Lecture Notes for Final Exam

Spontaneous Mutations--->occur in all cells.

Induced Mutations--->produced when a cell or organism is exposed to a mutagenic agent (mutagen).

Errors in DNA Replication

Normally A, C, G, and T are in the keto form.
(Figs. 19-1, 19-2)

Errors occur during DNA replication when rare imino forms of A and C or rare enol forms of T and G are added by DNA pol. Pair with the wrong base.

DNA pol III editing function removes mismatches when the rare forms change back unless polymerization has moved past the mismatch.

Mutations will result if editing does not occur unless the change is repaired by another mechanism called mismatch repair.

TRANSITIONS--->purine for the other purine or pyrimidine for the other pyrimidine.

TRANSVERSIONS--->Pyrimidine substituted for a purine and vice versa.

FRAMESHIFTS--->Caused by deletion or insertion of one to a few nts. Slipped mispairing during DNA replication. (Fig. 19-4)

SPONTANEOUS LESIONS--->Mutations can occur due to DNA damage.

A. Depurination--->When the glycosidic bond between the base and the sugar is broken.

Resulting apurinic site (AP site) can’t specify a complementary base during replication.
10,000/cell/generation in mammals. (DNA repair)

B. Deamination--->loss of an amino group from the base.

Deamination of dC yields dU. dU pairs with dA (GC--->AT transition). (DNA repair)

C. Oxidative Damage--->Byproducts of aerobic metabolism produces compounds that cause oxidative DNA damage.

MECHANISMS OF MUTAGENESIS

A. Replace a base in DNA--->Molecules which are similar in structure to bases (base analogs) but have different pairing properties can replace the normal base in the DNA during DNA replication.

B. Specific Mispairing--->Some chemicals alter the structure of a base resulting in mispairing during replication.

C. Intercalating Agents--->Chemicals that are planar which mimic bases and can slip in (intercalate) between bases in the double helix. Results in frameshifts (insertions or deletions).

D. Loss of Pairing due to Chemical Alteration--->Chemical structure is altered so it can't pair with any base--->Replication block.
Lethal unless block is bypassed.

UV light can cause several types of DNA damage.

(e.g.) UV light can generate cyclobutane pyrmidine dimers which distorts the structure of DNA such that base pairing is not possible.

Most carcinogens cause this type of DNA damage.

(i.e.) Chemical Alteration

Cancer can be caused by mutations in genes whose protein products regulate cell division. If this check point is abolished it will lead to uncontrolled cell division (i.e.) cancer.

(e.g.) p53--->» 50% of all cancerous tissues contain mutations in the p53 gene.

p53 arrests cell division when it recognizes DNA mismatches.

(i.e.) involved in cell cycle control.

DNA REPAIR

Several enzymatic systems exist to repair various types of DNA damage.

In humans several disorders are caused by defects in DNA repair systems which can lead to cancer.

Classes of Repair Pathways (Systems)

I. Avoidance of Errors Before they Happen

Some enzymes neutralize damaging compounds.

(e.g.) Detoxification of molecules that cause oxidative damage to DNA.

Superoxide dismutase converts oxygen radicals to hydrogen peroxide.

Then catalase converts hydrogen peroxide to water.

II. Direct Reversal of DNA Damage

A. Cyclobutane pyrimidine dimers (UV) Repaired by a photoreactivating enzyme (PRE).

Requires visible light for the enzyme to work.
(Fig. 19-28)

B. Removal of alkyl groups added to bases.

Alkyltransferases responsible for direct reversal.
(e.g.) Methyltransferase of E. coli . (Fig. 19-29)

III. Excision-Repair Pathways

A. General Excision Repair (See Fig. 19-32)

Removal of altered bases, along with a stretch of neighboring bases, and then repairing the gap by DNA synthesis.

E. coli--->Endonuclease cuts on both sides of the damaged base removing ssDNA containing the damaged base(s). Gap filled in by DNA pol I. DNA ligase seals the nicks.

