Final Study Flashcards
What is cancer?
a genetic disorder involving mutations in cells
Who does cancer affect?
everyone, but is increasingly becoming a disease of the elderly
Is cancer inherited?
it is not inherited, but certain inherited mutations can increase chances of cancer
What begins cancer (oncogenesis)?
begins with loss of cell cycle control forming a tumour
What happens to the tumour cells during cancer?
the Tumor cells undergo further changes that allow them to invade and disrupt other tissues causing cancer
What are the types of cancer?
- Carcinoma
- Sarcoma
- Leukemia
- Lymphoma
What is carcinoma?
a type of cancer that forms in epithelial tissue eg. skin
What is sarcoma?
a type of cancer that forms in connective tissue (eg. bone, cartilage)
What is leukemia?
a type of cancer that starts in blood-forming tissue (eg. bone marrow)
What is lymphoma?
a type of cancer that starts in the cells of the immune system (eg. B-cells)
What is a tumor suppressor gene?
a gene that encodes a protein that acts to regulate cell division
What happens when a tumor suppressor gene is inactivated by mutation?
the protein it encodes is not produced or does not function properly, resulting in uncontrolled cell division and development of cancer
What are examples of tumor suppressor genes?
p53 (or TP53) and RB (retinoblastoma)
Normally, what binds to the Start checkpoint and commits the cell to proceed to the S phase?
cyclin-dependent kinase (CDK), regulated by cyclin D
What does p53 regulate in mammals?
p53 regulates G1 to S checkpoint
What is p53?
a protein transcription factor that induces expression of p21
- it stops movement into S phase, and so causes cell cycle arrest
What is cell cycle arrest?
A regulatory process that halts progression through the cell cycle during one of the normal phases
What does p21 do?
inactivates CDK, stopping the cell to proceed to S phase
What happens when damage to DNA cannot be repaired?
p53 concentration increases to a level that will initiate apoptosis
What happens when p53 mutates?
allows a cell to rapidly acquire further gene mutations
What happens when p53 gene mutation is homozygous?
it has the ability to disrupt checkpoint, then the damaged DNA gets replicated and strand breaks occur that cause serious chromosomal abnormalities
What percentage of cancers are associated with mutations in p53?
approximately 50%
What is considered the “first strike” leading down the road to cancer?
disruptions in p53
What does RB do?
regulates G1 to S checkpoint
What controls cell cycle?
protein product RB
What happens when functional RB is absence?
S phase cannot be stopped, resulting in uncontrolled cell growth
- normally p53 would then become activated to induce apoptosis and prevent the cell from replicating
What does tumor of the eye involve?
mutation in tumor suppressor gene RB
What does inheritance of one copy of the RB allele result in?
increase chance of individuals to have cancer in the retina
Tumor of the eye can be:
hereditary or sporadic (not inherited by parents, but arises via a mutation)
Hereditary retinoblastoma is?
one mutant RB copy is inherited from a parent (first hit)
- mutation present in germ line (passed on)
- if the good RB becomes mutated (second hit), then retinoblastoma begins in that eye
- somatic
- recessive allele, but end effect is dominant
Sporadic retinoblastoma is?
requires two independent mutations of RB to begin oncogenesis (normal cells turn into cancerous cells)
- somatic mutation not passed on
What is the RB mutation?
autosomal dominant because there exists a strong likelihood that another mutation in the normal allele will eventually occur
- exhibits incomplete penetrance and variable expression
What are oncogenes?
mutant alleles that act dominantly to stimulate proliferation (increase in number)
- over 90% of cervical cancer in women is associated with these strains of HPV carrying oncogenes that inactivate the p53 and RB
- is the first step in precipitation of cancer (other mutations must follow)
What do some DNA viruses carry?
oncogenes
What is papilloma virus (HPV)?
