Genome Organization and Evolution

• DNA is associated with architectural proteins and packaged into chromosomes.

• But, genetic information has to be accessible for processes such as replication and transcription.

• Genomes are mosaic and reflect a complex evolutionary history.

Genome organization varies in different organisms

The domains of life

• Scientists first divided life into prokaryotes and eukaryotes.

• Comparison of 16S rRNA sequences and ribosome structure revealed the archaea.

• Three major groups of living things: eukaryotes, bacteria, archaea

Two models for the divisions of life

• Three domain tree: bacteria, eukaryotes, archaea

• Eocyte tree: bacteria and archaeaEukaryotes are a type of archaea derived from

ancestral cells called eocytes.

Viruses with DNA genomes

• Not considered organisms because they are not made of cells.

• But, viruses have a genome and they evolve.

Two classes of genomes• Small genomes of viruses, archaea,

bacteria (<10 Mb), and many unicellular eukaryotes (<20 Mb)– Protein-coding and RNA-coding sequences

occupy most of the nucleotide sequences.

• Large genomes of multicellular and some unicellular eukaryotes (>100 Mb)– The majority of the nucleotide sequence is

non-coding.

Packaging of the eukaryotic genome

The problem:

• How to fit 2 meters of DNA into a <10 µm space.

The solution:

• Double-stranded linear DNA molecules are packed into chromatin.

The way in which eukaryotic DNA is packaged in the cell nucleus is one of the wonders of macromolecular structure.

G. Michael Blackburn, Nucleic Acids in Chemistry and Biology (1990), p. 65

Diversity in the number of chromosomes that make up eukaryotic genomes

• Butterflies: >200 chromosomes• Kangaroos: 12• Humans: 46• Adder tongue fern: 1260• Male jack jumper ant: 1

Histones are small, positively charged proteins

1928: Albrecht Kossel isolated histones, small basic proteins, from the nuclei of goose erythrocytes.

1970s: Electron microscopic and biochemical studies showed that the fundamental packing unit of chromatin is the nucleosome.

Two types of histones:

• Highly conserved core histones.

• More variable linker histones.

Core histones

• Small, positively-charged, basic proteins.

• Molecular weight 11,000-16,000 daltons.

• Histones H2A, H2B, H3, and H4.

• Rich in arginine and lysine (basic amino acids)

• Bound to DNA in eukaryote chromosomes to form core octamers.

Linker histones

• Slightly larger, positively-charged, basic proteins.

• Molecular weight >20,000 daltons.

• Histones H1, H5, H1, etc.

• Occur between core octamers.

• Most eukaryotes package their genomes with histones.

• There are some exceptions:

– Dinoflagellates package their DNA with small basic non-histone proteins.

– Sperm DNA is compacted with basic proteins known as protamines.

Chapter 12: Organization in Chromosomes

20

Eukaryotic chromosomal organization

• Histone proteins– Abundant– Histone protein sequence is highly conserved among

eukaryotes—conserved function– Provide the first level of packaging for the chromosome;

compact the chromosome by a factor of approximately 7– DNA is wound around histone proteins to produce

nucleosomes; stretch of unwound DNA between each nucleosome

Chapter 12: Organization in Chromosomes

21

Eukaryotic chromosomal organization

• Nonhistone proteins– Other proteins that are associated with the chromosomes– Many different types in a cell; highly variable in cell types,

organisms, and at different times in the same cell type– Amount of nonhistone protein varies– May have role in compaction or be involved in other

functions requiring interaction with the DNA– Many are acidic and negatively charged; bind to the

histones; binding may be transient

Chapter 12: Organization in Chromosomes

22

Eukaryotic chromosomal organization

• Histone proteins– 5 main types

• H1—attached to the nucleosome and involved in further compaction of the DNA (conversion of 10 nm chromatin to 30 nm chromatin)

• H2A• H2B• H3• H4

– This structure produces 10nm chromatin

Two copies in each nucleosome ‘histone octomer’; DNA wraps around this structure1.75 times

Chapter 12: Organization in Chromosomes

23Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 8.17 A possible nucleosome structure

Chapter 12: Organization in Chromosomes

24Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 8.18 Nucleosomes connected together by linker DNA and H1 histone to produce the “beads-on-a-string” extended form of chromatin

10 nm chromatin is produced in the first level of packaging.

