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A F Stewart

Publications and source records attributed to A F Stewart.

At least 91 records · Page 5Linked to original sources
PubMed1995

The chromo shadow domain, a second chromo domain in heterochromatin-binding protein 1, HP1.

The chromo domain was originally identified as a protein sequence motif common to the Drosophila chromatin proteins, Polycomb (Pc) and heterochromatin protein 1 [HP1; Paro and Hogness (1991) Proc. Natl. Acad. Sci. USA, 88, 263-267; Paro (1990) Trends Genet., 6, 416-421]. Here we describe a second chromo domain-like motif in HP1. Subsequent refined searches identified further examples of this chromo domain variant which all occur in proteins that also have an N-terminally located chromo domain. Due to its relatedness to the chromo domain, and its occurrence in proteins that also have a classical chromo domain, we call the variant the 'chromo shadow domain'. Chromo domain-containing proteins can therefore be divided into two classes depending on the presence, for example in HP1, or absence, for example in Pc, of the chromo shadow domain. We have also found examples of proteins which have two classical chromo domains. The Schizosaccharomyces pombe SWI6 protein, involved in repression of the silent mating-type loci, is a member of the chromo shadow group. The similar modular architecture of SpSW16, HP1 and HP1-like proteins supports the model that the specificity of action of chromatin proteins is generated by combinations of protein modules.

Amino Acid Sequence↗
PubMed1995

Ligand-regulated site-specific recombination.

Site-specific recombination offers a potential way to alter a living genome by design in a precise and stable manner. This potential requires strategies which can be used to regulate the recombination event. We describe a strategy to regulate FLP recombinase activity which relies on expressing FLP as a fusion protein with steroid hormone receptor ligand binding domains (LBDs). In the absence of a ligand cognate to the LBD, the recombinase activity of the fusion protein is extremely low. Upon ligand administration, recombinase activity is rapidly induced. These results outline the basis for inducible expression or disruption strategies based on inducible recombination. Additionally, we have exploited the conditional nature of FLP-LBD fusion proteins to direct integration of a plasmid into a specific genomic site at frequencies approaching the frequency of random integration.

Animals↗
PubMed1995

Cell type-specific secretion of parathyroid hormone-related protein via the regulated versus the constitutive secretory pathway.

Parathyroid hormone-related protein (PTHrP) is endoproteolytically processed to yield a family of mature secretory forms. These include an amino-terminal, a mid-region, and a carboxyl-terminal form. Prior studies suggested that the mid-region form is secreted via the regulated secretory pathway, whereas the amino- and carboxyl-terminal forms are secreted via the constitutive pathway. Further, PTHrP is unusual in that it is produced under normal circumstances by neuroendocrine cell types as well as by prototypical constitutively secreting cell types. The potential for cell-specific secretory pathway use by PTHrP has not been explored. Using immunohistochemical and perifusion techniques, we demonstrate that all three PTHrP daughter peptides are secreted via the regulated pathway in neuroendocrine cells. In contrast, all three daughter peptides are secreted in a constitutive fashion by non-neuroendocrine cells. Thus, the secretion of PTHrP is unique in that it appears to be cell-specific. When it is expressed in neuroendocrine cells that contain the regulated pathway, it is secreted in a regulated fashion; when it is expressed in non-neuroendocrine cells, it defaults to the constitutive pathway. This phenomenon has not previously been described for a polypeptide hormone in naturally occurring cells.

Animals↗
PubMed1994

Distribution of alpha 1C-adrenergic receptor mRNA in adult rat tissues by RNase protection assay and comparison with alpha 1B and alpha 1D.

