Engineered Plant Minichromosomes

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Chapter 7

Engineered Plant Minichromosomes

Robert T. Gaeta and Lakshminarasimhan Krishnaswamy

Abstract

The advent of transgenic technologies has met many challenges, both technical and political; however,

these technologies are now widely applied, particularly for crop improvement. Bioengineering hasresulted in plants carrying resistance to herbicides, insects, and viruses, as well as entire biosyntheticpathways. Some of the technical challenges in generating transgenic plant or animal materials include: aninability to control the location and nature of the integration of transgenic DNA into the host genome,and linkage of transformed genes to selectable antibiotic resistance genes used in the production of thetransgene cassette. Furthermore, successive transformation of multiple genes may require the use of sev-eral selection genes. The coordinated expression of multiple stacked genes would be required for com-plex biosynthetic pathways or combined traits. Engineered nonintegrating minichromosomes canovercome many of these problems and hold much promise as key players in the next generation of trans-genic technologies for improved crop plants. In this review, we discuss the history of artificial chromo-some technology with an emphasis on engineered plant minichromosomes.

Key words:Engineered minichromosome, Artificial chromosome, Telomere-mediated truncation,Site-specific recombination

Minichromosomes describe a broad range of small chromosomesthat contain some or all of the elements essential for their replica-

tion and autonomous existence within a cell. In E. coli,minichro-mosomes can exist as small circular plasmids with little more thanan origin of replication (oriC) (1). In eukaryotes such as plantsand animals, they are often linear and require functional centro-meres and telomeres in addition to origins of replication and existseparately from the normal karyotype of a cell. Extrachromosomalgenetic entities such as minichromosomes (natural or engineered)have been reported in bacteria, yeast, mammals, birds, amphibians,

1. Introduction

James A. Birchler (ed.), Plant Chromosome Engineering: Methods and Protocols,Methods in Molecular Biology, vol. 701,DOI 10.1007/978-1-61737-957-4_7, Springer Science+Business Media, LLC 2011


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132 Gaeta and Krishnaswamy

protozoans, and plants (29). B chromosomes, which are commonin nearly 10% of plants species, represent the largest class of suchentities (10). Studies of minichromosomes have contributed sig-nificantly to our understanding of chromatin, chromosome struc-ture, and replication. Most recently, the term minichromosomehas been used to define small, engineered (artificial) chromo-somes in human cell lines (7, 11) and in the crop plant corn (Zeamays) (8, 9, 12). In this review, we use the term engineeredminichromosome to refer to artificial plant chromosomes derivedfrom the truncation of endogenous chromosomes.

Artificial chromosomes are nonintegrating vectors designed tocontain specific genetic components and have the capability forstably harboring large amounts of DNA (13). As with minichro-mosomes, they exist and segregate independent of the normalchromosome complement (karyotype) of the host cell. In lowerorganisms like bacteria and yeast, artificial chromosomes havebeen engineered for large-insert molecular cloning and utilizedfor a multitude of genomic and genetic analyses. In yeast, E. coli,and mammalian systems, artificial chromosomes have been avail-able for decades; however, engineered plant chromosomes arerecent developments. There are two approaches for engineering achromosome: the bottom-up approach and the top-downapproach (reviewed in ref. 14). The bottom-up approach involvesassembling the essential elements of a chromosome piece by piece(using telomere and or centromere sequences) and relies onde novo assembly. This method has been proven successful in

yeast and has been utilized in mammalian systems (1517). In thetop-down approach, artificial chromosomes are derived from thebreakage and truncation of preexisting endogenous chromo-somes. This method has been proven successful in both animaland plant systems (79, 11, 16, 18, 19).

Linear yeast artificial chromosomes (YACs) were constructedover 20 years ago in Saccharomyces cerevisiaeusing telomere, cen-tromere, and origin of replication sequences, and are capable ofmaintaining large DNA inserts of up to 2 Mb in length (15, 20).Bacterial artificial chromosomes (BACs) were created in E. coli,and P1 artificial chromosomes (PACs) were engineered in the P1bacteriophage, both of which are capable of maintaining up to300 kb of DNA (2125). The utility of these early engineeredchromosomes (YACs, BACs, and PACs) as vectors for large DNA

inserts cannot be understated, and they have played an essentialrole in the sequencing, mapping, and characterization of manylarge genomes. In addition, the early studies conducted by Murray

