PC12 cells. These results suggest that a bacterial cAMP responsive element may function also in eukaryotes as a cis-acting element in a cell type dependent manner. Results from gel mobility shift assays showed that a protein(s) exists that specifically binds to the lacCAP binding site in eukaryotic nuclear extracts. As one of the proteins binding to the above site, we have identified a 130 kDa protein by using the Southwestern method. Although a function of the 130 kDa protein has not yet been understood, there is a possibility that the 130 kDa protein may play a role in the regulation of cAMP-dependent gene expression.

Base Sequence↗
PubMed1991

Rat DNA sequence associated with a complex form of DNA polymerase alpha in nonregenerating liver interacts with a ubiquitous transcription/replication factor Oct-1.

A ubiquitous mammalian transcription factor, Oct-1 (also known as OTF-1, NF-A1, OBP100, or NFIII), stimulates the initiation of replication of adenovirus DNA, and may also be involved in the activation of some chromosomal replication origins. If this is true, binding sites for Oct-1 should be present within regions responsible for the initiation of DNA replication. In this study such a binding site has been identified within a 340bp fragment that was originally isolated from a minor fraction of DNA associated with a complexed form of DNA polymerase alpha from nonregenerating rat liver, and which shows autonomous replication sequence activity in a transient transfection assay. Northern blot analysis was used to show that Oct-1 mRNA is induced in regenerating rat liver 6-14 h after hepatectomy.

Animals↗