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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using Tn4431, a transposon that allows transcriptional fusions to a promoterless luciferase (lux) operon, we have isolated a nonpathogenic mutant of Xanthomonas campestris pv. campestris, i.e., JS111, that does not incite any of the black rot symptoms on all tested cruciferous host plants (J. J. Shaw, L. G. Settles, and C. I. Kado, Mol. Plant Microbe Interact. 1:39-45, 1988). In the study reported here, we determined that in contrast to the wild-type strain, JS111 is unable to induce a hypersensitive necrotic response on nonhost plants such as datura, tomato, and cucumber, suggesting that JS111 is a nonpathogenic, nonhypersensitive Hrp mutant. JS111 displayed culture growth rates, exopolysaccharide production, and protease, pectate lysase, cellulase, amylase, and phosphatase activities comparable to those of the wild-type strain. However, the growth of JS111 in host leaves was markedly attenuated. Coinoculation of JS111 with the wild-type strain in cauliflower or radish leaves rescued the growth deficiency of the mutant to normal levels. The locus mutated in JS111 was cloned and named hrpXc, and transcriptional and genetic complementation analyses of the hrpXc locus were conducted. The regulation of hrpXc expression was also investigated in vitro and in planta, using fusions to a lux or chloramphenicol acetyltransferase reporter gene. The hrpXc gene was found to be strongly induced in radish leaves. This is the first report and analysis of a hrp locus from a Xanthomonas species.
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PMID:A plant-inducible gene of Xanthomonas campestris pv. campestris encodes an exocellular component required for growth in the host and hypersensitivity on nonhosts. 216 73

To identify DNA regions important for basal and hormone-stimulated transcription of the rat PRL gene, a series of clustered point mutations were prepared within the immediate 5' flanking region. DNA fragments representing the wild-type and 19 different linker-scanner mutations of the PRL gene were each linked to a luciferase marker gene, and the DNA constructs were transferred into GH3 pituitary tumor cells by electroporation. Luciferase activity was determined 24 h after transfection in extracts from control cells or cells treated with 0.5 mM chlorophenylthio-cAMP, 100 nM TRH, or 100 nM phorbol myristate acetate. The individual clustered point mutations covered a region from just up-stream of the TATA box (position -30) to a position 193 basepairs up-stream from the start of transcription. Five regions in which mutations produced substantial decreases in both basal and cAMP-, TRH-, or phorbol ester-stimulated expression of the marker gene were detected. Three of these regions (positions -41 to -58, -113 to -124, and -149 to -156) correspond to previously identified binding sites for the pituitary-specific, homeobox protein, Pit-1/GHF-1. The fourth and fifth regions do not correspond to Pit-1/GHF-1-binding sites and presumably represent sites for an unidentified factor. Within these regions, sequences with some similarity to a consensus cAMP response element and an AP-2-binding site have been detected. These data confirm the importance of Pit-1/GHF-1 as a key factor in PRL gene transcription. In addition, the results suggest that additional transcription factors are probably required for efficient expression of the PRL gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1990 Oct
PMID:Clustered point mutation analysis of the rat prolactin promoter. 217 21

Expression of Photobacterium leiognathi bioluminescence genes under the control of lac, tac, tet promoters in Escherichia coli cells has been studied. The position of the genes for aliphatic aldehyde biosynthesis and for the synthesis of luciferase subunits was identified. The plasmid pBRPL1 has been constructed containing the system of bioluminescence genes devoid of promoter following the polylinker DNA fragment. The plasmid can be used for selection of promoter containing DNA sequences as well as for studying the promoters regulation in process of Escherichia coli cells growth.
Mol Gen Mikrobiol Virusol 1990 Feb
PMID:[Expression of Photobacterium leiognathi bioluminescence system genes in Escherichia coli]. 218 20

The acute-phase activation of the rat angiotensinogen (rAT) gene in liver cells is a transcriptional event mediated through an interleukin-1-inducible, NF kappa B-binding, cis-acting element (the acute-phase response element [APRE]). Using a cell culture model for the acute-phase response, we showed that the increase in angiotensionogen mRNA in H35 rat hepatoma cells requires costimulation with glucocorticoids and cytokines. Stably transfected rAT promoter-luciferase reporter genes were also activated by cytokines only in the presence of glucocorticoids. This permissive role of glucocorticoids is dependent on the expression of functional glucocorticoid receptors, because in HepG2 cells naturally deficient in such receptors, rAT gene-luciferase reporter constructs responded to interleukin-1 only when cotransfected with an expression vector for the glucocorticoid receptor. Point mutations in the two rAT gene glucocorticoid response elements located adjacent to the APRE led to loss of interleukin-1 inducibility. Induction of luciferase activity in transfected cells occurred even in the presence of cycloheximide, demonstrating that this synergistic response did not depend on new protein synthesis. Thus, a direct interaction between the interleukin-1-inducible NF kappa B-binding APRE and glucocorticoid response elements, located in cis, underlies the acute-phase activation of the rAT gene.
Mol Cell Biol 1990 Aug
PMID:The permissive role of glucocorticoids on interleukin-1 stimulation of angiotensinogen gene transcription is mediated by an interaction between inducible enhancers. 237 Aug 71

