Biomedical subjects

N Mermod

Publications and source records attributed to N Mermod.

34 records · Page 2Linked to original sources

PubMed1991

A pancreatic exocrine cell factor and AP4 bind overlapping sites in the amylase 2A enhancer.

A factor found in pancreatic exocrine cell lines and pancreatic nuclei binds selectively to the alpha-amylase 2A transcriptional enhancer. Pancreatic exocrine cell extracts protect asymmetrically an unusually large, 35 base pair region from DNase I digestion in vitro, suggesting the involvement of a multimeric DNA binding complex. We show that this region of the enhancer contains a major affinity recognition sequence for the HeLa transcription factor AP4. A 4 base pair mutation in the enhancer sequence shown previously to abolish activity in vivo [Boulet, A. M., Erwin, C. R., & Rutter, W. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3599-3603] abolishes AP4 binding in vitro and weakens but does not eliminate the binding of adjacent enhancer factors. Further, sequences similar to the AP4 binding site are found within a consensus sequence of most pancreatic exocrine genes (Boulet et al., 1986). We have identified three AP4 binding sites in the pancreatic elastase gene: one occurs in the consensus sequence of the enhancer. Thus, protein(s) with the binding selectivity of AP4 may play a role in the expression of the pancreatic exocrine gene family.

Amylases↗

PubMed1991

Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

CCAAT-Enhancer-Binding Proteins↗

PubMed1990

Protein-protein interactions between adenovirus DNA polymerase and nuclear factor I mediate formation of the DNA replication preinitiation complex.

The in vitro adenovirus (Ad) DNA replication system provides an assay to study the interaction of viral and host replication proteins with the DNA template in the formation of the preinitiation complex. This initiation system requires in addition to the origin DNA sequences 1) Ad DNA polymerase (Pol), 2) Ad preterminal protein (pTP), the covalent acceptor for protein-primed DNA replication, and 3) nuclear factor I (NFI), a host cell protein identical to the CCAAT box-binding transcription factor. The interactions of these proteins were studied by coimmunoprecipitation and Ad origin DNA binding assays. The Ad Pol can bind to origin sequences only in the presence of another protein which can be either pTP or NFI. While NFI alone can bind to its origin recognition sequence, pTP does not specifically recognize DNA unless Ad Pol is present. Thus, protein-protein interactions are necessary for the targetting of either Ad Pol or pTP to the preinitiation complex. DNA footprinting demonstrated that the Ad DNA site recognized by the pTP.Pol complex was within the first 18 bases at the end of the template which constitutes the minimal origin of replication. Mutagenesis studies have defined the Ad Pol interaction site on NFI between amino acids 68-150, which overlaps the DNA binding and replication activation domain of this factor. A putative zinc finger on the Ad Pol has been mutated to a product that fails to bind the Ad origin sequences but still interacts with pTP. These results indicate that both protein-protein and protein-DNA interactions mediate specific recognition of the replication origin by Ad DNA polymerase.

Adenoviruses, Human↗

PubMed1990

Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity.

Enhancer binding protein AP-4 is a transcription factor that activates both viral and cellular genes by binding to the symmetrical DNA sequence, CAGCTG. Here, we report the molecular cloning and characterization of human AP-4 cDNAs. The deduced amino acid sequence reveals that AP-4 is a helix-loop-helix (HLH) protein. Like other members of this family, the AP-4 HLH motif and the adjacent basic domain are necessary and sufficient to confer site-specific DNA binding. However, unlike other HLH proteins, AP-4 also contains two additional protein dimerization motifs consisting of leucine repeat elements LR1 and LR2. The analysis of various deletion and point mutants for their ability to dimerize in the presence or absence of DNA reveals several unusual features. Although the HLH basic region is sufficient for DNA recognition and binding, dimer formation between different truncated versions of AP-4 in solution requires an intact LR1 or LR2 domain. AP-4 is unable to form heterodimers with other helix-loop-helix family members such as the immunoglobulin enhancer binding factor, E12. In contrast, an AP-4 derivative, delta C222, which lacks LR1 and LR2 but retains an intact HLH, can form heterodimers with E12. Moreover, AP-4 molecules containing LR2 or LR1 are unable to form mixed dimers with carboxy-terminally truncated AP-4 molecules such as delta C222, but retain the ability to form complexes with longer versions of AP-4 that contain LR1 and/or LR2. Our findings strongly suggest that AP-4 contains multiple protein-protein interfaces that function to promote homodimer formation and restrict heterocomplexes. These findings provide a mechanism by which different members of the helix-loop-helix family of transcription factors can form functional dimers in a specific fashion with their appropriate partners to control transcriptional networks during cellular differentiation.

