Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants

Abstract

Mitochondria and chloroplasts (photosynthetic members of the plastid family of cytoplasmic organelles) in eukaryotic cells originated more than a billion years ago when an ancestor of the nucleated cell engulfed two different prokaryotes in separate sequential events. Extant cytoplasmic organellar genomes contain very few genes compared with their candidate free-living ancestors, as most have functionally relocated to the nucleus. The first step in functional relocation involves the integration of inactive DNA fragments into nuclear chromosomes, and this process continues at high frequency with attendant genetic, genomic, and evolutionary consequences. Using two different transplastomic tobacco lines, we show that DNA migration from chloroplasts to the nucleus is markedly increased by mild heat stress. In addition, we show that insertion of mitochondrial DNA fragments during the repair of induced double-strand breaks is increased by heat stress. The experiments demonstrate that the nuclear influx of organellar DNA is a potentially a source of mutation for nuclear genomes that is highly susceptible to temperature fluctuations that are well within the range experienced naturally.

DNA in the highly energetic cytoplasmic organellar genetic compartments of eukaryotes is subjected to high levels of stress damage from the oxygen free radicals produced during respiration and photosynthesis. Nonetheless, for various reasons, the rate of accumulation of mutations is slower in plant cytoplasmic organelles than in the nucleus (1, 2). The vast majority of mitochondrial and plastid (chloroplast) genes have vacated their ancestral prokaryote genetic compartment in favor of the nucleus (2), with very few remaining within extant organelles. The first step in gene relocation for the organelle genomes is DNA escape, followed by insertion into nuclear chromosomes, a process shown to occur remarkably frequently under normal physiological conditions in tobacco (3, 4) and yeast (5), although an experimental screen in the unicellular alga Chlamydomonas reinhardtii did not detect any such transpositions (6). These experimental studies, together with bioinformatic and genomic analyses (7–14), demonstrated that DNA transfer from cytoplasmic organelles is a frequent and continuous process in essentially all eukaryotes examined, although it may be very rare in organisms with very low organelle numbers (15). Although DNA transfer per se is very frequent in higher plants, genes that migrate are normally inactive in the nucleus because they lack the motifs required for nuclear expression. Nonetheless, on rare occasions nuclear integrants of organelle DNA (norgs) are activated by genomic rearrangements (16, 17), that effectively duplicate the gene. Over evolutionary time, these frequent nonfunctional and rare functional DNA transfer processes have resulted in the net loss of redundant genes in the cytoplasmic organelles and have added to the genetic complexity of the nucleus.

Environmental factors are known to modify mitochondria-to-nucleus DNA movement in yeast (5), and there is good evidence that the normal aging process in rats (18) and yeast (19) increases the rate of formation of nuclear integrants of mitochondrial DNAs (numts). Here we tested abiotic stress effects on cytoplasmic organelle DNA transfer to the nucleus in tobacco using sensitive transplastomic reporter genes designed for nucleus-specific expression.

Results

Transient and Integrative Expression of the gus Reporter Gene.

A nucleus-specific reporter gene was placed into the large single-copy region of tobacco plastome (plastid genome). In this genetic compartment, the reporter gene (gus) was not expressed, because it was driven by the 35S promoter and terminator and contained a nuclear intron (STLS2) (20), precluding translation in the plastid. In this transplastomic line (tp-gus), the reporter gus gene is expressed only when transferred to the nucleus—an events that is readily monitored by histochemical staining to detect the gus product, visualized as blue sectors in cells and tissues. Using this system in preliminary experiments, we investigated the effects of various stress conditions examined previously, including heat (21), salt (22), hydrogen peroxide (23), and paraquat (24), on plastid-to-nucleus DNA transfer. Heat stress was outstanding in increasing nuclear gus expression (Table 1, experiment 1). The number of blue-stained sectors on leaves and roots of seedlings grown at 25 °C for 3 wk, treated for 3 h at 45 °C, and allowed 2 d to recover at 25 °C was ∼2.5-fold greater than that of control seedlings. Two independent experiments using 2-wk-old plants confirmed this result in leaves of tobacco seedlings, with less pronounced effects seen in cotyledons and roots (Table 1, experiments 2 and 3). Extending the range of exposure time to heat stress showed a significant dose–response in gus expression events, with the number of blue sectors peaking in leaves after 5 h of heat stress (Fig. 1A). After longer heat stress, some blue sectors included large areas of the leaves (Fig. 1A).

Table 1.

