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Minichromosome technology
- 1. BY J. SRIVARSHA Ph.D (Ag.) GPB DOCTORAL SEMINAR II MINICHROMOSOME TECHNOLOGY IN PLANTS
- 2. 2 What is a minichromosome? Production of minichromosomes Behaviour of minichromosomes Attaching genes on MC Manipulation of genes Minichromosomes in plants Applications and Case studies Pros and Cons Future thrust
- 3. Department of Agril Botany 3 Brainstorming Issues of the Transgenic Era: Gene Stacking Random Gene Integration Position Variegation Effects Frequent Loss Of Gene Integrity Solution is MINICHROMOSOME TECHNOLOGY
- 4. Department of Agril Botany 4 DNA and histone proteins are packaged into structures called chromosomes. Origin of B chromosomes
- 5. Extremely small version of a chromosome; Linear/circular DNA ; associated proteins; Carries genes; transfers genetic information. Plasmids that replicate autonomously from OriC (Hiraga 1976). Resembles their chromosomal counterparts. Enriched with transposable/repetitive elements. Department of Agril Botany 5 (Hiraga 1976, Messer and Weigel 1996, Aakash et al. 2009)
- 6. Department of Agril Botany 6 ARS: autonomously replicating sequence, CEN: centromere sequence, TEL: telomere sequence S. Fujimoto and S. Matsunaga , Cytologia 81(3), (2016)
- 7. Only in mammalian cells. Individual components of MC- not understood. Centromeric and telomeric repeats - epigenetic. Exceptions in centromere function. Functional centromeres have been formed that do not contain centromere repeats at all (Fu et al., 2013). In barley, removal of centromeric repeats does not prevent centromere formation. Department of Agril Botany 7 Graham et al. 2015
- 8. Utilises existing chromosome within the genome. History: First demonstration – Human mammalian cells- transgene with an array of telomere repeat sequences- Farr et al. 1991. Utilizes the insertion of telomere sequences into the existing chromosome. Signals the new telomeres for truncation of chromosomes. Department of Agril Botany 8 Weichang Yu et al. 2007 (a)
- 9. Department of Agril Botany 9 Graham et al. 2015 Can be produced from both A and B chromosomes. Radiation induced chromosomal breakage. From B-chromosomes by Breakage-Fusion-Bridge cycle. Telomere mediated chromosomal truncation. Ac – Ds transposon system along with the Cre-lox recombination system (Murata et al., 2013). by creating satellite DNA-based artificial chromosome
- 10. Department of Agril Botany 10 Generalized scheme for the production of engineered minichromosomes in maize.
- 11. Department of Agril Botany 11 Telomere-mediated chromosomal truncation Blue bar - chromosome. Filled black circle - centromere. Orange triangle - transgene integration site. Red dotted line - transformed and newly seeded telomere. Green line - other transgene components such as the selection marker. Orange circle with a cross - the chromosomal breakage site. Birchler et al. 2008
- 12. Department of Agril Botany 12 Telomere- mediated chromosomal truncation: • Mechanism is not known; speculation: during transformation the telomere sequence is exposed and the cell recruits telomere elongation machinery to form a de novo telomere (Birchler et al., 2008). • In plants – first demonstration- maize- Yu et al. 2007. Arabidopsis (Nelson et al., 2011; Teo et al., 2011) barley (Kapusi et al., 2011) rice (Xu et al., 2012) • Agrobacterium- mediated successful in A. Thaliana. • Particle Bombardment- successful in maize – B chr.
