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G Ab

Publications and source records attributed to G Ab.

At least 19 recordsLinked to original sources
PubMed1998

The bZip transcription factor vitellogenin-binding protein is post transcriptional down regulated in chicken liver.

The vitellogenin-binding protein (VBP) is a member of the proline and acidic-region rich (PAR) family of bZip transcription factors. PAR is located N-terminally to the DNA-binding domain. VBP binds to specific sites within the 300-bp 5'-flanking region of the chicken-liver-specific estrogen-dependent very-low-density apolipoprotein gene (apoVLDL II). One of these binding sites (site D) resembles the albumin site D and is positioned in close proximity of the major estrogen-responsive element. Previous studies showed that VBP can bind simultaneously with the estrogen receptor to the putative complex regulatory element E1D. To investigate whether VBP is involved in apoVLDL II gene expression, we examined its capacity to enhance apoVLDL II transcription and its presence in liver. We show that VBP is capable of enhancing transcription in transfection experiments. However, VBP could not be detected in liver by Western-blots or immuno-electro mobility shift assays (EMSAs) using antibodies against different moieties of the protein. We examined the possible reduction in translation efficiencies due to a small upstream open reading frame in the VBP leader sequence, but did not find any. Although VBP binds to the proximal apoVLDL II promoter region and enhances transcription in co-transfection experiments, the protein is unlikely to be involved in apoVLDL II gene transcription because of its undetectable low level in liver nuclei.

Animals↗
PubMed1997

Differential stimulation by CCAAT/enhancer-binding protein alpha isoforms of the estrogen-activated promoter of the very-low-density apolipoprotein II gene.

The transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBP alpha and C/EBP beta) are highly expressed in liver and are believed to function in maintaining the differentiated state of the hepatocytes. C/EBP alpha appears to be a critical regulator of genes involved in metabolic processes. We are interested in the roles of C/EBP in the expression of the very-low-density apolipoprotein II (apoVLDL II) gene. This gene encodes an avian yolk protein, is induced by estrogens and is only expressed in liver. To examine the role of C/EBP in apoVLDL II expression, footprinting and electromobility-shift analysis were performed. For three of the protein-binding sites in the apoVLDL II promoter region, C/EBP alpha and C/EBP beta were identified as the major DNA-binding activities. For one of the C/EBP genes, C/EBP alpha, the effect of the gene products on apoVLDL II transcription was examined. From transfection experiments we conclude that maximal estrogen-dependent activity of the apoVLDL II promoter requires the dual action of the estrogen receptor and C/EBP. The level of activity is different depending on the nature of the C/EBP alpha translational isoform transfected, the full-length C/EBP alpha polypeptide being the most active isoform and the N-terminally truncated isoform being moderately active. The present results suggest a role of C/EBP alpha translational isoform ratio in the modulation of expression of C/EBP target genes, such as those involved in metabolic processes.

Animals↗
PubMed1997

The chicken CCAAT/enhancer binding protein alpha gene. Cloning, characterisation and tissue distribution.

We present the cloning and sequencing of the gene encoding the chicken CCAAT/Enhancer Binding Protein alpha (cC/EBP alpha). The coding region and 1.5 kb of 5' flanking DNA from a CpG island. Comparison of the chicken C/EBP alpha sequence to the homologous proteins of other species reveals several evolutionary conserved regions. cC/EBP alpha mRNA expression is restricted to a subset of tissues with high expression in liver, lung and small intestine. Recombinant cC/EBP alpha binds to its cognate C/EBP binding site as a homodimer or as a heterodimer with the related cC/EBP beta/NF-M.

Amino Acid Sequence↗
PubMed1996

Multiple steps in the regulation of transcription-factor level and activity.