- genital warts are caused by infection by a virus which causes disruption of the cell cycle, and active tissue growth (virus is pushing cells into S phase so that it can make more virus)
- but when the virus is done, the cells die and the wart will disappear; a new wart may appear in a different spot but no tumour is made
- however, accidental integration of viral DNA may also occur
- but since there is no virus, cells that normally would have been killed by the virus are not, and instead continue to replicate
- increases probability of mutations and cancer
- strains 16 and 18 carry oncogenes that make products that inactivate the protein products of p53 and RB
- in Ontario, 30-50% of sexually active people (ages 19-24) are infected
What causes cancer?
genetic mutation
- normally cells with damaged DNA are caught by p53 repair or apoptosis (tumor suppressed)
- mutations in p53 (2 hits) cell can’t detect damage and DNA gets replicated
What happens to telomeres during normal replication?
normally, most cells will only undergo a limited number of replication rounds as telomeres shorten each time, then die
What happens to telomeres when there is a mutation?
mutation in genes controlling telomerase expression allow telomeres to be repaired and cells become immortalized (this could happen as a result of mutations of p53 gene)
How many mutations must occur to change a normal cell to a cancerous one?
multiple mutations; analysis of cells from human colon cancers show that these cells contain mutations in at least 5-10 genes
What are some phenotypes of cancer cells?
- phenotypes for cancer include many types of cellular abnormalities
- normally, contact inhibition causes cells to stop growing once they make contact with one another; cancer cells show loss of cell-to-cell communication, and continue to proliferate
- cancer cells have autocrine stimulation
- metastasis: disruption of local tissue (basement membrane) and invasion into distant tissues
- angiogenesis: stimulate the growth of blood capillaries
How are changes in populations found?
studying allele frequencies, genotype frequencies and phenotype frequencies
What is phenotype frequency?
the proportion of individuals that are of a particular phenotype
What is genotype frequency?
the proportion of individuals in a population that are of a particular genotype
What is allele frequency?
the proportions of all copies of a gene in a population that are of a given allele type
What is the Hardy-Weinberg Law?
defines the relationships between genotype and allele frequencies within a generation and from one generation to the next
What are the 5 assumptions that the Hardy-Weinberg Law depends on?
- the population includes a very large, number of individuals that all have equal access to mating
- the individuals mate at random in the sense that each individual’s genotype at the locus of interest does not influence his or her choice of a mate
- no new mutations appear in the gene pool (the alleles in question do not change)
- there is no migration into or out of the population
- there are no genotype-dependent differences in the ability to survive to reproductive age and transmit genes to the next generation (natural selection is not occuring)
What is Hardy-Weinberg equlibrium?
hypothetical populations that satisfy all five assumptions that the Hardy-Weignberg Law depends on
What is the Hardy-Weinberg Equation?
p^2 + 2pq + q^2 = 1; a population is said to be Hardy-Weinberg equilibrium if it satisfies the equation
What happens if one of the H-W assumptions have been violated?
the population is not in genetic equilibrium; it is evolving for that gene (locus)
What does calculations using the Hardy-Weinberg law show?
- allele frequencies for a particular gene do not change after one generation in a hypothetical population at Hardy-Weinberg equilibrium (they remain stable)
- the genotypic frequencies p^2, 2pq, and q^2 will be determined by the allele frequencies
- the frequency of heterozygous carriers can be estimated based on observed frequencies of diseased and healthy individuals
How do you compute genotype frequencies?
- add up the number of individuals possessing the genotype and divide by the number of individuals in the sample (N)
How do you compute phenotype frequencies?
- add up the number of individuals possessing the phenotype and divide by the number of individuals in the sample (N)
How do you compute allele frequencies?
- translation of the genotype frequencies of one generation into the genotype frequencies of the next generation
A founding population of 2500 individuals has a genotype frequency of AA=2452, Aa=45, and aa=3. Is it in Hardy-Weinberg equilibrium?
