This document outlines a seminar on synthetic/artificial chromosomes. It begins by discussing the need for synthetic chromosomes due to limitations of traditional genetic engineering approaches. It then describes various methods that have been used to develop artificial chromosomes, including the bottom-up and top-down methods. Several case studies are presented where telomere-mediated truncation was used to produce engineered minichromosomes in plants with endogenous centromeres. The document concludes by discussing ways that engineered minichromosomes can be amended in planta, such as through site-specific recombination systems and zinc finger nucleases.

1. Department of Agril-biotechnology,
Orissa University of Agriculture & Technology,
BBSR.

2.  Why Synthetic/ Artificial Chromosome (AC)?
 Methods to develop AC
 Case study
 Amendments in AC
 Applications
 Summary Points
 Future Direction
 Conclusion
OUTLINES OF THE SEMINAROUTLINES OF THE SEMINAR

3. Need.....???
 Traditional genetic engineering
1) The transformation process
often results in the integration of foreign genes into an
endogenous gene and disrupts its function
2) Transgenes can be influenced by upstream or downstream
regulatory elements around them, that is, they are subject
to position effects under these circumstances
3) Only a single gene or a few genes can be expressed
4) Most importantly, Gene stacking or pyramiding
( Halpin, 2005)

4. First Generation Genetic Engineering
• Gene stacking- difficult
• Transgene position effects
• Insertion-site complexity
Second Generation Genetic Engineering
Synthetic Chromosome
 Delivery of large DNA sequences
 Complete metabolic pathways
 Genetic changes 100-kb to megabases (Mb)
Goyal et al., 2009
Need.....???

5. Artificial Chromosome:
It refers to any nonintegrating vector that is transmissible and has the
ability to harbor large amounts of DNA
 In bacteria – circular
 In yeast, animals & plants- linear
 First constructed in yeast and E. coli systems
(Clarke & Carbon, 1980)
 Requirements for chromosomal maintenance and stability
- Centromeres
- Telomeres
- Origins of replication
(Murray & Szostak, 1983)
Introduction

6. Requirement for Synthetic Plant Chromosome
 Basic requirements for Synthetic Plant Chromosome
1.Centromere
2. Telomere
3. Sufficient Chromatin
4. Selectable Marker transgene
Gaeta et al., Annu. Rev. Plant
Biol., 2012

7. Centromere Components In Plants
 Centromere – Requisite component of AC
 Contains tendem repeated sequences of various sizes, ~1 to 3 Mb
(Copenhaver, 2003)
 In grass family, there is small (156 bp) repeat sequences
(Jin et al., 2004)
 Study of mis-division of centromeres: The core functional B centromere is in the
range of 300 kb or larger in maize
(Jin et al., 2005)
 Retrotransposons
(Neumann et al., 2009)
 In maize, the tendem array size of centromere repeat C (CentC)- 700 kb to 2.7Mb
but the entire length is not necessarily associated with CENH3
(Wolfgruber et al., 2009)

8. Epigenetic Aspects Of Centromere Specification
 For construction of AC, Yeast as a model- old concept
 Centromere specification in yeast is unusual among
eukaryotes
 Centromeres in higher eukaryotes have diffuse organization
(Henicoff et al., 2001)
 Kinetochores form over apparently unique DNA, which is referred to as a
neocentromere
 Repeats are not a necessary determinant of the sites of kinetochore formation
(Malik &Henikoff, 2009)
 Mechanism by which centromeres are established, maintained, and function
remain a mystery
(Dalal, 2009)

9. Telomere Components In Plants
 Specialized structure which cap the ends of
eukaryotic chromosome
 Consist of highly conserved long array of short
tandemly repeated sequences
e.g. TTTAGGG in A. thaliana
TTAGGG in Homo sapiens
TTAGG in insects
 Average length: 3-40 kb on an average
(Burr et al., 1992)
 Functions: 1. Maintaining the structural integrity
2. Ensure complete replication of extreme ends of chromosome
Surovtseva et al., Mol. Cell, 2009

10. 1. Yeast Artificial Chromosome (YAC)
 YAC is plasmid into which yeast genes have
been inserted
 Developed by Murray & Szostak in 1983
 Capacity- 3000 kb
 YACs- Largest capacity vectors available
 Advantage: Can be used to express
eukaryotic proteins that require post
translational modification
 Disadvantages: 1. Less stable than BACs
2. Chimerism
Artificial Chromosome As Vectors
Murray and Szostak, Nature,
1983

11. 2. Bacterial Artificial Chromosome (BAC)
 Construction is based on F-plasmid
 Recombinants can be identified by Lac selection
 Capacity- 350 kb
 Uses: 1. Extensively used to sequence the genome of organisms
e.g.- HGP
2. Being utilized to a greater extent in modelling genetic diseases
e.g.- Alzheimer's disease, Cancer
3. Infectious viral clones
e.g.- herpesviruses, poxviruses
CONTD…CONTD…
Shizuya et al., Proc. Natl. Acad.
Sci., 1992

