sence of histidinol the rate of elongation of protein synthesis is slowed to about 50% that in untreated cells. This would agree very well with the results obtained ...
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sence of histidinol the rate of elongation of protein synthesis is slowed to about 50% that in untreated cells. This would agree very well with the results obtained by using cellfree systems prepared from these cells. The decrease in the rate of elongation accounts for most, but not all, of the decrease in the rate of protein synthesis seen in the intact cell. To account for all the decrease in the rate of protein synthesis, initiation rates would also have to be slowed. Preliminary experiments have shown that this is indeed the case, as the S-30s system from histidinoltreated cells form initiation complexes with [35S]methionyl-tRNA,"etapproximately 30% less efficiently than the control S-30s system. The overall decrease in the rate of protein synthesis is thus due to a combination of effects on the two processes. The initiation of protein synthesis in Krebs I1 ascites cells is thus less sensitive to an increase in the amount of uncharged tRNA than is the case in HeLa cells, and this probably reflects the different selective pressures to which these two cell lines have been subjected. I thank the Medical Research Council for a research studentship and for financial support.
Austin, S. A. & Kay, J. E. (1975) Biochirn. Biophys. Acta395,468-477 Fan, H. & Penman, S. (1970) J. Mol. Biol. 50,655-670 Vaughan, M. H. & Hansen, B. S. (1973) J. Biol.Chem. 248,7087-7096
Messenger Ribonucleic Acid Content of Phytohaemagglutinin-Treated Lymphocytes ROSEMARY JAGUS-SMITH and JOHN E. KAY Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton BNl SQC, U.K. Lymphocytes isolated from the blood of humans and other animals can be induced to change in vitro from a non-growing state to a proliferative state by the addition of phytohaemagglutinin. Over the first, 24h of phytohaemagglutinin stimulation there is a 710-fold increase in the rate of protein syntheis (Kay et al., 1971;Ahern & Kay, 1973). This increase is observed both in intact cells and in cell-free systems prepared from lymphocytes (Ahern & Kay, 1973; Kay et al., 1975). The increase is due to the mobilization of the relatively large proportion of inactive ribosomes in unstimulated lymphocytes into polysomes and represents an increase in the rate of initiation of protein synthesis (Kay et al., 1971; Ahern & Kay, 1973, 1975). This increased initiation could be due either to an increase in the number of mRNA molecules available or an increase in the utilization of existing mRNA molecules, or a combination of the two. Ahern et al. (1974) found from studies in cell-free systems that the low rate of initiation in unstimulated lymphocytes cannot be changed simply by providing additional mRNA, but is influenced greatly by the provision of specific initiation factors. This suggests that the low rate of initiation is not due simply to a lack of available mRNA. However, it is known that mRNA synthesis increases over the first 20h of phytohaemagglutinin treatment (Cooper, 1974; Hemmincki, 1975) so it is possible that mRNA concentrations also change. Lymphocytes used in the present experiments were purified from pig blood and maintained in culture as described by Kay et al. (1975). mRNA concentrations have been measured by the extent of hybridization of RNA to [3H]poly(U) (Miles Laboratories, Elkhart, IN, U.S.A.). RNA from lymphocytes was extracted by 0.1 % sodium dodecyl sulphate in 0.1 5 M-NacI/O.I M-Tris/HCI, pH9, and phenol/chloroform (1 :1, v/v) at 40°C. The prepared RNA was contaminated withDNA and this was removed by treatment with DNAase* [Sigma (London), Chemical Co., London S.W. 6, U.K.; electrophoretically pure]. * Abbreviation: DNAase, deoxyribonuclease.
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Fig. 1 . Egects of phytohaemagglutinin on mRNA in pig lymphocytes Each point represents the mean of three determinations. Samples (50pg) of RNA (1 mg/ml) from 20h-phytohaemagglutinin-treated lymphocytes ( 0 ) and untreated lymphocytes ( 0 ) were hybridized to increasing amounts of [3H]poly&J).Ribonucleaseresistant material was precipitated with trichloracetic acid, filtered and the radioactivity contained on each filter determined.
