scholarly journals Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway.

1987 ◽  
Vol 7 (9) ◽  
pp. 3138-3146 ◽  
Author(s):  
C F Clarke ◽  
R D Tanaka ◽  
K Svenson ◽  
M Wamsley ◽  
A M Fogelman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Molecular Cloning ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Mrna Levels ◽  
Hmg Coa Reductase ◽  
Transcription System ◽  
Coa Reductase

Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.

Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway

1987 ◽  
Vol 7 (9) ◽  
pp. 3138-3146
Author(s):  
C F Clarke ◽  
R D Tanaka ◽  
K Svenson ◽  
M Wamsley ◽  
A M Fogelman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Molecular Cloning ◽  
In Vitro Transcription ◽  
In Vitro Translation ◽  
Mrna Levels ◽  
Hmg Coa Reductase ◽  
Transcription System ◽  
Coa Reductase

Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.

scholarly journals Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807 ◽  
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

Sheep antiluteolytic interferon: cDNA sequence and analysis of mRNA levels

1989 ◽  
Vol 2 (1) ◽  
pp. 65-70 ◽  
Author(s):  
H.J. Stewart ◽  
S.H.E. McCann ◽  
A.J. Northrop ◽  
G.E. Lamming ◽  
A.P.F. Flint
Keyword(s):  
Amino Acid ◽  
Northern Blotting ◽  
In Vitro Translation ◽  
Major Constituent ◽  
Interferon Α ◽  
Mrna Levels ◽  
Blastocyst Development ◽  
Cdna Sequences

ABSTRACT A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-α family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.

scholarly journals Asian plantain (Plantago asiatica) essential oils suppress 3-hydroxy-3-methyl-glutaryl-co-enzyme A reductase expression in vitro and in vivo and show hypocholesterolaemic properties in mice

2007 ◽  
Vol 99 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Mi Ja Chung ◽  
Kuen Woo Park ◽  
Kyoung Heon Kim ◽  
Cheong-Tae Kim ◽  
Jun Pill Baek ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Ldl Receptor ◽  
Ldl Oxidation ◽  
Mrna Levels ◽  
Hmg Coa Reductase ◽  
Protein Levels ◽  
Plantago Asiatica ◽  
Coa Reductase

Asian plantain (Plantago asiatica) essential oil (PAEO) contains multiple bioactive compounds, but its potential effects on lipid metabolism have not been examined. PAEO was found to be mostly composed of oxygenated monoterpenes, with linalool as the major component (82·5 %, w/w), measured using GC–MS. Incubation of 0–200 μg PAEO/ml with HepG2 cells for 24 h resulted in no significant toxicity. Incubation with 0·2 mg PAEO/ml altered the expression of LDL receptor (+83 %; P < 0·05) and 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase ( − 37 %; P < 0·05), as assessed using RT-PCR. LDL oxidation was markedly inhibited by PAEO treatment due to the prevalence of linalool compounds in PAEO. Oral administration of PAEO for 3 weeks in C57BL/6 mice significantly reduced plasma total cholesterol and TAG concentrations by 29 and 46 %, respectively. The mRNA (+58 %; P < 0·05), but not protein, levels of the LDL receptor were significantly higher, whereas both mRNA and protein levels of HMG-CoA reductase were significantly lower ( − 46 and − 11 %, respectively; P < 0·05) in the liver of PAEO-fed than of control mice. The mRNA levels of CYP7A1 were marginally reduced in HepG2 cells, but not in mouse liver after PAEO treatment. Thus, PAEO may have hypocholesterolaemic effects by altering the expression of HMG-CoA reductase. Reduced TAG and oxidised LDL may provide additional cardiovascular protective benefits.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

Fluorescent primer-based in vitro transcription system of viral RNA-dependent RNA polymerases

2013 ◽  
Vol 433 (2) ◽  
pp. 92-94
Author(s):  
Qiang Wang ◽  
Leiyun Weng ◽  
Hongbing Jiang ◽  
Shijian Zhang ◽  
Tetsuya Toyoda
Keyword(s):  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Viral Rna ◽  
Transcription System

Accurate transcription of a plant mitochondrial gene in vitro

1991 ◽  
Vol 11 (4) ◽  
pp. 2035-2039
Author(s):  
P J Hanic-Joyce ◽  
M W Gray
Keyword(s):  
Mitochondrial Gene ◽  
Plant Mitochondria ◽  
In Vitro Transcription ◽  
Dna Templates ◽  
In Vitro System ◽  
Transcription System ◽  
Translation Initiation Codon ◽  
Mitochondrial Extract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

In vitro antioxidant, antiglycation, and enzymatic inhibitory activity against α-glucosidase, α-amylase, lipase and HMG-CoA reductase of Terminalia boivinii Tul.

2021 ◽  
pp. 102235
Author(s):  
Bongani Sicelo Dlamini ◽  
Carlos Eduardo Hernandez ◽  
Chiy-Rong Chen ◽  
Wen-Ling Shih ◽  
Jue-Liang Hsu ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Hmg Coa Reductase ◽  
Coa Reductase
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