Organization of the eukaryotic genomes

GenomeSize of genome? Nuclear / organelle genome

DNA: coding, non-coding, repetitive DNA

Complexity of genes

Transposable elements

Multigenes

Pseudogenes

Regulatory sequences for Transcription?

Density of genes?

Genome organization•Prokaryotes–Most genome is coding–Small amount of non-coding is

regulatory sequences

•Eukaryotes–Most genome is non-coding (98%)–Regulatory sequences– Introns–Repetitive DNA

Prokaryote genomes• Example: E. coli

• 89% coding

• 4,285 genes

• 122 structural RNA genes

• Prophage remains

• Insertion sequence (IS) elements

• Horizontal transfers

Prokaryotic genome organization:

• Haploid circular genomes (0.5-10 Mbp, 500-10000 genes)

• Operons: polycistronic transcription units• Environment-specific genes on plasmids and

other types of mobile genetic elements• Usually asexual reproduction, great variety of

recombination mechanisms• Transcription and translation take place in the

same compartment

Eukaryotic genome• Example: C. elegans

• 10 chromosomes

• 19,099 genes

• Coding region – 27%

• Average of 5 introns/gene

• Both long and short duplications

Eukaryotic genome organization

1. Multiple genomes: nuclear, plastid: mitochondria, chloroplasts

2. Plastid genomes resemble prokaryotic genomes

3. Multiple linear chromosomes, total size 5-10,000 MB, 5000 to 50000 genes

4. Monocistronic transcription units5. Discontinuous coding regions (introns and

exons)

Eukaryotic genome organization(contd.)

6. Large amounts of non-coding DNA 7. Transcription and translation take place in

different compartments8. Variety of RNAs: Coding (mRNA, rRNA,

tRNA), Non-coding (snRNA, snoRNA, microRNAs, etc).

9. Often diploid genomes and obligatory sexual reproduction

10.Standard mechanism of recombination: meiosis

Hierarchy of gene organization

Gene – single unit of genetic function

Operon – genes transcribed in single transcript

Regulon – genes controlled by same regulator

Modulon – genes modulated by same stimilus

Element – plasmid, phage, chromosome,

Genome

** order of ascending complexity

Finding genes in eukaryotic DNA

Types of genes include• protein-coding genes• pseudogenes• functional RNA genes: tRNA, rRNA and others

--snoRNA small nucleolar RNA--snRNA small nuclear RNA--miRNA microRNA

There are several kinds of exons:-- noncoding-- initial coding exons-- internal exons-- terminal exons-- some single-exon genes are intronless

Mitochondrial Genome

Limited autonomy of mt genomesmt encoded nuclear

NADH dehydrogase 7 subunits >41 subunitsSuccinate CoQ red 0 subunits 4 subunitsCytochrome b/c comp1 subunit 10 subunitsCytochrome C oxidase 3 subunits 10 subunitsATP synthase complex 2 subunits 14 subunitstRNA components 22 tRNAs nonerRNA components 2 components noneRibosomal proteins none ~80 Other mt proteins none mtDNA pol, RNA pol

Human Mitochondrial Genome

Small (16.5 kb) circular DNA

rRNA, tRNA and protein encoding genes (37)

1 gene/0.45 kb

Very few repeats

No introns

93% coding;

Genes are transcribed as multimeric transcripts

Recombination not evident

Maternal inheritance

What are the mitochondrial genes?

• 24 of 37genes are RNA coding– 22 mt tRNA– 2 mit ribosomal RNA (23S, 16S)

• 13 of 37 genes are protein coding

(synthethized on ribosomes inside mitochondria)

some subunits of respiratory complexes and oxidative phosphorylation enzymes

Two overlapping genes encoded by same strand of mt DNA (ATPase 8/ ATPase 6)

(unique example)

Two independent AUG located in Frame-shift to each other, second stop codon is derived from TA + A (from poly-A)

Mitochondrial codon table

22 tRNA cover for

60 positions via

third base wobble

AUA=ileUGA=stop

Human Nuclear Genome3200 Mb23 (XX) or 24 (XY) linear chromosomes30,000 genes1 gene/100kbIntrons in the most of the genes1.5 % of DNA is codingGenes are transcribed individuallyRepetitive DNA sequences (45%)Recombination at least once for each chrom.Mendelian inheritance (X + auto), paternal (Y)

REPEATS!!!!

C value paradox: why eukaryotic genome sizes vary

The haploid genome size of eukaryotes (called the C value)varies enormously.

