Foundations 1 Flashcards
What type of RNA is the most common and what is its function?
rRNA and it makes up ribosomes
Explain the metaphor to software and hardware used to describe mRNA, rRNA and tRNA.
mRNA is like a software which tells the machinery what it should produce
RRNA and tRNA are like hardware which allow ribosomes to fulfill the “commands” given by mRNA
What is the purpose of microRNA’s?
They regulate the amount of protein that is produced by interfering with mRNA preventing them from being translated
What type of bonds unite nucleotides in both DNA and RNA?
Phosphodiester bonds
Outline major differences between DNA and RNA. (6)
1 Ribose vs deoxyribose (2’OH in ribose)
2 Uracil vs Thiamine
3 Single Stranded vs double stranded
4 RNA is less stable due to 2’ OH
5 RNA can have secondary and tertiary structure
6 RNA has a higher amount of non-Watson Crick base pairing
Draw a diagram explaining the important function of palindromic sequences in RNA molecules.
They create hairpin structures:
X
X. X
UA
GC
CG
AUWhat is the open reading frame ORF?
The area between the start and stop codons which will actually be translated
Describe the modifications that take place to pre-mRNA before it becomes mature mRNA when possible give reasoning behind these modifications.(3)
1 Splicing- introns are removed leaving only exons
2 5’ cap is added consisting of 7 methyl guanosine linked by a triphosphate linkage- This prevents degradation of mRNA by phosphatases and nucleares and allows the ribosome to recognize it
3 3’ Poly A tail is added which protects the mRNA from degradation
Describe some of the main features of tRNA.(3)
1 CCA at 3’ end- amino acids are bound to the 2’ or 3’ OH of the A at the carboxyl group
2 Have a high number of unusually modified bases
3 Exhibit clover leaf secondary and L-shaped tertiary structure
What is non Watson-Crick base pairing and when can it occur?
Any base pairing between bases other than those which normally pair (AT and GC). It can occur between any two bases as long as they each have a hydrogen bond donor and a hydrogen bond acceptor and they come in close proximity of each other.
Why is the difference in stability between DNA and RNA important?
DNA is more stable which avoids the loss of genetic material over the life course of an organism
RNA is much less stable which makes it easier to regulate the amount of protein being produced at different times
Compare and contrast promoters and enhancers.
Promoters bind RNA polymerase so that it can begin transcription. Enhancers bind other protein factors which interact with RNA polymerase to increase the rate of transcription.
Promoters are like putting a car into drive- they allow transcription to begin at a regular slow speed and enhancers are like pushing the gas pedal, they cannot start transcription by themselves but they can increase the speed if transcription has been initiated.
Another major difference is location. Promoters are always located directly upstream from the start sequence while enhancers may be located anywhere including thousands of base pairs away from the start site and the DNA bends to bring them close to the transcription site.
Compare and contrast enhancers and silencers.
Enhancers upregulate transcription and silencers downregulate transcription.
They may both be located very far away from the start site
Define cis-acting elements and trans-acting elements and explain how they relate to enhancers, promoters, etc.
Cis-acting elements are short DNA sequences which attract certain proteins. These proteins which bind to cis-acting elements are trans-acting elements and they affect the rate of transcription by interacting with RNA polymerase.
Enhancers, promoters and silencers are made up of many cis-acting elements near each other.
Differentiate between general and sequence specific transcription factors.
General transcription factors are required for transcription to occur. These factors must be present at a location for RNA polymerase to bind and for transcription to occur so all promoters have these near the start site
Sequence-specific transcription factors up/down regulate RNA polymerase and may or may not be present in DNA elements
What are some of the major differences between transcription and replication besides the end product (3).
1 Transcription is de novo synthesis- meaning that it does not require a primer, instead a triphosphate A or G begins at the 5’ end
2 Transcription has a lower fidelity because errors are less costly
3 Only one strand is created in transcription in the 5’-3’ direction so only one strand of DNA is used as a template in the 3’-5’ direction
Explain the importance of the concept of a combinatorial mechanism in promotion of replication.
Cis acting elements are not unique to individual promoters, enhancers etc. Instead what is unique is the combination and arrangements of elements
In addition, all of the transcription factors relating to the specific cis-acting elements must be present for a promoter to function. This explains tissue specific gene expression because different factors are present in different cell types leading to only the expression of those genes whose factors are present.
Make up an example to illustrate the principle of modularity in transcription.
Transcription factors are made up of multiple domains such as a DNA binding domain and a regulatory domain. These domains if switched to another protein would still function.
