Comparison of in vivo and in vitro translation of Cowpea Mosaic Virus RNAs

The translation products from Cowpea Mosaic Virus (CPMV) RNAs obtained in two different cell-free systems were compared with the viral polypeptides synthesized in CPMV-infected cowpea protoplasts. It was shown that in both the wheat germ system and the rabbit reticulocyte lysate CPMV M component RNA was translated into two polypeptides of 105,000 and 95,000 dalton, which were not detected in CPMV-infected protoplasts. B component RNA however, gave different products depending on the system used. In the reticulocyte system this RNA was translated into a 200,000 dalton polypeptide which was further cleaved to give 170,000 and 32,000 dalton polypeptides. In the wheat germ system this processing step was lacking as only the 200,000 dalton product was formed. Since the 170,000 and 32,000 dalton polypeptides were also found in CPMV-infected protoplasts the two in vitro systems used apparently represent different stages of the expression of the B component RNA, thus providing a tool to study the mechanism of CPMV gene expression in vivo.     

Inorganic polyphosphate interacts with ribosomes and promotes translation fidelity in vitro and in vivo

Inorganic polyphosphate is a biological macromolecule consisting of multiple phosphates linked by high-energy bonds. Polyphosphate occurs in cells from all domains of life, and is known to play roles in a diverse collection of cellular functions. Here we examine the relationship between polyphosphate and protein synthesis in Escherichia coli. We report that polyphosphate associates with E. coli ribosomes in vitro. Characterization of this interaction reveals that both long-chain and short-chain polyphosphates interact with the ribosome. Intact 70S ribosomes, as well as 50S and 30S subunits, display a specific interaction with polyphosphate that is mediated primarily by contacts with ribosomal proteins. Additionally, we examined functional consequences of a ppk mutation, which severely reduces levels of intracellular polyphosphate. Extracts from ppk mutants contain lower levels of polysomes than wild-type cells, suggesting a defect in mRNA utilization or the mRNA-ribosome interaction. Ribosomes from wild-type and ppk mutant cells were isolated, and their activities were compared using a polyU RNA in vitro translation assay. While rates of polyphenylalanine synthesis are similar, use of ribosomes from ppk cells results in a misincorporation rate about five times higher compared with the rate observed when ribosomes from wild-type cells are used. Mistranslation rates in vivo were measured directly, and ppk mutants displayed higher readthrough frequencies for two different stop codons. Taken together, these results indicate that polyphosphate plays an important role in maintaining optimal translation efficiency in vivo and in vitro.     

In vivo and in vitro studies of phage T4 lysozyme mRNA translation]

Translation of phage T4 lysozyme mRNA is studied in vivo and in vitro. Polyribosomes, carrying growing lysozyme polypeptides, are found to be homogenous enough and to contain 6 ribosomes. Complete molecules of phage lysozyme, which possess an enzymatic activity and are similar to the native enzyme in its electrophoretic mobility in polyacrylamide gel, have been synthetized in vitro on RNA isolated from phage-infected cells. The efficiency of RNA translation in cell-free system is discussed on the model of synthesis of functionally active individual protein.     

Patterns of immunoenhancement and suppression induced by Chlamydia trachomatis in vivo and in vitro

We investigated the cycle of immune enhancement and suppression seen in mice infected with Chlamydia trachomatis by using in vivo and in vitro model systems. BALB/c mice injected intravenously with chlamydia reveal a three- to seven-fold increase in numbers of plaque-forming cells producing antibodies against sheep red blood cells (SRBC), when immunized with SRBC 0 to 5 days after chlamydia infection. When mice are injected with SRBC 10 to 15 days after initial chlamydia infection, the specific anti-SRBC plaque-forming cell response is suppressed two- to three-fold. In vitro, low numbers (2 to 5 X 10(6) bacteria/ml) of chlamydia stimulate potent proliferative responses by B lymphocytes while high numbers (25 X 10(6) bacteria/ml) of bacteria generate strong, general T suppressor activity. This model has important implications for regulation of immune responses that arise at different times during chlamydial infections, as well as for the potential effectiveness of chlamydial vaccines.     

