Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ubiquitin pathway targets proteins for degradation through the post-translational covalent attachment of the 76 amino acid protein ubiquitin to epsilon-amino lysyl groups on substrate proteins. Two instability determinants recognized by the ubiquitin pathway in Saccharomyces cerevisiae have been identified. One is described by the N-end rule and requires specific destabilizing residues at the substrate protein N-termini along with a proximal lysyl residue for ubiquitin conjugation. The second is a linear uncleavable N-terminal ubiquitin moiety. The ability of these two determinants to function in higher plants was investigated in tobacco protoplast transient transfection assays using DNA encoding variants of well characterized reporter enzymes as substrates: firefly luciferase that is localized to peroxisomes (pxLUC), a cytosolic version of LUC (cLUC), and Escherichia coli beta-glucuronidase (GUS). cLUC with phenylalanine encoded at its mature N-terminus was 10-fold less abundant than cLUC with methionine at its mature N-terminus. GUS with phenylalanine encoded at its mature N-terminus was 3-fold less abundant than GUS with methionine at its mature N-terminus. The presence of a uncleavable N-terminal ubiquitin fusion resulted in 50-fold lower protein accumulation of cLUC, but had no effect on GUS. Both instability determinants had a much larger effect on cLUC than on pxLUC, suggesting that these degradation signals are either unrecognized or poorly recognized in the peroxisomes.
Plant Mol Biol 1998 May
PMID:Engineering in vivo instability of firefly luciferase and Escherichia coli beta-glucuronidase in higher plants using recognition elements from the ubiquitin pathway. 961 5

In order to determine whether there are active genomic copies of the Anopheles gambiae transposon Ikirara, we developed an excision assay based on an internally deleted copy, Ikirara1. This element has 216 bp perfect inverted repeats at its termini, apparently caused a duplication of the dinucleotide TA at its insertion site between vitellogenin genes, and is thought to have been inserted recently at this location. The firefly luciferase gene on the E. coli tac promoter was inserted into Ikirara1 and used as a reporter to assess whether activities in an A. gambiae cell line could cause Ikirara excision. Excisions were observed at a rate of 0.038% in these experiments, but none was detected in controls. The five independent excision products examined gave identical sequences. Excisions were nearly precise, but left behind a footprint of 15 bp of the 3' inverted repeat of Ikirara1 between duplicated TAs. These excisions can be explained by a mechanism formally similar to that proposed for excision of mariner/Tc1 elements with cuts at the transposon ends staggered by 15 bases.
Insect Mol Biol 1998 Aug
PMID:Excisions of the Ikirara1 transposon in an Anopheles gambiae cell line. 966 73

DnaJ is a universally conserved heat shock protein involved in protein folding. DnaJ contains four conserved domains. The N-terminal 'J-domain' has been shown to be responsible for the recruitment of its specific DnaK partner protein. The 'Gly/Phe'- and 'Cys-rich' domains have been implicated in stabilizing interactions with DnaK. DnaJ is also able to interact independently with unfolded or native polypeptides. Very little is known regarding such binding/chaperone abilities, but it has been suggested that the least conserved carboxy-terminal domain could contribute to these properties. To gain insight into the biological activity of this fourth domain, we deleted two relatively conserved patches of amino acid residues, a 'G-rich' cluster and a 'G-D-L-Y-V' motif, resulting in the DnaJDelta[230-238] and DnaJDelta[242-246] mutant proteins respectively. Both mutant proteins are partially defective in stimulating the ATPase activity of DnaK and in preventing aggregation of firefly luciferase in vitro. Both mutants have lost the ability to regulate the sigma32-dependent heat shock response, as shown in vivo using a heat shock transcriptional fusion. Furthermore, and unlike wild-type DnaJ, DnaJDelta[242-246] is unable to assist the DnaK-dependent refolding of denatured luciferase. In agreement with these results, we found that DnaJDelta[242-246] is unable to restore either the temperature-sensitive phenotype or the motility defect of a dnaJ null mutation. Substitution of amino acids [242-246] by five alanines leads to similar phenotypic defects, suggesting that altering the 'G-D-L-Y-V' motif leads to partial loss of DnaJ activity. Our data clearly support a role in the intrinsic chaperone/substrate binding ability of the carboxy-terminal domain of DnaJ.
Mol Microbiol 1998 Oct
PMID:Genetic and biochemical characterization of mutations affecting the carboxy-terminal domain of the Escherichia coli molecular chaperone DnaJ. 979 Nov 78

