The PCR assay for targeted integration upstream of tDNAs in S. cerevisiae. The pVIT41 donor plasmid (Lauermann et al. 1997) contains a unique oligonucleotide-binding site referred to as SSB. LTRs are shown as triangles, with the wide part of the triangle representing the U3 region. After induction on galactose medium to express the Ty1, the Ty1 transcript is translated, reverse transcribed, and the resulting cDNA is integrated into the yeast genome in either orientation. Insertion events are detected by using a primer complementary to the SSB in the Ty1 element in combination with a tDNA-specific primer. PCR products labeled with an asterisk (*) are >6 kb long and are not generated by the PCR conditions used in these experiments.

Number of insertions upstream of each member of the t^Gly and t^Thr gene families. (A) Number of insertions upstream of each of the 16 genomic t^Gly genes. All t^Gly genes assayed had GCC as their anticodon. Roman numerals represent chromosome on which the copy exists; arabic numerals after the comma represent the copy number in cases where more than one t^Gly exists on the same chromosome. For example, t^Gly IV, 1, represents the first (5′-most on the Watson strand) of the two copies of t^Gly on chromosome IV. The name for this gene in the Saccharomyces genome database (http://www.yeastgenome.org/) is tG(GCC)D1. (B) Number of insertions upstream of each of the 11 genomic t^Thr genes. All t^Thr assayed had AGU as their anticodon and were named by the same conventions described for t^Gly.

Distribution of de novo insertions within 650 bases upstream of the TSSs of t^Gly and t^Thr gene families and individual preferred tDNAs. (A) Insertions upstream of the t^Gly family. (B) Insertions upstream of the t^Thr family. (C) Insertions upstream of individual tDNAs that received more than 15 insertion events. (Bottom, left) Composite of insertions upstream of all tDNA targets that received 15 or less insertion events. Insertions are plotted as described in the legend to Figure 4. Gray bars highlight the 36 bases (18 bases on each side) around each integration peak (–85, –170, –265, and –335).

The insertion pattern upstream of the t^Gly and t^Thr gene families is periodic and depends on the expression of the marked Ty1 element and the Ty1 integrase protein. Four primer pairs were used to identify Ty1 insertions upstream of t^Gly and t^Thr genes in both orientations. (A) PCR products from each primer combination resulting from galactose induction with the pVIT41 Ty1 donor plasmid, without induction or without the plasmid. (B) The periodic insertion pattern depends on the presence of wild-type Ty1 integrase and does not require the homologous recombination pathway. Inductions were completed in a Δrad52 strain. The pNB19 plasmid containing a mutation in the catalytic site of integrase (–) or pVIT41 (WT) were used as the source of Ty1.

Distribution of de novo Ty1 insertions and endogenous Ty retrotransposon sequences upstream of tDNA targets in the yeast genome. (A) Composite diagram of all sequenced de novo insertion events within 650 bases upstream of the TSS of 35 different genomic tDNAs (n = 803). Each line represents insertion events into a single base position. The frequency of insertion events into a position is shown vs. the distance upstream of the tDNA target. Insertions begin 65 bases upstream of the tDNA, shown at the far right, and distance increases leftward across the x-axis. Ty1 insertions amplified using the orientation 1 primer are shown above the x-axis; those amplified with the orientation 2 primer are shown below the line. (B) Position of endogenous Ty1, Ty2, and Ty4 LTRs in the yeast genome upstream of the mature 5′ processed end of the tDNA target.

Comparison of features around a hot and cold t^Gly target and effects on integration frequency. (A) Schematic of features surrounding the most preferred t^Gly target, t^GlyII (tG(GCC)B) from JB1217 and the least preferred target, t^GlyIV (tG(GCC)D1) from BY4741. (B) Various features were added into several positions around t^GlyIV. The t^Ile with 60 bases of upstream sequence and 46 bases of downstream sequence was amplified from JB1217 t^GlyII and inserted 60 bases upstream, 25 bases downstream, or 299 bases downstream of BY4741 t^GlyIV. The LTR downstream of t^GlyII (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 t^Gly. 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 t^Ile. When the 180 bases containing the t^Ile were added 25 bases downstream of t^Gly, the LTR shifts downstream by 180 bases, to 479 bases downstream of the t^GlyIV. The same PCR primer was used to amplify insertions upstream of t^Gly in every strain, and its distance from t^GlyIV shifts depending on the length of sequences inserted at the position 25 bases downstream of t^GlyIV. Histogram showing amounts of PCR product generated from PCR of insertions upstream of t^GlyIV 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 t^GlyIV. Two different transformants from every strain are shown. PCR of the actin gene was performed to control for amount of input genomic DNA.