For example, assume you had a transcription factor with a DNA binding domain that binds to an element that regulates expression of TNF alpha and that same transcription factor has a regulatory domain that enhances transcription. Then say you transfer the regulatory domain to a factor with a different DNA binding domain. NOw, the new transcription factor would cause the upregulation of transcription of whatever gene the binding domain binds to.
On what characteristics are transcription factors separated into families?
The sequences to which they bind
Explain what signal sequences are and their function in protein localization.
THey are a part of the amino acid sequence of a protein either at the N or C terminus that targets that protein for its destination and will often be removed after reaching the destination.
Diagram a nuclear pore.
Should show 2 membranes of nuclear envelope
Projections toward the outside of the nucleus that look like tentacles
Projections toward the inside from a “basket”
What is the purpose of nuclear pores?
They allow small hydrophilic molecules and ions to travel freely between the cytosol and the nucleus while allowing those larger molecules with the correct signals to travel as well.
Explain and diagram the process by which a protein destined for the nucleus arrives there
Proteins destined for the nucleus will contain a nuclear localization signal which is a short sequence of positively charged amino acids. This signal binds to nuclear import receptors in the cytosol. These receptors then bind to cytosolic fibriles of nuclear pores which transport protein through pores into nucleus
nuclear localization signal –> nuclear import receptors–> cytosolic fibriles–> pass through nuclear pore
Explain and diagram the process by which a protein gains entry into the mitochondria.
Proteins have an N-terminus signal sequence which binds to import receptors on the cytosolic side of the outer mitochondrial membrane. import receptors are bound to translocators which feed the protein through the outer membrane toward translocators on the inner membrane which will feed them through to the matrix
N-terminus signal sequence –> import receptors on membrane –> outer translocator –> inner translocator –> mitochondrial matrix
What differentiates free ribosomes from ER bound ribosomes?
They are structurally identical and it only depends on which protein they are currently translating. If it is bound for the ER the ribosome will move to the ER and if not the ribosome will remain free.
The ER acts as a distribution center for proteins going to what destinations? (5)
ER, golgi, lysosomes, endosomes and cell surface
How do proteins bound for the ER get there?
When the protein begins to be translated, the beginning N terminus will contain a signal sequence for the ER. The signal sequence is recognized by signal recognition particle (SRP) which binds to an SRP receptor on the ER membrane which allows the growing protein to pass through a translocator as it is translated.
signal sequence –> signal recognition particle (SRP) –> SRP receptor –> translocator–> ER
Compare and contrast soluble and embedded ER proteins.
Soluble proteins pass completely through the membrane as they are translated and end up free floating within the ER. These proteins are ultimately bound for the lumen of an organelle or else to be secreted outside of the cell completely.
Embedded proteins are held in the ER membrane after translation based on transmembrane portions. These proteins will be membrane bound in their destination and are transferred to their final location by vessicular transport.
Outline the process by which a protein becomes embedded in the ER membrane.
As it is being translated, a hydrophobic stop sequence occurs causing the translocator to “spit” the protein out into the surrounding membrane. This hydrophobic portion remains embedded into the membrane while the protein continues to be translated anchoring the protein there. A single protein may have several stop hydrophobic stop and start sequences causing it to make multiple passes through the membrane.
What protein removes the signal sequence from ER proteins?
Signal peptidase
Describe the symptoms and causes of Swyer syndrome or XY gonadal dysgenesis and how it relates to protein localization.
Symptoms: XY individuals form female external genitalia but no ovaries
Cause: non-functional SRY gene
Connection: May result from lack of nuclear localization signal on SRY gene causing the gene to not perform its function in the nucleus as a transcription factor
Describe the process of adding polyadenylation.
An endonuclease recognizes the sequence AAUAAA and cleaves at this site allowing polyadenylate polymerase to add a poly A tail.
What does codon degeneracy mean?
More than 1 codon code for the same amino acid
Contrast insertion and deletion mutations at the protein level to frameshift mutations
Insertion and deletion mutations at the protein level refer to addition or deletion of 1 or more amino acids without causing a frameshift whereas frameshift mutations occur when a number of nucleotides not divisible by 3 was added or deleted from the reading frame which shifts the reading frame causing major changes to the product.
What is a poly some?
A group of ribosomes that are working independently of each other to translate the same piece of mRNA.
Draw how you would represent monozygotic and dizogotic twins on a pedigree
One vertical line coming from the sibling line with two diagonal lines to individuals represents twins. Monozygotic twins will have a horizontal line between them while dizogotic twins will not
What is consanguinity and how would it be represented on a pedigree chart?