B. Specific Excision Repair

1. AP Endonuclease Repair Pathway

AP endonuclease removes AP site by breaking phosphodiester bonds at AP site. (Fig. 19-35)

General excision repair pathway takes over.

2. DNA Glycosylase Repair Pathway

DNA glycosylases recognize certain damaged bases and cleave the N-glycosidic bond between the base and the sugar leaving an AP site. (Fig. 19-34)
(e.g.) Uracil DNA Glycosylase

AP site is cleaved by AP endonuclease.
General excision repair pathway takes over.

IV. Postreplication Repair

Mismatch Repair---> (See Fig. 19-38)
(i.e.) DNA editing by DNA pol III did not occur.

1) Recognize mismatch.
2) Determine which mismatched base is incorrect.
3) Excise the incorrect base.
4) General excision repair takes over.

The second step is crucial!!!!!!!

DNA methylase methylates A residues following replication in the sequence: 5' GATC 3'

But it takes a few minutes for the newly synthesized strand to be methylated.

The old methylated strand is distinguished from the newly synthesized unmethylated strand.

An enzyme introduces a cleavage in the backbone of the unmethylated strand near the mismatch and adjacent to the nearest GATC sequence.

ssDNA gap is filled in by DNA pol I.

Ligase seals the nick.

 
Repair Defects and Human Disorders

Often lead to increased incidence of cancer.

Usually autosomal recessive (See table 19-5)

Xeroderma Pigmentosum caused by a defect (mutation) in one of the excision-repair enzymes. Most people die by the age of 30 from skin cancer.

Bloom syndrome--->lack of DNA ligase.

Greatly increased cancer rate (usually die by 30).

 
Chapter 20

Recombination occurs at regions of homology between chromosomes through the breakage and rejoining of DNA molecules.

General Homologous Recombination

Can occur at any large region of homology between chromosomes. (requires complementary nt sequence)
(Fig. 20-3)

Holliday Model

Heteroduplex molecules are formed during X-overs.

1. Creation of Branch (cross bridge)
2. Branch Migration
3. Resolution of the Holliday structure

Creation of the Branch (Fig. 20-4)

1. Two strands of the same polarity and sequence are cut. (i.e.) Both 5'-->3' or Both 3'-->5'

2. Free ends leave the complementary strands.

3. Ends associate with the complementary strands in the other duplex by base pairing.
(i.e.) 3’--->5’ with 5’--->3’ (See Fig. 20-22)

4. DNA ligation creates partial heteroduplexes.

Branch Migration--->occurs by strand transfer by unzipping the bases (breaking H-bonds) between the original duplex strands and rezipping the bases (forming H-bonds) between the X-over strands and the complementary strands of the other duplex.
(Fig. 20-4 and 20-5)

The ligated heteroduplex structure is termed the Holliday structure.
(See Fig. 20-19)

Resolution of Holliday Structure (Fig. 20-6)

The structure in 20-6a is the same structure shown in 20-4e but in extended form.

Rotate as shown in 20-6b resulting in 20-6c.

Resolution of the structure can occur by cleaving the strands in the horizontal or vertical plane (20-6d).

DNA ligation results in the recombinant DNA molecules (20-6f).

Horizontal cleavage does not result in a crossover of the flanking markers, but vertical cleavage does.
(i.e.) only vertical cleavage recognized as a X-over.

Note that in either case two regions of heteroduplex DNA exists.

Site-Specific Recombination--->Recombination at specific DNA sites.

(i.e.) specific DNA sequences are recognized by specific recombination enzymes (recombinases).

A. Phage Lambda Integration (Fig 20-27)

Lambda integrates into the E. coli chromosome between the gal and bio genes.

Requires a specific 15 bp sequence present in the Lambda and E. coli genomes.

Requires the lambda Int protein and an E. coli protein called Integration Host Factor (IHF).

Together these proteins form the recombinase.

Excision of lambda from the chromosome requires Int, IHF, and a lambda factor called Xis (excise).
 

B. Salmonella Phase Variation (Fig. 20-28)
Salmonella infects humans.

Site-specific recombination controls which of two alternate flagellin proteins are expressed.