- 13. Generally markers are inserted along with transgene. Mostly antibiotic resistance genes. Markers used till date: Npt II marker: Neomycin phosphotransferase II Resistance to glyphosate ( to express EPSPS) hph gene (encodes hygromycin B-kinase) bar Department of Agril Botany 13 Graham et al. 2015
- 14. Discovered in late nineties in plants (Buchiwicz 1997). Minichromosomes in Arabidopsis: In teleocentric line of A. thaliana, a minichromsome was identified through FISH approach and it revealed that it was from the short arm of chromosome number 4 (Murata et al.,2006). Size ~ 7.5Mb Recently, two more minichromosome (alpha,ß and∂ ) have also been discovered by the same research group. These two minichromosomes were found in a transgenic Arabidopsis plant produced by in planta vacuum infiltration technique. Department of Agril Botany 14 Weichang Yu et al. 2007 (a)
- 15. Department of Agril Botany 15 Minichromosome-like structures in wheat: • Origin is unknown Jerzy Buchowicz 1997 Structural organisation of a wheat minichromosome T- Telomere; A- ARS core; R- RAP 1 binding site D- Direct repeat with a motif common to ABA responsive elements and introns
- 16. Department of Agril Botany 16 Minichromosome technology in maize: • Only few maize varieties have B chromosomes. • Property of B-chromosome in maize discovered by Carlson and Roseman (1992) . • Rediscovered by Ronceret et al., in 2007. • Recently both A and B chromosomes are use. • Radiation induced chromosomal breakage is unstable. • Telomere mediated truncation method is reliable. • Repeated backcrossing to transfer into diploid background. Weichang Yu et al. 2007 (a)
- 17. B chromosomes - Preferred Dispensable Do not pair with any of the standard A chromosomes at meiosis Irregular modes of inheritance Have little effect on an individual’s phenotype High survival rate after telomere-associated truncation Department of Agril Botany 17
- 18. Department of Agril Botany 18Ronceret et al., 2007 BEHAVIOUR OF MINICHROMOSOMES
- 19. Department of Agril Botany 19 Behaviour during meiosis: Chromosome pairing in Pachytene larger minichromosomes were better at pairing. Han et al. 2007 also looked at the disjunction behaviour of minichromosomes in a situation with only one minichromosome present in a diploid cell. minichromosomes lost their autonomous ability for postmeiotic nondisjunction at the second pollen mitosis found in original B chromosomes. Localization pattern of SGO1 was reported. No differences between the MC and NC for the phosphorylation pattern of the H3 histone at Ser-10.
- 20. Department of Agril Botany 20 Distribution of Minichromosomes during the cell division cycle Klaus Ersfeld and Keith Gull, 1997
- 21. Department of Agril Botany 21 Materials and methods: •BAC library using DNA of maize inbred B73. •BAC clones with strong hybridization signals to one or more of the repetitive sequences were selected for MMC construction. •constructed circular MMC’s by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments. •Transferred to maize embryos by particle bombardment Carlson et al., 2007
- 22. Department of Agril Botany 22 Results: Screening for functional MMC’s: • 102 constructs bombarded; 66 gave rise to regenerated plants; •52 of these constructs -randomly chosen and characterized •FISH; arrested root tip cells in mitosis, and stained chromosome spreads with rhodamine and fluorescein-labelled probes corresponding to centromeric repeats and to MMC-encoded genes, respectively. •47/52 (90%) of the constructs evaluated with FISH were able to form an autonomous MMC, and 43/52 (with centromeric inserts ranging in size from 7 to 190 kb) gave rise to plants that contained only an autonomous MMC. •Increase in the rate of formation of autonomous MMC by the increase in the size of centomeric fragment.
- 23. Department of Agril Botany 23 Generation of Autonomous Minichromosomes
- 24. Department of Agril Botany 24
- 25. Department of Agril Botany 25 Conclusion: • MMC can be maintained autonomously. • Gene cassette is stably inherited for four generations. • MMC centromere sequences, could rely on the kinetochore and spindle machinery for faithful segregation, or could be inherited through alternative mechanisms.
