Genome organization

Eukaryotic genomes are complex and DNA amounts and organization vary

widely between species.

Genome OrganizationG

C value paradox:

The amount of DNA in the haploid cell of an organism is not related to its evolutionary complexity or number of genes.

Highly Repeated Sequences

There are different classes of eukaryotic DNA based on sequence complexity.

105 106 107 108 109 1010 1011 1012

basepairs

Amount of DNA in a Genome Does Not Correlate with

Complexity

How many genes do humans have?

Original estimate was between 50,000 to 100,000 genes

We now think humans have ~ 20,000 genes

How does this compare to other organisms?

Mice have ~30,000 genes

Pufferfish have ~35,000

Nematodes (C. elegans), have ~19,000

Yeast (S. cerevisiae) has ~6,000

The microbe responsible for tuberculosis has ~4,000

Single Copy Sequences

Exome

Even the Amount of DNA a Gene Spans Differs Among Species

Problems?• Some gene products are RNA (tRNA,

rRNA, others) instead of protein

• Some nucleic acid sequences that do not encode gene products (noncoding regions) are necessary for production of the gene product (protein or RNA).

• Eukaryotic genes are complex!

Gene Identification• Open reading frames• Sequence conservation

– Database searches– Synteny

• Sequence features– CpG islands

• Evidence for transcription– ESTs, microarrays

• Gene inactivation– Transformation, RNAi

Unique genes

Noncoding regions

• Regulatory regions– RNA polymerase binding site

– Transcription factor binding sites

• Introns

• Polyadenylation [poly(A)] sites

Splice Sites

• Eukaryotes only • Removal of internal parts of the newly

transcribed RNA.• Takes place in the cell nucleus• Splice sites difficult to predict

One gene, many proteins via alternative splicing , 3’ cleavage and polyadenlyation

Exon Shuffling

Trans-Splicing in Higher Eukaryotes

Gingeras, Nature (2009) 461, 206-211

Non-contiguous Transcription Generates An Enormous Number of Possible

Transcripts

Gingeras, Nature (2009) 461, 206-211

Six 2-exon co-linear combinations from four exons

325 combinations of 3-exons, non-colinear

• Reassortment of exons coding for ncRNA or protein domains could dramatically increase number of functional products beyond the number of ‘genes’

• Trans-splicing exists in higher eukaryotes as well as in lower ones like Trypanosomes

Blue: only co-linearRed: all combinations

Why genome size isn’t the only concern (size doesn’t matter?)

• More sophisticated regulation of expression?

• Proteome vastly larger than genome?

– Alternate splicing

– RNA editing

• Postranslational modifications?

• Cellular location?

• Moonlighting

Gene families

• E.g. globins, actin, myosin• Clustered or dispersed• Pseudogenes

Pseudogenes

• Nonfunctional copies of genes

• Formed by duplication of ancestral gene, or reverse transcription (and integration)

• Not expressed due to mutations that produce a stop codon (nonsense or frameshift) or prevent mRNA processing, or due to lack of regulatory sequences

Duplicated genes• Encode closely related (homologous) proteins• Formed by duplication of an ancestral gene

followed by mutation

Five functional genes and two pseudogenes

Paralogs vs Orthologs

• Different members of the globin gene family are paralogs, having evolved one from another through gene duplication. Paralogs are separated by a gene duplication event.

• Each specific gene family member (e.g. a specific gene in human) is an ortholog of the same family member in another species (e.g. mouse). Both evolved from an ancestral globin gene. Orthologs are separated by a speciation event.

• It is not always easy to distinguish true orthologs from paralogs , especially in polyploid organisms!

Protein - coding sequences less than 1.5% of the genome in humans!

Noncoding RNAs (ncRNA)

• Do not have translated ORFs• Small• Not polyadenylated

Functions of Known lncRNAs

Ponting et al, Cell (2009) 136, 629-641

• Transcriptional interference -lncRNA transcription turns off transcription of nearby gene

• Initiation of chromatin remodeling - lncRNA transcription turns on transcription of nearby gene

• Promoter inactivation - lncRNA binds to TFIIB and to promoter DNA

• Activation of an accessory protein - lncRNA binds to allosteric effector protein TLS and inhibits histone acetyltransferase, decreasing transcription

Functions of Known lncRNAs

• Activation of transcription factors - binding of lncRNA to Dlx2 activates Dlx5/6 activity

• Oligomerization of an accessory protein - lncRNA induces heat shock factor trimerization

• Epigenetic silencing of gene clusters -Xist RNA inactivates X chromosome

• Epigenetic repression of genes in trans -HOTAIR binds PRC2, leading to methylation and silencing of several genes in HOXD locus

• Transport of transcription factors -lnRNA NRON keeps NFAT out of nucleus

Ponting et al, Cell (2009) 136, 629-641

• ~97-98% of the transcriptional output of the human genome is ncRNA– Introns– Transfer RNAs (tRNA)

• ~ 500 tRNA genes in human genome– Ribosomal RNAs

• Tandem arrays on several chromosomes• 150-200 copies of 28S – 5.8S – 18S

cluster• 200-300 copies of 5S cluster

ncRNA

• The level of transcription from human chromosomes 21 and 22 is an order of magnitude higher than can be accounted for by known or predicted exons

• Almost half of all transcripts from well-constructed mouse cDNA libraries are ncRNAs (identified because they do not code for an open reading frame of larger than 100 codons)

Genome Organization - ncRNA

Repeat sequences – 50% or more of the genome

Repetitive DNA

• Moderately repeated DNA– Tandemly repeated rRNA, tRNA and histone genes

(gene products needed in high amounts)– Large duplicated gene families– Mobile DNA

• Simple-sequence DNA– Tandemly repeated short sequences– Found in centromeres and telomeres (and others)– Used in DNA fingerprinting to identify individuals

Segmental duplications

• Found especially around centromeres and telomeres

• Often come from nonhomologous chromosomes

• Many can come from the same source• Tend to be large (10 to 50 kb)• Unique to humans?

Repetitive DNA - Segmental duplications

Mobile DNA

• Moves within genomes• Most of the moderately repeated DNA

sequences found throughout higher eukaryotic genomes– L1 LINE is ~5% of human DNA (~50,000

copies)– Alu is ~5% of human DNA (>500,000 copies)

• Some encode enzymes that catalyze movement

Repetitive DNA – Highly repetitive satellite DNA