It represents that a mating couple are related by blood. It is represented by a double mating line instead of the normal single line
What does a diagonal line through a person on a pedigree chart mean?
That person is deceased
Draw your own pedigree chart.
Make sure it follows all of the normal rules (squares = male circle female, mating lines etc.)
Name the major components of chromatin (3)
DNA
Histones
Non-histone proteins
What is meant by saying that DNA-histone interactions are “fluid”?
They are not always exactly the same but are constantly changing leading to changes in gene expression patterns
What is a nucleosome?
A “bead” made up of 8 histones with DNA wrapped around it
What is cohesion and what proteins make it happen?
The binding of sister chromatids together. Cohesin protein complex accomplishes this by acting like a napkin ring around sister chromatids
Describe the changes that occur to the cohesin complex over the course of the mitosis and meiosis
Mitosis
- Cohesion loads onto sister chromatids at replication
- majority dissociates at prophase but some remains at the centromere until anaphase
Meiosis
- Loads along homologous pairs in meiosis 1
- Exists only between centromeres of sister chromatids in meiosis 2 similar to mitosis
Describe the role of condensin proteins
They cause the chromatin to condense at the beginning of mitosis
CDK protein triggers M phase and causes condensin to accumulate
Describe the major differences between euchromatin and heterochromatin
Euchromatin is less densely packed, more transcriptionally active and appears lighter than heterochromatin in G-banding
What is G-banding?
It is a way to visualize chromosomes. Trypsin partially digests histones and allows dye to bind to DNA so that they can be visualized. Each chromosome has a fairly standardized pattern of bands that allow geneticists to identify them. Each band is made up of 50-100 genes.
When in the cell cycle is best for G-banding and why?
During prometaphase. The chromosomes can be visualized then or during metaphase but the bands are clearer during prometaphase than metaphase
What is the centromere?
A repetitive sequence of DNA on each chromosome that allows the proteins of the mitotic spindle to bind during mitosis.
Describe the three subtypes of chromosomes based on centromere positioning
1 meta centric: centromere basically in the center
2 Submetacentric: centromere halfway between the center and one end
3 Afrocentric: centromere is near the end
Differentiate between the p and q arms of chromosomes.
The centromere divides the chromosome into 2 arms. The smaller arm is the p arm (petite) and the q arm is the larger arm
Explain the purpose of telomeres and the normal function of telomerase
Telomeres are repeats of a 6 base pair sequence (TTAGGG) that occur over and over near the end of the chromosome. They protect the coding regions of the chromosome from degradation since some DNA is lost in each cell cycle. Short telomeres trigger apoptosis. Telomerase is a protein that replaces telomeres and is active in germ cells but normally becomes inactive afterwards. Active telomerase is a trait of neoplastic (cancer) cells
What are the three types of chromosomal abnormalities?
Numerical: wrong number of chromosomes
Structural: changes to the structure of a given chromosome
Mosaicism: 2 or more genetically distinct cell lines within the same individual
Give the karyotype nomenclature for a normal female and a male with Down syndrome.
46,xx
47,xy,+21
Compare and contrast polyploidy and aneuploidy
Polyploidy involves the wrong number of copies of all chromosomes ie triploidy or 69 (n =3) while aneuploidy refers to an incorrect number of only some of the chromosomes but not all of them (most often just one) polyploidy occurs in 1% of all conceptions accounting for 10-20% of chromosomally abnormal miscarriages but is basically always lethal. Aneuploidy usually results in spontaneous abortion but in some cases can result in a live birth usually with serious defects
What is the most commonly occurring aneuploidy? Which ones can result in live births?
16 is the most common but basically always lethal
Trisomies 21, 18, and 13 can all result in live births as well as monosomy X or XXY
What are the most common causes of structural abnormalities of chromosomes?
1 Non homologous recombination
2 double strand breaks of sister chromatids
3 telomere instability
What is a translocation? Compare and contrast reciprocal translocations and Robertsonian translocations.
An exchange of info between non homologous chromosomes
A reciprocal translocation occurs when 2 non-homologous chromosomes essentially undergo crossing over
A Robertsonian translocation occurs when 2 Acrocentric chromosomes join at the centromere resulting in one large chromosome that is a combination of the other two and the loss of the small p arms of both chromosomes
What are the main clinical features of trisomy 21?
Down Syndrome Mild to moderate intellectual disability Characteristic facial appearance hypotonia (weak muscle tone) at birth Higher risk of: Gastroesophageal reflux Celiac disease Hypothyroidism Hearing/vision problems Leukemia Alzheimer’s
What are the common clinical features of Trisomy 18?