Linker DNA

H1 Histone octomer

Nucleosomes are the fundamental packing unit of chromatin

Beads-on-a-string: the 10 nm fiber

• Visualized by electron microscopy as 10-11 nm fiber after low salt extraction.

• Beads represent DNA wrapped around the histone core octamer.

• String represents the DNA double helix.

Nucleosomes

• Repeating structural element in eukaryotic chromosomes.

• Core octamer of histones plus one molecule of the linker histone.

• 180 bp DNA wound around.

Core histone octamer

• Dimer of histones H2A and H2B at each end.

• Tetramer of histones H3 and H4 in the center.

• 146 bp of negatively charged DNA wraps nearly twice around the positively charged histones.

Carboxyl (C) terminal end

• Extended histone-fold domain• Histone-histone interactions• Histone-DNA interactions

Amino (N) terminal charged “tails”

• Lysine-rich• Sites of many post-translational modifcations

Higher order structure: the 30 nm fiber

• Visualized by electron microscopy in higher salt.

• Two models:

1. Classic solenoid model

2. Currently favored zig-zag ribbon model

Further packaging of DNA involves loop domains

• Further compaction of the 30 nm fiber into loops that contain 50-100 kb of DNA.

• Insight into loop structure comes from studies of lampbrush chromosomes in amphibian oocytes.

• In interphase of the cell cycle, the packing ratio is 1000-fold.

Chapter 12: Organization in Chromosomes

32Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 8.20b Packaging of nucleosomes into the 30-nm chromatin fiber

Chapter 12: Organization in Chromosomes

34

Eukaryotic chromosomal organization

• Compaction continues by forming looped domains from the 30 nm chromatin, which seems to compact the DNA to 300 nm chromatin

• Human chromosomes contain about 2000 looped domains

• 30 nm chromatin is looped and attached to a nonhistone protein scaffolding

• DNA in looped domains are attached to the nuclear matrix via DNA sequences called MARs (matrix attachment regions)

Chapter 12: Organization in Chromosomes

35

Fig. 8.21 Model for the organization of 30-nm chromatin fiber into looped domains that are anchored to a nonhistone protein chromosome scaffold

Fully condensed chromatin: metaphase chromosomes

• Condensation requires ATP-hydrolyzing enzymes and the condensin complex.

• Packing ratio of 10,000-fold.

• Each chromosome is composed of one linear, double-stranded molecule of DNA.

Chapter 12: Organization in Chromosomes

37

Fig. 8.22 The many different orders of chromatin packing that give rise to the highly condensed metaphase chromosome

The centromere provides the site of attachment for segregation

during cell division

• A fully condensed metaphase chromosome consists of two sister chromatids connected at the centromere.

• From the centromere, the kinetochore captures spindle microtubules, which ensure that sister chromatids segregate correctly to daughter cells.

During mitosis, chromosomes:

• Condense• Congregate at the metaphase plate• Orient• Attach to microtubules• Are pulled apart

Centromere DNA typically is:

• Localized to a specific region of the chromosomes.

• Consists of many repeated DNA sequences spanning 0.1-4 Mb.

• Centromere DNA has little or no sequence conservation.

• Centromere location is specified by the formation of a specialized chromatin structure.

• The histone variant CenH3 triggers a complex network of interactions, leading to the fully assembled kinetochore.

Each chromosome must contain:

• A centromere• One or more origins of replication• A telomere at each end

Chromosome classification

• Metacentric: centromere in the middle• Acrocentric: centromere toward one end• Telocentric: centromere at the end

Autosomes and sex chromosomes

• Chromosomes are classified as sex chromosomes or autosomes.

