Selective inhibition of tubulin synthesis by amiprophos methyl during flagellar regeneration in Chlamydomonas reinhardi.

Amiprophos methyl (APM) is a strong, readily reversible and highly selective inhibitor of tubulin synthesis in Chlamydomonas reinhardi. The extensive induction of tubulin synthesis that accompanies flagellar regeneration in this organism is prevented by 3 to 10 micrometerAPM. When applied after induction has begun, APM causes a rapid cessation of tubulin synthesis. Translation studies in vitro indicate that the lack of tubulin production in APM-treated cells is not due to a direct inhibition of tubulin messenger RNA translation but rather to a selective depletion of tubulin messenger RNA.

Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration.

Flagellar regeneration in gametes of Chlamydomonas reinhardi is initiated within 15-20 min after flagellar amputation and proceeds at a rapid but decelerating rate until by 90 min flagellar outgrowth in 80-85% complete. Sufficient flagellar protein reserves exist in the cytoplasm to allow regeneration of flagella 1/3-1/2 normal length. Nevertheless, in vivo labeling with 14C-amino acids shows that microtubule protein and other flagellar proteins are synthesized de novo during flagellar regeneration. To determine whether tubulin is synthesized continuously by gametic cells or whether its synthesis is induced as a consequence of deflagellation, we have isolated polyribosomes from deflagellated and control cells, and analyzed the proteins produced by these polyribosomes during in vitro translation. Two proteins of 53,000 and 56,000 molecular weight which co-migrate with flagellar and chick brain tubulin on SDS-polyacrylamide gels and which selectively co-assemble with chick brain tubulin during in vitro microtubule assembly are synthesized by polyribosomes (or polyadenylated mRNA) from deflagellated cells. No microtubule proteins can be detected in the translation products synthesized by polyribosomes (or mRNA) from control cells, clearly indicating that deflagellation results in the induction ot tubulin synthesis. Kinetics of tubulin synthesis demonstrate that induction takes place immediately after deflagellation; polyribosomes bearing tubulin mRNA can be detected in the cytoplasm in as little as 15 min after removal of flagella. Maximal rates of tubulin synthesis occur between 45 and 90 min after deflagellation when approximately 14% of the protein being synthesized by the cell is tubulin. This estimate of tubulin synthesis based on in vitro translation data agrees well with in vivo measurements of flagellar tubulin synthesis. While high levels of tubulin production extend well beyond the period of rapid flagellar assembly, synthesis begins to decline after 90 min, and by 180 min after deflagellation only low levels of tubulin mRNA are detectable in polyribosomes.

Tubulin induction in C. reinhardii: requirement for tubulin mRNA synthesis.

Flagellar excision in Chlamydomonas reinhardii triggers a rapid and extensive induction of tubulin synthesis. Cloned plasmids, pFT beta 1 and pFT beta 2, carrying cDNA inserts complementary to beta-tubulin mRNA, have been prepared and used to demonstrate a direct requirement for tubulin mRNA synthesis during tubulin induction. Increased tubulin mRNA synthesis is detected within 5 min after deflagellation. During the 45 min peak period of tubulin synthesis, tubulin mRNA accumulates to levels 15- to 35-fold higher than those found in control (non-deflagellated) cells. In addition, there appears to be a direct correlation between tubulin mRNA concentrations and the levels of tubulin production during the induction and deinduction cycle that accompanies flagellar regeneration. Amiprophosmethyl (APM), a compound we reported earlier as a selective inhibitor of tubulin synthesis in deflagellated cells, is shown to block the accumulation of tubulin mRNA following flagellar excision and to cause the rapid loss of tubulin mRNA from cells treated at the peak of induction.

Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis.

Poliovirus RNA polymerase mutation 3D-M394T results in a temperature-sensitive defect in RNA synthesis.

Mutant ts10 is an RNA-negative temperature-sensitive mutant of Mahoney type 1 poliovirus. Mutant ts10 3D pol was purified from infected cells and was shown to be rapidly heat-inactivated at 45 degrees when compared to wild-type polymerase. Sequencing of mutant ts10 genomic RNA revealed a U to C transition at nt 7167 resulting in an amino acid change of methionine 394 of 3D pol to threonine. The 3D-M394T mutation was engineered into a wild-type infectious clone of poliovirus type 1. The resultant mutant virus, 3D-105, had a temperature-sensitive phenotype in plaque assays. The translation and replication of wild-type, ts10, and 3D-105 virion RNAs were all characterized in HeLa S10 translation-RNA replication reactions in vitro. The optimum temperatures for the replication of the wild-type and mutant viral RNAs in the HeLa S10 translation-replication reactions were 37 and 34 degrees, respectively. To characterize the temperature-sensitive defect in the replication of the mutant RNA, we used preinitiation RNA replication complexes which were formed in HeLa S10 in vitro reactions containing guanidine HCl. Negative-strand RNA synthesis in 3D-M394T mutant preinitiation replication complexes was normal at 34 degrees but was rapidly and irreversibly inhibited at 39.5 degrees. To differentiate between the initiation and elongation steps in RNA replication, we compared the elongation rates in mutant and wild-type replication complexes at 39.5 degrees. The results showed that the elongation rates for nascent negative strands in both the mutant and wild-type replication complexes were identical. Therefore, the results indicate that the heat-sensitive step in negative-strand synthesis exhibited by the 3D-M394T replication complexes is in the initiation of RNA synthesis and not in the elongation of nascent chains.