Comparison of features around a hot and cold tGly target and effects on integration frequency. (A) Schematic of features surrounding the most preferred tGly target, tGlyII (tG(GCC)B) from JB1217 and the least preferred target, tGlyIV (tG(GCC)D1) from BY4741. (B) Various features were added into several positions around tGlyIV. The tIle with 60 bases of upstream sequence and 46 bases of downstream sequence was amplified from JB1217 tGlyII and inserted 60 bases upstream, 25 bases downstream, or 299 bases downstream of BY4741 tGlyIV. The LTR downstream of tGlyII (68% identity to a query Ty1 LTR [http://www.public.iastate.edu/∼voytas/], Ty1 H3 5′ LTR) was inserted in both orientations either 299 or 25 bases downstream of tGly. Back-to-back LTRs (the first LTR is 68% identical and the second 87% identical to the reference LTR) were added at position 299. The LTR insertions were combined with insertion of tIle. When the 180 bases containing the tIle were added 25 bases downstream of tGly, the LTR shifts downstream by 180 bases, to 479 bases downstream of the tGlyIV. The same PCR primer was used to amplify insertions upstream of tGly in every strain, and its distance from tGlyIV shifts depending on the length of sequences inserted at the position 25 bases downstream of tGlyIV. Histogram showing amounts of PCR product generated from PCR of insertions upstream of tGlyIV in each strain after normalization to actin is shown at right. Histogram represents the mean of either eight or nine independent trials per strain (quantitation described in Methods section). Error bars, SEM. An asterisk (*) to the right of the bar indicates a statistically significant difference (P ≤ 0.01 by the Mann-Whitney U-test) in PCR product compared with the amount generated in the BY4741 strain. (C) Gels showing the products generated using the PCR assay of insertions upstream of tGlyIV. Two different transformants from every strain are shown. PCR of the actin gene was performed to control for amount of input genomic DNA.
