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G Schütz

Publications and source records attributed to G Schütz.

At least 19 recordsLinked to original sources
PubMed1992

At least three promoters direct expression of the mouse glucocorticoid receptor gene.

We have characterized the gene for the mouse glucocorticoid receptor. The gene spans approximately 110 kilobases, and glucocorticoid receptor transcripts are assembled from nine exons. Expression of the gene is controlled by at least three promoters, resulting in glucocorticoid receptor transcripts with different 5' nontranslated exons. One promoter is cell-specific, found to be active only in T lymphocytes. The other two promoters are active to various degrees in all cell lines and tissues so far analyzed and are located in a CpG island. The promoter activities are accompanied by DNase I hypersensitivity sites in chromatin. In contrast to a conservation of exon-intron structure, differences in promoter organization suggest a divergence between the evolution of regulatory and coding regions among members of the steroid receptor super-family.

Animals↗
PubMed1992

Transgenic mice generated by pronuclear injection of a yeast artificial chromosome.

Transgenic mice have become invaluable for analysing gene function and regulation in vivo. However, the size of constructs injected has been limited by the cloning capacity of conventional vectors, a constraint that could be overcome with yeast artificial chromosomes (YACs). We investigated the feasibility of making transgenic mice with YACs by pronuclear injection of a small YAC carrying a gene encoding tyrosinase. Use of a vector with a conditional centromere allowed fifteenfold amplification of the YAC in yeast and its recovery in high yield. The albino phenotype of the recipient mice was rescued demonstrating the correct expression of the tyrosine gene from the construct. Furthermore, the telomeric sequences added by the yeast integrated into the mouse genome and did not reduce efficiency of integration. Using this technique future experiments with longer YACs will allow the expression of gene complexes such as Hox and the globin gene clusters to be analysed in transgenic animals.

Animals↗
PubMed1992

Expression of the mouse tyrosinase gene during embryonic development: recapitulation of the temporal regulation in transgenic mice.

Pigment in mammals is produced in melanocytes of the skin derived from the neural crest and in the pigmented epithelial cells of the retina derived from the optic cup. Tyrosinase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1) is regarded as the key enzyme in pigment synthesis in both these cell types. In this study, we have investigated the temporal regulation of expression of the tyrosinase gene during early eye development and in the developing hair follicle of the mouse by in situ hybridization and have asked whether transgenes would precisely mimick this pattern. We show that the mouse tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression in melanocytes is detected from day 16.5 onwards. This cell type-specific and temporal expression is largely reproduced in transgenic mice carrying a tyrosinase minigene. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell-type specificity and developmental regulation in melanocytes and in the pigment epithelium.

Animals↗
PubMed1992

The rat poly pyrimidine tract binding protein (PTB) interacts with a single-stranded DNA motif in a liver-specific enhancer.

We characterized and purified a protein from rat liver which specifically binds to a DNA motif present in a liver-specific enhancer of the rat tyrosine aminotransferase (TAT) gene, when offered as single-stranded DNA. Binding is highly sequence-specific and coincides with a region known to be essential for function of the enhancer. Microsequencing revealed that this protein is the rat homologue of the mouse and human poly Pyrimidine Tract binding protein (PTB), which has been shown to bind to premRNA and may participate in RNA splicing. This finding was corroborated by subsequent Western blot experiments using a PTB-specific antibody. These findings indicate a possible dual role for this protein in RNA processing and transcription.

Amino Acid Sequence↗
PubMed1992

Multiple mRNA isoforms of the transcription activator protein CREB: generation by alternative splicing and specific expression in primary spermatocytes.

We have characterized cDNA clones representing mouse CREB (cyclic AMP responsive element binding protein) mRNA isoforms. These include CREB delta and CREB alpha, of which the rat and human homologues have been previously identified. Both encode proteins with CRE-binding activity and identical transactivation potential. The additional CREB mRNA isoforms potentially encode CREB related proteins. From the structural organization of the mouse CREB gene we conclude that the multiple transcripts are generated by alternative splicing. Furthermore we show that specific CREB mRNA isoforms are expressed at a high level in the adult testis. Expression of these isoforms is induced after commencement of spermatogenesis. In situ hybridization suggests that this expression occurs predominantly in the primary spermatocytes. Comparison of the CREB gene with the recently isolated CREM (cAMP responsive element modulator) cDNAs illustrates that the two genes have arisen by gene duplication and have diverged to encode transcriptional activators and repressors of the cAMP signal transduction pathway.

Amino Acid Sequence↗
PubMed1992

Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation.

Amino Acid Sequence↗
PubMed1992

The mouse CREB (cAMP responsive element binding protein) gene: structure, promoter analysis, and chromosomal localization.

