Abstract
G63, the major surface glycoprotein of Leishmania chagasi promastigotes, increases 11-fold in amount as promastigotes grow from logarithmic to stationary phase. Transcripts from three different classes of gp63 genes are differentially expressed during this development (Ramamoorthy, R., Donelson, J. E., Paetz, K. E., Maybodi, M., Roberts, S. P., and Wilson, M. E. (1992) J. Biol. Chem. 267, 1888-1895). We studied the effect of protein synthesis inhibitors on gp63 mRNAs. The steady state level of log class gp63 RNA, expressed primarily in logarithmic phase promastigotes, increased 16.5-fold after incubation in cycloheximide. A similar increase in log gp63 RNAs was caused by inhibitors that block different steps in translation. In contrast, the levels of stationary class gp63 RNA, expressed in stationary phase parasites, and constitutive class gp63 RNA, expressed throughout promastigote growth, increased only 2.3- and 1.5-fold, respectively. The latter was not statistically significant. Nuclear run-on assays showed that the cycloheximide effect was not due to an increased rate of transcription. However, the t1/2 of log RNAs was prolonged 6.5-fold after incubation in cycloheximide, in contrast to a 1.7-fold increase in the t1/2 of ATPase RNA, suggesting that cycloheximide specifically stabilizes log gp63 mRNAs. Thus, a highly labile negative regulatory protein, such as an RNase, may specifically target log gp63 RNAs for degradation.
Highlights
G63, the major surface glycoprotein of Leishmania mented in the case of p-tubulin, involves the co-translational chagusi promastigotes,increases 11-fold in amountas degradation of mRNAs by a ribosome-associated RNase
SynthesisAffects Leishmania Gp63 RNAs rapidly during promastigote development,and because Northern blots suggest that both transcripts arise from a polycistronic precursor2 [13], we examined whether these changes might require ongoing protein synthesis
We found that the steady state level of RNA transcripts from log gp63 genes increased 16.5-fold aftertheaddition of cycloheximide to promastigote cultures, whereas the amountsof stationary and constitutive gp63 gene transcripts increased by only small amounts.We examined each of the three potential mechanismfosr the induction of log RNAs by cycloheximide
Summary
Richard Pearson, M.D., at theUniversity of Virginia. Parasites were Effect of Cycloheximide and Other Translation Inhibitors on maintained in hamsters. 2.4 shows a reprebegan in 30 min (not shown) and was maximal by 11 h after sentative Northern blot demonstrating theffect of puromythe addition of cycloheximide to promastigote cultures Similar RNA samples were probed with aDNA fragment in such a protein, we used nuclear run-on assays to estimate generated by polymerase chain reaction from the unique 3’ the relative rates of transcription of the different gp gene region of a constitutiue gp cDNA.
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