- 26. Minichromosomes generated by telomere truncation may have two configurations: a) with transgene b) Without transgene This difference depends on the orientation of construct integration relative to the centromere of the recipient chromosome Minichromosomes with attached transgenes were characterized in detail for mitotic and meiotic behaviors, transmission frequencies, gene expression, and the ability to use site-specific recombination. Alternative method: Cobombardment of two different constructs. Department of Agril Botany 26 Weichang Yu et al. 2007 (a)
- 27. Department of Agril Botany 27 Manipulation of genes on minichromosomes • Principle: addition of genes by site-specific recombination. Adding genetic materials to minichromosomes by site-specific recombination and genetic manipulation of minichromosome copy numbers. Weichang Yu et al. 2007 (a)
- 28. Department of Agril Botany 28 Plant Materials: •HiII hybrid plants = HiII parent A with B chr. × parent B. Development of minichromosomes: By Telomere mediated chromosomal truncation. At the same time introducing site-specific recombination cassettes for future manipulations. Minichromosomes from truncated maize A chromosomes: Developed by using: Agrobacterium mediated transformation 1. two telomere-containing constructs, pWY76 and pWY86, that possess a bialophos herbicide resistance selectable marker (bar) 2. Cre/lox or FLP/FRT site-specific recombination cassettes 3. telomere repeats
- 29. Department of Agril Botany 29 Minichromosome R2 produced by telomere truncation of an A chromosome. Weichang Yu et al. 2007 (b)
- 30. Department of Agril Botany 30 Minichromosomes from B chromosomes: • Developed by using: Biolistic mediated transformation three telomere-containing plasmids, pWY76, pWY86 or pWY86-bar (a pWY86 derivative with a deletion of the selection bar gene expression cassette) a pAHC25 construct Gene Expression from Normal B and MiniB Chromosomes. • Two minichromosomes were transmitted to the F1 by the mechanism of nondisjunction at the second pollen mitosis when a male parent carrying the minichromosome and full- length B chromosomes was used in an outcross.
- 31. Department of Agril Botany 31 Targeted normal B and mini B chromosomes GUS expression from minichromosome 86B23 with the pAHC25 transgeneA. sh, shoot; em, embryo; en, endosperm. Weichang Yu et al. 2007 (b) 8614-A, 86B23,86B93
- 32. Department of Agril Botany 32 Transmission Test of Minichromosomes: • Plants with minichromosomes were selfed or cross-pollinated as a male to tester lines without the minichromosome. • Kernels from each cross were germinated in moisturized Vermiculite (Therm-O-Rock, New Eagle, PA) for 2–3 days at 30°C. • Root tip treatment and metaphase chromosome spreads were prepared . • Progeny of minichromosome containing plants were screened either by chromosome counts (for R2) or by FISH with a B repeat probe (miniB chromosomes)
- 33. Department of Agril Botany 33 Research findings: • Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. • miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. • Faithful transmission of miniB chromosomes ; but can be changed in dosage in the presence of normal B chromosomes.
- 34. Department of Agril Botany 34 In understanding physical features that determine chromosomal function. Mass production of pharmaceuticals. Gene stacking with desired genes. To develop a mini B chromosome-based genomic cloning system. APPLICATIONS
- 35. I. Epigenetics of centromere Specification II. Homologous gene targetting III. Issue of Centromere size Once a centromere has become inactive, it is inherited in this state potentially indefinitely (Phan et al. 2007). Recent developments with zinc finger nuclease gene targeting have shown the best promise for overcoming second obstacle. Department of Agril Botany 35 James A. Birchler (2010)
- 36. Addition of whole biochemical pathways to plants – confer new properties. Plant factories – for producing novel proteins inexpensively. To transfer combined properties from one variety to another. Department of Agril Botany 36 Birchler A. 2015
- 37. Department of Agril Botany 37 Pros: target transgenes - defined insertion position. add many genes in sequential manner. No chance of linkage drag. Less epigenetic effects reported. providing an independent linkage group that can be rapidly introgressed into various germplasms. Limited availability of B-chromosomes. Regulatory approval for truncated A- chromosomes. Less research on the ring chromosomes Lack of research in plants where the sexual-crossability is not possible. Limited knowledge on the meiotic transmissability.