Small genomes include:•Encephalotiozoon cuniculi (2.9 Mb)•A variety of fungi (10-40 Mb)•Takifugu rubripes (pufferfish) (365 Mb)(same number of genes as other fish or as the human genome, but 1/10th the size)•Human 3200 MbLarge genomes include:•Pinus resinosa (Canadian red pine)(68 Gb)•Protopterus aethiopicus (Marbled lungfish)(140 Gb)•Amoeba dubia (amoeba)(690 Gb)

viruses

plasmids

bacteria

fungi

plants

algae

insects

mollusks

reptiles

birds

mammals

Genome sizes in nucleotide base pairs

104 108105 106 107 10111010109

The size of the humangenome is ~ 3 X 109 bp;almost all of its complexityis in single-copy DNA.

The human genome is thoughtto contain ~30,000 genes.

bony fish

amphibians

C value paradox: why eukaryotic genome sizes vary

The range in C values does not correlate well with the complexity of the organism. This phenomenon is called the C value paradox.

Why?

Britten and Kohne (1968) identified repetitive DNA classes

Reassociation Kinetics = isolated genomic DNA, Shear, denature (melted), & measure the rates of DNA reassociation.

Repetitive DNA•Two types–Tandemly repetitive– Interspersed

repetitive

Tandem repeatsTandem repeats occur in DNA when a pattern of two or more

nucleotides is repeated and the repetitions are adjacent to each other

Form different density band on density gradient centrifugation

(from bulk DNA) -satellite Example:

A-T-T-C-G-A-T-T-C-G-A-T-T-C-G

Tandem repeats:– Satellite DNA:– Microsatellite:– Minisatellite:

Satellite DNA• Unit - 5-300 bp depending on species.

• Repeat - 105- 106 times.

• Location - Generally heterochromatic.

• Examples - Centromeric DNA, telomeric DNA. There are at least 10 distinct human types of satellite DNA.

Microsatellite DNA • Unit - 2-4 bp (most 2).

• Repeat - on the order of 10-100 times.

• Location - Generally euchromatic.

• Examples - Most useful marker for population level studies..

Minisatellite DNA• Unit - 15-400 bp (average about 20).

• Repeat - Generally 20-50 times (1000-5000 bp long).

• Location - Generally euchromatic.

• Examples - DNA fingerprints. Tandemly repeated but often in dispersed clusters. Also called VNTR’s (variable number tandem repeats).

Tandemly Repetitive DNA Can Cause Diseases:

• Fragile X Syndrome– “CGG” is repeated hundreds or even thousands of

times creating a “fragile” site on the X chromosome.

– It leads to mental retardation.

• Huntington's Disease– “CAG” repeat causes a protein to have long

stretches of the amino acid glutamine.

– Leads to a neurological disorder that results in death

Interspersed Repetitive DNA

• Interspersed repetitive DNA accounts for 25–40 % of mammalian DNA.

• They are scattered randomly throughout the genome.

• The units are 100 – 1000 base pairs long.

• Copies are similar but not identical to each other.

• Interspersed repetitive genes are not stably integrated in the genome; they move from place to place.

• They can sometimes mess up good genes

Interspersed Repetitive DNAThese are:

• Retrotransposons (class I transposable elements) (copy and paste), copy themselves to RNA and then back to DNA (using reverse transcriptase) to integrate into the genome.

• Transposons (Class II TEs) (cut and paste) uses transposases to make makes a staggered sticky cut.

Interspersed Repetitive DNA• Retrotransposons are:

long terminal repeat (LTR) Any transposon flanked by Long Terminal Repeats. (also called retrovirus-like elements). None are active in humans, some are mobile in mice.

long interspersed nuclear elements (LINEs) encodes RT and

short interspersed nuclear elements (SINEs) uses RT from LINEs. example Alu made up of 350 base pairs long, recognized by the RE AluI (Non-autonomous)

Long interspersed nuclear elements (LINEs ) 20% of genome

• LINE1 – active

(Also many truncated inactive sequences)

• Line2 – inactive

• Line 3 – inactive

RNA binding also endonuclease

LINEs prefer AT-rich euchromatic bands

Internal promoter

In everyone’s genome 60-100 copies of LINE1 are still capable of transposing,

and may occasionally cause the disease by gene disruption

Mechanism of LINE repeat jumpsFull length LINE transcript is generated from 5’-

UTR-based promoter

ORF1 and ORF2 translated into proteins that stay bound to LINE mRNA

ORF1/ORF2/mRNA complex moves back into the nucleus

5’ 3’

5’ 3’orf1orf2

5’ 3’orf1orf2

5’ 3’3’ 5’

3’ 5’