Codon-specific missense errors in vivo

We have developed a simple method for measuring the missense substitution of amino acids at specified positions in proteins synthesized in vivo. We find that the frequency of cysteine substitution for the single arginine in Escherichia coli ribosomal protein L7/L12 is close to 10(-3) for wild-type bacteria, decreases to 4 x 10(-4) in streptomycin-resistant bacteria containing mutant S12 (rpsL), and is virtually unchanged in Ram bacteria containing mutant S4 (rpsD). We have also found that the frequency of the cysteine substitution for the single tryptophan in E. coli ribosomal protein S6 is 3-4 x 10(-3) for wild-type bacteria, decreases to 6 x 10(-4) in streptomycin-resistant bacteria and is elevated to nearly 10(-2) in Ram bacteria.     

Multicolor in vitro translation

In vitro translation is a widely used tool for both analytical and preparative purposes. For analytical purposes, small amounts of proteins are synthesized and visualized by detection of labeled amino acids incorporated during translation. The original strategy of incorporating radioactively labeled amino acids, such as [35S]methionine or [14C]leucine, has been superseded by the addition of antigenic tags or the incorporation of biotin-labeled or BODIPY-FL-labeled amino acids. Such nonradioactive tags are easier to visualize after translation and do not pose a radiation hazard. Among the nonradioactive tags, BODIPY-FL-lysine offers the advantage that proteins that have incorporated this amino acid can be directly visualized after gel electrophoresis. We show here that multiple fluorophores introduced into proteins can considerably extend their usefulness, particularly for the comparison of in vitro-translated proteins from related sources. This technology can be applied in various situations, including the simplified detection of rare truncating mutations in clinical samples from cancer patients.     

Phosphorylation of mammalian translation initiation factor 5 (eIF5) in vitro and in vivo

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S initiation complex (40S•eIF3•AUG•Met-tRNA_f•eIF2•GTP) and, acting as a GTPase activating protein, promotes the hydrolysis of bound GTP. We isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate purified bacterially expressed recombinant rat eIF5. Physical, biochemical and antigenic properties of this kinase identify it as casein kinase II (CK II). Mass spectrometric analysis of maximally in vitro phosphorylated eIF5 localized the major phosphorylation sites at Ser-387 and Ser-388 near the C-terminus of eIF5. These serine residues are embedded within a cluster of acidic amino acid residues and account for nearly 90% of the total in vitro eIF5 phosphorylation. A minor phosphorylation site at Ser-174 was also observed. Alanine substitution mutagenesis at Ser-387 and Ser-388 of eIF5 abolishes phosphorylation by the purified kinase as well as by crude reticulocyte lysates. The same mutations also abolish phosphorylation of eIF5 when transfected into mammalian cells suggesting that CK II phosphorylates eIF5 at these two serine residues in vivo as well.     

The ribosome-associated inhibitor A reduces translation errors

Recently we have reported about a novel stress response protein (pY or RaiA) associated with Escherichia coli ribosomes that inhibits translation at the aminoacyl-tRNA binding stage. Here we show that leucine misincorporation during in vitro poly(U) translation is inhibited by this protein much stronger than the incorporation of phenylalanine. The miscoding counteraction by RaiA is especially strong at the concentrations of magnesium ions close to those observed in vivo and diminishes at higher magnesium concentrations. The results obtained suggest that the anti-miscoding activity of RaiA could be the main function of the protein, rather than the inhibition of translation. The role of the protein in adaptation of cells to environmental stress is discussed.     

