In vitro translation of rat liver and Novikoff hepatoma cytokeratin mRNAs

1986 ◽  
Vol 70 (1) ◽  
Author(s):  
WandaM. Krajewska ◽  
WarrenN. Schmidt ◽  
LubomirS. Hnilica
Keyword(s):  
Rat Liver ◽  
In Vitro Translation ◽  
Novikoff Hepatoma

scholarly journals Repression of the albumin gene in Novikoff hepatoma cells.

1982 ◽  
Vol 2 (3) ◽  
pp. 258-266 ◽  
Author(s):  
Y G Capetanaki ◽  
C N Flytzanis ◽  
A Alonso
Keyword(s):  
Rat Liver ◽  
Cdna Probe ◽  
Hepatoma Cells ◽  
In Vitro Translation ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Novikoff Hepatoma ◽  
Albumin Gene ◽  
Albumin Mrna

Novikoff hepatoma cells have lost their capacity to synthesize albumin. As a first approach to study the mechanisms underlying this event, in vitro translation in a reticulocyte system was performed using total polyadenylated mRNA from rat liver and Novikoff hepatoma cells. Immunoprecipitation of the in vitro translation products with albumin-specific antibody revealed a total lack of albumin synthesis in Novikoff hepatoma, suggesting the absence of functional albumin mRNA in these cells. Titration experiments using as probe albumin cDNA cloned in pBR322 plasmid demonstrated the absence of albumin-specific sequences in both polysomal and nuclear polyadenylated and total RNA from Novikoff cells. This albumin recombinant plasmid was obtained by screening a rat liver cDNA library with albumin [32P]cDNA reverse transcribed from immuno-precipitated mRNA. The presence of an albumin-specific gene insert was documented with translation assays as well as by restriction mapping. Repression of the albumin gene at the transcriptional level was further demonstrated by RNA blotting experiments using the cloned albumin cDNA probe. Genomic DNA blots using the cloned albumin cDNA as probe did not reveal any large-scale deletions, insertions, or rearrangements in the albumin gene, suggesting that the processes involved in the suppression of albumin mRNA synthesis do not involve extensive genomic rearrangements.

Repression of the albumin gene in Novikoff hepatoma cells

1982 ◽  
Vol 2 (3) ◽  
pp. 258-266
Author(s):  
Y G Capetanaki ◽  
C N Flytzanis ◽  
A Alonso
Keyword(s):  
Rat Liver ◽  
Cdna Probe ◽  
Hepatoma Cells ◽  
In Vitro Translation ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Novikoff Hepatoma ◽  
Albumin Gene ◽  
Albumin Mrna

Novikoff hepatoma cells have lost their capacity to synthesize albumin. As a first approach to study the mechanisms underlying this event, in vitro translation in a reticulocyte system was performed using total polyadenylated mRNA from rat liver and Novikoff hepatoma cells. Immunoprecipitation of the in vitro translation products with albumin-specific antibody revealed a total lack of albumin synthesis in Novikoff hepatoma, suggesting the absence of functional albumin mRNA in these cells. Titration experiments using as probe albumin cDNA cloned in pBR322 plasmid demonstrated the absence of albumin-specific sequences in both polysomal and nuclear polyadenylated and total RNA from Novikoff cells. This albumin recombinant plasmid was obtained by screening a rat liver cDNA library with albumin [32P]cDNA reverse transcribed from immuno-precipitated mRNA. The presence of an albumin-specific gene insert was documented with translation assays as well as by restriction mapping. Repression of the albumin gene at the transcriptional level was further demonstrated by RNA blotting experiments using the cloned albumin cDNA probe. Genomic DNA blots using the cloned albumin cDNA as probe did not reveal any large-scale deletions, insertions, or rearrangements in the albumin gene, suggesting that the processes involved in the suppression of albumin mRNA synthesis do not involve extensive genomic rearrangements.

Purification of mRNA coding for the enzyme deficient in hereditary tyrosinemia, fumarylacetoacetate hydrolase

1986 ◽  
Vol 64 (5) ◽  
pp. 489-493 ◽  
Author(s):  
Louis M. Nicole ◽  
Jean Paul Valet ◽  
Claude Laberge ◽  
Robert M. Tanguay
Keyword(s):  
Rat Liver ◽  
Relative Abundance ◽  
In Vitro Translation ◽  
Translation Product ◽  
Fumarylacetoacetate Hydrolase ◽  
Hereditary Tyrosinemia ◽  
Enzyme Deficient

As a step towards the cloning of the gene for fumarylacetoacetate hydrolase (FAH), we have purified the FAH mRNA from rat liver by specific immunoadsorption of polysomes. The relative abundance of this mRNA has been estimated to be 0.14%. The major in vitro translation product of the purified mRNA preparation is specifically precipitated by a rabbit anti-rat FAH antiserum and it is, furthermore, undistinguishable by criteria of mass and charge from purified rat FAH.

scholarly journals In vitro insertion of the 22-kD peroxisomal membrane protein into isolated rat liver peroxisomes.

