Telomere-mediated chromosomal truncation in maizeWeichang Yu, Jonathan C. Lamb, Fangpu Han, and James A. Birchler*

Division of Biological Sciences, University of Missouri, Columbia, MO 65211

Edited by Michael Freeling, University of California, Berkeley, CA, and approved September 27, 2006 (received for review July 9, 2006)

Direct repeats of Arabidopsis telomeric sequence were constructedto test telomere-mediated chromosomal truncation in maize. Twoconstructs with 2.6 kb of telomeric sequence were used to trans-form maize immature embryos by Agrobacterium-mediated trans-formation. One hundred seventy-six transgenic lines were recov-ered in which 231 transgene loci were revealed by a FISH analysis.To analyze chromosomal truncations that result in transgeneslocated near chromosomal termini, Southern hybridization analy-ses were performed. A pattern of smear in truncated lines was seenas compared with discrete bands for internal integrations, becausetelomeres in different cells are elongated differently by telomer-ase. When multiple restriction enzymes were used to map thetransgene positions, the size of the smears shifted in accordancewith the locations of restriction sites on the construct. This resultdemonstrated that the transgene was present at the end of thechromosome immediately before the integrated telomere se-quence. Direct evidence for chromosomal truncation came from theresults of FISH karyotyping, which revealed broken chromosomeswith transgene signals at the ends. These results demonstrate thattelomere-mediated chromosomal truncation operates in plant spe-cies. This technology will be useful for chromosomal engineeringin maize as well as other plant species.

karyotyping � transformation � chromosome engineering

Telomeres are the natural ends of chromosomes that protectthem from instability (1). They consist of tandem arrays of

a highly conserved sequence and associated proteins. The telo-meric sequences of many multicellular eukaryotes have beencloned (2–6). The telomere motifs are usually 5- to 8-nt long andare highly conserved among eukaryotic organisms (7). In mostplant species, the conserved sequence is TTTAGGG, which wascloned from Arabidopsis thaliana (2). Exceptions to the Arabi-dopsis type were reported in some species of Asparagales (8–11).These repeats recruit proteins that form a protective nucleo-protein complex. Also, the telomeric repeat can recruit thetelomerase for chromosome end-replication to counterthe resulting chromosome shortening, which is predicted fromthe normal chromosome replication (12).

The function of the cloned human telomeric sequence wasdemonstrated by re-introduction into mammalian cells (13). Aninteresting phenomenon was that introduced telomeric se-quences could produce chromosomes with new telomeres (13,14). The broken chromosomes were transmissible in cell lines aswell as through the germ line (13, 15). The telomere-mediatedchromosomal truncation technology has been adopted for theproduction of minichromosomes, which can be used as artificialchromosome platforms (15–20).

Telomeric repeats had been used in transformation of Arabi-dopsis (21). The construct had telomeric repeats at both ends ofthe transfer DNA (T-DNA) region and a low frequency oftransformation was observed. The telomeric sequence from theconstruct was not found in regenerated transgenic plants. In thisreport, we demonstrated that 2.6 kb of Arabidopsis telomericrepeats were efficient for telomere-mediated chromosomal trun-cation in maize. By analyzing plants regenerated from callus ofthe primary transformation before an intervening haploid ga-metophyte generation, deletions resulting from truncationscould be recognized and analyzed. We also show that internally

integrated telomeric sequences are stable in the genome. Withappropriate modification, this technology opens the possibilityfor plant artificial chromosome construction as a platform forgenetic engineering.

