b ch 2 part 1

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Part 1

The Structure of

Genes and Genomes

[ Chapter 2 in Griffiths et al. ]

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What’s on the Menu

What is the structure of DNA?

What is the organization of a gene?

What are chromosomes? What is chromatin?

What’s in a genome?

How are genes organized in the genome?

How are genomes different among living organisms?

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1. The Structure of DNA

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Nature of DNA

• Transformation (uptake of foreign DNA) in prokaryotes and eukaryotes has repeatedly

shown that DNA is the hereditary material.

• DNA is accurately replicated prior to each

cell division.

• DNA encodes proteins needed by the cell

and the organism.

• DNA is capable of mutation, providing raw

material for evolutionary change.

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The Griffith and Avery experiments (1928-1944)A

C

E

DNA is the hereditary material.

Negative controlexperiment: degrade DNA

with DNase -> virulence

not transmitted anymore

F

D

B

?

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The nucleotide

• Building block of DNA (and RNA)• Deoxyribose (pentose sugar), with 3’ –OH

• Phosphate (on 5’ carbon)

• Nitrogenous base – purine

• Adenine: A

• Guanine: G

– pyrimidine

• Thymine: T

• Cytosine: C

P CO

N

N

base

sugar

5’

3’OH

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The structure of DNA:

The Double Helix

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WATSON, J.D. & CRICK, F.H.C. A Structure for

Deoxyribose Nucleic Acid. Nature 171, 737-738 (1953)

“We wish to suggest a structure for the salt of deoxyribose

nucleic acid (D.N.A.). This structure has novel features which areof considerable biological interest.”

James Watson, Francis Crick and Maurice Wilkins, Nobel Prize 1962

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The double helix

• DNA normally consists of twoantiparallel polynucleotide chains

– sugar–phosphate backbone

• phosphodiester bonds

• 5’ to 3’ connection

– complementary base pairs

• A – T

• G – C

• hydrogen bonds

– 2 per A – T

– 3 per G – C

• 5’→ 3’ chain polarity

• Major and minor grooves (see

model)

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5’ 3’

3’ 5’

5’-AATTGGCCGATC-3’

3’-TTAACCGGCTAG-5’

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Figure 1.9 Genomes 3 (© Garland Science 2007)

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“We wish to suggest a structure for the salt of deoxyribose

nucleic acid (D.N.A.). This structure has novel features which areof considerable biological interest.”


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Blah, blah, blah...

???...

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Rosalind Franklin“The Dark Lady of DNA”

1920-1958

"The instant I saw the picture my mouth fell open and my pulse began

to race.... the black cross of reflections which dominated the picture

could arise only from a helical structure... mere inspection of the X-ray

picture gave several of the vital helical parameters.” -JD Watson

Franklin R & Goslind RG. Evidence for a 2-chain Helix in

the Crystalline Structure of Sodium Deoxyribonucleate.

Nature 172: 156 (1953)

X-ray diffraction photograph of DNA

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Rosalind Franklin

Franklin and Wilkins X-ray diffraction studies

revealed that DNA was helical and had two

distinctive regularities of 0.34 nm and 3.4 nm along

the axis of the molecule. In addition, it was shown

that DNA had a uniform thickness of 2 nm.

Maurice Wilkins

The DNA double helix is 2 nm wide.

A stack of 10 base pairs (= one turn of the

helix) have a linear length of 3.4 nm.

2 nm

3.4 nm

10 bp

2 nm

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Erwin Chargaff’s rules:

(early 1950’s)

1. The composition of DNA may

vary from one species to

another in the relative amount

of A, T, C, G

2: But for any DNA:

% A = % T

% C = % G

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Chargaff’s rules

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The model fitted:

(1) The X-ray diffraction data produced by Franklin(2) Chargaff’s rules

The structure also fulfilled the requirements for a

hereditary molecule:

(1) The ability to store information (coding capacity)(2) The ability to self-replicate (strand separation and

specificity of base pairing)

(3) The ability to change over time, ie. to mutate

(base substitution)

The double helix model of Watson & Crick: conclusions

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YIPEEE!

