The development of a phenotypic assay for monitoring the mutagenic effects of 5-methylcytosine deamination in Escherichia coli

Describes development of a pheotypic assay which monitors C to T transition mutations at the second C within CCAGG sequences in Escherichia coli

Human T Cell Leukemia Virus Type 1 Tax Protein Increases NF-κB Dimer Formation and Antagonizes the Inhibitory Activity of the IκBα Regulatory Protein

AbstractHuman T cell leukemia virus type 1 (HTLV-1) encodes a strong transcriptional transactivator, the Tax protein, that stimulates viral transcription through the long terminal repeat and also stimulates many cellular genes via the activation of host transcription factors. Previous studies have demonstrated that Tax activates NF-κB through binding to the Rel homology domain of NF-κB proteins. Tax was also shown to increase degradation of IκBα resulting in the induction of NF-κB DNA binding activity. We addressed the specificity and function of Tax interaction with members of the NF-κB/IκBα family by using EMSA, protein affinity chromatography, protein–protein crosslinking and co-immunoprecipitation assays. The results of the present study demonstrate that: (1) Tax enhances NF-κB binding to DNA 40- to 100-fold by increasing NF-κB dimer formation which can be detected in the absence of DNA; (2) Tax binds to all NF-κB DNA binding subunitsin vitroand to IκBα; (3) Tax physically associates with IκBαin vivo;and (4) Tax and IκBα have antagonistic effects on NF-κB binding and gene activity. These results suggest that Tax interaction with IκBα interferes with the formation of NF-κB–IκBα complexes and may play a role in targeting IκBα for degradation

Molecular analysis of HTLV-1 tax interactions with the NF-kB and IkBa transcription factors

The human T cell leukemia virus type I (HTLV-I), the etiological agent of adult T cell leukemia is the first human retrovirus linked to malignancy. The viral protein Tax, is essential for HTLV-I gene transactivation and responsible for oncogenic transformation. Tax is capable of indirectly transactivating numerous growth regulatory genes through interactions with host transcription factors. One of the targets of the Tax protein is the NF-kappaB/IkappaB family of transcriptional regulators. In HTLV-I Tax expressing cells, the maintenance of NF-kappaB activity is essential for cellular transformation. Previous studies demonstrated that Tax activated NF-kappaB through binding to the Rel homology domain of NF-kappaB. The objective of this work was to elucidate the mechanism by which Tax transactivates NF-kappaB. We addressed the specificity and function of Tax interaction with members of the NF-kappaB/IkappaBalpha family and demonstrated that: (1) Tax increased IkappaBalpha degradation, resulting in the induction of NF-kappaB DNA binding activity; (2) Tax enhanced NF-kappaB binding to DNA 40 to 100 fold by increasing NF-kappaB dimer formation which was detected in the absence of DNA; (3) Tax interacted with all NF-kappaB DNA binding subunits and with IkappaBalpha in vitro; (4) Tax physically associated with IkappaBalpha in vivo; and (5) Tax and IkappaBalpha had antagonistic effects on NF-kappaB binding and gene activity. These results suggested that Tax interaction with IkappaBalpha interferes with the formation of NF-kappaB-IkappaBalpha complexes and may play a role in targeting IkappaBalpha for degradation. To further characterize the interactions between Tax and IkappaB, co-immunoprecipitation experiments were performed that identified a complex in HTLV-I infected cells comprising Tax-IkappaBalpha and the HsN3 subunit of the 26S proteasome. Metabolic labeling and protein turnover studies indicate that Tax targets IkappaBalpha to the proteasome for degradation in