The switch controls the surface antigen (flagellin) enabling Salmonella to evade host immune system.

DNA region can be inverted by Hin recombinase by recognizing inverted repeats (IR).

hin gene is located in the invertible region.

H1--->Encodes flagellin H1.
H2--->Encodes flagellin H2.
rH1--->Encodes a repressor protein that blocks expression of the H1 gene.

In one orientation the promoter allows expression of the H2 and rH1 genes.
Thus, expression of H2 occurs and expression of H1 is repressed.

When in the reverse orientation the promoter is in the wrong orientation for expression of H2 and rH1.
Thus, no H2 flagellin or rH1 repressor.

Therefore, H1 gene is derepressed resulting in expression of H1 flagellin.

 
Chapter 21

Transposable Genetic Elements

Genetic elements than can move or "transpose" from one position to another on the chromosome, or to a different chromosome.

Barbara McClintock:  Nobel prize

Bacteria

1. Insertion Sequences (IS)
2. Transposons (Tn)

When IS elements insert in the middle of a gene it inactivates that gene.

(e.g.) IS1, IS2, IS3, etc...
 
 

tnp	tnp--->transposase

Transposons--->Composite elements composed of 2 IS elements and a drug resistance gene between them. (Fig. 21-9)

Transposons can insert into naturally occurring plasmids that can be transferred into other bacterial strains by transformation.

Can lead to multiple drug resistant strains of bacteria. (Problem in a clinical setting)

Eukaryotic Transposable Elements

Yeast--->Ty Elements--->Transpose through an RNA intermediate.

Drosophila

1. Copia elements
2. FB (foldback) elements
3. P elements--->can be used to introduce DNA into the Drosophila germ line.

Maize (corn)--->Ac/Ds elements

Retroviruses--->ssRNA animal viruses. (e.g.) HIV

1. Eject RNA genome.

2. Reverse transcriptase synthesizes dsDNA from the ssRNA.

    A. First DNA strand synthesized using RNA as template.

    B. Second DNA strand synthesized using the first DNA strand as template while simultaneously degrading the RNA strand.

3. Integration into the mammalian genome.

 
Chapter 22

The Extranuclear Genome--->probably in all eukaryotes.

Chloroplasts and Mitochondria contain unique genes linked in their own circular chromosome.

Inheritance patterns are different from nuclear genes.

Different inheritance pattern in reciprocal crosses.
(Fig. 22-3)

(i.e.) White female x green male--->all white
        Green female x white male--->all green

Maternal inheritance--->Phenotype of all progeny are identical to the female parent. Male phenotype is irrelevant.

Inheritance comes from the egg cytoplasm containing chloroplasts.

Pollen (sperm) only add their nuclear DNA to zygote.
 

Chloroplast Genome (Fig. 22-23)

Chloroplast function--->photosynthesis

A. Genes involved in translation

    1. Some ribosomal proteins.
    2. tRNAs
    3. rRNAs
    4. Initiation factor
    RNA can't be transported across membrane.

B. Photosynthesis and electron transport chain subunits.

C. Transcription (RNA polymerase)

Requires communication between nuclear and chloroplast genomes since several chloroplast proteins are nuclear encoded.

Communication is necessary to coordinate expression of all genes necessary for chloroplast function.

Nuclear encoded proteins are transported into the chloroplast.

Mitochondrial Genome--->Inheritance pattern is similar to chloroplasts. (i.e.) Maternal

Mitochondrial function--->produce ATP

Genes encoded in mtDNA (Fig. 22-20)

A. Translation

    1. rRNAs
    2. tRNAs

B. Electron Transport Chain

    1. Subunits of the enzyme ATPase.
    2. Other proteins involved in electron transport.

The remaining genes required for mt function are nuclear encoded. Proteins are transported into the mitochondria.

