Basic considerations in formulating a21st Century theory of the genome and

genome evolutionJames A. Shapiro

University of Chicagojsha@uchicago.edu, http:shapiro.bsd.uchicago.edu

• 40 years of molecular deconstruction ==> no units, only systems

• Contact with physicists ==> emphasis on basic principles

• Experience with transposable elements ==> genetic change notstochastic

What are theBasic Functions

of Cells?

Fraser et al. 1997. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi.Nature 390: 580-586.

• Transport• Metabolism• Growth (includes RNA,protein synthesis, DNAreplication, membranebiogenesis, etc.)• Division (includesdistribution of essential cellstructures & molecules)• Movement (includestropisms and motility)• Communication with othercells (multicellularity,symbiosis & pathogenesis)• Cognition, signaltransduction & informationprocessing (all the above)

SignalTransduction

in YeastMating

Mary J. Cismowski et al. Genetic screens in yeast to identify mammalian nonreceptor modulators ofG-protein signaling. Nature Biotechnology 17, 878 - 883 (1999).

Pheromone ResponseFrank van Drogen et al. MAP kinasedynamics in response to pheromones inbudding yeast. Nature Cell Biology 3,1051 - 1059 (2001).

What are basic properties ofbiological macromolecules?

• Stereospecificity• Structural flexibility• Multivalency (multiple

ligands)• Allostery• Reversible covalent

modifications (-CH3, ~PO3)• Weak interactions• Cooperativity• Modularity• Combinatorics

What functions does the genome fulfill?

• Expression (Protein & RNA coding) – transcription,chromatin

• Maintenance – replication & repair• Transmission to progeny cells – chromosome

movement/segregation at division• Restructuring – natural genetic engineering

(genome as RW storage organelle)

• NB – all functions involve nucleoprotein complexes,not naked DNA

What is the functional organization ofthe genome?

• Formatting by generic signals for compaction, attachment,transcription, replication, transmission, repair

• Networking by repeat sequences• Higher order “indexing” by chromatin domains, spatial

organization of chromosomes

DNA formatting to execute a cognitive algorithm - E. coli discriminates glucose and lactose

1. The algorithm: IF lactose present AND glucose not present AND cell can synthesizeactive LacZ (beta-galactosidase) and LacY (lactose permease), THEN transcribe lacZYAfrom lacP

2. The formatted DNA:

DNA formatting for chromosome transmissionto daughter cells - centromeres

B A Sullivan, M DBlower & G HKarpenDETERMININGCENTROMEREIDENTITY:CYCLICALSTORIES ANDFORKINGPATHS NatureReviews Genetics2; 584-596 (2001)

What are the sources of novelty inevolution?

•New protein functions - domain shuffling andaccretion•New adaptive systems (Hox complexes,

olfactory sensing, early embryonic functions) –duplication, reverse transcription, multipletranspositions•New species (novel cereals, Muntjac,

Drosophila) – hybridization, changes in ploidy,chromosome fusions, inversions•New kinds of cells (e.g. Gram-negative bacteria,

eukaryotes, plants) - symbiosis

Protein evolution by domain accretion

International Human Genome Sequencing Consortium. Initial sequencing and analysis of the humangenome. Nature 409, 860 - 921 (2001)

Most pairs of similar OR gene clusters (labeled in black) fall into established syntenic chromosomalregions (http://www.ncbi.nlm.nih.gov/Homology/index.html), but some (labeled in red) do not.Young, J. M. et al. Different evolutionary processes shaped the mouse and human olfactory receptor genefamilies. Hum. Mol. Genet. 11, 535-546 (2002)

Protein Amplification: Olfactory Receptors

Segmental Duplications in Arabidopsis

Segmentally duplicated regions in the Arabidopsis genome. Individual chromosomes are depicted as horizontal grey bars (withchromosome 1 at the top), centromeres are marked black. Coloured bands connect corresponding duplicated segments. Similarity betweenthe rDNA repeats are excluded. Duplicated segments in reversed orientation are connected with twisted coloured bands. The scale is inmegabases. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796 - 815 (2000).

Evolution of imprinted heterochromatin by insertions

Lippman Z, Gendrel AV, Black M, Vaughn MW, DedhiaN, McCombie WR, Lavine K, Mittal V, May B,Kasschau KD, Carrington JC, Doerge RW, Colot V,Martienssen R. Role of transposable elements inheterochromatin and epigenetic control. Nature. 2004 Jul22;430(6998):471-6.

