Polysome profiling

Measuring the Distribution of miRNP Components in Polysome Profiles 99... 

Genome-Wide Analysis of mRNA Polysomal Profiles with... 

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... 

We utilize three independent methods to assess a compound s effect in vivo on translation—metabolic 35S-methionine/cysteine labeling, polysome profile analysis, and effects on transfected reporter gene activity. 

From the above discussion, it appears that zinc may have its primary effect on zinc-dependent enzymes that regulate the biosynthesis and catabolic rate of RNA and DNA. In addition, zinc may also play a role in the maintenance of polynucleotide conformation. Sandstead et al. (99) observed abnormal polysome profiles in the liver of zinc-deficient rats and mice. Acute administration of zinc appeared to stimulate polysome formation both in vivo and in vitro. This finding is supported by the data of Femandez-Madrid, Prasad, and Oberleas (42), who noted a decrease in the polyribosome content of zinc-deficient connective tissue from rats and a concomitant increase in inactive monosomes. 

Pronezuk, A. W., Baliga, B. S., Triant, J. W., and Munro, H. N., Comparison of the effect of amino acid supply on hepatic polysome profiles in vivo and in vitro, Biochim. Biophys. Acta, 157, 204, 1968. 

Yokogoshi, H. and Yoshida A., Effect of feeding or intubation of tryptophan or methionine and threonine in hepatic polysome profiles in rats under meal-feeding or fasting conditions, Agric. Biol. Chem., 47, 373, 1983. 

A further parallelism between the modes of action of pyridoxine and pantothenic acid was evidenced by experiments demonstrating a reduction in the vivo incorporation of C -amino acids into the serum albumin fraction of pantothenic acid-deficient rats (Roy and Axelrod, 1971). However, despite this significant reduction of amino acid incorporation vivo, the hepatic polysomal profile of the pantothenic acid-deficient rats failed to show any variation from normal. The concentration of nascent peptides associated with the polyribosomes after a short pulse with C -amino acids also indicated that protein translation by the polysomes was normal. Studies of C -amino acid incorporation with the microsomal and supernatant fractions from control and pantothenic acid-deficient... 

These results indicated that thiaisoleucine may be incorporated in place of isoleucine into polypeptides,in competition with isoleucine,and that its incorporation does not impair the polypeptide synthesis. This was confirmed by the analysis of the polysomal pattern and of the polysomal-bound radioactivity. Rabbit reticulocyte lysates were incubated,in the presence of all the factors necessary for protein synthesis,with labelled isoleucine or leucine, and with or without thiaisoleucine. As shown in Fig.3 the polysomal profile remained unchanged in the presence or absence of thiaisoleucine,while the polysomal-bound radioactivity due to isoleucine was highly reduced in the presence of thiaisoleucine.On the other hand the polysomal-boiind radioactivity due to leucine was unaffected by the presence of thiaisoleucine. These results showed that the incorporation of thiaisoleucine into the polypeptide chain does not modify the ribosome run-off nor the elongation rate of the growing polypeptide chain. 

Pig.3. Polysomal profile (full lines,A250) smd polysomal bound radioactivity ("broken lines of reticulocyte lysates su bjected to sucrose density gradients after incubation with H-isoleucine in the absence (A) or in the presence (B) of 1 mM thiaisoleucine. Experimental details in (29). 

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