Plastid-to-nucleus DNA transfer in heat-stressed tobacco seedlings

			n	Mean	SD	Min	Max	P
Experiment 1	Leaf	Control	34	2.41	2.01	0	8	***
		Heat	34	6.05	5.31	1	19
	Cotyledon	Control	34	3.05	2.10	0	9	NS
		Heat	34	4.41	1.81	1	8
	Root	Control	34	5.5	4.03	0	14	***
		Heat	34	9.41	4.43	3	19
Experiment 2	Leaf	Control	39	0.76	1.06	0	5	***
		Heat	37	2.48	2.54	0	10
	Cotyledon	Control	39	3.84	1.82	0	9	NS
		Heat	37	3.89	1.69	0	7
	Root	Control	39	2.69	1.68	0	7	***
		Heat	37	4.40	2.56	0	11
Experiment 3	Leaf	Control	43	0.93	1.26	0	5	**
		Heat	42	2.76	4.28	0	23
	Cotyledon	Control	43	4.32	1.82	0	9	NS
		Heat	42	3.5	2.14	0	8
	Root	Control	43	3.72	2.41	0	10	NS
		Heat	42	4.59	2.93	0	11

The results of three independent experiments are shown. Transplastomic (tp-gus) plants grown at 25 °C for 3 wk (experiment 1) or 2 wk (experiments 2 and 3) were treated at 45 °C for 3 h and allowed to recover for 2 d under normal growth conditions before histochemical staining for gus expression. SD and P values (two-sample Independent t test) are shown. n, seedling numbers counted; mean, the mean number of blue sectors per seedling; min, the minimum number of blue sectors per seedling; max, the maximum number of blue sectors per seedling. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Fig. 1.

Plastid-to-nucleus DNA transfer in heat-stressed tobacco seedlings: time course of heat treatment. (A) Histochemical staining of whole seedlings. Seedlings were grown for 2 wk at 25 °C, treated at 45 °C for the indicated times, and allowed to recover at 25 °C for 2 d before staining for gus expression. (B) RT-PCR of gus mRNA with (+) and without (−) reverse transcriptase after heat stress (hr). The positive control gs1.1 (20) is a plant line containing a nuclear copy of gus. rpl25 mRNA RT-PCR was used as a technical and loading control. (C) Spliced gus mRNA levels relative to rpl25 mRNA. Gus mRNA was significantly increased in the 5 h sample compared with the 0 h control (P < 0.05, two-sample Independent t test). (D) The memory of heat stress. The 2-wk-old plants with two leaves and two cotyledons were treated at 45 °C for 0 or 5 h and allowed to recover for 1 mo before GUS staining. (Scale bars: 3 mm.)

Despite the nucleus-specific promoter in tp-gus, both sense and antisense gus RNAs are transcribed in the chloroplast from adjacent plastid promoters (25), but the nuclear intron (STLS2) precludes plastid expression (20). Thus, the design of the nucleus-specific gus gene allowed quantification of its spliced nuclear mRNA in the presence of excess unspliced chloroplast transcripts. A reverse primer, predominantly in the second exon but spanning the splice junction with the last three 3′ nucleotides in the first exon, together with an upstream primer, allowed specific PCR amplification of transcripts from which the intron had been removed (Fig. 1B). Under this regime, quantitative PCR demonstrated that spliced gus mRNA accumulation increased in leaf tissue in parallel with the time spent by the seedlings at 45 °C and correlated with the number and size of the blue areas after staining (Fig. 1C). We conclude that the reporter gene escaped from the chloroplast into the nucleus, where it was transcribed and spliced, and the mRNA was translated on 80S ribosomes in the cytoplasm.

Young leaves in plants treated for 5 h at 45 °C were almost completely blue-stained (Fig. 1A), suggesting that at the time of heat treatment they were at a developmental stage particularly susceptible to stress. This high expression of gus might be related to incorporation of reporter genes into the nuclear genome, resulting in mitotic lineages of transformed cells, or possibly to a large amount of plastid DNA entering the nucleus, resulting in massive transient expression. To determine the longevity of the elevation of gene transfer, 2-wk-old seedlings were treated for 5 h at 45 °C and then returned to standard growth conditions for 30 d. After this long recovery period, the first two leaves that were present at the time of heat stress showed markedly more blue sectors than controls (Fig. 1D), with no obvious increase in leaves that initiated and expanded after the stress treatment. This finding suggests that heat is a specific inducer of chloroplast DNA transfer. The observation of discrete leaf areas of persistently elevated gus expression strongly implies nuclear integration and stable expression, which suggests that considerable transient expression of the reporter gene explains the large, more diffuse, blue areas seen after 5 h of heat stress (Fig. 1A). However, it is very difficult to distinguish unequivocally between transient expression and nuclear integration using tp-gus (20), necessitating an alternative approach.

