pSV00CAT: low background CAT plasmid

Araki, Eiichi; Shimada, Fumiyuki; Shichiri, Motoaki; Mori, Masataka; Ebina, Yasuhiko

doi:10.1093/nar/16.4.1627

Cited by 84 publications

(44 citation statements)

References 2 publications

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“…Each fragment was filled in and inserted into the HindIII site of pSV00CAT (38), via a HindIII linker, to generate pMAZCAT1, pMAZCAT2, pMAZCAT3, pMAZCAT4, and pMAZCAT5, respectively. Internal deletion mutants of the MAZ promoter were created by amplification by the polymerase chain reaction, ligation of the appropriate DNA fragments, and insertion into the HindIII site of pSV00CAT to generate pMAZCAT2-d, pMAZCAT3-wt, pMAZCAT3-⌬I, pMAZCAT3-⌬II, and pMAZCAT3-⌬III, respectively.…”

Section: Methodsmentioning

confidence: 99%

Independent Repression of a GC-rich Housekeeping Gene by Sp1 and MAZ Involves the Same cis-Elements

Song

Ugai²,

Kanazawa

et al. 2001

Journal of Biological Chemistry

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The transcription factors Sp1 and MAZ (Myc-associated zinc finger protein) contain several zinc finger motifs, and each functions as both a positive and a negative regulator of gene expression. In this study, we characterized the extremely GC-rich promoter of the human gene for MAZ, which is known as a housekeeping gene. Unique symmetrical motifs in the promoter region (nucleotides ؊383 to ؊334) were essential for the expression of the gene for MAZ, whereas an upstream silencer element (nucleotides ؊784 to ؊612) was found to act in a position-dependent but orientation-independent manner. Sp1 and MAZ bound to the same cis-elements in the GC-rich promoter, apparently sharing DNA-binding sites. The relative extent of binding of Sp1 and MAZ to these cis-elements corresponded to the extent of negative regulation of the expression of the gene for MAZ in various lines of cells. Furthermore, novel repressive domains in both Sp1 (amino acids 622-788) and MAZ (amino acids 127-292) were identified. Suppression by Sp1 and suppression by MAZ were independent phenomena; histone deacetylases were involved in the autorepression by MAZ itself, whereas DNA methyltransferase 1 was associated with suppression by Sp1. Our results indicate that both deacetylation and methylation might be involved in the regulation of expression of a single gene via the actions of different zinc finger proteins that bind to the same cis-elements.

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Section: Methodsmentioning

confidence: 99%

Independent Repression of a GC-rich Housekeeping Gene by Sp1 and MAZ Involves the Same cis-Elements

Song

Ugai²,

Kanazawa

et al. 2001

Journal of Biological Chemistry

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“…Plasmids-A DNA fragment (from nucleotide (nt) Ϫ383 to minus]70) from the MAZ promoter was obtained by digestion with appropriate restriction enzymes, filled in, and inserted into the HindIII site of pSV00CAT (18), via a pHindIII linker, to generate pMAZCAT. pCMVSp1 and pGEX-Sp1 were kindly provided by Dr. R. Chiu (UCLA School of Medicine, Los Angelos, CA).…”

Section: Methodsmentioning

confidence: 99%

Two Consecutive Zinc Fingers in Sp1 and in MAZ Are Essential for Interactions with cis-Elements

Song¹,

Ugai²,

Ogawa³

et al. 2001

Journal of Biological Chemistry

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The zinc finger proteins Sp1 and Myc-associated zinc finger protein (MAZ) are transcription factors that control the expression of various genes. Regulation of transcription by these factors is based on interactions between GC-rich DNA-binding sites (GGGCGG for Sp1 and GGGAGGG for MAZ) and the carboxyl-terminal zinc finger motifs of the two proteins. Sp1 and MAZ have three and six zinc fingers, respectively, and the details of their interactions with ciselements remain to be clarified. We demonstrate here that Sp1 and MAZ interact with the same GC-rich DNA-binding sites, apparently sharing DNA-binding sites with each other. We found that the DNA binding activities of Sp1 and MAZ depended mainly on consecutive zinc fingers, namely the second and third zinc fingers in Sp1 and the third and fourth zinc fingers in MAZ. Furthermore, the interactions of the zinc finger proteins with the same cis-elements appear to play a critical role in the regulation of gene expression. It seems plausible that two consecutive zinc finger motifs in a zinc finger protein might be essential for interaction of the protein with DNA.The regulation of gene expression is mediated by the binding of transcription factors to cis-elements in promoter regions. The promoter regions of many eukaryotic genes contain GCrich sequences (1), and some of the most widely distributed promoter elements are GC boxes and related motifs. The zinc finger proteins Sp1 and Myc-associated zinc finger protein (MAZ) 1 are transcription factors that bind to GC-rich sequences, namely GGGCGG and GGGAGGG, respectively, to regulate the expression of various target genes.Sp1 was originally characterized as a ubiquitous transcription factor of 778 amino acids that recognized GC-rich sequences in the early promoter of simian virus 40 (2, 3). The DNA-binding domain of Sp1 consists of three contiguous C2H2-type zinc fingers (4). The amino-terminal region contains two serine-and threonine-rich domains and two glutamine-rich domains, which are essential for the transcriptional activity of Sp1 (5). The carboxyl-terminal domain of Sp1 is involved in synergistic activation and interactions with other transcription factors. Sp1 is considered to be a constitutively expressed transcription factor and has been implicated in the regulation of a wide variety of housekeeping genes, tissue-specific genes, and genes involved in the regulation of growth (6). It interacts with many factors, such as YY1 (7), E2F (8), and p300 (9). Moreover, Sp1-null mouse embryos exhibit severely retarded growth and die within 10 days after displaying a wide range of abnormalities (10).MAZ was first identified as a transcription factor that bound to a GA box (GGGAGGG) at the ME1a1 site of the c-myc promoter and to the CT-element of the c-myc gene (11). It is a zinc finger protein with six C2H2-type zinc fingers at the carboxyl terminus, a proline-rich region, and three alanine repeats. It is expressed ubiquitously, albeit at different levels in different tissues (12). It can regulate the expression of nume...