Amino Acid Sequence↗

PubMed1989

The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain.

Human CTF/NF-I consists of a family of CCAAT box binding proteins that activate both transcription and DNA replication. Analysis of cDNA mutants expressed in E. coli and Drosophila cells reveals that the N-terminal portion of CTF-1 is sufficient for site-specific DNA recognition, protein dimerization, and adenovirus replication. In contrast, transcriptional activation requires an additional C-terminal domain. Furthermore, this transcription domain efficiently activates a heterologous promoter, such as SV40, when fused to the DNA binding domain of Sp1. The CTF C-terminal region consists of an unusual type of transcriptional activation domain containing approximately 25% proline residues. We propose that this proline-rich domain represents a novel class of activators which are distinct from those containing either acidic or glutamine-rich activation motifs. This indicates that transcriptional activation is likely to be mediated by several different mechanisms. In addition, these results suggest that the interactions, and consequently the mechanisms, governing transcriptional activation by CTF are distinct from those mediating DNA replication.

Amino Acid Sequence↗

PubMed1988

Proteins bound at adjacent DNA elements act synergistically to regulate human proenkephalin cAMP inducible transcription.

Synthesis of the endogenous opioid precursor, proenkephalin, is regulated by neurotransmitters and membrane depolarization. These events act through second messenger dependent signal transduction pathways via a short inducible DNA enhancer to regulate transcription of the proenkephalin gene. Two DNA elements located within this enhancer are essential for the transcriptional response to cAMP and phorbol ester. Inactivation of either element by mutation or by alteration of their stereospecific alignment eliminates inducible enhancer activity. The promoter distal element, ENKCRE-1, in the absence of a functional adjacent ENKCRE-2 element, has no inherent capacity to activate transcription. However, in the presence of a functional ENKCRE-2 element, this element synergistically augments cAMP and phorbol ester inducible transcription. The promoter proximal element, ENKCRE-2, is essential for both basal and regulated enhancer function. Four different protein factors found in HeLa cell nuclear extracts bind in vitro to the enhancer region. ENKTF-1, a novel enhancer binding protein, binds to the DNA region encompassing ENKCRE-1. The transcription factors AP-1 and AP-4 bind to overlapping sites spanning ENKCRE-2, and a fourth transcription factor, AP-2, binds to a site immediately downstream of ENKCRE-2. The binding of ENKTF-1 to mutant ENKCRE-1 sequences in vitro correlates with the in vivo inducibility of the mutant elements suggesting that ENKTF-1 acts in combination with factors that recognize the ENKCRE-2 domain to regulate cAMP inducible transcription. Together, the two DNA elements, ENKCRE-1 and ENKCRE-2 and the protein factors with which they interact, play a critical role in the transduction and reception of signals transmitted from cell surface receptors to the proenkephalin nuclear transcription complex.

Cyclic AMP↗

PubMed1988

A family of human CCAAT-box-binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs.

The CTF/NF-I group of cellular DNA binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription as well as DNA replication. Molecular analysis of human CTF/NF-I complementary DNA clones reveals multiple messenger RNA species containing alternative coding regions, apparently as a result of differential splicing. Expression and functional analysis establish that individual gene products can bind to GCCAAT recognition sites and serve both as promoter-selective transcriptional activators and as initiation factors for DNA replication.

Adenoviridae↗

PubMed1988

Enhancer binding factors AP-4 and AP-1 act in concert to activate SV40 late transcription in vitro.

