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Poly(A) polymerase activity during cell cycle and erythropoietic differentiation in erythroleukemic mouse spleen
Abstract:
Poly(A) polymerase activity was studied in lysates of cultured murine erythroleukemic cells (Friend cells). Incorporation of ATP into acid-precipitable products is dependendent on the presence of Mn2+ or Mg2+ and of an RNA primer. The reaction is specific for ATP as the substrate (KM=290 290 micron, it is not inhibited by actinomycin D and only slightly interferred with by ethidium bromide. Cordycepin 5'-triphosphate and sodium pyrophosphate inhibit the enzyme activity. The chain length of the products of the reaction is dependent on the primer concentration and reaches up to 30 nucleotides. Poly(A) polymerase activity is low in resting (G1 phase) cells 75 nmol ATP incorporated/h per 10(6) cells) and increases to a level about twice as high in early S phase of the cell cycle. A possible model for regulation of enzyme activity is discussed. Polymerase activity in the early phase of erythropoietic differentiation of the cells induced by butyric acid does not show any difference in comparison to untreated controls. A decrease in enzyme activity to levels characteristic for cells in G1 phase accompanies shutdown of cell growth in the course of the ongoing differentiation. Analysis of the DNA content of the cells revealed that erythropoietic differentiation of Friend cells induced by butyric acid is characterized by arrest of the cells in G1 phase of the cell cycle. Poly(A) polymerase activity in erythroleukemic cells is thus controlled only by the phase of the cell cycle; it is not affected by changes in gene expression during erythroid differentiation.
Insights
Poly(A) polymerase activity in Friend cells is regulated by the cell cycle, peaking in S phase. This enzyme activity is not influenced by erythroid differentiation or gene expression changes.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Polyadenylation is a crucial post-transcriptional modification affecting mRNA stability and translation.
- Poly(A) polymerase (PAP) is the enzyme responsible for adding the poly(A) tail to RNA transcripts.
- Understanding PAP regulation is vital for comprehending gene expression control.
Purpose of the Study:
- To investigate the activity and regulation of Poly(A) polymerase in cultured murine erythroleukemic cells (Friend cells).
- To determine the influence of cell cycle phase and erythroid differentiation on PAP activity.
- To explore potential mechanisms controlling PAP enzyme activity.
Main Methods:
- Enzyme assays measuring ATP incorporation into acid-precipitable products using cell lysates.
- Analysis of PAP activity in different cell cycle phases (G1 and S).
- Assessment of PAP activity during butyric acid-induced erythroid differentiation.
- DNA content analysis to confirm cell cycle arrest.
Main Results:
- PAP activity requires divalent cations (Mn2+ or Mg2+) and an RNA primer, with ATP as the specific substrate.
- Activity is inhibited by cordycepin triphosphate and pyrophosphate.
- PAP activity is low in G1 phase and doubles in early S phase.
- Butyric acid-induced differentiation, characterized by G1 arrest, did not alter PAP activity in early stages but decreased it later.
- PAP activity correlated with cell cycle phase, not with differentiation-induced gene expression changes.
Conclusions:
- Poly(A) polymerase activity in Friend cells is primarily controlled by the cell cycle phase.
- Erythroid differentiation does not directly impact PAP activity; observed changes are linked to cell cycle arrest.
- Gene expression changes during differentiation do not modulate PAP levels or activity.