Polysomes, fractionation

The collected polysomal fraction also contains a significant amount ofRNases. An overload of RNases cannot be completed by the commonly used RNase Inhibitors (rRNasin, RNase Inhibitor, etc.) and will therefore lead to massive degradation. It is therefore recommended not to collect more than the amount indicated here. 

Figure 10.1 Experimental schemes for microarray analysis. All experimental schemes start with a separation step of the cell lysate by velocity sedimentation in a sucrose gradient (top scheme). Collection of the desired fractions is assisted by a continuous ultraviolet (UV) reading of the gradient (an example of such UV reading is shown in each section). This allows determination of the sedimentation position of the 40S, 60S, 80S, and polyribosomal complexes (2,3, and more).Three general ways for fraction collection and analysis are presented (sections A, B, and C) (A) Collection of two fractions (free and polysomes) and direct comparison between them, with the free mRNA fraction labeled with green dye and the polysome fraction labeled with red dye. (B) Collection of two fractions and indirect comparison between them by utilizing an unfractionated reference RNA. (C) Collection of multiple fractions (four in this case), where each fraction is compared to an unfractionated reference sample. The blue arrows indicate the addition of spike-in RNA to each fraction and to the reference RNA.

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). 

In a comparison of supernatant and ribosomal tRNA populations, Wettstein [55] had found that Leu-tRNAi, which accounts for more than 50% of coli Leu-tRNA, is present much less frequently in ribosomal fractions than is the second fraction (MAK peak 2) and that, in T4-infected cells, it is largely excluded from the ribosomal fraction. In an extension of this work, Sueoka and Kano-Sueoka [56] obtained polysomal, ribosomal, and supernatant fractions from noninfected cells, as well as from infected cells, 1.5, 3, 6, and 10 minutes after T2 infection. Transfer RNA was then isolated from the various fractions, deacylated, and reacylated with radioactive leucine. The proportion of the various Leu-tRNA species in each preparation was analyzed by MAK chromatography. As shown in Table I, the Leu-tRNAi content of the polysomal fraction, which amounted to 20% of total Leu-tRNA before infection, fell to 6% within 1.5 minutes after infection and then remained essentially constant up to 10 minutes, suggesting a reduced usage of this species and consequently of the CpUpG codon. 

Rakusanova et al. (1972) examined the pulse-labeled RNA that annealed with cell DNA (cRNA). Both production and turnover of nuclear cRNA were inhibited in cells infected with PRV. Only 10% of the cRNA detected in the cytoplasm of infected cells was found associated with a polysome fraction as compared to 40% in normal cells. The remaining 90% of cytoplasmic cRNA that was not associated with polysomes nevertheless contained poly(A). The implication is that both the synthesis and processing of cellular mRNA were interrupted in infected cells and that those molecules that entered the cytoplasm, although carrying poly(A), were not able to as-... 

Example polysome profiles from sucrose gradient fractionation of HeLa cell lysates, either untreated or treated with EDTA, are shown in Fig. 6.4A and B. The polysome profile of untreated HeLa lysates shows three defined peaks in less dense fractions (6 to 9), which correspond to the 80S, 60S, and 40S peaks (Fig. 6.4A). Treatment of lysates with 30 /iM EDTA results in ribosome dissociation leaving predominantly free 60S and 40S subunits... 

Collecting the correct fraction is greatly facilitated by the use of a continuous ultraviolet (UV) detector, such as the ISCO UA-6 system. The OD254 reading allows accurate determination of the sedimentation of various complexes (e.g., 40S, 80S, and various polysomal complexes Fig. 9.1, step 3a). This information can be used to correct for small variations in sedimentation. Importantly, in many cases, it enables the determination of the number of ribosomes on the mRNA or on the resulting fragments, thereby allowing more accurate conclusions. 

An inherent limitation of this design is that the information that it provides is in the form of a ratio (between the free and polysomes), and not the actual distribution in these fractions. This fact becomes a limitation when multiple treatments are compared, since the product of dividing one ratio by another does not necessarily reflect the extent of changes (see Example 10.1). A possible way to overcome this limitation is to extract... 

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. 

Poly(dT)-cellulose was used to isolate a poly(A) containing RNA fraction from membrane-bound polysomes of mouse myeloma tumour... 

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). 

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