1993 ◽  
Vol 123 (6) ◽  
pp. 1717-1725 ◽  
Author(s):  
P Diestelkötter ◽  
W W Just
Keyword(s):  
Membrane Protein ◽  
Rat Liver ◽  
Atp Hydrolysis ◽  
Firefly Luciferase ◽  
In Vitro Translation ◽  
Membrane Insertion ◽  
Peroxisomal Membrane ◽  
Permeabilized Cells ◽  
Peroxisomal Membrane Protein

The membrane insertion of the 22-kD integral peroxisomal membrane protein (PMP 22) was studied in a system in which peroxisomes isolated from rat liver were incubated with the [35S]methionine-labeled in vitro translation product of PMP 22 mRNA. Membrane insertion of PMP 22 was demonstrated by protease treatment of peroxisomes in the absence and presence of detergent. Approximately 35% of total in vitro translated PMP 22 became protease resistant after a 1-h incubation at 26 degrees C. Import was dependent on time and temperature, did not require ATP or GTP and was not inhibited by N-ethylmaleimide treatment of neither the soluble components of the translation mixture nor of the isolated peroxisomes. In contrast to these results it was recently shown that the import of the peroxisomal marker, firefly luciferase, into peroxisomes of permeabilized cells was dependent on ATP hydrolysis and was blocked by N-ethylmaleimide pretreatment of the cytosol-depleted cells (Rapp et al., 1993; Wendland and Subramani, 1993). Therefore, the present data suggest that insertion of PMP 22 into the peroxisomal membrane and translocation of firefly luciferase into peroxisomes follow distinct mechanisms. At low temperature binding of PMP 22 to the peroxisomal membrane was not influenced whereas insertion was strongly inhibited. Pretreatment of peroxisomes with subtilisin reduced binding to a low level and completely abolished insertion. Therefore it is suggested that binding is prerequisite to insertion and that insertion may be mediated by a proteinaceous receptor.

scholarly journals Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein.

1986 ◽  
Vol 103 (3) ◽  
pp. 767-776 ◽  
Author(s):  
N M Kumar ◽  
N B Gilula
Keyword(s):  
Gap Junction ◽  
Rat Liver ◽  
Human Liver ◽  
Single Copy ◽  
In Vitro Translation ◽  
Coding Region ◽  
Human Cdna ◽  
Junction Protein ◽  
Gap Junction Protein

An extended synthetic oligonucleotide (58-mer) has been used to identify and characterize a human liver gap junction cDNA. The cDNA is 1,574 bases long and contains the entire coding region for a gap junction protein. In vitro translation of the RNA products of this cDNA is consistent with it coding for a 32,022-D protein. Southern blot analysis indicates that the gap junction gene is present as a single copy, and that it can be detected in a variety of organisms using the human liver cDNA as a probe. The human cDNA has been used to screen a rat liver cDNA library, and a rat liver junction cDNA clone has been isolated. The rat liver clone is 1,127 bases in length, and it has strong sequence homology to the human cDNA in the protein-coding region, but less extensive homology in the 3'-untranslated region.

scholarly journals Heterogeneity and properties of hepatic dexamethasone-binding proteins

1979 ◽  
Vol 180 (1) ◽  
pp. 187-193 ◽  
Author(s):  
S L H Liu ◽  
T E Webb
Keyword(s):  
Rat Liver ◽  
Binding Proteins ◽  
Binding Protein ◽  
Novikoff Hepatoma ◽  
Liver Cytosol ◽  
Activated Complex ◽  
Simple Dissociation ◽  
Rat Liver Cytosol

Evidence from experiments in vivo and in vitro is presented for the presence of three species of dexamethasone-binding proteins in rat liver, which are identified by chromatography on Sepharose 6B or by isoelectric focusing. Although two of these species (DI and DII) possess properties characteristic of a true receptor, the third binding protein (i.e. DIII), which migrates most slowly on Sepharose 6B, but has stability properties similar to protein DII, exhibits a 3-fold lower affinity for dexamethasone and the activated complex neither binds to DNA-cellulose nor translocates to the nucleus. Only the predominant liver receptor (DI), which is eluted first from Sepharose 6B, is present in Novikoff-hepatoma cytosol, suggesting that the major and minor species are not interconverted through simple dissociation during their isolation. The binding activities of all three species in the liver cytosol increase approx. 2-fold in vivo after adrenalectomy and show a transient 2-fold fall in vivo after the administration of cortisol. These changes in vivo in protein DIII shows a marked lag compared with those in proteins DI and DII, which change in parallel. It is therefore proposed that rat liver cytosol contains two dexamethasone receptors and a dexamethasone-binding protein that may be derived from these receptors.

scholarly journals Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles.

1982 ◽  
Vol 94 (2) ◽  
pp. 297-307 ◽  
Author(s):  
M M Mueckler ◽  
H C Pitot
Keyword(s):  
Rat Liver ◽  
Large Fraction ◽  
In Vitro Translation ◽  
Frequency Distributions ◽  
Distinct Frequency ◽  
Membrane Bound ◽  
Different Populations ◽  
And Function

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.

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