HeLa cell ribosomes

Fig. 7.21. Dependence of mobility-gel concentration profiles of DNA on conformation. Samples are supercoiled SV40 DNA (sc), nicked circular SV40 DNA (NC) and HeLa cell linear double-stranded DNA. A single-stranded species, 28 S HeLa cell ribosomal RNA is also shown (Harley et al. 1973).

Jeanteur, P. H., and G. Attardi. 1969. Relationship between HeLa cell ribosomal RNA... 

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) ... 

In order to stimulate fmet incorporation by cell-free extracts, crude preparations of initiation factors prepared from the 0.5 M KGI wash of HeLa cell ribosomes were added to the protein-synthesizing reactions. The ribosomal salt wash from uninfected cells stimulated total fmet incorporation about two-fold at the concentrations utilized, and stimulated initiation at both sites eq ially. Ribosomal salt wash prepared from infected cells also stimulated total fmet incorporation to about the same extent as the uninfected cell preparation however, SDS-PAGE analysis of the fmet-labeled products revealed that only synthesis of the smaller polypeptide was increased. No stimulation of NCVPla synthesis occurred (Knauert and Ehrenfeld, submitted). 

Fig. 14. In vitro binding of poliovirus RNA to HeLa cell ribosomes. HeLa cells at a concentration of 1 XIO cells/ml were incubated for one minute at 37° C without and with 100 [xg/ml DEAE-dextran. The cells were centrifuged, washed once with 0.3 M NaCl, 0.03 M Na citrate, pH 7.0, resuspended at 3X10 cells/ml in 0.1 M NaCl, 0.01 M Tris, pH 7. 4, 3 mM MgClg and lysed by the addition of NP-40 (to 0.5%). Monosomes and ribosomal subunits were isolated as described by Zomzely, Roberts and Peache (1970), and incubated with labeled polio RNA (1 jig RNA/ribosomes from 7.2 X 10 cells) at 37° G for 2 minutes. Samples were layered on a 20 to 40% sucrose gradient and centrifuged for 210 minutes at 39,000 rpm in a Spinco SW 40 at 5° C. The gradients were collected from the bottom of the tube and the infectivity of the RNA determined with the agar cell suspension plaque assay (Koch, 1971). Upper panel infectivity in PFU.

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... 

Narciclasine (215) is an antitumor agent which exerts an antimitotic effect during metaphase by immediately terminating protein synthesis in eukaryotic cells at the step of peptide bond formation (97,101,141,142), apparently by interaction with the ansiomycin area of the ribosomal peptidyl transferase center (142). The alkaloid has also been found to inhibit HeLa cell growth and to stabilize HeLa cell polysomes in vivo (97). Although DNA synthesis was retarded by narciclasine, RNA synthesis was practically unaffected (97,142). Sev-... 

As mentioned above, one consequence of stalled RNA polymerase II at a DNA adduct is activation of transcription-coupled repair [27], This effect may depend on the type of polymerase, however, since the removal of some types of DNA damage is slower from RNA-polymerase I transcribed ribosomal DNA than from a nuclear gene [160], The lower level of repair in the nucleolus could also reflect the influence of other transcription factors, such as the HMG-domain protein UBF, which bind to cisplatin-mod-ified DNA [145]. When HeLa cells were exposed to cisplatin at concentrations which did not seem to affect nuclear transcription, inhibition of rDNA gene expression was associated with the redistribution of UBF, along with other factors responsible for rRNA transcription [138], These observations indicate how cisplatin might exert a combination of effects. Transcription is stopped due to titration of essential factors by the platinum-DNA adducts, and the same proteins could shield the lesions from the repair activity. 

It should be noted that Hagopian, Bosmann and Eylar emphasised as early as 1968 that the N-acetylgalactosaminyltransferases of Ehrlich ascites carcinoma and HeLa cells were associated with the ribosome-free (smooth) membranes of the endoplasmic reticulum. Hence this type of glycosyl transfer is associated with a different site from dolichol-dependent processes of assembly. This is discussed further in Chapter 6. 

Background The assanbly of the ribosomal complex depends on the presence of proteins called enkaryotic initiation factors (elEs). In mammals the largest of these is eIF3, an -800 kDa noncovalent complex comprising of 13 stoichiometric nonidentical subunits ranging in mass from 25 to 167 kDa. This paper describes the isolation and characterization of human eIE3 obtained from HeLa cells (an inunortal cell line derived from cervical cancer cells from Henrietta Lacks in 1951). 

Jeanteur, P., F. Amaldi, and G. Attardi. 1968. Partial sequence analysis of ribosomal RNA from HeLa cells. II. Evidence for sequences of non-ribosomal type. J. Molec. Biol., 33 757-775. 

Warner, J. R., R. Soeiro, H. S. Birnboim, M. Girard, and J. E. Darnell. 1966. Rapidly labeled HeLa cell nuclear RNA. I. Identification by sedimentation of a heterogeneous fraction separated from ribosomal precursor RNA. J. Molec. Biol., 19 349-361. 

Willems, M., E. Wagner, R. Laing, and S. Penman. 1968. Base composition of ribosomal RNA-precursors in the HeLa cell nucleolus further evidence of non-conservative processing. J. Molec. Biol., 32 211-220. 

