Prof. Neil Jonesrnj@aber.ac.uk

IBERS - Institute of Biological, Environmental and Rural Sciences

Barbara McClintock’s controlling elements: the full story

A major event in the history of genetics was the discovery in 1947 that genes could transpose.

Published 1951: Cold Spring Harbor Symp Quant Biol 16: 13-47 The story was not believed for 20 years, until restriction enzymes (1970s), made it possible to clone IS elements from bacteria.

McClintock was isolated from the Genetics Community for more than 20 years after publishing her work.

She was award the Nobel Prize in 1983

1902-1992

The story is timeless

Structure of the maize kernel

Embryo 2n

aleurone layer (3n)- develops pigment

endosperm (3n)

Pericarp is maternal tissue

endosperm /aleurone products of fertilisation

Pigment characters scored without growing seeds

pericarp

Chromosomes of maize

Photo: Bill Sheridan, University of North Dakota

Pachytene stage of meiosis

ABCCBA

ABC

ABC

AB

C

9S AB

C

pachytene

ABC

AB

CC

BA

A

AB

B

C

C

Chiasma

metaphase I tetrad

microspore

(n)

x

x

1 2 3 4

1 unbalanced 2 > 90% balanced

3,4 non-male transmission deficient (due to genetic constitution)

replication / fusion of sticky ends

Male meiosis (x=sticky end)

invertedduplication

How to make a sticky end without losing anything ?

xx

xx

pollen grain mitosis pollen introduced into crosses HOW BEHAVE ??

xx

tubenucleus

Sticky ends – gametophyte- in plants gametes are produced by mitosis

microspore

(n)

Chromatid breakage-fusion-bridge cycle [BFB]

x

HEALING of broken end almost immediately after fertilisation

Endosperm - BFB

bridge (B)

x

x

breakage (B)

replicationand fusion (F)

sperm

embryo sac

spermx

xEndosperm- BFB

EMBRYO

..based on cytological observations and on the use of markers

Sticky ends from male parent only

C

c

Wx

wx

X

(amylose, blue)

(amylopectin, red)

xx

Wx

Wx

Wx

Wx

C Wx c wx c Wx

Reading phenotypes: size of spots = stage of loss “spots within spots” (wx / Wx by iodine stain)

♂

♀♀

Replication followed by B-F-B and off-centre breaks

9S

Wx

CC

CCC

C

Wx

triploid endosperm

Endosperm variegation due to the chromatid BFB

(purple kernels)

(colourless)

Chromosome breakage-fusion-bridge cycle [BFB]

x x1

2

3

4 x xx x

Zygote no healing

fusion at telophase – before replication (prevents chromatid BFB)

5

prophase

6

7oreither

anaphase

both parents generating sticky ends

Chromosome BFB cycle continues during early development.Healing >> later in development. Tried use chromosome BFBto induce small internal deficiencies in 9S to study mutations.Endosperm 3 chrs >>independent chromatid BFB cyclesWHAT DOES THIS HAVE TO DO WITH TRANSPOSITION?

‘Earthquake ear’ of 1944

C

CI Wx

wx

♂♀9S

Wd

wdxx

This cross was made to induce internal deficiencies in 9S. The chromosome BFB was taking place in this F1 early in development, and this triggered a ‘genetic earthquake’.

earthquake ear of 1944

670 KERNELS 590 germinated; 134 died as seedlings;456 transferred to the field; 73 died;383 PLANTS - SELFED OR FIXED IN 1945

ONE of the selfed plants, which must have beenheterozygous CI//C, gave a few variegated kernels, UNEXPECTED NO STICKY ENDS INTRODUCED

A new type of aleurone variegation appeared.It had a uniform pattern of coloured spots (C)of similar size. It seemed that CI was beingeliminated in some cells at a particular rate,and at a particular stage in development.

