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.

Elevated in vitro translation of a 25-kDa protein in renal cell carcinoma

1991 ◽  
Vol 69 (8) ◽  
pp. 561-565 ◽  
Author(s):  
Gilles Paradis ◽  
Josée Gaudreau ◽  
Gilles Frenette ◽  
Michel Thabet ◽  
Roland R. Tremblay ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Normal Tissue ◽  
Sodium Dodecyl ◽  
In Vitro Translation ◽  
Translation Product ◽  
Primary Translation Product ◽  
Rabbit Reticulocyte Lysate System

As a first step in understanding the changes in protein synthesis that occur in renal cell carcinoma, we have prepared poly(A)+ RNA from surgically removed tumors and from their normal tissue counterpart. These RNAs were then translated in vitro in the rabbit reticulocyte lysate system and the synthesized labeled polypeptides were separated by one- and two-dimensional gel electrophoresis. A major 25-kDa primary translation product was observed with all renal cell carcinomas. The synthesis of this protein was barely detectable with the RNA from normal tissue adjacent to the tumor. To determine if this protein could be further processed (removal of signal peptide and (or) core glycosylation), canine pancreatic microsomal membranes were added to the system. This addition resulted in the formation of a vertical row of three additional spots, with the same isoelectric point as the primary translation product and with molecular masses ranging from 27 to 31 kDa. The 31-kDa protein was retained on Concanavalin A. After digestion with endoglycosidase H, it was no longer visible on sodium dodecyl sulfate gels and a new 27-kDa band was generated suggesting that the mature protein was indeed a glycoprotein. Future experiments will be aimed at identifying this protein and examining its potential value as a marker of renal cell carcinoma.Key words: renal cancer, post-translational modifications, glycosylation, tumor markers.

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.

The in vitro translation product of the murine λ 5 gene contains a functional signal peptide

1988 ◽  
Vol 25 (8) ◽  
pp. 687-693 ◽  
Author(s):  
Jan Jongstra ◽  
Jenny Jongstra-Bilen ◽  
George F. Tidmarsh ◽  
Mark M. Davis
Keyword(s):  
Signal Peptide ◽  
In Vitro Translation ◽  
Translation Product

OPSIN mRNA ISOLATION FROM BOVINE RETINA AND PARTIAL SEQUENCE OF THE IN VITRO TRANSLATION PRODUCT

1980 ◽  
Vol 343 (1 Precursor Pro) ◽  
pp. 347-355 ◽  
Author(s):  
David S. Papermaster ◽  
Yigal Burstein ◽  
Israel Schechter
Keyword(s):  
In Vitro Translation ◽  
Partial Sequence ◽  
Translation Product ◽  
Bovine Retina

In vitro translation of mRNA for arginine esterase, the major secretory protein of dog prostate, and in vitro processing of the translation product

1985 ◽  
Vol 63 (7) ◽  
pp. 705-710 ◽  
Author(s):  
Pierre Chapdelaine ◽  
Jean Y. Dubé ◽  
Gilles Frenette ◽  
Roland R. Tremblay
Keyword(s):  
Molecular Weight ◽  
Signal Peptide ◽  
Seminal Plasma ◽  
Gel Electrophoresis ◽  
In Vitro Translation ◽  
Translation Product ◽  
Two Dimensional ◽  
Single Chain ◽  
Two Dimensional Gel Electrophoresis

Poly(A)+ rich RNA was isolated from prostate of adult dogs and translated in the rabbit reticulocyte lysate cell-free protein-synthesizing system. Two-dimensional gel electrophoresis of the translation products showed that a protein with a molecular weight of 31 000 was predominantly synthesized. This protein was immunoprecipitated with antibodies directed against purified arginine esterase from dog seminal plasma. mRNA isolated from the prostate of animals castrated for 1 or 2 weeks was unable to direct the synthesis of arginine esterase. However, the synthesis of the enzyme could be stimulated by androgens in castrated animals, presumably by increasing prostatic concentrations of arginine esterase mRNA. The single chain translation product could be further processed in vitro by the addition of dog pancreas microsomes and purified arginine esterase. This procedure yielded split chains of arginine esterase which had identical electrophoretic mobilities as seminal plasma enzyme by two-dimensional gel electrophoresis. When prostatic tissue slices were incubated with tunicamycin, the unglycosylated arginine esterase obtained had a lower molecular weight than the in vitro translation product, suggesting that a signal peptide had been removed in the living cells. These results indicate that arginine esterase processing may include the following steps: removal of a signal peptide, glycosylation, and splitting of the polypeptide chain by active arginine esterase in the secretory granules or outside the cell.

Sign in / Sign up

Export Citation Format

Share Document