Transcript Document

Barbara McClintock’s controlling elements:
the full story
Prof. Neil Jones
[email protected]
IBERS - Institute of Biological, Environmental and Rural Sciences
The story is timeless
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
Structure of the maize kernel
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
Chromosomes of maize
Pachytene stage of meiosis
Photo: Bill Sheridan, University of North Dakota
How to make a sticky end without losing anything ?
Male meiosis (x=sticky end)
A
B
C
C
B
A
A
B
inverted
duplication
A
C
C
B
B
9S
A
A
B
C
B
A
C
C
1 2
(n)
3 4
C
B
x
A
C
C
B
Chiasma
microspore
B
C
A
x
B
A
A
replication / fusion
of sticky ends
1 unbalanced
2 > 90% balanced
3,4 non-male transmission deficient
(due to genetic constitution)
pachytene
metaphase I
tetrad
Sticky ends – gametophyte
-
in plants gametes are produced by mitosis
tube
nucleus
x
x
x
x
xx
pollen grain mitosis
microspore
(n)
pollen
introduced into crosses
HOW BEHAVE ??
Chromatid breakage-fusion-bridge cycle [BFB]
Sticky ends from male parent only
x
x
sperm
sperm
replication
and fusion (F)
embryo sac
x
Endosperm- BFB
x
x
EMBRYO
Endosperm - BFB
HEALING of
broken end almost
immediately after
fertilisation
breakage (B)
bridge (B)
..based on cytological observations and on the use of markers
Endosperm variegation due to the chromatid BFB
9S
(colourless)
c
wx
(amylopectin, red)
♀♀
♂
X
(purple kernels)
C
Wx
triploid
endosperm
(amylose, blue)
Wx
C
C
C
Wx
C
C
Wx
C
x
x
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)
Chromosome breakage-fusion-bridge cycle [BFB]
both parents generating sticky ends
1
x
x
5
fusion at telophase – before replication
(prevents chromatid BFB)
2 Zygote
no healing
6
prophase
3
anaphase
7
4
x
x
x
x
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
x
CI = colour inhibitor
wd
CI Wx
Wd
C
wx
‘Earthquake ear’ of 1944
♂
♀
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
A new type of aleurone variegation appeared.
It had a uniform pattern of coloured spots (C)
of similar size. It seemed that CI was being
eliminated in some cells at a particular rate,
and at a particular stage in development.
CONTROLLED BREAKAGE
she sensed that these were something special –
BREAKAGE WAS TAKING PLACE
Seeds grown and studied - additional markers
ONE of the selfed plants, which must have been
heterozygous CI//C, gave a few variegated kernels,
UNEXPECTED NO STICKY ENDS INTRODUCED
(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
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
x
CI = colour inhibitor
wd
CI Wx
Wd
C
wx
‘Earthquake ear’ of 1944
♂
♀
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 CI was being
eliminated in some cells at a particular rate,
and at a particular stage in development.
CONTROLLED BREAKAGE
She sensed that these were something special
BREAKAGE WAS TAKING PLACE
Grown and studied – using additional markers
ONE of the selfed plants, which must have been
heterozygous CI//C, gave a few variegated kernels,
- UNEXPECTED NO STICKY ENDS INTRODUCED
(Pale yellow = colourless)
Discovery of Ds – dissociation locus
Breakage without sticky ends – but where was it taking place ?
Markers:
CI colour inhibitor
C coloured aleurone
c colourless aleurone
CI > C > c (dominance)
Sh
sh
Bz
bz
Wx
wx
normal endosperm
shrunken endosperm
purple aleurone
bronze aleurone
amylose, blue starch
amylopectin, red starch
CI
Sh
Bz
Ds
no markers, no BFB
Wx
x
x
♂
♀♀
C
sh
bz
wx
CI 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.
Discovery of Ac
C
CI
ds
♀
♂
x
C
CI
ds
C
ds
CI
Ds
Ac
ac
Ds
Ds
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 CI 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.
