CHAPTER 19THE ORGANIZATION AND CONTROL OF

EUKARYOTIC GENOMES

=> Larger than prokaryotes

Not all 25,000 genes are active in all cells

Junk DNA anyone?Junk DNA anyone?

• Only 25,000 genes in humans - 3% of total DNA in cell (YIKES!)

• Rest of it (97%) - junk????? (noncoding DNA)

96% similar to humans)

•Prokaryotes- most of the DNA in a genome codes for protein (or tRNA and rRNA), with a small amount of noncoding DNA, primarily regulators.•Eukaryotes - most of the DNA (about 97% in humans) does not code for protein or RNA.

1) Some noncoding regions are regulatory sequences (these are promotors and enhancers that can increase binding of RNA polymerase to DNA).

2) Other are introns.3) Finally, even more of it

consists of repetitive DNA, present in many copies in the genome.

““Noncoding” DNA and what it does in Noncoding” DNA and what it does in the eukaryotic genomethe eukaryotic genome

• Know 2 types of Repetitive DNA:

1)TANDEMLY REPETITIVE DNA (AKA SATELLITE DNA - *3 types) - 10 to 15% of DNA

2) INTERSPERSED REPETITIVE DNA - 25 - 40% of DNA

REPPPPETITIVE DNA IN EUKARYOTESGTTACGTTACGTTAC….repeated 10 to 10 million times (Satellite DNA)

• 1) SATELLITE DNA/TANDEMLY REPETITIVE DNA

• These sequences (1 to 10 base pairs) are repeated up to a million times in series.

• GTTACGTTACGTTAC….• 3 types:a) Regular Satellite -100,000 - 10 millb) Minisatellite -100 -100,000 repeatsc) Microsatellite - 10 to 100 repeats -Very important for forensics - helps

figure out uniqueness of a person’s DNA

REPPPPETITIVE DNA IN EUKARYOTES

• A number of genetic disorders are caused by abnormally long stretches of tandemly repeated nucleotide triplets within the affected gene.

CAGRepeat

CAG Repeat SizeMedian Age at Onset * (years)(95% confidence interval)

39 66 (72-59)

40 59 (61-56)

41 54 (56-52)

42 49 (50-48)

43 44 (45-42)

44 42 (43-40)

45 37 (39-36)

46 36 (37-35)

47 33 (35-31)

48 32 (34-30)

49 28 (32-25)

50 27 (30-24)

You know that antisocial neighbor -May be a microsatellite problem!

• Satellite DNA plays a structural role at telomeres and centromeres. This is important! You don’t want non-repetitive DNA in telomeres because?

CSI Lab on this coming up- CSI Lab on this coming up- Microsatellites – more repeats but Microsatellites – more repeats but

really short!really short!

• Are only 1-10 nucleotides long and are repeated only 10-100 times in the genome

• Used in DNA fingerprinting (forensics)

• What, more “junk”?• (2) About 25-40% of most mammalian

genomes consists of interspersed repetitive DNA.

-One common family of interspersed repetitive sequences, Alu elements, is transcribed into RNA molecules with unknown roles in the cell.

-Alu sequences may help alternate RNA splicing

-Transposons are interspersed repetitive DNA

Table 19.1bottom

Out of 25,000 genes Out of 25,000 genes what gets expressed what gets expressed

depends upondepends upon::• Type of cell: Not all

genes are expressed in all cells (epigenetics controls it)

• Development period: During embryonic development certain genes may be expressed that are not expressed in adults (and viceversa)

Gene families - collection of Gene families - collection of genes that may be genes that may be

identical/nonidenticalidentical/nonidentical

• Most genes are present as a single copy per haploid set of chromosomes

• Multigene families exist as a collection of identical or very similar genes (exceptions).

• These likely evolved from a single ancestral gene.

• The members of multigene families may be clustered or dispersed in the genome.

Gene families have Gene families have evolved by duplication of evolved by duplication of

ancestral genesancestral genes

• Identical genes are multigene families that are clustered tandemly.

• Evolution - first duplicate a gene and then mutate the copy; result : original copy is still there, mutated gene - could make a new protein = new function (natural selection acts on it)

• Nonidentical genes have diverged since their initial duplication event.

Pseudogenes- DNA segments that have sequences similar to real genes but that do not yield functional proteins - remnants of evolution or?

1) Rare mutations (between 1/106 and 1/105

nucleotides )2) Gene amplification – selective DNA

replication of some genes to increase protein expression (ex. after chemotherapy)

3) Transposons/Retrotransposons-(Jumping genes)50% - Corn10% Human(Not inherited)4)Gene rearrangement

Did you know your genome Did you know your genome changes continually in changes continually in your your

lifetimelifetime??

Transposon moved into the purple color gene destroying its activity

• Altering genomes during your lifetime? continued

1) Rare mutations2) Gene amplification -

temporary increase (selective loss also possible) in number of gene copies

3) Transposons and retrotransposons

4) Gene rearrangement in Immunoglobin genes Fig. 19.5

B lymphocytes (WBC) produce immunoglobins, or antibodies, that specifically recognize and combat viruses, bacteria, and other invaders.

