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PubMed1996

Promoter elements and second messenger pathways involved in transcriptional activation of tyrosine hydroxylase by ionomycin.

Membrane depolarization, or agents which increase intracellular calcium, elicit transcriptional activation of tyrosine hydroxylase (TH). In this study we analyze the factors involved in the regulation of the TH promoter by a calcium ionophore. PC12 cells were transiently transfected with plasmids containing wild type or mutated 5' flanking sequences of the rat TH gene, fused to bacterial chloramphenicol acetyl transferase (CAT). Point mutations introduced into the consensus cAMP-regulatory element (CRE) abolished the induction of CAT by ionomycin indicating that it is essential for mediating the calcium response. An intact and functional AP1 site did not confer calcium inducibility when the CRE/CaRE sequence was mutated. The extent and kinetics of the increase in intracellular calcium as well as the induction of CAT activity under the control of TH promoter by ionomycin were similar in PC12 cells and in the A123.7, protein kinase A (PKA) deficient cell line. In both cell lines addition of ionomycin rapidly increased the phosphorylation of transcription factor CREB at Ser-133. These results suggest that the activation of TH transcription by ionomycin does not require PKA. However, KN62 an inhibitor of Ca2+/calmodulin dependent (CaM) kinases prevented the induction indicating possible involvement of CAM kinases in the calcium response.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2002

The outermost lysine in the S4 of domain III contributes little to the gating charge in sodium channels.

We investigated the contribution the four outermost basic residues (K1, R2, R3, R4) in segment 4 of domain III in the human cardiac Na channel (hH1a, Na(V)1.5) to the total gating charge (Q(max)). Each of the four basic residues were mutated individually to a cysteine. In addition, R2 was also mutated to a glutamate. All mutant channels were transiently expressed with the alpha1 subunit in fused tsA201 cells. We used the relative reduction in Q(max) caused by anthopleurin-A (ApA) toxin, a site-3 toxin known to inhibit the movement of gating charge associated with domain IV, to estimate the size of the contribution from each basic residue. Studies of the toxin's ability to inhibit gating charge in mutant channels showed that R2 contributed 19-20% to the Q(max), R3 contributed 10%, and K1 and R4 made almost no contribution. In contrast to the outermost basic residue in the S4 of Shaker K channels and in the S4 of domain IV in hH1a, the outermost charge (K1) in domain III of Na channels is outside the voltage field.

Amino Acid Sequence↗
PubMed1997

Ubiquitous transcription factors NF1 and Sp1 are involved in the androgen activation of the mouse vas deferens protein promoter.

Transcription of the mouse vas deferens protein (MVDP) gene is stimulated by androgens and we have previously shown that in a 162 bp fragment, located at position -121 to +41, a TGAAGTtccTGTTCT sequence functions as an androgen-dependent enhancer. To determine which factors are involved in the hormonally regulated MVDP gene transcription, we have used DNase I footprinting and band-shift assays to examine in vitro binding of proteins to the enhancer and promoter sequences and have determined the functional significance of the recognized DNA sequences in transient transfection assays. Studies using recombinant proteins such as the DNA binding domain of the androgen receptor (AR-DBD) and Sp1, and crude cellular extracts from T47D and vas deferens epithelial cells (VDEC) showed that in addition to AR-DBD, the transcriptional activators NF1 and Sp1 interact with the -121/+41 fragment in a specific manner. Transient transfection assays revealed that site-directed mutations in the NF1 and Sp1 binding elements strongly reduced (NF1) or abolished (Sp1) androgen induced expression. The results demonstrate that the -121/+41 sequence is a composite site for the androgen receptor mediated transactivation of the MVDP gene.

Aldehyde Reductase↗
PubMed1997

Androgen induction of the SVS family related protein MSVSP99: identification of a functional androgen response element.

The gene encoding MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids) is specifically expressed in the mouse seminal vesicle under androgenic control. To study hormonal regulation, fragments of the 5'-flanking region, extending from -2365 to +16 were linked to the chloramphenicol acetyl transferase (CAT) gene and cotransfected with an androgen receptor expression vector into CV-1 cells. A minimal region (-387 to +16) was sufficient for full androgen induction. Further deletion, up to nt-261, almost completely abolished androgen inducibility. DNase I footprinting and band-shift assays, using the DNA binding domain of the androgen receptor (AR-DBD), revealed three AR binding sites: two putative androgen response elements (AREs) occurring at positions -361 (AREd) and -208 (AREp), and an androgen receptor binding region (ARBR) located between positions -317 and -293. Transient transfection assays revealed that site-directed mutation in AREp abolished androgen induced expression, whereas mutation in AREd or in ARBR had no effect. The results demonstrate that AREp is a functional sequence that must cooperate with additional cis-acting elements, located between -387 and -261, for androgen induction of the MSVSP99 gene.