Blah, blah, blah...

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DNA: summary

• Units of measurement – base pair (bp)

– kilobase (kb)

– megabase (Mb)

• Replication: each strand serves as templatefor synthesis of complement, using rules of

base pairing

• Information: specified by sequence of

nucleotides; may be copied into RNA

• Mutation: replacement, insertion, deletionof nucleotide results in altered sequence

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2. The Structure of Genes

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Structure of genes

• Gene = transcriptional unit

• Gene may encode coding RNA (mRNA) OR

non-coding RNA (tRNA, rRNA, miRNA...)

• Gene is a functional element of the chromosome and is transcribed intoRNA at the correct time and place in development or cell cycle

• To some researchers, gene actually includes its adjacent regulatory

region(s) such as the promoter (remember, definition of gene may

vary)

DNA encoding functional RNApromoter

RNA primary transcript

gene

TSS=Transcription

start site

TTS=Transcription

termination site

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Eukaryotic genes: introns and exons

• Intron: noncoding region of gene, excised from

primary RNA transcript (=intron splicing)• zero to many intron per eukaryotic gene

• variable length, may represent most of gene length

• Function of introns poorly understood (no general function

known, but they often contain functional regulatory sequences)

• Exon: coding region of gene (sequence is

included in mature transcript)

E1 E2I1 I2 E3 I3 E4

E1 E2 E3 E4

Primarytranscript

Mature transcript

nuclear processing steps,

including splicing

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Introns are only present in eukaryotic genes(but they may be absent in some eukaryotic genes)

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Because of the abundance and large size of introns,

some eukaryotic genes can attain huge sizes

An extreme example: human dystrophin gene, 2.5 Mb long

(1.5% of entire chr. X), 78 introns !

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3. The Structure of

Genomes

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The nature of genomes

Genomics: study of structure and function of genomes

• Nuclear Genome (very variable size, especially in

eukaryotes)

• Organellar genomes

– chloroplast, mitochondrion – derived by endosymbiosis from bacterial ancestors

• Plasmids

– symbiotic DNA molecules, not essential but often useful to

the organism (antibiotic resistance) – mostly circular in prokaryotes

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The Prokaryotic genome

• Usually circular DNA, often a singlemolecule per cell (= 1 single chromosome)

• Gene-dense: genes are close together with

little intergenic spacer

• Genes are often organized in operon

– tandem cluster of coordinately regulated genes

– Several genes transcribed as single mRNA

• No spliceosomal introns

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The Lactose Operon

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A ‘simple’ genome:

the bacterial genome§ Unicellular organism

§ Single, circular

chromosome

§ Compact genome, gene

dense, 90% coding DNA

§ ~500 to ~5,000 genes,

depending on species

E. coli

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Mycoplasma: one of the smallest gen

- Extremely streamlined: 580 kb

- ~500 genes

- Gene-dense: 90% is coding DNA

- Very short intergenic regions

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Viral genomes• Virus=Replicating, infectious but nonliving particle, can only

replicate within a host cell (and can move from cell to cell) – Genome=nucleic acid

– Encodes multiple proteins

Many viruses cause infectious diseases

• Viral Genome = DNA or RNA

– single-stranded or double-stranded

– linear or circular

• Generally compact genomes with little spacer DNA, containingfrom a few to many genes (note: some viral genes contain introns, likeeukaryotic genes. Recently giant ‘mimivirus’ discovered, 1.1 Mb long)

• Unknown origin of viruses, but some appear to have evolvedfrom mobile genetic elements. The latter are normally containedwithin the host genome but they can acquire infectious capacityallowing them to escape the cell (e.g. retroviruses originate fromretrotransposons)

Note: In prokaryotes,

viruses are referred

to as bacteriophages.