At present, leptin is quantitated using immuno-assays that measure leptin mass. Leptin biological activity is determined using protocols that measure feed consumption and weight reduction. These in vivo protocols are semi-quantitative and require large quantities of leptin. We describe a rapid, sensitive and quantitative in vitro assay for leptin using HEK-293 cells stably co-transfected with the leptin receptor Ob-Rb isoform and a STAT-inducible promoter regulating the firefly luciferase cDNA. The assay, performed in a 96-well format, has an EC50 of 150 pM and is linear from 3 to 700 pM of leptin. We demonstrate that the assay is capable of measuring leptin in plasma samples. We demonstrate that bacterially-expressed, recombinant leptin and in vivo expressed leptin are equipotent. Furthermore, we demonstrate that a leptin-derived peptide, leptin fragment 22-56, previously shown to be capable of reducing feed intake following ICV injection does not act directly through the leptin receptor.
Mol Cell Endocrinol 1998 Aug 25
PMID:A rapid, quantitative functional assay for measuring leptin. 980 56

Translation of the upstream open reading frame (uORF) in the 5' leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control.
Mol Cell Biol 1998 Dec
PMID:The evolutionarily conserved eukaryotic arginine attenuator peptide regulates the movement of ribosomes that have translated it. 981 38

The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the sigma32 subunit of RNA polymerase, and negative modulators. Modulators are the DnaK chaperone system, which inactivates and destabilizes sigma32, and the FtsH protease, which is largely responsible for sigma32 degradation. A yet unproven hypothesis is that the degree of sequestration of the modulators through binding to misfolded proteins determines the level of heat shock gene transcription. This hypothesis was tested by altering the modulator concentration in cells expressing dnaK, dnaJ and ftsH from IPTG and arabinose-controlled promoters. Small increases in levels of DnaK and the DnaJ co-chaperone (< 1.5-fold of wild type) resulted in decreased level and activity of sigma32 at intermediate temperature and faster shut-off of the heat shock response. Small decreases in their levels caused inverse effects and, furthermore, reduced the refolding efficiency of heat-denatured protein and growth at heat shock temperatures. Fewer than 1500 molecules of a substrate of the DnaK system, structurally unstable firefly luciferase, resulted in elevated levels of heat shock proteins and a prolonged shut-off phase of the heat shock response. In contrast, a decrease in FtsH levels increased the sigma32 levels, but the accumulated sigma32 was inactive, indicating that sequestration of FtsH alone cannot induce the heat shock response efficiently. DnaK and DnaJ thus constitute the primary stress-sensing and transducing system of the E. coli heat shock response, which detects protein misfolding with high sensitivity.
Mol Microbiol 1998 Nov
PMID:Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli. 982 22

We have cloned the rat fibroblast growth factor-2 (FGF-2) promoter region including 1058 base pairs (bp) of 5'-flanking DNA. Complete sequencing of this promoter region revealed a 74 bp domain between nucleotides-793 and-720 that was greater than 97% A/G-rich. A repeat of the sequence 5'-AGGGAGGG-3' separated by 11 bp was located at the core of this domain. A 37 bp A/G-rich oligonucleotide containing these AGGG-repeat sequences was synthesised, and tested for function on a minimal herpes simplex virus thymidine kinase (TK) promoter, fused to the firefly luciferase gene (TKp.luc), in transiently transfected neonatal rat cardiac myocytes. Promoter activity was stimulated approximately 3 fold in the presence of AGGG-repeat sequences. This effect was neither tissue or species-specific since TK promoter activity was increased approximately 11 fold in both rat and human glial tumor cells. Four specific complexes (Cl-4) were detected between neonatal rat heart nuclear proteins and the 37 bp A/G-rich oligonucleotide by gel mobility shift assay. Competition with excess unlabelled 37 bp A/G-rich oligonucleotide revealed that two complexes represented very high affinity/specificity interactions (C2 > C4) while Cl and C3 were of lower affinity. As a result, competition with up to a 25 fold molar excess of 37 bp A/G-rich oligonucleotide led to the loss of C2 and C4, and a corresponding and transient increase in the levels of Cl and C3, which themselves were reduced with more competitor oligonucleotide. The AGGG-repeat resembles the 5'-gGGGAGGG-3' sequence previously implicated in the response of the atrial natriuretic factor promoter to the alpha-adrenergic agonist, phenylephrine. Although an additional 1.5 fold increase in TK promoter activity was detected in the presence of the 37 bp A/G-rich oligonucleotide with phenylephrine treatment of transfected myocytes, this effect was not statistically significant. Furthermore, there was no difference in the gel mobility shift (Cl-4) pattern obtained with the 37 bp A/G-rich oligonucleotide and nuclear protein isolated from neonatal rat cardiac myocytes grown in the presence or absence of norepinephrine. These data suggest that the A/G rich sequences in the rat FGF-2 gene 5'-flanking DNA, including the AGGG-repeat, are able to confer stimulatory activity on a promoter in a tissue- and species-independent manner, but alone are not able to induce a significant phenylephrine response in neonatal rat cardiac myocytes.
Mol Cell Biochem 1998 Nov
PMID:An A/G-rich motif in the rat fibroblast growth factor-2 gene confers enhancer activity on a heterologous promoter in neonatal rat cardiac myocytes. 982 22