This review focuses on the regulation of transcription factors, many of which are DNA-binding proteins that recognize cis-regulatory elements of target genes and are the most direct regulators of gene transcription. Transcription factors serve as integration centres of the different signal-transduction pathways affecting a given gene. It is obvious that the regulation of these regulators themselves is of crucial importance for differential gene expression during development and in terminally differentiated cells. Transcription factors can be regulated at two, principally different, levels, namely concentration and activity, each of which can be modulated in a variety of ways. The concentrations of transcription factors, as of intracellular proteins in general, may be regulated at any of the steps leading from DNA to protein, including transcription, RNA processing, mRNA degradation and translation. The activity of a transcription factor is often regulated by (de) phosphorylation, which may affect different functions, e.g. nuclear localization DNA binding and trans-activation. Ligand binding is another mode of transcription-factor activation. It is typical for the large super-family of nuclear hormone receptors. Heterodimerization between transcription factors adds another dimension to the regulatory diversity and signal integration. Finally, non-DNA-binding (accessory) factors may mediate a diverse range of functions, e.g. serving as a bridge between the transcription factor and the basal transcription machinery, stabilizing the DNA-binding complex or changing the specificity of the target sequence recognition. The present review presents an overview of different modes of transcription-factor regulation, each illustrated by typical examples.

Animals↗
PubMed1995

Cloning and characterisation of a nuclear, site specific ssDNA binding protein.

Estradiol inducible, liver-specific expression of the apoVLDL II gene is mediated through the estrogen receptor and a variety of other DNA-binding proteins. In the present study we report the cloning and characterisation of a single-strand DNA binding protein that interacts with the lower strand of a complex regulatory site, which includes the major estrogen responsive element and a site that resembles the rat albumin site D (apoVLDL II site D). Based on its binding specificity determined with electro-mobility shift assays, the protein is named single-strand D-box binding factor (ssDBF). Analysis of the deduced 302 amino acid sequence revealed that the protein belongs to the heteronuclear ribonucleoprotein A/B family (hnRNP A/B) and resembles other known eukaryotic single-strand DNA binding proteins. Transient transfection experiments in a chicken liver cell-line showed that the protein represses estrogen-induced transcription. A protein with similar binding characteristics is present in liver nuclear extract. The relevance of the occurrence of this protein to the expression of the apoVLDL II gene is discussed.

Amino Acid Sequence↗
PubMed1995

The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal.

Here we describe the identification of the targeting sequence of peroxisomal amine oxidase (AMO) of H. polymorpha. Deletion analysis revealed that essential targeting information is located within the extreme N-terminal 16 amino acids. Moreover, this sequence can direct a reporter protein to the peroxisomal matrix of H. polymorpha. The N-terminal 16 amino acids of AMO contain a sequence with strong homology to the conserved PTS2 sequence. Therefore, AMO is considered to be a PTS2 protein.

Amine Oxidase (Copper-Containing)↗
PubMed1995

Review: methylotrophic yeasts as factories for the production of foreign proteins.

In this contribution we discuss the potential of methylotrophic yeasts as hosts for the high level production of valuable foreign proteins. Recent relevant achievements on the intracellular production or secretion of proteins are summarized. Special attention is paid to a specific advantage of the use of methylotrophic yeasts, namely the possibility of accumulating the foreign gene products inside peroxisomes. This approach may be of major advantage when the protein product is toxic for the host cell and, also, to protect these proteins from undesired side-effects such as proteolysis or aggregation.

Amino Acid Sequence↗
PubMed1994

Translation start site multiplicity of the CCAAT/enhancer binding protein alpha mRNA is dictated by a small 5' open reading frame.

The CCAAT/enhancer binding proteins (C/EBP) alpha and beta of the bZIP family of transcription factors each occur as multiple forms due to translation initiation at different in-frame AUG codons from the same messenger RNA. The C/EBP alpha mRNAs of chicken, rat and Xenopus all contain a small 5' open reading frame (5'ORF) whose size (18 nucleotides) and distance (seven nucleotides) to the C/EBP alpha cistron has been conserved in vertebrate evolution. The present studies shows that the small 5'ORF is crucial to the leaky scanning mechanism of ribosomes causing a fraction of them to ignore the first C/EBP alpha AUG codon and to start at internal AUGs. Our data challenge the view that translational start site multiplicity is mainly governed by the sequence context of the potential initiation codons. Western analysis showed that the two major chicken C/EBP alpha translation products, the full-length cC/EBP alpha-42 which acts a trans-activator in liver and the N-terminally truncated cC/EBP alpha-29 which lacks transcription activation potential, occur in a fixed ratio which is similar in different expressing tissues, like liver, lung and small intestine. The presence of a similar, thusfar unnoticed, small ORF 5' to the major initiation codon of C/EBP beta mRNA suggests that start site multiplicity from this mRNA may be governed by the same mechanism.

