LECTURE 18: TRANSPOSABLE ELEMENTS
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Transcript of LECTURE 18: TRANSPOSABLE ELEMENTS
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 AuditoriumSaturday, November 18th
8:00 am to 2:30 pm
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.
LECTURE 21 TRANSPOSABLE ELEMENTSQUIZ #5
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
EUKARYOTESclass 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
EUKARYOTESclass 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
EUKARYOTESclass 1. retrotransposons
gag: maturation of RNA genome
pol: reverse transcriptase
env: protein coat (viral gene only)
EUKARYOTESclass 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
EUKARYOTESclass 2. DNA transposons
mechanisms similar to those in bacteria Drosophila P-elements 1st characterized discovered
~ hybrid dysgenesis
EUKARYOTESclass 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
EUKARYOTESclass 2. DNA transposons
hybrid dysgenesis mechanism in Drosophila
EUKARYOTESclass 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
x
P-ELEMENTS & YEAST GAL4 SYSTEM
w– E X/
w– /
GAL4 w+
GFP w+
/ GAL4 w+ w– E X
/ w– GFP w+
greenfluorescentprotein
P-ELEMENTS & YEAST GAL4 SYSTEM
CYTOPLASMICSIGNAL
NUCLEARSIGNAL
P-ELEMENTS & YEAST GAL4 SYSTEM
MUSHROOM BODYKENYAN CELL
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
transposonsgenes
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