Cell Cycle

During M phase an abrupt change in the biochemical state of the celloccurs at the transition from metaphase to anaphase; a cell can pause in metaphase before this transition point, but once the point is passed, the cell will carry on smoothly to the end of mitosis and through cytokinesis into interphase.

The Cell CycleDuring development from stem to fully differentiated, cells in the body alternately divide (mitosis) and "appear" to be resting (interphase). This sequence of activities exhibited by cells is called the cell cycle.Interphase, which appears to the eye to be a resting stage between cell divisions, is actually a period of diverse activities. Those interphase activities are indispensible in making the next mitosis possible. Interphase: Interphase generally lasts at least 12 to 24 hours in mammalian tissue. During this period, the cell is constantly synthesizing RNA, producing protein and growing in size. By studying molecular events in cells, scientists have determined that interphase can be divided into 4 steps: Gap 0 (G0), Gap 1 (G1), S (synthesis) phase, Gap 2 (G2). Gap 0 (G0): There are times when a cell will leave the cycle and quit dividing. This may be a temporary resting period or more permanent. An example of the latter is a cell that has reached an end stage of development and will no longer divide (e.g. neuron).Gap 1 (G1): Cells increase in size in Gap 1, produce RNA and synthesize protein. An important cell cycle control mechanism activated during this period (G1 Checkpoint) ensures that everything is ready for DNA synthesis. (Click on the Checkpoints animation, above.)

S Phase: To produce two similar daughter cells, the complete DNA instructions in the cell must be duplicated. DNA replication occurs during this S (synthesis) phase.

Gap 2 (G2): During the gap between DNA synthesis and mitosis, the cell will continue to grow and produce new proteins. At the end of this gap is another control checkpoint (G2 Checkpoint) to determine if the cell can now proceed to enter M (mitosis) and divide.

Mitosis or M Phase: Cell growth and protein production stop at this stage in the cell cycle. All of the cell's energy is focused on the complex and orderly division into two similar daughter cells. Mitosis is much shorter than interphase, lasting perhaps only one to two hours. As in both G1 and G2, there is a Checkpoint in the middle of mitosis (Metaphase Checkpoint) that ensures the cell is ready to complete cell division. Actual stages of mitosis can be viewed at Animal Cell Mitosis.

http://www.cellsalive.com/mitosis.htm

Regulation of the Cell CycleThe length of the cell cycle varies depending upon the type of cell and is extremely important in many aspects of biology, especially development.. The timing differences are primarily in the G1 phase, which may become very long (as in G0 phase). Variation in the length of the cell cycle depends upon cell cycle checkpoints which control the cell’s progression.

Restriction point

http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-19/1930.jpg

These controls make certain that the cell’s machinery is operating properly with the correct timing. In addition, the cell cycle control mechanisms must make certain that each phase of the cycle is completed properly such that the next steps are prepared for. The control system must be able to respond to certain conditions that may affect the cell cycle. The cell cycle checkpoints determine if a cell is ready to progress to the next stage. Late in the G1 phase, the G1 checkpoint determines if the cell will enter the following S phase. In animals, the G1 checkpoint or restriction point, is largely controlled by growth factors. The G2 checkpoint determines if the cell will enter the M phase and requires the proper completion of DNA synthesis. The third cell cycle checkpoint is the spindle assembly checkpoint which occurs between metaphase and anaphase and requires the proper attachment of all the chromosomes to the spindle apparatus.

The essential processes, such as DNA replication andmitosis and cytokinesis, are triggered by a central cell-cycle control system. The control system is drawn as an indicator that rotates clockwise, triggeringessential processes when it reaches specific points on the outer dial.

• The cell-cycle control system is based on two key families of proteins. The first is the family of cyclin-dependent protein kinases (Cdk for short), which induce downstream processes by phosphorylating selected proteins on serines and threonines.