2. Engineered(Artificial)

Chromosomes


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133Engineered Plant Minichromosomes

and Szostak (15) were instrumental in determining the minimalrequirements for chromosome maintenance and stability (i.e.,centromeres, telomeres, and replication origins) in budding yeast.Unfortunately, these early artificial chromosomes did not func-tion autonomously in mammalian and plant cells, which haveunique requirements for proper replication, segregation, andtransmission. However, one study successfully utilized YACs for

vectoring mammalian genes into mice (26).Circular human artificial episomal chromosomes (HAECs)

were first reported by Sun et al. (27) and were used for cloningand analyzing large genomic inserts up to 330 kb. While this sys-tem allowed for the random cloning of human DNA in humancells, it relied on viral sequences for replication and was not anefficient platform for the development of engineered chromo-somes for use in gene therapy. Harrington et al. (16) constructedhuman artificial chromosomes (HACs) by introducing humancentromere, telomere, and genomic DNA into human cells, whichresulted in both de novo artificial chromosomes (bottom-upmethod) and minichromosomes. These were the first linear,mitotically stable chromosomes developed for mammalian sys-tems. YACs containing human centromere and telomere sequences

were later utilized for assembling mammalian artificial chromo-somes (MACs) (17). In other studies, telomere-mediated chro-mosome truncation was used in human cell lines to generateminichromosomes (7, 11, 18, 19, 28). In these studies, humantelomere arrays (TTAGGG) were introduced into human celllines and truncated X and Y-chromosomes were selected. Theresearch by Heller et al. (11) resulted in Y-chromosome derivedminichromosomes that contained little more than a centromere.In these studies, truncating constructs were detectable at the ter-mini of truncated chromosomes by fluorescent in situ hybridiza-tion (FISH) and/or RFLP analysis, suggesting that telomeresequences resulted in healing of the chromosome ends. In mammals,minichromosomes may prove to be effective vectors for genetherapies (reviewed in ref. 29).

Most recently, telomere truncation was applied successfully inplants to generate engineered minichromosomes in maize (8, 9).In these experiments, minichromosomes were derived from

A-chromosomes and supernumerary B chromosomes. Theminichromosomes transmit both mitotically and meiotically. Thetruncating constructs contained visual marker (e.g., DsRed) andselection transgenes, as well as functional site-specific recombina-tion sites (i.e., LoxP and FRT sites). This marked the first demon-stration that transgenes could be attached to engineered plantminichromosomes along with specific sequences for facilitating

targeted recombination. The limitations of plant engineeredchromosomes are yet to be fully tested, and they promise to fur-ther our understanding of basic chromosome biology, while


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simultaneously providing a platform for the next generation oftransgenic technologies in agriculture. These qualities will be use-ful for facilitating the agricultural needs of the growing humanpopulation and for increasing the plasticity of crop germplasm inthe face of an uncertain global climate.

Eukaryotic chromosomes have three essential regions: telomere,centromere, and origins of replication (30). Telomeres consist oftandem repeats of simple, short, G-rich sequence (e.g., TTTAGGGin plants), and associated proteins. The primary structure is con-served over a wide range of organisms. The telomere cap protectsthe ends of chromosomes from sticking to each other, and alsofrom degradation by the action of exonucleolytic enzymes.Centromeres are also characterized by tandem repetitive sequencesand associated proteins. In contrast to the telomere, the centrom-eres consist of several types of repetitive DNA sequences, whichare not widely conserved among diverse organisms. In addition tothe primary structure of DNA, epigenetic factors also have a sig-nificant role in determining centromere function. The centromereregion is also where the kinetochore assembles. Kinetochores arecomposed of a dynamic complex of proteins that provide the sitesof attachment to spindle fibers. The centromere, therefore, isessential for segregation of the chromosomes to the spindle polesduring mitosis and meiosis. Origins of replication are the locations

where DNA replication is initiated, and eukaryotic chromosomeshave numerous origins along their length. The sequences of repli-cation origins have been characterized in yeast (31, 32); however,initiation of replication in higher eukaryotes does not seem tocorrelate with specific DNA sequences, but rather it appears to bedetermined epigenetically (30).

