Unsaturated fatty acid auxotrophs of Eschericha coli have been divided into two distinct cistrons by extract complementation and genetic complementation based on abortive transduction. Lesions in one cistron result in the loss of the ,B-hydroxydecanoyl thioester dehydrase which produces the first biosynthetic intermediate in unsaturated fatty acid formation. Evidence is presented which indicates that lesions in the second cistron result in the lack of a second enzyme specifically involved in the biosynthesis of unsaturated fatty acids.
The composition of a soluble glycopeptide obtained after the lysis of cell walls of Staphylococcus aureus by an acetylmuramidase has been reported. This glycopeptide has been degraded sequentially by two peptidase preparations, containing six enzyme activities, viz. two glycine bridge splitting enzymes, glycine and Lalanine aminopeptidases, and glycine and D-alanine
Encephalomyocarditis viral RNA has previously been shown to outcompete host cellular mBNA for translation in vitro in crude and fractionated protein synthe sizing systems. In the present communication it is shown that the competition is regulated byan initiation factor or complex of factors, and not the 40S initiation complex per se. The factor primarily involved is the murine equivalent of a component present in a partially purified preparation of rabbit initiation factor M3. Both the murine and rabbit factors are clearly messenger discriminatory. In initial studies on the translation of encephalomyocarditis (EMC) viral RNA and cellular mRNA in crude cell-free extracts from MOPC-460 cells it was observed that EMC RNA could strongly outcompete cellular mRNA for translation (1). In a recent communication it was shown that mRNA competition in vitro occurs during the initiation of protein synthesis at or prior to the formation of the first peptide bond (t, 2). It was also found that the 40S ribosome does not preferentially bind to EMC RNA in the presence of host mRNA, suggesting that the ribosome per se is not the site of mRNA competition (t, 2). In view of this result it seemed likely that an initiation factor (or complex of factors) that has a strong preference for EMC RNA might be the site of competition. In the present communication we report results supporting this hypothesis, and show that the factor responsible for most of the messenger specificity is the murine equivalent of a factor present in a partially purified preparation of initiation factor MS (IF-M3) from rabbit reticulocytes (3).
METHODSThe preparation of viral and cellular mRNAs, the preparation of a fractionated protein synthesizing system from Fig. 1A, which is an autoradiogram of a polyacrylamide gel electropherogram of the [3aS]methionine-labeled polypeptides produced in reaction mixtures programmed by globin and EMC mRNAs, added separately or together. As noted previously (*, 2), it is also evident here that the inclusion of EMC RNA in the reaction mixture strongly suppresses globin mRNA translation, whereas a large molar excess of globin mRNA has little or no effect on translation of EMC RNA (compare lanes 4 and 6, or lanes 5 and 7, of Fig. IA). Moreover, it is clear that the addition of excess pH 5 enzyme does not relieve this suppression (lane 7, Fig. 1A). In contrast, excess crude initiation factors (ribosomal salt wash) promote the partial resumption of globin synthesis, and in this sense cause the competition to be relieved (lane 8, Fig. 1A).To show that this effect is specific for globin synthesis and is not due to a general increase in the translation of all mRNAs, we quantitated results of experiments similar to those shown in Fig. IA
Heat-shock proteins (hsp) were elicited when mycelia of the Downs strain and the more virulent G184A and G222B strains of Histoplasma capsulatum were shifted up to temperatures which induced the mycelial-to-yeast transition (34-40 "C). The classes of the major hsp which increased in synthesis in each strain were similar. However, the pattern of synthesis of these proteins at the different temperatures in Downs differed from those in the G184A and G222B strains: hsp synthesis in Downs peaked at 34 "C, whereas in G184A and G222B it was highest at 37 "C.
The relative initiation rates for encephalomyocarditis virus mRNA and host mRNA's in infected cells were measured using two independent techniques. In both cases the results showed that viral mRNA initiates at a much higher rate than host mRNA'S. This difference was observed midway in the infectious cycle, well before virus-induced cytopathic effects (leakage of low-molecular-weight metabolites, failure to exclude trypan blue) were apparent. These results confirm that encephalomyocarditis viral mRNA is a more efficient initiator than host mRNA's in vivo, as has previously been demonstrated in in vitro experiments.
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