Prof. Neil Jones 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: 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 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 A BC ABC A B C 9S A B C pachytene A B C A B C C B A A A B B C C Chiasma metaphase I tetrad microspore (n) x x unbalanced 2 > 90% balanced 3,4 non-male transmission deficient (due to genetic constitution) replication / fusion of sticky ends Male meiosis (x=sticky end) inverted duplication How to make a sticky end without losing anything ?

x x xx pollen grain mitosis pollen introduced into crosses HOW BEHAVE ?? x x tube nucleus 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) replication and fusion (F) sperm embryo sac sperm x x Endosperm- 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) x x Wx C Wx c wxc 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 CC C C triploid endosperm Endosperm variegation due to the chromatid BFB (purple kernels) (colourless)

Chromosome breakage-fusion-bridge cycle [BFB] xx x x xx Zygote no healing fusion at telophase – before replication (prevents chromatid BFB) 5 prophase 6 7 oreither anaphase both parents generating sticky ends 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?

‘Earthquake ear’ of 1944 C CICI Wx wx ♂ ♀ 9S Wd wd x x 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 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 I //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 C I 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 C I = 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 CICI Wx wx ♂ ♀ 9S Wd wd x x 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 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 I //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 C I 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 C I = colour inhibitor (Pale yellow = colourless)

Discovery of Ds – dissociation locus C I Bz (Sh Wx) C bz (sh wx) ♂ x x C CICI wx Wx bz Bz sh Sh 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 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 I Ds Discovery of Ac x ♀ C ds C I 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 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. Ac ac

Inheritance of Ac C I Ds x ac Ac ac Ac ac Ds C I Ds C I C C ds C 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 C I C Sh ds Ds Wx wx sh Bz bz + ac ac ac + Ac ac ac + Ac Ac ac + Ac Ac Ac 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. ♂ ♀

C I Sh Bz Wx Ds C sh bz wx x Ac Change of state of Ac C I 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 – 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 Identical Ac alleles from 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 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 F1

Mutation of the C locus – the interpretation (intellectual leap) “footprint”mutant c m-1 locus Ds C locus DsAc C (a) (b) (c) purple colourless The position of Ds had also moved and was inseparable from the new c m-1 locus. The site of chromosome breaks had moved (transposed). 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 Ds now had new function > mutation. Other mutable loci later.. Transposition was discovered!

Ds Transposition C CICI wx Wx bz Bz sh Sh Ds 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 sectors Wx x x Bz Sh Bz Wx Bz Sh CICI CICI CICI C BzWx shbzwx 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 C I Transposition of Ac in early studies Ac was not linked with 9S markers: Ac wx Bz Sh C Ds ac Ds Wx Bz Sh C I 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 sh C x C I Sh Bz Wx Ds Ac x bz wx sh C C 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 (unlinked) (linked) ac Ac

The Ac – Ds family of ‘controlling elements’ Ds Ac Ds 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 Cohesive ends 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.

CAGGGATGAAA Exon Ds Ds2d1 Ds2d2 Ds6 transcription of transposase gene TTTCATCCCTA Cloning McClintock’s elements Ac 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. Petersburg 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 What is a telomere? 2007 Epigenetics 2009 What is a gene? 2009 Epigenesis to Epigenetics 2012 Chromosomes without genes revisited McClintock’s controlling elements: the full story Acknowledgements Dynasty Foundation for financial support