Animals↗
PubMed1998

Estrogen receptor does not directly regulate the murine Muc-1 promoter.

Muc-1 is a heavily O-glycosylated, type 1 membrane glycoprotein present on the surface of polarized secretory uterine epithelial cells. Previous studies have shown that treatment of ovariectomized mice with 17-beta-estradiol (E2) strongly induces Muc-1 mRNA expression in an estrogen receptor (ER)-mediated fashion in the uterus. In this study, the 5.4 kb Muc-1 gene promoter has been isolated from a mouse genomic library and the proximal 1.85 kb region has been sequenced. Sequence analysis revealed the presence of one potential full estrogen response element (ERE) (GCTCGCGGTGACC) located at -748 to -735 bp in the Muc-1 promoter and several potential ERE half sites. Electrophoretic mobility shift assays (EMSA) showed that neither ERalpha nor ERbeta bind efficiently to this sequence. Transient cotransfection assays using constructs containing various deletion mutations of the 5' Muc-1 flanking sequences showed that E2 had no direct stimulation on promoter-driven reporter in NMuMG cells or primary mouse uterine epithelial cells, but did stimulate a consensus ERE CAT-reporter gene activity. In addition, E2-treatment of Weg-ER cells, a mouse uterine epithelial cell line stably expressing human ERalpha, did not restore endogenous Muc-1 expression or activate Muc-1 promoter-driven CAT activity. These results indicate that regions of the Muc-1 gene promoter within -1838 to +43 bp do not respond to E2 and ER stimulation and that ER alone is not sufficient to restore Muc1 gene expression. Deletion analyses also revealed that the sequence between -73 and +43 bp of the Muc-1 promoter is the minimal promoter region required for maximal Muc-1 promoter activity. Collectively, these results demonstrate that ER does not directly regulate the 1.85 kb murine Muc-1 gene promoter. Therefore, E2 control of uterine Muc-1 gene expression is likely to be indirect, i.e. mediated by stromal cell-derived factors.

Animals↗
PubMed2002

Huntington's disease transgenic mice are resistant to global cerebral ischemia.

Excitotoxicity plays a key role in ischemic neuronal death and is also one of the candidate mechanisms contributing to neurodegeneration in Huntington's disease (HD). Unexpectedly we have now found that transgenic mice expressing exon 1 of a mutant human HD gene (R6/1) are protected against global cerebral ischemia (GCI), installed by temporary bilateral occlusion of the carotid arteries. Whereas wild type mice showed a substantial neuronal damage in the hippocampus following 15, 20 and 60 min of GCI, transgenic mice were partially protected after 15 and 20 minutes of hypoxemia. This tolerance to ischemia is not blocked by pretreatment of mice with cycloheximide, an unspecific protein synthesis inhibitor. We conclude that this form of tolerance to ischemia in HD transgenic mice--although somewhat reminiscent of ischemic tolerance after ischemic preconditioning--is therefore independent of short term expression of endogenous neuroprotective proteins.

Animals↗
PubMed1997

In vivo analysis of the drosophila light-sensitive channels, TRP and TRPL.

We have tested the proposal that the light-sensitive conductance in Drosophila is composed of two independent components by comparing the wild-type conductance with that in mutants lacking one or the other of the putative light-sensitive channel subunits, TRP and TRPL. For a wide range of cations, ionic permeability ratios in wild type were always intermediate between those of trp and trpl mutants. Effective channel conductances derived by noise analysis in wild type were again intermediate (17 pS; c.f. 35 pS in trp and 4 pS in trpl) and also showed a complex voltage dependence, which was quantitatively explained by the summation of TRPL and TRP channels after taking their different reversal potentials into account. Although La3+ partially blocked the light response in wild-type photoreceptors, it increased the effective single channel conductance. The results indicate that the wild-type light-activated conductance is composed of two separate channels, with the properties of TRP- and TRPL-dependent channels as determined in the respective mutants.

Animals↗
PubMed2003

Regulation of CDPK isoforms during tuber development.

CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.

Amino Acid Sequence↗
PubMed1997

Genetic interaction between PARP and DNA-PK in V(D)J recombination and tumorigenesis.

Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are DNA break-activated molecules, Although mice that lack PARP display no gross phenotype and normal DNA excision repair, they exhibit high levels of sister chromatid exchange, indicative of elevated recombination rates. Mutation of the gene for DNA-PK catalytic subunit (Prkdc) cases defective antigen receptor V(D)J recombination and arrests B- and T-lymphocyte development in severe combined immune-deficiency (SCID) mice. SCID V(D)J recombination can be partly rescued in T-lymphocytes by either DNA-damaging agents (gamma-irradiation and bieomycin) or a null mutation of the p53 gene, possibly because of transiently elevated DNA repair activity in response to DNA damage or to delayed apoptosis in the absence of p53. To determine whether the increased chromosomal recombination observed in PARP-deficient cells affects SCID V(D)J recombination, we generated mice lacking both PARP and DNA-PK. Here, we show that thymocytes of SCID mice express both CD4 and CD8 co-receptors, bypassing the SCID block. Double-mutant T-cells in the periphery express TCR beta, which is attributable to productive TCR beta joints. Double-mutant mice develop a high frequency of T-cell lymphoma. These results demonstrate that increased recombination activity after the loss of PARP anti-recombinogenic function can rescue V(D)J recombination in SCID mice and indicate that PARP and DNA-PK cooperate to minimize genomic damage caused by DNA strand breaks.

Animals↗
PubMed1998

The antibiotic rifampicin is a nonsteroidal ligand and activator of the human glucocorticoid receptor.

The glucocorticoid receptor (GR) belongs to a superfamily of ligand-regulated nuclear steroid hormone receptors. The steps in the signal transduction pathway leading to the biological effects of glucocorticoids (GCs) include sequentially binding of the steroid to the GR ligand binding domain (LBD), receptor transformation, nuclear translocation and either positive or negative gene transactivation. Rifampicin (RIF) is a macrocyclic antibiotic used as an antituberculosis agents. As the incidence of tuberculosis has been increasing, in part because of the AIDS epidemic, a growing number of patients are being exposed to the adverse effects of this antibiotic. Indeed, this compound, as are the GCs, is often implicated in noxious drug interactions, because of its strong ability to induce drug-metabolizing enzymes. Moreover, in humans, RIF, as are the GCs, has been described as a potential immunodepressor, associated notably with the reduction of mitogenic responsiveness of human peripheral blood lymphocytes. Here, we report that RIF activates the human glucocorticoid receptor (hGR). Transient expression of wild-type, deleted or mutated GRs; sucrose density gradient sedimentation; and the BIAcore technique strongly suggest that RIF binds to the receptor with the physiological consequence that this antibiotic acts as an immunodepressor. Given the wide use of RIF in the treatment of coinfection of tuberculosis and HIV, this report is highly relevant to current medical practice.

Animals↗
PubMed2006

Distinct roles for de novo versus hydrolytic pathways of sphingolipid biosynthesis in Saccharomyces cerevisiae.

Saccharomyces cerevisiae produces the sphingolipid ceramide by de novo synthesis as well as by hydrolysis of complex sphingolipids by Isc1p (inositolphosphoceramide-phospholipase C), which is homologous with the mammalian neutral sphingomyelinases. Though the roles of sphingolipids in yeast stress responses are well characterized, it has been unclear whether Isc1p contributes to stress-induced sphingolipids. The present study was undertaken in order to distinguish the relative roles of de novo sphingolipid biosynthesis versus Isc1p-mediated sphingolipid production in the heat-stress response. Ceramide production was measured at normal and increased temperature in an ISC1 deletion and its parental strain (ISC1 being the gene that codes for Isc1p). The results showed that Isc1p contributes specifically to the formation of the C24-, C24:1- and C26-dihydroceramide species. The interaction between these two pathways of sphingolipid production was confirmed by the finding that ISC1 deletion is synthetically lethal with the lcb1-100 mutation. Interestingly, Isc1p did not contribute significantly to transient cell-cycle arrest or growth at elevated temperature, responses known to be regulated by the de novo pathway. In order to define specific contributions of ISC1, microarray hybridizations were performed, and analyses showed misregulation of genes involved in carbon source utilization and sexual reproduction, which was corroborated by defining a sporulation defect of the isc1Delta strain. These results indicate that the two pathways of ceramide production in yeast interact, but differ in their regulation of ceramides of distinct molecular species and serve distinct cellular functions.

Gene Deletion↗
PubMed1998

Dominant negative ATF1 blocks cyclic AMP-induced neurite outgrowth in PC12D cells.