小鼠体内外受精植入前胚胎全基因组甲基化模式比较

为考察体外受精、操作及培养环境对体外受精的小鼠植入前胚胎全基因组DNA甲基化模式的影响,本研究以体内受精的植入前胚胎作为对照,采用间接免疫荧光法检测小鼠体内外受精植入前胚胎基因组DNA甲基化模式.实验结果表明,体外受精各期植入前胚胎呈现出与之相应时期的体内受精植入前胚胎不同的DNA甲基化模式和水平,原核期甲基化水平较高,2-4-、8-细胞期明显降低,而桑葚胚和囊胚期又略有升高.各期体外受精植入前胚胎的基因组DNA甲基化水平都比同时期体内受精胚胎的甲基化水平低.本实验结果部分显示了体外受精、操作及培养环境可能对正常的DNA甲基化模式产生影响,造成体外受精植入前胚胎甲基化模式异常.     

The stem-loop binding protein is required for efficient translation of histone mRNA in vivo and in vitro

Metazoan replication-dependent histone mRNAs end in a conserved stem-loop rather than in the poly(A) tail found on all other mRNAs. The 3' end of histone mRNA binds a single class of proteins, the stem-loop binding proteins (SLBP). In Xenopus, there are two SLBPs: xSLBP1, the homologue of the mammalian SLBP, which is required for processing of histone pre-mRNA, and xSLBP2, which is expressed only during oogenesis and is bound to the stored histone mRNA in Xenopus oocytes. The stem-loop is required for efficient translation of histone mRNAs and substitutes for the poly(A) tail, which is required for efficient translation of other eucaryotic mRNAs. When a rabbit reticulocyte lysate is programmed with uncapped luciferase mRNA ending in the histone stem-loop, there is a three- to sixfold increase in translation in the presence of xSLBP1 while xSLBP2 has no effect on translation. Neither SLBP affected the translation of a luciferase mRNA ending in a mutant stem-loop that does not bind SLBP. Capped luciferase mRNAs ending in the stem-loop were injected into Xenopus oocytes after overexpression of either xSLBP1 or xSLBP2. Overexpression of xSLBP1 in the oocytes stimulated translation, while overexpression of xSLBP2 reduced translation of the luciferase mRNA ending in the histone stem-loop. A small region in the N-terminal portion of xSLBP1 is required to stimulate translation both in vivo and in vitro. An MS2-human SLBP1 fusion protein can activate translation of a reporter mRNA ending in an MS2 binding site, indicating that xSLBP1 only needs to be recruited to the 3' end of the mRNA but does not need to be directly bound to the histone stem-loop to activate translation.     

The role of mRNA competition in regulating translation. III. Comparison of in vitro and in vivo results

Competition of encephalomyocarditis virus, reovirus, and L-cell mRNAs for a message-discriminatory component was studied in vitro. The data were analyzed qualitatively to determine the relative initiation efficiencies among the various mRNAs. The effects of potassium chloride concentration, magnesium acetate concentration, and m7G methylation on mRNA competition in vitro were also studied. These results were correlated with translation rates in vivo for the same mRNAs, to determine if the sites of competition in vitro and in vivo are the same. It was found that under a particular set of magnesium acetate and potassium chloride concentrations, the order of mRNA initiation efficiencies was the same both in vivo and in vitro, suggesting that the same limiting message-discriminatory factor is regulating initiation rates in both cases. This can only be accomplished in a competitive situation when RNA is in molar excess relative to the discriminatory component.     

RNA secondary structure and in vitro translation efficiency

Cell-free protein synthesis is a promising technology featuring many advantages compared to in vivo expression techniques. However, most proteins are still synthesized in vivo due to relatively low protein yields commonly achieved in vitro, especially in the batch mode of reaction. In Escherichia coli S30 extract-based cell-free systems protein yields are supposed to be partially limited by a secondary structure formation of the mRNA. In this study we checked promising members of various classes of RNA chaperones and several different RNA helicases on their ability to enhance in vitro translation. The data clearly show that the addition of none of these factors provides a general solution to the problem. However, protein yields can be increased in presence of a microRNA hybridizing with the 5′ untranslated region of mRNAs, possibly by inducing structural changes improving accessibility of the Shine Dalgarno sequence for the ribosomes.     

Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy

BACKGROUND: Because the process of protein translation is an event of sparse molecules, the measurement requires high sensitivity. One of the candidates for studying the molecules is fluorescence correlation spectroscopy (FCS), which gleans quantitative information from fluctuating fluorescence signals in a diluted solution. METHODS: Using FCS, the translation products of expression plasmid for green fluorescent protein (GFP) and its fusion proteins were measured in vitro and in vivo. RESULTS: In in vitro translation, the number of products increased linearly for 90 min upon concentration of the plasmid. The autocorrelation function for GFP was fitted with a one-component model with a diffusion time of 0.18 ms, which was identical to the value expected from the molecular weight. In the cases of GFP- tagged hypoxia-inducible factor-1 alpha and glucocorticoid receptor, each fitting result was significantly improved with a two-component model. The slow component with a diffusion time of 6 ms appeared to be related to the ribosome or polysome. In response to the addition of dexamethasone, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in the focal point. CONCLUSIONS: FCS permits monitoring of the number of molecules translated in vitro and in vivo, the translation rate, and the molecular weight.     

IRES-mediated translation of the carboxy-terminal domain of the horizontal cell specific connexin Cx55.5 in vivo and in vitro

Background

Changes of the interneuronal coupling mediated by electrical synapse proteins in response to light adaptation and receptive field shaping are a paramount feature in the photoreceptor/horizontal cell/bipolar cell (PRC/HC/BPC) complex of the outer retina. The regulation of these processes is not fully understood at the molecular level but they may require information transfer to the nucleus by locally generated messengers. Electrical synapse proteins may comprise a feasible molecular determinant in such an information-laden signalling pathway.

Results

Connexin55.5 (Cx55.5) is a connexin with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the PRC/HC/BPC complex in form of hemichannels where light-dependent plasticity occurs. Here we provide evidence for the generation of a carboxy-terminal domain of Cx55.5. The protein product is translated from the Cx55.5 mRNA by internal translation initiation from an in-frame ATG codon involving a putative internal ribosome entry site (IRES) element localized in the coding region of Cx55.5. This protein product resembling an 11 kDa domain of Cx55.5 is partially located in the nucleus in vivo and in vitro.

Conclusion

Our results demonstrate the generation of a second protein from the coding region of Cx55.5 by an IRES mediated process. The nuclear occurrence of a fraction of this protein provides first evidence that this electrical synapse protein may participate in a putative cytoplasmic to nuclear signal transfer. This suggests that Cx55.5 could be involved in gene regulation making structural plasticity at the PRC/HC/BPC complex feasible.     

Patterns of mineralization in vitro

Summary Various patterns of mineralization are found in the organism during fetal and postnatal development. Different findings and theories have been published in the literature with regard to the mechanisms of mineralization, many of which are controversely discussed. In the present study the different patterns of mineralization observed in the organoid culture system of fetal rat calvarial cells were investigated by electron microscopy. In organoid culture, calvarial cells grow and differentiate at high density, and deposition of osteoid and mineralization of the matrix occur to a very high extent. Different types of mineralization could be observed more or less simultaneously. It was found that hydroxyapatite crystals were formed at collagen fibrils as well as in the interfibrillar space. Mineralization was frequently seen in necrotic cells and cellular remnants as well as in extra-and intracellular vesicles. Addition of bone or dentin matrices or the artificial hydroxyapatite Interpore 200 to the cells caused an increased mineralization in the vicinity and on the surface of the matrices with and without participation of collagen. On previously formed mineralized nodules, an apposition of mineralizing material appeared due to matrix secretion by osteoblasts. It is concluded that initiation of mineralization occurs-at least in vitro-at every nucleation point under appropriate conditions. These mineralization foci enlarge by further apposition as well as by cellular secretion of a mineralizing matrix. Furthermore, cell necroses may liberate mineralizable vesicles. All these patterns of mineralization are the result of different activities of one cell type.