Edward Syndrome
Slow intrauterine growth/low birth weight
Heart defects
Small abnormally shaped head
Small jaw and mouth
Clenched fist with overlapping fingers
Most die within a month and those that don’t usually have severe intellectual disabilities
What are the common clinical features of Trisomy 13?
Patau Syndrome
Severe intellectual disabilities
Many physical abnormalities especially along the midline: cleft lip/palate, micropthalmia (small underdeveloped eyes), extra fingers, toes
Heart and spinal chord defects
What is Turner Syndrome? What are the common clinical features?
Monosomy X
Results in female sex characteristics
Short stature
Early loss of ovarian function and infertility
Most do not undergo puberty without hormone treatment
30% have extra skin folds on the back of the neck
1/3 coarctation (unusually narrow) of the aorta
What is Klinefelter’s syndrome and what are the common clinical features?
XXY karyotype
Male sex characteristics
Taller than average
Infertile
small testes —> low testosterone —> delayed puberty
Can develop some feminine feature such as breasts, feminine belly fat placement patterns, hip structure, etc.
What is the cause of most aneuploidies?
Nondisjunction during Meiosis 1 of oogenesis
What are two main exceptions to the central dogma?
1 Reverse transcriptase - goes backward from RNA to protein
2 RNA as the final product- tRNA, miRNA, snRNA, rRNA are not meant to be translated into protein
What are the 3 ways that gene expression can be controlled by physical modifications to DNA?
1 DNA/gene loss
2 DNA/Gene amplification
3 DNA rearragement- Ex: immunoglobulin subunits can be rearranged to create many unique proteins
What are 3 real world examples of DNA methylation in gene expression?
1 Globin gene is methylated in all cells besides red blood cells
2 X-inactivation
3 Imprinting
What DNA sequence is usually methylated and why is that?
CpG islands or regions with a high number of C’s followed by G’s. The reason is because C’s are generally methylated and this sequence is a palindrome that allows both strands to be methylated
Draw and explain how imprinted DNA stays methylated across cell divisions.
Each division there one strand comes from the parent cell and one new strand. The parent strand if it was methylated will remain methylated and the new strand is not methylated. This is referred to as hemimethylated. All hemimethylated cells are recognized and methylated.
Summarize the different ways that red blood cell expression is controlled through various mechanisms. (4)
1 All DNA is deleted from mature RBC’s stopping expression entirely
2 Globin gene is methylated in all cells except red blood cells to keep it from being expressed
3 As RBC’s mature, the chromatin becomes more condensed limiting expression
4 When iron is not present, Globin mRNA’s are prevented from being translated since they will not be able to form hemoglobin
Label all of the following inhibitors of translations as affecting eukaryotes, prokaryotes or both: Ricin Kanamycin Tetracycline Puromycin Cycloheximide Streptomycin Gentamicin Neomycin Diphtheria toxin Erythromycin Chloramphenicol Rifamycin Alpha amantin Actinomycin D
Ricin: E Kanamycin P Tetracycline P Puromycin B Cycloheximide E Streptomycin P Gentamicin P Neomycin P Diphtheria toxin E Erythromycin P Chloramphenicol P Rifamycin P Alpha amantin E Actinomycin D B
Compare and contrast Loss of function and gain of function mutations and the diseases they cause.
Loss of function refers to a mutation which reduces or eliminates the function of a protein. These disorders are generally recessive since one functional copy is often enough to prevent a disorder. An example of this is Duchenne muscular dystrophy. However in the case of proteins that are required in high quantities, loss of function disorders may be dominant as is the case for osteogenesis imperfecta
Gain of function mutations are when the mutation causes the protein to have a function that the normal protein did not have either within the same pathway as is the case in achondroplasia or in an unrelated pathway as is the case in Huntington’s disease. These disorders are generally dominant with the main exception being sickle cell anemia.
Describe the clinical presentations of osteogenesis imperfecta
Group of disorders characterized by brittle bones
Type 1 = milder leads to frequent fractures of long bones with any exertion which heal normally
Type 2 = more severe, many fractures present at birth and patients normally die within a few weeks of birth
Describe the mutations that cause osteogenesis imperfecta type 1 and 2.
