Barbara McClintock’s controlling elements: the full story
IBERS - Institute of Biological, Environmental and Rural Sciences
Prof. Neil Jones rnj@aber.ac.uk

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

Pericarp is maternal tissue endosperm /aleurone products of fertilisation aleurone layer (3n)
- develops pigment endosperm (3n)
Embryo 2n pericarp
Pigment characters scored without growing seeds

Pachytene stage of meiosis
Photo: Bill Sheridan, University of North Dakota

Male meiosis (x=sticky end)
A B C C B A inverted duplication
C B
9S
A
C B A
C
Chiasma
B
A
A
B
C
C
B
A
B
A
C
C
B
A
A
B
C
C
B
A
1 2
3 4 x microspore
(n) x replication / fusion of sticky ends
1 unbalanced
2 > 90% balanced
3,4 non-male transmission deficient
(due to genetic constitution) pachytene metaphase I tetrad

in plants gametes are produced by mitosis x x microspore
(n)
x x tube nucleus x x
introduced into crosses
HOW BEHAVE ??

Sticky ends from male parent only sperm x sperm x replication and fusion ( F ) embryo sac x x
Endosperm- BFB x
EMBRYO
HEALING of broken end almost immediately after fertilisation
Endosperm - BFB breakage ( B ) bridge ( B )
..based on cytological observations and on the use of markers

Wx
C
C x
Wx x
9S
(colourless) c
X
(purple kernels) C wx (amylopectin, red)
Wx (amylose, blue)
♀♀
♂ triploid endosperm
C
C
C
C
Wx
Wx
Wx
Wx
Replication followed by B-F-B and offcentre breaks c wx C Wx c Wx
Reading phenotypes: size of spots = stage of loss
“spots within spots” ( wx / Wx by iodine stain)

4
3
1
both parents generating sticky ends x x
5
2 Zygote no healing fusion at telophase – before replication
(prevents chromatid BFB)
6 prophase x x x x anaphase
7 either or
Chromosome BFB cycle continues during early development.
Healing >> later in development. Tried use chromosome BFB to induce small internal deficiencies in 9S to study mutations.
Endosperm 3 chrs >>independent chromatid BFB cycles
WHAT DOES THIS HAVE TO DO WITH TRANSPOSITION?

9S x wd
C I = colour inhibitor
C
I
Wx x
Wd C wx
♂
♀
earthquake ear of 1944
A new type of aleurone variegation appeared.
It had a uniform pattern of coloured spots ( C ) of similar size. It seemed that C
I was being eliminated in some cells at a particular rate, and at a particular stage in development.
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 ’.
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 been heterozygous C
UNEXPECTED
I // C , gave a few variegated kernels,
NO STICKY ENDS INTRODUCED
CONTROLLED BREAKAGE she sensed that these were something special
–
BREAKAGE WAS TAKING PLACE
Seeds grown and studied - additional markers
(Pale yellow = colourless)

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
Genetic ‘earthquake’
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

9S x wd
C I = colour inhibitor
C
I
Wx x
Wd C wx
♂
♀
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 ’.
670 KERNELS
590 germinated; 134 died as seedlings;
456 transferred to the field; 73 died;
383 PLANTS - SELFED OR FIXED IN 1945 earthquake ear of 1944
New type of aleurone variegation appeared.
With a uniform pattern of coloured spots ( C ) of similar size. It seemed that C I was being eliminated in some cells at a particular rate, and at a particular stage in development.
ONE of the selfed plants, which must have been heterozygous C I // C , gave a few variegated kernels,
- UNEXPECTED NO STICKY ENDS INTRODUCED
CONTROLLED BREAKAGE
She sensed that these were something special
BREAKAGE WAS TAKING PLACE
Grown and studied – using additional markers
(Pale yellow = colourless)

Breakage without sticky ends – but where was it taking place ?
Ds
Markers:
C I colour inhibitor
C coloured aleurone c colourless aleurone
C I > C > c (dominance)
Sh normal endosperm sh shrunken endosperm
Bz purple aleurone bz bronze aleurone
Wx amylose, blue starch wx amylopectin, red starch
C
I
C
Sh sh
Bz bz
Wx wx x x no markers, no BFB
♂
♀♀
C
I
Bz (Sh Wx)
C bz (sh wx)
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.