• The second is a family of specialized activating proteins, called cyclins, that bind to Cdk molecules and control their ability to phosphorylate appropriate target proteins

There are two main classes of cyclins: mitotic cyclins, which bind to Cdk molecules during G2 and are required for entry into mitosis, and G1 cyclins, which bind to Cdk molecules during G1 and are required for entry into S phase

The events that drive the cell into mitosis are as follows: Mitotic cyclin accumulatesgradually during G2 and binds to Cdk to form a complex known as M-phase-promoting factor (MPF). This complex is at first inactive, but through the action of other enzymes that phosphorylate and dephosphorylate it, it is converted to an active form. The ultimate activation of MPF is almost explosive. This is believed to be due to a positive feedback mechanism whereby active MPF increases the activity of the enzymes that activate MPF: thus the concentration of active MPF builds up at an accelerating pace until a critical flashpoint is reached, whereupon a flood of active MPF triggers the downstream events that propel the cell into mitosis. MPF is inactivated equally suddenly by the degradation of mitotic cyclin at the metaphase-anaphase boundary, enabling the cell to exit from mitosis..

The two key subunits of MPF.

Principle CDKs and cyclins active at each stage of mammalian cycle

G1 CDK: CDK2,3,4,6 Cyclins D1-3CDK:CDK2 Cyclins: E class [ two CDK-cyclin systems are active in

G1 of the cell cycle.CDK2-cyclinE complex required for G1-S transiionThe other CDKs and D cyclins are responsible for interpreting growth signals for the environment And act at the restriction point to channel the cell into either late G1 or G0 phase.]

S CDK:CDK2Cyclins : A class required for DNA replication

G2/M phase CDK:CDK1Cyclins : A and B class

The mammalian CDK1-cyclin B complex is MPFM phase cyclins are required for Mitosis

Cyclins and Cdk proteins in the standard vertebrate cell cycle. Vertebrates have many different cyclin genes and many different cdk genes. Their products act in different cyclin/Cdk combinations at different stages of the cycle. The diagram shows only a few of thesemolecules and is speculative. The roles of Cdk2 and cyclin A, in particular, are still uncertain

Summary of feedback, size, and damage controls in the cell cycle. The red T barsrepresent checks on progress of the cell-cycle control system arising from intracellular processesthat are uncompleted or deranged.

MUTATIONSChanges in DNA that affect genetic

information

Gene Mutations

• Point Mutations – changes in one or a few nucleotides– Substitution

• THE FAT CAT ATE THE RAT• THE FAT HAT ATE THE RAT

– Insertion• THE FAT CAT ATE THE RAT• THE FAT CAT XLW ATE THE RAT

– Deletion• THE FAT CAT ATE THE RAT• THE FAT ATE THE RAT

What is a mutation?

• Mutation – a change in the DNA of an organism• Germ-line mutation – occur in gametes of organism

– Passed on to offspring, do not affect the organism

• Somatic mutation – mutations in the organism’s body– Affect the organism, but not passed on to offspring

– Ex. Skin cancer, leukemia, any cancer

Gene Mutations• Frameshift Mutations – shifts the

reading frame of the genetic message so that the protein may not be able to perform its function.– Insertion

• THE FAT CAT ATE THE RAT• THE FAT HCA TAT ETH ERA T

– Deletion• THE FAT CAT ATE THE RAT• TEF ATC ATA TET GER AT

H

H

Chromosome Mutations• Changes in number and structure of entire

chromosomes

• Original Chromosome ABC * DEF

• Deletion AC * DEF

• Duplication ABBC * DEF

• Inversion AED * CBF

• Translocation ABC * JKL

GHI * DEF

Chromosome Mutations

• Down Syndrome– Chromosome 21 does not

separate correctly.

– They have 47 chromosomes in stead of 46.

– Children with Down Syndrome develop slower, may have heart and stomach illnesses and vary greatly in their degree of inteligence.


