Electrophoretically homogeneous myeloma light chain mRNA and its translation in vitro

mRNA coding for the light chain of a myeloma protein has been purified to give one band in acrylamide gel electrophoresis. This pure RNA (S～13.5) could be translated $\text{in}\text{vitro}$ into the light chain in a heterologous cell-free translation system. The light chain synthesized $\text{in vitro}$ is apparently slightly larger than the light chain secreted by the tumor.     

Synthesis of metallothionein in a polysomal cell-free system.

A new covalent chromatography system utilizing Activated Thiol Sepharose 4B was employed to quantitate the content of thionein chains synthesized in a polysomal cell-free system. Liver polysomes from zinc injected rats directed the translation of more thionein-like polypeptide chains than polysomes from control rats. The increase was similar to the stimulation in MT synthesis $\text{in vivo}$ following a zinc injection. This evidence supports the concept that metallothionein synthesis is regulated by changes in the pool of translatable thionein mRNA.     

Lactate dehydrogenase-C mRNA: Its isolation and invitro translation

Lactate dehydrogenase-C (LDH-C) mRNA was purified from $\text{DBA}\text{2}$ mouse testes and translated $\text{in}\text{vitro}$. First, the LDH-C synthesizing polysomes were isolated by double immunoprecipitation using specific anti-LDH-C and anti-horse immunoglobulin antibodies. Extraction of mRNA was made from the isolated polysomes using hot sodium dodecyl sulfate-phenol method at alkaline pH. In a wheat germ cell-free translation system, the mRNA coded for a polypeptide chain that could be immunoprecipitated with specific anti LDH-C antibody and comigrated with authentic LDH-C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

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.     

Identification of the covalently bound flavin of L-gulono-gamma-lactone oxidase.

A specific mRNA for a structural lipoprotein of the $\text{Escherichia}\text{coli}$ outer membrane was translated in a wheat germ cell-free protein synthesizing system, S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) had neither stimulative nor inhibitory effect on the translation. When the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two peaks appeared at the appearent molecular weights of about 15,000 and about 7,500. Both products were cross-reactive with antiserum against the lipoprotein.     

In vitro translation of mRNA from rat pheochromocytoma tumors, characterization of tyrosine hydroxylase

Studies are presented which demonstrate that rat pheochromocytoma tumors are a convenient material for the preparation of tyrosine hydroxylase mRNA. Total pheochromocytoma poly(A)⁺mRNA has been extracted from tumors, then translated in a reticulocyte lysate cell-free system. Neo-synthesized tyrosine hydroxylase has been identified by direct immunoprecipitation followed by sodium dodecyl sulfate acrylamide gel electrophoresis. The proportion of this specific mRNA has been calculated; it represents 0.15 per cent of the total poly(A)⁺mRNA. The molecular weight of the $\text{in}\text{vitro}$ synthesized tyrosine hydroxylase is 62,000.     

Translation of endogenous message in embryonic chick erythroid cell polysomes in a cell-free protein-synthesizing system

Polysomes from 5.5-day and 6.5-day embryonic chick erythroid cells contain messenger RNA (mRNA) which can be translated into products in a cell-free protein-synthesizing system. The products of the cell-free system coelectrophoresed with carrier globin chains from the cells from which the polysomal pellet was isolated. Wheat germ S30 fraction increases by 1.5--2.0 fold [3H] leucine incorporation into trichloroacetic acid-precipitable material directed by the endogenous mRNA on chick erythroid cell polysomes. The wheat germ probably provides a specific factor or factors absent in a shortage in the incubation medium.     

Translation of mouse interferon mRNA in Xenopus laevis oocytes and in rabbit reticulocyte lysates

Mouse interferon mRNA, extracted from NDV (Newcastle disease virus)-induced L-929 cells has been translated with high efficiency in $\text{Xenopus laevis}$ oocytes and rabbit reticulocyte lysates. The translational efficiency of a crude RNA extract was 10 640 interferon units/mg RNA/hour for the $\text{Xenopus}$ oocytes and 4 012 interferon units/mg RNA/hour for the reticulocyte lysates. The translation product fulfilled the usual criteria for mouse interferon, $\text{viz.}$ species specificity and neutralization by specific anti-mouse interferon antiserum. Upon injection of crude interferon mRNA into $\text{Xenopus}$ oocytes, interferon activity appeared both in the oocyte homogenates and the oocyte incubation medium. When analyzed by velocity sedimentation in formamidesucrose, the mouse interferon mRNA showed a rather sharp peak halfway between the 4 S and 18 S RNA markers, as could be expected from a mRNA which codes for a 20,000 dalton protein.     

The role of ribosomes in stabilizing bacteriophage T4 deoxynucleotide kinase mRNA.

In the present investigation, an approach toward defining the role of ribosomes in stabilizing functional messenger RNA in cell-free extracts is described. The data presented show that initiation of protein synthesis is necessary for maximal functional stability of bacteriophage T4 deoxynucleotide kinase mRNA $\text{in vitro}$ and suggest that much of the stability is attained by interaction of the deoxynucleotide kinase mRNA initiation site with a 30S ribosomal subunit. Data is also presented which suggest that any of several $\text{E}$. $\text{coli}$ ribonucleases could serve as a messenger ribonuclease $\text{in vivo}$.     