12. 3. Human Artificial Chromosome (HAC)
 HAC is synthetically produced vector DNA, possessing the characteristics
of human chromosome
 Developed by Harrington et al., in 1997
 Size- 1/10th
to 1/5th
of human chromosome- 6 to 10 Mb
 Mitotically stable for up to six months
 Advantage: 1. Can carry human genes that are too long
2. Can be used for gene therapy
3. Useful in expression studies as gene transfer vectors
CONTD…CONTD…
Harrington et al., Nat. Genet. 1997

14. Bottom-up Method
 Building chromosomes by de novo assembling their component parts
 This approach has been used for assembly of HAC
(Harrington et al., 1997)
 Upon delivery, DNA is subjected to rearrangements resulting in AC larger than
the original construct
(Lim & Farr, 2004)
 Not yet used in plant due to high complexity & limited understanding about plant
centromere organization
(Houben & Schubert, 2007)

15. Top-down Method
 Based on chromosome fragmentation or truncation
 Achieved by- 1. Irradiation
2. Telomere mediated truncation
Irradiation:
Example- An unstable maize minichromosome comprising part of the short arm of
chromosome 10 has been recovered as a result of pollen irradiation
(Brock and Pryor, 1996)
Disadvantage: Unstable

16. Telomere-mediated Truncation
 Aim- To whittle away the chromosome arms using transformation of telomere
repeats
 It bypasses the complications of the epigenetic aspects of centromere specification
 It works robustly in plants & can be used to produce engineered minichromosomes
with endogenous centromeres
 Construct- Genes of interest, Site-specific recombination cassettes, Telomere
repeats
(Yu et al., 2006)

17. B Chromosome Based Minichromosomes
We can produce A & B chromosome based minichromosomes by this method
but B chromosome based minichromosome is interesting-
Reasons:-
1 Naturally occurring supernumerary chromosome
2. Basically inert, small size – no phenotype
(Jones et al., 2003)
3. No developmental & transmission problem
4. Easy detection of B chromosome derivatives
(Kato et al., 2005)
5. No report of recombination with A chromosome set
 Minimal detrimental effect on host genome

18. Gaeta et al., Annu. Rev. Plant Biol., 2012

19. J. A. Birchler et al.,2010

20. J. A. Birchler et al.,2010

21. J. A. Birchler et al.,2010

22. J. A. Birchler et al.,2010

24. RESULTS...
Fig. 1. Chromosomal truncation constructs pWY76 and pWY86 and the control construct pWY96.
Tvsp- terminator from soybean vegetative storage protein gene; Bar- bialophos resistance gene as a selection marker
gene; P35S- 35S promoter from cauliflower mosaic virus; Tnos- Nos terminator from Agrobacterium; Tmas- Mas
terminator from Agrobacterium; Pnos- Nos promoter 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
Arabidopsis . Arrows designate the direction of transcription.
Development of Constructs

25. Construct Transgenic
Event
Truncation
locations
Pollen
Abortion
pWY86 (A) B77 3 S truncation +
pWY76 (B) T87 1 S truncation +
pWY86 (C) B37 4 L truncation +
pWY86 (D) B44 4 L truncation +
Recovered transgenic lines- 126
Transgenic events- 220 (T93, B83 & 44)
Transgenic Events
Pollen abortion: + = 50% pollen abortion

26. CONTD…
Fig. 2. Cytological detection of chromosomal truncations. Metaphase chromosomes were hybridized with pWY96 probe
(red) and mixtures of repetitive sequence 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 ransgenic event B77 with a chromosome 3 short-arm truncation. (Inset) The chromosome 3 pair
with (Left) and without (Right) the transgene (red). (B) pWY76 transgenic event T87 with a chromosome 1 short-arm
terminal-knob truncation. (Inset) Chromosome 1 homologues with (Upper) and without (Lower) the transgene (red). (C and
D) pWY86 transgenic events B37 and B44 with truncations 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.)
pWY86 B77 3S T pWY76 T87 1S T
pWY86 B37 4L T pWY86 B44 4L T

27. CONTD…
Fig. 4. Telomere sequences detected in internal chromosome locations.
Metaphase chromosomes of a pWY86 transgenic line were probed with a pWY96 probe (red) and a telomere
probe (green). Chromosomes were stained with DAPI. Arrowheads indicate the internal telomere (green) and
transgene (red) signals. Arrowheads in the enlarged images from the top to the bottom panels denote merged
transgene (red) and telomere (green) signals, and telomere only (green) and transgene only (red). (Scale bar,
10 m.)