Extension of the neuronal process is a crucial step for establishment of the neuronal network. As CREB preferentially forms heterodimers with ATF1 in PC12D cells, we examined the roles of the CREB/ATF1 heterodimer on cyclic AMP (cAMP)-induced neurite extension, using originally constructed ATF1RL, which has a point mutation at the DNA binding domain of ATF1. Transient expression of ATF1RL suppressed the protein kinase A/CREB-induced expression of the CRE reporter gene as expected. Treatment with forskolin elicited a relatively poor mRNA induction for immediate early genes in PC12D-ATF1 RL cells, a PC12D cell line stably expressing ATF1RL, in comparison with the parental PC12D cells. Furthermore, the PC12D-ATF1RL cells were proved to be defective at cAMP-induced neurite outgrowth. In contrast, both the gene expression and the differentiation after nerve growth factor treatment noted in PC12D-ATF1RL cells were at the same levels as those in the parental cells. These data provide us the first evidence that links CREB/ATF1 to the cAMP-induced differentiation of PC12 cells.

Activating Transcription Factor 1↗
PubMed1999

The release of acetylcholine receptor inducing activity (ARIA) from its transmembrane precursor in transfected fibroblasts.

Acetylcholine receptor inducing activity (ARIA) is made by motoneurons and is released at the neuromuscular synapse to stimulate the synthesis of acetylcholine receptors by skeletal muscle. ARIA is derived from a transmembrane precursor (pro-ARIA) via proteolytic cleavage of the ectodomain. We studied requirements in the amino acid sequence at the cleavage site with various substitution and deletion mutations. Wild type (WT) and mutant proteins were transiently expressed in COS cells, and release of ARIA into the conditioned medium was measured by tyrosine phosphorylation of its receptor, p185, in L6 cells. Removal of all potential cleavage sites between the extracellular epidermal growth factor domain and the transmembrane domain by substitution and small deletions (<11 amino acid residues out of 21) did not significantly reduce ARIA release, whereas larger deletions abolished it. We propose that cleavage occurs independently of amino acid sequence at a short distance from the epidermal growth factor domain, unless sterically hindered by the nearby secondary structure. A mutant with shorter cytoplasmic domain ("c" isoform) released significantly less ARIA than the WT ("a" isoform), suggesting that the c isoform may be suitable for signaling through direct cell-cell contact. Alternatively, proteolytic conversion of the a isoform to the c isoform may rapidly down-regulate release of ARIA.

Amino Acid Sequence↗
PubMed2004

NF-kappaB participates in the corticotropin-releasing, hormone-induced regulation of the pituitary proopiomelanocortin gene.

Corticotropin-releasing hormone is a main regulator of mammalian stress response by stimulating pituitary proopiomelanocortin (POMC) gene expression, and thus adrenocorticotropic hormone (ACTH) secretion, which then causes glucocorticoid release from the adrenal. In a recent study in the pituitary corticotroph cell line AtT20, oxidative stress stimulated the activity of nuclear transcription factor B (NF-kappaB), whereas corticotropin-releasing hormone (CRH) inhibited both the constitutive and the oxidative stress-induced NF-kappaB DNA-binding activity. To further investigate the role of NF-kappaB on the CRH-induced pituitary POMC gene activation, AtT20 cells were transiently transfected with a POMC-luciferase construct mutated at an NF-kappaB binding site. After treatment with CRH, intracellular POMC-luciferase activity was significantly higher from the stimulation observed with transfection of the parental POMC-luciferase construct. In agreement with a previous report, CRH inhibited the constitutive NF-kappaB DNA-binding activity in AtT20 cells, as shown by electrophoretic mobility-shift assay, as soon as within 15 min of treatment. These effects of CRH were blocked by the CRH-R1 antagonist CP154,256. Our findings provide evidence that the regulation of corticotroph NF-kappaB activity by CRH is related to the activation of the pituitary POMC gene and, thus, may play an important role in stress response.

Animals↗
PubMed2004

The use of transient chromatin immunoprecipitation assays to test models for E2F1-specific transcriptional activation.

The E2F family of transcription factors regulates the expression of genes involved in cell cycle progression, DNA synthesis, repair, and recombination, and a variety of other cellular processes. Although E2F proteins are often redundant in function, specificity of binding and activity can occur. For example, E2F1, but not other E2F family members, was shown previously to bind the murine carboxylesterase promoter in chromatin immunoprecipitation studies. This promoter region lacks a consensus E2F binding site, suggesting that E2F1 may be recruited to the DNA in a unique fashion. To further investigate this E2F1-specific binding, we have employed a "transient chromatin immunoprecipitation" approach. Using various deletions and mutations of the promoter region, we localized the E2F1-specific binding site and demonstrated that it was required for E2F1-mediated transcription of the carboxylesterase promoter. The identified site was similar to the 8-bp consensus E2F site but differed from the consensus at a crucial position. To address whether E2F1 directly bound to this non-consensus site, we demonstrated that the DNA binding domain of E2F1 is necessary for E2F1-mediated activation of the carboxylesterase promoter. Interestingly, a "UP" mutation of the site, making it more similar to the consensus element, did not improve the ability of E2F1 to bind the promoter. Rather, E2F1 could no longer bind to the carboxylesterase promoter that contained the consensus E2F site. We propose a model in which E2F1-specific regulation of the carboxylesterase promoter requires both E2F1/DNA interactions and protein-protein interaction between E2F1 and a factor that binds adjacent to the non-consensus site.