ResultsPlasmid Construction and Agrobacterium-Mediated Gene Transforma-tion. Telomere-mediated chromosomal truncation has been re-ported in mammalian cells (13). In this report, a 2.6-kb directrepeat of Arabidopsis telomeric sequence was used in twoconstructs, pWY76 and pWY86, to test the ability of telomericsequences to cause chromosomal truncations. In addition to thetelomeric repeats, promoter-less lox66-GFP or lox71-DsRed wasincluded in the constructs, pWY76 and pWY86, respectively, forfuture manipulations by site-specific recombination systems. Ahygromycin phosphotransferase gene (HPT) was reported as aselection marker gene for maize transformation (22). We in-cluded a HPT gene-expression cassette in pWY76, althoughunder our conditions this selection marker did not work well. InpWY86, a Pnos-FRT-FLP-Tnos gene-expression cassette wasincluded for potential manipulations using the FRT�FLP site-specific recombination system (23). A third construct, pWY96,which contains the elements common to both pWY76 andpWY86 but lacks the 2.6-kb telomere sequence (Fig. 1) was usedas a control to test the function of telomeric sequences and wasused as a FISH probe to detect all transgenes from pWY76,pWY86, and pWY96 transformations.

The three constructs were used to transform maize immatureembryos by an Agrobacterium-mediated gene transformationmethod (24) from July, 2004 to December, 2005. The totalnumber of transgenic events were 93, 83, and 44 for theconstructs pWY76, pWY86, and pWY96, respectively. Trans-genic lines were analyzed as described below.

Chromosomal Truncations in Maize Transgenic Lines. A maize karyo-typing method developed by Kato and colleagues (25) was usedto localize transgenes and to screen for cases of chromosomaltruncations. It has been shown that karyotypes of HiII parent Aand parent B as well as chromosomal aberrations can be easilydetected by this method (26). To localize transgenes and todetect chromosomal truncations, metaphase chromosomes fromT0 transgenic plant root tips were probed with a pWY96 probeplus the karyotyping mixture (25). Transgene locations andstructural chromosomal aberrations were recorded (Table 1,which is published as supporting information on the PNAS website). One hundred twenty-three trangene loci were observed inthe 93 transgenic plants with construct pWY76, 57 of which werefound at distal regions of chromosomes (near chromosometermini). The distal chromosomal position of a transgene re-

Author contributions: W.Y., J.C.L., and J.A.B. designed research; W.Y. and F.H. performedresearch; J.C.L. and F.H. contributed new reagents�analytic tools; W.Y., J.C.L., F.H., andJ.A.B. analyzed data; and W.Y., J.C.L., F.H., and J.A.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: DSB, double-strand break; NHEJ, nonhomologous end-joining; T-DNA,transfer DNA.

*To whom correspondence should be addressed. E-mail: birchlerj@missouri.edu.

© 2006 by The National Academy of Sciences of the USA

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vealed by FISH on highly condensed metaphase chromosomescannot distinguish whether chromosomal truncation has oc-curred. However, chromosomal truncations should all havedistal transgenes. Similarly, in the 83 transgenic plants with thepWY86 construct, 108 transgenic sites were observed with 61distal loci. In the control, 58 transgenes were characterized in 44transgenic plants with the construct pWY96, in which 11 distalloci were observed. A chromosomal truncation was revealed byFISH when the loss of a chromosomal segment was observed. InFig. 2, we show the deletion of the short arm of chromosome 3in event B77 (Fig. 2 A), the loss of the short arm of chromosome

1 terminal knob in event T87 (Fig. 2B), and the loss ofsubtelomeric 4–12-1 sequence (25) from the long arm of chro-mosome 4 in events B37 and B44 (Fig. 2 C and D). In all thesecases, the deletion of a chromosomal segment was correlatedwith the integration of the transgene at the correspondingterminal position. However, many truncations might not berevealed by this method. For example, small deletions caused bytruncation might not be observed on highly condensed somaticchromosomes.

Because of the limitations of the cytological analysis, a South-ern hybridization method was performed on selected transgenic

Fig. 1. Chromosomal truncation constructs pWY76 and pWY86 and the control construct pWY96. LB, T-DNA left border; RB, T-DNA right border; Tvsp,terminator from soybean vegetative storage protein gene; Bar, bialophos resistance gene as a selection marker gene; TEV, tobacco etch virus 5� untranslatedregion; P35S, 2� 35S promoter from cauliflower mosaic virus; Tnos, Nos terminator from Agrobacterium; Tmas, Mas terminator from Agrobacterium; Pnos, Nospromoter from Agrobacterium; Pmas1�, Mas promoter from Agrobacterium; lox and FRT, site-specific recombination sites; HPT, hygromycin B-resistance gene;GFP, green fluorescent protein gene; DsRed, red fluorescent protein gene; FLP, recombinase gene; Telomeres, telomere units of pAtT4 isolated from Arabidopsisthaliana (2). Arrows designate the direction of transcription.

