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PubMed2002

Optimising gene repair strategies in cell culture.

Gene repair, the precise modification of the genome, offers a number of advantages over replacement gene therapy. In practice, gene targeting strategies are limited by the inefficiency of homologous recombination in mammalian cells. A number of strategies, including RNA-DNA oligonucleotides (RDOs) and short DNA fragments (SDFs), show promise in improving the efficiency of gene correction. We are using GFP as a reporter for gene repair in living cells. A single base substitution was introduced into GFP to create a nonsense mutation (STOP codon, W399X). RDOs and SDFs are used to repair this mutation episomally in transient transfections and restore green fluorescence. The correction efficiency is determined by FACS analysis. SDFs appear to correct GFP W399X in a number of different cell lines (COS7, A549, HT1080, HuH-7), although all at a similar low frequency ( approximately 0.6% of transfected cells). RDOs correct only one of our cell lines significantly (HT1080-RAD51), these cells overexpress the human RAD51 gene; the bacterial RecA homologue. The GFP W399X reporter is a fusion gene with hygromycin (at the 5' end), this has allowed us to make stable cell lines (A549, HT1080) to study genomic correction. Initial studies using our correction molecules show only low efficiencies of genomic repair ( approximately 10(-4)). Polyethylenimine (PEI) is used to deliver RDOs and SDFs into mammalian cells in culture for our study. We have used fluorescently labelled RDOs and SDFs to study the effectiveness of this process. FACS analysis of transfected nuclei implied efficient delivery (>90%) both with SDFs and RDOs. However, confocal fluorescence microscopy suggests that a large proportion of the complexed RDO/SDF appears to remain outside the nucleus (or attached to the nuclear membrane). On the basis of these data we are assessing new delivery methods and factors that may alter recombination status to optimise gene repair.

Animals↗

PubMed2006

Association of a mutation in TRPV3 with defective hair growth in rodents.

DS-Nh mice and WBN/Kob-Ht rats are spontaneous hairless mutant rodent strains. These animals develop spontaneous dermatitis under normal conditions. The non-hair Nh and Ht phenotypes are inherited in an autosomal dominant fashion, and the Nh mutation possesses a high potency for penetration. We previously reported that genes involved in dermatitis and hairlessness did not segregate from each other. Here, we carried out genetic analysis to identify the genes responsible for these hairless mutations. An amino-acid substitution at the same position in one gene was detected in DS-Nh mice and WBN/Kob-Ht rats: Gly573 to Ser (Nh mutation) or Gly573 to Cys (Ht mutation), located in the transient receptor potential (TRP) cation channel subfamily V member 3 (TRPV3) gene. Mutated TRPV3 was expressed in skin keratinocytes of DS-Nh mice. Histopathological analyses revealed that mast cells in skin lesions were increased in both rodents compared to their age-matched parent strains, and that this may partially be due to hairlessness and dermatitis. We concluded that TRPV3 was the gene responsible for Nh and Ht mutations, and that mutation in TRPV3 possibly correlated with increased mast cell numbers.

Alopecia↗

PubMed1992

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily.

Amino Acid Sequence↗

PubMed2001

Molecular basis for selectivity of high affinity peptide antagonists for the gastrin-releasing peptide receptor.

Few gastrointestinal hormones/neurotransmitters have high affinity peptide receptor antagonists, and little is known about the molecular basis of their selectivity or affinity. The receptor mediating the action of the mammalian bombesin (Bn) peptide, gastrin-releasing peptide receptor (GRPR), is an exception, because numerous classes of peptide antagonists are described. To investigate the molecular basis for their high affinity for the GRPR, two classes of peptide antagonists, a statine analogue, JMV594 ([d-Phe(6),Stat(13)]Bn(6-14)), and a pseudopeptide analogue, JMV641 (d-Phe-Gln-Trp-Ala-Val-Gly-His-Leupsi(CHOH-CH(2))-(CH(2))(2)-CH(3)), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimeric or mutated receptors were transiently expressed in Balb/c 3T3. Only substitution of the fourth extracellular (EC) domain of the GRPR by the comparable NMBR domain markedly decreased the affinity for both antagonists. Substituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 amino acid differences between the GRPR and NMBR in this domain were exchanged. The substitutions of Thr(297) in GRPR by Pro from the comparable position in NMBR, Phe(302) by Met, and Ser(305) by Thr decreased the affinity of each antagonist. Simultaneous replacement of Thr(297), Phe(302), and Ser(305) in GRPR by the three comparable NMBR amino acids caused a 500-fold decrease in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagonist. Receptor modeling showed that each of these three amino acids faced inward and was within 5 A of the putative binding pocket. These results demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagonists. They demonstrate that Thr(297), Phe(302), and Ser(305) of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-pi interactions and hydrogen bonding are important for their high affinity interaction.