Fig. 1 shows a comparison of the hybridization of [3H]poly(U) to RNA prepared from phytohaemagglutinin-treated lymphocytes with that to RNA from untreated lymphocytes. Although the phytohaemagglutinin-treated culture yielded twice as much RNA as the untreated cultures containing the same number of cells, hybridization of the same amount of RNA (50pg) was compared fromphytohaemagglutinin-treated lymphocytes and untreated lymphocytes. Because more than 80 % of cell RNA is ribosomal, this gives a measure of the number of mRNA molecules per ribosome. There is twice as much hybridization of [3H]poly(U)to RNA from phytohaemagglutinin-treated lymphocytes as to the same amount of RNA from untreated lymphocytes, i.e. there are twice as many mRNA molecules per ribosome, assuming that the size of polyadenylate sequences remain the same after stimulation. Because phytohaemagglutinin-treatedpig lymphocytescontain twice as much RNA as untreated lymphocytesthis means that there is approximately four times more mRNA per cell in phytohaemagglutinin-stimulated lymphocytes than in unstimulated lymphocytes. Table 1 shows the time-course for the effects of phytohaemagglutinin on mRNA concentrations. It also shows the effect of phytohaemagglutinin on rates of protein synthesis and RNA contents of lymphocytes. Expressed as hybrid c.p.m./50pg of RNA these data are an indication of the number of mRNA molecules per ribosome. Expressed as a percentage of the initial value, the data reflect the amount of mRNA per cell. At times soon after phytohaemagglutinin addition (between 0 and 4h) there is a twofold increase in protein synthesis known to reflect a twofold increase in mRNA utilization; the increased initiation of protein synthesis must take place on existing mRNA. This could be caused by an increased availability of initiation factors or by the mRNA already present becoming available for utilization after phytohaemagglutinin treatment. 1976
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Table 1. Time-course of fhe effect ofphytohaemagglutinin on mRNA content Lymphocyte cultures were incubated with phytohaemagglutinin for the times indicated. Time after phytohaemagglutinin addition (h) 0 2
4 12 16 20
[14C]Leucine incorporation (c.p.m./h per 1.5 x 106 cells) 966 1370 2090 5070 7220 10800
RNA content (Pg/cell> 1.5
1.8 1.4 1.5 2.2 3.0
[3HlPol~(U) [3H1Pol~ W) hybrid hybrid (% of zero-hour (c.p.m./5Opg value) of RNA) 3070 100 2970 103 3200 100 214 6620 6350 305 6490 428
Between 12 and 20h the rate of protein synthesis doubles. The amount of mRNA per cell also double during this time, following closely the increase in RNA content per cell, so that the number of mRNA molecules per ribosome remain constant during this period. Although it is clear that the initial increases in rates of protein synthesis are not caused by increases in mRNA concentrations, it is not apparent whether the later increases in mRNA concentrations are necessary for the observed increases in protein synthesis or are part of a co-ordinated growth control mechanism used by the lymphocyte to anticipate future needs. An investigation of the localization of mRNA between mRNA-protein complexes in the cytoplasm and those associated with polysomes should clarify this. We thank the M.R.C. for financial support. Ahern, T. & Kay, J. E. (1973) Biochim. Biophys. Acta 331,91-101 Ahern, T. &Kay, J. E. (1975) Exp. Cell Res. 92,513-715 Ahern, T., Sampson, J. &Kay, J. E. (1974) Nature (London)248,519-521 Cooper, H. L. (1974) Cold Spring Harbor Conf CellProliferation 1,769-783 Hemmincki, K. (1975) Exp. Cell Res. 92,191-200 Kay, J. E., Ahern, T. & Atkins, M. (1971) Biochim. Biophys. Acta 247,322-334 Kay, J. E., Ahern, T., Lindsay, V. J. & Sampson, J . (1975) Biochim. Biophys. Acta 378,241-250
Molecular Equilibria in Ribonucleic Acid Polymerase AILSA M. CAMPBELL Department of Biochemistry, University of Glasgow,Glasgow G12 8QQ, Scotland, U.K.
RNA polymerasemolecules in solution were studied by laser light scattering by previously described techniques (Campbell, 1976). The refractive increment at 632.8nm was 0.169 ml/g. At 20°C in 0.05~-Tris/HCI,pH8, 0.1 mM-dithiothreitol, 0.1 m ~ - E D T A(disodium salt) and O.~ M - K Conly ~ , the monomeric polymerase was present in solution at all protein concentrations and there was some repulsion between molecules. When the KCI concentration was lowered to 0.1 8~ a curve of apparent molecular weight against concentration showed that the weight-average mol.wt. rose from 450000 at l00pg/ml to 700000 at 600pg/ml. This was best described by a K,,,. value in the region of 1061itre/mol. Below loO,xg/ml, further dissociation of the enzyme was observed. This was attributed to the loose association of the sigma subunit (Travers, 1975). Combination of the polymerase with bacteriophage PM2 DNA led to the formation of large complexes with up to one polymerase molecule for every 56 base-pairs. At concentrations above 50fig/ml all the polymerase molecules in solution were found bound to DNA and no unbound molecules remained in solution. The evidence suggests strongly
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