The murine thyrotropic MGH101A tumor is characterized by absent thyrotropin (TSH) beta gene expression and altered thyroid hormone (T3) regulation of the alpha-subunit. Comparison of the promoter structures of both alpha and TSH beta subunit genes from MGH101A with the promoter in expressing TtT-97 thyrotropes revealed no detectable differences. Transfection of the TSH beta promoter from MGH101A linked to luciferase showed minimal expression in primary or cloned MGH101A cells, or L-cells. However, a 6- to 10-fold increase in expression was exhibited in transfected thyrotropes. For the alpha gene, promoter activity was highest in thyrotropes and in cloned MGH101A cells, 5-fold lower in MGH101A tumors, and 10-fold lower in L-cells. Both promoters were not substantially affected by T3 treatment in MGH101A cells. In thyrotropes, promoter activity was inhibited 62.5% and 57.7% by 10 nM T3 treatment for the TSH beta and alpha genes, respectively. DNase I protection showed that factors from TtT-97 but not from MGH101A cells interacted with regions in the TSH beta promoter, while nuclear extracts from each tumor demonstrated at least one protein-DNA interaction with the alpha-subunit promoter. These studies suggest that the molecular defects in the MGH101A tumor are related to the absence of trans-acting factors and are not a result of altered primary gene structure.
Mol Cell Endocrinol 1990 Jun 18
PMID:TSH subunit gene promoters from a murine alpha-subunit producing tumor function normally. 237 87

Synthesis and metabolism of the epidermal growth factor (EGF) receptor are extensively regulated to modulate cellular responses to ligand. To study regulation of EGF receptor gene expression, the 5' region of the gene was isolated from a human placental genomic library. A 5' proximal 1.1-kilobase fragment (-1100 to -19 relative to the ATG translation start site) and subfragments of this were subcloned in both forward and reverse orientations into the luciferase expression vector pSVOAL delta 5' and transfected into human cell lines. Luciferase activity was stimulated by treatment of transfected HeLa cells with EGF, 12-O-tetradecanoylphorbol 13-acetate (TPA), (Bu)2 cAMP, retinoic acid, and dexamethasone. Deletion analysis indicated full retention of activity after removal of the -1100 to -485 region (-485 to -19 fragment), but a 5-fold reduction in activity on removal of the -485 to -153 region (-153 to -19 fragment). Despite a reduction in basal activity, the proximal 134-basepair fragment retained responses to all inducers. Additivity was observed in response to maximal concentrations of TPA plus retinoic acid and of TPA plus (Bu)2 cAMP; the response to a combination of four inducers exceeded that to the RSV-LTR strong promoter. Differences in stimulated responses were observed in various recipients, with hepatoma HepG2 cells lacking responses to (Bu)2 cAMP and glioblastoma T98G cells lacking responses to EGF and TPA. These results indicate that a 134-basepair DNA fragment closely adjacent to the translation start site contains elements responsible for directing basal and stimulated expression of the EGF receptor gene.
Mol Endocrinol 1989 Feb
PMID:Regulation of epidermal growth factor receptor gene expression. 254 Apr 31

To determine the rat PRL (rPRL) promoter sequences that mediate pituitary-specific and cAMP-induced gene expression in vivo, various lengths of the rPRL promoter were ligated to the luciferase reporter gene and introduced into pituitary and non-pituitary cell lines. A 30-fold increase in rPRL promoter activity was observed in GH4 rat pituitary tumor cells compared to nonpituitary Rat2 fibroblast and HeLa cervical carcinoma cells. About 45% of this cell-specific promoter activity was competed by a plasmid containing the -67 to -45 rPRL promoter region, which is the most proximal binding site for a lactotroph-specific factor. Compared to a -425 rPRL construct, transfection with rPRL 5'-end points of -212, -178, and -127 contained 23%, 45%, and 1%, respectively, of luciferase activity. Forskolin stimulation resulted in a 10-fold induction of all the rPRL promoter fragments tested. Of note, a -127 deletion which was devoid of any basal promoter activity was also induced 10-fold by forskolin. The forskolin effect was abolished when GH4 rat pituitary cells were cotransfected with a plasmid encoding a protein kinase A inhibitor, indicating protein kinase A is involved in the activation mechanism. These data document that both positive and negative effectors influence basal rPRL promoter activity. Furthermore, the minimum sequences required for pituitary-specific rPRL promoter activity are altered by intracellular cAMP levels. Taken together, the data indicate that hormone-activated and cell-specific factors may interact to establish a particular setpoint for rPRL gene expression.
Mol Endocrinol 1989 May
PMID:Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo. 254 56

