ABSTRACT
The murine translation initiation factor eIF4A is encoded by two genes: eIF4A(I), expressed in all mouse tissues, and eIF4A(II), a gene preferentially expressed in organs with low proliferative capacity. To investigate the hypothesis that regulation of the eIF4A isogenes is dependent upon cellular growth status, steady state expression of eIF4A(I) and eIF4A(II) mRNAs was quantitated in asynchronous cell populations and in cultures synchronized by nutrient starvation. Our data showed that changes in cell growth state were responsible for striking differences in eIF4A isogene-specific regulation. eIF4A(I) mRNA was 10-fold more abundant than eIF4A(II) in growing cells. In growth arrested cells eIF4A(I) mRNA levels remained unchanged, whereas eIF4A(II) mRNA levels increased approximately 3-fold. Following serum stimulation of growth arrested cells, eIF4A(I) mRNA levels increased 3- to 10-fold; conversely, eIF4A(II) mRNA levels decreased 2- to 3-fold. Thus, eIF4A(I) mRNA is synthesized and translated most efficiently in growing cells while eIF4A(II) mRNA synthesis and translation is associated preferentially with the growth-arrested (quiescent) state. This difference in expression patterns likely enables the cell to maintain required levels of this factor throughout its life cycle.
Subject(s)
Peptide Initiation Factors/genetics , Animals , Cell Division/genetics , Cell Division/physiology , Cell Line , Eukaryotic Initiation Factor-4A , Gene Expression Regulation , Interphase/genetics , Interphase/physiology , L Cells , Mice , Peptide Initiation Factors/biosynthesis , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Azaserine causes DNA damage in stationary-phase cells. In our investigation of this damage, we used strains of Escherichia coli differing in repair capabilities to study azaserine-induced DNA damage, detected as DNA strand breaks by sucrose gradient sedimentation techniques. Reduced sedimentation in alkaline and neutral sucrose gradients indicated the presence of both alkali-labile sites and in situ strand breaks. Azaserine induced DNA single-strand breaks (SSBs) abundantly in all but the recA strain, in which SSBs were greatly reduced. Treatment of purified DNA with azaserine from bacteriophages T4 and PM2 produced no detectable SSBs. Several other studies also failed to detect DNA damage induced directly by azaserine. Increased levels of beta-galactosidase were induced in an E. coli strain possessing a rec::lac fusion, providing further evidence for azaserine induction of the recA gene product. In addition, azaserine induced adaptation against killing but not against mutagenesis in wild-type E. coli strain.
Subject(s)
Azaserine/toxicity , DNA, Bacterial/genetics , Escherichia coli/genetics , Mutation/drug effects , Cell-Free System , DNA Repair , Gene Expression Regulation/drug effects , Hydrolysis , Rec A Recombinases/geneticsABSTRACT
The molecular mechanism of DNA injury by mild heat was investigated using matched isogenic mutants of E. coli. On heating at 52 degrees C for 1 h, the number of DNA single-strand breaks (SSBs) detected by the alkaline sucrose gradient sedimentation technique was consistently smaller in mutants NH5016 and BW2001, both deficient in the AP (apurinic/apyrimidinic) endonuclease of exonuclease III, as compared with their wild-type parent AB1157. The greater number of SSBs in the wild type was accompanied by more extensive cell death as compared with the AP-deficient mutants. Heating of endonuclease-free DNA systems, viz., T4 phage and T4 DNA, at 52 degrees C for up to 4 h did not result in any detectable SSB. Apparently, cellular injury by mild heat is self-inflicted through an AP-endonuclease-mediated process and hence depends on the cell's genetic complement of AP endonuclease. Mild heat is believed to activate the nucleolytic attack, and the resultant DNA-strand breaks, if not repaired, will eventually lead to cell death.