Fig. 2. Cytological detection of chromosomal truncations. Metaphase chromosomes were hybridized with pWY96 probe (red) and mixtures of repetitivesequence probes, CentC (green), knob (green), subtelomere 4–12-1 clone (green), and Cent4 (white). Arrows denote the transgene truncation sites (white arrows)and the corresponding sites on the homologues (gray arrows). (A) pWY86 transgenic event B77 with a chromosome 3 short-arm truncation. (Inset) Thechromosome 3 pair with (Left) and without (Right) the transgene (red). (B) pWY76 transgenic event T87 with a chromosome 1 short-arm terminal-knobtruncation. (Inset) Chromosome 1 homologues with (Upper) and without (Lower) the transgene (red). (C and D) pWY86 transgenic events B37 and B44 withtruncations of chromosome 4 long-arm terminal subtelomeric 4–12-1 sequence (green). (Insets) Chromosome 4 homologues with (Upper) and without (Lower)the transgene (red). Chromosome 4 can be identified by the Cent4 hybridization (white) at their centromeres. (Scale bar, 10 �m.)

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lines that had distal transgenes to characterize potential trun-cations. Chromosomal truncations result in synthesis of newtelomeres at broken ends to maintain chromosome stability. Theseeded telomeres are heterogeneous in size because the telom-erase adds different numbers of telomeric repeats to the chro-mosomal termini in each cell (13). A Southern hybridization willreveal the heterogeneous telomeres as a smear if DNA isdigested with a restriction enzyme that cleaves a nearby internalsite. Genomic DNAs from T0 transgenic plants with distaltransgene locations were digested with HindIII or EcoRV, andSouthern blots were hybridized with 32P-labeled GFP gene or aFLP gene fragment (Fig. 1). We also probed these blots with a32P-labeled bar gene as a control to test whether the nontelo-meric left border sequence could cause truncations. Discretebands were observed when probed with the bar gene because notelomere is associated with this internal fragment. In contrast,smears were observed when the blot was probed with the GFPgene probe for pWY76 transgenic plants or a FLP gene fragmentprobe for pWY86 transgenic plants because there are no re-striction sites on the telomere side of the probe homology. Wecharacterized a total of 56 transgenic lines that had transgeneslocalized near the ends of chromosomes and found that 3 of 17pWY76 transgenic lines (containing 22 distal transgenes) and 6of 27 pWY86 transgenic lines (containing 31 distal transgenes)had the smear patterns of hybridization (17.0%). If this ratio canbe applied to the number of lines with distal transgenes, we canexpect a minimum of 20 truncations in 118 distal transgene locifrom 231 recovered insertions, an efficiency of 8.7% chromo-somal truncation by the two constructs with telomeric sequence.Smear patterns were observed for two events (B37 and B77) thatwere characterized by FISH and that had obvious truncations(Fig. 2 A and C). As a control, all of the 10 lines with 11 distaltransgene loci from the total of 58 insertions that were trans-formed with pWY96 gave a discrete band when probed witheither the left-border bar probe or the right-border FLP probe,an indication that no truncations occurred (data not shown).Thus, from the 58 insertions of the control construct, none couldbe documented as a truncation. These results indicate that thetelomeric sequence per se in the experimental constructs is thecause of chromosomal truncations.