CONTROLLED BREAKAGEshe sensed that these were something special – BREAKAGE WAS TAKING PLACE Seeds grown and studied - additional markers

CI = colour inhibitor

(Pale yellow = colourless)

Cytological disturbances

150 plants grown from earthquake ear - fixed for pachytene analysis:

Deficiencies in chromosome 9 Duplications of 9S Telocentrics Isochromosomes Breaks in chromosomes other than 9 Inversions Knob fusions

Plus:32 newly arising stable mutants, due to small deficiencies, and several unstable mutants affecting sectors of the plant phenotype – controlled, and taking place at different Times in development

Genetic ‘earthquake’

‘Earthquake ear’ of 1944

C

CI Wx

wx

♂♀9S

Wd

wdxx

This cross was made to induce internal deficiencies in 9S. The chromosome BFB was taking place in this F1 early in development, and this triggered a ‘genetic earthquake’.

earthquake ear of 1944

670 KERNELS 590 germinated; 134 died as seedlings;456 transferred to the field; 73 died;383 PLANTS - SELFED OR FIXED IN 1945

ONE of the selfed plants, which must have beenheterozygous CI//C, gave a few variegated kernels, - UNEXPECTED NO STICKY ENDS INTRODUCED

New type of aleurone variegation appeared.With a uniform pattern of coloured spots (C)of similar size. It seemed that CI was beingeliminated in some cells at a particular rate,and at a particular stage in development.

CONTROLLED BREAKAGEShe sensed that these were something special BREAKAGE WAS TAKING PLACE Grown and studied – using additional markers

CI = colour inhibitor

(Pale yellow = colourless)

Discovery of Ds – dissociation locus

CI Bz (Sh Wx)C bz (sh wx)

♂xx

C

CI

wx

Wx

bz

Bz

sh

ShDs

♀♀

Markers:CI colour inhibitorC coloured aleuronec colourless aleurone

CI > C > c (dominance)

Sh normal endosperm sh shrunken endospermBz purple aleuronebz bronze aleuroneWx amylose, blue starchwx amylopectin, red starch

Breakage without sticky ends – but where was it taking place ?

Sectoring not uniform: controlled breakage and loss of all four dominant markers at the same time ….also plant markers ..

Pachytene breaks in 9S in some plants, in one of the homologues and acentric fragments seen.Break always at the same site - junction of the euchromatin and heterochromatin – Ds locus.What was Ds? How were breaks controlled in relation to development – some breaks late in development.

no markers, no BFB

C ds

C ds

CI Ds

CI Ds

Discovery of Ac

x♀

C ds

CI Ds

♂ The first clue about control of DSKernels found without variegationin plants expected with Ds breaks.All progeny were expected to beheterozygous with variegated kernels due to loss of the CI allele:only half variegated – a 1:1 ratio.One of the parents must have beenheterozygous for another factor.She called it Activator or Ac.

Breeding tests, using appropriate Ds stocks, confirmed that Ac was inherited independently of Ds and acted as a dominant allele in crosses.

Ac

ac

Inheritance of Ac CI Ds

CI Dsx

ac

ac

ac

Ac

ac

Ac

ac

ac

Ds

CI

Ds

CI

C

C

ds

ds

C ds1/2

1/2

ds C

Breeding tests, using appropriate Ds stocks, confirmed that Ac was inherited independently of Ds and acted as a dominant allele in crosses:

Ac//ac x Ac//ac 1Ac Ac:2Ac ac:1ac ac Ac//ac x ac//ac 1Ac ac:1ac ac

Ac//Ac x ac//ac all Ac ac

Dosage effect of Ac CI

C

Sh

ds

Ds Wx

wx sh

Bz

bz

+ ac ac ac

+ Ac ac ac

+ Ac Ac ac

+ Ac Ac Ac

0

1

2

3

Evidence for the controlling effect of Ac came from varying the dosage of Ac in the triploid endosperm: 0, 1, 2, or 3 doses.The dosage of Ac controlled when Ds breakage would take place, but how did frequency of breaks alter in development.

♂

♀ ♀

CI Sh Bz Wx Ds

CI Sh Bz Wx Ds

C sh bz wx

C sh bz wx

xAc

Ac

Change of state of Ac

CI Sh Bz Wx Ds

C sh bz wx

Ac

several plants

The effect of Ac varied in different plants, different ears of one plant, and different parts of a single kernel.

The formation of sectorial kernels, due altered times of breakage, indicated changed forms of Ac – mimickedthe Ac dosage effect.

Further breeding tests showed thatthe altered kernels were due to change in the state of Ac, and also a change in the number of Ac elements.Ac controlled the time of breakage of Ds and Ac could change its state

Identical Ac allelesfrom this cross

Sectorial kernel

- Ac

+ Ac

C Sh wx Ds

c sh wx ds

½

½

Ac

Transposition of Ds in 1947

♂

ac♀♀ (12)

While trying to map Ds in its standard location an unexpected event took place at the C locus.It changed to a new mutable form cm-1.Male parent was Ac/ac Female parent had no Ac or Ds elements.Half kernels expected purple – no AcOther half variegated with colourless - with Ac.Found in all 12 EARS, but 1/4000 was different.