Alteration of ribosomal protein S2 in kasugamycin-resistant mutant derived from Escherichia coli AB312

A new type of kasugamycin-resistant mutant has been isolated from $\text{E. coli}$ K12, strain AB312 (Hfr, $\text{lac,thr,leu,thi,strA,fus}$). In a cell-free protein-synthetic system, the resistance is localized in the ribosome but not in the supernatant fraction. On initiation complex formation, the resistance is associated with the washed ribosome but not with initiation factors. In reconstitution of the 30S ribosomal subunit, the resistance is due to the protein(s) but not to 16S RNA. In two-dimensional electrophoresis, protein S2 is deficient in the 30S ribosomal subunit of kasugamycin-resistant mutant. The results indicate that the kasugamycin-resistance is attributed to alteration of ribosomal protein S2.     

alpha-Fetoprotein biosynthesis and hepatocellular differentiation

In anemia of the Belgrade rat ($\text{b}\text{b}$) reticulocytes contain less than half of the normal amount of mRNA for seven adult rat globin chains. cDNA hybridization measurements of the relative sizes of polysomal and nonpolysomal pools of globin mRNAs in these cells show that 45% of all globin mRNA molecules are not used at any given time in protein synthesis. This implies a translational control which ensures a production of globin chains in a correct ratio despite a severe mRNA unbalance.     

Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [¹⁴C]- or [³H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·10⁴ and 2.1·10⁴ were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with $\text{N-[}{}^3\text{H]ethylmaleimide}$ or $\text{N-[}{}^{14}\text{C]ethylmaleimide}$, and the ³H to ¹⁴C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.     

Crosslinking of proteins to ribosomal RNA in HeLa cell polysomes by sodium periodate.

HeLa cell polysomes were oxidized with sodium periodate and reduced with sodium borohydride to induce covalent crosslinks between ribosomal RNA and nearby proteins. We proved that RNA was tryly crosslinked to protein in oxidized, and not in control, samples using denaturing cesium trichloroacetate density gradients and phenol extraction. By both one- and two-dimensional gel analysis, we found that protein S3a can be crosslinked to 18S RNA, protein L3 to 28S RNA, and proteins L7′ and L23′ to 5.8S RNA. Because of the specificity of the periodate reaction, and since we were able to crosslink protein S1 to 16S RNA in $\text{Escherichia}$,$\text{coli}$ 30S ribosomal subunits, it is likely that we have crosslinked proteins to the 3′OH ends of HeLa polysomal RNAs.     

Base-paired interaction, in vitro, between hen globin 9S mRNA and eukaryotic ribosomal RNAs.

Hen globin 9S mRNA complexes efficiently with mouse sarcoma 18S rRNA, and to a lesser extent with 28S rRNA, but not with tRNA. The mRNA-18S rRNA complex is dissociated under conditions that lead to disruption of hydrogen bonds, and exhibits a biphasic thermal denaturation curve with Tms at $\text{ca.}$ 39° and 58° in 0.15 $\text{M}$ NaCl-0.03 $\text{M}$ Tris-HCl, pH 7.5. Hen globin mRNA also interacts with 18S rRNAs from various other eukaryotes, and the melting profile and Tm of the complex formed with hen 18S rRNA is very similar to that of the complex formed with mouse sarcoma 18S rRNA.     

Nature of eukaryotic proteins required for joining of 40S and 60S ribosomal subunits

A mixture of 40S and 60S subunits from salt-washed rabbit reticulocyte ribosomes fails to promote methionyl-puromycin synthesis under conditions in which an AUG-40S-Met-tRNA_i initiation complex, but not an 80S complex, is readily formed. This suggests that the inability of the system to form methionyl-puromycin is due to failure of the subunits to join. When $\text{Artemia salina}$ 60S subunits are substituted for their reticulocyte counterparts, the resulting hybrid system readily forms an 80S initiation complex and synthesizes methionyl-puromycin. Activity of the reticulocyte 60S subunits can be restored by factors IF-M2A and IF-M2B. This suggests that one or both of these factors may be 60S proteins, essential for subunit joining, that may be removed from ribosomes by salt washing procedures.     

Importance of polysomal mRNA-associated polypeptides for protein synthesis initiation in yeast

The polysomal mRNA from the cell-free system of the yeast Saccharomyces cerevisiae, in the absence of exogenous energy, binds to the 40S ribosomal subunit thus forming a 48S preinitiation complex which, with energy added, is converted into 80S initiation complex. By using ribosomes with a high affinity to polysomal mRNA (pmRNA) from an edeine-resistant mutant of S. cerevisiae in place of wild-type ribosomes, increased quantities of the 48S preinitiation complex are obtained. The pmRNA is found associated with several polypeptides having molecular masses of 115-98 kDa, 72 kDa, 60 kDa and 51 kDa. These polypeptides, labelled with 125I, interact with 40S and 80S ribosomes and are essential for the formation of the 48S and 80S initiation complexes inasmuch as deproteinized pmRNA alone cannot initiate the process. In addition, other polypeptides present in the cytosol are required to carry out the above-mentioned steps of protein synthesis.