A total of 13 chromosomal truncations were revealed by eitherFISH or Southern hybridization (Table 1). To confirm furtherthe terminal nature of the transgenes, three restriction enzymeswere chosen to digest the genomic DNAs, and the blots werehybridized with the 32P-labeled FLP gene fragment (Fig. 3A). Wepredicted a shift in smear size according to the relative positionof the restriction sites if chromosomal truncation had occurred,because the end of the telomere can be regarded as a fixed endof the restriction fragment (13). The HindIII sequence is themost distal restriction site, and should produce a smear with thelowest size, whereas the largest smear should be produced bySmaI digestion, which cleaves the most proximal site (Fig. 3A).As expected, candidate truncated events (B37 and B77) pro-duced the shifted smear patterns (Fig. 3B, lane 1–3 and 7–9),whereas two events without smears, one with a distal insertion(B2) and one with an internal insertion (B21) produced discretebands (Fig. 3B, lane 4–6 and 10–12). Because an EcoRV site wasalso present distal to the telomere sequence in the transforma-tion plasmid, the smeared EcoRV restriction fragments indicatethat the sequences distal to the telomere were deleted during thetruncation process. This result confirmed that the transgenes inthe two lines with smear patterns of hybridization were locatedat the ends of chromosomes. The additional discrete bands foundfor these truncated lines likely result from other internal inte-grations of the transgene or its fragments. As a control, the samedigested blot produced all discrete bands when probed with thebar gene (data not shown), which ruled out the possibility oftruncations by the nontelomeric sequences in these constructs.

Stable Integration of Telomeric Sequences at Internal Locations. Todetermine whether telomeric sequences were lost in the inter-nally integrated lines, metaphase chromosome spreads from sixrandomly selected transgenic maize lines that had internaltransgenes (eight transgene loci) were probed with a telomereprobe (green) and a pWY96 probe (red). Internal telomeresignals were detected in all eight internal transgene locations,and they overlapped with the transgene signals (Fig. 4). Thisresult suggested that telomeric sequences were present at mostof the transgene locations. However, the loss of sequencesadjacent to the right border was detected by a Southern blot inone example (Fig. 3, lane 11). The telomeric sequence in theconstructs was flanked by two EcoRV sites (Fig. 3A). Thedigestion of genomic DNA with EcoRV should generate afragment of �4 kb if there were no sequences missing. Thesmaller size of the EcoRV band (�4 kb) showed that somesequences within the two EcoRV sites were lost. One transgenicline with an internal telomeric sequence was grown for fourgenerations, but no changes in either the host chromosome orthe telomere signal intensity were observed. Similar results werereported for mammalian cells by Barnett and colleagues (14),who found that internal telomeric repeats in two clones werestable after 300 population doublings and suggested that te-lomere-mediated chromosomal truncation occurred very soon

Fig. 3. Chromosomal truncations revealed by a Southern blot with multipleenzymatic digestions. (A) Restriction map of the T-DNA region of the pWY86construct with restriction sites (top line) and their positions relative to theright border (RB) (bottom line). Bars show the position of bar and FLP probes.(B) Restriction mapping of the positions of four transgenes by a Southern blot.Genomic DNAs from pWY86 events B37 (lanes 1–3), B2 (4–6), B77 (lanes 7–9),and B21 (lanes 10–12) were digested with HindIII (lanes 1, 4, 7, and 10),EcoRV(lanes 2, 5, 8, and 11), and SmaI (lanes 3, 6, 9, and 12). The Southern blotwas hybridized with the 32P-labeled FLP probe. DNA fragment sizes wereindicated at the left-hand side. Telomere smears are indicated by arrowheads.Discrete bands found for B37 and B77 events likely result from other copies ofthe transgene inserted elsewhere in the genome.

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after integration if indeed the two processes were not occurringcoincidently.

Phenotypes of Transgenic Plants with Truncated Chromosomes. Chro-mosomal truncations create deficient chromosomes. However,the sporophytes of the plants with chromosomal truncationswere indistinguishable from the other transgenic plants. It isknown that large deletions in maize, such as deficiency forseveral chromosome arms, can result in abnormal phenotypes inotherwise diploid plants (27). The observed result may indicatethat large deletions in some chromosomes may be selectedagainst during tissue culture and plant regeneration. In fact, theonly large truncation recovered in 175 diploid lines was onchromosome arm 3S (Fig. 2 A and Table 1). Chromosomaltruncations affect the gametophytes, because pollen abortionwas frequently observed in transgenic lines with truncatedchromosomes (Table 1). For example, pollen abortion wasobserved in the B77 event with a chromosome arm 3S trunca-tion, and the truncated chromosome was not recovered in thenext generation because of the lethality of the gametes with the3S deficiency (Table 1). However, it should be noted that pollenabortion could also be caused by mutational insertions of thetransgene into a gametophytically vital gene, and, indeed, weobserved pollen abortion in transgenic plants that had only atransgene located internally (Table 1).