Studies of gene regulation are greatly facilitated by the ability to transfect DNA into cultured cells. We examined a variety of transfection techniques to optimize transient expression of the human glycoprotein hormone alpha-gene in primary pituitary cells and subsequently investigated the regulation of alpha-promoter transcription. Expression vectors driven by either the rous sarcoma virus-chloramphenicol acetyl transferase (RSVCAT) or the human alpha-gene (alpha CAT) promoters were transfected into cultures of dispersed female rat pituitary cells using calcium phosphate (CaPO4), diethylaminoethyl-dextran, lipofection, and electroporation procedures. CAT activity was optimal using the CaPO4 technique, resulting in 511 +/- 49% and 57 +/- 5% conversion/100 micrograms protein/4 h for RSVCAT and alpha CAT, respectively. Immunohistochemical analyses of alpha CAT expression using anti-CAT monoclonal antibodies demonstrated that the alpha-gene promoter is expressed in pituitary cells, predominantly if not exclusively, in gonadotropes and thyrotropes. Hormonal regulation of alpha-promoter activity was assessed using both the CAT and the luciferase (LUC) reporter systems. alpha-Promoter activity was significantly (P less than 0.001) stimulated by 8-bromo-cAMP (217% increase), GnRH (75% increase), GnRH agonist analog (141% increase), and TRH (75% increase). The expression of control plasmids (RSVLUC, TKLUC, pOLUC) was not affected by treatment with these agents. We conclude that CaPO4-mediated transfection allows analyses of transient gene expression in primary pituitary cells. The alpha-promoter directs expression specifically in pituitary cells, predominantly gonadotropes and thyrotropes. alpha-Gene transcription is stimulated by GnRH, TRH, and 8-bromo-cAMP.
Mol Endocrinol 1989 Oct
PMID:Regulation of transfected glycoprotein hormone alpha-gene expression in primary pituitary cell cultures. 255 99

Pituitary thyrotroph cells specialize in the synthesis of TSH, and thus represent a model to study cell-specific gene expression. We have used the murine TSH beta (mTSH beta) gene promoter and TSH-producing and nonproducing transplantable tumors derived from murine thyrotroph cells, referred to as TtT-97 and MGH 101A, respectively, to identify nuclear factors which selectively interact with the mTSH beta gene. DNase I protection analyses demonstrate that factors present in TtT-97 nuclear extracts bind with high affinity to five separate sites in the TSH beta promoter region, denoted as distal D1 (-253 to -227) and proximal, P1 (-76 to -68), P2 (-106 to -98), P3 (-126 to -112), and P4 (-142 to -131) footprints. By contrast, non-TSH beta expressing thyrotroph cell nuclear extracts and L-cell nonpituitary cell extracts did not appear to footprint the D1 site; whereas the nonpituitary nuclear extracts revealed minimal DNase I protection in the P1-P4 regions. These data show that the distal D1 site is thyrotroph specific and contains a 6 base pair direct repeat sequence (5'-AGATAT-3'). Factor occupancy of the D1 site is protein dependent, occurs rapidly (less than 15 sec), is destabilized by 170 mM KCl, and results in an associated DNase I hypersensitive region. A double-stranded oligonucleotide spanning the D1 footprint competes only the distal factor binding region. Transfection of plasmid constructs containing progressive 5'-deletions of the mTSH beta promoter linked to the reporter gene luciferase into primary TtT-97 cells demonstrate a marked decrease in activity between the regions -270 and -79, which contains the D1 region.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1989 Jul
PMID:Identification of thyrotroph-specific factors and cis-acting sequences of the murine thyrotropin beta subunit gene. 279 1

A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI--BglII fragment between the kanamycin resistance (Kmr) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Kmr transfer frequency of 10(-6) per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8 x 10(6) cpm for a cell density of 10(3) colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.
Mol Gen Genet 1988 Jul
PMID:Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium. 285 9


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