How does primary genome structure change? -Natural Genetic Engineering: the cellular

toolbox for genome restructuring

• Homologous recombination• Non-homologous end-joining• Site-specific recombination• DNA transposons (large-scale rearrangements)• Retrotransposons and reverse transcription (small-

scale rearrangements)• Homing and retrohoming introns and inteins• Mutator polymerases

The MammalianImmune System:

An EvolvedRapid Evolution

System

D. C. van Gent, J. H. Hoeijmakers, R. Kanaar,Chromosomal Stability And The DNA Double-Stranded Break Connection 2, 196 (2001)

Somatic hypermutation &class switch recombination- transcription directed

Tasuko Honjo, Kazuo Kinoshita, and Masamichi Muramatsu. 2001. Molecular Mechanism of Class Switch Recombination: Linkage withSomatic Hypermutation. Annu. Rev. Immunol.;

Temporal & metabolic regulation of naturalgenetic engineering

0 10 200

100

200

MCS2 (2 subclones)

MCS1366 (4 subclones)

Days/32

Tota

l fus

ion

colo

nies

lacZaraB

Derepression(42C, starvation)ClpPX, Lon RpoS

MuA, HU, IHF

Strand transfer

Replication (exponential growth)

ClpX

DNA processing(RpoS-, Crp-dependent functions?)

lacZaraB

araB-lacZ fusion

STC = strand transfer complex

araB lacZ

U118

U118

lacZaraBCDC/Target complex

U118araB lacZ

Adjacent inversion (precludes fusion)

U118

Transposasome formation

MuB for replication(Crp-dependent starvation-induced functions inhibitand/or replace MuB?)

ClpX

Shapiro, J.A. 1997b. Genome organization, natural genetic engineering, and adaptive mutation. Trends in Genetics 13, 98-104

Leaf wounding and retrotransposon transcription

http://www-biocel.versailles.inra.fr/Anglais/03Transposon.html

The expression of the tobacco Tnt1 retrotransposon is induced bywounding : the expression of the LTR-GUS construct is detected by a bluestaining surrounding injury points in transgenic tomato (A), tobacco (B)and Arabidopsis (C) plants.

M.-A. Grandbastien et al. Stress activation and genomic impact of Tnt1 retrotransposons in Solanaceae.Cytogenetic and Genome Research 2005;110:229-241

Targeting of natural genetic engineering

Shapiro, JA. 2005. A 21st Century View Of Evolution: Genome System Architecture, Repetitive DNA, And Natural Genetic Engineering. Gene345: 91-100

Known molecular mechanisms:

• Sequence recognition by proteins (yeast mating-type switching,ribosomal LINE elements, homing introns, VDJ joining);• Protein-protein interaction wth transcription factors or chromatinproteins (Ty retrotransposon targeting);• Sequence recognition by RNA (reverse splicing of group IIretrohoming introns);• Transcriptional activation of target DNA (somatic hypermutation;class-switch recombination).

Unknown mechanisms:

• Telomere targeting of certain LINE elements in insects;• HIV & MLV targeting upstream of transcribed regions;• P factor homing directed by transcription, chromatin signals.

Yeast Ty5 targeting

S. Sandmeyer. Integration by design. PNAS, May 13, 2003; 100(10): 5586 - 5588.

Advantages of non-random searches ofgenome space at evolutionary crises

• Genome changes occur under stress or other conditions, when they aremost likely to prove beneficial;

• Multiple related changes can occur when a particular natural geneticengineering system is activated;

• Rearrangement of proven genomic components increases the chancethat novel combinations will be functional;

• Targeting can increase the probability of functional integration andreduce the risk of system damage;

• Rearrangements followed by localized changes provide opportunitiesfor fine tuning once novel function has been achieved.

What general principles operate in genomefunction and genome reorganization?

• All genome functions are interactive (no Cartesiandualism, genome always in communication with restof cell);

• Every genome component operates as part of acomplex information-processing system (no “onegene-one trait” correlation);

• Genome systems are organized and integrated intocell networks by repetitive DNA;

• Genome change is a regulated biological function;• Natural genetic engineering processes are subject to

biological feedback at multiple levels.