`
`PCT/NL02/00390
`
`58
`
`SV40 promoter (FIG 11): the yield from the highest-expressing STAR6 clone is
`
`2-3 fold greater than the best pSDH-SV40 clones, and 6 STAR clones (20% of
`
`the population) have yields higher than the best STAR-less clones. In the
`
`context of the Tet—Off promoter under inducing (low doxycycline)
`
`concentrations, STAR6 also improves the yield and predictability of transgene
`
`expression (FIG 12): the highest-expressing STARS clone has a 20-fold higher
`
`yield than the best pSDH—Tet clone, and 9 STARG clones (85% of the
`
`population) have yields higher than the best STAR-less clone. It is concluded
`
`that this STAR element is versatile in its transgene-protecting properties,
`
`10
`
`since it functions in the context of various biotechnologically useful promoters
`
`of transcription.
`
`Example 16
`
`15
`
`STAR element function can be directional
`
`While short nucleic acid sequences can be symmetrical (e. g. palindromic),
`
`longer naturally-occurring sequences are typically asymmetrical. As a result,
`
`the information content of nucleic acid sequences is directional, and the
`
`sequences themselves can be described with respect to their 5’ and 3’ ends. The
`
`directionality of nucleic acid sequence information affects the arrangement in
`
`which recombinant DNA molecules are assembled using standard cloning
`
`techniques known in the art (Sambrook et al., 1989). STAR elements are long,
`
`asymmetrical DNA sequences, and have a directionality based on the
`
`orientation in which they were originally cloned in the pSelect vector. In the
`
`examples given above,using two STAR elements in pSDI—I vectors, this
`
`directionality was preserved. This orientation is described as the native or 5’-3’
`
`orientation, relative to the zeocin resistance gene (see FIG 13). In this example
`
`the importance of directionality for STAR function is tested in the pSDH-Tet
`
`vector. Since the reporter genes in the pSDH vectors are flanked on both sides
`
`by copies of the STAR element of interest, the orientation of each STAR copy
`
`20
`
`25
`
`30
`
`

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