• Cri-du-chat– Deletion of material on 5th

chromosome– Characterized by the cat-like

cry made by cri-du-chat babies– Varied levels of metal

handicaps

Sex Chromosome Abnormalities

• Klinefelter’s Syndrome– XXY, XXYY, XXXY

– Male

– Sterility

– Small testicles

– Breast enlargement

Sex Chromosome Abnormalities• XYY Syndrome

– Normal male traits

– Often tall and thin

– Associated with antisocial and behavioral problems

Sex Chromosome Mutations

• Turner’s Syndrome– X

– Female

– sex organs don't mature at adolescence

– sterility

– short stature

Significance of Mutations• Most are neutral

• Eye color

• Birth marks

• Some are harmful• Sickle Cell Anemia

• Down Syndrome

• Some are beneficial• Sickle Cell Anemia to Malaria

• Immunity to HIV

What causes mutations?• Mutagens – agent that causes

mutations to occur within a cell. – Ex. Ionizing radiation, Base analogs,

Intercalating agents, and Bromine

Types of mutations• Chromosome mutations – changes in the structure

of a chromosome or loss of an entire chromosome.– Deletion – loss of piece of DNA due to chromosomal

breakage– Duplication – Chromosomes steal part of homologs and

have both alleles for each gene involved– Inversion – piece of DNA breaks off and reattaches itself

in opposite direction– Translocation – chromosome breaks off and reattaches to

a non-homologous chromosome– Nondisjunction – chromosome does not properly

separate from its homolog during meiosis

Nondisjunction

• Results in gametes receiving to many or too few chromosomes

• Ex. Down Syndrome = trisomy of chromosome 21

What kind of chromosomal mutation is this?

Original chromosome

A. Duplication

B. Translocation

C. Inversion

D. Deletion

Gene Mutations

• May involve a large section of DNA or a single nucleotide within a codon

• Point mutation – the substitution or change of a single nucleotide

• Insertion or Deletion – one nucleotide is removed from or added to a sequence

• Frame shift mutation – occurs when codons are incorrectly grouped

DNA sequence

↓

mRNA sequence

↓

Polypeptide

Gene mutations which affect only one gene

Transcription

Translation

© 2010 Paul Billiet ODWS

http://www.saburchill.com/IBbiology/bio_hp.html

DNA (antisense strand)

mRNA

Polypeptide

Normal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids

The antisense strand is the DNA strand which acts as the template for mRNA transcription


Point Mutation

• Can result in – No effect - the

protein structure is not changed

– Missense – one amino acid is replaced by another

– Nonsense – prematurely stop codon in amino acid sequence

Point mutation

• Ex. Sickle cell anemia –mutation in a single nucleotide that causes the malformation of the hemoglobin molecule which carries oxygen to our cells

Mutations: SubstitutionsSubstitution mutation

GGTCACCTCACGCCA

↓

CCAGUGGAGUGCGGU

↓

Pro-Arg-Glu-Cys-Gly

Substitutions will only affect a single codonTheir effects may not be serious unless they affect an amino acid that is essential for the structure and function of the finished protein molecule (e.g. sickle cell anaemia)

Normal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids


The genetic code is degenerate

A mutation to have no effect on the phenotype

Changes in the third base of a codon often have no effect.


No changeNormal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids

Substitution mutation

GGTCTTCTCACGCCA

↓

CCAGAAGAGUGCGGU

↓

Pro-Glu-Glu-Cys-Gly


DisasterNormal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids

Substitution mutation

GGTCTCCTCACTCCA

↓

CCAGAAGAGUGAGGU

↓

Pro-Glu-Glu-STOP


Mutations: Inversion

Normal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids

Inversion mutation

GGTCCTCTCACGCCA

↓

CCAGGAGAGUGCGGU

↓

Pro-Gly-Glu-Cys-Gly

Inversion mutations, also, only affect a small part of the gene


Mutations: Additions

Normal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids

Addition mutation

GGTGCTCCTCACGCCA

↓

CCACGAGGAGUGCGGU

↓

Pro-Arg-Gly-Val-Arg

A frame shift mutation


Mutations: Deletions

Normal gene

GGTCTCCTCACGCCA

↓

CCAGAGGAGUGCGGU

Codons

↓

Pro-Glu-Glu-Cys-Gly

Amino acids

Deletion mutation

GGTC/CCTCACGCCA

↓

CCAGGGAGUGCGGU

↓

Pro-Gly-Ser-Ala-Val

A frame shift mutation


Insertion and

Deletion

The removal or addition of a nucleotide base to a sequence usually results in a frameshift mutation.

