F Zimmermann

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.

With the availability of several recombinant erythropoietin (Epo) reagents, it has been possible to undertake a systematic study of the relative Epo sensitivity of late erythroid colony-forming units (CFU-E) in 8.5-day embryos, 13.5-day fetal liver, and adult bone marrow of the mouse. All Epo preparations tested, including one from impure sheep and a highly purified human native Epo preparation, produced parallel, but displaced, dose-response curves when Epo concentration was plotted against percent CFU-E response calculated from the optimal Epo concentration. It was found that the CFU-E derived from 8.5-day embryos demonstrated the greatest Epo sensitivity which decreased in fetal liver and adult bone marrow CFU-E populations. Modifications to the culture system allowed CFU-E to be stimulated with as little as 0.003 mU/mL, equivalent to approximately 0.03 fg Epo. Under these culture conditions, no evidence for apoptosis was found, although a normal programmed cell death function cannot be ruled out.

Sequential virus isolates from an HIV-1-infected woman treated orally with 3'-azido-3'-deoxythymidine (AZT) for over two years showed a 10-fold reduced sensitivity for AZT after 8 months and a 100-fold resistance after 24-32 months of drug therapy. These AZT-resistant mutants were totally sensitive in vitro to other reverse transcriptase (RT)-inhibitors like the AZT-analogue 3'-fluoro-3'-deoxythymidine (FdT) or the chemically less related nucleoside analogue 2',3'-dideoxycytosine (ddC). Even the benzodiazepin derivative 4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)-imidazo [4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), a new drug specific for HIV-1 RT, was inhibitory for these virus strains. Moreover, compounds with different modes of action, e.g. polysulfated polyxylan, exhibited full antiviral activity as well. Thus, AZT resistance seems to be highly specific and should allow to develop further drugs to be used when AZT resistance has emerged. 5.9 kb fragments of the 5'-genomic halves of these sequential HIV-isolates were amplified by PCR and cloned. DNA sequence analysis revealed that the RT gene of the two highly AZT-resistant isolates carried two of the mutations described by Larder et al. [Science 246, (1989)], the Lys 70----Arg and the Thr 215----Tyr transitions. The isolate obtained after 32 months of AZT-therapy in addition contained a third mutation at position 67 (Asp----Asn); in contrast to Larder's report, no mutation was found at position 219. Thus, although these virus isolates showed at least a 100-fold reduced susceptibility for AZT in vitro, the four mutations postulated to be relevant for highly resistant strains were only partially confirmed.