Product of ORF2 cut ds DNA

Freed 3’ serves as a primer for LINE reverse transcription from 3’ UTR

ORF2 and ORF1 function• ORF1 keeps ORF2 and LINE mRNA bound together and

retracted into nucleus

• ORF2 (endonuclease) cut dsDNA to provide free 3’ end as a primer to LINE 3’UTR

• ORF2 (reverse transcriptase) makes cDNA copy of LINE mRNA, which becomes

integrated into chromosomal DNA (as it bound to it by former 3’ freed end)

TTTT A is ORF1 cleavage site, that is why integration prefers AT rich regions

Short interspersed nuclear elements (SINE) 13% of genome

• Non-autonomous (no RT)

• 100-400 bp long;

• No open reading frames (no start/stop codon)

• Derived from tRNA (transcribed with

RNA pol III, leaving internal promoter)

• Depend on LINE machinery for its movement

AluI - elements

• Derived from signal recognition particle 7SL

• Internal promoter is active, but require appropriate flanking sequence for activation

• Integrates in GC rich sequences• Only active SINE in the human genome

Diseases caused by Alu-integration

• Neurofibromatosis (Shwann cell tumors), • haemophilia,• breast cancer, • Apert syndrome (distortions of the head and face

and webbing of the hands and feet), • cholinesterase deficiency (congenital myasthenic

syndrome) • complement deficiency (hereditary angioedema)

• α-thalassaemia

• Several types of cancer, including Ewing sarcoma, breast cancer, acute myelogenous leukaemia

Genes• About 30,000 genes, not a particularly

large number compared to other species.

• Gene density varies along the chromosomes: genes are mostly in euchromatin,

• Most genes (90-95% probably) code for proteins. However, there are a significant number of RNA genes.

Gene familiesA gene family is a group of genes that share

important characteristics. These may be

• Structural: have similar sequence of DNA building blocks (nucleotides). Their products (such as proteins) have a similar structure or function.

• Functional: have proteins produced from these genes work together as a unit or participate in the same process

Gene families (structural)1. Classical gene families (overall

conservativeness) Histones, alpha and beta-globines

2. Gene families with large conservative domains (other parts could be low conservative) HLH/bZIP box transcription factors

3. Gene families with short conservative motifs e.g. DEAD box (Asp-Glu-Ala-Asp), WD (Trp-Asp) repeat

Gene families (functional)1 Regulatory protein gene families

2 Immune system proteins

3 Motor proteins

4 Signal transducing proteins

5 Transporters

6 Unclassified families

Multigene familiesSome genes are Transcribed (But Don't Make Proteins)• The entire family of genes probably evolved from a single

ancestral gene.– Famous examples: rRNA, globin genes – Four different pieces of rRNA are used to make up a

ribosome: 18S, 5.8S, 28S, and 5S.– It turns out that three of these rRNAs (18S, 5.8S, 28S, )

occur in the genome as a gene (on chrom 13, 14, 15, 21, 22) & transcribed together. (one 5S on chrom. 1)

– The entire multigene family is repeated nearly 300 times in clusters on five different chromosomes!

• It makes sense to have many repeats of this multigene family because each cell needs many ribosomes for protein synthesis

Multigene family: rRNA Genes

• RNA polymerase I synthesizes 45S which matures into 28S, 18S and 5.8S rRNAs

• RNA polymerase II synthesizes mRNAs and most snRNA and microRNAs.

• RNA polymerase III synthesizes tRNAs, rRNA 5S and other small RNAs found in the nucleus and cytosol.

tRNA genes (497 nuclear genes + 324 putative pseudogenes)

• Humans have fewer tRNA genes that the worm (584), but more than the fly (284);

• Frog (Xenopus laevis) has thousands of tRNA genes;

• Number of tRNA genes correlates with size of the oocytes;

In large oocytes lots of protein needs to be sythesized simultaneously.

Fascinating world of RNAs coding & non-coding

Non-coding RNAs• tRNA & rRNA

• 4.5S & 7S RNA (Signal Recognition Particles)

• snRNA – Pre-mRNA splicing

• snoRNA – rRNA modification

• siRNA – small interfering RNA

• gRNA – guide RNA in RNA editing

• Telomerase RNA – primer for telomeric DNA synthesis

• tmRNA is a hybrid molecule, half tRNA, half mRNA

• Xist: The X chromosome silencing is mediated by Xist – a 16,000 nt long ncRNA

• shRNA (small heterochromatic RNAs ): expresses only one allele while other is silenced

• LNA Locked Nucleic Acid

• piRNA Piwi-interacting RNA

Protein-coding Genes• Genes vary greatly in size and organization.• Intron less: Some genes don’t have any introns.