3T3 Cells↗

PubMed2001

The unique proline of the Prochlorothrix hollandica plastocyanin hydrophobic patch impairs electron transfer to photosystem I.

A number of surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in amino acids located in the amino-terminal hydrophobic patch of the copper protein, which presents a variant structure as compared with other plastocyanins. The single mutants Y12G, Y12F, Y12W, P14L, and double mutant Y12G/P14L have been produced. Their reactivity toward photosystem I has been analyzed by laser flash absorption spectroscopy. Plots of the observed rate constant with all mutants versus plastocyanin concentration show a saturation profile similar to that with wild-type plastocyanin, thus suggesting the formation of a plastocyanin-photosystem I transient complex. The mutations do not induce relevant changes in the equilibrium constant for complex formation but induce significant variations in the electron transfer rate constant, mainly with the two mutants at proline 14. Additionally, molecular dynamics calculations indicate that mutations at position 14 yield small changes in the geometry of the copper center. The comparative kinetic analysis of the reactivity of plastocyanin mutants toward photosystem I from different organisms (plants and cyanobacteria) reveals that reversion of the unique proline of Prochlorothrix plastocyanin to the conserved leucine of all other plastocyanins at this position enhances the reactivity of the Prochlorothrix protein.

Bacterial Proteins↗

PubMed2001

Thrombin stimulation of the vascular cell adhesion molecule-1 promoter in endothelial cells is mediated by tandem nuclear factor-kappa B and GATA motifs.

The goal of this study was to delineate the transcriptional mechanisms underlying thrombin-mediated induction of vascular adhesion molecule-1 (VCAM-1). Treatment of human umbilical vein endothelial cells with thrombin resulted in a 3.3-fold increase in VCAM-1 promoter activity. The upstream promoter region of VCAM-1 contains a thrombin response element, two nuclear factor kappaB (NF-kappaB) motifs, and a tandem GATA motif. In transient transfection assays, mutation of the thrombin response element had no effect on thrombin induction. In contrast, mutation of either NF-kappaB site resulted in a complete loss of induction, whereas a mutation of the two GATA motifs resulted in a significant reduction in thrombin stimulation. In electrophoretic mobility shift assays, nuclear extracts from thrombin-treated endothelial cells displayed markedly increased binding to the tandem NF-kappaB and GATA motifs. The NF-kappaB complex was supershifted with anti-p65 antibodies, but not with antibodies to RelB, c-Rel, p50, or p52. The GATA complex was supershifted with antibodies to GATA-2, but not GATA-3 or GATA-6. A construct containing tandem copies of the VCAM-1 GATA motifs linked to a minimal thymidine kinase promoter was induced 2.4-fold by thrombin. Taken together, these results suggest that thrombin stimulation of VCAM-1 in endothelial cells is mediated by the coordinate action of NF-kappaB and GATA transcription factors.

Amino Acid Motifs↗

PubMed2001

Regulation of the accumulation and function of p53 by phosphorylation of two residues within the domain that binds to Mdm2.

The function and stability of the tumor suppressor p53 are tightly controlled by the negative regulator mouse double minute 2 (Mdm2), which binds to p53, blocking DNA binding and targeting p53 for proteosome-mediated degradation. Following DNA damage or cellular stress, p53 is phosphorylated within the Mdm2 binding domain on threonine 18 and serine 20. To analyze the roles of these phosphorylation events, residues 18 and 20 were mutated to alanines. Transient transfection into p53-null cells demonstrated that the T18A protein can be expressed stably, but the S20A protein is very unstable, precluding further analysis. When expressed stably at low basal levels in p53-null human fibroblasts or fibrosarcoma cells, the T18A mutant accumulated 5-10-fold less well than wild-type p53 following exposure to UV. Analysis of p53-dependent transcription following UV revealed that the phosphorylation of threonine 18 is required for transactivation of the p21, Hdm2 (the human ortholog of Mdm2), and GADD45 genes. The phosphorylation of serine 33, another early event following DNA damage, is not required for p53 accumulation or p53-dependent transactivation following UV.