- 38. Reliable prediction of the performance and expression of transgenic traits. MC may enable the construction of multigene pathways to produce pharmaceuticals and other industrial products in plants. Further research to be carried out regarding the inheritance updates in ring chromosomes too. More research regarding multiple site specific recombination cassette is to be done. Should be broadened to other plant species. Department of Agril Botany 38
- 39. MC: they may behave strongly, but they get the work done. stable platform for stacking of transgenes. Possibility of inheritance as a stable unit. Doubled haploid breeding + MC technology - easy introgression of genes. As a tool to test the reaction of transgenes in multiple genetic backgrounds. Department of Agril Botany 39
- 40. “Science knows no country, because knowledge belongs to humanity, and is the torch which illuminates the world.” -Louis Pasteur THANK YOU
Editor's Notes
- Gene stacking refers to the process of combining two or more genes of interest into a single plant. Gene pyramiding and multigene transfer are other monikers in the scientific literature referring to the same process. The combined traits resulting from this process are called stacked traits. Integration at random sites results in unpredictable transgene expression due to position effect variegation, variable copy number from tandem integrations, and frequent loss of gene integrity as a result of unpredictable breakage and end joining. AS A STEP TOWARDS IMPROVING TRANSGENIC CROP PRODUCTION In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes.
- THE growth, development, and reproduction of an organism relies on genetic material that is organized into chromosomes. The chromosome complement carried by all members of a species is composed of essential chromosomes referred to as the A chromosomes. A subset of individuals within a species may also possess extra chromosomes that are nonessential and not members of the standard A chromosome set. These supernumerary chromosomes are commonly referred to as B chromosomes.
- A minichromosome is an extremely small version of a chromosome, the thread-like linear or circular DNA and associated proteins that carry genes and functions in the transfer of genetic information. They depend on functional DnaA and DnaC products, de novo protein synthesis and RNA polymerase mediated transcription for initiation of bi-directional replication; thereby resembling their chromosomal counterparts. First identified by Blackburn and Gall (1978) in the macronuclei of Tetrahymena thermophila, and from then referred to as minichromosomes.
- The first one involves the de novo assembly of artificial chromosomes with artificial compositions of cloned chromosomal components delivered into cells (bottom-up approach). The second one involves truncation of endogenous chromosomes to create small chromosomes (top-down approach). Bottom-up Approach: de novo assembly of cloned chromosomal elements Centromeric and telomeric sequences a selective marker gene (to identify transformation) genomic DNA that contains a replication origin. This method is well established in yeast (Murray and Szostak, 1983), mammalian cells (Harrington et al. 1997).
- In fact, there are instances where functional centromeres have been formed that do not contain centromere repeats at all (Nasuda et al., 2005; Gong et al., 2009; Gonzalez et al.,2013; Fu et al., 2013). Additionally, as previously mentioned, removal of centromeric repeats from barley does not prevent centromere formation. As a result, it is largely believed that the regulation of regional centromeres is epigenetic in nature.
- The lack of knowledge of how to activate silent centromere is the serious impediment to de novo artificial chromosome construction. However, engineered minichromosomes produced by telomere truncation can be easily created in many plants with a single construct because the telomere sequence in plants is conserved. This method also bypasses the issue of the Epigenetic complications of activating centromere function inherent in the de novo approach.
- This approach was an answer to the epigenetic complications. Chromosome arms are truncated by two independent transgene events. The first event truncates the long arm and the second event truncates the short arm to result in a minichromosome with only the centromeric region remaining. small deletions probably remove sequences from the right border and expose the telomere repeats. The exposure of the telomere repeats probably converts the DNA double strand break repair mechanism to that of de novo telomere elongation during integration.
- megabases, where 1 Mb = 106 bases
- However it is known that plants growing under natural environmental conditions, the chromosomal DNA will be exposed to lesions which results into fragmentations. These fragmentations will contain telomere repeats, ori and regulatory elements to replicate autonomously. 28bp inverted terminal repeats with heptamer units The presence of yeast type ARS core element, RAP1 binding site, two inphase ORF’s and 22 bp intriguing direct repeats will further makes the cloned sequence resembling the functional, small genetic entity.
