Polysome preparation

Figure 12A shows typical polysome structures released by detergent extraction of purified mitochondria prepared without the use of EDTA and with a buffer containing Mg +. These structures are to be compared with free cytoplasmic polysomes prepared from the postmitochondrial supernatant fraction without the use of detergent (Fig. 12C). That some membrane material is still associated with Triton-X-100-extract ed mitochondria-bound polysomes is suggested by the fact that rapidly sedimenting material comprising predominantly larger polysome structures appears in the gradient following treatment of the Triton-extracted preparation with deoxycholate (Fig. 12B). No similar effect is observed on the optical density profile of free cytoplasmic polysomes prepared without the use of detergent (Fig. 12C).

Fig. 12. Comparison of mitochondria-bound and free cytoplasmic polysomes. Polysomes were prepared from isolated mitochondria by extracting with 2% Triton X-100. Free cytoplasmic polysomes were obtained without the use of detergent. Where indicated, polysomes preparations were made in 1% sodium deoxycholate before centrifugation for 40 min at 227,861gmax in a Beckman SW50-1 rotor through a 10-50% sucrose gradient. The position of the 80 S monosomes is indicated by the arrows. (From Kellems et alJ )...

There are numerous protocols for polysomal gradients preparations that differ mainly at the step for harvesting the cells, and the gradient composition and separation times. The protocol presented later was optimized for isolation of polysomal mRNA from the yeast Saccharomyces cerevisiae, yet many steps will be similar to other eukaryotes and the procedure can easily be modified for other organisms. We will use this protocol as a template on which we will indicate and highlight points that are critical for the microarray analysis. Generally, the RNA isolated by this protocol can be used for analysis by DNA microarray, Northern blot, or RT-PCR. 

Alternatively, poor efficiencies of inhibitor mRNAs may be due to their Incorporation into rlbonucleoproteln particles (RNPs) such as found in sea urchin embryos (21). Newly made mRNA in the embryos is found in RNPs and they apparently have "weak" template activities while in these particles. The presence of newly synthesized tomato mRNA in similar particles might explain the apparently low translational efficiencies noted herein. The use of chaotropic buffers in the preparation of tomato leaf mRNA (11) would not differentiate between free or polysome-bound mRNAs and those complexed in RNPs. If an RNP or similar particle is involved, then its role must be a temporal one since a second wound does not repeat the phenomenon (Fig. 4). 

Cell-free systems capable of synthesising polypeptides have been prepared from protoscoleces of E. granulosus (7), larval T. crassiceps (588) and H. diminuta (633). In general, these studies have demonstrated that protein synthesis in cestodes, although showing some specificity, is similar to that in mammals in that it requires polysomes, amino acid adenylates, aminoacyl-tRNAs, pH 5 fraction, ATP, GTP, magnesium and either sodium or potassium ions. 

Incubate the reticulocytes with [3H]leucine, which will be incorporated into proteins. Prepare electron microscope autoradiographs and count silver grains per cell and the number of polysomes. The latter appear as rosettes of five ribosomes in these cells. A statistical comparison between the number of polysomes and the amount of protein synthesized during the incubation time (proportional to the number of silver grains) indicates whether there are nonactive polysomes. In fact, many of the polysomes are inactive i.e., they are switched off (see Chap. 17 for the control of protein synthesis). 

Hew and Yip (1976) have shown that a 6-10 S, poly(A)-rich RNA from the flounder contained mRNA for the synthesis of the antifreeze protein. When injected into Xenopus oocyte, the RNA preparation from the fish liver polysomes stimulated a 4-fold incorporation of [3H]alanine into the antifreeze protein fraction (Table XXII). 

The less highly purified ribosome preparations contained polysomes, which are more active in protein synthesis than single ribosomes. 

Fig. 14. Polysomal mRNP from rat liver. A. Sucrose gradient analysis of the polyribosomes preparation treated with EDTA. Labeling period, 40 minutes. Centrifugation was at 21.000 rpm for 16 hours. —A A Cts/min -------, absorbance. B. CsCI equilibrium gradient analysis of the poly-...

Nuclear and polysomal fractions were prepared from embryos labeled in vitro with [ HJuridine in the presence of actinomycin D at a concentration (0.04 /ig/ml) sufficient to inhibit rRNA synthesis as described by Schultz et al. (1973a). 

When RNA prepared from 3T6 polysomes is injected into Xenopus oocytes, the hydroxyproline proline ratio rises (Table II). RNA from... 

In the work described above, the collagen messenger activity was detected in total RNA prepared from a mixture of polysomes covering a wide range of sizes. The majority of the RNA preparation is ribosomal, and it is likely that collagen mRNA constitutes only a small proportion of the remainder, but appears to be detectable. 

Lynch et al. (47) made use of such an antiserum to isolate vWf mRNA from polysomes of cultured endothelial cells. From the enriched mRNA, single-stranded cDNA probes were derived. A cDNA library made from endothelial cell mRNA larger than 28S rRNA was screened with this probe. Nine clones out of 3000 were positive and appeared to share a common region. The longest clone of 2.4 kbp hybridized to an endothelial cell mRNA species that was about 9.5 kb in size. When mRNA from this partial cDNA clone was prepared with the SP6 RNA polymerase system and translated in a rabbit reticulocyte lysate, several polypeptides were produced that reacted with a specific polyclonal rabbit antibody against vWf, and also with four out of a collection of 30 monoclonal antibodies raised against vWf. Final proof that the cDNA clone isolated by Lynch et al. was an authentic vWF cDNA clone was obtained by comparing the sequence of the 3 end of the clone with the sequence of the carboxy-terminal 10 amino acids (270). 

There are two predominant proteins (M.Wt. approximately 50>000 and 7Qf000) in polysomal mRNPs isolated from a wide variety of mammalian sources (reviewed in reference 59) Other proteins are present in minor amounts, and are far from consistent between different preparations. Those who wish to see complications at every level of mammalian protein synthesis like to hint that these minor components could be protein specific to particular mRNA species, perhaps involved in regulating the translation of the mRNA, whilst the 50,000 and J8,000 dalton proteins would be seen as invariant components common to all mRNAs. This idea seems unlikely to be true, at least as a generality, since there is no sign of the putative mRNA-specific proteins in mRNP preparations from cells... 

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