DiscussionTelomere-mediated chromosomal truncation in higher eu-karyotes was first reported by Farr and colleagues (13). When500 bp of telomeric repeat was introduced into mammalian cells,6 of 27 transgenes were located at chromosome ends. However,telomere-mediated chromosomal truncation occurred more fre-quently (24 of 44) when 1.0-kb telomeric sequence was used (14).Hanish and colleagues (28) found that all constructs that hadtelomeric sequence from 0.8 to 3.2 kb seeded new telomeresefficiently. In this article, we demonstrated that a 2.6-kb telo-meric sequence was efficient in producing chromosomal trun-cations in plants. By analyzing materials between the time oftransformation and the first haploid gametophyte generation by

FISH and Southern hybridization, truncations could be identi-fied and characterized.

A previous report noted that, in human cells, nontelomericsequences located to the 3� of the telomeric sequence did notaffect telomere-mediated chromosomal truncation (14). In thiswork, we cloned the 2.6-kb telomeric sequence into the multiplecloning sites of an existing binary vector pTF101.1 (29), whichhad �300 bp of vector sequence before the T-DNA right border.The T-DNA right border was found to be essential for T-DNAtransfer from Agrobacterium to the plant genome (30). Whereasfrequent and large deletions are observed at sequences adjacentto the left border during T-DNA integration, T-DNA sequencesat the right border are integrated with few deletions (31). It isreasonable to assume that all telomeric sequences in our exper-iment were present during the transformation. Indeed, weobserved telomeric sequences by FISH in all tested lines that hadan internal transgene. However, the presence of vector sequenceat the right border did not prevent at least some telomere-mediated chromosomal truncation in this experiment. TheSouthern blot with EcoRV digestion (Fig. 3, lanes 2 and 8)showed smear patterns of hybridization, which indicated that abreakage must have occurred within the telomeric sequence, andthe nontelomeric vector sequence at the 3� of the T-DNA hadbeen removed. The differences in size of the telomere containingfragments between events (Fig. 3) might be attributed to adifferent size of such deletions within the transformed telomererepeats or differential additions of new telomere repeats.

Although telomere-mediated chromosomal truncation wassuccessful, the frequency of such events was low as comparedwith that in mammalian cells. There may be several reasons forthis observation. First, plants might require a longer telomericsequence for efficient chromosomal truncation. Efficient chro-mosomal truncation can be achieved by 500- to 800-bp telomericsequence in mammalian cells (13, 14). However, we observedonly a moderate frequency of chromosomal truncations with 2.6kb of telomeric sequences (8.7% minimum efficiency). Second,telomere-mediated chromosomal truncation was performed inmammalian cell lines, but we used a standard plant transforma-tion process during which truncated chromosomes must passthrough plant regeneration on a selection medium. Becauselarge chromosomal deficiencies can have detrimental effects onplant growth, some truncated chromosomes might be selectedagainst during the plant-regeneration process and thus be elim-inated. Third, neocentromere activity was observed in mamma-lian chromosomes, which helps to preserve the truncated frag-ment from being lost during mitosis (20). In contrast, we did notrecover any non-centromere-associated chromosomal fragmentsin our transformations.

Telomeric Sequences During Transformation: Integration or Seeding aNew Telomere. Telomeric sequences are mainly localized atchromosome ends. However, they are also found at someinternal locations (32–36). The internal telomeric sequenceswere thought to have originated from recombination, ancestralchromosome fusions, or double-strand break (DSB) repairs (35,37). For example, sequence analysis revealed internal telomericsequences at intrachromosomal break sites, in many cases,accompanied by deletions or random sequence additions (35).Integrated telomeric sequences in the maize genome by genetictransformation (this report) can also produce stably maintainedinternal telomeric sequences. These internal telomeric se-quences were monitored over four generations and were stillstable. Although telomeric sequences are the preferential se-quence substrate for telomerase, nontelomere sequences canalso be used by the telomerase for additions of telomeresequence in mammalian cells (28) as well as in plants (38). Forexample, it has been reported that telomerase could add newrepeats to the nontelomeric sequences at the end of introduced