- These minichromosomes were transferred to a diploid background by repeated backcrossing and were stably maintained. Brock and Pryor (1996) reported minichromosome in maize as a consequence of gamma irradiation of pollen. However unstable. So far healed chromosomal broken segments have been reported only in barley (Wang, 1992)
- B chromosomes, the often neglected components of the karyotypes of numerous plant and animal species, could become a major player for the generation of engineered chromosomes because of their unique features. Dispensable = able to replace or replaceable Only in the case of a high number, B chromosomes can reduce vigor (Puertas, 2002). Thus, engineered mini-Bs allow the study of gene dosage effects. The survival rate after telomere-associated truncation was higher for B than for A chromosomes (Yu et al., 2007), most likely because most Bs are genetically inert. Constitutive transgene expression from A and B chromosome–derived minichromosomes suggests that inactivation of genes on B chromosomes, if it occurs, it is at least not a rapid process. To achieve viability of plants with an A chromosome–derived minichromosome, the truncation event should take place in a polyploid or (for the target chromosome) aneuploid background. Maize A-derived minichromosomes were faithfully transmitted from one generation to the next, whereas the meiotic transmission rate of B-type minichromosomes varied from 12 to 39% via the male parent (Yu et al., 2007), comparable to the transmission rate of mini-B chromosomes generated by breakage-fusion-bridge cycles (Kato et al., 2005). The fact that B chromosomes are similar to normal chromosomes but devoid of genes makes them ideal as gene delivery vehicles because transgenes can be inserted into them without disrupting endogenous genes, which is often the case on normal genecarrying chromosomes, and they can be added and removed without any consequences for the organism. B chromosomes have an interesting propensity to accumulate in large copy numbers. They often undergo nondisjunction in the second pollen mitosis, which is followed by a preferential fertilization of the B chromosome containing sperm (Jones et al., 2007).
- Sister chromatids separate at anaphase I in contrast to the normal B chromosome, whose sisters remain attached until meiosis II. This type of behavior is typical of small chromosomes in maize. One of the most important characteristics of a useful minichromosome is its stability through cell divisions and, in particular, its behavior during meiosis.
- Chromosome pairing in Pachytene: They found that minichromosomes had lost the ability to pair with both their B and chromosome 9 progenitors. Han et al. 2007: Two classes of minichromosomes could be distinguished on this basis. The first class showed regular disjunction behavior typical for univalent chromosomes (normal or B). The univalent moved to one pole during meiosis I, and sister chromatids separated at anaphase II. The second class showed an abnormal disjunction in which sister chromatids separated at meiosis I (Figure 1B). Losing autonomous ability: This mechanism of accumulation is known to require the tip of B chromosome long arm, which was absent from the minichromosomes (Han et al. 2007). This property could be restored in trans by the addition of a full-size B chromosome. Faced with the unusual behavior of minichromosomes, Han et al. Initiated a study to understand its basis. In several lines showing premature sister chromatid separation, they followed the localization pattern of SGO1, which is thought to protect pericentromeric cohesion in meiosis I. In maize afd1 mutants, when sister chromatids separate prematurely during anaphase I, SGO1 is not detected (Hamant et al., 2005). However, in the minichromosomes, the localization of SGO1 was normal, even though sister chromatids separated in meiosis I. They also did not find any differences between the minichromosomes and normal chromosomes for the phosphorylation pattern of the H3 histone at Ser-10, which is known to correlate with the status of sister chromatid cohesion (Kaszas and Cande, 2000).
- The distribution of minichromosomes during the cell division cycle. (A) In interphase, minichromosomes were asymmetrically distributed near the nuclear periphery (see also right cell in D). (B) After formation of a spindle, minichromosomes congregated in the nuclear center. (C to E) During progression of mitosis, the minichromosomes separated into two entities and relocated to the poles of the spindle. The minichromosomal signal is shown in red, the antibody to tubulin in green, and the total DNA in blue. The third and fourth frame of each row represent the merged signals and the phase-contrast image, respectively. The kinetoplasts as markers for cell cycle progression are labeled by arrows (first row only). Bar, 10 micrometer.
- Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes.
