Effect of an ecdysteroid on DNA synthesis, DNA polymerase α and thymidine kinase activities ofDrosophila melanogaster cells in vitro

Munsch, Nicole; Cade-Treyer, D

doi:10.1007/bf01959738

Cited by 4 publications

(2 citation statements)

References 20 publications

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“…Deletion of 30 amino acids at the C terminus decreased the activity to about 1% of rDm-dNK, indicating the loss of a domain essential for catalysis. Up-regulation of a TK activity parallel to the activity of polymerase ␣ during proliferation has been reported for a D. melanogaster cell line (40). It is speculative whether the C terminus with the putative phosphorylation site is involved in this up-regulation, but our data clearly show the ability of this region to suppress deoxyribonucleoside phosphorylation, while providing broader substrate specificity.…”

Section: Discussionsupporting

confidence: 62%

Functional Expression of a Multisubstrate Deoxyribonucleoside Kinase from Drosophila melanogaster and Its C-terminal Deletion Mutants

Munch‐Petersen¹,

Knecht²,

Lenz³

et al. 2000

Journal of Biological Chemistry

116

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The occurrence of a deoxyribonucleoside kinase in Drosophila melanogaster (Dm-dNK) with remarkably broad substrate specificity has recently been indicated (Munch-Petersen, B., Piskur, J., and Søndergaard, L. (1998) J. Biol. Chem. 273, 3926 -3931). To prove that the capacity to phosphorylate all four deoxyribonucleosides is in fact associated to one polypeptide chain, partially sequenced cDNA clones, originating from the Berkeley Drosophila genome sequencing project, were searched for homology with human deoxyribonucleoside kinases. The total sequence of one cDNA clone and the corresponding genomic DNA was determined and expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified and thrombin cleaved recombinant protein phosphorylated the four deoxyribonucleosides with high turnover and K m values similar to those of the native Dm-dNK, as well as the four ribonucleosides and many therapeutical nucleoside analogs. DmdNK has apparently the same origin as the mammalian kinases, thymidine kinase 2, deoxycytidine kinase, deoxyguanosine kinase, and the herpesviral thymidine kinases, but it has a unique C terminus that seems to be important for catalytic activity and specificity. The Cterminal 20 amino acids were dispensable for phosphorylation of deoxyribonucleosides but necessary for full activity with purine ribonucleosides. Removal of the C-terminal 20 amino acids increased the specific activity 2-fold, but 99% of the activity was lost after removal of the C-terminal 30 amino acids.

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Section: Discussionsupporting

confidence: 62%

Functional Expression of a Multisubstrate Deoxyribonucleoside Kinase from Drosophila melanogaster and Its C-terminal Deletion Mutants

Munch‐Petersen¹,

Knecht²,

Lenz³

et al. 2000

Journal of Biological Chemistry

116

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“…Interruption of S2 cell proliferation by the addition of 20‐hydroxyecdysone is accompanied by an arrest of DNA synthesis, deduced from the measurement of tritium labeled thymidine incorporation performed previously (Gvozdev et al . 1974; Rosset 1978; Munsch & Cade‐Treyer 1982). To test the effect of ecdysone hormone on dPCNA promoter activity in S2 cells, we conducted transient luciferase expression assays with −168PCNA‐luc plasmid in the presence or absence of 20‐hydroxyecdysone.…”

Section: Resultsmentioning

confidence: 99%

Antagonistic regulation of the Drosophila PCNA gene promoter by DREF and Cut

Seto

Hayashi²,

Kwon³

et al. 2006

Genes to Cells

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The gene promoter of Drosophila proliferating cell nuclear antigen ( dPCNA ) contains several transcriptional regulatory elements, such as upstream regulatory element (URE), DNA replicationrelated element (DRE, 5′ ′ ′ ′ -TATCGATA), and E2F recognition sites. In the present study, a yeast one-hybrid screen using three tandem repeats of DRE in dPCNA promoter as the bait allowed isolation of a cDNA encoding Cut, a Drosophila homolog of mammalian CCAAT-displacement protein (CDP)/Cux. Electrophoretic mobility shift assays showed that Cut bound to both DRE and the sequence 5′ ′ ′ ′ -AATCAAAC in URE, with much higher affinity to the former. Measurement of dPCNA promoter activity by transient luciferase expression assays in Drosophila S2 cells after an RNA interference for Cut or DREF showed DREF activates the dPCNA promoter while Cut functions as a repressor. Chromatin immunoprecipitation assays in the presence or absence of 20-hydroxyecdysone further showed both DREF and Cut proteins to be localized in the genomic region containing the dPCNA promoter in S2 cells, especially in the Cut case upon induction of differentiation. These results indicate that Cut functions as a transcriptional repressor of dPCNA gene by binding to the promoter region in the differentiated state, while DREF binds to DRE to promote expression of dPCNA during cell proliferation.

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Hormonal Control of Development of Insects

Echalier¹

2018

Drosophila Cells in Culture

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Effect of an ecdysteroid on DNA synthesis, DNA polymerase α and thymidine kinase activities ofDrosophila melanogaster cells in vitro

Cited by 4 publications

References 20 publications

Functional Expression of a Multisubstrate Deoxyribonucleoside Kinase from Drosophila melanogaster and Its C-terminal Deletion Mutants

Functional Expression of a Multisubstrate Deoxyribonucleoside Kinase from Drosophila melanogaster and Its C-terminal Deletion Mutants

Antagonistic regulation of the Drosophila PCNA gene promoter by DREF and Cut

Hormonal Control of Development of Insects

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