Figure 4.

Expression of chimeric IC70/ARS gene constructs. (A) A promoterless ARS gene (shaded) was joined to IC70 flanking sequences (−430 to +1) to form 70F/ARS, or to a larger fragment spanning the IC70 5′-UTR as well (−430 to +430) to form 70FU/ARS. By removing the first intron from 70FU/ARS, 70FUΔI/ARS was obtained. 70+5′I/ARS and 70–5′I/ARS resulted from the insertion of the first intron upstream of 70FUΔI/ARS. Arrowheads indicate the orientation of intron sequences. (B) RNA isolated before deflagellation (ND) and 30 min after deflagellation (DF) from untransformed cells (C) and from selected transformants of 70F/ARS and 70FU/ARS was probed with ARS and CBLP. Endogenous ARS gene expression was totally repressed by the presence of SO₄²⁻ in the medium (cont. lanes), 70F/ARS was expressed but not induced, whereas 70FU/ARS transcripts increased after deflagellation. (C) The 70F/ARS blot from panel B was stripped and reprobed with IC70 and CBLP to reveal a normal induction of the endogenous IC70 gene. (D) RNA from two 70FUΔI/ARS transformants probed with ARS and CBLP. Loss of the intron eliminates deflagellation inducibility. (E) The same blot as panel D stripped and reprobed with IC70. (F) RNA from a control strain and from strains transformed with 70+5′I/ARS and 70−5′I/ARS probed with ARS and CBLP. Insertion of the intron restores deflagellation inducibility.