Communication is required between the mitochondrial and nuclear genomes to coordinate expression. (Fig. 22-21)

Mitochondrial Genetic Code

Humans

1. AUA (isoleucine in nucleus, methionine in mt)
2. UGA (stop in nucleus, tryptophan in mt)
3. AGA & AGG (stop codons) Total of 4

Changes are different in other organisms. (e.g.) yeast

Mitochondria and chloroplasts arose in eukaryotic cells by the engulfment of primitive prokaryotic cells.

Evolution led to their present function.

Mitochondrial Diseases in Humans     (Fig. 22-25)

Caused by mutations in some of the genes encoding subunits of the electron transport chain resulting in defects in ATP production.

Results in defects of the brain, heart, muscle, kidney, and liver.

Condition is maternally inherited (egg cytoplasm).

Chapter 23

Developmental Gene Regulation

Fertilized egg--->adult organism

Regulatory proteins (genes) are responsible for differential gene expression throughout development and in various cell types.

Regulation of Transcription Initiation

Mechanisms exist to modulate when, where, and how much transcript is made in a given cell. Involves different classes of promoters, enhancers and silencers.

Tissue-Specific Enhancers

1. Transcription factor that binds to enhancer is only present in some cell types.

2. Transcription factors only active in some cells.

3. Other proteins block transcription factor binding.

4. Multiple enhancers, some only used at different times in development or only in certain cells.

Post-Transcriptional Regulation

3’ untranslated region (3’ UTR) of mRNA can affect mRNA stability and efficiency of translation.

Post-Translational Regulation--->Regulatory protein phosphorylation state controls activity.

Alternative mRNA Splicing

Different mRNA splicing patterns give rise to related but different proteins in different cell types.

(i.e.) Same gene but different proteins.

Exon 1--Intron 1--Exon 2--Intron 2--Exon 3
 

Tissue 1	Tissue 2
Exon 1--Exon 2--Exon 3	Exon 1--Exon 3

In tissue 2, both introns and exon 2 are removed as a single intron.

Gene Rearrangement

A. Gene Amplification

Eggshell production in Drosophila ( See Fig. 23-2)

Need large quantities of eggshell proteins in a short period of time.

Copy number of the eggshell genes increased through DNA rearrangements that only occur in follicle cells.

Eggshell genes amplified 20-80 fold by a specialized replication mechanism just before they need to be transcribed.

Extra copies only present when needed.

B. Antibody (Ab) Production

Antibodies contain 2 identical subunits, each containing light and heavy chains, held together by disulfide bridges. (Fig. 23-23)

Each Ab only recognizes a specific antigen.

Constant regions similar in all Abs. Variable and hypervariable regions differ.

Each B lymphocyte only makes a single Ab and each different B lymphocyte makes a different Ab.
Not enough DNA in genome to encode each Ab.

About 100-200 variable, 5 joining and 1 constant segment in the light chain gene. (Fig. 23-24)

Site-specific recombination events delete all but one V and J segment resulting in a V-J fusion.
RNA splicing results in a V-J-C fusion.
(200 x 5 = 1,000 combinations of light chains)

Heavy chains encode about 20 diversity segments in addition to the V and J segments. V-D-J-C fusion.
(1000 x 20 = 20,000 combinations of heavy chains)

Any light chain can pair with any heavy chain.
(1,000 x 20,000 = 20,000,000 Ab combinations)

Thus, Gene rearrangements allow Ab diversity.

 
Chapter 24

The Cell Cycle (G₁-S-G₂-M)

Rates of cell division are regulated to ensure sufficient cells to replace dying ones, and to prevent production of excess cells.

How does the cell "know" when to divide?

Progression of one stage of the cell cycle to the next depends on protein complexes consisting of a cyclin and a cyclin-dependent protein kinase (CDKs).

Cyclins only expressed at specific cell cycle stages.

Protein kinases phosphorylate specific proteins.

Cyclins tether the CDK to the target protein so it can be phosphorylated.

The timing of gene expression of different cyclins results in phosphorylation of different proteins at different times.

Phosphorylation initiates a chain of events leading to the activation of transcription factors that promote transcription of genes required for the next stage of the cell cycle.