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“…An HBV DNA fragment that contains the core domain of the enhancer 1 and the X promoter region through the translational initiation codon of the X protein (nt 989 to 1253) were cloned into pSV00CAT (1). This reporter plasmid is equivalent to pXStNcCAT (11).…”

Section: Chronic Hepatitis B Virus (Hbv) Infection Is a Major Globalmentioning

confidence: 99%

Nuclear Respiratory Factor 1 Plays an Essential Role in Transcriptional Initiation from the Hepatitis B Virus X Gene Promoter

Tokusumi

Zhou

Takada

2004

J Virol

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The X gene of hepatitis B virus (HBV) is one of the major factors in HBV-induced hepatocarcinogenesis and is essential for the establishment of productive HBV replication in vivo. Recent studies have shown that the X gene product targets mitochondria and induces calcium flux, thereby activating Ca ؉ -dependent signal transduction pathways. However, regulatory mechanisms of X gene expression have remained unclear. Previous studies had localized a minimal promoter activity to a 21-bp GC-rich sequence located 130 bp upstream of the X protein coding region and showed that there was a cellular protein bound to this DNA. Interestingly, the 21-bp sequence identified as an X gene minimal promoter does not contain any previously identified core promoter elements, such as a TATA box. To better understand the mechanisms of transcriptional initiation of the X gene, we set out to biochemically purify the binding protein(s) for the 21-bp DNA. We report here the identification of the X gene minimal promoter-binding activity as nuclear respiratory factor 1 (NRF1), a previously known transcription factor that activates the majority of nucleus-encoded mitochondrial genes and various housekeeping genes. Primer extension analyses of the X mRNAs show that mutations at the binding site specifically inactivate transcription from this promoter and that a dominant-negative NRF1 mutant and short interfering RNAs inhibit transcription from this promoter. Therefore, NRF1 specifically binds the 21-bp minimal promoter and positively contributes to transcription of the X gene. Simultaneous activation of the X gene and mitochondrial genes by NRF1 may allow the X protein to target mitochondria most efficiently.Chronic hepatitis B virus (HBV) infection is a major, global cause of hepatocellular carcinoma (HCC). The X gene of this virus has been indicated to be a viral oncogenic factor (reviewed in references 5 and 27). The X gene product exerts a pleiotropic effect on diverse cellular functions including transcriptional regulation (27), apoptosis (13,35,37,40), DNA repair (18), and the proteasome function (34, 44). These effects of the X gene product are thought to be mediated by interaction with cellular proteins and activation of signal transduction pathways (27). Recent findings also established a connection between the X gene and mitochondrial function. The X protein was shown to cause calcium ion (Ca ϩ ) flux by targeting mitochondria, consequently activating proline-rich tyrosine kinase 2 (Pyk2) and then the Ca ϩ -dependent Src kinase signaling pathway (2). Consistent with this finding, the X protein has been shown to be colocalized with mitochondria (26) and to change mitochondrial membrane potential, resulting in release of cytochrome c from mitochondria. These observations may explain why the X gene deregulates cellular control of apoptosis (35, 36). In the natural viral life cycle, the X gene is essential for the establishment of productive HBV replication and infection in vivo (6,45).To understand mechanisms of HBV-induced hepatocarci...

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pSV00CAT: low background CAT plasmid

Cited by 84 publications

References 2 publications

Independent Repression of a GC-rich Housekeeping Gene by Sp1 and MAZ Involves the Same cis-Elements

Independent Repression of a GC-rich Housekeeping Gene by Sp1 and MAZ Involves the Same cis-Elements

Two Consecutive Zinc Fingers in Sp1 and in MAZ Are Essential for Interactions with cis-Elements

Nuclear Respiratory Factor 1 Plays an Essential Role in Transcriptional Initiation from the Hepatitis B Virus X Gene Promoter

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