This is caused by a reduction in the amount of collagen which is important in bone formation
Collagen is made up of 2 Alpha 1 chains and 1 Alpha 2 chain
Type 1 - mutation results in no production of alpha 1 —> heterozygotes produce 1/2 normal alpha 1 and therefore 1/2 normal collagen
Type 2 - point mutation to alpha 1 chain that makes any collagen chain with defective alpha 1 chain non-functional. This leads to 3/4 of collagen becoming non-functional and only 1/4 of the normal amount of collagen
In what ways are serious genetic disorders perpetuated even though affected members may be unable to reproduce?
1 Novel mutations and mosaicism/chimerism, a novel mutation could occur which is only present in some fo the cells which could make it so that the phenotype is not present but if the mutation is present in any germline cells, it could be passed on.
2 For recessive disorders, carriers could be at an advantage in some ways such as sickle cell
Compare and contrast mosaicism and chimerism
Both result in different genetic cell lines within the same organism but the mechanism is different.
Mosaicism: Mutation occurs early in cell divisions of the embryo. The earlier the mutation occurs, the more cells will be affected
Chimerism: two genetically distinct zygotes fuse to form one organism early in cell division. Often as a result of one zygote holding a fatal abnormality that would otherwise cause spontaneous abortion
Describe the clinical presentations of achondroplasia.
Autosomal dominant Gain of function mutation to the fibroblast growth factor receptor (FGFR3) gene. Mutation leads to ligand independent stabilization of dimers activating downstream signaling. Results in lack of normal growth in long bones but otherwise healthy individuals.
80-90% are de novo mutations
What is anticipation in genetics and what explains it?
A genetic disease that occurs earlier and more severely in each successive generation. This is explained by triplet repeat expansion where a disease is caused by an abnormally high number of repeats of a 3 nucleotide sequence. The number of repeats generally increases each generation and the higher number of repeats the more sever the disorder.
Describe the clinical manifestations of Huntington’s disease
Autosomally dominant gain of function disease caused by expansion of the number of CAG repeats in a specific gene.
Normal is 10-30 repeats
Affected is 36+
Average onset is 37 years with 100% penetrance by age 80
Begins with mild motor function deficiencies memory loss and progresses to uncontrollable movements, extreme personality changes, severe memory loss and severe psychiatric conditions until the patient is immobilized and cognitively non functional. Death 10-20 years after onset.
Describe the clinical manifestations of Spinal Muscular atrophy (SMA).
Autosomal recessive loss of function mutation to Survival motor neuron 1 (SMN).
Leads to motor neuron degeneration the severity of which is dependent on the number of copies of SMN2 that an individual has.
Type 1: born with little muscle tone - death within 2 years
Type 2: sits but never stands childhood lethal
Type 3 and 4: most mild with some adult survival but generally loss significant mobility
Recent gene therapy has been successful to date in treating SMA
Describe the clinical manifestations of Duchenne muscular dystrophy (DMD)
X-linked recessive disorder
Delayed motor development, proximal muscle weakness, classic microscopic myopathy can changes, muscle fiber breakdown results in high serum levels of muscle enzymes such as creatine kinase, dilated cardiomyopathy, wheelchair bound by 12, typically die by late 20’s
Define allelic heterogeneity, locus heterogeneity, and clinicalheterogeneity.
Allelic: same phenotype caused by different mutations to the same gene
Locus: Same phenotype caused by mutations to different genes often within the same pathway
Clinical: Very different phenotypes resulting from mutations in the same gene
What patterns does mitochondrial inheritance follow?
Similar to X-linked dominant transmission, variability in symptoms based on mixed population of normal and affected mitochondria
Mostly affect aerobic tissues (nervous system, eyes, cardiac and skeletal muscle)
Describe how chromosome analysis or karyotype works, what it can be used to detect and what it cannot be used to detect.
A chemical is added to cells to arrest them in metaphase and cells are dyed and dropped onto a microscope slide so that chromosomes and banding patterns can be analyzed.
Used for- detection large chromosomal abnormalities such as aneuploidy, translocations, deletions, duplications etc.
Can’t be used for Uniparental disomy, methylation changes or abnormalities smaller than 5Mbps
How does Fluorescent in situ hybridization (FISH) work? What can it and can’t it be used for?
Fluorescent probes are created to be complimentary to certain DNA regions. They are then hybridized to the cellular DNA and visualized.
Can tell: Deletions, duplications, translocations and mosaicism (limited by size of probes and knowledge of the target)
Can’t tell: Uniparental disomy or methylation changes
How does Array CGH work? What can it and can’t it be used for?
Oligonucleotide probes are placed on a glass slide. Patient and control DNA are labeled with different colors and then allowed to hybridized with probes. The fluorescent patterns are then compared.
