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)

Mdj1p, a DnaJ homolog in the mitochondria of Saccharomyces cerevisiae, is involved in the folding of proteins in the mitochondrial matrix. In this capacity, Mdj1p cooperates with mitochondrial Hsp70 (mt-Hsp70). Here, we analyzed the role of Mdj1p as a chaperone for newly synthesized proteins encoded by mitochondrial DNA and for nucleus-encoded proteins as they enter the mitochondrial matrix. A series of conditional mutants of mdj1 was constructed. Mutations in the various functional domains led to a partial loss of Mdj1p function. The mutant Mdj1 proteins were defective in protecting the tester protein firefly luciferase against heat-induced aggregation in isolated mitochondria. The mitochondrially encoded var1 protein showed enhanced aggregation after synthesis in mdj1 mutant mitochondria. Mdj1p and mt-Hsp70 were found in a complex with nascent polypeptide chains on mitochondrial ribosomes. Mdj1p was not found to interact with translocation intermediates of imported proteins spanning the two membranes and exposing short segments into the matrix, in accordance with the lack of requirement of Mdj1p in the mt-Hsp70-mediated protein import into mitochondria. On the other hand, precursor proteins in transit which had further entered the matrix were found in a complex with Mdj1p. Our results suggest that Mdj1p together with mt-Hsp70 plays an important role as a chaperone for mitochondrially synthesized polypeptide chains emerging from the ribosome and for translocating proteins at a late import step.
Mol Cell Biol 1996 Dec
PMID:Role of the mitochondrial DnaJ homolog Mdj1p as a chaperone for mitochondrially synthesized and imported proteins. 894 61

RNase protection assays and primer extension analysis have been used to locate a major transcription start site 960 bp upstream from the translational start of the PfPCNA coding sequence. A second, minor, site is situated a further 40 bp upstream. Intraerythrocytic parasite stages were transiently transfected with constructs containing a firefly luciferase reporter gene under the transcriptional control of variously modified elements of the PfPCNA 5' flanking sequence. These experiments identified a 470 bp region essential for promoter activity, which contains the physically mapped transcriptional start sites. In addition, a region between 290 and 620 bp upstream of the transcriptional start sites is required for efficient promoter activity.
Mol Biochem Parasitol 1996 Nov 25
PMID:Physical and functional mapping of the transcriptional start sites of Plasmodium falciparum proliferating cell nuclear antigen. 894 86

Manipulation of gene expression in Giardia lamblia, one of the most ancient eukaryotes, may provide insights into the evolutionary transition from prokaryotes to eukaryotes. Two recent successes in transient expression of the firefly luciferase (luc) gene in G. lamblia were mediated by a 5'-untranslated region (UTR) of the Giardia glutamate dehydrogenase (gdh) gene and a giardiavirus (GLV) genomic transcript, respectively. We now report a stable coexpression of luc gene with a neomycin phosphotransferase (neo(r)) gene in G. lamblia. An in vitro transcript of the construct pC670-Neo; containing the neo(r) encoding region flanked with the 5'670 nucleotides (nt) and the 3'2022 nt portion of GLV positive strand RNA, was electroporated into G. lamblia trophozoites that were infected with GLV. G418-resistant Giardia trophozoites were cloned, and the neo(r) mRNA in these clones was found to increase with increasing G418 pressure. This drug resistance remained stable upon continuous in vitro cultivation in the absence of G418 for over 15 days. Another plasmid pNeo/GDH/Luc, was constructed by inserting luc gene downstream from the neo(r) gene and the 193 nt 5' portion of gdh gene in pC670-Neo, and its bicistronic in vitro transcript was introduced into GLV-infected G. lamblia by electroporation. The transfectants demonstrated G418-resistance and persistent luciferase activity at levels parallel to the amount of G418 used for selection, peaking at a level of several thousand-fold above the background. Taken together, these data indicate that the neo(r) gene provides an effective selection marker for transformation of Giardia trophozoites, and the bicistronic RNA transfection vector may open the way for functional analysis of other genes in Giardia.
Mol Biochem Parasitol 1996 Dec 02
PMID:Stable coexpression of a drug-resistance gene and a heterologous gene in an ancient parasitic protozoan Giardia lamblia. 901 Aug 44