Sequential activation of different CDK-cyclins leads to sequential activation of transcription factors and in turn progression of the cell cycle.
CDK-cyclin-binding proteins inhibit the kinase activity of the CDK until the cell is ready to go to the next stage of the cell cycle. (e.g.) p53/p21

Various checkpoints serve as monitors of the status of DNA replication, spindle apparatus formation, etc.

The key is the negative regulators that inhibit the kinase activity of the CDK-cyclin complexes.

Intercellular Communication

Cells communicate with each other via signal transduction pathways. (Fig. 24-17b)

Signal Transduction--->A small molecule (ligand) is released from one cell and interacts with a membrane bound receptor of another cell. Results in phosphorylation of the receptor.

Often the next step in propagating the signal is to activate a G-protein (Fig. 24-18).

G-proteins cycle between GDP bound (inactive) and GTP bound (active) forms.

The active G-protein leads to phosphorylation of transcription factors leading to genes being turned on or off.
(Often occurs through a phosphorylated intermediate)

CANCER

Cancerous cells are uncoupled from the regulatory mechanisms that keeps cell proliferation in check.

Caused by multiple mutations in a single cell that causes it to proliferate out of control.

Some of these mutations are inherited, others originate in the somatic cell lineage.

Dominant Oncogene Mutations

Mutations resulting in proteins that are activated when they shouldn’t be.

Typically these proteins are components of intracellular communication pathways such that the cell always behaves as if it is receiving a signal to proliferate. (e.g.) Ras (Fig. 24-25)

Ras (G-protein) mutations results in Ras always being in the GTP bound (active) form. Results in the continuous propagation of the signal that promotes cell proliferation.
 

Recessive Tumor Suppresser Genes

Mutations in genes whose proteins normally contribute to the inhibition of cell proliferation.
(2 classes)
A. Proteins involved in inhibiting progression of the cell cycle. (i.e.) inhibitor protein is inactive.
B. Proteins involved in the repair of DNA damage.

p53 is a DNA-binding regulatory protein that indirectly monitors DNA damage and prevents progression of the cell cycle until the DNA is repaired.

p53 mutations in >50% of cancerous tissues.

p53 indirectly recognizes mismatches in damaged DNA and activates p21. p21 binds to and inhibits the kinase activity of CDK-cyclin complexes, thereby blocking cell cycle progression. (Fig. 24-11)

The block in the cell cycle continues until the mismatches are repaired.

In the absence of functional p53, cell division occurs in the absence of DNA repair leading to an increase in mutations.

This increases the chance of mutations in other genes involved in controlling the cell cycle, leading to uncontrolled cell growth--->cancer.

Chapter 25

DEVELOPMENTAL GENETICS

Single cell (fertilized egg)--->adult organism composed of thousands, millions or trillions of cells organized into tissues and organs.

Developmental biology--->study of the processes responsible for the transfiguration of a fertilized egg to an adult.

Cell determination--->Cells adopt specific fates or the capacity to differentiate into specific types of cells (gradual process).

(i.e.) periodic decisions are made in each lineage to more exactly specify the fates of the daughter cells.

In general, the same basic set of regulatory proteins, govern the major developmental events in many, if not all, higher animals.

Totipotent--->a cell that is totally uncommitted as to its cell fate. (e.g.) zygote (fertilized egg)

At what point do cells begin to become irreversibly committed to particular cell fates?

Many highly differentiated organisms can regenerate new organs and tissues.
(e.g.) starfish arms, damaged human liver

PLANTS

The growth of an entire plant from a single cell can be achieved in the laboratory.

(e.g.) carrot from a single cell
Not observed in nature.

Totipotency in animal cells

Frog (Rana)

1. Remove nucleus from unfertilized egg resulting in an enucleated egg.
2. Inject nucleus from a blastula stage cell or from a later stage of development (gastrula).
3. Blastula nucleus resulted in the development of a normal adult frog but not the gastrula nucleus.

