Number of MTX-resistant subclones with each type of palindrome analyzed in all transformants. Nine, eight, and four transformants of noIR, 79IR, and 229IR respectively were subjected to DSB induction and MTX selection. The number of MTX-resistant subclones from cells with DSB (Right) and without DSB (Left) are indicated by the lengths of the bars. The presence of various types of palindromes in the MTX-resistant subclones of every transformant was determined by Southern hybridization analysis, and their fractions are indicated by using different shadings. Many subclones from transformants 79IR-2, -8, and -28 contained short IR-mediated palindromes. 229IR-16 and -17 contained only IR-mediated palindromes. Transformants of noIR produced a lower number of MTX-resistant subclones than 79IR transformants (79IR+DSB vs. noIR+DSB, P = 0.02). The “others” category includes subclones with unrearranged DNA, other types of rearranged DNA, or mixtures of DNA types that might include palindromes.

PCR amplification and sequencing of the bisulfite-modified DNA from the palindromic center. (A) PCR amplification. Nested PCR was used to amplify the junctions of the rearranged DNA. The locations of the primers used, including those for the positive controls, are indicated. DNA from 79IR-8 parental cells (P) and MTX-resistant subclones 2, 4, 5, 6, and 8 were treated with bisulfite and amplified. Subclones 2, 4, and 8 produced an ≈500-bp PCR fragment with the palindromic structure diagrammed. Subclone 5 produced an ≈700-bp fragment. There is no amplification product from subclone 6 or a parental transformant. (B) Sequence of the short IR-mediated palindrome from subclone 2. PCR fragments described above were cloned and sequenced (bottom line, bisulfite modified) and compared with the predicted palindromic sequence (middle line). Bisulfite treatment converts C in the middle lines to T in the bottom line. The parental nonpalindromic sequence in this region is also shown (top line). The sequence upstream of the 79-bp IR is shown in lowercase. The two divergent arrows indicate the locations of the 79-bp IR, and the dashed line indicates the nonpalindromic center. A schematic drawing is shown at the top. (C) Sequence of the end-to-end joining palindrome from subclone 5. The sequence after bisulfite modification (bottom line) was shown along with the predicted sequence (top line). The two palindromic arms are joined head to head at the I-SceI cleaved site (asterisk) with 9 bp and 8 bp of deletions from the two sides (end-to-end joining palindrome), which is shown in the parenthesis. Note that two pairs of the short IR (arrows), including the nonpalindromic center (dashed line), are present in this sequence, compared with only one in the sequence in B.

(A) Intramolecular recombination model of palindrome formation. For simplicity, one possible mode of recombination, single-strand annealing version, is shown (19, 20). After a DSB, one strand of a short IR is exposed by 5′ to 3′ degradation, followed by intrastrand pairing, to generate a hairpin molecule, which is converted to a large palindrome by bi-directional replication. Lines represent DNA sequences, and a triangle represents one repeat of a short IR. (B) pDhfrSce constructs. Each construct has an I-SceI recognition sequence and either a 79-bp IR (pD79IRSce), 229-bp IR (pD229IRSce), or noIR (pDnoIRSce). Each short IR has a 29-bp nonpalindromic center. Simian virus 40 (SV40), SV40 enhancer element and origin of replication; MPL, adenovirus major late promoter; TPL, tripartite leader from adenovirus late mRNA and small intervening sequence; pA, polyadenylation signal from SV40 early region.

Palindrome formation after DSB induction in 79IRSce transformants. (A) Restriction maps of the expected structures. In the original construct, the AfiII-KpnI fragment detectable by the probe indicated is 1.8 kb. If palindrome formation occurs after cleavage at the I-SceI site, this KpnI site will be lost, and the fragment will be replaced by a 3.4-kb AfiII fragment. Solid lines represent sequences of the transgene. (B) Southern blotting analysis of DNA from MTX-resistant subclones. Subclones from transformants 79IR-8 and 79IR-28 are shown as examples. After DSB, many subclones produced the 3.4-kb AfiII fragment indicative of the palindrome predicted. In some subclones (e.g., subclones 5 and 10 from 79IR-8), a slightly larger fragment is seen that represents a variant palindrome. In other subclones (e.g., subclones 12 and 13 of 79IR-8 and subclones 2 and 5 of 79IR-28), multiple fragments, including the 3.4-kb fragment, were detected. They could contain a mixture of rearrangements or be a mixture of cells with different rearrangements and were not counted as palindromes in Fig. 2. Size markers (200-bp ladder) are shown on both sides of the gel. P, parental transformant. (C) Snap-back analysis of the palindromic DNA. The 3.4-kb fragment is always accompanied by a l.7-kb fragment of lower intensity, which could be a snap-back form of the palindrome. After heat denaturation and rapid cooling (boiled), the 3.4-kb fragment disappears and the 1.7-kb fragment becomes dominant. (D) Extension of palindromic structure outside of the transgene. DNA from the parental transformant 79IR-8 (P), subclones with a short IR-mediated palindrome (2, 4), and end-to-end joining palindrome (5, 10) were digested with SfiI or PstI and probed with the DHFR gene. In all cases, only one fragment was detected, suggesting that both arms of the palindrome are identical to that of the parental transformant and extend beyond the transgene. Solid lines represent sequences of the transgene, and dotted lines represent the flanking genomic sequence. A 1-kb ladder is used as size marker.

Intrachromosomal amplification of DHFR. (A) Subclones with short IR-mediated palindrome (subclones 2 and 4) and end-to-end joining palindrome (clone 5) isolated in 0.2 μM MTX from transformant 79IR-8 were further selected with 3.2 μM MTX, and resistant subclones were isolated after 10 days of growth. The genomic DNA was digested with KpnI and AfiII and analyzed by Southern blotting. (B) Intrachromosomal palindrome formation of DHFR transgene in subclone 2. Chromosomes shown are from three different cells. FISH was performed by using DNA fragment from pD77IRSce and 5 kb of the flanking genomic sequence as a probe. Each signal (red) probably represents one palindrome of the DHFR transgene. (C) In the highly amplified Sce2–1 subclone, large blocks of signals were seen in different cells on the same chromosome, which, in some cases, showed fused sister chromatids (Left).