On the cell-free system of protein synthesis from mammalian mitochondria

Summary Some properties of a submitochondrial cell-free system for protein synthesis are described. The system was prepared from rat liver mitochondria lysed with Triton X-100, and the lysate was characterized by a linear rate of [¹⁴C]amino acid incorporation for 15–20 min with subsequent decline in activity. The incorporation reaction was inhibited by chloramphenicol and was in-sensitive to cycloheximide. Poly(U) addition stimulated [¹⁴C]phenylalanine incorporation by the preincubated submitochondrial system. Upon the addition of 7.5S mRNA that was iso-lated from mitochondria the major translation product was identified as a hydrophobic poly-peptide which in some properties (solubility in chloroform-methanol mixture) was similar to one of polypeptides synthesized by the sub-mitochondrial system on endogeneous mRNAs.     

Nuclear formation in a Drosophila cell-free system

A cell-free preparation obtained from 0- to 5-h-old Drosophila melanogaster embryos induces chromatin decondensation and nuclear formation from demembranated Xenopus sperm. Newly formed nuclei have a peripheral lamina, a double membrane, and structures resembling pore complexes. Indirect immunofluorescence analyses demonstrate the association of Drosophila lamins and DNA topoisomerase II with newly assembled nuclei.     

Cyclic AMP-protein-DNA complex formation in mammalian cell-free systems.

A cytoplasmic protein fraction from KB and Chinese hamster ovary cells (CHO-Kl) was shown to bind in vitro to cAMP and subsequently to DNA-cellulose. This protein complex was not found in DE-52 purified CHO-K1 cAMP-dependent protein kinases. The complex appeared to exist as a small fraction of the total cAMP binding proteins, preferred native to denatured DNA and exhibited multiple sedimentation coefficients in glycerol gradients. This complex, after elution from the DNA cellulose column, was shown to have bound specifically to [3H]-cAMP which could be displaced by non-radioactive cAMP in competitive binding assays.     

Enhanced differential synthesis of proteins in a mammalian cell-free system by addition of polyamines.

Addition of the polyamines spermidine, spermine, or putrescine to a fractionated mammalian cell-free protein-synthesis system programmed by a variety of mRNAs results in a 3- to 5-fold stimulation of amino acid incorporation over that found in the absence of added polyamine. The mRNAs used as template were adenovirus mRNA, globin 9s mRNA, and RNA from the bacteriophages R17, Qbeta, and MS2. The relative amounts of 10 adenovirus polypeptides synthesized in vitro are altered by the addition of polyamines to the translation system to reflect more closely the relative amounts of these polypeptides synthesized in vivo. This qualititive improvement in translation products on addition of polyamines allow the analysis of a number of products which are at best only marginally synthesized in the absence of added polyamines. The low level of synthesis due to endogenous mRNA is stimulated by spermidine and spermine but a lesser extent by putrescine.     

Regulation of apoptosis by BH3 domains in a cell-free system

Background: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins.Results: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system.Conclusions: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.     

The formation of immunogenic RNA-antigen complexes in a cell-free system

The formation of complexes between ribonucleic acid (RNA) and solubilized bacteriophage T2 in vitro is readily demonstrable. The ability to evoke antibody formation against T2 in lymph node cultures is not a property of all such complexes but rather is restricted to those formed in the presence of cell sap (100,000g supernatant). Further requirements for the formation of immunogenic RNA-antigen complexes include the use of RNA and of cell sap derived from organs, tissues or cells implicated as functional in the immune response. The activity of cell sap resides in a thermolabile, nondialyzable fraction which is presumed to be protein. Kinetic studies and observations on optima of temperature and pH suggest an enzymatic process and the presumed substrate of the enzyme(s) is the antigen part of the complex. The antibody response elicited by suitable complexes formed in the cell-free system closely resembles that observed in response to RNA-antigen complexes extracted from peritoneal macrophages which had been incubated with intact T2 phage. This resemblance extends to magnitude of the response, its kinetics and class of the antibody formed.     

On the nature of -galactosidase synthesized by DNA-directed cell-free system

Summary The -galactosidase product of the DNA-directed cell-free system for the synthesis of protein of the lac operon, developed by Zubay and his colleagues, has been purified to radioactive homogeneity and compared to wild-type -galactosidase. When analyzed by sucrose gradient centrifugation, SDS-gel electrophoresis, and kinetic analysis, the purified cell-free enzyme behaves identically to the purified wild-type -galactosidase.     

