A simple method for the purification of reticulocyte globin messenger ribonucleic acid

A relatively simple and inexpensive method has been developed for the preparation of highly purified rabbit reticulocyte globin mRNA. After phenol extraction, polysomal RNA was chromatographed on Sigmacell type 38 cellulose and Sepharose 4B. The resulting mRNA preparation has a purity in excess of 90%. No selective loss of either alpha or beta globin mRNA is observed.     

Translation of messenger RNA fractions extracted from free and membrane bound rat forebrain ribosomes in a rabbit reticulocyte cell-free system

Messenger RNA fractions were obtained from free and membrane bound rat forebrain ribosomes by alkaline phenol extractions. These RNA fractions stimulated protein synthesis in a cell-free rabbit reticulocyte system partially dependent on the addition of exogenous mRNA. The polypeptide products of protein synthesis with RNA fractions derived from free and membrane bound brain ribosomes and reticulocyte ribosomes were compared by polyacrylamide gel electrophoresis and found to have different distributions.     

Translation of myosin heavy chain messenger ribonucleic acid in an eukaryotic initiation factor 3- and messenger-dependent muscle cell-free system.

A cell-free protein synthesis system has been prepared from embryonic chick muscle; this system is dependent on initiation factor eukaryotic initiation factor 3 (eIF-3) and mRNA for efficient translation. Highly purified chick muscle eIF-3 has been fractionated into "core" and discriminatory components. In the presence of core eIF-3 from chick muscle or rabbit reticulocytes, myosin heavy chain mRNA is translated less efficiently than globin mRNA present in an equimolar concentration. When the discriminatory components are added to core eIF-3 from either source, myosin mRNA is translated with a greater efficiency. Thus, chick muscle eIF-3 contains components which allow it to recognize and stimulate specifically the translation of myosin mRNA in a muscle cell-free protein synthesis system.     

Endogenous messenger ribonucleic acid-directed polypeptide chain elongation in a cell-free system from the yeast Saccharomyces cerevisiae.

An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respcetively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 muM pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes invitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35-S]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.     

A fractionated cell-free system for analysis of prophase nuclear disassembly

We describe a cell-free system in which a postribosomal supernatant (s140) from metaphase Chinese hamster ovary (CHO) cells induces prophase-like changes in isolated CHO cell nuclei, including chromatin condensation, and nuclear envelope and lamina disassembly. These events are strongly promoted by gamma-S-ATP and an ATP-regenerating system, and do not take place with an s140 derived from G2-phase cells. The metaphase cell s140 also induces disassembly of an isolated nuclear lamina fraction that is depleted of membranes, chromatin, and nuclear pore complexes. Disassembly of the isolated lamina is accompanied by phosphorylation of the major lamina proteins (lamins A, B, and C) to levels characteristic of metaphase cells. Kinetic analysis of lamina depolymerization indicates that cooperativity may be involved in this process. The biochemical properties of in vitro lamina disassembly suggest that the activity that depolymerizes the lamina during mitosis is soluble in metaphase cells, and support the notion that this activity is a lamin protein kinase.     

The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

A cell-free protein-synthesizing system has been prepared from Saccharomyces cerevisiae by differential centrifugation of lysed spheroplasts. The preparation, a modified 100,000 x g supernatant fraction, contains ribosomes and monosomes, ribosomal subunits, translation factors, and aminoacyl-tRNA synthetases, but no polysomes. After removal of small amounts of remaining mRNA with micrococcal nuclease, protein synthesis is stringently dependent on the addition of mRNA, as well as amino acids and an energy-generating system. The 5'-cap analogue, 7-methylguanosine 5'-phosphate, inhibits translation of several natural mRNAs, but has no effect on chain elongation. Incubation of the polysome-free extract with natural mRNA leads to the formation of protein-synthesizing polysomes and eventually, to the release of protein; the molecular weight of the protein synthesized in the presence of BMV (brome mosaic virus) RNA is consistent with that of BMV coat protein.     

Neurospora crassa glutamine synthetase. Translation of specific messenger ribonucleic acid in a cell-free system derived from rabbit reticulocytes.

The total reticulocyte lysate cell-free protein-synthesizing system was incubated in the presence of Neurospora crassa RNA. With the aid of an antibody directed against purified N. crassa glutamine synthetase, the synthesis of a specific protein was detected. This protein precipitates with antiglutamine synthetase using both direct and indirect procedures, migrates with the same molecular weight as the monomer of N. crassa glutamine synthetase when subjected to acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and chromatographs as N. crassa glutamine synthetase on anthranilate-bound Sepharose. These data indicate the translation of the mRNA that codes for N. crassa glutamine synthetase. This RNA behaves as poly(A)-containing material when fractionated on oly(U)-Sepha-rose.     

Translation of avian myeloblastosis virus RNA in a reticulocyte cell-free system.

AMV-RNA was translated into a precursor polypeptide of 76,000–80,000 daltons in a reticulocyte cell-free system. Besides this high molecular weight precursor, several smaller precursor polypeptides and the four major internal structural viral proteins were also synthesized. These virus-specific translation products were detectable after immunoprecipitation with antiserum against the gs-antigens of AMV.