Two alpha 1-adrenergic receptor (AR) subtypes have been defined by pharmacological studies in rat tissues, the alpha 1A and the alpha 1B, whereas three alpha 1-ARs have been cloned, alpha 1B, alpha 1C, and alpha 1D. It has been reported that alpha 1C mRNA is absent in all rat tissues, making uncertain the correspondence of this cloned subtype, if any, to the native alpha 1-ARs defined by pharmacological criteria. In the present study, a partial alpha 1C-AR cDNA was obtained from rat cardiac myocytes using RT-PCR with degenerate primers. A sensitive RNase protection assay was used to map the distribution of alpha 1C mRNA in adult rat tissues, in comparison with alpha 1B and alpha 1D. alpha 1C mRNA was abundant in heart, brain, aorta, vena cava, vas deferens, submaxillary gland, lung, and kidney; was detected at lower levels in prostate, parotid gland, and skeletal muscle; and was undetectable in liver and spleen. alpha 1B and alpha 1D mRNAs were present in most of the same tissues. In contrast to alpha 1C, however, alpha 1B and alpha 1D were both present in spleen; alpha 1B was the sole alpha 1-AR mRNA in liver; and alpha 1D mRNA was not detected in submaxillary gland, a tissue known to be enriched in the pharmacological alpha 1A. We conclude that the distribution of alpha 1C-AR mRNA in rat tissues is compatible with the idea that the alpha 1C corresponds to the classical native alpha 1A-AR. Although many tissues contain all three alpha 1-AR mRNAs, distinct tissue-specific expression is evident.

Amino Acid Sequence↗
PubMed1994

Muscle-enriched TEF-1 isoforms bind M-CAT elements from muscle-specific promoters and differentially activate transcription.

M-CAT elements mediate cardiac- and embryonic skeletal muscle-specific expression of the cardiac troponin T gene and a number of other cardiac-specific genes. M-CAT binding factor was shown to be related to cloned human TEF-1, a transcriptional regulator of the SV40 viral enhancer. Here we describe the cloning of TEF-1 from chick heart and the identification of several novel isoforms. We show that TEF-1 mRNA is considerably enriched in cardiac and skeletal muscle, consistent with a proposed role in muscle gene transcription. The predominant TEF-1 isoforms, TEF-1A and a novel isoform TEF-1B, bind M-CAT elements with high affinity and in a sequence-specific manner. We further demonstrate that the C-terminal portion of TEF-1B, which contains the 13-amino acid exon that distinguishes this isoform, can activate transcription when linked to a heterologous DNA binding domain, while the same domain of TEF-1A cannot. Therefore, isoforms of TEF-1 may play different roles in the regulation of M-CAT-dependent promoters in striated muscle cells.

Amino Acid Sequence↗
PubMed1994

Cloning of the rat alpha 1C-adrenergic receptor from cardiac myocytes. alpha 1C, alpha 1B, and alpha 1D mRNAs are present in cardiac myocytes but not in cardiac fibroblasts.

alpha 1-Adrenergic receptor (AR) activation in cardiac muscle has several different physiological effects that might be mediated through different alpha 1-AR subtypes. Two alpha 1-AR subtypes have been cloned from the rat, the alpha 1B and the alpha 1D; both are present in adult rat heart. A third subtype, the alpha 1C, cloned from the cow and human, was reported to be absent in the rat. However, we recently found alpha 1C mRNA in adult rat heart by using a partial alpha 1C cDNA. Thus, all three cloned alpha 1-AR subtypes are present in the heart, but it is unknown whether each is expressed in cardiac myocytes or in cardiac fibroblasts. In the present study, the full-length rat alpha 1C-AR was cloned from cultured neonatal cardiac myocytes. alpha 1C mRNA transcripts of 3, 9.5, and 11 kb were present in adult rat heart by Northern blot analysis. alpha 1B-, alpha 1C-, and alpha 1D-subtype mRNAs were each present in isolated adult and neonatal cardiac myocytes by RNase protection assay. In addition, cultured neonatal cardiac myocytes expressed the three alpha 1-AR subtype mRNAs. In contrast, none of the alpha 1-AR mRNAs was detected in cultured neonatal cardiac fibroblasts. In addition, alpha 1-ARs were absent in fibroblasts by [3H]prazosin binding and norepinephrine-stimulated [3H]inositol phosphate production. The absence of alpha 1-ARs in cardiac fibroblasts differs from beta-adrenergic and angiotensin II receptors, which are present in both cardiac fibroblasts and cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Parathyroid hormone-related protein as a prohormone: posttranslational processing and receptor interactions.