We previously established a stable expression system in MCF-7 cells for the detection of (anti)estrogenic activity by assaying the reporter enzyme activity of firefly luciferase. In this cell line (called MVLN), the bioluminescent response can be measured either in the cellular homogenate, or in intact living cells. Here we present various potential experimental uses of this cellular model. First, we used this cell line to screen natural or synthetic molecules classified as full or partial (anti)estrogens and observed that their behavior towards our model was identical to that expected. Moreover, the bioluminescent response was in agreement with the natural responses like cellular proliferation or stimulation of the progesterone receptor. We then demonstrated the inhibitory effects of retinoic acid and 1,25 dihydroxyvitamin D3, two molecules which do not compete with estradiol for its receptor. We thus deduced that with this cell line an "antiestrogenic" effect which occurred at any step of the estrogenic action, might be detected. Finally, we showed that detection of luciferase activity in intact living cells was particularly helpful for investigating the evolution of estrogenic activity. For instance, we observed that long-term treatment of MVLN cells with an antiestrogen irreversibly decreased the bioluminescent response by more than 90%. This phenomenon affected all cells equally and could not be reversed, even by long-term estradiol treatment. We therefore conclude that this chimeric response faithfully reflects estrogenic action in the cell and can be used to develop different aspects of the endocrine research.
J Steroid Biochem Mol Biol 1993 Sep
PMID:MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity. 983 84

Muscle and melanoma tissue of fish in the genus Xiphophorus were examined for their ability to take up and express foreign DNA. Supercoiled plasmid DNA containing a firefly luciferase reporter gene with expression driven by the cytomegalovirus enhancer and thymidylate kinase promoter was directly injected into the muscle or melanoma of individual Xiphophorus. Expression levels gradually increased to a maximum at 6 days after injection in both tissues, and this level was maintained for at least 10 days after injection. In both muscle and melanoma, there was a clear relationship between dose injected and reporter gene activity, with maximal expression at a dose of 20 microg of plasmid injected. At higher doses expression levels declined, suggesting the possibility that the uptake mechanism can be inhibited by high concentrations of DNA. Histochemical localization using a beta-galactosidase construct revealed high expression of the enzyme in isolated muscle fibers. The activity of a second coinjected reporter gene, sea pansy (Renilla reniformis) luciferase, was highly correlated with the activity of the firefly luciferase reporter gene in both tissues (R2 >.940), suggesting that the majority of variation between samples results from variation in overall DNA uptake between individuals. When firefly luciferase activity is expressed as a function of activity of the coinjected reporter, the variation between samples is greatly reduced. As a result, small differences in activity between constructs can be detected. This demonstrates the usefulness of the system for gene expression analysis in vivo.
Mol Mar Biol Biotechnol 1998 Dec
PMID:Efficient gene transfer into Xiphophorus muscle and melanoma by injection of supercoiled plasmid DNA. 989 13

We have analyzed two tandem promoters, separated by only about 400 bp, of the purple (pr) gene of Drosophila melanogaster, by fusing them to the firefly luciferase reporter gene and employing a transient expression assay with Drosophila S2 cells. Both the distal promoter and the proximal promoter were found to function in S2 cells and an about 700 bp long region (-270 to +421), containing both promoters, was sufficient to effect maximal promoter activity. When the two promoters were analyzed separately, the distal promoter was found to be much stronger in its function than the proximal promoter. At least three different kinds of cis elements near the transcription start site appear to play crucial roles in driving constitutive expression from the distal promoter. On the other hand, only a single cis element, which may play a role in tissue-specific expression, appears to be important for the activity of the proximal promoter in S2 cells. We propose that the clustering of important cis elements near the transcription start sites may be responsible for the selective regulation of the two tandem promoters.
Mol Cells 1998 Dec 31
PMID:An analysis of two tandem promoters of the Drosophila purple gene. 989 20


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