sodium-dodecyl-sulfate and Carcinoma--Krebs-2

The polypeptides of encephalomyocarditis, Mouse-Elberfeld and type 5 rhinoviruses behave similarly when chromatographed on calcium phosphate (brushite), each being eluted by a linear phosphate buffer gradient containing sodium dodecyl sulphate in three major peaks, CI, C2 and C3. Analysis of the peaks by polyacrylamide gel electrophoresis suggests that the major capsid polypeptides of these three picornaviruses elute in the order: delta (peak CI), gamma with (peak C2) and alpha (peak C3).

Topics: Animals; Calcium Phosphates; Carbon Radioisotopes; Carcinoma, Krebs 2; Cell Line; Chromatography; Electrophoresis, Polyacrylamide Gel; Encephalomyocarditis virus; Enterovirus; Genetic Variation; HeLa Cells; Humans; Maus Elberfeld virus; Mice; Molecular Weight; Peptides; Rhinovirus; Sodium Dodecyl Sulfate; Viral Proteins

Passage of cell-free extracts of rabbit reticulocytes through heparin-Sepharose affinity columns results in the loss of the ability of the effluent to initiate protein synthesis. This is shown by the loss of response to added rabbit globin mRNA or to inhibitors of initiation of protein synthesis, such as heparin and aurin tricarboxylic acid, and by recovery of initiation activity by addition of protein retained and subsequently eluted from the columns. The effluent retains, however, the ability to elongate protein chains. Only 0.8% of the applied cell extract protein binds to heparin-Sepharose columns. This bound protein, which can be recovered by increasing the salt concentration of the eluting buffer, has initiation factor activity equal to that of a crude initiation factor preparation obtained from rabbit reticulocyte ribosomes by extraction with 0.5 M KCl. The protein patterns on polyacrylamide gels of the initiation factors prepared by either method are very similar and indicate a protein mixture, which may represent a complex. These data confirm that heparin interacts specifically with initiation factos, and indicate that heparin-Sepharose chromatography will simplify procedures for the preparation of initiation factors.

Topics: Animals; Aurintricarboxylic Acid; Blood Proteins; Carcinoma, Krebs 2; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Female; Globins; Heparin; Leucine; Mice; Peptide Initiation Factors; Rabbits; Reticulocytes; RNA, Messenger; Sepharose; Sodium Dodecyl Sulfate

Topics: Adenosine Monophosphate; Animals; Carcinoma, Krebs 2; Cell-Free System; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Hydrocortisone; Immunodiffusion; Liver; Mice; Polynucleotides; Protein Biosynthesis; Rats; RNA, Messenger; Sodium Dodecyl Sulfate; Transcription, Genetic; Tryptophan Oxygenase

Topics: Adenoviridae; Animals; Carcinoma; Carcinoma, Krebs 2; Cell Line; Cell-Free System; Centrifugation, Density Gradient; Chromatography, DEAE-Cellulose; Chromatography, Ion Exchange; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Hydroxyapatites; Magnesium; Methionine; Mice; Mouth Neoplasms; Peptide Fragments; Phosphates; Polyribosomes; Protein Biosynthesis; Sodium Dodecyl Sulfate; Spectrophotometry, Ultraviolet; Sulfur Radioisotopes; Tritium; Trypsin; Uridine; Viral Proteins

1. When prostate polyribosomes are labelled with radioactive precursors in vivo and subsequently dissociated with sodium dodecyl sulphate, a heterogeneous 6-15S RNA species may be identified that possesses all of the distinctive properties of mRNA. 2. Apart from the selective incorporation of 5'-fluoro-orotic acid into this 6-15S RNA component, it is bound by nitrocellulose filters under experimental conditions where only poly(A)-rich species of RNA are specifically retained. Most importantly, however, only the 6-15S RNA fraction is capable of promoting the incorporation of amino acids into peptide linkage in an mRNA-depleted cell-free system derived from ascites-tumour cells. 3. With the development of a simpler method for labelling the total RNA fraction of the prostate gland in vitro, the poly(A)-enriched RNA fraction may be readily isolated by adsorption and elution from oligo(dT)-cellulose. The synthesis of the poly(A)-enriched 6-15S RNA fraction is stringently controlled by androgens in a highly tissue- and steroid-specific manner. 4. From an analysis of the proteins synthesized in the ascites cell-free system in the presence of the poly(A)-rich RNA fraction, it appears that protein synthesis in the prostate gland is stimulated in a rather general way, even during the earliest phases of the androgenic response. This conclusion may require modification when more specific means of analysis are available than those used in the present investigation. 5. The implications of these findings to the mechanism of action of androgens are discussed.