Can tell: deletions and duplications
Can’t tell: Balanced rearrangements, UDP, Anything smaller than 10-30Kbps, changes in methylation
What can PCR and microsatellite PCR be used for?
Microsatellite PCR targets short repeats that occur a different number of times for different people.
Can be used for: Crime scene identification, paternity testing, changes in methylation and UDP
How does Sanger sequencing work? What can it be used for and what can’t it be used for?
It is like PCR but it also includes labeled dideoxyribonucleotides which terminate the chain and be used to tell which base is present at each location.
Can tell: Small alterations in targeted regions
Can’t tell: UDP or methylation (clinically )
How does multiple probe ligations MLPA work and what can it and can’t it be used for?
Nucleotide probes are fused to universal PCR primers and hybridized with DNA. Then PCR is performed which amplifies only those probes that hybridized.
Can tell: Small deletions and duplications no larger than one gene
Can’t tell: Single base mutations, UDP, methylation, translocations and inversions
Describe how massively parallel sequencing works and what its potential uses are.
The genome is fragmented and bound to adapters then ligated to a flow cell. Bridge amplification is used to create clusters of identical DNA strands. Fluorescently tagged reversibly terminating nucleotides are added and lasers are used to tell what base was added at each location. They are then removed and cycled through for each location until a sequence is created.
This can sequence much more information including a whole exime at a much lower cost than Sanger sequencing but is more error prone.
Can be used to find small abnormalities and could eventually be used to look for larger ones.
What are the major differences between hemoglobin and myoglobin?
Hemoglobin is a tetramer and myoglobin is a tetramer this causes hemoglobin to exhibit cooperativity and have a lower oxygen affinity in the tissues than myoglobin. Because of this difference, hemoglobin is used to transport oxygen throughout the body and myoglobin is used to store oxygen to be used during exercise in skeletal muscles
What structural changes occur to hemoglobin as a result of O2 binding?
In the deoxygenated state, the iron atom of hemoglobin is pulled out of the plane of the heme ring and when oxygen binds it pulls the whole helix to which iron is bound so that the iron is back in the plane. This difference is characterized as the T-state (deoxygenated) and the R-state (oxygenated).
What interactions are destabilized in the R state vs the T state of hemoglobin?
Several ionic interactions between amino acids that stabilize alpha-alpha and alpha1-beta2 and alpha2-beta1 subunit interactions in the T-state are no longer possible in the R state due to the conformational changes that take place
How does CO binding affect hemoglobin?
CO binds at the oxygen binding site with 300 times more affinity than oxygen blocking O2 from binding. CO is constantly present in small amounts in the body but Hb bound CO is removed in a matter of hours
Describe the cooperativity of hemoglobin.
The T-state of hemoglobin has a lower affinity for oxygen than the R-state. When 2 oxygen molecules are bound to a Hb the two states are in equilibrium. However when 1 O2 is bound the T-state becomes favored and when 3 are bound the R state is favored. This increases the affinity for oxygen in high concentrations of O2 and decreases affinity for O2 at low concentrations
What are the 3 allosteric inhibitors of hemoglobin and how does each affect it?
All three lower the affinity for oxygen by favoring the T-state
1pH- low pH causes H+ ions to bind to hemoglobin favoring the T-state. Low pH results from exercise
2 2,3BPG- occurs as a short term result of high altitude
3 CO2 - also occurs as a result of exercise
Describe the natural Hemoglobin variants.
Hemoglobin A is the major type present in adults consisting of two alpha and two beta subunits
Hemoglobin A2 is present in smaller concentrations in adults and is made up of 2 alpha and 2 delta subunits
Hemoglobin F is found in fetuses and consists of 2 alpha and 2 gamma subunits
Embryonic hemoglobin consists of 2 alpha and 2 epsilon subunits
Pre-embryonic hemoglobin consists of 2 zeta and two epsilon subunits
Fetal and embryonic hemoglobin variants have higher O2 affinity than adult hemoglobin
Describe alpha thalassemia.
Any mutation to the alpha subunit of hemoglobin which causes insufficient production of hemoglobin
Most are deletions
1 deletion = silent carrier w/o symptoms
2 deletions = alpha thalassemia trait and mild anemia
3 deletions = hemoglobin H disease- moderate to marked anemia but not lethal
4 deletions = hemoglobin Bart’s no O2 carrying capacity, neonatal death
Describe beta thalassemia.