Two activated matrices have been developed to determine whether immobilization chemistry can be used to orient proteins on a support. Restriction endonuclease EcoRI from Escherichia coli RY13 (E.C.3.1.23.13) was used as a model in these studies. Thiol-activated Sephadex G-10 was used to couple the EcoRI endonuclease through its free sulfhydryl, while amino-activated Sephadex G-10 was used to couple it randomly through its free carboxyl groups. To determine whether the enzyme was immobilized randomly or specifically, both lower and higher molecular weight substrates were used. The polymerase chain reaction amplified multiplied cloning site region of pBluescript KS obtained using T3 and T7 primers was considered as the small substrate. The plasmid SP64 containing firefly luciferase gene was the large substrate. Immobilized EcoRI preparations were characterized with respect to repeated usage and storage stability. The EcoRI immobilized on thiopropyl-Sepharose 4B could be stored for over 14 days at 4 degrees C without observable loss of activity. In an independent experiment the same gel was used thrice repeatedly without any discernible loss of activity.
J Mol Recognit
PMID:Oriented immobilization of restriction endonuclease EcoRI. 917 57

Direct injection of plasmid DNA into the myocardium of several species has been shown to be useful for studying cardiac gene expression. However, despite a better understanding of mouse genetics and the availability of several disease models in mice, gene injection with plasmid DNA into the mouse heart has not been reported. In this study, we demonstrate a simple and reproducible method for gene transfer into the mouse heart via direct injection of plasmid DNA. A firefly luciferase gene, driven by the RSV promoter, was used to quantitatively determine the spatial and temporal characteristics of gene transfer. Luciferase gene expression was stable for 8 weeks and showed a dose-dependent response over a range of 0.3-3 micrograms of input DNA. Inter-animal variability was low and gene expression was restricted to the left ventricle, near the site of injection. This method was also demonstrated to be suitable for detecting the expression of structural genes under the control of cellular promoters. Immunohistochemistry was used to detect the expression of an epitope-tagged myosin heavy chain driven by a rat alpha-myosin heavy chain promoter. Thus, naked DNA injection into the mouse heart results in a highly reproducible expression of constructs with either viral or cellular promoters. It is a relatively inexpensive and efficient means of studying cardiac gene regulation in vivo and a useful tool for screening the potential transgenes before generating transgenic mice.
J Mol Cell Cardiol 1997 May
PMID:Direct gene transfer into the mouse heart. 920 34

Osteocalcin is a hormonally regulated calcium-binding protein made almost exclusively by osteoblasts. In normal cells, osteocalcin expression requires ascorbic acid (AA), an essential cofactor for osteoblast differentiation both in vivo and in vitro. To determine the mechanism of this regulation, subclones of MC3T3-E1 preosteoblasts were transiently transfected with 1.3 kb of the mouse osteocalcin gene 2 promoter driving expression of firefly luciferase. AA stimulated luciferase activity 20-fold after 4-5 days. This response was stereospecific to L-ascorbic acid and was only detected in MC3T3-E1 subclones showing strong AA induction of the endogenous osteocalcin gene. Similar results were also obtained in MC3T3-E1 cells stably transfected with the osteocalcin promoter. A specific inhibitor of collagen synthesis, 3,4-dehydroproline, blocked AA-dependent induction of promoter activity, indicating that regulation of the osteocalcin gene requires collagen matrix synthesis. Deletion analysis of the mOG2 promoter identified an essential region for AA responsiveness between -147 and -116 bp. This region contains a single copy of the previously described osteoblast-specific element, OSE2. Deletion and mutation of OSE2 in DNA transfection assays established the requirement for this element in the AA response. Furthermore, DNA-binding assays revealed that MC3T3-E1 cells contain OSF2, the nuclear factor binding to OSE2, and that binding of OSF2 to OSE2 is up-regulated by AA treatment. Taken collectively, our results indicate that an intact OSE2 sequence is required for the induction of osteocalcin expression by AA.
Mol Endocrinol 1997 Jul
PMID:Ascorbic acid-dependent activation of the osteocalcin promoter in MC3T3-E1 preosteoblasts: requirement for collagen matrix synthesis and the presence of an intact OSE2 sequence. 921 58

A nuclear tRNA(Lys) gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (CAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gln, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNA(Lys) and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNA(LysCUA) from non-replicative vectors promoted 10-40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNA(Lys) suppressor genes from a geminivirus vector capable of replication promoted 30-80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gln and Glu codons.
Plant Mol Biol 1998 Jan
PMID:Nonsense and missense translational suppression in plant cells mediated by tRNA(Lys). 948 71