Integrative Expression of the neo Reporter Gene.

To assess the effect of heat stress on nuclear integration, we used a second transplastomic plant (tp-neo) containing a copy of neo-STLS2 (3) encoding NPTII in both inverted repeats of the plastome. These plants show a low level of kanamycin resistance compared with WT when grown under standard conditions (3, 4). This partial resistance is probably due to the same leakage of plastid DNA to the nucleus as described above for gus, resulting in a few cells producing NPTII protein, which protects the entire seedling to some extent. To test for nuclear integration frequency, 2-wk-old tp-neo plants were treated at 45 °C for 5 h and allowed to recover at 25 °C for 2 d, after which 400 leaf explants (each 30 mm²) were placed on regeneration medium containing 400 μg/mL of kanamycin to overcome the low level of resistance in tp-neo (4). Most leaf explants from heat-treated plants remained green, and 45 resistant shoots were selected as soon as they became visible over 120 d (Fig. 2). When a resistant shoot was transferred to new medium, the entire explant was removed from the experiment, necessarily resulting in a gradual decrease in the number of cells in the screen. All leaf explants from an equal number of non–heat-treated control seedlings were bleached much more quickly, except for 13 resistant shoots that emerged over the same 120 d period (Fig. 2).

Fig. 2.

Heritable plastid-to-nucleus DNA transfer. Examples of plates showing the progression of heat-treated and control transplastomic (tp-neo) leaf explants regenerating under kanamycin selection. The 2-wk-old seedlings were treated for 0 h (Left) or 5 h (Right) at 45 °C and then maintained under standard growth conditions for 48 h to allow recovery. Leaf explants (∼30 mm²) were placed on culture plates containing plant regeneration medium supplemented with 400 μg/mL of kanamycin. As resistant shoots emerged over 120 d (left-hand labels), they were transferred to 0.5× MS medium to root and subsequently transferred to soil. Plates are 11-cm Petri dishes.

Based on cell number estimates (17), the frequency of kanamycin-resistant shoots emerging from the screen equated to 1 neo transfer to the nucleus in ∼1.4 million cells in the control, compared with 1 in ∼400,000 cells after heat stress—an ∼3.5-fold increase. All of the resistant shoots were recovered, and the majority grew into mature plants, designated the kr6 series. Back-crosses of mature kr6 genotypes to WT females were used to replace the transplastome with that of WT in the progeny, some of which were tested for segregation of the kanamycin resistance phenotype (Table S1). Of 14 kr6 lines tested genetically (7 from controls and 7 from heat-treated tissues), 9 showed the expected 1:1 ratio in their progeny characteristic of a nuclear hemizygote, whereas the other 5 showed various significant departures from expectation, consistent with previous experiments (26). No differences were observed between the heat-treated (kr6h) and control (kr6c) lines (Table S1).

Anatomical Effects of Heat Stress.

To identify the reasons for the large increase in plastid-to-nucleus DNA transfer induced by heat stress, we examined the integrity of chloroplasts. A transplastomic tobacco line pMSK56 (27) that encodes and expresses GFP in its chloroplasts was treated at 45 °C for 5 h. In seedlings without heat treatment, GFP fluorescence colocalized with chlorophyll autofluorescence in chloroplasts of stomatal guard cells and underlying mesophyll tissues (Fig. 3A). In contrast, GFP and chlorophyll distribution was widespread after heat stress, suggesting disruption of chloroplast membranes and release of DNA into the cytosol and intercellular spaces, with a consequent potential for plastid DNA fragments to transfect nuclei (Fig. 3B).

Fig. 3.

Confocal laser scanning microscopy images of tobacco guard cells. Epidermal strips with underlying mesophyll cells from control (A) and heat-treated (B) pMSK56 tobacco plants containing gfp in the chloroplast genome (27). GFP fluorescence (green, Left) and chlorophyll autofluorescence (red, Middle) superimposed on the interference contrast images (Right). (Scale bars: 25 μm.)

Use of Double-Strand Break Repair to Monitor Escape of Cytoplasmic Organellar DNA to the Nucleus.

