Polysomes cell extracts

The viral RNAs used as mRNAs in all of these studies were extracted from purified virions. It is known that these RNAs carry a covalently-linked small protein at their 5 termini which is absent from intracellular viral mRNAs extracted from polysomes in infected cells. The latter molecules terminate in 5 pUp (29, 30, 31), and thus, in this respect, they are different from the virion ENA molecules used for to vitro translation. It is claimed, however, that the 5 terminal protein is rapidly removed in the HeLa cell extracts (27) as well as in the reticulocyte lysate (V. Ambros and B. Baltimore, personal communication), to generate an apparently authentic mRNA. 

Figure 6. Polysome breaicdown in cell extract incubated with 2 oligo(A). The treatment of the cells and the composition of the incubations are described in Figure 2, with the only exception that dsMA was substituted by 2 5 oligo(A) to a final concentration equivalent to 1 xM ATP. A, not incubated B, 1 hr incubation G, 1 hr incubation with added 2 5 oligo(A). 

Incubation of an HeLa cell extract with 2 5 oligo(A) resulted in extensive polysome breakdown (Figure 6), whereas no significant changes in polysome pattern could be detected in a control incubation. 

In this method the cells are lysed by incubation in a hypotonic solution, which leaves the nuclei intact. The cell debris and nuclei are pelleted by centrifugation leaving the cytoplasmic RNA free from DNA in the supernatant. The RNA is released from the polysomes by incubation with proteinase K and the protein extracted into phenol/chloroform. The RNA is then precipitated from the aqueous phase using ethanol. 

Azahypoxanthine prevented cloning of human malignant HeLa S3 cells, but not of normal human cells, whereas 8-azaadenine prevented cloning of either type. °° 8-Azainosine (1 fiM) inhibited colony formation by human epidermoid (type 2) carcinoma cells in culture. In cell-free extracts of HeLa cells, protein synthesis was inhibited by the incorporation of 8-aza-guanine into the mRNA of polysomes, in which it blocked formation of peptide bonds. ... 

Approximately 25% of the total polysomes in poliovirus-infected and uninfected HeLa cells are membrane-bound (10). The membrane-bound polysomes are about five times more active in translation per unit mass than free polysomes, as determined by incorporation of amino acids in cell-free extracts (11). The exact role of the membrane in this process has not yet been defined. 

Recent evidence points to the presence of protease activity-associated with polysomes and ribosomes when extracts of uninfected cells are assayed (refs. 27 32, Figure j). Characteristic of infection of cells by poliovirus is drastic, rapid inhibition of protein synthesis. Poliovirus infection also depresses the ribosomal protease activity (27, 29, 55) Ribosomes from uninfected cells have been reported to possess an autoproteolytic activity (31, 32), and this has been confiimed by two-dimensional gel analysis (Figure 4) Poliovirus infection of HeLa cells reduces the autoproteolysis of isolated 808 ribosomes markedly (not shown). The inhibition of HeLa cell ribosomal protease activity requires protein synthesis, but proceeds in the presence of guanidine (55) ... 

We have very little idea about the stoichiometry of the two major polysomal mRNP proteins, or their location on the mRNA, apart from the fact that the 8,000 dalton protein seems to be associated with the poly A tract at the 5 end (59) The function of the proteins is even more obscure. In cell-free translation assays the activity of polysomal mRNP is no greater than that of deproteinised mRNA (20). This result is open to the qualification that in crude cell-free systems the added deproteinised mRNA mi t pick up proteins from the cell-free extract and thereby be effectively converted into polysomal mRNP particles, but since the same result is obtained in highly fractionated systems (11) this reservation can probably be discounted. The major polysomal mRNP proteins are distinct from all seven recognised initiation factors (ll), and initiation complex formation is found to require the same set of seven factors regardless of whether polysomal mRNP or deproteinised mRNA is used (11). In short, there is no evidence that these proteins play a role in mRNA translation. 

Figure 2. Polysome breakdown in extracts of interferon-treated and control cells incubated with dsEHA. The cells were treated 1 7 hr with 100 units/ml of interferon. Both control and interferon-treated cells were incubated 1 hr with 1 iig/ml of cycloheximide to increase polysome size according to Fan and Penman (l ) Extracts prepared from these cells were incubated 1 hr at 30 U with the components described in Figure 1, 1 mM ATP, 0.1 mM spars onQTCin and no added dsEHA in A and C or 10 jig/ml of poly(I) poly(C) in B and B. The samples were centrifuged 90 min at 40,000 rpm on 15-40% sucrose gradients and the A260 analyzed with a recording spectrophotometer.

Further studies of Millward and his colleagues indicate that reovirus mRNAs associated with polysomes at late times postinfection are not capped and that the proportion of uncapped mRNA increases as the infection progresses (Skup et al., 1981 Zarbl and Millward, 1983). The proportion of capped reovirus mRNAs decreases simultaneously. This transition from capped to uncapped mRNAs corresponds temporally with the synthesis of progeny transcripts and the capacity of cell-free extracts from infected cells to translate uncapped mRNAs. 

The amount of jSRNA began to increase in the early blastula stage which correlated with the accumulation of the cytoplasmic mRNA which contained poly (A). The maximum amount of aRNA (25% of the total heterogenous nuclear RNA) was obtained in the middle blastula stage (Dubroff and Nemer, 1975/76). The ratio of mRNA s with and without poly (A) sequences correlated with the increasing of size of free polysomes in the sea urchin embryo (Nemer and Surrey, 1975/76 Nemer et al., 1975). It is possible to translate different mRNA fractions extracted from sea urchin and amphibian embryos in the cell-free system (Ruder-man and Pardu, 1977). 

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