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1918 Influenza pandemics caused 20-30 million deaths

The flu virus, influenza: a single-stranded RNA virus

Hemaglutinin

Neuraminidase

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The swine flu virus, H1N1 (ssRNA virus)

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Conceptual structure of HIV, a ssRNA

virus using reverse-transcription

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The replicative

cycle of HIV-1

retrovirus

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Restricted tovertebrates

Example: HIV-1

Widespread in eukaryotes

Example: Ty1 element in yeast,

By acquisition of envelope gene, a

retrotransposon can gain infectious

capacity, it becomes a retrovirus

(example of an intermediate: gypsy in

fruit flies)

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Eukaryotic genome: 2 or 3 genomes per cell

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The Mitochondrial Genome

- Circular - Resembles a reduced

prokaryotic genome in

terms of organization and

gene numbers- Encodes genes mostly

involved in the production

of energy (oxydative

phosphorylation) and in

translation (tRNAs, rRNA)

- Maternally inherited

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Human(mammal)

16.5 kb

Yeast

(fungus)

75 kb

Marchantia(moss)

186 kb

Mitochondrial genomes can vary in

size between species

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The Chloroplast Genome (plants)

- Circular

- Resembles a reduced

prokaryotic genome in

terms of organization and

gene numbers

- Encodes genes mostlyinvolved in photosynthesis

and electron transport

- ‘Maternally’ inherited

(transmitted through theseed)

- Do not vary much in sizeMarchantia (moss)

CpDNA 121 kb

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4. The structure of

eukaryotic chromosomes

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Eukaryotic nuclear genome: chromosomes (1)

• Linear structure

• Chromosome number is conserved within species but greatlyvaries between species

• Ploidy refers to number of complete sets of chromosomes – haploid (1n): one complete set of chromosomes (e.g. yeast)

– diploid (2n): two sets of chromosomes (e.g. most animals)

– polyploid (≥3n): more than two sets (e.g. many plants, a few animals)

• In diploids, chromosomes come in homologous pairs(homologs) – structurally similar (i.e size and position of centromere)

– same assortment of genes (homologous genes)

– may contain different alleles for each gene: each gene exist either ashomozygote state (same two alleles) or heterozygote state (two differentalleles)

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In humans, somatic cells have 2n = 46 chromosomes

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Remy’skaryotype

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Human chromosomes 11 and 17

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• Cytogenetics: microscopic study of chromosomes

• Variable centromere position

– telocentric: centromere at end

– acrocentric: centromere close to end

– metacentric: centromere in middle – For human chromosomes: p arm is shortest, q arm is

longest

• Telomere: end of chromosome

• Nucleolar organizer region (NOR): The chromosomal regionaround which the nucleolus forms (contains rRNA genetandem array)

• Chromomere (or knob): small bead-like region of condensedchromatin visible during meiosis and mitosis

Eukaryotic nuclear genome:

chromosomes (2)

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Maize chromosomes (2n = 20)

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• Considerable difference in size and in the

number of genes carried on chromosomes, both

between and within species

• Genes may occupy only a minor fraction of a

chromosome (extreme case is human Y)


chromosomes (3)

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0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

Chromosome size (Mb)

Gene density (per 10 Mb)

1 2 3 4 5 6 7 8 9 10 1112 13 141516 17 18 19 20 21 22 X Y

Human chromosomes: size and gene density

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• Heterochromatin – densely stained regions of highly condensed

DNA

– mostly made of non-coding repetitive DNA, low

gene density and transcription activity• Euchromatin:

– poorly stained, less compact chromatin

– contains most transcribed genes

Note: Polytene chromosomes

– replicated, unseparated chromosomes

– present in certain tissues of dipteran insects (salivary glands)


chromosomes (4)

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The chromosomes of maize (corn)

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Microscopic view of atomato chromosome

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Organization of Nuclear DNA

• Highly organized, various degrees of coiling

• Nucleosome – fundamental unit of chromatin

– 146 bp of DNA wrapped twice around histone core(histone octamer)

• histones are highly conserved proteins

• H2A, H2B, H3, H4

• Chemical modification of histones underlie

changes in chromatin compaction

– Nucleosome forms a 10 nm fiber

– 6 nucleosomes coil to form Solenoid, 30 nm fiber

• Higher order coiling – solenoid loops attach to scaffold (SAR, MAR)