-Millions of types of Antibodies can be produced depending on what the infectious agent is - how?

-Immunoglobins have constant and variable region

-100s of gene segments code for the variable region of the antibody.

-DNA segments are put together to create an endless combination of constant and variable regions - gene rearrangement occurs in your lifetime!

TRANSCRIPTION

RNA Polymerase makes premRNA using the ‘elves’ - transcription factors (proteins)

Promotor

TRANSLATION

Many protein ‘factors’ are involved in translation as well

Levels of control –goals…..• 1) Changing DNA physically =>’ mRNA

making’ affected• 2) Changing access to DNA Promotor • 3) If mRNA is made; How long mRNA

hangs around; change which protein is made from one mRNA - (splicing); don’t use the mRNA

• 4) Change/destroy the protein after its made

How is How is gene expressiongene expression controlled? controlled?

•That is = if/what protein is made? How can you control this?

• Gene expression control = which genes are “on”• Levels of control – • 1) chromatin (DNA) packing and chromatin modification

- change access sites on DNA for RNA Polymerase so that its binding decreases/increases (epigenetics - layer of control above the genome - NOVA Video)

• 2) Transcription - when DNA makes mRNA• 3) Post-transcriptional - RNA processing, translation• 4) Post-translational - various alterations to the protein

product.

How is How is gene expressiongene expression controlled in you? (IMPORTANT)controlled in you? (IMPORTANT)

•When is the gene active (on or off)? That is what protein is made? How can you control this?

Fig. 19.7

1a) Level of packing is one way that gene expression is regulated.– Densely packed areas are inactivated.

(Heterochromatin)– Loosely packed areas are being

actively transcribed. (Euchromatin) -

- during mitosis

- during Interphase

• Interphase - chromatin fibers highly extended

• Mitosis - chromatin coils and condenses to form short, thick chromosomes.

Chromatin structure is based Chromatin structure is based on successive levels of DNA on successive levels of DNA

packingpacking

INTERPHASE

MITOSIS

• Histone proteins are responsible for the first level of DNA packaging.

• Their positively charged amino acids bind tightly to negatively charged DNA.

Beads on a string = a nucleosome, in which DNA winds around a core of histone proteins

Which stage do you see ‘beads on a string’? (Interphase)Are genes active? - Yes transcribed into mRNA!

• Next level of packing - ‘30 nm solenoid fiber’ – nucleosome fiber

• Has (DNA + HISTONES) with 6 nucleosomes per turn

Which stage do you see ‘30 nm fiber’? (Mitosis)Are genes active? - Yes transcribed into mRNA!

• The 30 nm fiber forms looped domains attached to a scaffold of nonhistone proteins.

Which stage do you see ‘looped domains’? (Mitosis)Are genes active? -No

• Chemical modifications of DNA bases:• A) DNA methylation is the attachment

by specific enzymes of methyl groups (-CH3) Inactive DNA is highly methylated compared to DNA that is actively transcribed.– Genomic imprinting is related to DNA methylation

1b) Chromatin 1b) Chromatin modifications (epigenetics)modifications (epigenetics)

DNA Methylation - add a methyl group to DNA Methylation - add a methyl group to make DNA less accessiblemake DNA less accessible

to RNA Polymeraseto RNA Polymerase

1b) Chromatin 1b) Chromatin modificationsmodifications

B) Histone acetylation (addition of an acetyl group -COCH3) and deacetylation

Acetylated histones grip DNA less tightly = ?More access to RNA Polymerase! SO,….

Epigenetics - DNA methylation and histone acetylation may be responsible for a lot of traits that are not just related to whether you have the gene/not. Example: If your gene is methylated you may never express the trait!

2) Control of Transcription – very 2) Control of Transcription – very important - to make or not make important - to make or not make

mRNAmRNA

Control elements - noncoding DNA segments that regulate transcription by binding transcription factors that are needed for RNA Polymerase binding. (TATA Box -Promotor, Activators in bacteria - ‘Enhancers’ in Eukaryotes, Repressors in bacteria - ‘Silencers’ in Eukaryotes)

• How can a DNA control element 100s of basepairs upstream of a gene regulate the access to RNA Polymerase?

• Bending of DNA enables transcription factors, activators (like steroid hormones), bound to enhancers to contact the complex at the promoter.

Fig. 19.9

Mostly positive gene regulation in eukaryotes!

• The hundreds of eukaryotic transcription factors follow only a few basic structural principles.– Each protein generally has a DNA-

binding domain that binds to DNA and a protein-binding domain that recognizes other transcription factors.

Fig. 19.10

• A) RNA processing – alternative splicing - controls which protein is made from one mRNA - mix-n-match introns/exons

3) Post-transcriptional mechanisms 3) Post-transcriptional mechanisms - so mRNA is made, what next?- so mRNA is made, what next?