- Clones enriched in satellite sequences, centromeric retroelements, and other repetitive sequences were chosen to assess whether they can form MMCs when delivered to plant cells.This unexpectedly high rate of recovering autonomous MMCs suggests that embryogenic maize tissue readily establishes MMCs from purified DNA and that the BAC clones that yielded transformed plants contained sequences that efficiently promote MMC formation. In vitro Cre-lox recombination was used to fuse selected BAC clones to a circular vector containing a plant selectable marker (nptII) and a cell-autonomous reporter gene (nuclear-expressed DsRed), forming circular constructs. While some MMC constructsintegrated (see below), we considered MMCs autonomous when (i) 70% of the cells examined (n 15) contained signals that were clearly distinct from the DAPI-stained host chromosomes, (ii) integrated signals were not detected, and iii) the fluorescent probe corresponding to the MMCencoded genes colocalized with the probe to repetitive centromeric DNA, suggesting an intact construct and making it unlikely that the signal was due to noise. Figure explanation: Centromere fragments across a wide size range enable autonomous MMC inheritance. For each size category, the percentage of transformation events (total ¼ 52) that yielded only an autonomous MMC (white bars) or both an autonomous and integrated MMC in the same cell (grey bars) is shown; the number of MMCs in each category is noted parenthetically; error bars indicate standard error.
- (A–H) Metaphase chromosome spreads from MMC1 event V-1: (A–D) T1 plant; (B–D) correspond to the region denoted by the arrowhead in (A); (E–H) T2 plant. DNA is stained with DAPI ([B F], blue) and labeled with FISH probes specific for the DsRed and nptII gene cassette ([C, G], green); or centromere sequences ([D, H], red). (I, J) Event V-4 with autonomous and integrated copies of MMC1 (I); pCHR758 (noncentromeric control) (J). Autonomous minichromosomes (arrowheads); integrated constructs appear as pairs of FISH signals (arrows); size bar, 5 lm. (K) Centromere fragments across a wide size range enable autonomous MMC inheritance. For each size category, the percentage of transformation events (total ¼ 52) that yielded only an autonomous MMC (white bars) or both an autonomous and integrated MMC in the same cell (grey bars) is shown; the number of MMCs in each category is noted parenthetically; error bars indicate standard error.
- aM, monosomic for MMC1; D, disomic for MMC1; WT, wild-type maize. bp-Value calculations based on chi-square distributions with 1 degree of freedom. p-Values significantly different from expectations (v2 p , 0.05) are indicated with an asterisk. p-Values were not calculated for expectations of 1:0 and are noted as NA. cLoss rates calculated as the difference between the expected and observed numbers of DsRed positive progeny, expressed as percent of the expected (assuming Mendelian assortment). dCrosses derived from a single V-1 plant that demonstrated sectoring in the T1 generation; loss was confirmed by PCR. We tested the ability of these MMCs to confer inheritance by crossing T0 transformants to wild type, growing the progeny without selection, and monitoring nuclear-localized DsRed fluorescence. (A) Fluorescent detection of nuclear-localized DsRed in MMC1 maize leaf; size bar, 50 lm. (B, C) Detection of DsRed sectors in a T2 plant leaf from event V-1 under (B) bright-field and (C) fluorescence microscopy; size bars, 0.5 mm. (D) high magnification view of image shown in (C) with the corresponding sector, comprising all cell layers, indicated by an asterisk; the edge of a sector that comprises only the adaxial cell layer is indicated by arrowheads, cells with typical DsRed expression are indicated by arrows. Size bar, 50 lm. (E) MMC consisting of a pCHR758 backbone and a centromere-derived insert, gene expression cassettes (grey), centromeric inserts (box), BglII restriction sites (arrowheads), and probes used for FISH and Southern blot analyses are indicated
- Overall, the GC content of the BLASTN (http://www.ncbi. nlm.nih.gov/BLAST/Blast.cgi) was used to assess sequence similarity, GENSCAN (http://genes.mit.edu/GENSCAN.html) to predict promoters and open reading frames, and repeat finder (http://tandem. bu.edu/trf/trf.basic.submit.html) to analyze CentC satellites.e MMC1 centromeric insert is 48%. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact.