A founding population of 2500 individuals is equal to 5000 alleles. We know p = A, q = a, and p+q=1. We calculate the allele frequencies in a hypothetical first generation. p(A) = (2452(2)+45)/5000 = 0.99. q(a) = (3(2) +45)/5000=0.01. We assume the first generation is in H-W equilibrium p^2 + 2pq +q^2 = 1. In order to get the genotype frequencies, we do AA=(0.99)^2 = 0.98, Aa = 2(0.99)(0.01) = 0.019, aa = (0.01)^2 = 0.0001. Then the expected number of individuals in 2500 is AA = 0.98 x 2500 = 2450, Aa = 0.019 x 2500 = 2450. aa = 0.0001x2500 = 0.25, which are identical to the genotype frequency in the founding population, the founding population is in H-W equilibrium.
What is genetic drift?
unpredictable, chance fluctuations in allele frequency that have no effect on survival
- the smaller the population size, the greater the effects of drift
- in small populations, chance dictates that some gametes unite more often than others (eg. coin toss)
What is most of the genetic variation observed in large natural sexually-reproducing populations arise from?
- recombination due to sexual reproduction
- allele shuffling during meiosis
- spontaneous, random mutations, genetic drift, migration, and assortive mating account for very little contributions
Does natural selection produce variation?
no, but it can select for certain alleles
What is genetic natural selection?
interactions between genetically determined phenotypes and environmental conditions that cause differential reproduction of certain genotypes
What does genetic natural selection result in?
genetically based adaptive traits appearing with a greater frequency (changes in allele frequencies) after many generations (evolution)
What is fitness (w)?
an individual’s relative ability to survive and transmit its genes to the next generation
two basic components: viability and reproductive success
What is natural selection?
the process that progressively eliminates individuals whose fitness is low and chooses individuals of high fitness to survive and become the parents of the next generation
What is relative fitness (RF)?
ranges from 0 to 1; used to calculate the relative fitness for a given genotype
- if all 3 genotypes have a relative fitness of 1, then selection is not occurring
Why is selection unable to reduce the frequency of a recessive lethal allele to zero?
because when the level of the allele in the population is low, the incidence of homozygotes will be rare
What is mutation?
heritable changes in base sequences that modify the information content of DNA
- occur at a low frequency all the time, due to natural processes of deamination, UV light, and errors by DNA polymerase
What is a wildtype allele?
the allele(s) that dominates in a wild population
- any allele present in more than 1% of the population
Are wildtype alleles dominant or recessive?
can be both
What is dominant and recessive a reflection of?
what goes on at the molecular level; a dominant allele usually masks the presence (effect) of the recessive allele
What is a polymorphic gene?
more than one wildtype allele can exist for a gene
How many different base substitutions can occur?
12
What is a transition base substitution?
purine for purine; pyrimidine for pyrimidine
What is a transversion base substitution?
purine for pyrimidine; pyrimidine for purine
What is reciprocal translocation?
part of one chromosome has been replaced with a region from another chromosome and vice versa (changing places)
How would you estimate rates of mutation for a particular gene?
can use inbred homozygous individuals for a particular trait. breed large numbers of offspring and count how many show an alternative phenotype for the trait (eg. mouse colour)
In multicellular organisms, mutation in what type of cells will be passed along future offspring?
germ cells
What happens to mutations in somatic cells?
the mutations in somatic cells are only passed to daughter cells in that individual
Who are the scientists that performed the Luria-Delbruck ‘Jackpot’ Fluctuation Test?
Salvador Luria and Max Delbruck
What is the Luria-Delbruck Experiment?
they set up small vials of bacterial culture and let them grow for a specified amount of time then add equal portions of each culture to plates containing media along with phage, and observed the results
What were the results of the Luria-Delbruck Experiment?
resistance occurs as a result of random, spontaneous mutations that happen at various time points before exposure to the selective agent (selective pressure is not causing the mutation)
mutations occurring early in the growth of the liquid culture, resulted in many resistance colonies when plated; those occurring late resulted in few resistant colonies (fluctuations in numbers obtained)
What is a mutagen?
any physical or chemical agent that raises the frequency of mutations above the spontaneous rate (eg. X rays)
What are purines?
A and G
What are pyrimidines?