Animals↗
PubMedDate not supplied

The cloned promoter of the human DNA beta-polymerase gene contains a cAMP response element functional in HeLa cells.

The mammalian DNA beta-polymerase (beta-pol) gene is constitutively expressed in cultured cells as a function of growth stage and DNA replication, but is expressed in rodents in a tissue-specific fashion. As revealed by transient expression experiments with wild-type and mutated beta-pol promoter fusion genes, the cloned human beta-pol promoter is transcriptionally regulated by signals acting through the single palindromic sequence (GT-GACGTCAC) known as an ATF/CRE-binding site centered at position -45 in the core promoter. Although the mere presence of the ATF/CRE palindromic sequence in a promoter does not always confer cAMP responsiveness or protein binding over and around the ATF/CRE sequence, we find that agents that increase cAMP levels (forskolin and IBMX) in HeLa cells activate the beta-pol promoter; activation also can be observed by coexpression of the protein kinase A catalytic subunit. Experiments with mutagenized beta-pol promoters indicate that the ATF/CRE-binding site mediates these effects. Thus, the ATF/CRE-binding site in the context of this TATA-less constitutive promoter is able to respond to the kinase A signal transduction pathway.

Base Sequence↗
PubMed1993

The Caenorhabditis elegans spe-6 gene is required for major sperm protein assembly and shows second site non-complementation with an unlinked deficiency.

Caenorhabditis elegans spermatozoa move by crawling. Their motility requires thin cytoskeletal filaments assembled from a unique cytoskeletal protein, the major sperm protein (MSP). During normal sperm development the MSP is segregated to developing sperm by assembly into filaments that form a paracrystalline array in a transient organelle, the fibrous body-membranous organelle. Mutations in the spe-6 gene cause sterility because they lead to defective primary spermatocytes that do not form spermatids. In these mutant spermatocytes the MSP fails to assemble into fibrous body filaments. Instead, the unassembled MSP distributes throughout the cytoplasm and nucleus. Thus, the spe-6 gene product is necessary for normal MSP localization and assembly during sperm development. In addition to their MSP assembly defect, spe-6 mutant spermatocytes arrest meiosis at diakinesis although their spindle pole bodies still replicate and separate. This results in spermatocytes with four half-spindles surrounding condensed, but unsegregated, chromosomes. All four spe-6 alleles, as well as a chromosome III deficiency that deletes the spe-6 gene, fail to complement two small overlapping chromosome IV deficiencies, eDf18 and eDf19. This non-allele-specific second site non-complementation suggests a concentration-dependent interaction between the spe-6 gene product and products of the gene(s) under eDf18 and eDf19, which include a cluster of sperm-specific genes. Since MSP filament assembly is highly concentration-dependent in vitro, the non-complementation might be expected if the sperm-specific gene products under eDf18 and eDf19 were needed together with the spe-6 gene product to promote MSP assembly.

Animals↗
PubMed2000

An inbreeding model of associative overdominance during a population bottleneck.

Associative overdominance, the fitness difference between heterozygotes and homozygotes at a neutral locus, is classically described using two categories of models: linkage disequilibrium in small populations or identity disequilibrium in infinite, partially selfing populations. In both cases, only equilibrium situations have been considered. In the present study, associative overdominance is related to the distribution of individual inbreeding levels (i.e., genomic autozygosity). Our model integrates the effects of physical linkage and variation in inbreeding history among individual pedigrees. Hence, linkage and identity disequilibrium, traditionally presented as alternatives, are summarized within a single framework. This allows studying nonequilibrium situations in which both occur simultaneously. The model is applied to the case of an infinite population undergoing a sustained population bottleneck. The effects of bottleneck size, mating system, marker gene diversity, deleterious genomic mutation parameters, and physical linkage are evaluated. Bottlenecks transiently generate much larger associative overdominance than observed in equilibrium finite populations and represent a plausible explanation of empirical results obtained, for instance, in marine species. Moreover, the main origin of associative overdominance is random variation in individual inbreeding whereas physical linkage has little effect.

Animals↗