- THE NEXT STEP: MAKING USE OF ENGINEERED PLANT MINICHROMOSOMES
- By crossing plants containing a miniA chromosome that contained a promoterless lox-DsRed gene with transgenic plants that express the Cre recombinase from a 35S-lox-Cre cassette, recombination occurred between the two nonhomologous chromosomes with the activation of the red fluorescence gene (DsRed) and also the placement of other genetic materials to the minichromosome. Explanation for picture: A minichromosome and a normal chromosome are first targeted with lox recombination sites (orange lines), and crossed together. Recombination (1) between these two chromosomes takes place by expressing the Cre recombinase from either the normal chromosome or the minichromosome to add genetic material to the minichromosome. The minichromosome can be retrofitted (2) with more genes by repeated recombination with other site-specific recombination systems (blue lines) as described by Ow [22]. The copy number of engineered minichromosome can be manipulated to maximize gene expression or to enrich the cloned chromosomal fragment (3). Chromosomes are represented as double lines, and centromeres and telomeres are represented with green and red dots, respectively.
- HiII parent A line with B chromosomes was developed by recurrent back-cross of B chromosome containing plants to the HiII parent plants. HiII parent A line with B chromosomes was selfed to allow the accumulation of B chromosomes. Progeny with the multiple B chromosomes were self pollinated or crossed by HiII parent B to produce immature embryos for genetic transformation.
- Centromere and NOR are labeled green; truncating transgene is labeled red. Arrowhead denotes R2. Insets in A shows transgene (Top), CentC (Middle), and the merged (Bottom) images of R2. Inset in B shows an enlarged merged image of R2. (C–E) R2 minichromosome in meiotic cells. CentC and knob are labeled green. Transgene is labeled red. R2 was not paired with other chromosomes at pachynema (C), diakinesis (D), and metaphase I (E). (F) Homozygote of R2. Enlarged images of R2 are shown in Insets. Transgenes are labeled red. Arrowheads denote chromosomes enlarged in Insets. (Scale bars, 10 micrometers). Explanation: minichromosome was derived from chromosome 7 by truncation of the long arm, as revealed bykaryotyping probes. It was recovered in an otherwise tetraploid plant and was named R2. :; as a result of large chromosomal deletion; In fact, gametophyte abortion was not observed, and this minichromosome was recovered in the progeny after crossing the tetraploid by a diploid plant, which reduced the ploidy level in the progeny to triploid. The triploids that contained this minichromosome were again crossed by diploid plants of the transformation recipient inbred line to reduce the ploidy level to diploid. Five aneuploid plants that contained thisminichromosome were rescued by embryo culture.These aneuploid plants contained one to four trisomes and were again crossed by normal diploid plants to produce diploid plants with the minichromosome. When examined in this diploid background, R2 was never found to pair with chromosome 7 or any other chromosome during meiosis in 30 meiotic prophase cells examined (C–E). The lack of pairing of R2 with its progenitor is probably because it is too small to synapse with the normal chromosome pair. This characteristic of small chromosomes has been reported previously. This fact illustrates that small chromosomes have minimal chance of recombination with the normal set and, thus, can be used as starting materials for plant engineered chromosomes. R2 is stable during both mitosis and meiosis, and homozygotes with a pair of R2 could be selected in the progeny of selfed plants.
- The pAHC25 plasmid has a strong maize ubiquitin promoter driving the bar selection marker gene and also has beta glucuronidase (GUS) gene expression cassette. All miniB chromosomes can transmit through meiosis.
