Isolation and molecular weight of pure feather keratin mRNA

Biologically active feather keratin mRNA which directs the synthesis of most or all the keratin polypeptide chains, M.W. 10,000, was prepared either by cellulose chromatography or by dissociation of mRNP particles with Na dodecyl sulphate. Both preparations gave 1 RNA band of MW 250,000 on formamide-acrylamide gels indicating the presence of long untranslated segment(s) whose length has been conserved during the evolution of individual keratin genes.     

Translation of immunoglobulin mRNAs in a wheat germ cell-free system

An unfractionated wheat germ cell-free system will efficiently translate immunoglobulin messenger RNAs from four murine myelomas. The system responds as well to immunoglobulin mRNA as to globin mRNA and translates mRNAs for both heavy and light immunoglobulin chains. The mRNAs for both kappa and lambda chains are translated into polypeptides 1700–2000 daltons larger than the authentic light chains. Chain completion is poor with most mRNAs, but improves when the reactions are done at KCl concentrations considerably higher than the optimum for maximal incorporation of radioactivity. Mammalian transfer RNA stimulates translation of all mRNAs tested.     

Translation of glutamate dehydrogenase mRNA in a reticulocyte cell-free system

Messenger RNA template activity for glutamate dehydrogenase was detected in poly(A)-rich RNA extracted from rat liver polysomes. Enzyme synthesized in cell-free reticulocyte system was detected by measuring enzyme activity in the translation incubation mixture using dual wavelength spectrophotometric technique. The translation product was also identified by a partial purification of the labeled synthesized enzyme and by coelectrophoresis with the carrier enzyme preparation from mitochondrial matrix.     

Translation of bacteriophage T4 mRNA into active lysozyme by a wheat germ cell-free system.

T4 bacteriophage mRNA for lysozyme was extracted from T4 phage infected $\text{E. coli}$ cells, partially purified by column chromatography, and translated in a heterologous cell-free protein synthesizing system prepared from wheat germ. The translation product was confirmed by SDS polyacrylamide gel electrophoresis and enzymatic activity — bacteriolysis as tested with $\text{Micrococcus luteus}$. The specific activity of the enzyme prepared was 660 U/mg.     

The terminal structures of feather keratin mRNA

Terminal labeling of embryonic feather keratin mRNA with [³H]KBH₄ followed by digestion with ribonuclease T₁ and T₂, alkaline phosphatase, snake venom phosphodiesterase, and nucleotide pyrophosphatase and analysis of the products by high voltage paper electrophoresis, indicated the presence of the sequence m⁷G(5)ppp(5)N at the 5-end of the mRNA. Ribonuclease T₁ and A digests of the terminally labeled, and also of unlabeled mRNA followed by fractionation on denaturing polyacrylamide gels indicated the presence of polyadenylate tracts ranging in size from 45 to 165 nucleotides at the 3-end of the mRNA.     

Control of feather keratin synthesis by the availability of keratin mRNA

The relative timing of the synthesis of keratin and its mRNA in the developing chick embryo feather has been examined. Study of both active mRNA in polysomes, and of the total number of mRNA sequences in the tissue, leads to the conclusion that the rate-limiting step in the synthesis of keratin is the accumulation in the cytoplasm of its mRNA.     

Translation of collagen messenger RNA in a cell-free system derived from wheat germ.

A cell-free system for synthesizing protein from wheat germ was used to translate the messenger RNA extracted from 16-day embryonic chick calvaria. A part of the product had properties similar to collagenous peptides and served as a substrate for prolyl hydroxylase, an enzyme specific for collagen. The level of potassium was critical for the synthesis of high molecular weight products with properties similar to pro-alpha-chains. The potassium concentration for optimal protein synthesis, as judged by maximum incorporation of [3H]proline into acid precipitable material, was considerably lower than the concentration required for the synthesis of high molecular weight collagenous peptides.     

Translation in a cell-free system of mRNA from term placenta extracted by guanidine hydrochloride

Human term placenta RNA and polyadenylated mRNA were prepared using guanidine HCl and oligo (dT)-cellulose affinity chromatography. Both fractions translated in a wheat germ cell-free system showed, under optimal condition of K+ and Mg++ ions and spermidine, about 9 times activity for RNA and 15-25 times for poly(A+) mRNA greater than the control. Homogenization of the tissue at high speed compared to that at low speed improved 4-fold activity. Analysis of tritiated products by SDS-Polyacrylamide gel electrophoresis and detected by fluorography showed more than ten different intensity bands ranging between 12 and 66 kD. According to the results obtained, guanidine HCl is an advantageous procedure for the extraction of RNA from this nuclease-rich tissue compared with that obtained with phenol extraction, in both activity and in larger translation products.     

Translation of albumin messenger RNA in a cell-free protein-synthesizing system derived from wheat germ.

Purified rat liver albumin mRNA directed the synthesis of albumin in a mRNA-dependent cell-free protein-synthesizing system derived from wheat germ extracts. The [3H]leucine-labeled in vitro translation product reacted with antibodies specific for albumin and co-migrated with authentic 14C-labeled serum albumin during gel electrophoresis in the presence or absence of sodium dodecyl sufate. Higher concentrations of potassium and magnesium ions were required for the translation of albumin mRNA than for total liver mRNAs. These requirements were consistent for the purified albumin as well as when it was a component in the liver mRNA mixture. At the higher potassium or magnesium concentrations, only intact albumin molecules were synthesized, whereas lower concentrations of these ions caused the production of antibody-reactive fragments. These fragments were apparently the result of premature termination of peptide synthesis and not due to endogenous proteolytic activity.     

Multi-hour translation of mRNA in a cell-free system

In this study, we demonstrate that mRNA molecules can serve as an efficient template for cell-free translation through a combination of methods to protect them from nucleolytic digestion. Removal of major endonucleases activity from cell extract, the addition of a stemloop structure at the 3??-end of the mRNA and continuous reloading of ribosomes onto mRNA were found to be crucial for maintaining the functional integrity of mRNA during cell-free synthesis. When these three approaches were combined, mRNA-directed protein synthesis continued over 15 h, leading to the production of 2.6 mg/mL of encoded protein. The methods for direct translation of mRNA presented herein will provide a useful option for deciphering genetic information, including the fields of mRNA display and materialization of metagenomic information.     

Translation of mRNA extracted from human placenta in a wheat germ cell free system

The mRNA were isolated from total RNA extracted from placenta by affinity chromatography on poly U Sepharose 4 B and were tested with wheat germ cell free system. The neosynthesized hPL is isolated by specific immunoprecipitation using 2 antibodies from the CEA (hPLK1 and hPLK3). It represented 7% of the total radioactive synthesized proteins. Two components were separated by gel electrophoresis. One is the natives hormone while the other which is the major component, migrated with a molecular weight of 25 000. These results are in accordance with a functionally cell free system which could be used study the non histone protein's role in the hPL synthesis.