Topics: Amino Acids; Animals; Carbon Radioisotopes; Carcinoma, Krebs 2; Castration; Cell Fractionation; Centrifugation, Density Gradient; Electrophoresis, Polyacrylamide Gel; In Vitro Techniques; Injections, Intraperitoneal; Male; Organ Specificity; Orotic Acid; Polyribosomes; Prostate; Protein Biosynthesis; Rats; RNA, Messenger; RNA, Ribosomal; RNA, Transfer; Sodium Dodecyl Sulfate; Sulfur Radioisotopes; Testosterone; Tritium; Uridine

In a very active cell-free system containing polysomes derived from human placenta and a cell-sap fraction prepared from ascites tumor cells, the synthesis of the hormone human placental lactogen (HPL) was detected. The identification was based on the following: (a) The in vitro synthesized protein labeled with [(35)S]methionine migrated at the same rate as authentic HPL on sodium dodecyl sulfate-polyacrylamide gels and (b) tryptic fingerprint analysis of the labeled protein yielded peptides having the same mobilities as seen with the same analysis of purified HPL. The amount of HPL synthesized in a cell-free system containing polysomes derived from term placenta was about 10% of the total proteins synthesized and in a comparable system containing first trimester ribosomes the level of synthesis was about 5%. These data suggest the potential for quantitating the HPL mRNA activity as a function of the period of gestation and for isolating the mRNA itself.

Topics: Animals; Autoradiography; Carcinoma, Krebs 2; Cell Fractionation; Cell-Free System; Electrophoresis, Polyacrylamide Gel; Female; Gestational Age; Humans; Methionine; Placenta; Placental Lactogen; Pregnancy; Ribosomes; RNA, Messenger; Sodium Dodecyl Sulfate; Sulfur Radioisotopes; Time Factors

A highly active in vitro system for the translation of globin mRNA, resulting in more than 10 rounds of translation, is described. The reconstituted system consists of native small ribosomal subunits of rabbit reticulocytes (as a source of initiation factors as well as small ribosomal subunits), large subunits derived from rat liver polysomes by the puromycin-KCl procedure, and a pH 5 fraction obtained from a Krebs ascites cell high speed supernatant. In this system no differences were found between globin messenger ribonucleoprotein and globin mRNA.

Topics: Animals; Carcinoma, Krebs 2; Cell Fractionation; Electrophoresis, Polyacrylamide Gel; Globins; Hydrogen-Ion Concentration; Liver; Mice; Nucleoproteins; Peptide Chain Initiation, Translational; Peptide Initiation Factors; Polyribosomes; Potassium Chloride; Protein Biosynthesis; Puromycin; Rabbits; Reticulocytes; Ribosomes; RNA, Messenger; Sodium Dodecyl Sulfate

Topics: Animals; Binding Sites; Carbon Radioisotopes; Carcinoma, Krebs 2; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Hydroxyapatites; Kinetics; Mice; Molecular Weight; Organ Specificity; Peptide Chain Elongation, Translational; Peptide Elongation Factors; Phenylalanine; Phosphorus Radioisotopes; Protein Binding; Ribosomes; Sodium Dodecyl Sulfate; Species Specificity; Spectrophotometry, Ultraviolet; Sucrose; Tritium

