Figure 6

Identification of the polyA addition site of the HBZ transcript. (A), PolyA+ RNA and total RNA from 293T cells transfected with 5 μg K30-3'/4089 or ACH were analyzed by RT-PCR with the primers 21-5 and 20-19 (20–27 for ACH-transfected cells). Controls were performed for DNA contamination (lane 2) and autopriming (lane 3). CTL represents PCR amplification conducted in the absence of cDNA or RNA samples. M = 100 bp marker (the asterisk indicates the 600 bp band). (B) RNA samples from 293T cells transfected with 5 μg K30 or HTLV-I-infected MJ cells were analysed by 3' RACE. Amplified products were run next to a 100 bp marker (M). (C) Position of the polyA addition site (indicated with arrow) next to a consensus polyA signal and a GU-rich consensus sequence. The structure of the HBZ mRNA with the most representative HBZ spliced variant (SP1) and the 3' polyA tail is shown below. Dark boxes represent the coding portion of the transcript. The complete proviral DNA and the former HBZ ORF are also shown below. ( D ) HTLV-I sequences taken from GenBank were compared with polyA signals (position 3821–3880) located on the antisense strand of the K30 proviral DNA (accession number L03561). Comparisons were focussed on the AATAAA polyA signal, the cleavage site deduced from our 3'RACE results and the GT-rich sequence (underlined in the K30 proviral DNA sequence). GenBank accession numbers are provided for each compared HTLV-I proviral DNA clones.