• The number, size, and shape of the chromosomes make a species-specific set or karyotype.

Examples of diversity in sex chromosome systems

• Humans: XX (female) and XY (male)• Birds: ZW (female) and ZZ (male)• Insects: XX (female), and X (male)• Duck-billed platypus: XXXXX,XXXXX

(female) and XXXXX, YYYYY (male)

Organization and expression of the genetic material

Heterochromatin: chromatin that is condensed suppresses transcription

Euchromatin: chromatin that is more open and allows for gene activation

• Euchromatin is uncoiled and active, whereas heterochromatin remains condensed and is inactive.

Figure 12.12

G-banding is due to differential staining

along the length of each chromosome.

Eukaryotic gene expression is regulated at three levels

• DNA sequence: DNA-binding proteins associate with regulatory elements in the DNA.

• Chromatin structure: changes in the way the DNA is wrapped around the histones.

• Nuclear architecture: positioning of chromosomes in “territories” in the nucleus.

• Early insights into how chromatin structure changes during transcription have come from studies of polytene chromosomes.

• Chromosome puffs represent sites of high transcriptional activity.

5.4 The majority of the eukaryotic genome is noncoding

C-value paradox

• The observation that the amount of DNA in the haploid genome is not related to an organism’s evolutionary complexity.– e.g. wheat has 16,000 Mb of DNA, while

humans only have 3,200 Mb.

• Most genomic DNA consists of various classes of repetitive DNA sequences.

Organization of the human genome

• Less than 40% of the human genome is comprised of genes and gene-related sequences.

• Intergenic DNA consists of unique or low copy number sequences and moderately to highly repetitive sequences.

Repetitive DNA sequences are divided into two major classes

• Interspersed elements

• Tandem repetitive elements

Interspersed elements are primarily transposable elements

Genome-wide repeats that are primarily degenerate copies of transposable elements

• Short interspersed nuclear elements (SINEs)

• Long interspersed nuclear elements (LINEs)

Tandem repetitive sequences are arranged in arrays with variable

numbers of repeats

Three subdivisions based on length

• Satellite DNA

• Minisatellites

• Short tandem repeats (STRs)

Satellite DNA

• Very highly repetitive DNA with repeat lengths of one to several thousand base pairs.

• Buoyant density during density gradient centrifugation differs from that of the bulk of the DNA.

Figure 12.14

5.5 Lateral gene transfer in the eukaryotic genome

• Lateral or horizontal transfer is the transfer of DNA between two different species, especially distantly related species.

• Important mechanism for bacterial evolution; in particular, through movement of transposable elements.

• Evidence is accumulating for the importance of lateral transfer in fungi, animal, and plant evolution.

Organelle genomes reflect an endosymbiont origin

• Both mitochondria and chloroplasts contain their own genetic information.

• Endosymbiont hypothesis: both organelles are derived from primitive, free-living, bacterial-like organisms.

• Inherited independently of the nuclear genome.

• Uniparental mode of inheritance: organelles are only contributed from the maternal gamete.

Chloroplast DNA (cpDNA)

• Circular (?) or linear (?) double-stranded DNA molecule.

• 120-160 kb

• Multiple copies (20-40) per organelle.

• Different buoyant density and base composition compared with nuclear DNA.

Mitochondrial DNA (mtDNA)

• Typically a circular, double-stranded DNA molecule.

• Linear in yeast and some other fungi.

• In animals, typically 16-18 kb.

• In plants, 100 kb to 2.5 Mb.

• Multiple copies (several to 30) per organelle.

Mitochondrial DNA and disease

• Defects in mtDNA can lead to degenerative disorders, e.g.

Leber’s hereditary optic neuropathy (LHON)

Kearns-Sayre syndrome

• Heteroplasmy leads to differences in the severity and the kind of symptoms.