Cell-free extracts from Krebs ascites cells and rabbit reticulocytes synthesized a variety of viral-specific proteins when programmed with several different kinds of Sindbis viral RNAs. The RNAs included purified virion RNA (42S) and two species (26S and "33S") of purified intracellular viral messenger RNAs from viral-infected BHK cells. Proteins formed in vitro were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rate-zonal centrifugation in urea-sucrose gradients, two-dimensional tryptic peptide fingerprints, and immunoprecipitation with rabbit anti-Sindbis virus serum. The only major identifiable protein formed in vitro was viral capsid, but the relative amount of capsid produced was determined by the mRNA, the source of cell-free extract, and the components of the cell-free system. Virion RNA directed synthesis of larger-molecular-weight proteins than did intracellular viral RNAs, and some of this protein was distinct from that formed by the smaller viral RNAs. Indirect evidence is presented for in vitro synthesis of viral envelope proteins.

Topics: Animals; Autoradiography; Carcinoma, Krebs 2; Cell-Free System; Centrifugation, Zonal; Electrophoresis, Polyacrylamide Gel; Hemagglutinins, Viral; In Vitro Techniques; Precipitin Tests; Rabbits; Reticulocytes; RNA, Messenger; RNA, Viral; Sindbis Virus; Sodium Dodecyl Sulfate; Sulfur Radioisotopes; Tritium; Viral Proteins

Topics: Anemia; Animals; Carcinoma, Krebs 2; Centrifugation, Density Gradient; Chromatography, Gel; Evaluation Studies as Topic; Methods; Mice; Nucleoproteins; Phenylhydrazines; Polyribosomes; Rabbits; Reticulocytes; RNA, Messenger; RNA, Ribosomal; Sodium Dodecyl Sulfate; Spectrophotometry, Ultraviolet

Topics: Animals; Carcinoma, Krebs 2; Cell-Free System; Chickens; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Evaluation Studies as Topic; Female; Globins; Leucine; Methods; Ovalbumin; Oviducts; Phenylhydrazines; Polyribosomes; Protein Biosynthesis; Rabbits; Reticulocytes; Ribosomes; RNA, Messenger; Sodium Dodecyl Sulfate; Spectrophotometry, Ultraviolet; Tritium

Growth hormone has been synthesized in a cell-free system derived from Krebs II ascites cells, under the direction of RNA prepared from rat pituitary tumor (GC) cells. Growth hormone synthesized in the cell-free system was identified by precipitation with antiserum against growth hormone developed in baboon, followed by electrophoretic analysis of the dissolved precipitate on sodium dodecyl sulfate-polyacrylamide gels. RNA from both the membrane and the post-membrane fractions of the cytoplasm of GC cells stimulated protein synthesis in the cell-free system, but only RNA from the membrane fraction was found to direct the synthesis of growth hormone.

Topics: Animals; Carbon Radioisotopes; Carcinoma, Krebs 2; Cell-Free System; Electrophoresis, Polyacrylamide Gel; Growth Hormone; Immunoassay; Immunoelectrophoresis; Kinetics; Leucine; Methionine; Papio; Peptide Biosynthesis; Phosphocreatine; Phosphoenolpyruvate; Pituitary Neoplasms; Rats; RNA, Neoplasm; Sodium Dodecyl Sulfate; Sulfur Radioisotopes

Topics: Animals; Carbon Isotopes; Carcinoma, Krebs 2; Cattle; Cell-Free System; Electrophoresis, Paper; Electrophoresis, Polyacrylamide Gel; Encephalomyocarditis virus; Globins; Kinetics; Methionine; Neoplasm Proteins; Peptide Initiation Factors; Poly U; Potassium; Protein Biosynthesis; Puromycin; Rabbits; Reticulocytes; Ribosomes; RNA, Messenger; RNA, Neoplasm; RNA, Viral; Sodium Dodecyl Sulfate

A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described. The method depends upon annealing poly(adenylic acid)-rich mRNA to oligothymidylic acid-cellulose columns and its elution with buffers of low ionic strength. Biologically active rabbit globin mRNA has been purified by this procedure and assayed for its ability to direct the synthesis of rabbit globin in a cell-free extract of ascites tumor. Inasmuch as various mammalian mRNAs appear to be rich in poly(adenylic acid) and can likely be translated in the ascites cell-free extract, this approach should prove generally useful as an initial step in the isolation of specific mRNAs.

