Transcript GENETICS – BIO 300

LECTURE 18: TRANSPOSABLE ELEMENTS
chapter 13
exam 2 & grades
general ideas
discovery in maise
prokaryotes
eukaryotes
dynamic & plentiful
host regulation
The Biology Graduate Student Association Presents
A forum to present current and prospective research
projects in the field of Biology
Barrick Museum Auditorium
Saturday, November 18th
8:00 am to 2:30 pm
LECTURE 21 TRANSPOSABLE ELEMENTS
QUIZ #5
you need a piece of paper and a pen or pencil...
write your name and student number at the top...
give brief answers for the questions below...
Q1: Transposable elements were first
discovered in _________.
Q2: Name the 2 classes of eukaryotic
transposons and describe some of
their defining features.
TRANSPOSABLE ELEMENT QUESTIONS
why were they 1st discovered in corn but 1st isolated
from E. coli?
how do they confer antibiotic resistance in bacteria?
why the classification as DNA or RNA transposons?
what do autonomous and non-autonomous mean?
what are the implications for the human genome with
 50% of it derived from transposable elements
GENERAL IDEAS
Barbara McClintock
1920s  1980s (nobel in 1984)
mostly ignored for decades
 50% of human genome
2 types
class 1. retrotransposons
transcribe
class 2. DNA elements
excise
DISCOVERY IN MAISE
Barbara McClintock
1920s  1980s (Nobel in 1983)
“Indian” corn, 10 chromosome pairs
Dissociation (Ds) factor on # 9 commonly broken
Activator (Ac) unlinked factor required for breaks
could not map to constant place
lots of bizarre kernel phenotypes, not ~ parents
DISCOVERY IN MAISE
mosaicism through the activity of Ds movement
during mitosis, results in patchy tissue
chromosome breakage & loss
all linked genes affected
DISCOVERY IN MAISE
mosaicism through the activity of Ds movement
during mitosis, results in patchy tissue
unstable insertions & excission
single gene affected only, e.g. C gene
DISCOVERY IN MAISE
mosaicism through the activity of Ds movement
during mitosis, results in patchy tissue
size of mutant sector ~ time of event
DISCOVERY IN MAISE
autonomous & nonautonomous elements
C gene, allele called c-mutable(Ds) or c-m(Ds)
Ds stable without Ac
Ds excision is dependent on Ac
Ds is nonautonomous
C gene, allele called
c-mutable(Ac) or c-m(Ac)
Ac always unstable
Ac is autonomous
Ac can transform  Ds
Ds = Ac mutant
DISCOVERY IN MAISE
autonomous & nonautonomous elements
Ds & Ac are members of a transposable
element family
many other families discovered in maize
autonomous elements encode information
necessary for the transposition of themselves
and nonautonomous members of their family
PROKARYOTES
molecular biology of transposable elements first
characterized in bacteria
insertion sequence (IS) elements
transposable
can block gene & operon function (polar)
e.g., gal operon in E. coli
PROKARYOTES
are the gal mutants all the same?... NO
several different IS sequences
inserted in different places
all encode transposase enzyme
multiple copies,  can recombine
F factor
PROKARYOTES
2 types of bacterial transposons
1. composite: genes (e.g., drug resistance)
sandwiched between inverted IS sequences...
in this case, these are called inverted repeat
(IR) sequences
PROKARYOTES
2 types of bacterial transposons
2. simple: genes sandwiched between IR sequences
genes include transposase
IR sequences are short (<50 bp) & do not
encode transposase
PROKARYOTES
transposons can tranpose (jump) to & from
plasmids & chromosomes
implications for drug resistance
PROKARYOTES
basic mechanism of tranposition
transposase makes staggered cuts in host DNA
element inserts
host DNA repair fills
in gaps
in this example, it
generates 5-bp direct
repeats on either side
called target-site
duplications
PROKARYOTES
2 types (at least) of transposition mechanisms
1. replicative: copy remains in original site
2. conservative (nonreplicative): excision only
PROKARYOTES
2 types (at least) of transposition mechanisms
1. replicative: copy remains in original site
recombination event
EUKARYOTES
2 types of eukaryotic transposons
class 1. retrotransposons
transcription mechanism
class 2. DNA transposons
excision mechanism
EUKARYOTES
class 1. retrotransposons
resemble single stranded RNA retroviruses
copied into DNA using reverse transcriptase
inserts into host
transcribes new
viral genome &
proteins  new