In this paper we report the isolation and characterization of the mouse CREB gene. It is composed of 11 exons and 10 introns and spans a region of 70 kb. BR-A and BR-B, the two alpha-helical regions of the proposed basic DNA binding domain of CREB, are encoded separately on exons 10 and 11. The mouse CREB gene is expressed from a promoter that is situated in a CpG island. The promoter contains no TATA or CCAAT box homologies but has a number of putative binding sites for the acidic transcriptional activator Sp1 and a 9/11 match with the initiator region. Transcriptional start site mapping identified five major start sites spread over at least 41 nucleotides. Northern blot analysis indicated that expression of the CREB gene is almost ubiquitous with expression at differing levels of multiple transcripts. Testis expressed a predominant RNA species of approximately 1.6 kb. The CREB gene was found to be single copy in the mouse and well conserved through evolution. Finally Creb-1, the CREB locus, was mapped to the proximal region of mouse chromosome 1.

Amino Acid Sequence↗
PubMed1992

Physical mapping of the albino-deletion complex in the mouse to localize alf/hsdr-1, a locus required for neonatal survival.

The albino-deletion complex in the mouse defines a genetically well-characterized region of chromosome 7 in which a number of loci essential for normal development and viability reside. One locus, designated alf or hsdr-1, is necessary for neonatal survival. Its absence results in hypoglycemia associated with biochemical and ultrastructural abnormalities in hepatocytes and proximal tubule cells of the kidney. We constructed a long-range physical map of the region defined by the proximal segment of the albino-deletion complex as a step toward localizing alf/hsdr-1. Sixteen markers, including 11 whose isolation is described here and in the accompanying paper (A. Schedl et al., 1992, Genomics 14, 288-297), were ordered on a panel of albino-deletion DNAs and their distribution was examined by pulsed-field gel electrophoresis. The resulting approximately 4300-kb physical map covers the entire region absent from the prototypic alf/hsdr-1 deletion c14CoS, estimated as approximately 3600 kb. Since the deletion c11DSD complements and overlaps most of c14CoS, alf/hsdr-1 was mapped at the proximal extreme of c14CoS, approximately 3000 kb from the albino locus. The density of CpG islands was found to be very heterogeneous across the region mapped.

Albinism↗
PubMed1992

Chromosome jumping from flanking markers defines the minimal region for alf/hsdr-1 within the albino-deletion complex.

The locus alf/hsdr-1, defined by the albino-deletion complex on mouse chromosome 7, is essential for neonatal survival. Animals homozygous for a subset of the deletions die shortly after birth due to impaired gene expression in liver parenchymal cells and kidney proximal tubular cells. Here, we describe a detailed analysis of the region containing alf/hsdr-1 by means of chromosome jumping from flanking markers. Three chromosome jumping libraries based on the restriction enzymes XmaI and SalI were constructed. Isolation of eight jumping clones distributed over 450 kb allowed more than 240 kb to be cloned in genomic lambda and cosmid libraries. Five of the probes map within the minimal genetic interval for alf/hsdr-1, which is defined by the proximal borders of the deletions c10R75M and c11DSD. The breakpoints of these deletions were precisely mapped, which allowed alf/hsdr-1 to be localized to a 310-kb interval.

Albinism↗
PubMed1992

Deficiency of an enzyme of tyrosine metabolism underlies altered gene expression in newborn liver of lethal albino mice.

Mice homozygous for albino deletions encompassing the locus alf/hsdr-1 die shortly after birth. Lethality is thought to be the consequence of hypoglycemia, which results from the failure to activate hormone-dependent genes in liver and kidney encoding enzymes important for gluconeogenesis. Within the region in which alf/hsdr-1 has been defined by physical mapping, we identified the gene encoding fumarylacetoacetate hydrolase (FAH), an enzyme of tyrosine metabolism. Lack of FAH activity should lead to accumulation of toxic tyrosine metabolites. In man, genetically determined FAH deficiency is the primary defect in tyrosinemia type I, a fatal liver disease of infants. Northern blot and in situ hybridization analysis of mouse tissues showed that the cell types that normally express FAH correspond to those that exhibit a phenotype in alf/hsdr-1 deletion mice. Moreover, we could mimic aspects of the alf/hsdr-1 deletion phenotype in vitro by treating primary hepatocyte cultures with an intermediate of tyrosine metabolism. These findings strongly suggest that alf/hsdr-1 encodes FAH and that absence of FAH is responsible for neonatal lethality in albino deletion mice. Mechanisms by which this metabolic defect might bring about alterations in gene expression characteristic of the alf/hsdr-1 deletion phenotype are discussed.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of the mouse tyrosinase gene: pigment cell-specific expression in transgenic mice.

Tyrosinase is the key enzyme in melanin synthesis, and is expressed in the pigment epithelium of the retina, a cell layer derived from the optic cup; and in neural crest-derived melanocytes of skin, hair follicle, choroid, and iris. The tyrosinase gene has been cloned and shown to map to the well-characterized c-locus (albino locus) of the mouse. Subsequent studies demonstrated that a functional tyrosinase minigene was able to rescue the albino phenotype in transgenic mice. The transgene was expressed in a cell type-specific manner in skin and eye. During development of the mouse, the tyrosinase gene is expressed in the pigment epithelium of the retina as early as day 10.5 of gestation. In the hair follicle, tyrosinase gene expression is detected from day 16.5 onwards. This cell-type-specific expression is largely reproduced in transgenic mice. Our results suggest that sequences in the immediate vicinity of the mouse tyrosinase gene are sufficient to provide cell type-specificity and developmental regulation in melanocytes and the pigment epithelium.