Inheritance of Ac
C
ds
ac
CI
Ds
Ac
CI
Ds
ac
x
C
ac
ds
C
ds
Ac
CI
Ds
ac
C
ds
ac
CI
Ds
ac
1/2
1/2
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
Ac//Ac x ac//ac
1Ac ac:1ac ac
all Ac ac
Dosage effect of Ac
♂
♀♀
CI Sh Bz
Wx Ds
C
wx ds
sh bz
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
0
+ Ac ac ac
1
+ Ac Ac ac
2
+ Ac Ac Ac
3
Change of state of Ac
Ac
CI Sh Bz Wx Ds
C sh bz wx
x
Ac
CI Sh Bz Wx Ds
Identical Ac alleles
from this cross
Ac
C sh bz wx
CI
Sh Bz Wx Ds
C sh bz wx
Sectorial kernel
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 – mimicked
the Ac dosage effect.
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
Transposition of Ds in 1947
Ac
C
Sh
wx Ds
½
½
ac
- Ac
c
sh
wx ds
♂
♀♀
(12)
F1
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.
+ Ac
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:
● 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
Mutation of the C locus – the interpretation
(intellectual leap)
(a)
Ds
C locus
purple
The site of chromosome breaks
had moved (transposed).
Ds
(b)
“footprint”
mutant cm-1 locus
Ac
(c)
Ac
Ds
C
The position of Ds had also
moved and was inseparable
from the new cm-1 locus.
colourless
Purple spots would only appear
if Ac was also present …
and …. dosage effect of Ac
Evidence consistent with cm-1
arising from transposition of Ds
and 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
CI
Ds
Sh
Ac
Bz
Wx
♂
♀♀
Ac
C
sh
bz
wx
Exceptions:
Wx
CI
Sh
Bz
Wx
C
sh
bz
wx
CI
Bz
Wx
Bz
Sh
Sh
Bz
Sh
C sh bz Wx
Bz
Sub-sectors:
C sh bz wx
Reveal new position of Ds
Wx
Sub-sectors:
C sh Bz Wx
C sh bz Wx
C sh bz wx
x
Sh
CI
C Sh Bz Wx
Wx
x
twin sectors
Transposition of Ds
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
Transposition of Ac
in early studies Ac was not linked with 9S markers:
C
sh
bz wx
♀
CI Sh Bz Wx Ds
Ac
C Sh ac
ac
x
C
sh
bz wx
wx Ds
in later crosses linkage was sometimes found:
C
sh
bz
CI Sh Bz Wx Ds Ac
wx
♀
x
C
sh
bz
wx
C Sh Bz wx Ds ac
20%
Position varied in different crosses (ears):
CI Sh Bz Wx Ds
Ac
Ac
Ac
♂
♂
Transposition of Ac explains some unexpected events
Ac//Ac x ac//ac
Expected: Ac//ac (usually found)
Unexpected:
Ac
ac
ac
Ac
Ac
ac//ac
(loss)
Ac
Ac
Ac//Ac
(unlinked)
Ac
Ac
Ac//Ac
(linked)
Ac
Ac
Ac
The Ac – Ds family of ‘controlling elements’
Ac
Ds
Ac
Ds
Ac
Ac
Ac activates breakage
at Ds. Loci may be on
different 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
Ac
Ac
Ds
Cohesive
ends
Ds
Where did they
come from?
They were present
all the time.
The genome shock
in the ‘earthquake’
ear activated them
from being buried
in heterochromatin
somewhere in the
genome.
Cloning McClintock’s elements
CAGGGATGAAA
TTTCATCCCTA
Ac - 4563 bp
Exon 1
2
3
4
5
transcription of transposase gene
Ds
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.
Lectures in St. Petersburg
1995
2000
2001
2002
2003
2005
2006
2006
2007
2009
2009
2012
2012
Advances in B chromosome research
Physical mapping of plant chromosomes
Genetically modified crops
Challenging genome integrity
Chromosomes without genes
What is a centromere?
Order and chaos in the plant nucleus.
What is a telomere?
Epigenetics
What is a gene?
Epigenesis to Epigenetics
Chromosomes without genes revisited.
McClintock’s controlling elements: the full story
Acknowledgements
Dynasty Foundation for financial support