Mutations of haemoglobin • Haemoglobin is a tetramer = 2 and 2 -chains• The genes for these polypeptides are found on

different chromosomes• The -chain gene is found on chromosome 11• The -chain gene is found on chromosome 16• The nucleotide sequences have been worked out• Several inherited diseases occur on the -chain,

which contains 146 amino acids.


haemoglobin sense strand cDNA sequence

• cDNA (complementary DNA) is obtained by back-transcribing the mRNA used to translate the polypeptide

• So cDNA has no introns

• This is done using reverse transcriptase enzyme.


ATG GTG CAT CTG ACT CCT GAG GAG AAG TCT GCC GTT ACT GCC CTG TGG GGC AAG GTG AAC GTG GAT GAA GTT GGT GGT GAG GCC CTG GGC AGG CTG CTG GTG GTC TAC CCT TGG ACC CAG AGG TTC TTT GAG TCC TTT GGG GAT CTG TCC ACT CCT GAT GCT GTT ATG GGC AAC CCT AAG GTG AAG GCT CAT GGC AAG AAA GTG CTC GGT GCC TTT AGT GAT GGC CTG GCT CAC CTG GAC AAC CTC AAG GGC ACC TTT GCC ACA CTG AGT GAG CTG CAC TGT GAC AAG CTG CAC GTG GAT CCT GAG AAC TTC AGG CTC CTG GGC AAC GTG CTG GTC TGT GTG CTG GCC CAT CAC TTT GGC AAA GAA TTC ACC CCA CCA GTG CAG GCT GCC TAT CAG AAA GTG GTG GCT GGT GTG GCT AAT GCC CTG GCC CAC AAG TAT CAC TAA

Methionine initiator

Nonsense terminator© 2010 Paul Billiet ODWS

Mutation Codon Change to DNA sense strand

Change in Amino Acid

S (sickle cell anaemia)

6 GAG to GTG Glu to Val

C (cooley’s syndrome)

6 GAG to AAG Glu to Lys

GSan Jose 7 GAG to GGG Glu to Gly

E 26 GAG to AAG Glu to Lys

MSaskatoon 63 CAT to TAT His to Tyr

MMilwauki 67 GTG to GAG Val to Glu

OArabia 121 GAA to GTA Glu to Val


Sickle Cell Anaemia

Blood smear (normal)Image Credit: http://lifesci.rutgers.edu/~babiarz/

Sickle cell anemiaImage Credit: http://explore.ecb.org/

What affect does this mutation have on the function of the protein?

• Proteins are folded in a specific fashion according to the amino acid sequence it contains.

• This would cause the function of the protein to be severely reduced or not functional at all.

Hemophilia

A sex-linked, recessive genetic disorder that affects the individuals ability to clot blood.

Mutations

• Any change in the DNA sequence of an organism is a mutation.

• Mutations are the source of the altered versions of genes that provide the raw material for evolution.

• Most mutations have no effect on the organism, especially among the eukaryotes, because a large portion of the DNA is not in genes and thus does not affect the organism’s phenotype.

• Of the mutations that do affect the phenotype, the most common effect of mutations is lethality, because most genes are necessary for life.

• Only a small percentage of mutations causes a visible but non-lethal change in the phenotype.

Mutations are Random• A central tenet of biology is that the flow of information from DNA to protein is

one way. DNA cannot be altered in a directed way by changing the environment. Only random DNA changes occur.

• The “fluctuation test”, an early experiment in bacterial genetics (Luria and Delbruck, 1943) showed that variations in bacterial phenotypes are due to pre-existing mutations and not due to physiological changes induced by environmental conditions.

• A large batch of E. coli is infected with phage T1. Most are lysed by the phage, but a few survive. Are the survivors a few rare individuals who managed to induce their T1-protection mechanisms on time, or are they pre-existing mutants?