Animals↗
PubMed1994

The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins).

Two main types of peroxisomal targeting signals have been identified that reside either at the extreme C terminus (PTS1) or the N terminus (PTS2) of the protein. In the methylotrophic yeast Hansenula polymorpha the majority of peroxisomal matrix proteins are of the PTS1 type. Thus far, for H. polymorpha only amine oxidase (AMO) has been shown to contain a PTS2 type signal. In the present study we expressed H. polymorpha AMO under control of the strong endogenous alcohol oxidase promoter. Partial import of AMO into peroxisomes was observed in cells grown in methanol/(NH4)2SO4-containing medium. However, complete import of AMO occurred if the cells were grown under conditions that induce expression of the endogenous AMO gene. Similar results were obtained when the heterologous PTS2 proteins, glyoxysomal malate dehydrogenase from watermelon and thiolase from Saccharomyces cerevisiae, were synthesized in H. polymorpha. The import of PTS1 proteins, however, was not affected by the growth conditions. These results indicate that the reduced rate of AMO import in (NH4)2SO4-grown cells is not due to competition with PTS1 proteins for the same import pathway. Apparently, AMO is imported via a separate pathway that is induced by amines and functions for PTS2 proteins in general.

Amine Oxidase (Copper-Containing)↗
PubMed1994

Binding of a bZip protein to the estrogen-inducible apoVLDL II promoter.

Activation of the very low density apolipoprotein II (apoVLDL II) gene in chicken liver by estrogen results in the binding of a variety of nuclear proteins including members of the steroid receptor superfamily and the bZip superfamily to the immediate 5' flanking region. In the present study, we have identified a bZip protein from chicken liver as one of the potential binding activities. Its cognate cDNA was cloned from an expression library using a recognition site DNA probe corresponding to part of the apoVLDL II promoter region. By footprinting and gel shift analysis with the recombinant protein from a prokaryotic expression system we have established that the protein binds to at least three different sites in the apoVLDLII promoter region. One of these sites partially overlaps with the major estrogen response element of the gene. Despite the proximity of their binding sites, the estrogen receptor and the bZip protein can bind simultaneously to the very region. Possible implications of this intimate arrangement of binding sites for the activation of the apoVLDL II promoter are discussed.

Animals↗
PubMed1994

Cis-acting elements reinforcing the activity of the estrogen-response element in the very-low-density apolipoprotein II gene promoter.

The gene coding for chicken very low density apolipoprotein II (apoVLDLII) is expressed exclusively in liver in response to estrogen. Previous work in our laboratory identified several protein binding sites, identified by the letters A to F, and their cognate factors within the first 300 bp flanking the gene. Here we present an extensive functional analysis of the apoVLDLII promoter by gene transfer experiments using a chicken hepatoma cell line and cultured non-hepatic cells. Deletion analysis revealed that the -301 to -163-bp promoter region, comprising elements E1, E2 and F, is sufficient for strong estrogen-dependent expression. Mutation analysis demonstrated that efficient transcription requires the interplay of the major estrogen response element E1 with several other cis-acting elements. Analysis of individual protein binding sites showed that element E1 is sufficient by itself to confer weak estrogen-induced transcription from the apoVLDLII promoter, and that additional promoter elements are required for full estrogen-responsiveness. Elements F and B1 were capable of strongly potentiating the activity of element E1. In general, the activity of certain cis-acting elements appeared to be strongly promoter-context dependent. Cultured non-liver cells expressed transfected VLDL-CAT reporter plasmids in the presence of cotransfected estrogen receptor expression vector in a hormone-dependent way, indicating that for the control of tissue specificity the 5'-proximal promoter region is not sufficient.