As an alternative to using transplastomic plants to screen for organellar DNA transfer to the nucleus, we developed a method to determine the sequences at double-strand breaks (DSBs) repaired by nonhomologous end-joining, which is involved in the majority of integration events (17). In nuclear transgenic lines, we conditionally induced expression of the rare-cutting endonuclease I-SceI to cause DSBs at two restriction sites separated by a short spacer region that we also introduced at a second nuclear locus. The spacer region contained the dao1 gene, which potentially facilitates both positive and negative selection of seedlings in Arabidopsis (28), but selection was not used in the experiments reported here. Importantly, however, spacer excision enabled unequivocal recognition of digested and repaired loci after I-SceI digestion, and analysis of more than 300 unique DSB repair junctions was achieved by single-molecule (sm) PCR. By diluting template DNA until each reaction received no more than one template molecule, smPCR circumvented such problems as template jumping and preferential amplification of small products (29). This made it possible to amplify rare, large template molecules (corresponding to insertion events) from a population of much smaller, more frequent template molecules (corresponding to repair events not involving insertion). Seven insertions were observed after heat stress, four of which were identical to clusters of mitochondrial (mt) DNA from up to four disparate regions of the organellar genome (Table 2). In contrast, no insertions of mtDNA were seen in DNA from an equal number of untreated plants, suggesting that copious amounts of mtDNA were liberated into the cytoplasm after heat treatment, some of which were incorporated into nuclei. No integrants of plastid DNA were observed; the remaining three insertions were not of cytoplasmic organellar origin (Table 2).

Table 2.

Characterization of sequences inserted after DSB repair

Insertion	Length, bp	Insertion origin
A5	338	Mitochondrial (298061–298400)*
D7	102	Nuclear; DSB flanking region
D5	358	Mitochondrial (57336-57413 [408086–408166]†, 24465–24390, 56185–56304, 29727–29810)*
F5	165	Nuclear; similar to copia-like retro transposon (e = 1.1 × 10−14)
F8	229	Mitochondrial (187992–188220)*
G5	118	Nuclear; similar to tobacco EST EG649865.1 (e = 2 × 10−30)
G12	211	Mitochondrial (152588–152685, 181465–181435, 164031–163950)*

*Numbers in brackets correspond to the equivalent nucleotides in the tobacco mitochondrial genome (46).

^†Alternative coordinates are given in square brackets.

Insertions D5 and G12 (Table 2) were patchworks of sequences originating from noncontiguous regions of mtDNA. It was thought that these could possibly be insertions of preexisting numts, rather than de novo mtDNA; thus, PCR primers were designed to amplify integrant D5 (Fig. S1A), with primer D5F located within mitochondrial sequence 57336–57413 and primer D5R in mitochondrial sequence 29727–29810. No product was amplified from WT DNA unless it was spiked with an equimolar amount (i.e., one copy per tobacco nuclear genome) of the original D5 smPCR product (Fig. S1B), indicating that the sequence is not present in WT DNA. A control PCR using nucleus-specific primers confirmed the technical quality of DNA samples (Fig. S1C). Therefore, D5 represents a de novo mtDNA integrant that was inserted at an experimentally induced I-SceI DSB. Together, the lack of any mtDNA insertions in an equal number of single molecules from control plants and the patchworking of the integrants suggest the availability of multiple small fragments of mtDNA after heat stress that are regularly incorporated during nuclear DSB repair.

The reason why larger fragments of mtDNA were not seen may be related to their relative difficulty of amplification. However, PCR was demonstrably capable of amplifying considerably larger inserts, suggesting that mtDNA was extensively fragmented. Thus, it appears that chloroplast DNA is not present in such large amounts of small fragments, although it is clearly available as large molecules sufficient to carry the entire neo and gus selectable markers (Figs. 1 and 2), as well as substantial flanking plastomic regions (17, 30). Most of these latter inserts would have been too large to emerge from the smPCR screen.

Discussion

The experimental demonstration of DNA transfer from the chloroplast to the nucleus (3, 4) showed such a high frequency as to make its implications controversial (31, 32). Therefore, it is even more surprising to find that the rate of transfer is increased up to 10-fold by mild temperature stresses applied over short periods. Tobacco plants grow optimally at daytime temperatures of ∼25 °C, but crops regularly encounter much higher temperatures in nature. The temperatures reached in leaf cells at ambient temperatures exceeding 40 °C may be even higher as a result of radiant heating effects, although these effects would be countered somewhat by the evaporative cooling effects of transpiration. Nonetheless, the complex leaf canopy of any species is certain to provide for a large range of temperatures in different microclimates, and we would expect to see equally complex cellular responses, including cell-to-cell variation in plastid and mtDNA escape and insertion into the nucleus.