Base Sequence↗

PubMed2003

The trypanosomatid signal recognition particle consists of two RNA molecules, a 7SL RNA homologue and a novel tRNA-like molecule.

Trypanosomatids are ancient eukaryotic parasites affecting humans and livestock. Here we report that the trypanosomatid signal recognition particle (SRP), unlike all other known SRPs in nature, contains, in addition to the 7SL RNA homologue, a short RNA molecule, termed sRNA-85. Using conventional chromatography, we discovered a small RNA molecule of 85 nucleotides co-migrating with the Leptomonas collosoma 7SL RNA. This RNA molecule was isolated, sequenced, and used to clone the corresponding gene. sRNA-85 was identified as a tRNA-like molecule that deviates from the canonical tRNA structure. The co-existence of these RNAs in a single complex was confirmed by affinity selection using an antisense oligonucleotide to sRNA-85. The two RNA molecules exist in a particle of approximately 14 S that binds transiently to ribosomes. Mutations were introduced in sRNA-85 that disrupted its putative potential to interact with 7SL RNA by base pairing; such mutants were unable to bind to 7SL RNA and to ribosomes and were aberrantly distributed within the cell. We postulate that sRNA-85 may functionally replace the truncated Alu domain of 7SL RNA. The discovery of sRNA-85 raises the intriguing possibility that sRNA-85 functional homologues may exist in other lower eukaryotes and eubacteria that lack the Alu domain.

Animals↗

PubMed2003

Characterization of regulatory elements and methylation pattern of the autoimmune regulator (AIRE) promoter.

Defects in the AIRE gene cause a monogenic autoimmune syndrome APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy), which is characterized by loss of self-tolerance to multiple organs. In concordance with its role in immune tolerance, AIRE is most strongly expressed in thymic epithelial cells and in cells of monocytic-dendritic lineage. The AIRE protein has been shown to function as a transcriptional regulator, however, the mechanisms regulating AIRE gene expression are not known. Here we have characterized the AIRE promoter region by identifying a minimal promoter region within 350 bp of the translation initiation codon. Electrophoretic mobility shift assays and transient transfections with mutated promoter constructs revealed a functional TATA box (-163 to -153) and binding sites for transcription complexes AP-1 (-307 to -296), NF-Y (-213 to -202), and Sp1 (-202 to -189). The presence of a 390-bp CpG island within the proximal promoter suggested that cytosine methylation has a role in transcriptional regulation of AIRE, which was supported by in vitro methylation experiments of promoter constructs. Sodium bisulfite sequencing showed a less methylated status of AIRE promoter in the thymic epithelial cell line TEC1A3 compared with HeLa and monocytic cells U937 and THP-1. Real-time PCR analysis showed that treatment with 5-aza-2'-deoxycytidine (5-azaCdR), a DNA methyltransferase inhibitor, up-regulated AIRE transcript levels in TEC1A3, U937, and HeLa cells and that even greater activations in TEC1A3 and U937 cells were observed using combined treatments with deacetylase inhibitor trichostatin A. These results suggest that AIRE gene expression is modulated through modifications in chromatin methylation and acetylation.

Base Sequence↗

PubMed2003

Ume1p represses meiotic gene transcription in Saccharomyces cerevisiae through interaction with the histone deacetylase Rpd3p.