In order for engineered chromosomes to replicate, be stablymaintained, and perpetuate over successive generations of celldivision, it is imperative that they have functional centromeres,origins of replication, and telomeres. As stated above, minichro-mosomes can be generated by two different approaches (Fig. 2;reviewed in ref. 33): the top-down approach, where minichro-mosomes are generated by inducing truncation of endogenouschromosome followed by de novo telomere synthesis, and thebuild up or bottom-up approach, where minichromosomesare generated through de novo assembly of centromere repeatsequence, telomere repeat sequence, and filler DNA that are

introduced into the cell (Figs. 1and 2). Both techniques havebeen applied in plant and animal systems. One advantage ofgenerating minichromosomes by the top-down approach is

3. Methodsfor EngineeringMinichromosomesin Plants


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that endogenous chromosome-derived minichromosomes have adefined structure and organization and are therefore more stableand amenable to further modifications. In contrast, the structureof de novo assembled minichromosomes is variable and poorlyorganized because of random incorporation of genomic DNAsequences from the native chromosomes.

There are several ways to generate minichromosomes from endog-enous chromosomes. Brock and Pryor generated minichromo-somes from maize chromosome 10 following gamma irradiationof pollen (34). The broken chromosomes generated by thismethod were thought to undergo healing during DNA repairand result in relatively unstable minichromosomes. Zheng andcolleagues described a range of stable minichromosomes in a studyof the chromosome type breakagefusionbridge cycle (BFB cycle)(3537). When the two centromeres of a dicentric chromosomeare pulled towards opposite poles during meiotic or mitotic anaphase,a bridge is formed across the metaphase plate. The chromosomebridge breaks at random, and de novo repair of the broken chromo-

some ends can result in the formation of minichromosomes. Katoand colleagues adapted this genetic tool as a top-down approach togenerate several B chromosome-derived minichromosomes,

3.1. Top-Down

Approaches

to Minichromosome

Assembly in Plants

Fig. 1. Generation of minichromosome by top-down (a) and bottom-up (b) strategies. (a) In the top-down approach,

minichromosomes are formed by inducing truncation of endogenous chromosomes. In telomere-mediated truncation, a

transgene construct carrying telomere repeat arrays is transformed into the cell. Integration of the transgene could trun-

cate the chromosome, and the introduced telomere sequence serves as seed for de novo telomere synthesis at the site

of truncation. (b) In the bottom-up strategy, minichromosomes are assembled piece by piece from individual chromo-

some elements. When DNA sequences of centromeric repeats, telomeric repeats, and filler DNA are transformed into the

cell, these may assemble into a functional minichromosome.


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which were analyzed structurally and cytologically (38). In eachof these examples, minichromosomes were generated entirelyfrom endogenous chromosomes without introducing any foreignDNA sequences. However, these minichromosomes require sub-sequent targeting with transgenes if they are to be used as vectorsor platforms for biosynthetic pathways.

In contrast, telomere-mediated truncation using an engineered

construct offers the distinct advantage that minichromosomeformation is coincident with transgene delivery. In a strategysimilar to what has seen success earlier in mammalian celllines, Yu et al. (8, 9) transformed maize embryos with DNAconstructs containing telomere repeat arrays. Both biolistic andAgrobacterium-mediated transformation methods were used. Inthese experiments, several transformation events were recoveredin which the integrating transgene resulted in truncation of thechromosome (Fig. 2). Chromosome truncation and minichro-mosome formation was not observed when a similar construct

lacking telomere sequence was used for transformation. While theexact mechanism of telomere-mediated truncation is not known,it is presumed that during transgene integration into the genome,

Fig. 2. Telomere-mediated truncation of chromosome 1 in maize. A partial metaphase

spread is shown in which a normal chromosome 1 (Chr 1) and truncated chromosome 1

(Chr 1-trunc) are shown (see arrows). Fluorescent in situ hybridization (FISH) was

performed using centromere (CentC) and subtelomeric repeat (4-12-1) probes (labeled

green), as well as a probe homologous to the truncating construct (labeled red). The

truncated chromosome was derived from telomere-mediated truncation using the con-

struct WY86 as described by Yu et al. (8). In this example, the short arm of chromosome 1has been lost, and the truncating construct labeled in redcan be seen.


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the telomere repeat array serves as a seed for de novo telomeresynthesis, leading to truncation and capping of the chromosome.

In these studies, minichromosomes were frequently recov-ered from the truncation of B chromosomes (9). In maize andmany other plants, B chromosomes are naturally occurringsupernumerary chromosomes whose presence has little effecton phenotype when present in low numbers (3941). Theseminichromosomes were meiotically transmissible through malegametes at a frequency of 1239%. Because B chromosomes arenot known to carry any genes, truncation was unlikely to result indeficiencies. Bar and GUS genes showed cytological colocaliza-tion with the truncating construct on the B-derived minichromo-somes. Expression of Bar was evidenced by transgene-inducedresistance to bialaphos selection, and GUS protein expression wasdetectable in several tissues including leaves, roots, shoots, maturekernel embryos, and endosperm. These data suggest that B chro-mosomes will provide useful platforms for assembling minichro-mosomes in plants.