Homoplasmy

• All of the mtDNA within cells of an individual are identical.

Heteroplasmy• Mutation occurring in one copy of mtDNA can

result in both mutant and normal mtDNA within the same cell.

• An individual may have some tissues enriched for normal mtDNA and others enriched for mutant mtDNA.

Intercompartmental DNA transfer

• A special form of lateral gene transfer.

• Associated with the gradual loss of an endosymbiont’s independence on the path to becoming an organelle.

Known types of interorganelle transfer:

• Mitochondrion to nucleus• Chloroplast to nucleus• Chloroplast to mitochondrion• Nucleus to mitochondrion• Mitochondrion to chloroplast

• Eukaryotic genomes are mosaic―the product of a complicated evolutionary history.

• Most human genes were transferred from an endosymbiont:– Genes of archael origin are involved in

information processing.– Genes of bacterial origin are associated with

metabolism and cell structure.– The proteins that make the nuclear envelope

are encoded by genes of both archael and bacterial origin.

5.6 Prokaryotic and viral genome organization

Bacterial genome organization

• A single, covalently closed circular DNA molecule.

• Condensation involving histone-like proteins into a structure called a nucleoid.

• Further condensation into supercoiled domains.

Histone-like or nucleoid-associated proteins

• HU (heat-unstable protein)

• IHF (integration host factor)

• HNS (heat-stable nucleoid structuring)

• SMC (structural maintenance of chromosomes)

• Lateral gene transfer provides a source of genetic material for bacteria.

• This allows for their rapid response to changing environments.– e.g. In Japan, a human gut bacterium has

acquired a gene from a marine bacterium that encodes an enzyme involved in digesting the seaweed used to wrap sushi.

Plasmid DNA

• Small, double-stranded circular or linear DNA molecules.

• Carried by bacteria, some fungi, and some higher plants.

• Extrachromosomal, independent, and self-replicating.

Plasmids from bacteria

• Small, covalently closed circular DNA molecules.

• Carriers of resistance to antibiotics.

• Vehicles for genetic engineering.

Archael genome organization• One double-stranded circular DNA molecule (0.5

to 5.5 Mb)

• Some archaea have two distinct histones, each with a single histone fold domain.

• 60 bp of DNA wraps around a histone tetramer.

• Some archaea use non-histone packaging proteins.

• The evolutionary origins of histones can be traced back to the archael histones.

• A “doublet histone” in some archaea may represent an intermediate in the transition from archael to eukaryotic histones.

Viral genome organizationBacteriophages and mammalian DNA

viruses

• Double-stranded linear, single-stranded circular, and double-stranded circular genomes.

• Model systems for molecular biology.

• Provide a cloned set of genes on a single DNA molecule.

Prokaryotic viruses (phages)

Bacteriophage (bacterial viruses)

• Genome typically consists of a single DNA molecule, largely devoid of associated proteins.

• Commonly used bacteriophages in molecular biology:

Bacteriophage (double-stranded linear genome)

M13 (single-stranded circular genome)

• Many recent advances in the study of bacteriophages and viruses of archaea.

• Metagenomics: the sequencing of genomes of entire populations within the “virosphere.”

• Isolation of many new virus-host systems of major environmental importance.

• Many phage genes have no known functions or homologs: “ORFans.”

• But, viruses and cellular organisms also share a common gene pool by lateral gene transfer.

Mammalian DNA viruses

• Infect mammalian cells and make use of the host machinery for their replication.

• Genomes come in a diversity of forms:

Human papilloma virus (circular, double-stranded)

Simian virus 40 (circular, double-stranded)

Adenovirus (linear, double-stranded)

• Little is known about how many mammalian DNA viruses package their genome into the viral capsid.

• Some encode their own basic proteins.

• Simian virus 40 (SV40) uses host cell histones (H2A, H2B, H3, and H4).