About transposable genetic elements

1. DEPARTMENT OF GENETICS AND PLANT BREEDING
COURSE TITLE: PLANT BIOTECHNOLOGY
COURSE CODE: PLB 608
CREDIT HOUR: 3 (2+1)
PRESENTATION ON: TRANSPOSABLE GENETIC ENZYMES
PRESENTED BY: AJAY KUMAR YADAV
EXAM ROLL NO: PLB-01M-2022
CLASS ROLL NO: PLB-07M-2022
M. Sc. Ag. 1st Year 1st Semester
AGRICULTURE AND FORESTRY UNIVERSITY (AFU)
FACULTY OF AGRICULTURE
RAMPUR, CHITWAN

2. • Biotechnology is broadly defined as the science of using living organisms, or the
products of living organisms, for human benefit.
• Term “Biotechnology” was first coined by a Hungarian Agricultural Engineer
Karoly Ereky (“The father of biotechnology”) in 1919.
• Plant Biotechnology is a technique used for development of desired varieties of
plants by manipulating its genetics.
INTRODUCTION

3. Fields of plant Biotechnology:
• Plant Tissue culture
• Plant Genome sequencing
• Genetic structures and mechanisms
• Genetic engineering (Methods for transgenic biotechnology)
• Molecular markers/ Marker assisted selection and bioinformatics
• Gene Editing/Genome Editing
• Synthetic Biology
INTRODUCTION Cont…

4. OBJECTIVES:
• To know about biotechnology and plant biotechnology
• To know about transposable genetics
METHODOLOGY:
• This paper is solely based on literature review. It was prepared by Collecting
Secondary data from different Source such as journals, E- books, articles, thesis
and Related Websites.
INTRODUCTION Cont…

5. • Identified in Prokaryotes and all Eukaryotes:
(with exception of parasitic Plasmodium falciparum)
- Animals 3-45%, Fungi 2-20%, Plants 10-80%.
• More abundant in eukaryotic genomes than prokaryotes.
• Transposons make up the major content of eukaryotic genomes.
~50% of genomes of Human, Chimp, Mouse, Ape
~ 10% of several fish species
~ 12 % of C. elegans genome
~75% of Maize genome
~85% of Barley genome
~98% of Iris genome
WHAT ARE TRANSPOSONS?
Iris brevicaulis Iris fulva

6. • A discrete sequence in the genome that are mobile and able to insert itself at a new location in
the genome, without having any sequence relationship with the target locus.
• Term was given by Hedges and Jacob (1947)
• Other names viz., Jumping genes, Junk DNA, Selfish DNA, Molecular parasites, Mobile
genetics elements and Controlling elements.
• These elements comprise at least 45% of the human genome while coding sequences occupy
<3%.
• Transposons are segments of DNA that can move around to different positions in the genome
of a single cell.
• In the process, they may cause mutations and increase (or decrease) the amount of DNA in the
genome of the cell, and if the cell is the precursor of a gamete, in the genomes of any
descendants.
TRANSPOSON (TRANSPOSABLE ELEMENT):

7. • Cold Spring Harbor Laboratory, NY.
• Nobel Prize in Physiology and Medicine (1983)
• “Discovery of mobile genetic elements”
• Her discovery of jumping genes, through an analysis of genetic instability in
Maize.
• The instability involves chromosome breakage and was found to occur at sites
where transposable elements were located i.e. at C locus of 9th chromosome.
BARBARA McCLINTOCK (1902-1992) EXPERIMENT:

8. Genetic marker that McClintock followed is C’ i.e. a dominant inhibitor of aleurone coloration.

9. Unexpected corn kernel phenotypes in the offspring of her corn crosses:

10. There must be two elements to the process:
• An element that actually caused the mutation: The dissociator (Ds), located on
the short arm of chromosome 9 (where the breaks occurred).
• An additional element that controlled the activity of the first element: The
activator (Ac)
Dr. McCLINTOCK’S CONCLUSIONS

