Studies of the expression of tumor-type poly(A) polymerase and the development of an accurate RNA polymerase II-directed in vitro transcription system
Total RNA was isolated from normal liver and Morris hepatoma 3924 A by a guanidine-HCI method. Poly(A+) RNA and poly(A-) RNA were separated by chromatography on oligo(dT)-cellulose. The various RNA fractions were translated using a rabbit reticulocyte lysate system. Following translation, labeled proteins were immunoprecipitated with anti-hepatoma poly(A) polymerase IgG or control IgG, subjected to polyacrylamide gel electrophoresis under denaturing conditions and visualized by autoradiography. Hepatoma poly (A+) RNA-directed translation yielded a unique 35 kDa protein after immunoprecipitation with immune IgG. Poly(A-) RNA from the hepatoma also contained the translatable sequences for this 35 kDa protein. Liver poly (A+) RNA-directed translation products did not contain detectable levels of this radiolabeled polypeptide. Since the antibodies demonstrated specificity for tumor-type poly(A) polymerase in both radioimmunoassays and Western blot analysis, it is concluded that the 35 kDa radiolabeled protein corresponds to tumor-type poly(A) polymerase. In an attempt to determine if tumor-type poly(A) polymerase is formed from a large precursor, hepatoma 7777 cells were metabolically labeled with (/sup 35/S) methionine. Lastly, a nuclear extract was prepared form the hepatoma and fractionated on a SEAE-Sephadex column. Transcription was sensitive to low levels of ..cap alpha..-amanitin, demonstrating that it was directed by RNA polymerase II.
- Research Organization:
- Pennsylvania State Univ., University Park (USA)
- OSTI ID:
- 6956579
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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PROTEINS
AUTORADIOGRAPHY
RNA POLYMERASES
BIOCHEMISTRY
ION EXCHANGE CHROMATOGRAPHY
TRANSCRIPTION
ELECTROPHORESIS
HEPATOMAS
IN VITRO
LIVER CELLS
METHIONINE
SULFUR 35
TUMOR CELLS
AMINO ACIDS
ANIMAL CELLS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMISTRY
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
DISEASES
DRUGS
ENZYMES
EVEN-ODD NUCLEI
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
NEOPLASMS
NUCLEI
NUCLEOTIDYLTRANSFERASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
SEPARATION PROCESSES
SOMATIC CELLS
SULFUR ISOTOPES
TRANSFERASES
550201* - Biochemistry- Tracer Techniques