Figure 5.

The length of the 3′ unique sequence drastically affects Alu retrotransposition capability. (A) Constructs with 30 homogenous As and variable 3′ unique region lengths (0–126 bp) were evaluated by transient transfection of HeLa cells. The activity of these Alu elements under both exogenous (black bars) and endogenous (white bars) conditions of ORF2p is shown. The A30-0, an Alu with 30 homogenous As immediately followed by a terminator (TTTTT), was arbitrarily selected as the 100%; the asterisk (*) indicates significant difference from A30-0, P < 0.01 exogenous, P < 0.03 endogenous (Students paired t-test). (B) Northern blot analysis of poly(A) selected RNA extracts was performed from cells transfected with the A30-0 control and the variable A30 3′ unique constructs. The unspliced (open arrowhead) and spliced (black arrow) neo-tagged Alu transcripts are indicated. The spliced Alu transcript from the variable length constructs were normalized to cyclophillin (C, loading control) and expressed relative to the A30-0 construct (designated as 1.00). The mean ± SD for the quantitation results for each construct are indicated below (n = 3). (C) Histogram of the length distribution of the 3′ unique sequence of young and old Alu elements. The distribution of the length of 3′ unique sequence (defined as the sequence between A-tail and the first four Ts in the 3′ genomic flank) of a subset of randomly selected AluSx (n = 289) and Ya5 (n = 227) families is shown as the frequency subdivided into bins of various sizes. (•) Alu elements containing a premature terminator within their internal dimeric sequence are included in this bin; Sx (n = 25), Ya5 (n = 1).