Colour pattern was reversed. Tests indicated:● cm-1 had reverted to C in this kernel. ● Reversion in chromosome with Ac in male parent in F1.● Ds breaks present in chromosome with new cm-1 locus● Location of Ds had also moved – inseparable from cm-1

F1

Mutation of the C locus – the interpretation(intellectual leap)

“footprint” mutant cm-1 locus

Ds

Ds C locus

DsAc

Ac C

(a)

(b)

(c)

purple

colourless

The position of Ds had alsomoved and was inseparablefrom the new cm-1 locus.

The site of chromosome breaks had moved (transposed).

Purple spots would only appearif Ac was also present …and …. dosage effect of AcEvidence consistent with cm-1

arising from transposition of Dsand its insertion into C – moving out from cell clones .Not explained by Ds breakage

Ds now had new function > mutation.

Other mutable loci later ..Transposition was discovered!

Ds Transposition

C

CI

wx

Wx

bz

Bz

sh

ShDs

Ac

Ac

Exceptions:

C Sh Bz Wx

Sub-sectors: C sh Bz Wx C sh bz Wx C sh bz wx

C sh bz Wx

Sub-sectors: C sh bz wx

twin sectorsWx x

x

WxBz

ShSh

Bz

Wx

Wx

Bz

Bz

Sh

Sh

CI

CI

CI

C

Sh Bz Wx

sh bz wx

Reveal new position of Ds

♂

♀♀

C Sh Bz Wx

Ds

Transposition of Ds

With an adequate means of detection it is possible to show that Ds can transpose to numerous other sites within the chromosome complement

(i) insertion into new loci (new mutations)(ii) kernels with new patterns of variegation(iii) in the absence of Ac the Ds locus is stable, and can be mapped by recombination analysis

Ds could also change its state – NOT SHOWN

ac

CI

Transposition of Ac in early studies Ac was not linked with 9S markers:

Ac

wx Bz Sh C Ds ac

Ds Wx Bz Sh CI

in later crosses linkage was sometimes found:

Sh Bz Wx Ds Ac

wx ac Sh Ds C

20%

Position varied in different crosses (ears):

bz

C sh bz wx

wx sh C

x

CI Sh Bz Wx Ds

Ac Ac Ac

x bz wx sh C

C sh bz wx

♀ ♂

♀ ♂

Transposition of Ac explains some unexpected events

Ac//Ac x ac//ac

Expected: Ac//ac (usually found)

Unexpected:

ac//ac(loss)

Ac//Ac

Ac//Ac

(unlinked)

(linked)

ac ac

Ac Ac

Ac

Ac

Ac

Ac

Ac

Ac Ac Ac

The Ac – Ds family of ‘controlling elements’

Ds

Ds

Ac

Ac

Ac

Ac

Ac

Ac

Ds

Ds

Ac activates breakageat Ds. Loci may be ondifferent chromosomes.

Ac can promote its own transposition, or that of Ds, to another site either on the same chromosome or on a different one.

Ds cannot move unless Ac is present in the same cell.

Ac is AUTONOMOUS Ds is NON-AUTONOMOUS

Cohesive ends

Where did they come from?

They were presentall the time.

The genome shockin the ‘earthquake’ ear activated them from being buried in heterochromatin somewhere in the genome.

CAGGGATGAAA

Exon 1 5 4 3 2

Ds

Ds2d1

Ds2d2

Ds6

transcription of transposase gene

TTTCATCCCTA

Cloning McClintock’s elements Ac - 4563 bp

Changes of state: – insertion into another gene, change of methylation at target site, transposase doubles up as repressor of transposition. Not thought to have role in development.DNA transposons make the genome dynamic: - increase in number if transpose before replication.Transposon promoter may insert next to gene and change its pattern of expression, causing alternative splicing.

Lectures in St. Petersburg1995 Advances in B chromosome research2000 Physical mapping of plant chromosomes2001 Genetically modified crops 2002 Challenging genome integrity2003 Chromosomes without genes2005 What is a centromere?2006 Order and chaos in the plant nucleus.2006 What is a telomere?2007 Epigenetics2009 What is a gene?2009 Epigenesis to Epigenetics2012 Chromosomes without genes revisited.2012 McClintock’s controlling elements: the full story

AcknowledgementsDynasty Foundation for financial support