Albinism↗
PubMed1992

Multiple effects on liver-specific gene expression in albino lethal mice caused by deficiency of an enzyme in tyrosine metabolism.

alf/hsdr-1 is a locus in the mouse defined by albino deletions to be essential for neonatal viability. Homozygous deletion of alf/hsdr-1 leads to a pleiotropic phenotype in liver and kidney, including impaired perinatal activation of hormone-dependent genes, and the induction of detoxifying enzymes and early-response genes. To elucidate the molecular basis of this complex phenotype, we have identified the gene mapping at alf/hsdr-1 by positional cloning, using overlapping albino locus deletions to define the location of alf/hsdr-1. The gene encodes fumarylacetoacetate hydrolase, FAH, an enzyme of tyrosine metabolism. Genetically determined FAH deficiency in man leads to a severe liver failure in infants. In mice, we find that the normal sites of expression of FAH correlate tightly with cell-types which display abnormalities in albino lethal mice. The identification of the Fah gene as a candidate for alf/hsdr-1 offers a novel explanation for the complex phenotype, one into which all aspects can be accommodated. The phenotype can now be understood as a sequence of responses to toxic electrophilic metabolites.

Animals↗
PubMed1991

The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-dependent protein kinase.

The tissue-specific extinguisher locus TSE1, a dominant negative regulator of transcription in somatic cell hybrids, acts via a cAMP response element (CRE) to repress activity of a hepatocyte-specific enhancer. Guided by the antagonism between TSE1 and cAMP-mediated signal transduction, we identified the regulatory subunit RI alpha of protein kinase A (PKA) as the product of the TSE1 locus. The evidence derives from concordant expression of RI alpha mRNA and TSE1 genetic activity, high resolution mapping of the RI alpha gene and TSE1 on human chromosome 17, and the ability of a transfected RI alpha cDNA to generate a phenocopy of TSE1-mediated extinction. The mechanism of TSE1/RI alpha-mediated extinction involves repression of basal PKA activity, reduced phosphorylation of CREB at Ser-133, and a corresponding reduction of in vivo protein binding at the target CRE.

Animals↗
PubMed1991

The mouse tyrosinase promoter is sufficient for expression in melanocytes and in the pigmented epithelium of the retina.

The mouse c locus encodes tyrosinase (monophenol monooxygenase; monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1), the key enzyme in melanin synthesis, which is expressed in the pigment epithelium of the retina and in melanocytes derived from the neural crest. To define regulatory regions of the gene that are important for cell type-specific expression, a deletion series of the tyrosinase 5' region was fused to a chloramphenicol acetyltransferase (CAT) reporter gene and electroporated into tyrosinase-expressing and -nonexpressing cell lines. We show that 270 base pairs 5' of the transcriptional start site is sufficient for CAT expression in a human and a mouse melanoma cell line. This 5' flanking fragment, when cloned in the context of a tyrosinase minigene construct and injected into fertilized eggs of an albino mouse strain, is sufficient for cell type-specific expression in mice. The transgenic mice were pigmented in both skin and eyes. In situ hybridization analysis shows that the 270-base-pair regulatory region contains elements sufficient for specific expression of the transgene both in the pigmented epithelial cells of the retina, which are derived from the optic cup, and in neural crest-derived melanocytes.

Animals↗
PubMed1991

Analysis of CpG methylation and genomic footprinting at the tyrosine aminotransferase gene: DNA methylation alone is not sufficient to prevent protein binding in vivo.

Specific DNA sequences from several DNase I hypersensitive sites located upstream of the tyrosine aminotransferase (TAT) gene are bound by ubiquitous nuclear factors in vitro. Genomic footprinting has shown, however, that proteins are excluded from their potential binding sites in cells where the gene is inactive and that the absence of in vivo footprints is correlated with CpG methylation and altered chromatin structures at these sites. In vitro, interactions of proteins with sequences of the TAT gene, including binding of the transcription factor CREB to the cAMP-responsive element (CRE), are prevented by a methylated CpG dinucleotide in the respective binding sites, suggesting that methylation of DNA might be sufficient to exclude proteins from their sites in vivo. To test directly whether the absence of in vivo footprints is the result of DNA methylation, we treated two different cell lines with 5-azacytidine to demethylate CpG dinucleotides. While genomic sequencing confirmed demethylation at two widely separated regions upstream of the TAT promoter, no footprints appeared in these cell lines, even though proteins capable of binding these sites in vitro were present in the nuclei. Thus, the simple model whereby protein exclusion in vivo is caused solely by DNA methylation is not appropriate in this case. The nucleosomal organization of the potential binding sites suggests that chromatin structure is a dominant determinant in maintaining the inactive state of these sites.

Azacitidine↗