Huberman, J. A., and G. Attardi. 1967. Studies of fractionated HeLa cell metaphase chromosomes. I. The chromosomal distribution of DNA complementary to 28S and 18S ribosomal RNA and to cytoplasmic messenger RNA. J. Molec. Biol., 29 487-505. 

Nonribosomal RNA appears in the cytoplasm in the form of ribonu-cleoprotein particles called informosomes (Spirin et ah, 1964 Spirin, 1969). Such ribonucleoprotein particles, which are mostly not bound to ribosomal subunits, have been found in a variety of tissues. They are in part incorporated into polyribosomes (see Lebleu et ah, 1971 Knochel and Tiedemann, 1972 Bonanou-Tzedaki et ah, 1972). The appearance of messenger-ribonucleoprotein complexes in the polysomes is a slow process as compared to the synthesis of nuclear RNA. In chick embryos newly synthesized messenger (nonribosomal) RNA is detectable in polyribosomes after about 30 minutes. The time sequence of incorporation of mRNA into polyribosomes does not change when the RNA synthesis is completely blocked after 15 minutes by actinomycin D (Knochel, 1972b). In HeLa cells mRNA appears in the polysomes after a 15-20 minutes lag phase (Penman et ah, 1968). 

The enzyme has been found ubiquitously in the nuclei of a great variety of eukaryotic cells, including human [11] and plant [12]. However, three previous studies have presented evidence that the enzyme is also localized in the cytoplasm. Roberts et al. [13] have found the enzyme associated with polysomes and ribosomes of HeLa cells. A poly(ADP-ribose) synthetase was also reported to occur in rat liver mitochondria [14]. More recently we found a significant level of enzymatic activity associated with testis mitochondria [15, 16]. The present paper is a follow-up of this study and it presents evidence that after the nuclear fraction, the most important level of poly(ADP-ribose) synthetase in spermatogenic cells is associated with the micro-somal-ribosomal fraction. 

A cytoplasmic poly(ADPR) polymerase activity was first detected by Roberts et al. in 1975 [1]. The enzyme associated with ribosomes and polysomes in HeLa cells requires both DNA and histones for activity. A mitochondrial enzyme has been described in rat liver and in testis (for review see [2, 3]). Finally, the existence of an active poly-(ADPR) polymerase associated with ribonucleoproteins in the microsomal-ribosomal fraction of testis has been reported. The enzyme shows a considerable activity without exogenous DNA and is insensitive to RNase A [3]. 

Another theory to explain the shut-off, proposed by Cooper et al. (4Q) involves the synthesis of viral proteins which have an affinity for the small ribosomal subunit and the 5 end of viral mENA. The viral protein would repress the synthesis of cellular proteins by combining with the 4OS subunit, thereby blocking its link with host mENA. By also binding to the 5 end of viral ENA, the proteins would facilitate the attachment of viral ENA to the 40s subunit and increase the translation of viral ENA. Viral proteins have been found to co-sediment with the 4OS subunits of HeLa cells infected with poliovirus (49)> of Ehrlich ascites tumor cells infected with EMC virus (50) and of L-cells infected with mengovirus (5 ) poliovirus infected cells, the viral proteins co-sedimenting with ribosomes were identified as VPO, VP1 and VP3, all structural proteins (49) Both structural and non-structural proteins were found associated with ribosomes from EMC... 

In the second step, the virus synthesizes an inhibitor that inactivates an initiation factor and by the mid-point of infection, most ribosomes are directed towards translating viral mENA. This two step process could explain the differential effect of such perturbations as exposure to high salt concentrations and histidinol treatment on cellular and viral mENA translation and the inactivation of initiation factor eIF-4B after infection of HeLa cells with poliovirus. 

Figure 4 Binding of Met-tENA to 40 S ribosomal subunits in cells infected with EMC. At the times post-infection indicated in the top left corner of each panel, HeLa cells infected as described in Figure 1 were collected by centrifugation, resuspended in I/4 vol of methionine-free medium, incubated 2 min with 0.1 mg/ml of emetine and then with 20 iG /ml of ( 35s ) methionine. The cells were washed and lysed to prepare a cell extract, which was fractionated by sucrose gradient centrifugation for 16 hr at 24,000 rpm (11). The position of ribosomes and subunits is indicated. Fractions were counted as previously described...

Figure 6. Electrophoretic analysis of proteins associated with native 40 S ribosomal subunits of control and EMC-infected HeLa cells. Extracts were prepared from mock-infected and infected cells as described in Figure 2 0.5 ml of extract from control cells (b) or from cells infected 2 hr (a) or 3 hr (c) with EMC were layered on 15-50% sucrose gradients in 30 mM KC), 2mM Mg(OAc)p, 20 mM HHPES-KOH, pH 7 4i 1 mM dithiothreitol, and centrifuged 17 hr at 50f000 rpm. Fractions corresponding to the 40 S peak were combined, precipitated with 10% trichloroacetic acid, washed with acetone/ether (2/3) and ether, dissolved in sample buffer and fractionated on 12,5% polyacrylamide gels as previously described (14) The gels were stained with Goomassie Blue. Markers were run in parallel to assign M to the bands indicated by arrows.

Proteolytic Activity of HeLa Cell Polysomes and Ribosomes... 

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