3) Post-3) Post-transcriptional transcriptional mechanismsmechanisms

B) Life span of a mRNA molecule

Prokaryotic mRNA molecules degraded by enzymes after only a few minutes.

Eukaryotic mRNAs endure typically for hours or even days or weeks.

G AAAAA

5’ Cap Leader Trailer Poly A tail

L T

3) Post-transcriptional 3) Post-transcriptional mechanismsmechanisms

G AAAAA

5’ Cap Leader Trailer Poly A tail

L T

C) Translation - can be blocked by regulatory proteins that bind to 5’ leader region of mRNA.(prevents attachment of mRNA to ribosomes)

Protein factors required to initiate translation = simultaneous control of translation of all the mRNA in a cell.

• Processing of polypeptides to yield functional proteins.– This may include cleavage, chemical

modifications, and transport to the appropriate destination.

• Regulation may occur at any of these steps.

4) Post-translational 4) Post-translational mechanismsmechanisms

• The cell limits the lifetimes of normal proteins by selective degradation.

• Proteins intended for degradation are marked by the attachment of ubiquitin proteins.

• Giant proteosomes recognize the ubiquitin and degrade the tagged protein.

Fig. 19.12

CANCER REVIEW- read on CANCER REVIEW- read on your own - use these your own - use these

animationsanimations                                                            

• Cell cycle CONTROL events don’t work• Spontaneous mutations or

environmental influences (carcinogens)• Cancer-causing genes – oncogenes

(retroviruses), proto-oncogenes (in other organisms).

• What happens when proto-oncogenes/oncogenes are turned ‘ON’? (Ras gene)

• Cell will divide without stopping

Cancer results from Cancer results from genetic changes that affect genetic changes that affect

the cell cyclethe cell cycle

• Malignant cells often have significant changes in chromosomes

Fig. 19.13

• Tumor-suppressor genes -normal products inhibit cell division, repair DNA, control adhesion (p53).

• Mutations to these tumor suppressor genes = cancer

Are there genes that Are there genes that prevent cancer?prevent cancer?

Oncogene proteins and Oncogene proteins and faulty tumor-suppressor faulty tumor-suppressor

proteinsproteins

• The p53 gene, named for its 53,000-dalton protein product, is often called the “guardian angel of the genome”.

• Damage to the cell’s DNA acts as a signal that leads to expression of the p53 gene.

• The p53 protein is a transcription factor for several genes.– It can activate the p21 gene, which halts

the cell cycle.– It can turn on genes involved in DNA

repair.– When DNA damage is irreparable, the

p53 protein can activate “suicide genes” whose protein products cause cell death by apoptosis.

• More than one somatic mutation is generally needed to produce the changes characteristic of a full-fledged cancer cell.

• If cancer results from an accumulation of mutations, and if mutations occur throughout life, then the longer we live, the more likely we are to develop cancer.

3. Multiple mutations 3. Multiple mutations underlie the development underlie the development

of cancerof cancer

• Colorectal cancer, with 135,000 new cases in the U.S. each year, illustrates a multi-step cancer path.

• The first sign is often a polyp, a small benign growth in the colon lining with fast dividing cells.

• Through gradual accumulation of mutations that activate oncogenes and knock out tumor-suppressor genes, the polyp can develop into a malignant tumor.

Fig. 19.15

• About a half dozen DNA changes must occur for a cell to become fully cancerous.

• These usually include the appearance of at least one active oncogene and the mutation or loss of several tumor-suppressor genes.– Since mutant tumor-suppressor alleles are

usually recessive, mutations must knock out both alleles.

– Most oncogenes behave as dominant alleles.

• In many malignant tumors, the gene for telomerase is activated, removing a natural limit on the number of times the cell can divide.

• Viruses, especially retroviruses, play a role is about 15% of human cancer cases worldwide.– These include some types of leukemia,

liver cancer, and cancer of the cervix.

• Viruses promote cancer development by integrating their DNA into that of infected cells.

• By this process, a retrovirus may donate an oncogene to the cell.

• Alternatively, insertion of viral DNA may disrupt a tumor-suppressor gene or convert a proto-oncogene to an oncogene.

• The fact that multiple genetic changes are required to produce a cancer cell helps explain the predispositions to cancer that run in some families. – An individual inheriting an oncogene or a

mutant allele of a tumor-suppressor gene will be one step closer to accumulating the necessary mutations for cancer to develop.

• Geneticists are devoting much effort to finding inherited cancer alleles so that predisposition to certain cancers can be detected early in life.– About 15% of colorectal cancers involve

inherited mutations, especially to DNA repair genes or to the tumor-suppressor gene APC.• Normal functions of the APC gene include

regulation of cell migration and adhesion.

– Between 5-10% of breast cancer cases, the 2nd most common U.S. cancer, show an inherited predisposition.• Mutations to one of two tumor-suppressor

genes, BRCA1 and BRCA2, increases the risk of breast and ovarian cancer.