– --if protection is a physiological condition induced by the presence of the phage, then the percentage of survivors will be the same among all small batches of the cells; all cells are genetically identical.

– if protection is due to pre-exisiting mutations, then some small batches will have many survivors (descendants of T1-resistant mutants), while most other batches will have few or no survivors (there were no T1-resistant mutants in the original batch).

• Result: some batches had many survivors, but most batches had few or none. T1-resistant mutants existed in the population before T1 was added.

Fluctuation Test

Types of DNA Change• The simplest mutations are base changes, where one base is

converted to another. These can be classified as either: – --“transitions”, where one purine is changed to another purine (A -> G,

for example), or one pyrimidine is changed to another pyrimidine (T -> C, for example).

– “transversions”, where a purine is substituted for a pyrimidine, or a pyrimidine is substituted for a purine. For example, A -> C.

• Another simple type of mutation is the gain or loss f one or a few bases.

• Larger mutations include insertion of whole new sequences, often due to movements of transposable elements in the DNA or to chromosome changes such as inversions or translocations.

• Deletions of large segments of DNA also occurs.

Types of Mutation• Mutations can be classified according to their effects on the protein (or

mRNA) produced by the gene that is mutated.• 1. silent mutations (synonymous mutations). Since the genetic code is

degenerate, several codons produce the same amino acid. Especially, third base changes often have no effect on the amino acid sequence of the protein. These mutations affect the DNA but not the protein. Therefore they have no effect on the organism’s phenotype.

• 2. missense mutations. Missense mutations substitute one amino acid for another. Some missense mutations have very large effects, while others have minimal or no effect. It depends on where the mutation occurs in the protein’s structure, and how big a change in the type of amino acid it is. – Example: HbS, sickle cell hemoglobin, is a change in the beta-globin gene,

where a GAG codon is converted to GUG. GAG codes for glutamic acid, which is a hydrophilic amino acid that carries a -1 charge, and GUG codes for valine, a hydrophobic amino acid. This amino acid is on the surface of the globin molecule, exposed to water. Under low oxygen conditions, valine’s affinity for hydrophobic environments causes the hemoglobin to crystallize out of solution.

More Types of Mutation• 3. Nonsense mutations convert an amino acid into a stop codon. The effect

is to shorten the resulting protein. Sometimes this has only a little effect, as the ends of proteins are often relatively unimportant to function. However, often nonsense mutations result in completely non-functional proteins.– an example: Hb-β McKees Rock. Normal beta-globin is 146 amino acids long.

In this mutation, codon 145 UAU (codes for tyrosine) is mutated to UAA (stop). The final protein is thus 143 amino acids long. The clinical effect is to cause overproduction of red blood cells, resulting in thick blood subject to abnormal clotting and bleeding.

• 4. Sense mutations are the opposite of nonsense mutations. Here, a stop codon is converted into an amino acid codon. Since DNA outside of protein-coding regions contains an average of 3 stop codons per 64, the translation process usually stops after producing a slightly longer protein.– Example: Hb-α Constant Spring. alpha-globin is normally 141 amino acids

long. In this mutation, the stop codon UAA is converted to CAA (glutamine). The resulting protein gains 31 additional amino acids before it reaches the next stop codon. This results in thalassemia, a severe form of anemia.

Frameshifts and Reversions• Translation occurs codon by codon, examining nucleotides in groups of 3. If a

nucleotide or two is added or removed, the groupings of the codons is altered. This is a “frameshift” mutation, where the reading frame of the ribosome is altered.

• Frameshift mutations result in all amino acids downstream from the mutation site being completely different from wild type. These proteins are generally non-functional.

– example Hb-α Wayne. The final codons of the alpha globin chain are usually AAA UAC CGU UAA, which code for lysine-tyrosine-arginine-stop. In the mutant, one of the A’s in the first codon is deleted, resulting in altered codons: AAU ACC GUU AAG, for asparagine-threonine-valine-lysine. There are also 5 more new amino acids added to this, until the next stop codon is reached.