- GUS EXPRESSION: FOR KNOWING ABOUT THE EFFICIENCY OF GENE DELIVERY SYSTEM Fig. 2. Targeted normal B and miniB chromosomes. (A–C) Mitotic chromosomes of 86-14 (A), 86B93 (B), and 86B23 (C). Transgenes are labeled red, the B chromosome-specific repeat that identifies the centromeric region of B chromosome is labeled green, and chromosomes are stained blue with DAPI. Arrowheads denote intact or truncated B chromosomes with transgenes. Inset in C shows the merged (Top), B-repeat (Middle), and transgene (Bottom) images of 86B23. (D and E) Minichromosome 86B23 at pachynema (D) and anaphase I (E) of meiosis. B chromosome-specific repeat is labeled red and knobs are labeled green. Arrowheads denote minichromosomes. The sister chromatids of the minichromosome separate at anaphase I (E). (F) Progeny of the minichromosome 86B23. Two minichromosomes were transmitted to the F1 by the mechanism of nondisjunction at the second pollen mitosis when a male parent carrying the minichromosome and full-length B chromosomes was used in an outcross. B chromosome-specific repeat is labeled green. Arrowheads denote the minichromosomes. Enlarged images of minichromosomes (A–E) are shown in Insets. (Scale bars, 10 m.)
- This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.
- Active centromeres are always associated with a replacement histone H3 that is specific to nucleosomes in active centromeres (Henikoff et al., 2001; Zhong et al., 2002). All inactive centromeres lack this histone variant (Han et al., 2006) and are no longer capable of conditioning an active kinetochore. The initial production of engineered minichromosomes in plants had two obstacles to overcome. First of all, plant centromeres exhibit an epigenetic component to their specification (Nasuda et al., 2005; Han et al., 2006) as do those from most other eukaryotes.
- In practical applications, they might be used to combine many useful traits on an independent chromosome that shows no linkage with the remainder of the genome and that will allow easy transfer of the transgene set into multiple lines. The potential exists to add whole biochemical pathways to plants to confer new properties to them, to use plants as factories to produce novel proteins or metabolites in large quantities inexpensively, and to transfer combined traits from one variety to another very quickly, among other possibilities.
- Pros: Long breeding programs are often required to introgress an integrated transgene into desired germplasm, while eliminating undesirable linked loci. Because an MMC forms an independent linkage group, these programs could be accelerated, allowing products to appear in the marketplace sooner. Engineered minichromosomes provide the ability to target transgenes to a defined insertion position for predictable expression on an independent chromosome. This technology promises to provide a means to add many genes to a synthetic chromosome in sequential manner. An additional advantage is that the multiple transgenes will not be inserted into the normal chromosomes and thus will not exhibit linkage drag when converging the transgenes to different germplasm nor will they be mutagenic. Cons: While this telomere-truncation approach can deliver both transgenes and sequences that promote site-directed integration, its utility for commercial applications may be limited—most commercial maize hybrids lack B chromosomes, and the duplications needed to maintain truncated A chromosomes may prove challenging for regulatory approval. It remains to be tested whether microinjection or microcell-mediated transfer will broaden the application of engineered chromosomes in cases where the transfer of plant minichromosomes via sexual crossing is not feasible.
- Moreover, the performance and expression of transgenic traits will likely become more predictable and reliable as MMC design rules are understood. Extensions of this minichromosome technology beyond traditional agriculture may enable the construction of multigene pathways to produce pharmaceuticals and other industrial products in plants. No epigenetic effects reported. MMC if optimized to commercial performance levels, it will provide an unprecedented opportunity to deliver gene combinations (‘‘stacks’’) that confer valuable traits to corn varieties. Because an MMC forms an independent linkage group, these programs could be accelerated, allowing products to appear in the marketplace sooner. Long breeding programs are often required to introgress an integrated transgene into desired germplasm, while eliminating undesirable linked loci.
- In summary, the future for engineered plant chromosomes as a fascinating new tool for basic research on chromosomes and biotechnology is promising.The Han et al. study shows that minichromosomes often do not behave in the same way as normal chromosomes: they do not always pair and frequently undergo premature sister chromatid separation. However, in their abnormal way, they are still meiotically stable, and equational segregation of sister chromatids in meiosis I can alleviate the problem of reduced pairing. The fact that minichromosomes often do not pair may not be such a bad thing after all. If chromosomes do not pair, they also do not recombine, so two similar but not identical minichromosomes might be placed in one cell with no danger that their contents will eventually shuffle. In summary, the future for engineered plant chromosomes as a fascinating new tool for basic research on chromosomes and biotechnology is promising.