Given our monitoring of the nuclear expression of entire functional nuclear reporter genes as they escape from the transplastid, we necessarily expected all other parts of the plastome to be involved equally even though they remained invisible to the screen. Therefore, an astonishing array of undetectable transpositional events must occur that would substantially change the clonal nature of the somatic cells of individual plants and make them heterogeneous chimeras containing nuclei that are highly genetically variable with respect to recent integrants of cytoplasmic organellar DNA.

The sensitivity of the two transplastomic lines used in the present study was the key to revealing this unique facet of nuclear mutation and evolution. Although these transplastomics are initially able to show only DNA movement per se, subsequent rearrangements within the nucleus have been shown to activate prokaryotic genes in ways that recapitulate real endosymbiotic evolution (16, 17). The profound global consequences of the processes that are made possible by initial DNA transfer are fully revealed by bioinformatic analyses showing how endosymbiotic evolution has contributed thousands of genes to the eukaryotic nucleus (9, 10, 33–35). This deluge of cytoplasmic organellar DNA into the nucleus (31) clearly results in a “trial-and-error” system of genomic rearrangements after which a few integrants gain functionality. Thus, a large majority are quickly removed in whole or part (26), whereas a small minority have profoundly changed gene disposition in eukaryotes and added to the gene content and heterogeneity of the cell.

These transplastomic assays have shown that plastomic DNA is transferred to the nucleus in very large, sometimes scrambled (17), sections, and bioinformatic evidence shows that the process also involves small pieces of the DNA (8, 9, 11, 17). However, there is no experimental evidence of small de novo inserts of plastid DNA analogous to the mitochondrial sequences that we demonstrated by DSB induction and smPCR. The lack of a system for transforming the mitochondrial genome of plants precludes a screen for large mtDNA integrants, although there is ample bioinformatic evidence of their existence (11, 36).

The insertion of organelle DNA at sites of DSB repair in yeast was first reported more than a decade ago (37). Although indirect evidence of the same process occurring in some higher organisms has been published (17, 38), the present study has been able to demonstrate directly that organelle DNA integrates at DSBs in multicellular eukaryotes. In previous experiments (39, 40), in the absence of stress, no cytoplasmic organelle DNA insertions were observed at sites of nuclear DSBs. Taken together, these results suggest the presence of a large amount of available organelle DNA at the time of DSB repair after heat stress. There is some previous evidence that mitochondrial membrane potential is perturbed in cucumber (41) and Arabidopsis (42) by much more extreme heat shock at 55 °C for short periods, suggesting damage to mitochondrial ultrastructure that may be associated with mtDNA release into the cytosol.

Given that the environments studied are by no means extreme, these results have far-reaching consequences for nuclear evolution in organisms that must withstand ambient temperatures. The ingress of cytoplasmic organellar DNA in tobacco grown in ideal environments is already considered remarkably high (31), and it must be mutagenic at rates equivalent to or exceeding any other causes (43). Clearly, eukaryotes evolved in the real world of highly variable growth conditions, and they must have been subject to massive oscillations in the rate of bombardment with cytoplasmic organellar DNA. The possibility of approaching increased fluctuations in global environments points to the importance of understanding the processes involved.

Materials and Methods

Plant Lines.

Tobacco (Nicotiana tabacum) plants were grown under standard conditions (16-h light, 8-h dark cycle at 25 °C) in sterile jars or 11-cm Petri dishes on 0.5× MS medium (44) or in compost in growth rooms with a 14-h light/10-h dark and 25 °C-day/18 °C-night growth regime. The transplastomic tobacco lines were described previously: tp-neo (3), tp-gus (20), and pMSK56 (27).

Heat Stress Treatments.

In a pilot experiment, 3-wk-old seedlings were treated for 3 h at 45 °C (21), followed by recovery at 48 h under standard conditions. Subsequent experiments used 2-wk-old seedlings grown at 45 °C for various times (3, 4, 5, and 6 h), followed by recovery at 48 h or 30 d under standard conditions. In all experiments, control plants were maintained at 25 °C.

Analysis of GUS Activity.

Histochemical GUS staining was done as described previously (20). The numbers of blue spots from seedlings with and without stress treatment were scored blind and analyzed with the two-sample independent t test using SPSS software. Images were captured using an Olympus Provis AX70 microscope.

PCR Assays.