• A “reversion” is a second mutation that reverse the effects of an initial mutation, bringing the phenotype back to wild type (or almost).

• Frameshift mutations sometimes have “second site reversions”, where a second frameshift downstream from the first frameshift reverses the effect.

– Example: consider Hb Wayne above. If another mutation occurred that added a G between the 2 C’s in the second codon, the resulting codons would be: AAU ACG CGU UAA, or asparagine-threonine-arginine-stop. Note that the last 2 codons are back to the original. Two amino acids are still altered, but the main mutational effect has been reverted to wild type.

mRNA Problems• Although many mutations affect the protein sequence directly, it is

possible to affect the protein without altering the codons.• Splicing mutations. Intron removal requires several specific sequences.

Most importantly, introns are expected to start with GT and end in AG. Several beta globin mutations alter one of these bases. The result is that one of the 2 introns is not spliced out of the mRNA. The polypeptide translated from these mRNAs is very different from normal globin, resulting in severe anemia.

• Polyadenylation site mutations. The primary RNA transcript of a gene is cleaved at the poly-A addition site, and 100-200 A’s are added to the 3’ end of the RNA. If this site is altered, an abnormally long and unstable mRNA results. Several beta globin mutations alter this site: one example is AATAAA -> AACAAA. Moderate anemia was the result.

Trinucleotide Repeats• A fairly new type of mutation has been described, in which a particular codon is

repeated.• During replication, DNA polymerase can “stutter” when it replicates several

tandem copies of a short sequence. For example, CAGCAGCAGCAG, 4 copies of CAG, will occasionally be converted to 3 copies or 5 copies by DNA polymerase stuttering.

• Outside of genes, this effect produces useful genetic markers called SSR (simple sequence repeats).

• Within a gene, this effect can cause certain amino acids to be repeated many times within the protein. In some cases this causes disease

• For example, Huntington’s disease is a neurological disease that generally strikes in middle age, producing paranoia, uncontrolled limb movements, psychosis, and death. Woody Guthrie, a folk singer from the 1930’s, had this disease.

• The Huntington’s disease gene normally has between 11 and 33 copies of CAG (codon for glutamine) in a row. The number occasionally changes. People with HD have 37 or more copies, up to 200). The rate of copy number change is much higher in HD people--too many copies makes the repeated sequence more subject to DNA polymerase stuttering during meiosis.

• Interestingly, the age of onset of the disease is related to the number of CAG repeats present: the more repeats, the earlier the onset.

Germinal vs. Somatic Mutations

• Back up to the phenotype level• Mutations can occur in any cell. They only affect future

generations if they occur in the cells that produce the gametes: these are “germinal” or “germ line” mutations.

• Mutations in other cells are rarely noticed, except in the case of cancer, where the mutated cell proliferates uncontrollably. Mutations in cells other than germ line cells are “somatic” mutations.

• A human body contains 1013 - 1014 cells approximately. The average mutation rate for any given nucleotide is about 1 in 109. That is, on the average 1 cell in 109 has that particular nucleotide altered. This means that virtually every possible base change mutation occurs repeatedly in our body cells.

Retinoblastoma

• Retinoblastoma is a hereditary form of cancer that illustrates the interaction between somatic and germinal mutations.

• This disease affects the retinoblasts, cells that are precursors to the retinal cells. Retinoblasts exist in the eyes until about 3 years of age. Thus, retinoblastoma always occurs by about this age.

• There are 2 forms of retinoblastoma: the hereditary form, which is almost always bilateral (affects both eyes), and the spontaneous form, which almost always affects just one eye. Neither parent has the disease in spontaneous cases.

• Why should the hereditary form affect both eyes while the spontaneous form affects only 1 eye?

RB Explanation

• The retinoblastoma gene, Rb, is a “tumor suppressor” gene. Individual cells become cancerous if they lack this gene, or if both copies are defective mutants: Rb- Rb-.

• The mutation rate for the Rb gene is about 10-6, which means that about 1 copy in 106 will spontaneously go from Rb+ to Rb-.

