HeLa cell extract

Legagneux, V., Morange, M., Bensaude, O. (1990). Heat-shock and related stress enhance RNA polymerase II C-terminal-domain kinase activity in HeLa cell extracts. Eur. J. Biochemistry 193, 121-126. 

Figure 6. The c-mos negative regulatory element (NRE). Nucleotide positions of the NRE are shown relative to the spermatocyte transcription start site, taken as 280 base pairs upstream of the c-mos ATG (see Fig. 4). The endpoints of the NRE are defined by deletions that allow c-mos expression in NIH 3T3 and other somatic cells. Mutations of the sequences designated by boxes 1,2, and 3 also allow c-mos transcription in NIH 3T3 cells, indicating that these sequences represent functional elements within the NRE. Boxes 1 and 2 are similar to sequences upstream of the protamine (Prot) promoter that inhibit in vitro transcription in HeLa cell extracts. A sequence just upstream of box 2 is also similar to a putative repressor-binding site in the regulatory region of Pgk2.

Mixture of HDAC isoforms from HeLa cell extract... 

One of the most important sugar lesion is the 3 -phosphoglycolate that is typically formed in the presence of O2 (for its excision by purified HeLa cell extracts see Winters et al. 1992). Specifically deuterated nucleoside triphosphates were used for incorporation into dsDNA by PCR (Balasubramanian et al. 1998). Hydroxyl radicals were generated by a Fenton reaction, and the yields of free 3 -phosphate end groups, 3 -phosphoglycolate and 5 -aldehyde were measured. Depending on the position of the deuteration, the yields vary with respect to a non-deuterated sample (Table 12.9). 

Nuclear extract (see Section 3.1.1, for HeLa cell extract preparation)... 

Hernandez, N. and Keller. W. (1983). Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts. Cell 35, 89-99. 

Several years ago a number of groups demonstrated that the Sm binding sequence of the C. elegans SL RNA was functional since the C. elegans SL RNP was precipitable from C. elegans extract with human Sm antisera. Furthermore, the SL RNA became precipitable with Sm antisera when incubated in HeLa cell extracts (28). It was subsequently established that assembly of the Ascaris SL RNA into an Sm snRNP was an absolute prerequisite for its participation in trans-splicing in vitro (see below) (29). 

In addition, we present methods for the preparation of HeLa cell extracts that can support import in vitro. In general, we find for both proteins and snRNPs that the Xenopus egg extract is the most efficient in terms of the speed of nuclear import and the final level of intranuclear accumulation. The Xenopus oocyte extract, HeLa cell extract, and reticulocyte lysates are generally less efficient in our hands. We have not been able to reconstitute nuclear import using extracts from Drosophila early embryos (C. Dingwall, unpublished). 

Cell extracts prepared at different times after infection of HeLa cells with EMC virus have been used to study the mechanism of shut-off. (These studies were made possible by the observation of Weber al. (11) that HeLa cell extracts supplemented with haemin and other components necessary for m vitro protein s3nithesis were active in initiation. 

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.

There are very clear evidences of a role of host proteases in the replication of certain RNA viruses, particularly the myxo- and paramyxoviruses (see ref. 1 for a review). By comparison, the evidence for participation of cellular proteases in picornavirus replication is less direct. The cleavage of nascent picornavirus polyproteins is probably carried out by cellular enzymes which are associated with membrane-bound polyribosomes. This was concluded from both in vivo (26) and cell-free studies (22, 25, 26). With poliovimis polyprotein as substrate, uninfected HeLa cell extracts were able to produce primary cleavage products with the molecular weights and antigens of intermediates in the normal cleavage process. Little or no production of capsid polypeptides was reported (25). 

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. 

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. 

The apparently paradoxical observation that high concentrations of GTP not only restore translation in gel-filtered reticulocyte lysates (Jagus and Safer, 1981a), in lysates deprived of heme (Balkow et al., 1975 Ernst et al., 1976), incubated in the presence of dsRNA (Ernst et al., 1976), or subjected to high temperature (Mizuno, 1977), as well as in HeLa cell extracts (Weber et al., 1977), but actually block formation of the heme-controlled inhibitor (Balkow et al., 1975) is in... 

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