PubMed · 5440203
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G M Komrower. 1970. Phenylketonuria. Some current problems.. https://doi.org/10.1136/adc.45.239.2
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We previously demonstrated that hybrid retrotransposons composed of the yeast Ty1 element and the reverse transcriptase (RT) of HIV-1 are active in the yeast Saccharomyces cerevisiae. The RT activity of these hybrid Ty1/HIV-1 (his3AI/AIDS RT; HART) elements can be monitored by using a simple genetic assay. HART element reverse transcription depends on both the polymerase and RNase H domains of HIV-1 RT. Here we demonstrate that the HART assay is sensitive to inhibitors of HIV-1 RT. (-)-(S)-8-Chloro-4,5,6, 7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1, 4]-benzodiazepin-2(1H)-thione monohydrochloride (8 Cl-TIBO), a well characterized non-nucleoside RT inhibitor (NNRTI) of HIV-1 RT, blocks propagation of HART elements. HART elements that express NNRTI-resistant RT variants of HIV-1 are insensitive to 8 Cl-TIBO, demonstrating the specificity of inhibition in this assay. HART elements carrying NNRTI-resistant variants of HIV-1 RT can be used to identify compounds that are active against drug-resistant viruses.
The helper T lymphocyte precursor (HTLp) assay is of value for predicting graft-vs.-host disease after allogeneic bone marrow transplantation. The assay is based on limiting dilution analysis and requires detection of the amount of interleukin 2 (IL-2) produced by one activated T cell. IL-2 is detected by 3H-thymidine (3H-TdR) incorporation into the IL-2 dependent cell line, CTLL-2. Detection of IL-2 in the whole culture volume of the wells in the microtiter plates increases sensitivity, but requires elimination of 3H-TdR incorporation into the responder cells in the HTLp assay. We have compared the ability of X-radiation and ultraviolet B (UV-B) radiation to eliminate 3H-TdR incorporation. X-irradiation of the cells reduced 3H-TdR incorporation by 90% with doses up to 400 Gy, but the incorporation was still 10 times higher than in wells with stimulator cells only. UV-B irradiation (with Philips TL 12 tubes) of the cells with > or = 2 J/cm2 decreased 3H-TdR incorporation to the level of wells with stimulator cells only. Neither X-irradiation nor UV-B irradiation of the cultures affected IL-2 produced in the assay or human recombinant IL-2 measured by 3H-TdR incorporation into the IL-2 dependent cell line, CTLL-2. HTLp frequencies determined after 25 Gy X-irradiation were higher (mean 1.5, range 1.02-2.2 times higher) than HTLp frequencies determined after UV-B irradiation with 2 J/cm2.
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