• The retina contains about 108 retinoblasts.

• So, since we are diploid, a cell must go from Rb+ Rb+ to Rb+ Rb- and then to Rb- Rb- to become cancerous. This requires 2 independent mutations. Since the chance of 2 independent events is the product of the individual chances, the chance of a Rb+ Rb+ cell becoming Rb- Rb- is 10-6 * 10-6 = 10-12. Given that there are 108 retinoblasts per person, this would occur about 1 time in 10,000 people. This is about the rate of occurrence of spontaneous RB. That is, about 1 person in 10,000 will have 1 cell that is homozygous mutant, resulting in RB in one eye only.

More RB Explanation

• People with hereditary RB inherit one mutant allele. Every cell in their bodies starts out Rb+ Rb-. It takes only a single somatic mutation to convert a cell to Rb- Rb-.

• Given that the mutation rate is about 10-6 and the number of cells per retina is about 108, it is almost a certainty that multiple tumors will start in both eyes of people with hereditary retinblastoma.

Determining the Human Mutation Rate

• Not easy. Need to look at dominant or co-dominant mutations, because humans can’t be test-crossed.

• One study in Michigan looked for dominant mutations known to be caused by a single gene, with no known phenocopies that are fully expressed and highly penetrant. They looked at achondroplasia (dwarfism) and retinoblastoma.

• Achondroplasia: found 7 new (non-hereditary) cases among 242,257 births, for a rate of 1.4 x 10-5 new mutations per allele per generation.

• Retinoblastoma: found 52 new cases among 1,054,985 births, for a rate of 2.3 x 10-5 new mutations per allele per generation.

• Protein electrophoresis: found 4 new alleles among 1,226,099 examined, for a rate of 3.3 x 10-6 new mutations per allele per generation. A bit lower than for the dominant mutations, but these genes are smaller.

More on Mutation Rate

• In 1945, at the end of World War 2, the US detonated 2 nuclear weapons over Hiroshima and Nagasaki Japan.

• Extensive studies were done of the genetic effects on the survivors. A number of genes were examined by looking at the proteins they produced by gel electrophoresis.

• Radiation levels: a dose of 1 Sievert (Sv) is equal to 100 rem in the old terminology. – A dose of radiation that would kill 50% of people within 60days is about

5 Sv. – Natural exposure in Chicago are is about 1 milliSievert (1 mSv) per year.– Natural exposure in Denver (5000 foot altitude) is about 1.8 mSv/year.– radiation workers are permitted up to 20 mSv per year.– average Hiroshima survivor: 200 mSv

Hiroshima Study

• In the largest biochemical genetics study, 3 new mutations affecting migration rates of proteins on electrophoresis gels were found among 667,404 alleles examined among Hiroshima survivors. Also, 3 new alleles were found among 466,881 alleles examined in the control group.

• No easily detected change in mutation rate. Possible to estimate radiation dose needed to double human mutation rate at 4 Sv (with lethal dose of 5 Sv).

• However, cancer of all types is increased among survivors and continues high to the present.

Detecting Mutagens

• Radiation and certain chemical compounds are “mutagens”: they cause mutation.

• Cancer is caused by somatic mutations, and so mutagens are also carcinogens.

• Testing for mutagenicity is a key step is development of pharmaceutical drugs.

• Simple test using bacteria (Salmonella, a close relative of E. coli) developed by Bruce Ames: the “Ames test”.

Ames Test• Start with Salmonella that are his-, auxotrophs

unable to make their own histidine. They will only grow if histidine is added to the growth medium.

• Add compound to be tested to growth medium, count number of colonies growing. These are revertants, which have been mutated back to wild type.

• In many cases, mutagens need to be activated, converted to mutagenic state, by enzymes in the liver that are meant to detoxify dangerous compounds. Liver extracts are often added to the growth medium to accomplish this.

• Test isn’t perfect: Salmonella are prokaryotes, and we have complex biochemistries that modify foreign compounds. But, it is a good initial screen.

Cell Cycle

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