Most common example is the histone genes. • Some genes are quite huge: dystrophin (associated

with Duchenne muscular dystrophy) is 2.4 Mbp and takes 16 hours to transcribe. More than 99% of this gene is intron (total of 79 introns).

• Highly expressed genes usually have short introns

• Most exons are short: 200 bp on average. Intron size varies widely, from tens to millions of base pairs.

Pseudogenes• Pseudogenes are defective copies of genes. They have lost

their protein-coding ability – have stop codons in middle of gene – they lack promoters, or – truncated – just fragments of genes. – accumulation of multiple mutations

• Processed pseudogenes copied from mRNA and incorporated into the chromosome but lack of protein-coding ability (no intron/ poly-A tail present/ no promoter)

• Non-processed pseudogenes are the result of tandem gene duplication or transposable element movement. When a functional gene get duplicated, one copy isn’t necessary for life.

Processed pseudogenes

1. Complexity2. Gene number3. DNA amount

Why so small amount of genes we, humans, kings of nature, have?

Human 30,000 genesDrosophila – 13,000Nematode – 19,000

Potential of proteome and transcriptome diversity is so great that it is no need for increase of amount of genes

51

Solution 2 to the N-value paradox:We are counting the wrong things, we should count other We are counting the wrong things, we should count other genetic elements (e.g., genetic elements (e.g., smallsmall RNAsRNAs).).

Solution 1 to the N-value paradox:Many protein-encoding genes produce more than one Many protein-encoding genes produce more than one protein product (e.g., by protein product (e.g., by alternative splicingalternative splicing or by or by RNA RNA editingediting).).

Solution 3 to the N-value paradox:We should look at We should look at connectivityconnectivity rather than at rather than at nodesnodes..

These should be exciting and should stimulate the next generation of genomic investigation.

Solutions ?

The ENCODE project(Encyclopedia of DNA Elements)

The ENCODE project(Encyclopedia of DNA Elements)

Goal of ENCODE: build a list of all sequence-based functional elements in human DNA. This includes:

► protein-coding genes

► non-protein-coding genes

► regulatory elements involved in the control of gene transcription

► DNA sequences that mediate chromosomal structure and dynamics.

1977 first viral genome (Sanger et. Al. bacteriophage fX174; 11 genes)

1981 Human mitochondrial genome16,500 base pairs (encodes 13 proteins, 2 rRNA, 22 tRNA)Today, over 400 mitochondrial genomes sequenced

1986 Chloroplast genome 156,000 base pairs (most are 120 kb to 200 kb)

1995 Haemophilus influenzae genome sequenced

1996 Saccharomyces cerevisiae (1st Euk. Genome)and archaeal genome, Methanococcus jannaschii.

Chronology of genome sequencing projects

1997 More bacteria and archaeaEscherichia coli 4.6 megabases, 4200 proteins (38% of unknown function)

1998 Nematode Caenorhabditis elegans (1st multicellular org.)97 Mb; 19,000 genes.

1999 first human chromosome: Chrom 22 (49 Mb, 673 genes)

2000 Drosophila melanogaster (13,000 genes); Plant Arabidopsis thaliana & Human chromosome 21

2001: draft sequence of the human genome(public consortium and Celera Genomics)

Chronology of genome sequencing projects

09_25_Chromosome22.jpg

First microbial genome was completely sequenced in 1995 by The Institute for Genomic Research (TIGR)

Fleishmann, R.D. et al. 1995. Science 269:496-512.

Genome of Haemophilus influenzae Rd

single circular chromosome 1,860,137 bp

Outer circle – coding sequences with database matches

40% of genes at the time had no match in the databases

Some more statistics• Gene density 1/100 kb (vary widely);

• Averagely 9 exons per gene

• 363 exons in titin (molecular spring for elasticity of muscle) gene

• Many genes are intronsless

• Largest intron is 800 kb (WWOX gene)

• Smallest introns – 10 bp

• Average 5’ UTR 0.2-0.3 kb

• Average 3’ UTR 0.77 kb

• Largest protein: titin: 38,138 aa

INTRONLESS GENES

• Interferon genes

• Histone genes

• Many ribonuclease genes

• Heat shock protein genes

• Many G-protein coupled receptors

• Some genes with HMG boxes

• Various neurotransmitters receptors and hormone receptors

Smallest human genes

Percentages describe

exon content to the length of the gene

Typical human genes

Extra Large human genes

Presumable functions of human genes

Genes within genes

Neurofibromatosis gene (NF1) intron 26 encode :

OGMP (oligodendrocyte myelin glycoprotein), EVI2A and EVO2B, (homologues of ecotropic viral intergration sites in mouse)