11. Transposition of Ds Element Disrupts Gene Controlling Kernel Color:

12. • c/c = White kernels and C/- = Purple kernels
• Kernal color alleles/traits are “unstable”.
• If reversion of c to C occurs in a cell, cell will produce purple pigment and a spot.
• Earlier in development reversion occurs, the larger the spot.
• McClintock concluded “c” allele results from a non-autonomous transposon
called “Ds” inserted into the “C” gene.
• (Ds = dissassociation)
• Autonomous transposon “Ac” controls “Ds” transposon.
• (Ac = activator).
• Ac element is autonomous while Ds element is non-autonomous.
McCLINTOCK’S DISCOVERY OF TRANSPOSONS IN CORN:

13. • Ac is 4,563 bp with 11 bp ITRs and 1 transcription unit encoding an 807 amino
acid Transposase.
• Ac activates Ds; Ds varies in length and sequence, but possesses same ITRs as Ac.
• Many Ds elements are deleted or rearranged version of Ac; Ds element derived
from Ac.
• Ac/Ds are developmentally regulated; Ac/Ds transpose only during chromosome
replication and do not leave copies behind.
McCLINTOCK’S DISCOVERY OF TRANSPOSONS IN CORN Cont… :

14. • Exist as multiple copies in the genome.
• Insertion site of element does not have extensive homology to the transposon.
• Termini are an inverted repeat (ITR).
• Encode “Transposase” that promote movement.
GENERAL CHARACTERISTICS OF TE:

15. • Footprints: when some elements move, leave behind duplicated target sequence
(footprint), which can still affect the gene (only partial restoration of gene
function).
• Two-element systems: mobility of one element depends on another.
FEATURES UNIQUE TO PLANT TRANSPOSONS:

16. Structure of Ac and its Transposase:

17. 1. Transposition: The process by which these sequences are copied and inserted
into a new site in the genome.
2. Segments of the genome that are capable of moving around to different
locations.
• Transposable elements usually are flanked by repeated sequences.
• Often carry “Transposase genes” that confer the transposition ability.
TRANSPOSITION OF TES:

18. 1. Non-replicative Transposition:
• Conservative transposition is also known as the ‘cut and paste’ mechanism and
involves the excision of the element from one site and its integration at another
site in the genome.
• Generally, in this mechanism there is no increase in the copy number of the
element.
2. Replicative Transposition:
• In replicative transposition, the element is duplicated, one copy remains at the
donor site while the new copy integrates at the target site.
• Retro-element transposition is replicative because the RNAs from the elements
are converted to cDNA for integration at the target site.
MECHANISM OF TRANSPOSITION: 2 Types

19. • Both these type of transposition differs only for the timing of donor cut reaction.
In both cases, the initiation step is a donor cut at 3' end of the element providing
free ends for interaction with the target site.
• In the conservative mechanism, the donor DNA is released by an additional cut at
the 5’ end of the element thereby creating a gap in the donor DNA.
• For replicative transposition, the 3' end of the donor is physically joined to target.
By repair replication, the duplication of element is achieved.
• This type of transposition is reported to occur in bacteriophage Mu, Tn5 and
retroviruses.
• If the 5’ donor cut is before the strand exchange, then the element is released
and the process is conservative and if it occurs after strand exchange, the
element is directly transferred from donor site to target site.
MECHANISM OF TRANSPOSITION: Replicative Transposition Cont.

20. BASIC CLASSIFICATION OF TE:
Source: - Lodish et al., Molecular Cell Biology, 7th ed.
“Copy-and-paste”
“Cut-and-paste”

21. CATEGORIES OF TES BY TRANSPOSITION:

22. • In prokaryotes, three types of transposable elements have been reported.
1. IS elements:
• IS elements are relatively small in size (~760 to 2500 bp).
• They can insert in the bacterial and viral chromosomes as well as in plasmids and
episomes. They can cause unstable but strongly polar mutations in E. coli.
• About 100 different IS elements have been identified in different bacteria and
their plasmids.
• They consist of a central region flanked by imperfect inverted terminal repeats of
varying size and sequence.
• The central region may contain 1-3 open reading frames encoding genes that are
essential for the transposition process, e.g. transposase.
TYPES OF TRANSPOSABLE ELEMENTS:

23. 2. Composite or compound transposons (Tn):
• The central region of these transposable elements contains some nonessential genes, e.g.
antibiotic resistance which is flanked by two IS elements.
• They are designated as Tn.
• The transposase may be provided by one or both IS elements.
• The IS elements may be in the same or opposite orientation, thus the composite transposon
appears to have long direct or long inverted repeats.
3. Complex transposons (Tn):
• Complex transposons are similar to IS elements but contain genes essential for transposition
along with nonessential genes.
• These genes are located in the central region which is flanked by short inverted terminal repeats.
• They are generally 5000bp in size and contain inverted terminal repeats of ~386 bp at both ends.
TYPES OF TRANSPOSABLE ELEMENTS Cont…

24. • In eukaryotes, the transposable elements are of two types:
1. Class I - which mobilize using RNA intermediate (termed as retrotransposons)
2. Cass II- which directly mobilize as DNA (termed as transposons)
Class I transposons or Retrotransposons have RNA intermediate. These are of two types:
(a) Retrovirus- like elements:
• The basic structure of retrovirus-like element has central region coding for two genes which is flanked
by long terminal repeats (LTRs).
• The LTRs are oriented in the same direction and bounded by short inverted repeats.
• The two genes encode for a structural protein of the virus capsule and a reverse transcriptase/ integrase
enzyme.
• The transposition process involves transcription of DNA sequence, reverse transcription of the RNA,
synthesis of double-stranded DNA from RNA and insertion at a new target site in the chromosome.
• Retrovirus carries a third gene coding for the protein of the virus envelope.
TYPES OF TRANSPOSABLE ELEMENTS Cont…

25. (b) Retroposons:
• These elements also move through RNA intermediate but do not contain direct or
inverted repeats at their termini.
• They possess a long stretch of A-T base pairs at one end of the DNA.
• In Drosophila, several retroposons are reported which are known to occur at the
end of chromosomes and participates in the replication of telomeres.
• LINE-1 retroposon is the transposable element reported in humans.
TYPES OF TRANSPOSABLE ELEMENTS Cont…

26. BASIC TE SUBCLASSES IN PLANT GENOMES:
“copy-and-paste” “cut-and-paste”
'rolling circle'

27. EXAMPLES: TRANSPOSON IN PLANTS

28. • They provide a means for genomic change and variation, particularly in response
to stress (McClintock’s "stress" hypothesis)-(1983, Nobel lecture, Science
226:792)
• The movement of a transposable element can generate mutations or
chromosomal rearrangements and thus affect the expression of other genes -
Federoff, N. 1984
• No known examples of an element playing a normal role in development.
BIOLOGICAL SIGNIFICANCE OF TRANSPOSONS:

29. 1. Gene Mutation
• Inactivation of genes by insertion.
• They cause mutation which is used in the production of different colour of grapes, corn
and other fruits.
2. Gene Structural Modification After Insertion
• A TE inserts itself into a functional gene, it will probably damage it.
• Insertion into exons, introns, and even into DNA flanking the genes can destroys or alter
the genes activity.
3.Gene Movement
4. Gene Creation- Pseudo gene Creation
• Fragments in a single TE or fusion of a TE with the regulatory and/or coding regions of a
host gene by transposases.
EFFECTS CAUSED BY TRANSPOSONS:

30. 5. Gene Regulation
• Because TEs routinely carry their own regulatory modules that determine the
different expression
• Patterns for TE genes, it is not surprising that TE insertion near a gene often alters
its regulation.
EFFECTS CAUSED BY TRANSPOSONS Cont…

31. • As cloning vehicles: Transformation vectors for transferring genes between
organisms. Also drug resistance genes encoded by many transposons are useful
in the development of plasmids as cloning vehicles.
• Transposons mutagenesis: Use of transposons to increase rate of mutation due
to insertional inactivation
USES OF TRANSPOSONS:

32. • In shaping the structure of chromosomes.
• In modulating the expression of genes.
• In converting RNA molecules to DNA.
• In changing positions of DNA sequence in genome.
• In carrying gene for enzyme that catalyzes transposition.
• In carrying antibiotic resistance genes.
ROLES OF TRANSPOSABLE ELEMENTS: They Have Roles:

33. • Transposable elements can be used as a genetic tool for the analysis of gene
expression and protein functioning.
• These are used in genetic engineering to insert or remove specific genetic
sequences, and also to cause frame shift mutation.
• TEs are also a widely used tool for mutagenesis of most experimentally tractable
organisms.
• These are used for the reconstruction of phylogenies by the means of
presence/absence analyses.
• They can act as biological mutagen in bacteria like Arabidopsis thaliana,
Escherichia coli.
APPLICATION OF TRANSPOSABLE ELEMENTS:

34. • In the process of inserting into the genome, transposons can
• Interrupt the normal coding of DNA,
• Creating gene mutations with a variety of effects.
• They may turn nearby genes off, preventing their ability to create protein, or they may turn
them on, increasing the amount of protein made.
• There is evidence that transposons aren’t just “Selfish genes” intent on
replicating themselves or genomic “Junk” that provides no benefit to the host.
They may play a creative role in building new functional parts of the genome.
• Recent research has shown that transposons may help plants respond and adapt
to environmental stress by regulating other genes.
• In bacteria, transposons often carry genes that impart resistance to antibiotic
substances, helping the bacteria survive.
SO ARE TRANSPOSONS GOOD OR BAD?

35. Transposable element may alter gene expression: Gene expression may be altered
by the presence of a transposable element.
• An insertion may obliterate the reading frame (phenotypic effects).
• A transposable element may contain regulatory elements (effects on
transcription of nearby genes).
• Transposable elements may contain splice sites (effects on RNA processing even if
the element is in an intron).
TRANSPOSONS ROLE IN PLANT GENETIC ENGINEERING:

36. Regulation of antokyan synthesis genes.
TE affected expression of TF VvmybA1:

37. Red orange: Ruby – myb TF (regulation of antokyan genes)
• Activated by TE insertion.
• Cold induced expression in fruits.
TE affected gene expression:

38. Maize: – inactivation of CCT (photoperiod response) by CACTA-like element (DNA
TE) insertion to promoter.
• Allowed cultivation in temporal climate (long-day flowering)
• Block of branching (TE enhancer -OE of inhibitor)
TE affected gene expression:

39. • Technique proposed by:- Walbot, 1992
• Loss-of-function mutations.
• Transposon tagging is a gene cloning strategy that relies on the transposon to provide a
DNA “tag” with a known sequence
• The transposon sequence is used to identify DNA sequences adjacent to the
transposable element.
• Tagging an interrupted gene with transposon followed by recognition of its inactivation
on the basis of a phenotype and cloned, using transposon sequence as a probe.
• Performed by Phenotypic screening, Verification of co-segregation and isolation of
gene.
• Transposable elements are useful tags only if the sequences of the elements are known.
TRANSPOSON TAGGING AND GENE ISOLATION:

40. SCHEMATIC OF A TRANSPOSON-TAGGED MUTATION:

41. TRANSPOSON TAGGING AND GENE CLONING:

42. • Tos17 is one kind of copia-like retrotransposons in rice (Hirochika et al. 1996),
which can duplicate and paste to elsewhere in the genome.
• Tos17, a rice retrotransposon has been used for large scale mutagenesis.
• Tos 17 is activated only under tissue culture conditions and is not active under
normal conditions.
• By the tissue culture of the rice callus, the transcription of tos17 is activated and
the reverse transcript DNA fragments are integrated into new places in the
genome which creates disruption of genes.
• There are only two copies of Tos 17 under normal conditions in Japanica rice and
5-30 transposed copies are found in plants regenerated from tissue culture.
Tos17 in rice genome:

43. Generation of tos17 insertion mutant lines by tissue culture:

44. TRANSPOSONS USED AS TAGS:

45. List of some genes isolated using transposon tagging in plants:

46. REFERENCES:
Nishihara M., Yamada E., Saito M., Fujita K., Takahashi H. and Nakatsuka T. (2014).
Molecular characterization of mutations in white-flowered torenia plants;
BMC Plant Biology, 14:86
BIOLOGY: John W. Kimball, Tufts University & Harvard University, 10.4
Islam, T. (2022). Transposable genetic element introduction. Noakhali Science &
Technology University.
MOLECULAR BIOLOGY OF THE GENE: James D. Watson, Tania A. Baker, Stephan P.
Bell and others, 5th edition, Pearson- Benjamin Cummings.