28. AMENDING ENGINEERED MINICHROMOSOMES IN PLANTS
In planta Amendments
Zinc Finger NucleasesZinc Finger Nucleases
Durai et al., Nucleic Acids Res., 2005

29. Site-specific Recombination Systems
 Cre/loxP system
 Bacteriophage P1
 Cre (Cyclization recombination) recombinase
 loxP (locus of X-over of P1), 34-bp
 Two directly repeated loxP sites - deletion
 Two inverted loxP sites – inversion
Gilbertson, Trends Biotechnol., 2003
Fig-loxP Site

30. Inversion Deletion
CONTD…

31. Zinc Finger Nucleases (ZFNs)
 ZFNs are artificial RE generated by fusing a zinc finger DNA- binding domain to
a DNA-cleavage domain from FokI
Townsend et al., Nature,
2009
 It can be engineered to target desired
DNA sequences
 Non-specific cleavage domain: from
the type IIs RE FokI is used
 ZF DNA-binding domain: recognize
between 9 and 18 bp
 Uses: 1.Disabling an allele
2.Deletion of intervening sequences

32. (a) A diagrammatic engineered minichromosome generated by telomere-mediated chromosome truncation. The
minichromosome contains a terminal transgene locus (red ), which contains a promoter (Pro) driving expression of a
selection gene (S gene). The transgene cassette also contains a pair of directly oriented loxP sites flanking the selection
gene, as well as 3’ telomere repeat sequences (Telo). Crossing a line containing this minichromosome to one that
expresses Cre recombinase will lead to excision of the selection gene, and will leave a single wild-type loxP site. In a
subsequent transformation, a circular construct (donor molecule 1) containing a loxP site, an attP site (PP), a promoterless
S gene, a novel gene of interest (GOI-1), and two specific sites recognized by a zinc finger nuclease (ZF1) can be
introduced into the minichromosome-containing cell along with a plasmid expressing Cre recombinase.
Mechanisms To Amend Minichromosome Platforms

33. CONTD…
(b) The first novel gene has been added to the minichromosome transgene locus as a result of the previous Cre
recombination event. A specific zinc finger (targeting the ZF1 sites) could be used to delete the intervening sequences. In
a second round of gene stacking, a second donor plasmid (donor molecule 2) could be introduced into cells containing
the minichromosome along with an integrase enzyme that specifically leads to recombination between attP and attB sites.
In this round of integration, the donor molecule contains a promoterless selection gene, a second gene of interest (GOI-
2), novel attachment sites attB and attb (BB and bb, respectively), and a zinc finger nuclease recognition sequence.
Gaeta et al., Annu. Rev. Plant Biol., 2012

34. (c) The result of the previous integration event involves introduction of the second gene of interest. The result of
recombination between attachment sites BB and PP results in the site PB, which is not acted upon by the
integrase.
Similarly to panel b, a zinc finger nuclease could again be used to delete extraneous sequences. A third donor cassette
could be used and recombination could be targeted to the bb site (not shown). In each recombination event involving the
integrase, the target sites are destroyed, and thus attB and attP could be alternately used for sequential addition to the
minichromosome.
CONTD…
 At any point, we can delete all sequences between the two loxP sites that flank
the entire cassette by crossing this line with another line that expresses Cre
recombinase

35.  The artificial chromosome construction system can be used as powerful
mutagen
(Brown et al.,
2000)
 It can also be used for gene stacking in plants, which is currently considered
as challenging for plant biotechnology
(Halpin., 2005)
 Functional genomic studies could use minichromosomes as a platform for
adding specific genes or doses of chromosomal segments
 Can be used in sequencing projects
POTENTIAL APPLICATIONS

36.  AC platforms can provide solution to the stable expression & maintenance of
multiple transgenes in one genome
 It represents a potential powerful research tool for understanding chromosome
structure & function
 They can be useful for mass production of foreign proteins, pharmaceuticals, or
useful metabolites in plants
(Daniell et al., 2009)
 The inheritance of multiple foreign genes as a unit
CONTD…

37.  Next generation vectors for human gene therapy & plant genetic engineering
(Yu et al., 2007)
 Can be used in site-specific recombination or retrofitting the minichromosomes
with additional foreign genes
(Ow., 2007)
 AC could be easily introduced or removed from a genotype by genetic crosses
and would facilitate introgression of transgenes to different genetic backgrounds
 It is possible that an important application of AC in plants will be combine them
with haploid breeding
(Ravi et al., 2010)
CONTD…

38. Summary Points…
 Synthetic chromosomes in plants are likely to have more applications than in
other taxa because of the ease with which they can be manipulated throughout
their life cycle
 Telomere-mediated chromosomal truncation works robustly in plants
 Plant centromeres have an epigenetic component to their specification, which
constrains the approaches to producing engineered minichromosomes
 Site-specific recombination systems and zinc finger nucleases provide means
to amend and grow synthetic plant chromosomes

39.  It might be possible to develop a mini B chromosome-based genomic cloning
system for capturing large chromosome fragments
 Site-specific recombination systems & zinc finger nucleases are valuable tools
for marker gene removal & gene targeting. Such technologies could be applied
to AC
 The mechanism involved in de novo telomere formation (DNTF) & capping is
entirely unknown & are worthy for future study
Future Direction…Future Direction…