Ume1p is a member of a conserved protein family including RbAp48 that associates with histone deacetylases. Consistent with this finding, Ume1p is required for the full repression of a subset of meiotic genes during vegetative growth in budding yeast. In addition to mitotic cell division, this report describes a new role for Ume1p in meiotic gene repression in precommitment sporulating cultures returning to vegetative growth. However, Ume1p is not required to re-establish repression as part of the meiotic transient transcription program. Mutational analysis revealed that two conserved domains (NEE box and a WD repeat motif) are required for Ume1p-dependent repression. Co-immunoprecipitation studies revealed that both the NEE box and the WD repeat motif are essential for normal Rpd3p binding. Finally, Ume1p-Rpd3p association is dependent on the global co-repressor Sin3p. Moreover, this activity was localized to one of the four paired amphipathic-helix domains of Sin3p shown previously to be required for transcriptional repression. These findings support a model that Ume1p binding to Rpd3p is required for its repression activity. In addition, these results suggest that Rpd3-Ume1p-Sin3p comprises an interdependent complex required for mediating transcriptional repression.

Amino Acid Sequence↗

PubMed2006

The genetic spectrum of a population-based sample of familial hemiplegic migraine.

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura and transient hemiplegia. FHM mutations are known in three genes, the CACNA1A (FHM1) gene, the ATP1A2 (FHM2) and the SCN1A (FHM3) gene and seem to have an autosomal-dominant mode of inheritance. The aim of this study was to search for FHM mutations in FHM families identified through a screen of the Danish population of 5.2 million people. FHM patients were diagnosed according to the International Classification of Headache Disorders and all FHM patients had a physical and neurological examination by a physician. A total of 147 FHM patients from 44 different families were identified; 43 FHM families participated in this study. Linkage analysis of these families shows clear linkage to the FHM locus (FHM1) on chromosome 19, supportive linkage to the FHM2 locus whereas no linkage was found to the FHM3 locus. Furthermore, we sequenced all exons and promoter regions of the CACNA1A and ATP1A2 genes and screened for the Q1489K mutation in the SCN1A gene. CACNA1A gene mutations were identified in three of the FHM families, two known FHM mutations, R583Q and T666M and one novel C1369Y mutation. Three FHM families were identified with novel mutations in the ATP1A2 gene; a family with a V138A mutation, a family with a R202Q mutation and a family with a R763C mutation. None of the Danish FHM families have the Q1489K mutation in the SCN1A gene. Our study shows that only 14% (6/42) of FHM families in the general Danish population have exonic FHM mutations in the CACNA1A or ATP1A2 gene. The families we identified with FHM mutations in the CACNA1A and ATP1A2 genes were extended, multiple affected families whereas the remaining FHM families were smaller. The existence of many small families in the Danish FHM cohort may reflect less bias in FHM family ascertainment and/or more locus heterogeneity than described previously.

Adolescent↗

PubMed1981

Mutagenesis of intact parvovirus H-1 is expressed co-ordinately with enhanced reactivation of ultraviolet irradiated virus in human and rat cells treated with 2-nitronaphthofurans.

The exposure of human or rat cells to non-toxic concentrations of two 2-nitronaphthofuran derivatives activated co-ordinately the transient expression of mutator and repair activities. These activities gave rise to both an increase in the mutagenesis (enhanced mutagenesis, EM) and survival (enhanced reactivation, ER) of unirradiated and u.v.-irradiated parvovirus H-1 used as respective probes. The kinetics of expression was the same for mutator and repair activities and for the two chemicals studied. The dose-responses of these activities were also parallel for a given chemical, but one of the furan derivatives exerted its inducing effect at concentrations 20-25 times lower than the other derivative. Both EM and ER were depressed by cycloheximide, and inhibitor of de novo protein synthesis. This is the first report which shows that chemicals can enhance the mutagenesis of undamaged DNA by activating the expression of mutator functions in mammalian, including human, cells. The ability of the two 2-nitronaphthofuran derivatives to trigger EM and ER was found to correlate with their reported mutagenicity in a conventional bacterial test system.

Animals↗

PubMed1998

Regulation of expression of nuclear and mitochondrial forms of human uracil-DNA glycosylase.