Topics: Acrylamides; Animals; Carcinoma, Krebs 2; Cell-Free System; Centrifugation, Density Gradient; Chromatography; Chromatography, Ion Exchange; Electrophoresis; Globins; Leucine; Methods; Neoplasm Proteins; Polynucleotides; Rabbits; Reticulocytes; RNA, Messenger; RNA, Ribosomal; Sodium Dodecyl Sulfate; Thymine Nucleotides; Tritium

A cell-free system derived from Krebs II ascites tumor has been used to assay biologically active mRNA for myeloma (MOPC-41) light chain during its purification by oligothymidylate-cellulose chromatography and sucrose gradient centrifugation. The purified mRNA directs the synthesis of a product that yields tryptic peptides corresponding to those derived from authentic myeloma protein and that forms a specific immunoprecipitate with antibody directed against the MOPC-41 protein. The fact that the light-chain mRNA anneals to oligothymidylic acid-cellulose suggests that it, like several other eukaryotic mRNAs, contains a region rich in adenylic acid residues. The most active fractions of light-chain mRNA, representing about 0.1% of the RNA originally extracted from membrane-bound myeloma polysomes, sediment as a discrete peak with an s(20,w) of about 13, roughly corresponding to an RNA molecule containing 850 bases. The results suggest that the light-chain mRNA is monocistronic and that it contains about 200 more bases than would be necessary to encode the variable and constant regions of a single light-chain molecule.

Topics: Adenine Nucleotides; Animals; Carbon Isotopes; Carcinoma, Krebs 2; Centrifugation, Density Gradient; Chromatography, Affinity; Chromatography, Ion Exchange; Goats; Leucine; Multiple Myeloma; Myeloma Proteins; Peptides; Precipitin Tests; Ribosomes; RNA, Messenger; RNA, Neoplasm; Sodium Dodecyl Sulfate; Thymine Nucleotides; Tritium; Trypsin

Single-stranded reovirus RNA, synthesized in vitro by reovirus cores, functioned as messenger RNA in cell-free extracts prepared from several mammalian cells: Krebs II mouse ascites cells, mouse L cells, Chinese hamster ovary cells, HeLa cells, and rabbit reticulocytes. As shown by acrylamide gel electrophoresis, all eight polypeptides known to be specified by reovirus were synthesized in the reticulocyte system. In the other extracts, from 5 to 7 complete virus proteins were made.

Topics: Animals; Autoradiography; Carbon Isotopes; Carcinoma, Krebs 2; Cell Line; Cricetinae; Electrophoresis; Female; HeLa Cells; L Cells; Leucine; Methionine; Mice; Ovary; Rabbits; Reoviridae; Reticulocytes; RNA, Messenger; RNA, Viral; Sodium Dodecyl Sulfate; Sulfur Isotopes; Tritium; Viral Proteins

Topics: Animals; Carcinoma, Krebs 2; Cellulose; Chickens; Chromatography; Electrophoresis, Disc; Globins; Kinetics; Leucine; Macromolecular Substances; Ovalbumin; Oviducts; Peptide Initiation Factors; Precipitin Tests; Protein Biosynthesis; Rabbits; Reticulocytes; Ribosomes; RNA, Messenger; Serine; Sodium Dodecyl Sulfate; Tritium

Topics: Amino Acid Sequence; Amino Acids; Animals; Autoradiography; Carbon Isotopes; Carcinoma, Krebs 2; Cell-Free System; Centrifugation, Density Gradient; Electrophoresis, Polyacrylamide Gel; Humans; Immunoglobulin Fragments; Methionine; Microsomes; Protein Biosynthesis; RNA, Messenger; RNA, Neoplasm; Sodium Dodecyl Sulfate; Sulfur Isotopes