Animals↗
PubMed1994

9-cis-retinoic acid represses estrogen-induced expression of the very low density apolipoprotein II gene.

The chicken very low density apolipoprotein II (apoVLDLII) gene is estrogen-inducible and specifically expressed in liver. We examined the possible involvement of the retinoid X receptor (RXR) and its ligand 9-cis-retinoic acid (9-cis-RA) in the activation of the apoVLDLII promoter. We first concentrated on a potential RXR recognition site, which deviates at only one position from a perfect direct A/GGGTCA repeat spaced by one nucleotide (DR-1) and was earlier identified as a common HNF-4/COUP-TF recognition site. However, band shift analysis revealed that this imperfect DR-1 motif does not interact with RXR alpha-homodimers. In accordance with this observation we found that this regulatory element does not mediate transactivation through RXR alpha in the presence of 9-cis-RA. However, our experiments revealed another, unexpected, effect of 9-cis-RA. Instead of stimulating, 9-cis-RA attenuated estrogen-induced expression of transfected estrogen-responsive VLDL-CAT reporter plasmids. This repression appeared to take place through the main estrogen response element (ERE) of the gene. Importantly, 9-cis-RA also strongly repressed the estrogen-induced expression of the endogenous apoVLDLII gene in cultured chicken hepatoma cells.

Animals↗
PubMed1993

Estrogen-inducible and liver-specific expression of the chicken Very Low Density Apolipoprotein II gene locus in transgenic mice.

We have examined the chicken Very Low Density Apolipoprotein II (apoVLDL II) gene locus in transgenic mice. A DNA fragment composed of the transcribed region, 16 kb of 5' flanking and 400 bp of 3' flanking sequences contained all the information sufficient for estrogen-inducible, liver-specific expression of the apoVLDL II gene. The far-upstream region contains a Negative Regulating Element coinciding with a DNaseI-hypersensitive site at -11 kb. In transgenic mice, the NRE at -11 kb is used for downregulating the expression to a lower maximum level. The NRE might be used for modulating apoVLDL II gene expression, and may be involved in the rapid shut-down of the expression after hormone removal.

Animals↗
PubMed1993

Human catalase is imported and assembled in peroxisomes of Saccharomyces cerevisiae.

To study the conservation of peroxisomal targeting signals, we have determined the intracellular localization of human peroxisomal catalase when expressed in yeast. Using immunofluorescence, differential centrifugation and immunoelectron microscopy, we show that the protein is targeted to the peroxisomes of the heterologous cell and assembled in its active tetrameric form. These data show the conservation of the catalase targeting signal and import specificity between human and yeast peroxisomes.

Amino Acid Sequence↗
PubMed1993

Peroxisomal amine oxidase of Hansenula polymorpha does not require its SRL-containing C-terminal sequence for targeting.

Amine oxidase (AMO) is a peroxisomal matrix protein of Hansenula polymorpha, which is induced during growth of the yeast in media containing primary amines as a sole nitrogen source. The deduced amino acid sequence of the protein contains an SRL sequence at nine amino acids from the C-terminus. In this study, we have examined the possible role of the SRL motif in sorting of AMO to peroxisomes by mutating the corresponding gene sequence. For this purpose, we have developed a DNA construct that is specifically integrated into the AMO locus of the H. polymorpha genome, placing the mutant gene under the control of the endogenous AMO promoter and eliminating expression of the wild-type gene. Analysis of a stable transformant, containing the desired gene configuration, showed that mutation of the C-terminal sequence neither interfered with correct targeting of the protein into the peroxisome nor displayed significant effects on its activity. From this, it was concluded that the SRL-containing C-terminus is not essential for peroxisomal targeting of AMO in H. polymorpha.

Amine Oxidase (Copper-Containing)↗