– form larger diameter fibers

Chromatin is a highly dynamic structure

A haploid set of

humanchromosomes

consists of about

3 feet of DNA !

ll i f A d d hi f i l

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Illustration of DNA wrapped around histones, forming a nucleosome

Ill i f DNA d d hi f i l

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Illustration of DNA wrapped around histones, forming a nucleosome

H2A

H3

H2B

H4

H1

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Solenoid

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Electron micrograph of chromosome

shows long DNA loops emanating

from the protein scaffold (at the

bottom of the pic) . Note that there are

only loops -no ends- at the top of the

pic.

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5. Genome landscapes and

Comparative Genomics

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Prokaryotes and eukaryotes have very

different genome landscapes

• In prokaryotes, genes are compactly

arranged, with little or no spacer sequences

in between (short intergenic regions) = most

of the genome is coding DNA• In eukaryotes, there is considerable spacer

DNA between genes (large intergenic

regions) and within genes (introns) = most

of the genome is ‘non-coding’ DNA

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E k t

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– Where is non-coding DNA? In introns, intergenicregions, centromeric regions, telomeric regions

– the majority of non-coding DNA is repetitive DNA

= identical or nearly identical repeated units

- two types of repetitive DNA:

• Tandem repeats (e.g. DNA at centromeres andtelomeres)

• Interspersed repeats

– Most interspersed repeats are derived frommobile genetic elements (aka transposable elements)

Eukaryote genomes:

A whole lotta non-coding DNA

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Genome Size

• In eukaryotes, most of the cell DNA isfrom the nuclear genome

• Genome size is measured in pg or Mb(1pg ~ 1000 Mb) human genome is ~3.2 pg

• Nuclear genome size is extremelyvariable among eukaryote species

• ‘C-value paradox’ : no obvious

correlation between genome size andorganism complexity

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The ‘C-value paradox’

Genome size does NOT correlate with

organismal complexity

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Extensive variation in genome size (= C-value)

within and among the main groups of life

Gregory 2005

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C-value of eukaryotic nuclei varies ~200,000-fold, but

there is only ~20 fold variation in the number of protein-coding genes

Encephalitozoon cuniculi : 2.8 Mb, 2,000 genes

Navicola pelliculosa (diatom): >690,000 Mb (probably less than 40,000 genes)

-> Variation in gene numbers cannot explain

variation in genome size among eukaryotes

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• Most of variation in genome size is due tovariation in the amount of repetitive DNA (mostly

derived from TEs)• TEs accumulate in intergenic and intronic regions

Transposable Elements and

genome size

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The amount of TE correlate positively with

genome size

P l a s m

o d i u m

S l i m

e m o l d

B u d d i n

g y e a s t

F i s s i o

n y e a s t

N e u r

o s p o r

a

A r a b i d

o p s i s

B r a s s i c a R i c e M

a i z e

N e m

a t o d e

D r o s o p h i l a

M o s q u

i t o

S e a s

q u i r t

Z e b r

a f i s h F u g u

M o u s e

H u m

a n

0

500

1000

1500

2000

2500

3000 Genomic DNA

TE DNA

Protein-coding

DNA

Mb

Feschotte & Pritham 2006

Th ti f t i di d ith

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TEs

Protein-coding

genes

The proportion of protein-coding genes decreases with genome

size, while the proportion of TEs increases with genome size

Gregory, Nat Rev Genet 2005

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• Variation in gene numbers cannot explainvariation in genome size among eukaryotes

• Most of variation in genome size is due tovariation in the amount of non-coding, repetitiveDNA (mostly transposable elements, TEs)

• TEs accumulate in intergenic and intronic

regions

Repetitive DNA and genome size

Contrasted Genome Landscapes

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Transposable Element

Contrasted Genome Landscapes

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What have we learned

from the humangenome sequence?