Minichromosomes were also generated by telomere-mediatedtruncation of normal chromosomes (A chromosomes) in maizediploids (8, 9). When A chromosome truncations were detectedin regenerated plants, large truncations were rare, possibly becausethe loss of large chromosomal segments was selected against dur-ing tissue culture and plant regeneration. Furthermore, A-derivedminichromosomes were not transmitted meiotically in diploids,presumably because of deficiencies caused by loss of genes duringtruncation; however, in a spontaneous autotetraploid plant, aminichromosome derived from truncation of the long arm ofchromosome 7, (named R2), was successfully recovered in prog-enies (9). This was done by backcrossing the minichromosome-containing line into diploid plants for three generations. Thisminichromosome could be stably inherited through both mitosisand meiosis.

Minichromosome platforms will have greater utility if they canbe modified in vivo. For example, several genes for desirable traits

could be introduced in successive generations and targeted to thetransgene cassettes on the minichromosome. To test this possibility

Yu et al. (8, 9) utilized a site-specific recombination system (Crelox (see ref. 42)). The telomere truncation constructs used includeda lox71 sequence upstream of a promoterless marker gene, DsRed.

When crossed to a plant carrying 35Slox66Cre expression cas-sette at the tip of chromosome arm 3L, expression of the site-specificrecombinase facilitated recombination between the lox71 on thetruncated chromosome and lox66 on chromosome 3. This placedthe 35S promoter upstream of DsRed gene located on the minichro-

mosomes and activated expression of the gene. This demonstratedthe feasibility of using site-specific recombination systems to intro-duce new sequence to minichromosomes.


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There have been two reports of minichromosomes claiming to beengineered by the bottom-up approach in maize. Carlson et al.introduced a circular DNA with maize centromeric repeatsequences, a selection marker gene, a phenotypic marker gene,and filler DNA (12). It was claimed that the introduced DNAfunctioned in vivo to form autonomous circular chromosomesreferred as Maize minichromosome (MMCs). However, theFISH, genetic, and molecular data did not clearly establish theautonomous nature of the transgene, but rather the high fre-quency of meiotic transmission (~50% as a hemizygote and ~93%as a homozygote) is inconsistent with the circular nature of sucha claimed tiny chromosome and suggests the transgenes mayactually have integrated into the maize genome (reviewed in ref.43). Furthermore, previous studies have suggested that unpairedmonosomic chromosomes and other small chromosomes tend tobe lost at a high frequency (44, 45). In addition, previous studieshave indicated that circular chromosomes suffer loss and instabil-ity with initiation of the BFB cycle (36, 37).

Ananiev et al. transformed maize with native centromeric seg-ments, origins of replication, selectable marker genes, and telomericrepeats (46). Although the data clearly confirm the presence ofminichromosomes in this case, the true cause of their formation,

whether de novo assembly or telomere-mediated truncation result-ing from the introduced telomere sequences, was not resolved.Indeed, the introduction of circular constructs without telomererepeats only resulted in stable integrations into the chromosomes

without centromere function. Further studies are needed to confirmthe feasibility of this approach in plant systems.

The two widely adapted techniques for transformation in plantsinclude biolistic (particle bombardment) and Agrobacterium-mediated transformation (reviewed in ref. 47). Both techniquesare effective for generating minichromosomes by telomere-medi-ated truncation in maize (8, 9). However, both techniques posecertain challenges. Biolistics is a valuable transformation toolespecially for plants that are recalcitrant to Agrobacterium-mediated transformation and involves bombarding plant cells

with gold particles coated with DNA to be transformed. The fre-quency of transformation and truncation is more efficient by thebiolistics method (9); however, an inherent disadvantage withthis technique is that transgenes often insert as tandem repeats

and at multiple locations in the genome. Such an arrangementcould lead to silencing of genes on the transgene cassette. It ispossible that such tandem arrays can be resolved. For example,

3.2. The Bottom-Up

Approach to

Minichromosome

Assembly in Plants

4. Methodsfor TransformingMinichromosome-Generating DNA

Constructs


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Srivastava et al. demonstrated that when transgenes contain a loxsite, concatameric transgene insertions could be resolved to singlecopy during site-specific recombination between the outermostsites in the array (48). A similar strategy could be applied toresolve tandem copies of transgene cassettes used for telomere-mediated truncation. The issue of transgene insertion at multiplelocations in the genome can be resolved easily by backcrossingthe transformant to a parental genotype followed by self-pollina-tion in successive generations.