Promoters PA and PBin the UNG gene and alternative splicing are utilized to generate nuclear (UNG2) and mitochondrial (UNG1) forms of human uracil-DNA glycosylase. We have found the highest levels of UNG1 mRNA in skeletal muscle, heart and testis and the highest UNG2 mRNA levels in testis, placenta, colon, small intestine and thymus, all of which contain proliferating cells. In synchronized HaCaT cells mRNAs for both forms increased in late G1/early S phase, accompanied by a 4- to 5-fold increase in enzyme activity. A combination of mutational analysis and transient transfection demonstrated that an E2F-1/DP-1-Rb complex is a strong negative regulator of both promoters, whereas 'free' E2F-1/DP-1 is a weak positive regulator, although a consensus element for E2F binding is only present in PB. These results indicate a central role for an E2F-DP-1-Rb complex in cell cycle regulation of UNG proteins. Sp1 and c-Myc binding elements close to transcription start areas were positive regulators of both promoters, however, whereas overexpression in HeLa cells of Sp1 stimulated both promoters, c-Myc and c-Myc/Max overexpression had a suppressive effect. CCAAT elements were negative regulators of PB, but positive regulators of PA. These results demonstrate differential expression of mRNAs for UNG1 and UNG2 in human tissues.

Alternative Splicing↗

PubMed1996

Essential role of NF-kappa B in transactivation of the human immunodeficiency virus long terminal repeat by the human cytomegalovirus 1E1 protein.

The 72 kDa 1E1 protein of human cytomegalovirus (HCMV) is one of a few viral regulatory proteins expressed immediately after infection of a host cell. Although it is now well-established that 1E1 is a potent transcriptional activator of the human immunodeficiency virus (HIV) long terminal repeat (LTR), the identity of the nucleotide sequence responsive to 1E1 remains elusive and the molecular mechanism of this interaction is not well-understood. We have constructed various LTR mutants and tested them for their ability to be activated by 1E1 using transient transfection assays. Mutations in the NF-kappa B sites, of either a few changes in the nucleotide sequence or a deletion of the entire region, abrogated 1E1-driven transactivation. Deletion of the Tat-responsive element (TAR) had no significant effect on reporter expression. Mutations in the Sp1 sites or the TATA box significantly lowered LTR activity, but this is probably due to an effect on the general transcription system, as these elements are also required for the transactivation of the LTR by many stimulators including Tat, tumour necrosis factor alpha (TNF-alpha). E1A/E1B and phorbol myristate acetate (PMA). In addition, gel retardation analysis demonstrated that NF- kappa B activity was significantly increased in human T lymphoid H9 and monocytic U937 cell lines constitutively expressing 1E1. Taken together, these data suggest that NF- kappa B plays a central role in the 1E1 transactivation of the HIV LTR.

Base Sequence↗

PubMed1991

C1- and R-dependent expression of the maize Bz1 gene requires sequences with homology to mammalian myb and myc binding sites.

Tissue-specific expression of the maize anthocyanin Bronze-1 (Bz1) gene is controlled by the products of several regulatory genes. These include C1 or Pl and R or B that share homology to the myb proto-oncogenes and myc-like genes, respectively. Bz1 expression in embryo tissues is dependent on C1 and an R-sc allele of R. Transient expression from mutated and deleted versions of the Bz1 promoter fused to a luciferase reporter gene was measured in C1, Rscm2 embryos after gene transfer by microprojectiles. This analysis revealed that the sequences between -76 base pairs (bp) and -45 bp and a 9-bp AT-rich block between -88 bp and -80 bp were critical for Bz1 expression. The -76 bp to -45 bp region includes two short sequences that are homologous to the consensus binding sites of the myb- and myc-like proteins. Site-specific mutations of these "myb" and "myc" sequences reduced Bz1 expression to 10% and 1% of normal, respectively. Additionally, a trimer of a 38-bp oligonucleotide containing these myb and myc sites increased the expression of a cauliflower mosaic virus 35S minimal promoter by 26-fold. This enhancement was dependent on both C1 and R. Because the sites critical for Bz1 expression are homologous to the myb and myc consensus binding sequences and the C1 and R proteins share homology with the myb and myc products, respectively, we propose that C1 and R interact with the Bz1 promoter at these sites.

Base Sequence↗

PubMed2000

Transcriptional induction of heme oxygenase-1 gene expression by okadaic acid in primary rat hepatocyte cultures.