Fig. 4. Telomere sequences detected in internal chromosome locations.Metaphase chromosomes of a pWY86 transgenic line were probed with apWY96 probe (red) and a telomere probe (green). Chromosomes were stainedwith DAPI. Arrowheads indicate the internal telomere (green) and transgene(red) signals. Arrowheads in the enlarged images from the top to the bottompanels denote merged transgene (red) and telomere (green) signals, andtelomere only (green) and transgene only (red). (Scale bar, 10 �m.)

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telomeric sequences (28). Indeed, the maize telomerase can addthe TTTAGGG unit to many oligonucleotide primers in an invitro chromosome-healing assay (38). In addition, de novo telo-mere formation was observed as a chromosome-healing mech-anism in somatic cells during a chromosome breakage–fusion–bridge cycle (39, 40). Another example of chromosome healingwas reported when DSBs were generated by endonuclease, andthe healing resulted in chromosomal truncations (41). In all thesecases, new telomeres were seeded on chromosomal breakagesites without the presence of telomeric sequences. However, thepresence of telomeric sequences at the breakage sites signifi-cantly increases the new-telomere seeding efficiency. Chromo-somal DSBs can be repaired by different mechanisms, mostimportantly by the nonhomologous end-joining (NHEJ) pathwayand telomere healing (35). NHEJ is a general chromosome-repair mechanism used by both plants and animals to join DSBsto a fusion product. On the contrary, telomere healing involvesseeding of new telomere sequence at the end of a DSB and givesrise to a broken chromosome. It has been proposed that telomerehealing may occasionally be involved in NHEJ of DSBs (35). Inaddition, essential components are shared by telomeres and theDSB-repair machinery (42). For example, many proteins in-volved in DSB repair are also found in telomere maintenance(43). Agrobacterium-mediated T-DNA integration has beenshown to occur through a NHEJ mechanism (44). In a generalAgrobacterium-mediated gene transformation, single-strand T-DNAs are generated in the bacteria and subsequently trans-ferred to plant cells. The single-strand T-DNA is replicated inthe plant nucleus to form a dsDNA intermediate, which could berecognized as a DSB and repaired by the NHEJ pathway to giverise to an integration event. However, the telomeric sequence inthe T-DNA region might change this process, probably byspecifically recruiting telomerase. Thus, a transition from aNHEJ pathway to telomere healing results in the seeding of anew telomere at the integration site and the truncation of thechromosome. The observation that both internal integration andchromosomal truncation events were present in our transforma-tions with transgenes carrying the telomeric sequences supportsthe transition model from a NHEJ pathway to the telomere-healing pathway. It is reasonable to argue that the presence ofthe telomeric sequence and the DSB act in concert to makechromosomal truncation more efficient. It is conceivable thattelomeric sequences integrated at an internal site are stable,because the telomere sequence is probably not recognizable ornot accessible to the telomerase without a DSB. Our data andthose of others (13), that both truncations and integrations wereobserved in a single transformed cell, seem to support thishypothesis.

Telomere-mediated chromosome truncation has been dem-onstrated to produce minichromosomes in mammalian cells (13,14, 17, 18, 20). By using two telomere-mediated chromosomaltruncation constructs, we demonstrate that 2.6 kb of directArabidopsis telomeric repeats were able to produce chromo-somal truncations in maize. We have also modified this tech-nique to produce artificial minichromosomes in maize.

Materials and MethodsPlasmid Construction. Construction of a 2.6-kb direct repeat ofArabidopsis telomeric sequence, construction of plasmids, andbacterial strains used in this study are described in SupportingText and Table 2, which are published as supporting informationon the PNAS web site.

Gene Transformation. Maize HiII (45) immature embryos weretransformed by the Agrobacterium-mediated gene-transforma-

tion method (24). Transgenic plants were grown in the green-house at 26°C�20°C under 16�8 h day�night conditions.

