PowerPoint Presentation General Properties of Transposable Elements

1
(No Transcript)
2
DNA and Retrotransposable elements
Retrotransposable element
DNA transposable element
3
General Properties of Transposable Elements

• Move - from one position in the genome to another
  position in the genome
• same chromosome
• different chromosome
• Insertion - can disrupt gene (cause mutation)
• Excision (removal of transposable element)
• perfect restores activity to gene
• imperfect stable (permanent) mutation
• Genes trapped within a transposable element move
  around the genome with the element -
  hitch-hiker or passenger gene
• - this type of unit or entity is called a
  transposon

Distribution most organisms - phage, bacteria,
fungi, plants, animals
4
Biological implications and applications

• Drug resistant strains
• medical epidemiology
• biological weapons
• Movement and insertion
• cause mutations
• cause chromosomal rearrangements
• Mechanism to promote speciation?
• Practical applications
• clone genes gene tagging
• gene transfer transformation or gene therapy

5
Discovery - a little history

• Discovered
• Corn - Barbara McClintock Marcus Rhoades (late
  1940s/early 1950s)
• Drosophila - Yuichiro Hiraizumi, and Mel Green
  (mid 1950s)
• noticed high spontaneous mutation rate in certain
  strains, which they called mutator strains
• (high mutation rate 10-3 to 10-4 vs.
  normal 10-6 to 10-7)
• the mutations that occurred were often unstable
• Initially dismissed by many as an oddity of corn
  or Drosophila
• Discovered in bacteria in 1970s

6
Bacteria Insertion Sequences (IS elements)
1st found as unstable mutations in the
galactose operon
i
o Gal 4
K T
E o
Positive regulator
repressor
kinase transferase epimerase
galactose gal-1-PO4 UDP-gal
UDP-glucose

• Mutation in T gene (transferase)
• Polar - also causes loss of K function
• Unstable - reverts from gal- to gal
• rate of reversion is not increased by treatment
  with a mutagen
• \ not a missense or frameshift
  mutation
• Hypothesis mutation is due to the insertion of a
  mobile genetic element

7
Cloning bacterial IS element lgal vs. lgalIS
8
General Structure of IS Transposable Elements

• Size 768 to 5700 bp long
• Inverted repeats at the termini
• same sequence opposite orientation
• 5 to 41 bp long
• example

• 3. Complete elements encode transposase
  protein(s)
• product essential for mobility of the element
• 4. Deleted or partial elements
• lack transposase function
• can respond to transposase protein if its
  provided from another source

9

• Deleted or partial TEs often lack transposase
  function but can respond (move) if
• Both ends of the element are intact, and
• functioning transposase protein is provided

transposase gene
IR

• Deleted element
• 1. not mobile on its own
• 2. mobile in presence of a functioning
  transposase

mRNA
transposase protein
(trans-acting factor)
10
Bacteria - Insertion Sequences
Insertion elements length
inverted repeat Sequence E. coli (bp)
(bp) length (bp)
location blue genophore red plasmid
11
Summary

• Insertion elements are able to move from one
  position to another in the genome
• Multiple copies per genome (implies increase in
  )
• Complete or intact elements - encode a protein,
  or proteins, that are necessary for movement
  transposase
• Defective elements - can respond to transposase,
  provided from elsewhere, if the ends of the TE
  are intact

12
Insertion of a DNA Transposable Element
Observation the genomic sequence to the Left
Right of the inserted                       TE
is identical (tandem duplication of the
sequence) Implies the TE target site is
tandemly duplicated as consequence of
insertion Hypothesis Transposable Elements
insert by making a staggered cut
13
Insertion of a DNA Transposable Element
14
Model of transposition
15
Transposition process Co-integrant Resolution
X
Reciprocal exchange (cross-over) within TE
16
Genetic characteristics of TEs

• Insertion of TE may cause mutation
• 1. insertion into a non-coding region no
  obvious alteration in function
• 2. insertion into coding region mutation -
  disrupts coding info. product

• Excision of TE
• 1. precise precise removal of TE restore
  original DNA seq. function
• 2. imprecise lead to deletion of material or a
  permanent (stable) mutation
•                        (the remnant TE is
  immobile)

Transposons TEs can carry different genes -
size variable 500bp to gt20,000bp (limits?) -
can capture pieces of DNA between terminal
repeats - when
element moves, hitch-hiking gene moves TE
moves as a unit transposon
17
Examples of bacterial transposons
(Standard transposon)
18
Resistance Transfer Factor - an example
19
TEs in multi-cellular organisms