(i.e.) blastula nucleus--->totipotent
(i.e.) gastrula nucleus--->differentiated

African clawed toad (Xenopus)

Nuclei from highly differentiated tadpole intestinal cell allowed complete development of an enucleated egg.

Sheep (Hello Dolly!)--->cloned a sheep using the nucleus from a differentiated adult somatic cell and an enucleated egg.

Human Cloning???!!!

Pattern Formation--->Establishing the body plan.

C. elegans--->nematode (roundworm)

Adult is composed of only a few thousand cells.

embryonic stage-->4 larval stages-->adult (50 hrs)

Cell lineage has been traced from egg to adult.

At the 16 cell stage the dorsal-ventral (top-bottom) (D/V) and anterior-posterior (head-tail) (A/P) axes of the nematode are established.

Cell position is also important because neighboring cells communicate to each other via signal transduction.

The potential fate of a cell becomes progressively restricted as cell divisions continue.
(See Fig. 24-5--->cell lineage or fate maps)

Drosophila

Oogenesis--->generation of the egg cell.

Stem cell--->primary oocyte-->16 cells, one of which becomes the oocyte itself.

The other 15 cells are nurse cells that dump their cytoplasmic contents into the oocyte.
(Syncitium is formed by nuclear division without complete cell division.)
Polar granules (RNA & protein) form at the posterior pole of the oocyte.

Then pole cells form at the posterior end, which form the entire germ line of the fly.

Formation of the Body Plan

The Drosophila larva is highly differentiated along the A/P & D/V axes.

Segmentation Pattern--->10 hrs after fertilization 14 body segments are formed along the A/P axis.

(3 head, 3 thoracic, 8 abdominal)
Each segment gives rise to body parts of the adult.

How are segmentation patterns established?

The egg contributes localized gene products that establish polarity along the A/P & D/V axes, which ultimately determines cell fates.

Cell fate is determined during development by the selective local activation of a set of master regulatory proteins due to the concentration gradient of localized determinants (RNA & protein) established in the egg. (e.g.) polar granules

A/P Concentration Gradients (Fig. 25-21)

bcd (bicoid)--->bcd mRNA localized to the anterior tip of the embryo.

bcd protein diffuses forming a concentration gradient from anterior to posterior.

nos (nanos)--->nos mRNA localized to posterior.

nos protein diffuses forming a concentration gradient from posterior to anterior.

Similar gradients define the D/V axis.

Gradients of protein products establish polarity (different geographic positions) along the A/P & D/V axes of the Drosophila embryo.

Hierarchy of Gene Expression

As development continues a hierarchy of gene expression establishes the number of body segments, then subsegments, then segment identity, etc...

Segmentation Pattern

bcd encodes a transcriptional activator that activates a set of genes called gap genes.

1. hb (hunchback)--->expressed if high [bcd].

2. kr (Kruppel)--->expressed if low [bcd].

3. kni (Knirps)--->expressed if bcd is absent.

Gap genes encode the next layer of regulatory proteins (transcription factors).

Gap genes regulate expression of the pair-rule genes, which encode regulatory proteins that turn on expression of segment-polarity genes.

Some of the segment-polarity genes also encode regulatory proteins while others encode different classes of proteins.

bcd--->gap--->pair-rule--->segment-polarity.

A similar cascade occurs for the nos gene
(Fig. 25-21) and the D/V axis.

Parallel cascades establish segment identity.

Homeotic Genes--->mutations in homeotic genes change the segmental identity into that of another.

(i.e.) same number of segments but a duplication of one segment with another segment missing.

All homeotic genes encode transcription factors.

Gap gene proteins activate homeotic genes.

Thus, the number and identity of segments are determined in the early embryo.

The antp mutation (antennapedia) results in legs instead of antenna in the head.

(See the figure on the first page of chapter 25)

Another mutation doubles the number of wings (bithorax). (See Fig. 25-22)

Applications to Higher Animals

Homeotic (segment identity) genes exist in humans and mice, etc... (i.e.) homologous genes

Developmental strategies in animals are ancient.

Animals as divergent as Drosophila and humans develop using the same regulatory switches.