Agrobacterium-mediated transformation is fairly efficient in awide range of dicotyledonous plants, and several monocotyledonousplants. The advantages of Agrobacterium-mediated transforma-tion include lower transgene copy number and stable gene expres-sion (4951). However, the transformation efficiency is muchlower in comparison to particle bombardment. In addition, noB-chromosome truncations were recovered in experimentsusing Agrobacterium-mediated transformation of maize (8).Nevertheless, it is promising that the list of plants transformablebyAgrobacteriumis steadily growing. This includes several agro-nomical and commercially important plants such as rice, wheat,maize, sorghum, and barley (52).

When a truncating DNA construct is introduced into a plant cell,there are several possible outcomes: the transgene could get insertedinto the chromosome, it could truncate a chromosome whileinserting, or the introduced DNA could get enzymatically degraded.

Yu et al. (8) reported that when truncation constructs were trans-formed into maize, 231 independent transgenic plants were recov-ered, of which 118 insertions were at a distal position on thechromosome. Fifty-six distal insertions were further tested, andnine resulted in truncation. With a variety of possible outcomes, itis imperative to test and confirm putative truncation events.

The process of localizing transgenes has been simplified withthe recent advance in fluorescent in situ hybridization (FISH) inmaize (53, 54). DNA probes targetting transgenes and sequencesspecific to each of the maize chromosomes can be labeled withfluorescent molecules and hybridized onto metaphase chromo-some spreads from root-tip preparations, allowing the location ofa transgene in the genome to be determined. However, the cur-rent limits of resolution of the FISH technique cannot distinguishbetween a terminal location of the transgene (as in case of true

truncation) and a nontruncating distal insertion very close to thechromosome end. For these reasons, other methods of verifica-tion are helpful. Southern blot hybridization can be applied to

5. Detection and

Confirmation ofMinichromosomes


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determine whether a distal insertion is a truncation event.Genomic DNA from the plants are digested with a restrictionenzyme that cuts the truncating construct closer to the telomerearray, and hybridization is performed using a probe specific to theDNA distal to the enzyme site. Truncation events in which thetransgene cassette lies adjacent to the newly formed telomere willappear as a smear on the autoradiograph, reflecting the variablesize of telomeres in the minichromosome (8). On the otherhand, a nontruncating transgene insertion will likely appear as adistinct band.

The terminal position of the truncating transgene could alsobe tested by a PCR-based technique called Primer extensiontelomere repeat amplification (PETRA) (55). In this two-steptechnique, first an adapter oligo with the 3end complementaryto the G-rich single strand overhang at the end of the telomere isused to generate a copy of the complementary strand. This productis PCR-amplified in the second step using an oligo with theadapter sequence and a primer complementary to the truncatingconstruct. The presence of the truncating construct in the ter-minal position alone will amplify the 3end of the construct andtelomere.

Plants carrying minichromosomes will be of commercial and/or agronomic interest only if the minichromosome is stablethrough successive mitotic cycles and if it can pass through thegametophyte. Genetic and cytological analysis at different stagesof development of the plant and at successive generations canhelp determine the utility of any given minichromosome.Biotechnological utility crucially depends on the transcriptionallyactive or inactive state of the transgenes on the minichromosome,stable transmission, and amenability (see below).

Genetic engineering has tremendous potential for revolutionizingagriculture. Plant biotechnology has resulted in the developmentof plants with resistance to herbicides, insects, and viruses, as wellas plants carrying entire biosynthetic pathways (56). Transgenicexpression of multiple genes is required for complex biosyntheticpathways or combined traits, and both genes and regulatory ele-ments need to be stacked for these purposes. For instance, idealplants might carry resistance for multiple insect and fungal pests,as well as tolerance to temperature extremes, water stress, or saltconcentrations. However, traditional transformation techniques

have drawbacks. They tend to generate random genomic integra-tions that may suffer from position effect variegation or insertionsthat may disrupt endogenous genes. Consequently, transgenic

6. Next-GenerationEngineered

Minichromosomesin Plants and TheirApplications


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events require intensive screening for stable integration, meiotictransmission, and function, as well as extensive backcrossing forpurification. Movement of transgenes from one genetic back-ground to another is limited by linkage to undesirable genes orQTL. Currently, several alternatives to traditional transformationexist, including: episomal viral vectors (57), organellar transfor-mation (58, 59), and engineered minichromosomes (8, 9, 12). Inthe following sections, we discuss the distinct advantages of engi-neered chromosomes.