• Virtually all multicellular organisms have TEs
• - e.g., corn, snapdragons, C. elegans,
  Drosophila, mouse, humans, etc.
• Most organisms have many different kinds of TEs
• - e.g., Drosophila has more than 30 different
  kinds of TEs
• Examine one class of element as an example
• - P transposable element in Drosophila
• Examine it historically
• - the way it was discovered and characterized
• biology
• genetics
• technological applications

20
P transposable element and Hybrid Dysgenesis
Hybrid Dysgenesis - ill or bad
generation (male) wild population (nature) x
lab. strain (female) F1 offspring
(morphologically normal but panoply of aberrant
traits - reduced fertility (sometimes
completely sterile) - chromosome
rearrangements in germ-line - high spontaneous
mutation rate - germ-line - recombination in
males (absence of recomb. in males is an oddity
in D. m.)
Note Hybrid Dysgenesis effects observed in
gonadal (germ-line) tissue
21
Reciprocal Crosses between lab wild populations
laboratory strain wild population wild
population laboratory strain
x
x
(M strain)
(P strain)
(M strain)
(P strain)
F1 morphologically normal, but
physiologically defective hybrids (dysgenic
progeny)
F1 normal flies
F2 normal flies
no offspring
22
Crosses - - - - - - genotypes and cytotypes
P males M females
(lab. strain) M males P females (lab.
strain) P males P females M males
M females (lab. strain) (lab. strain)
23
High mutation rate
F1 dygenic male
female
5 genes y w ct ras
sn Y all alleles y w ct
ras sn
Test cross
expect all female offspring
observe very high freq. of sn, ras, and y
mutation 1/1,000 high frequency of w and ct
1/10,000
24
Mutations induced by hybrid dysgenesis are
unstable
phenotypes
snu snstable sn
wild-type revertant (sn/sn- genotype)
snu snstable sn
snstable
sn
snu snstable sn
snstable
sn
snstable
sn
snstable
snstable
sn
sn
snstable
sn
snu snstable sn
snu unstable sn mutation TE inserted into sn
locus sn sn precise excision of TE from sn
locus snstable stable sn mutation imprecise
excision of TE
25
P element cloned early in 1980s

• white eye mutation (wP insert) recovered from
  hybrid dysgenic cross
• white gene had been cloned previously
• construct a genomic library from wP insert mutant
  strain
• use the previously cloned white gene as a probe
  to identify bacteria that contain the wP insert
  mutant cloned DNA

• plate library out - colonies on plate
• blot onto filter paper
• lyse fix DNA to paper
• probe - hybridize with 32P labeled, cloned w
  gene
• expose to x-ray film (position of grains identify
  the colony with the wP insert)

26
P transposable element
Size - 2.9 kb (2907bp) Inverted repeats at
termini 31bp long
transposase gene - 4 exons
IR
IR
4 open reading frames (exons) ORF bp
length AA 1 354 118 2
714 238 3 792 264 4
654 218 Complete element encodes transposase
protein - mobilizes either an intact of defective
P element Consensus target sequence for
insertion G61G44C55C83A72G50A72C55
27
P x M crosses revisted
P strains - have P elements P cytotype
(repressor inhibits mobility)
M strains - lack both P elements and the
repressor that stops mobilization
P elements move induce new mutations, which
cause hybrid dysgenesis phenotypes
28
Why are P elements found in the wild populations
but not in laboratory strains?

• recent invasion of wild-populations
• stochastic loss from laboratory populations
• assembled de novo

29
Occurrence of P elements ()
Decade collected
P contains P element(s) M no P elements
detected

• invaded natural populations recently
• - then, it appears to have spread through
  populations

• Can P elements spread through a population?
• Where did P elements come from?
• What influence do P elements have on natural
  populations?

30
Can P transposable elements spread throughout a
population?

• at start, 0.5 of genomes have P elements 99.5
  have none
• allow random mating sample population at each
  generation

Result within 8 generations virtually all
individuals contain one or more P elements \ P
elements appear to spread rapidly within a
population
31
Where did P elements (in D.m.) come from?

