More recently, Anderson et al. [ 92] have attempted unsuccessfully to demonstrate peptide synthesis using a 322-nucleotide construct containing the most conserved regions of the peptidyl transfer centre, very close to the proposed ancestral peptidyl transferase sequence of Bokov and Steinberg [ 9].
Deletion of the sequence encoding the first, second or third to fourth LRRs (nucleotides 193 264, construct D2; nucleotides 265 345, construct D3; nucleotides 346 474, construct D4, Fig. 7A) still resulted in a significant decrease of cwp1-au5 mRNA in the pNUPF1HA co-transfectants relative to the control cell line (Fig. 7B).
This nucleotide polygene construct encodes a scrambled Nef variant lacking biological activity [28], [36].
To determine whether artificial addition of a poly(A) tract could stimulate the elimination of DSR-containing transcripts, we inserted poly dA) sequences of various lengths between the DSR and the snu2 terminator on Construct 3, as is schematically shown in Figure 3D (Construct 3-A10, 10 nucleotides; Construct 3-A50, 50 nucleotides; and Construct 3-A100, 100 nucleotides).
Residual luciferase reporter activity in the carcinoma cells was completely abolished by deletion of an additional seven nucleotides: the construct −43/+114 had the same negligible activity as a promoterless luciferase reporter (Fig. 3).
A further reduction in promoter activity was observed in the − 54 nucleotides truncated construct which only contains the IL-8 TATA box (p < 0.05; Fig. 2A).
No further reduction in promoter activity was observed in the − 54 nucleotides truncated construct which only contains the CXCL8 TATA box (Fig. 2C; P < 0.01).
13– 15 The composition vector method uses informative strings (ie, short nucleotide sequences) to construct phylogenies.
Orthologous protein sequences were aligned using MUSCLE[ 50] and PAL2NAL[ 51] was used to construct nucleotide alignments.
We used VGF-homologous and TK gene nucleotide sequences to construct a phylogenetic tree for comparison with other poxviruses.
We used ClustalW (http://www.clustal.org) and MEGA4 software (http://megasoftware.net/) to align nucleotide sequences and construct a phylogenetic tree (neighbor-joining method, 1,000 bootstraps) from the obtained HA fragment.