Engineered minichromosome platforms derived by top-downor bottom-up approaches will be useful in facilitating the next gen-eration of transgenic crop plants and will offer new solutions tosome of the challenges associated with classical transformation.Minichromosomes could be used as biofactories for metabolite pro-duction, as well as for basic studies of chromosome biology. Theycan be developed in one line or cultivar, and introgressed into relatedcultivars without the problems associated with cis linkage drag orposition effects. As an example, a naturally occurring (nontrans-genic) minichromosome discovered inArabidopsiswas easily trans-ferred from one ecotype to another by simple backcrossing (60).

Using top-down approaches they may be synthesized fromchromosomes of the normal karotype (A chromosomes) or super-numery B chromosomes in maize (8, 9); however, A-chromosometruncations are difficult to recover meiotically from diploids, andsuccessful recoveries have occurred only in spontaneous tetra-ploids (see above). It is possible that trisomics and addition linescould serve as targets for deriving specific A-chromosome derivedminichromosomes. In such lines, the diploid chromosome balance

would be restored upon truncation of the extra chromosome,possibly favoring their recovery. B chromosomes offer a uniqueand efficient platform for assembling minichromosomes by thetop-down method, in that they exist autonomously from theKaryotype (A genome), are essentially phenotypically inert, andexist in hundreds of plant species (61, 62). Minichromosomesengineered from B chromosomes in maize have the added benefit

of nondisjunction during the second pollen mitosis, and preferen-tial fertilization of the egg cell during pollination (reviewed ref.63). These properties would facilitate rapid accumulation or seg-regation of minichromosomes, allowing for copy number controland rapid removal when they are no longer needed in a particulargenetic background. Control over dosage would allow for maxi-mal production of some biomolecules that may have industrial,pharmaceutical, or nutritional value. It is possible that high copynumbers of transgenes on minichromosomes would experiencecosuppression, but currently, there are no data on these limita-

tions. Minichromosomes could also be outfitted with pollen-lethal genes, which would prevent them from spreadingunwittingly by pollen dispersal.


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methods. Telomere-mediated truncation of endogenous plantchromosomes will probably be quite an amenable method forminichromosome production, since the telomere-repeat sequenceis highly conserved. Using the top-down approach, initialminichromosomes could potentially be trimmed furtherthrough successive truncation to contain little more thancentromeres, transgene cassettes, and telomeres (reviewed in ref.63). In a similar way, the bottom-up approach would have thebenefit of minimization and control over the overall genetic con-tent of the entire synthesized chromosome. However, becausecentromere sequences are often species-specific, a single platform

would be difficult to move from one species to another.

In conclusion, engineered (artificial) chromosomes have come along way in the last quarter century. From yeast and bacteria tomammals and plants, chromosome vectors have played an impor-tant role in our understanding of chromosome biology and haveproven invaluable in genetic and genomic analyses. Future engi-neered minichromosomes hold great promise for furthering theseunderstandings and promise to expand upon classical transgenicresearch; however, there remain many unanswered questionsregarding the basic biology of chromosomes, both normal andengineered. Some of the questions future studies can addressinclude: (1) what conditions contribute to optimal formation,stability, and transmissibility of engineered plant minichromo-somes; (2) what are the benefits and drawbacks to minichromo-somes derived via the top-down or bottom-up approach, andhow can these methods realize their greatest potential; (3) whatare the size restrictions for engineered chromosomes; (4) what isthe maximum copy number of minichromosomes that can bemaintained by a cell, and how does copy number affect the expres-sion of transgene cassettes and transmissibility; (5) what is theeffect of genetic background, i.e., do different host genotypescontain genetic variation contributing to the formation and func-tioning of minichromosomes; (6) what methods will prove mostfruitful for amending minichromosomes through the in vivoremoval and replacement of selection genes, genes of interest,and promoters and other regulatory elements; (7) can minichro-mosomes be built up to contain extensive and elaborate biosyn-thetic pathways, in which regulator and response genes faithfullyintegrate to recapitulate the production of complex metabolites;

and (8) will it be possible to transfer genes and chromosomalfragments between minichromosome and host genome in a site-specific manner?

7. Conclusionsand FutureQuestions


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Acknowledgments

Research supported by National Science Foundation grant DBI

0701297 to James Birchler.

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