Cell-free translation reticulocyte lysate

The hybrid arrest of in vitro translation using some rabbit reticulocyte lysate preparations may not be reliable without added RNase H (41). Freshly prepared lysates are reported to contain 1-2% of the level of RNase H present in actively dividing cells, an amount sufficient to achieve near 100% cleavage of hybrid mRNAs (39). In contrast to some reticulocyte lysates, the wheat germ cell-free translation system apparently contains a sufficient amount of endogenous RNase H activity. 

To obtain maximal protein productivity, it is necessary to construct an expression clone in which a protein coding region (open reading frame, mature region, domain, etc.) obtained from a cDNA of interest is inserted into the MCS of the pTD 1 vector. Typically, expression of the target protein at about 35-50 pg per mL of the translation reaction mixture can be obtained by using mRNA transcribed from the expression clone and the Transdirect insect cell kit. Furthermore, the expression clone can be effectively combined with other eukaryotic cell-free protein synthesis systems, such as rabbit reticulocyte lysate and wheat germ systems (tee Note 3). 

Fig. 10. Cell-free synthesis of t-PA glycoforms. The niRNA coding for t-PA was translated in a rabbit reticulocyte lysate in the presence of dog pancreas microsomes. Microsonies were isolated posttranslationally and the translocated, glycosylated products were separated by SDS-PAGE. Translation was carried out under conditions that either prevented (lane 2) or allowed (lane 3) proper folding of the t-PA molecule, yielding enzymatically active protein that was sensitive to natural inhibitors and stimulators.

One way of searching for the presence of inhibitors of polypeptide initiation in infected cells was to add cytoplasmic fractions from virus infected cells to a cell-free system from rabbit reticulocytes. This system initiates the synthesis of new polypeptide chains at a very high rate. Cytoplasm from poliovirus infected HeLa cells, but not from uninfected cells, inhibited protein synthesis in the reticulocyte lysate (59) The inhibitor was isolated and identified as double-stranded (ds) RNA (60). To study the effect of ds RNA on host and viral protein synthesis, a cell-free system from HeLa cells was developed which initiated translation on endogenous cellular or viral mRNA. When added to this system, the ds RNA was found to inhibit the translation of both cellular and viral mRNAs (61). Furthermore, measurement of the amount of ds RNA present in cells early in infection (61, 62) revealed that an insufficient quantity was present to act as a direct agent of protein synthesis inhibition. 

In order to identify initiation factors involved in mRNA competition, a number of studies have employed reconstituted cell-free systems (Golini et al., 1976 Ray et al., 1983). A major problem with such systems is that certain components may be present in excess, while others may be limiting or partially inactivated, precluding efficient initiation. However, meaningful studies of translation initiation frequency in vitro can only be done in systems where ribosomes can cycle rapidly and repeatedly over mRNA. To date, the only cell-free systems that translate mRNA at high efficiency are the reticulocyte lysate (see Jackson, 1982) and the micrococcal nuclease-treated reticulocyte lysate (Pelham and Jackson, 1976). The latter system offers several advantages over reconstituted cell-free systems. It responds to translational control signals (see below), it is capable of extensive and efficient initiation in conditions more likely to be representative of protein synthesis in intact cells, and except for mRNA, it contains all other components for translation in a proportion much closer to that of the intact cell. 

Clearly, it will be necessary to demonstrate, first, relief of translational competition in an efficient and more native cell-free system, such as the micrococcal nuclease-treated reticulocyte lysate, and under conditions where the total number of initiation events does not change, and second, selective and specific binding of eIF-4A or CBP-II to individual mRNA species, before it can be concluded with certainty that these proteins can act as targets of mRNA competition. 

Most of the information on the regulation of eIF-2 activity comes from studies of reticulocyte lysates. It appears, however, that very similar, if not identical mechanisms operate in a variety of mammalian cell types. Initiation of translation in a cell-free system from HeLa... 

The first evidence for control of translation in adenovirus-infected cells came when mRNA isolated from infected cells harvested during the late phase was translated in heterologous, cell-free systems, usually derived from reticulocyte lysates such mRNA preparations direct the synthesis of quite substantial quantities of cellular proteins in addition to large amounts of viral structural proteins (see, for example, Anderson et ai, 1974 Lewis et ai, 1975 Paterson et ai, 1977). The mRNA was prepared from cells collected at a time... 

On the basis of our previous results discussed above, early viral transcripts were primary candidates in the inhibition of host protein synthesis. Since in vitro transcription produces only the early species of viral transcripts (Kates and Beeson, 1970), we acquired these early species of viral RNA by means of in vitro transcription by viral cores and tested their effect on the translation of various exogenous mRNAs in in vitro cell-free systems rendered messenger dependent. The results from such experiments (Coppola and Bablanian, 1983) revealed that transcripts prepared in vitro of either 8-10 S or 4-7 S size classes inhibit globin, HeLa, and hamster cell mRNA translation in a reticulocyte cell-free protein-synthesizing system. Inhibition was observed not only under conditions where in vitro viral transcripts by themselves were capable of producing viral polypeptides, but also when low concentrations of in v/7ro-synthesized transcripts were used which were incapable of synthesizing polypeptides as determined by polyacrylamide-gel electrophoresis. In contrast, the transcripts synthesized in vitro by vaccinia virus cores had no inhibitory effect on the translation of cytoplasmic RNA obtained from vaccinia virus-infected cells at early times after infection. Therefore, this inhibitory effect, like that seen in vaccinia virus-infected cells, was selective. The vaccinia virus transcripts, synthesized in vitro, also inhibited encephalomyocarditis virus mRNA translation in the cell-free reticulocyte lysate system indicating that this inhibition does not require... 

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