RNA preparation was performed using an RNeasy Plant Mini Kit (Qiagen), and genomic DNA contamination was removed using an Ambion TURBO DNA-Free Kit (Invitrogen). Reverse-transcription (RT) was then performed using an Advantage RT-for-PCR Kit (Clontech) with random hexamer primers. All kits were used in accordance with the manufacturers’ instructions. PCR amplification was performed using Taq DNA polymerase (Invitrogen) in accordance with standard protocols. Standard RT-PCR amplification of gus cDNA was done using primers 5′-ATACCGAAAGGTTGGGCAG-3′ and 5′-TTCACACAAACGGTGATACGTA-3′, and amplification of RPL25 cDNA was done using primers 5′-AAAATCTGACCCCAAGGCAC-3′ and 5′-GCTTTCTTCGTCCCATCAGG-3′ (17). Quantitative real-time PCR was performed as described previously (45). The primers used for gus quantitative PCR were 5′-ATACCGAAAGGTTGGGCAG-3′ and 5′-TTCACACAAACGGTGATACGTA-3′. For relative quantification, RPL25 mRNA, amplified with primers 5′-CCCCTCACCACAGAGTCTGC-3′ and 5′-AAGGGTGTTGTTGTCCTCAATCTT-3′, served as an internal standard (45).

Fluorescence Microscopy.

Leaves of pMSK56 were cut into sections, placed on a glass slide, and viewed by confocal scanning laser microscopy (Leica SP5 spectral scanning confocal microscope). Images were captured as described previously (27).

Analysis of Kanamycin Resistance.

Leaf explants (∼30 mm²) from plants grown in sterile jars were placed in 11-cm Petri dishes containing plant regeneration medium supplemented with 400 μg/mL of kanamycin (4). Resistant shoots were transferred to 0.5× MS medium to root and then transferred to soil in a controlled environment chamber with a 14-h light/10-h dark and 25 °C-day/18 °C-night growth regime. For seedling selection, surface-sterilized seeds were grown on 0.5× MS medium containing 150 μg/mL of kanamycin. All plates were incubated at 25 °C with 16-h light/8-h dark cycle. Significance of deviation from the expected Mendelian ratio was determined using the χ² test.

Analysis of DNA DSB Repair Junctions.

Seedlings of DSB induction lines D4A2 and D14A2 were grown in tissue culture jars. At 2 wk after germination, plants were heat-treated in a controlled temperature chamber for 5 h at 45 °C. Immediately after, or 1 d after heat stress, DSBs were induced by spraying ∼1–2 mL of 0.7 M ethanol into tissue culture jars. Plants were left for 4 d to allow time for I-SceI expression, creation of DSBs, and DSB repair.

After recovery from heat stress, DNA was prepared from leaf tissue and used as template for single-molecule (sm) PCR (29). Single-molecule PCR was performed using LongAmp taq DNA polymerase (New England Biolabs) and HincII (which cuts three times within the spacer region) digested DNA as a template. Reactions were conducted in a volume of 2 μL containing 0.3 mM dNTPs, 0.4 μM primers (DSBF1: 5′-GATAGTGACCTTAGGCGACTTTTGAACG-3′; DSBR1: 5′-TCCCCTGATTCTGTGGATAACCGT-3′), 0.2 U LongAmp taq DNA polymerase, 1× LongAmp buffer, and 110–130 pg of template DNA. Reactions were overlaid with mineral oil to prevent evaporation. Cycle conditions were as follows: initial denaturation at 95 °C for 30 s; followed by 45 cycles of 95 °C for 20 s, 59 °C for 20 s, and 65 °C for 4 min; followed by a final extension at 65 °C for 10 min. After PCR, 18 μL of water was added to each reaction; 5 μL was analyzed by standard agarose gel electrophoresis, and the remainder was used in subsequent sequencing. Insertion events (smPCR products larger than ∼850 bp) were sequenced directly using primers DSBF1 and DSBR1.

Insert D5 Amplification.

PCR was performed using Taq DNA polymerase (Roche) in accordance with the manufacturer's instructions. The D5 insertion PCR used primers D5F (5′-GGAAGCGAGATTGGATTGACG-3′) and D5R (5′-TGGGTCAGGGTCAGATACGGA-3′), with WT DNA as a template. As a positive control, WT DNA spiked with one copy per diploid genome of the original D5 smPCR product was used as template. The L25 gene was amplified from WT DNA using primers L25F (5′-AAAATCTGACCCCAAGGCAC-3′) and L25R (5′-GCTTTCTTCGTCCCATCAGG-3′) (17). Controls with no template DNA were included for each primer pair.