Any of over 100 mutations to the beta subunit of hemoglobin which causes insufficient hemoglobin production
Clinically heterogenous
From mild to major (Cooley’s)
Cooley’s = transfusion dependent, infants are listless and pale, grow slowly and may be jaundiced, internal organs can be enlarged
Describe Heinz body hemolytic anemias
Results from point mutations in the hydrophobic center of the hemoglobin usually in the beta subunit
Results in desaturation of the molecule and coagulation within the erythrocytes (Heinz bodies)
Describe Sickle cell anemia.
Results from a single point mutation causing an amino acid change from glutamate to valine in beta subunit of hemoglobin that causes them to become “sticky” in T state resulting in sickle shaped RBC’s
What is the average lifespan of a normal RBC?
120 days
What is a reticulocyte?
The step before a mature RBC in Erythropoiesis. You can often see them on blood smears they will appear larger than erythrocytes and purple with a mesh-like network of rRNA. They normally make up about 1% of RBC’s
Why could a count of reticulocytes be important? And what is the best way to measure them?
They can be measured in a complete blood count. They are useful to understand the rate of Erythropoiesis
If a patient has a higher or lower than normal amount of reticulocytes what could that indicate?
Higher: blood loss or hemolytic anemias
Lower: vitamin deficiency or Myelodysplastic syndromes
Outline the important components of the RBC cellular structure.
Biconcave discs with central pallor 1/3 of diameter- this increases the surface area for gas exchange
Unique membrane and cytoskeleton allow for flexibility and deform ability which is important for fitting through blood vessels
No nucleus
What is a schistocyte?
Fragmented RBC’s that look like triangles or helmets. Are caused by shearing or mechanical force
Only present under severe pathological conditions such as artificial heart valves or microangiopathic hemolytic anemia, malignant hypertension
Describe the shape and lifespan of sickle cell RBC’s.
They are crescent shaped with pointed ends. Typical lifespan is 10-20 days
What are spherocytes? What causes them?
RBC’s shaped like spheres with no central pallor due to loss of membrane. This causes decreased surface area to volume ratio. Can result from pathology such as burns and autoimmune hemolytic anemia or it can be hereditary
What causes hereditary Spherocytosis?
An autosomal dominant gene usually affecting the cytoskeleton protein ANK1 (ankyrin)
What are target cells and when are you likely to see them?
Abnormal RBC’s with too much membrane increasing the surface area to volume ratio to higher than normal. THey are seen in liver disease and after removal of the spleen and sometimes in thalassemia
What are ovalocytes? When are you likely to see them?
They are elongated oval shaped RBC’s. They look kind of like sickle cells but with rounded edges. They are most commonly seen in iron and vitamin B12 deficiencies as well as hereditary ovalocytosis
What causes hereditary elliptocytosis/ovalocytosis?
Autosomal dominant gene in the cytoskeleton protein spectrum
What is basophilic stippling and when does it usually present?
RBC’s with small blue dots dispersed throughout which are caused by clusters of rRNA
OFten seen in lead and arsenic poisoning, alcoholism and thalassemia
What conclusions did Anfinsen make about protein folding?
All of the information necessary for proper folding is inherent in the primary structure and there is no energy necessary for them to properly fold
What is Levinthal’s paradox and what realization did it lead to?
There is not enough time for proteins to try every conformation and choose the best one. This leads to the idea that there is a predetermined pathway
Why is crowding in cells a challenge for proper protein folding?
Interactions of amino acids with other proteins toward aggregation competes with proper folding. When folding takes place in a crowded environment there are many other proteins around with which the protein may aggregate
Explain the excluded volume effect and how it makes it so that the effective concentration inside cells is smaller than the actual volume.
In a crowded cell, a small molecule could fit into essentially any part of the open space. However larger molecules require a larger space which excludes much of the open space because. This makes the protein act as though the concentration was higher than it actually is (effective concentration vs actual concentration).
What proteins are at an elevated risk for aggregation?
New, unfolded proteins
Mutated proteins
Nuclear proteins
Proteins that become unfolded as a result of stress conditions
What mechanism do cells use to overcome the challenge to proper protein folding that crowding presents?
Helper proteins such as chaperones which protect the unfolded protein from unwanted interactions and allow it to fold properly
What is the function of peptidyl-prolyl cis trans isomerase (PPI)
It catalyze the inter conversion of cis and trans isomers of proline peptide bonds allowing for better protein folding
What is the role of protein disulfide isomerase (PDI) in protein folding?
IT catalyze disulfide bond formation
What are the three broad classes of chaperone proteins briefly describe each.
Small heat shock proteins- very small, ATP independent aid in the formation of oligomeric complexes
Low MW - relatively small, ATP dependent, bind to hydrophobic regions until they can properly fold
High MW- large proteins, ATP dependent, form a cage-like structure around the protein preventing it from interacting with anything else and allowing it to fold
What are the two main classes of protein folding diseases? Give an example of each.