C, T, U
What is depurination?
removal of a purine base, allows substitution of another base during replication
What does deamination cause?
deamination (by nitrous oxide) causes transition substitutions during replication
- half the daughter cells will have the mutation
What is ionizing radiation?
x-rays and gamma rays break the DNA backbone (deletion occurs)
What happens during UV radiation?
within-strand C-bonds formed, creating thymine dimers, causing a kink in the DNA strand, preventing replication of that strand by DNA polyermase
What repairs thymine dimers?
Photolyase (activated by absorption of blue light) cleaves thymine dimers
- this repair mechanism does not occur in eukaryotes
What is nucleotide excision repair?
found in prokaryotes and in eukaryotes, this system removes larger lesions of damaged DNA
- higher risk for mutations since polymerase makes mistakes during repair
Where is a hot-spot for mutations?
adjacent thymines
What is apoptosis?
too much damage to repair causes epidermal cells to die all at once, hence peeling
- some mutations derail apoptosis so some damaged cells don’t die (this can have serious consequences)
What is Xeroderma Pigmentosum?
an autosomal recessive mutation, results in inactive endonuclease, excision repair cannot occur, and there is no treatment available
What does oxidation cause?
a transversion mutation (half the daughter cells will have the mutation)
How often do replication of DNA errors occur?
extremely rare, generally occurring once per 109 base pairs
How is the rate of replication errors kept low?
due to correction or proofreading activity of DNA pol I and III which reduces the error rate from one in 106 bases down to 109 bases copied
What is DNA Polymerase Mismatch Repair?
- DNA polymease has 3’-5’ xonuclease activity that can recognize and remove mispaired bases during synthesis
- the 5’-3’ nucleotide addition function then adds the correct nucleotide
eg. adds base A to G, when its supposed to be base C
What is Methyl-directed mismatch repair?
- bacterial recognition and repair system that can fix mutations after DNA replication
- relies on tagging parental strands with methyl groups
- eukaryotic cells also have a mismatch repair system, but the tag is not yet known
When are mutations unable to be corrected?
unequal crossing-over and transposon movement (DNA sequences that move from one location on the genome to another) can change the information content of DNA
- one chromosome ends up with a duplication while the other ends up with a deletion
- these changes are not susceptible to excision or mismatch repair
What happens during red colourblindness?
deletion in red gene; no transcription; no red photoreceptor protein
What happens during green colourblindness?
deletion in green gene; no transcription; no green photoreceptor protein
What happens during red and green colourblindness?
unequal crossing-over; no transcription; no red or green photoreceptor
What happens during blue cone monochromacy?
deletion of long-range control element; no transcription of red or green genes; no red or green photoreceptor protein
What is transposons?
unequal crossing-over and transposon movement can change the information content of DNA
- these types of changes are not susceptible to excision or mismatch repair
What is chromosomal rearrangements?
mutations (heritable changes in the DNA sequence) occurring on a large scale
What is deletions?
removes DNA (information) from the genome
- homozygosity for gene deletions is often, but not always, lethal
- heterozygosity for gene deletions is often detrimental
What is gene dosage?
the number of times a given gene is present in the genome
- a deletion of an entire gene(s) or even a segment of the gene results in a single dose of that gene product in an otherwise diploid individual (who normally relies on two doses)
What is Cri-du-chat Syndrome?
deletion on the short arm of C5 (an acrocentric chromosome)
- heterozygotes have a small head, wide-spaced eyes, round face and cognitive deficiencies
What is duplications?
duplications increase the copy number of a particular chromosomal region
What is tandem duplications?
lie adjacent to each other, either in the same order or in reverse order
What is nontandem duplicatios?
two or more copies of regions that are not adjacent to each other and that may lie far apart on the same chromosome or different chromosomes
What is fragile X syndrome?
second leading cause of mental retardation (next to Down’s syndrome)
What is fragile X syndrome caused by?
caused by a large number of duplications of a CGG repeat on the end of the X chromosome. this prevents the gene downstream of that region from being transcribed
What does unequal crossing over between duplications cause?
increases or decreases gene copy number
What does duplication often give rise to?
duplications often give rise to new genes, creating gene families
eg. beta-globin gene family:
- duplication of beta-globin gene and subsequent selection gave rise to myoglobin (in muscle) which has a greater affinity for O2 than hemoglobin
Does inversions and translocations result in a gain or loss of genetic content?