This study was conducted to investigate quantitatively the luciferase activity of gene constructs with viral and hybrid enhancers and promoters in bovine preimplantation embryos by using firefly luciferase reporter genes. In Experiment I, to examine the stability of the luciferase, bioluminescence intensity of bovine embryos injected with the luciferase gene driven by the SV40 early promoter and enhancer (SVEluc) was measured with a luminometer at 2 days after microinjection. The results indicated that the bioluminescence could be analysed at any time within 30 min because the luciferase activity was constant during the measurement period from 5 to 30 min. In Experiment II, the luciferase expression of fertilized oocytes injected with four gene constructs (TKEluc, TK6WEluc, SVEluc, and Miwluc) was analysed by using a photon imaging system at 2 or 6 days following microinjection. The results from Experiment II indicated that the reporter gene governed by the Miw promoter (RSV LTR and chicken beta-actin promoter) was expressed more intensively in bovine morulae and blastocysts than three other gene constructs. In Experiment III, the effect of SV40 enhancer was investigated when fused downstream to the luciferase cDNA of the Miwluc vector. The results showed that SV40 enhancer further activated the luciferase activity of the Miw promoter in bovine preimplantation embryos. It was concluded, therefore, that the Miw promoter together with the SV40 enhancer would confer the strongest expression of the firefly luciferase reporter gene among the gene constructs tested in preimplantation bovine embryos.
Mol Reprod Dev 1998 Apr
PMID:Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos. 950 87

The A1 adenosine receptor (A1AR) contributes to the cytoprotective action of adenosine under conditions known to generate reactive oxygen species (ROS). Pharmacological manipulation of A1AR expression has been shown to modulate this cytoprotective role. In this study, we provide evidence that ROS generated could increase the expression of the A1AR and thereby offset the detrimental effects of ROS. Incubation of DDT1MF-2 smooth muscle cells with ROS-generating chemotherapeutic agents, such as cisplatin (2.5 microM) or H2O2 (10 microM), elicited an increase in A1AR expression within 24 hr. The induction by H2O2 was reduced by the ROS scavenger catalase but not superoxide dismutase. Inhibition of nuclear factor kappa B (NF kappa B) by pyrrolidine dithiocarbamate (200 microM), dexamethasone (100 nM), or genistein (1 microM) abrogated the cisplatin-mediated increase in A1AR. Cisplatin promoted rapid translocation of NF kappa B (but not AP-1) to the nucleus, as detected by electrophoretic mobility shift assays and by Western blotting. A putative NF kappa B sequence in the A1AR promoter effectively competed with labeled kappa B probe for binding in nuclear preparations derived from DDT1MF-2 cells. Transient transfection of DDT1MF-2 cells with the A1AR promoter coupled to firefly luciferase reporter gene led to cisplatin-inducible and pyrrolidine dithiocarbamate-sensitive luciferase activity, suggesting the presence of functional NF kappa B binding site(s) in the A1AR promoter sequence. Treatment of cells with (R)-phenylisopropyladenosine (1 microM), an agonist of the A1AR, reduced cisplatin-mediated lipid peroxidation, which was reversed after blockade of the A1AR. These data suggest that ROS can increase the expression of the A1AR by activating NF kappa B regulatory site(s) on this gene and thereby enhance the cytoprotective role of adenosine.
Mol Pharmacol 1998 Apr
PMID:Oxidative stress increases A1 adenosine receptor expression by activating nuclear factor kappa B. 954 56

We have developed a stable DNA transfection vector pRANneo for genetic manipulation of the primitive protozoan Giardia lamblia. pRANneo was constructed by replacing the protein coding region of a Giardia ran gene with a bacterial neomycin phosphotransferase gene (neo). This plasmid was electroporated into G. lamblia, and the transfectants were selected by G418. pRANneo replicated episomally to approximately 80 copies per G. lamblia trophozoite as demonstrated by dot hybridizations, Southern hybridizations and transformations of the DpnI-treated plasmids into Escherichia coli. pRANneo/GDHluc was then constructed by incorporation of a luciferase expression system into pRANneo to persistently express firefly luciferase in G. lamblia under G418 selection. The NEO and luciferase proteins were detected in the transfected G. lamblia cells by Western blottings. The level of luciferase activity and the plasmid copy number correlated with the concentration of G418. Removal of G418 from the transfectant culture resulted in gradual loss of the plasmid and luciferase activity. The stable DNA transfection system should provide a valuable tool for genetic studies of G. lamblia.
Mol Biochem Parasitol 1998 Apr 01
PMID:Stable DNA transfection of the primitive protozoan pathogen Giardia lamblia. 957 16


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