Pollen Abortion. Pollen abortion was examined in the greenhousein the morning with a pocket microscope (46).

FISH. Root tips from young T0 transgenic plants were collected.To harvest good root tips with the most dividing cells, transgenicplants were grown in small inserts in a tray until they had threeto five leaves and were then transferred to large pots. Root tipsfrom fast growing roots were harvested the next morning,treated, and fixed, and metaphase chromosomes were preparedaccording to the method described by Kato and colleagues (25).

FISH was performed according to the method described byKato and colleagues (25). pWY96 plasmid DNAs were labeledwith Texas red-dCTP (PerkinElmer Life Sciences, Boston,MA) by nick translation, and 50 ng of pWY96 probe was mixedwith a simplified karyotyping probe mix, which contained 0.5ng Alexa Fluor 488-dCTP- (Invitrogen, Carlsbad, CA) labeledknob (47) and 1 ng of Alexa Fluor 488-dCTP-labeled CentC(48) to localize the gross positions of transgenes. When achromosomal truncation was suspected, a full karyotypingmixture (25) was used that contained 5 ng of Cy5-dCTP-(PerkinElmer) labeled Cent4 (49), 1 ng of Alexa Fluor 488-dCTP-labeled CentC (48), 50 ng of Alexa Fluor 488-dCTP-labeled microsatellite 1–26-2 clone (25), 100 ng of Alexa Fluor488-dCTP-labeled pMTY9ER telomere-associated sequence(50), 0.1 ng of Alexa Fluor 488-dCTP-labeled NOR-173 (51),100 ng of Coumarin-dCTP- (Enzo Life Sciences, Farmingdale,NY) labeled knob (47), and 50 ng of Coumarin-dCTP-labeled5S 2–3-3 clone (25). If truncation of a specific chromosome wassuspected, probes specific for that chromosome were used. Forexample, the subtelomere 4–12-1 clone probe was used tocharacterize truncations on chromosome arms 2S, 4L, 4S, 5S,and 8L (25). To detect internal telomere sequences, 50 ng ofTexas red-dCTP-labeled pWY96 probe (red) was mixed with20 ng of Alexa Fluor 488-dCTP-labeled telomere (green) andhybridized to metaphase chromosomes of transgenic plants.Image capture and processing were performed according tothe method of Kato and colleagues (25).

Southern Hybridization. Genomic DNAs from selected transgenicplants were isolated with Plant DNAzol reagent (Invitrogen)according to the manufacturer’s instructions. Twenty micro-grams of genomic DNA from each sample was digested with 100units of restriction enzyme (New England Biolabs, Beverly, MA)in 30-�l volume at 37°C overnight and fractionated in 0.8%agarose gel at 25 volts overnight, denatured, and transferred toa Qiabrane nylon membrane (Qiagen, Chatsworth, CA). DNAhybridization with [32P]dCTP-labeled probes and x-ray autora-diograph were conducted according to the method of Sambrookand Russell (52).

We thank Dr. E. Richards (Washington University, St. Louis, MO) forproviding the pAT4 Arabidopsis telomere clone; Dr. R. P. Niedz (U.S.Department of Agriculture–Agricultural Research Service, Ft. Pierce,FL) for providing the pARS101 clone; Dr. J. E. Schoelz (University ofMissouri) for providing the pDsRed2 clone; Dr. Z. Zhang (University ofMissouri) for providing the pMECA, pSLJ7292, and pFGC1008 clonesand Agrobacterium tumefaciens EHA101 and EHA105 strains; Dr. K.Wang (Iowa State University, Ames, IA) for providing the binary vector,pTF101.1; K. Wang (Iowa State University) and H. Kaeppler (Universityof Wisconsin, Madison, WI) for training W.Y. at a National ScienceFoundation-sponsored Maize Transformation Workshop; and the PlantTransformation Core Facility and the Sears Greenhouse at the Univer-sity of Missouri for providing space and equipment for maize genetransformation. This work was supported by the Monsanto–MU Pro-teomics Grants Program.

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