no, just a change in distribution (location) but they are still mutations
What is inversions?
inversions are a half-circle (180 degree) rotation of a chromosomal region that can occur following two double strand breaks (radiation or within chromosome cross-overs)
What is paracentric inversion?
inversions that exclude the centromere
What is pericentric inversion?
inversions that include the centromere
What is the sole difference in human chromosomes and chimps?
by only a pericentric inversion (including the centromere) on chromsome 4
Inversions can also result from?
from rare crossovers between related DNA sequences present in two different locations on the same chromosome in inverted orientation
What happens if one end of an inversion lies within a coding region of DNA?
may result in a mutant phenotype
What is translocation?
large scale chromosomal rearrangements in which part of one chromosome either becomes attached to another chromosome or parts of two different chromosomes trade places
What is reciprocal translocation?
parts of two different chromosomes trade places
What is Robertsonian Translocation?
reciprocal translocation arising from breaks at or near the centromeres of two acrocentric chromosomes, results in one large and one small chromosome; the small chromosome is usually lost
What is Familial Down’s Syndrome?
Robertsonian translocation between C14 and C21 in the germ cell. if this gamete mated with a normal gamete, the offspring would have “partial” trisomy 21
What is the difference in chromosome number between humans (2n = 46) and the greater apes (2n = 48) a result of?
a result of a Robertsonian translocation sometime in our evolutionary history
What is transposable elements?
small segments of DNA which are able to move from one position to another in the genome
- can be present in thousands of copies; about 40% of the human genome consists of transposable elements
What are the characteristics of transposable elements?
- found in virtually all organisms
- need not be sequences that benefit or harm the organism
- many are regarded as selfish, carrying only information required for their own propagation
- genetic symbionts
What are the two kinds of transposable elements that prokaryotes carry?
insertion sequences (IS) and transposons (Tn)
What is insertion sequences (IS)?
DNA moves directly by cut and paste
- simple structure, possessing terminal inverted repeats
- code for a transposase enzyme, which recognizes inverted repeats, cuts the host DNA for IS movement
What are the steps for insertion of IS into target DNA?
- staggered cleavage of target DNA
- insertion of IS
- synthesis of DNA fills gap (eg. direct duplication of target site)
What is transposons (Tn)?
- DNA moves directly by cut and paste
- complex structure, more than one gene
- code for a transposase enzyme, which cuts at the IRs and cuts the host DNA for movement
- composite Tns = 2 IS elements flanking one or two genes
What are the two kinds of transposable elements that eukaryotes carry?
transposons (Tn) and retrotransposons
- classified on the basis of how they move around in the genome
What is transposons?
- DNA move directly by cut and paste, and does not copy itself
- their ends are inverted repeats, usually between 10-200 bp long
- carry a transposase gene (and one or 2 other genes)
What is retrotransposons?
- not cut-and-paste, but makes a copy of itself
- also called retroposons
- transpose via an RNA intermediate; uses reverse transcriptase to copy the RNA into a double-stranded DNA molecule for insertion into the genome
- carry features of elements known to exist in some virsues
- the structure of retrotransposons is similar to that of retrovirus genomes, suggesting an evolutionary link
What are the two major classes of retroposons for mammals?
- LINEs (long interspersed elements)
- SINEs (short interspersed elements)
What are the two major classes of retroposons for humans?
L1 (LINE), 6.4 kb; 20,000 copies
Alu (SINE), 0.28 kb; 500,000 copies
What is composite insertion sequences?
genetic transposition (insertion/deletion) by excision of sequences between nearby inverted repeat sequences from two or more transposons, followed by insertion in a new location
What is genomic library?
- contains all sequences
- contains same information no matter what cell type from the organism is used to construct the library
- used from prokaryotes mostly (no introns) or sequencing projects
What is cDNA library?
- contains only exon sequences
- information is conditional upon cell type
- used for eukaryotes mostly (to study expressed sequences)
What is clone isolation?
screen colonies to find the clone of interest
What is DNA Probe?
