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