The simian virus 40 (SV40) transcriptional enhancer is composed of multiple cis-acting DNA sequence motifs, each individually having a two- to fourfold effect on the efficiency of transcription. When various distinct cis-elements act in combination, however, a dramatic enhancement of transcription initiation often results. SV40-enhancer A-domain sequences were previously shown to be important for early and late transcription in vivo. Here we report the isolation of the enhancer binding factor AP-4, which recognizes a motif in this domain. Purified AP-4 activates SV40 late transcription in vitro, and this stimulation is augmented by the addition of transcription factor AP-1 which binds to adjacent sequences in the A-domain, suggesting coordinate action of the two factors for transcriptional enhancement. AP-1 also represses late transcription from a major in vitro start site which is poorly used in vivo, indicating that AP-1 can act as both a positive and negative regulator of SV40 late transcription. Thus by manipulating the levels of different trans-acting factors in vitro, we can recreate the pattern of SV40 late initiation observed during the viral lytic cycle in vivo.

Base Sequence↗

PubMed1988

Regulation of gene expression by interferons: control of H-2 promoter responses.

The magnitude of the response to interferons and the requirement for individual elements in the promoter of the H-2Dd gene were shown to be cell-specific and dependent on the type of interferon used. Three DNA sequences in the promoter were found to bind murine nuclear factors. Two of these sequences are in functionally defined enhancer regions and also bind to the transcription factor AP-1. The third sequence is part of the region involved in interferon regulation and is homologous to the enhancer element of the interferon beta gene. A model for interferon regulation of H-2 promoters is discussed.

Animals↗

PubMed1987

The xylS gene positive regulator of TOL plasmid pWWO: identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon.

The Pseudomonas putida TOL plasmid pWWO carries an operon that specifies a meta-cleavage pathway for the catabolism of benzoate and toluates whose transcription is positively regulated by the xylS gene product. Stimulation of transcription of the operon is thought to result from activation of this protein by pathway substrates/effectors. In the present study, overexpression of the xylS gene has led to identification of the regulator as a 33 kDa protein. Overexpression of xylS also resulted in partially constitutive, i.e. effector-independent expression of the meta-cleavage operon. Determination of the polynucleotide sequence of the xylS gene revealed amino acid sequence homology with several DNA binding proteins, particularly with the araC products of Escherichia coli and Salmonella typhimurium and with the nifA and ntrC products of Klebsiella pneumoniae. Homologous sequences were mainly located in an alpha-helix-turn-alpha-helix domain of the polypeptide. Interestingly, amino acid sequence homology was also found with sigma factors of E. coli (ntrA and htpR products) and Bacillus subtilis (spoIIG and phage SPOI Gp34 products) and other RNA polymerase core-interacting proteins, such as the E. coli nusA product.

Amino Acid Sequence↗

PubMed1987

Regulatory circuits controlling transcription of TOL plasmid operon encoding meta-cleavage pathway for degradation of alkylbenzoates by Pseudomonas.

TOL plasmid pWWO of Pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. The 'upper' operon encodes enzymes for the oxidation of toluene to benzoate and xylenes to toluates, whereas the meta-cleavage operon specifies the further oxidation of benzoate and toluates. Transcription of the upper pathway operon is positively regulated by the XylR protein, which is activated by toluene/xylenes and their alcohol catabolic products, in combination with the NtrA protein, a sigma factor. Expression of the meta-operon is positively controlled by the XylS protein which is activated by meta-pathway substrates, and is independent of NtrA protein. Expression of the meta pathway is also induced by toluene/xylene-activated XylR protein via a cascade regulatory system in which this protein in combination with NtrA protein stimulates transcription from the xylS gene promoter. Hyper-production of XylS protein in turn provokes high level expression of the meta-operon, which is independent of meta-pathway substrates. The two promoters, which are activated by the XylR and NtrA proteins, the upper pathway promoter and the xylS gene promoter, exhibit three regions of homology centred at -12(5'-TTGCATG-3'), -24(5'-TGGCPuT-3') and -45(5'-TAAAATAAGPuPuCGPuTC-3'), with respect to their principal transcription initiation points. The possible physiological significance of activated XylR-protein-induced expression of the meta-operon through amplification of XylS protein levels is considered.

Base Sequence↗

PubMed1987

Roles of the divergent branches of the meta-cleavage pathway in the degradation of benzoate and substituted benzoates.