3-Hydroxypropyl amide formation from 1-aminocyclopropane-1-carboxylic acid by a cell-free ethylene-forming system from olive leaves

The low ethylene yield in a cell-free ethylene-forming system from olive tree leaves ( Olea europaea L. cv. Picual) was investigated. During the incubation, 1-aminocyclopropane-1-carboxylic acid (ACC) was extensively transformed into 3-hydroxypropyl amide (HPA). Enzyme extract, Mn²⁺ and oxygen are responsible for this reaction. Horseradish peroxidase (EC 1.11.1.7) can substitute for the enzyme extract in this reaction. HPA formation could be one reason for the poor in vitro conversion efficiency of ACC to ethylene.     

Use of mutant cells to localize thymidine kinase in a mammalian cell-free system

The ability of nuclei preparations of Chinese hamster cell lines Don-C and B14I50 (the latter having greatly reduced thymidine kinase activity) to incorporate radioactive thymidine into DNA was measured. By placing the nuclei of one cell line in the cytoplasmic extract of the other cell, we were able to demonstrate that the thymidine kinase activity was largely restricted to the cytoplasm of the Don-C cells. The kinetics of isotope incorporation also suggested that the B14I50 nuclei contained a greatly reduced pool of thymidine triphosphate, compared with the Don-C nuclei.     

Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E₃ from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.     

Analysis of nuclear RNA processing and transport by temperature perturbation of a cell-free system from mammalian cells

The similarity of the Arrhenius plots relating temperature to messenger RNA (mRNA) transport from intact and membrane-denuded rat liver nuclei demonstrates that the ATP and cytosol-dependent transport is independent of the lipid phase of the nuclear membrane. This temperature dependence of RNA release was confirmed for alpha 2u-globulin mRNA by use of a recombinant DNA probe. Ribosomal RNA (rRNA) release showed a similar temperature dependence, suggesting that both mRNA and rRNA share a common temperature-sensitive step. The kinetics of RNA release at different temperatures suggest that RNA transport from mammalian cell nuclei is a rate-controlled rather than a graded unlocking phenomenon. The processing of mRNA precursors also exhibits a temperature dependence as shown by the linear increase in the ratio of total alpha 2u-globulin RNA to alpha 2u-globulin precursor as a function of time at 30 degrees C but not at 14 degrees C in spite of residual transport at the lower temperature. This temperature dependence of mRNA processing was confirmed by Northern blot analysis of the nuclear RNA following a 45 min incubation. Thus, both the processing and transport of RNA show temperature-sensitive steps when analyzed in cell-free systems derived from mammalian cells.     

A cell-free mammalian protein-synthesizing system stimulated by RNA from avian myeloblastosis virus.

Carcinoma cells, oncornavirus-infected cells and fetal bovine tissue provide salt wash ribosomal factors capable of responding to avian myeloblastosis virus (AM virus)-RNA and stimulating the incorporation of amino acids into proteins as well as catalyzing the binding of N-acetylated (35S) methionyl-tRNA. The exogenously dependent amino acid incorporation system is stimulated by the high molecular weight species of AM virus-RNA only, particularly the fraction containing polyadenylate (poly(A)) residues; the system is also markedly inhibited by the low molecular weight AM virus-RNA species. Activity for the exogenous system displays very definite divalent/monovalent cation optima and requires the presence of mammalian transfer RNA.     

Recombinant DNA formation in a cell-free system from Xenopus laevis eggs

A cell-free system is described which formed very high levels of recombinant DNA structures in 4 hr at 26°C. It consisted of a single fraction of a high speed supernatant prepared from an extract of unfertilized eggs of the frog Xenopus laevis. This fraction eluted at 0.16?0.18 M Tris homogenization buffer from a DEAE-cellulose column. When two partially homologous supercoiled DNA molecules of different contour lengths were incubated simultaneously in this system, high levels of heterologous figure eight DNA structures were formed and observed by electron microscopy. Subsequent cleavage of the newly formed figure eight structures with Bam HI and Eco RI restriction endonucleases gave rise to “α structures” and “χ structures.” The observed figure eight structures presumably represent the recombination intermediate predicted by the Holliday model for genetic recombination.