Since the elucidation of the structures of the three human PRHrP isoforms in 1987, information has rapidly accured which indicates that the role of PTHrP in normal physiology will prove to be crucial as well as exceedingly complex. The importance of the role of PTHrP in normal physiology is underscored by its broad tissue expression, by its intense evolutionary conservation, by its extremely early expression after fertilization of the ovum, and by the lethal consequences of PTHrP gene disruption. The complexity of the role of PTHrP in normal physiology increases almost monthly. This complexity is reflected in the broad tissue distribution of the peptide, its complex transcriptional regulation and mRNA instability motifs, and its multiple transcripts and isoforms. It is now clear that additional complexity exists at the level of posttranslational processing. Expression of the PTHrP gene leads to the tissue-specific processing and secretion of an increasingly complex family of derivative peptides, each with its own repertoire of cognate receptors, signal transduction pathways, and physiological consequences. Further elucidation of the posttranslational processing pathways and mechanisms can be anticipated in the coming years, coupled with a corresponding elucidation of multiple PTHrP receptors, their specific signal transduction pathways, and their unique physiological roles. The role of PTHrP in causing HHM is now clearly established. Work in the coming decade will focus on the normal physiological roles played by PTHrP.

Amino Acid Sequence↗
PubMed1994

Cross-talk modulation of signal transduction pathways: two mechanisms are involved in the control of tyrosine aminotransferase gene expression by phorbol esters.

Transcription of the rat tyrosine aminotransferase gene (TAT) is stimulated in liver by glucocorticoid hormones or by cAMP-increased protein kinase A activity via enhancers located 2.5 kilobases (kb) and 3.6 kb upstream of the start site of transcription. The proteins mediating induction have been characterized, and protein binding in the two enhancer regions has been analyzed in vivo and in vitro. The TAT gene is therefore a useful model system with which to study cross-talk between different signal transduction pathways. We find that activation of the second messenger pathway leading from protein kinase C to the transcription factor AP-1 by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) impairs induction of the TAT gene both by glucocorticoid hormones and cAMP. The effects of TPA treatment on chromatin structure of the TAT gene and protein-DNA interactions in vivo were assayed. Under conditions in which TPA impairs glucocorticoid induction of TAT mRNA, the glucocorticoid receptor and other proteins binding within the glucocorticoid-inducible enhancer occupy their binding sites, indicating that inhibition occurs at a later step necessary for transcriptional stimulation. On the other hand, inhibition of cAMP induction correlates with reduced occupancy of the cAMP response element in vivo.

Animals↗
PubMed1993

Evolution of endothermy in fish: mapping physiological traits on a molecular phylogeny.

Mackerels, tunas, and billfishes (suborder Scombroidei and Teleostei) provide an ideal taxonomic context in which to examine the evolution of endothermy. Multiple origins and diverse strategies for endothermy exist among these fish. Here a molecular phylogeny of the Scombroidei has been determined by direct sequencing of a portion of the mitochondrial cytochrome b gene. The distribution of endothermic species within this proposed genealogy indicates that the ability to warm the brain and retina arose independently in three lineages, each time in association with a movement into colder water. This suggests that the evolution of cranial endothermy in fish was selected in order to permit thermal niche expansion and not selected for increased aerobic capacity.

Animals↗
PubMed1993

Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle.

Ecdysone in Drosophila has been a paradigm for steroid hormones since its ability to induce gene activity directly was demonstrated by its effects on moulting and polytene chromosome puffing. The ecdysone receptor (EcR) was recently confirmed as a member of the nuclear receptor superfamily by cloning and characterization in a Drosophila cell line. Here we show that EcR needs to heterodimerize with either the retinoid X receptor (RXR) or its Drosophila homologue, ultraspiracle (USP), for DNA binding and transactivation. These results place the ecdysone receptor in the heterodimerizing class of the nuclear receptor superfamily and demonstrate that the role of RXR/USP as a central and promiscuous partner in mediating the activity of these receptors is highly conserved. Whereas EcR-USP DNA-binding activity is unaffected by hormone, EcR-RXR DNA-binding activity is stimulated by either ecdysteroid or 9-cis-retinoic acid, demonstrating that hormone can play a role in heterodimer stabilization.

Animals↗