The TOL plasmid-specified meta-cleavage pathway for the oxidative catabolism of benzoate and toluates branches at the ring cleavage products of catechols and reconverges later at 2-oxopent-4-enoate or its corresponding substituted derivatives. The hydrolytic branch of the pathway involves the direct formation of 2-oxopent-4-enoate or its derivatives, whereas the oxalocrotonate branch involves three enzymatic steps effected by a dehydrogenase, an isomerase, and a decarboxylase, which produce the same compounds. Evidence is presented which shows that benzoate and p-toluate can, under certain circumstances, be catabolized by the hydrolytic branch. However, in a fully functional pathway, only m-toluate is dissimilated via this branch, and benzoate and p-toluate are catabolized almost exclusively by the oxalocrotonate branch. The biochemical basis of this selectivity was found to reside in the high affinity of the dehydrogenase for ring fission products derived from benzoate and p-toluate and its inability to attack the ring fission product derived from m-toluate. Although isomerization of 4-oxalocrotonate occurs spontaneously in vitro, enzymatic isomerization was found to be essential for effective functioning of this branch of the pathway in vivo.

Benzoates↗

PubMed1986

Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria.

A pKT231-based broad-host-range plasmid vector was constructed which enabled regulation of expression of cloned genes in a wide range of gram-negative bacteria. This vector, pNM185, contained upstream of its EcoRI, SstI, and SstII cloning sites the positively activated pm twin promoters of the TOL plasmid and xylS, the gene of the positive regulator of these promoters. Expression of cloned genes was induced with micromolar quantities of benzoate or m-toluate, the inexpensive coinducers of the pm promoters. Expression of a test gene, xylE, which specifies catechol 2,3-dioxygenase, cloned in this vector was tested in representative strains of a variety of gram-negative bacteria. Regulated expression of xylE was observed in most strains examined, and induced levels of enzyme representing up to 5% of total cellular protein and ratios of induced:noninduced levels of enzyme up to a factor of 600 were observed. The level of xylE gene expression in different bacteria tended to be correlated with their phylogenetic distance from Pseudomonas putida.

Drug Resistance, Microbial↗

PubMed1986

Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.

Bacterial Proteins↗

PubMed1984

Transcription of the TOL plasmid toluate catabolic pathway operon of Pseudomonas putida is determined by a pair of co-ordinately and positively regulated overlapping promoters.

Expression of the meta-cleavage pathway operon of TOL plasmid pWW0 of Pseudomonas putida is positively regulated by the xylS gene product. We have sequenced the promoter region of this operon and localized the transcription initiation sites. Two overlapping promoters, designated Pm1 and Pm2, are responsible for the positively regulated expression of the meta-pathway operon. Mutants of P. putida were isolated that expressed the meta-cleavage pathway operon constitutively. Several plasmid-located mutations that led to constitutivity were characterized by sequencing and the transcription initiation sites on mutant plasmids localized. This resulted in the identification of newly created promoters whose functioning did not require the xylS product. Comparison of the promoter sequences obtained suggests a tentative consensus sequence for promoters of P. putida which is significantly different from that of E. coli.

Base Sequence↗

PubMedDate not supplied

A regulatory network for the efficient control of transgene expression.

BACKGROUND: Expression of heterologous genes in mammalian cells or organisms for therapeutic or experimental purposes often requires tight control of transgene expression. Specifically, the following criteria should be met: no background gene activity in the off-state, high gene expression in the on-state, regulated expression over an extended period, and multiple switching between on- and off-states. METHODS: Here, we describe a genetic switch system for controlled transgene transcription using chimeric repressor and activator proteins functioning in a novel regulatory network. In the off-state, the target transgene is actively silenced by a chimeric protein consisting of multimerized eukaryotic transcriptional repression domains fused to the DNA-binding tetracycline repressor. In the on-state, the inducer drug doxycycline affects both the derepression of the target gene promoter and activation by the GAL4-VP16 transactivator, which in turn is under the control of an autoregulatory feedback loop. RESULTS: The hallmark of this new system is the efficient transgene silencing in the off-state, as demonstrated by the tightly controlled expression of the highly cytotoxic diphtheria toxin A gene. Addition of the inducer drug allows robust activation of transgene expression. In stably transfected cells, this control is still observed after months of repeated cycling between the repressed and activated states of the target genes. CONCLUSIONS: This system permits tight long-term regulation when stably introduced into cell lines. The underlying principles of this network system should have general applications in biotechnology and gene therapy.

Animals↗