- short single-stranded stretches of DNA of known composition
- from 25 to several thousand nucleotides in length
- to visualize them, the probes are labeled, usually with 32P or fluorescent dyes
- used in the identification of clones that contain complementary DNA sequences
What is the Southern Blot Analysis used for?
used to show the location of a gene within a larger fragment of DNA
What are the steps for Southern Blot Analysis?
- transfer of DNA fragments from agarose gel to nitrocellulose filter paper (preserves relative positions of DNA fragment)
- perform hybridization between a labelled probe and DNA on the filter
- expose to x-ray film to identify hybrid
What is polymerase chain reaction (PCR)?
- artificial DNA xeroxing
- with the use of 2 specific oligonucleotides (primers), any fragment of DNA can be amplified from 1 to approx. 1 billion copies in as little as 2 hours
- has uses in DNA cloning, DNA sequencing, making DNA probes, identification of organisms without Southern blotting
What is the method of PCR?
- design two primers that are complementary to each end of the sequence of interest
- mix the primers, template DNA, thermostable DNA polymerase, and the 4 nucleotide bases in a microtube
- begin thermocycling procedure:
- denature
- anneal
- extend
What does each cycle of PCR result in?
results in an exponential duplication of the strands (amplification)
What is paleomolecular biology?
DNA fragments can give information about early human and animal remains
What is forensic science?
- using DNA fragments to identify criminals
- if done correctly, DNA samples can be used to defend or convict suspects
What is DNA sequencing?
provides the highest resolution of a cloned DNA fragment: a complete description of its genetic information at the level of the nucleotide
What is modern detection?
automation of DNA sequencing using a laser and fluorescence detector
What does the polyacrylamide gel do?
- polyacrylamide gels can resolve strands that differ in length by a single nucleotide
- read teh sequence of synthesized strand from the gel
- template strand has complementary sequence and opposite orientation
What is molecular cloning?
-how fragments of DNA are cloned to make recombinant protein products
- recombinant proteins are formed when DNA from one species is transplanted into another species
- protein products are used in medicine, food production, etc
What are restriction enzymes?
restriction enzymes are endonucleases that exist naturally in prokaryotes
- they restrict what type of DNA can exist in the host
What is the function of restriction enzymes in the natural host?
to recognize foreign DNA sequences and cut it into pieces for further degradation by host exonucleases
What is insulin?
a recombinant protein, it was the first therapeutic product to be synthesized using recombinant DNA; at first in E. coli, later in yeast
What is type II REN?
they recognize inverted palindromic sequences in DNA. each REN has a unique recognition sequence. when they cut within these sequences, they leave either cohesive (sticky) ends or blunt ends
What are blunt ends (RsaI)?
cut right down the middle
What are sticky 5’ ends (EcoRI)?
cut at top then bottom
What are sticky 3’ ends (KpnI)?
cut at bottom then at top
How is the recognition site always read?
always read 5’ to 3’
What is electrophoresis?
the movement of charged molecules in an electric field
What does gel electrophoresis do?
since DNA in solution is weakly acidic (negative charge), gel electrophoresis distinguishes DNA fragments according to size
- the larger fragments move more slowly through the gel matrix than smaller fragments
What is a restriction map?
shows the order of different size fragments within a clone
- consist of linear (or circular) diagrams of the sites along a DNA segment at which one or another restriction enzyme cleaves the molecule
What is molecular (DNA) cloning?
the process of using living cells to make many exact replicas of a fragment (or fragments) of foreign DNA
What are the steps for molecular (DNA) cloning?
- take a DNA fragment and insert the fragment into a specialized chromosome-like carrier termed a vector
- transfer the vector with its insert into cells. copies will be made through replication. these are therefore known as DNA clones
What are Plasmids?
(usually) circular double-stranded DNA.
- each has an origin of replication, several unique sites for restriction enzymes and a gene refereed to as a selectable marker. they can be engineered to have these elements.
- typically 2-4 kb in size, with an ability to carry inserts yp to 15 kb in length