viral particles
called provirus
when integrated
EUKARYOTES
class 1. retrotransposons
resemble single stranded RNA retroviruses
similar structure
& gene content
flanked by long
terminal repeat
sequences (LTRs)
100s of bp long
these are also called LTR-retrotransposons
EUKARYOTES
class 1. retrotransposons
gag: maturation of
RNA genome
pol: reverse
transcriptase
env: protein coat
(viral gene only)
EUKARYOTES
class 1. retrotransposons
Ty elements in yeast
copia-like elements in Drosophila
10-100 positions in genome
cause known mutations, e.g., wa
w+
wa w1118
EUKARYOTES
class 2. DNA transposons
mechanisms similar to those in bacteria
Drosophila P-elements 1st characterized
discovered
~ hybrid
dysgenesis
EUKARYOTES
class 2. DNA transposons
mechanisms similar to those in bacteria
Drosophila P-elements first characterized
discovered ~ hybrid dysgenesis
P-strains have 30 - 50 P-element copies / genome
2.9 kb wild type element, 31 bp inverted repeats
defective elements are smaller
tranposase gene has 3 introns + 4 exons
EUKARYOTES
class 2. DNA transposons
hybrid dysgenesis
mechanism in
Drosophila
EUKARYOTES
class 2. DNA transposons
action of Ac element
in maise
EUKARYOTES
DNA transposons, gene discovery & manipulation
controlled use of engineered P-elements
2 element system:
1. 2-3:
transposase source
disrupted terminal IR sequences
stable (immobilized)
2. bullet:
deleted transposase gene
inserted genes of interest (e.g. markers)
mobilized only in combination with #1
EUKARYOTES
DNA transposons, gene discovery & manipulation
controlled use of engineered P-elements
genes of interest
inserted in bullet
gene transfer... 
re-mobilization
EUKARYOTES
DNA transposons, gene discovery & manipulation
controlled use of engineered P-elements
insertional mutagenesis
provide transposase for 1 generation
cross away & screen for new mutants
use P-element sequence to probe for gene
= transposon tagging
enhancer trap mutagenesis
finds functional regulatory sequences
GAL4 system (binary, 2 bullets)
tool for gene manipulation
P-ELEMENTS & YEAST GAL4 SYSTEM


w–
w–
/
/
E
GAL4 w+
X
x
GFP w+
P-ELEMENTS & YEAST GAL4 SYSTEM
w–
/
w–
/
E
X
GAL4 w+
GFP w+
green
fluorescent
protein
P-ELEMENTS & YEAST GAL4 SYSTEM
MUSHROOM BODY
KENYAN CELL
CYTOPLASMIC
SIGNAL
NUCLEAR
SIGNAL
DYNAMIC & PLENTIFUL
DNA content of organism  C-value
lack of correlation with biological complexity 
C-value paradox
DNA repeat sequences make up large fraction of
eukaryotic genomes
genome size correlates with amount of DNA derived
from transposable elements
e.g., ~ half of the human genome is derived from
transposable elements
DYNAMIC & PLENTIFUL
human genome
long interspersed nuclear elements (LINES)
autonomous, retrotranspose, no LTRs
short interspersed nuclear elements (SINES)
nonautonomous, ~ lines w/o rev. transcriptase
Alu element ~ 10% of genome
DYNAMIC & PLENTIFUL
human genome
~ 20  as much DNA derive from transposable
elements as protein-encoding DNA
intron insertions remain only  spiced out
presumably initially also in exons  mutations &
negative selection
typical pattern in humans...
DYNAMIC & PLENTIFUL
human genome
class 1 transposons (LINES, SINES) cause some
hereditary diseases in humans, e.g.,
hemophilia A
neurofibromatosis
breast cancer
class 2 transposons (DNA)
low mutation rate (0.2 % or 1 in 500 known)
DYNAMIC & PLENTIFUL
plants (e.g. grasses)
synteny: similar gene content & organization
vastly different genome sizes due to transposons
safe havens: strategy of insertion in other
transposons, minimize negative effect on host
transposons
genes
DYNAMIC & PLENTIFUL
yeast
small genome, 70% exons
Ty LTR-retrotransposons
targeted insertions to benign sites
encoded integration enzyme
DYNAMIC & PLENTIFUL
Drosophila
telomeres are transposable elements!
HeT-A & TART non-LTR retrotransposons (LINES)
telomerase is a reverse transcriptase
RNA template for telomere DNA synthesis
HOST REGULATION
Ac activity reversible
lost of activity reappeared in later generations
epimutations: changes in chromatin structure
HOST REGULATION
transgene silencing
cosuppression: transformed gene & endogenous
homologous genes both silenced
unknown defense mechanism?
SPEND SOME TIME WITH...
key questions revisited (p.446-447)
summary (p.447)
terminology (p.447-8)
unsolved problems (p.449)...
2, 3, 5, 7, 11