scholarly journals Analysis of Efficiency and Fidelity of HIV-1 (+)-Strand DNA Synthesis Reveals a Novel Rate-limiting Step during Retroviral Reverse Transcription

2000 ◽  
Vol 276 (9) ◽  
pp. 6711-6719 ◽  
Author(s):  
Matthias Götte ◽  
Masanori Kameoka ◽  
Nathan McLellan ◽  
Luciano Cellai ◽  
Mark A. Wainberg
Keyword(s):  
Dna Synthesis ◽  
Reverse Transcription ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Hiv 1

scholarly journals Promoter activity of Tat at steps subsequent to TATA-binding protein recruitment.

1997 ◽  
Vol 17 (12) ◽  
pp. 6898-6905 ◽  
Author(s):  
H Xiao ◽  
J T Lis ◽  
K T Jeang
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Transcriptional Unit ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Adenovirus E1b ◽  
Rnap Ii ◽  
Rate Limiting ◽  
Hiv 1

Artificial recruitment of TATA-binding protein (TBP) to many eukaryotic promoters bypasses DNA-bound activator function. The human immunodeficiency virus type 1 (HIV-1) Tat is an unconventional activator that up-regulates transcription from the HIV-1 long terminal repeat (LTR) through binding to a nascent RNA sequence, TAR. Because this LTR and its cognate activator have atypical features compared to a standard RNA polymerase II (RNAP II) transcriptional unit, the precise limiting steps for HIV-1 transcription and how Tat resolves these limitations remain incompletely understood. We thus constructed human TBP fused to the DNA-binding domain of GAL4 to determine whether recruitment of TBP is one rate-limiting step in HIV-1 LTR transcription and whether Tat functions to recruit TBP. As a control, we compared the activity of the adenovirus E1b promoter. Our findings indicate that TBP tethering to the E1b promoter fully effected transcription to the same degree achievable with the potent GAL4-VP16 activator. By contrast, TBP recruitment to the HIV-1 LTR, although necessary for conferring Tat responsiveness, did not bypass a physical need for Tat in achieving activated transcription. These results document that the HIV-1 and the E1b promoters are transcriptionally limited at different steps; the major rate-limiting step for E1b is recruitment of TBP, while activation of the HIV-1 LTR requires steps in addition to TBP recruitment. We suggest that Tat acts to accelerate rate-limiting steps after TBP recruitment.

Isotope-dilution studies of the effects of 5-fluorodeoxyuridine and hydroxyurea on the incorporation of deoxycytidine and thymidine by cultured thymus cells

1976 ◽  
Vol 54 (3) ◽  
pp. 238-248 ◽  
Author(s):  
F. W. Scott ◽  
D. R. Forsdyke
Keyword(s):  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Isotope Dilution ◽  
Inhibitory Effect ◽  
Intact Cells ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Radioactive Labelling ◽  
Salvage Pathways ◽  
Rate Limiting

From experimentally induced changes in the slope (Vmax) and intercept (pool) of isotope-dilution plots inferences may be drawn on the position and regulation of rate-limiting steps affecting the incorporation of pyrimidine deoxynucleosides by intact cells. 5-Fluorodeoxyuridine (FdUrd; 1 μM) reduced the Vmax of radioactive labelling with deoxy[5-3H]cytidine; this was reversed by thymidine (19 μM) suggesting that FdUrd makes the concentration of deoxythymidine triphosphate (dTTP) rate-limiting for DNA synthesis. With deoxy[U-14C]cytidine the reversal of FdUrd inhibition by thymidine was only partial; this was in keeping with (i) deoxy[U-14C]cytidine labelling both cytosine and thymine in DNA, and (ii) a continuing inhibition of thymidylate synthetase by FdUrd in the presence of thymidine (19 μM).The deoxycytidine competitor pool was increased by cytidine (10–50 μM) and decreased by (i) thymidine (19 μM), (ii) hydroxyurea (50 μM) and (iii) deoxycytidine (12 μM, in the presence of FdUrd). It is suggested that these pool-decreasing agents, or their derivatives (e.g., dTTP), inhibit ribonucleotide reductase and hence prevent the entry of pyrimidine ribonucleotide derivatives into the deoxycytidine competitor pool; because of this pool decrease, radioactive labelling with deoxy[5-3H]cytidine was enhanced by thymidine (19 μM) and hydroxyurea (50 μM). However, at the latter hydroxyurea concentration, labelling with [Me-3H]thymidine was inhibited, due to a decrease in the Vmax of the rate-limiting step for thymidine incorporation (probably thymidine kinase). This sensitivity of labelling with [Me-3H]thymidine to inhibition by hydroxyurea (50 μM) was reduced by adding FdUrd to prevent the accumulation of dTTP. At high hydroxyurea concentrations (0.1–1.0 mM), labelling with deoxy[5-3H]cytidine was also inhibited, due to a decrease in Vmax of the rate-limiting step, which was probably at the level of DNA polymerase.The results suggest that hydroxyurea inhibits DNA synthesis by making the concentration of purine deoxynucleotides rate-limiting. Pyrimidine deoxynucleotides are formed in sufficient quantities from deoxycytidine by way of salvage pathways. Indeed, dTTP accumulates and inhibits thymidine kinase, thus amplifying the inhibitory effect of hydroxyurea on labelling with [Me-3 H]thymidine.

Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

1978 ◽  
Vol 39 (02) ◽  
pp. 496-503 ◽  
Author(s):  
P A D’Amore ◽  
H B Hechtman ◽  
D Shepro
Keyword(s):  
Endothelial Cells ◽  
Ornithine Decarboxylase ◽  
Decarboxylase Activity ◽  
Aortic Endothelial Cells ◽  
Ornithine Decarboxylase Activity ◽  
Rate Limiting Step ◽  
Bovine Aortic Endothelial Cells ◽  
Limiting Step ◽  
Rate Limiting ◽  
Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

Dynamics of hepatic and peripheral insulin effects suggest common rate-limiting step in vivo

Diabetes ◽  
1993 ◽  
Vol 42 (2) ◽  
pp. 296-306 ◽  
Author(s):  
D. C. Bradley ◽  
R. A. Poulin ◽  
R. N. Bergman
Keyword(s):  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting
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