• P elements did not exist prior to the radiation
  of the Drosophila species groups
• - Willistoni Species Group is the probable
  source of the D.m. P element

Horizontal Transfer of Genetic information - P
element moved from Willistoni Group to D. m.
32
What influence do P elements have on natural
populations?
Observation - hybrid dysgenesis can result in
sterility, or at least                       reduc
ed fertility, among hybrids Possibility -
transposable elements present in one population
but                   absent in another may lead
to, or promote,                   reproductive
isolation between the two populations Reproductive
isolation promotes speciation
Therefore, TEs may help promote speciation (note
the strength, or accuracy, of this conclusion
rests on the validity of statement 2)
33
Applications of P elements

• Transformation of Drosophila - insert cloned
  constructs into the somatic and germ-line tissues
  of the fly
• Cloning specific loci - tag specific gene by
  the insertion of a P element. Since the P
  element is cloned, use the cloned P element to
  clone the P-tagged gene.

34
Transformation of Drosophila - 1st multicellular
organism to be transformed

• P elements insert into the genome
• splice your gene of interest into the P element -
  replace transposase gene
• when P element inserts, so does the
  hitch-hiking gene

transposase gene construct
termini removed wings clipped provides
transposase but itself cannot insert
syncytial embryo genotype ry-/ry-
wild-type eyes genotype ry-/ry- ry
Result 1-20 of the flies are transformed (ry)
35
Transformation of D. m. - book fig. part 1
36
Transformation of D. m. - book fig. part 2
37
Use TEs to clone a gene - gene tagging
1. Create H.D. individuals - F1 between
defective P element strain P helper strain
P-defective strain P helper strain (cannot
move by itself) (immobile source of
transposase IRs-missing)
2. Cross H.D. F1 males to females homozygous for
recessive mutant allele of your gene of interest
F1 hybrid homo. rec. for your gene
                  of interest (ygoi/ygoi)

• Recover mutant flies with the following genotype
  P-tagged allele
• ygoi

Expect mostly / ygoi Score for mutant
flies ygoiP-tagged/ygoi
4. Clone the P-tagged allele by making a
genomic library of the mutant        strain
probing for colonies that contain the P-tagged
construct
38
The basic properties of Ac/Ds in Corn and P
element in Drosophila are similar
Ds incomplete element cannot move on its own
Ds incomplete element can respond to
transposase provided in trans
Ac intact element        therefore mobile
39
Summary

• Insertion elements are able to move from one
  position to another in the genome
• Multiple copies per genome (implies increase in
  )
• Complete or intact elements - encode a protein,
  or proteins, that are necessary for movement
  transposase
• Defective elements - can respond to transposase,
  provided from elsewhere, if the ends of the TE
  are intact
• Insertion can cause mutations and promote
  chromosomal rearrangements
• Transposable elements have several practical
  applications
• Used as a mutagen and to clone genes
• Used to insert a cloned gene (from any source)
  into the organism in which the TE is normally
  resident

40
Retroviruses and Retrotransposable elements

• Transposable elements are contained and function
   within the cell
• replicate and move within the cell
• Retroviruses viruses not transposable elements
• infect host cells
• replicate within cell
• produce progeny virions which escape the cell to
  infect   neighboring cells

Lets compare retroviruses and retrotransposable
elements
41
Retrovirus life cycle

• single stranded RNA viruses
• replicate by forming a dsDNA intermediate

42
General Structure of Retrovirus
LTR
LTR
mRNA

• Size 7 to 10 kb long
• LTR Long Terminal Repeat (identical sequences
  at termini)
• direct repeat. length of LTR 250 - 1400 bp
  (viral specific)
• gag gene 2000 bp long, encodes proteins
  assoc. with RNA in virus core
• pol gene 2900 bp long, encodes reverse
  transcriptase
• env gene 1800 bp long, encodes envelope
  proteins
• Transcription - initiated from the promoter in
  left LTR

43
Retroviruses can disrupt host gene function

• by inserting into a gene - disrupt gene function
• by inserting next to and activating gene
  expression from viral promoter

if the host gene promotes cell division, when
virus invades new cell it will stimulate cell
division - viral oncogene
44
Retrotransposable elements

• Replicate and move via an RNA intermediate
• Example Ty elements in yeast
• Ty1element is 5600 bp long
• Ty 1 terminal repeats (called d elements) are
  340 bp long direct repeats
• Ty 1 insertion causes 5 bp tandem duplication of
  target site
• Ty elements move at a low rate, 1 move in 20
  generations
• 35 copies of Ty elements in the yeast genome

45
Examples of transposable elements in the human
genome
46
Transposable elements are found in all biological
systems. Q. Where did transposable elements
come from?
47
virus
transposable elements
(e) Nomad transposable element in Drosophila