Heme oxygenase (HO) catalyzes the rate-limiting enzymatic step of heme degradation and regulates the cellular heme content. The gene expression of the inducible isoform of HO, HO-1, is up-regulated in response to various agents causing oxidative stress. To investigate the regulatory role of protein phosphatases in the hepatic regulation of HO-1 gene expression, primary cultures of rat hepatocytes were treated with okadaic acid (OA), which specifically inhibits the serine threonine protein phosphatases 1 and 2A. Both protein synthesis and mRNA expression of HO-1 were induced by OA in cultured hepatocytes, but not in cultured tissue macrophages of rat liver. The HO-1 mRNA induction by OA occurred in a time- and concentration-dependent manner. Simultaneous treatment with OA plus dibutyryl cAMP caused a synergistic up-regulation of steady-state levels of HO-1 mRNA, and the specific protein kinase A inhibitor KT5720 markedly reduced the OA-dependent HO-1 mRNA induction. In contrast, the dibutyryl cAMP-dependent induction of the phosphoenolpyruvate carboxykinase mRNA expression and enzyme activity was inhibited by simultaneous treatment with OA in hepatocytes. The induction of the HO-1 gene expression by OA was transcriptional as determined by studies with actinomycin D, nuclear run-off assay, and measurement of the half-life of HO-1 mRNA. Luciferase reporter constructs containing DNA sequences of the rat HO-1 promoter 5'-flanking region were up-regulated by OA in transiently transfected hepatocytes. Mutation of the cAMP response element/activator protein-1 (-665/-654) site obliterated the OA-dependent induction, suggesting that this element is involved in the transcriptional induction of the rat HO-1 gene by OA.

Animals↗

PubMed1997

Activation of the adenovirus major late promoter by transcription factors MAZ and Sp1.

Multiple binding sites for the transcription factors MAZ and Sp1 within the adenovirus type 5 major late promoter have been identified by DNase I protection studies. In the proximal region of the promoter, both MAZ and Sp1 interact with GC-rich sequences flanking the TATA box. Two MAZ binding sites are centered at -18 and -36 relative to the transcriptional initiation site. Sp1 bound only to the -18 GC-rich sequence. Several sites of interaction were also evident in the distal region of the promoter. Both MAZ and Sp1 interacted with a sequence centered at -166, and MAZ bound weakly to an additional site centered at -130. Overexpression of MAZ or Sp1 activated expression from the major late promoter in transient expression assays. Mutational analysis of the GC-rich sequences in the major late promoter suggested that a primary target of MAZ activation is the GC-rich sequences flanking the TATA sequence, whereas Sp1 requires the distal GC-rich sequence elements to stimulate gene expression. This activation is enhanced by the adenovirus E1A protein, and evidence for interaction between E1A and both transcription factors was obtained by using an immunoprecipitation assay. Activation by MAZ and Sp1 also was observed in transfection studies using the complete adenovirus type 5 genome as the target. Increased levels of late mRNA from both the L1 and L5 regions were observed when MAZ or Sp1 expression plasmids were transfected with viral DNA. Unexpectedly, activation of the major late promoter by MAZ and Sp1 was detected irrespective of whether the viral DNA could replicate.

Adenoviridae↗

PubMed1992

Transcription of the mouse secretory protease inhibitor p12 gene is activated by the developmentally regulated positive transcription factor Sp1.

We have previously shown that a trans-acting protein produced in some tissue culture cells positively control the transcriptional activity directed by the mouse p12 promoter. This nuclear protein exerts its positive activity by interacting with a regulatory sequence designated p12.A and located between the TATA and CCAAT box elements on the p12 gene promoter. Using DNase I and dimethyl sulfate methylation interference footprinting techniques coupled with gel retardation assays, we found evidence that the protein which binds to the p12.A element is the well-known transcription factor Sp1. Mutational analysis in transient transfection assays confirmed the positive activity exerted by this protein in every cell line tested. In agreement with this observation, we detected a p12.A-Sp1 binding activity in nuclear extracts prepared from all cell lines used. However, a similar binding activity could not be detected in a number of nuclear extracts prepared from normal mouse tissues. In this report, we provide the evidence that the lack of Sp1-binding activity results from the degradation of Sp1 in the kidney, liver, and pancreas of the mouse.

Animals↗