2001: first

draft of the

human

genome

sequence

Most of the Human Genome does not code

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Coding

Non-coding

1.5%

Most of the Human Genome does not code

for proteins

Half of the Human Genome is derived from

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Coding

Non-coding

1.5%

TE-derived

DNA

48.5%

Half of the Human Genome is derived from

Transposable Elements (TEs)

The human Genome Browser at UCSC

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A snapshot of the Human Genome

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A snapshot of the Human Genome

TEs

Genes

Conservation in other species

TEs are the most rapidly changing components of the

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TEs

Genes

Human-

specific TE

Ape-

specific TE

Primate-specific

TE

Cons-

erved

Exon

TEs are the most rapidly changing components of the

genome

Rapid changes in genome size in the grasses

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430 Mb 750 Mb2500 Mb

Genome size:

4800 Mb

Rapid changes in genome size in the grasses

~50 myr

~10 myr

Figure adapted from Sue Wessler

The maize genome: tiny gene islands floating on

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RIPRIP RIP RIP

gene A gene B genes C & D

The maize genome: tiny gene islands floating on

an ocean of repetitive DNA

Cluster of Repetitive DNACluster of Repetitive DNA

A typical maize chromosome

Nested LTR retrotransposons

Expansion of intergenic regions in maize by

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San Miguel et al. (1996) Nested Retrotransposons in the Intergenic Regions of the

Maize Genome. Science 274: 765-768

(+ other studies from Bennetzen lab)

Expansion of intergenic regions in maize by

accumulation of LTR-retrotransposons

Retrotransposon amplification has resulted in the

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San Miguel et al. (1998) The paleontology of intergene

retrotransposons of maize, Nature Genet. 20:43-45

Retrotransposon amplification has resulted in the

doubling of the maize genome in the last ~6 myr

Variation in TE activity triggers rapid changes

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430 Mb 750 Mb 2500 MbGenome size:

4800 Mb

in genome size in grasses

Genes

TEs

~50 myr

~10 myr

Comparati e genomics

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Comparative genomics

• Study of similarities and differences among

genomes

• Many genes are shared among all living things or

between related groups

• Study of genes in model organisms provides usefulinformation regarding genes in other organisms

• Large genome projects produce considerable

amount of information

– Requires computer analysis and development of newsoftware to analyze the avalanche of data (bioinformatics)

2001 fi

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What have we learned

from the humangenome sequence?

2001: first

draft of the

human

genome

sequence

1996: S. cerevisiae 1998: C. elegans 2000: D. melanogaster 2000: A. thaliana 2001: H. sapiens

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2002: S. pombe 2002: P. falciparum 2002: A. gambiae2002: F. rubripes

2002: M. musculus

2004: R. norvegicus

2002: O. sativa

2002: C. intestinalis

2004: T.pseudonana

2004: T.nigroviridis

2004: B. mori

2003: N. crassa2003: C. familiaris

2005: E. histolytica 2005: P. troglodytes

Genome sequences can be aligned and compared

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Genome sequences can be aligned and compared

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Human-Mouse

genome

comparison

A snapshot of the human genome browser at UCSC

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p g

Genes

THIS WEEK’S MENU

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What is the structure of DNA?

What is the organization of a gene?

What are chromosomes? What is chromatin?

How is DNA organized at the chromosome and chromatin level?

How are genes organized in the genome?

What makes the genome of a prokaryote and a eukaryote different?

What’s in a genome?

How are genomes different among eukaryotes?

Overview

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Overview• Each species has a uniquely fundamental set of

genetic information, its genome.

• The genome is composed of one or more DNAmolecules, each organized as a chromosome.

• The prokaryotic genome is generally compact andmade of a single circular chromosome.

• The eukaryotic genome consists of one or two setsof linear chromosomes confined to the nucleus.

• A gene is a segment of DNA that is transcribedinto a ‘functional’ RNA molecule.

• Introns interrupt many eukaryotic genes.

• Eukaryotic genomes are littered with repetitiveDNA (mostly derived from transposable elements)

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