♀
Ac
C ds
C ds
C
C
I
Ds x ds
C
I
Ds
C
I
Ds ac
♂
The first clue about control of DS
Kernels found without variegation in plants expected with Ds breaks.
All progeny were expected to be heterozygous with variegated kernels due to loss of the C I allele: only half variegated – a 1:1 ratio.
One of the parents must have been heterozygous 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.

C
C ds
ac C I x ac C I
Ds Ac ds Ds ac
C ds Ac
1/2
C I
C
Ds ds ac ac
1/2
C I Ds 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 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

♂
♀ ♀
C
I
Sh Bz
C sh bz
Wx Ds wx ds
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.
+ ac ac ac
+ Ac ac ac
+ Ac Ac ac
+ Ac Ac Ac
2
3
0
1

Ac
Ac
C
I
Sh Bz Wx Ds
C
I
Sh Bz Wx Ds
Identical Ac alleles from this cross
Ac x
C sh bz wx several plants
C
I
C sh bz wx
Sh Bz Wx Ds
The effect of Ac varied in different plants, different ears of one plant, and different parts of a single kernel.
C sh bz wx The formation of sectorial kernels, due altered times of breakage, indicated changed forms of Ac – mimicked the Ac dosage effect.
Sectorial kernel
Further breeding tests showed that the 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

½
½
Ac C Sh wx Ds
♂
♀♀
(12)
F1 ac c sh wx ds
- Ac
+ Ac
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 c m-1 .
Male parent was Ac/ac
Female parent had no Ac or Ds elements.
Half kernels expected purple – no Ac
Other half variegated with colourless - with Ac.
Found in all 12 EARS, but 1/4000 was different .
Colour pattern was reversed.
Tests indicated :
● c m-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 c m-1 locus
● Location of Ds had also moved – inseparable from c m-1

(a)
(intellectual leap)
Ds C locus
The position of Ds had also moved and was inseparable from the new c m-1 locus.
purple
Ds
The site of chromosome breaks had moved ( transposed ).
(b)
(c)
“footprint”
Ac mutant c m-1 locus
Ds colourless
Purple spots would only appear if Ac was also present … and …. dosage effect of Ac
Evidence consistent with c m-1 arising from transposition of Ds and its insertion into C
– moving out from cell clones .
Not explained by Ds breakage
Ac C Ds now had new function > mutation.
Other mutable loci later ..
Transposition was discovered!

Ac
Ac
C
I
Sh
Ds
Bz
Wx
C sh bz wx
Exceptions:
C
I
Sh Bz Wx
C sh bz wx
Reveal new position of Ds
C
I
C
I
Wx
Bz
Sh
Sh
Bz
Wx
♂
♀♀
Wx
Bz
Sh
Sh
Bz x
Wx x
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 sectors

Ds
C Sh Bz Wx
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

in early studies Ac was not linked with 9S markers:
C sh bz wx C
I
Sh Bz Wx Ds
♀ x
C sh bz wx C Sh ac wx Ds
♀ in later crosses linkage was sometimes found:
C sh bz wx
C sh
C
I
Sh Bz Wx Ds Ac x bz wx C Sh Bz wx Ds ac
20%
Position varied in different crosses (ears):
C
I
Sh Bz Wx Ds
Ac
♂ ac
♂
Ac Ac Ac

Ac // Ac x ac // ac
Expected: Ac // ac (usually found)
Unexpected:
Ac ac // ac
(loss)
Ac
Ac
Ac // Ac
(unlinked)
Ac
Ac
Ac // Ac
(linked)
Ac ac
Ac ac
Ac
Ac Ac

Ac
Ac
Ac
Ac
Ac
Ds
Ds
Ds
Ac
Ds
Ac activates breakage at Ds . Loci may be on different chromosomes.
Where did they come from?
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
They were present all the time.
The genome shock in the ‘earthquake’ ear activated them from being buried in heterochromatin somewhere in the genome.

CAGGGATGAAA Ac - 4563 bp
Exon 1 2 3 transcription of transposase gene
Ds
TTTCATCCCTA
4 5
Ds2d1
Ds2d2
Ds6
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.

1995 Advances in B chromosome research
2000 Physical mapping of plant chromosomes
2001 Genetically modified crops
2002 Challenging genome integrity
2003 Chromosomes without genes
2005 What is a centromere?
2006 Order and chaos in the plant nucleus.
2006 What is a telomere?
2007 Epigenetics
2009 What is a gene?
2009 Epigenesis to Epigenetics
2012 Chromosomes without genes revisited.
2012 McClintock’s controlling elements: the full story
Acknowledgements
Dynasty Foundation for financial support