Loss of function and gain of function
Cystic fibrosis is an example of loss of function because the disease is caused by misfolded and ineffective CFTR protein
Systemic amyloidosis is an example of gain of function because proteins that wouldn’t normally aggregate do forming plaques that damage tissues
Explain how alpha-1 antitrypsin deficiency is both a loss of function and gain of function disease.
Normally, alpha-1 antitrypsin coats and protects the lungs from damage. However the misfolded protein does not fulfill this role and leaves the lungs to be damaged (loss of function) in addition, misfolded protein accumulates in the liver causing liver damage (gain of function)
What is congo red?
It is a dye that is used to diagnose systemic amyloidosis. It turns fluorescent green when positive for amyloidosis
What protein structure often defines systemic amyloidosis aggregates?
Cross Beta sheets
Briefly differentiate between the three main types of systemic amyloidosis.
Primary- the most common, caused by overexpression of light chain immunoglobulin which aggregates
Secondary occurs as a result of chronic inflammation
Familial is hereditary based on a mutation to the TTR protein
What are prions?
Misfolded proteins which when in contact with properly folded versions of that protein act as a template for the properly folded protein to take on the misfolded form. Thus they are like protein infections
Compare and contrast foldases and chaperone proteins.
Both are used by cells to promote proper folding of proteins under crowded conditions. Foldases actively catalyze reactions related to folding while chaperones similar protect the protein from interacting with other proteins to allow it to fold by itself
How much DNA is found within the nuclear genome?
About 6000 Mbps
Where does most of the variability between the DNA of individuals occur?
In repeated DNA elements
Describe the structure of mitochondrial DNA
Circular similar to bacteria, contains about 37 genes 5-10 copies of genome per mitochondrion
How much of the variation in gene expression between individuals is explained by SNP’s?
83%
What differentiates between a SNP and a mutation?
A SNP is relatively common, occurring in at least 1% of the population while mutations are more rare
How much of the variation in gene expression between individuals is explained by copy number variants?
17%
What are copy number variations? What roles do we know that they play?
Deletions or duplications of sections of DNA of 1kb to 1Mb in size. They are important in the normal function of special senses and play a role in diseases such as cancer, autism, schizophrenia
What is a tandem repeat? What are the 3 types?
A specific pattern of nucleotides repeated over and over right next to each other. They occur mostly in non-coding regions
1 satellite DNA
2 mini satellite DNA
3 microsatellite DNA
Describe the two main types of satellite DNA.
Alpha-satellites = centromeres
Beta satellites - variable regions on acrocentric chromosomes. They occur near the centromeres
Differentiate between Satellite DNA, Microsatellite DNA and Minisatellite DNA.
All are tandem repeats and they are different classifications based on size
Satellites are larger than 100bp
Mini satellites are 10-100 bp
Microsatellite are 2-4 bp
What type of tandem repeat are telomeres?
Minisatellites because they are. 6 bp repeats
Why are mini satellites extremely variable?
They are hotspots for recombination and replication errors because of their similarity
What is a splice site mutation?
A mutation that occurs in the 2bp before or after an exon that determines splicing. These are almost always harmful
Give an example of the nomenclature of all of the following:
A nonsense mutation
A frameshift mutation resulting in a premature stop codon
A point mutation in cDNA
A point mutation in genomic DNA
A single amino acid change
A mutation to the splice site before an exon
A mutation to the splice site after an exon
P.Val58* C.397delAG (p.Gly47Ilefs*80) C.A786C G.G987T P.Val430Leu G.746+G>T G.754-T>C
Compare and contrast exogenous and endogenous DNA damage.
Exogenous occurs when the damaging factors comes from without the cell while endogenous is when the damaging factor comes from within the cell
What are the 2 main types of exogenous DNA damage and what kind of damage does each one cause?
1 UV- irradiation- tends to cause thymine dimers which are repaired by nucleotide excision repair
2 Alkylating agents- tend to affect guanine residues and may add small or large chains that can distort the helix and cross link strands. Alkylating agents tend to cause cancer
Describe depurination.
Adeline’s and guanine are often spontaneously lost during replication. This is one major cause of mosaicism
Describe deamination of cytosine.
An amine group is lost from cytosine turning it into uracil. Because uracil bonds with adenine, the base is changed in the next replication. This is a common cause of endogenous DNA mutations
How can reactive oxygen species cause DNA damage?
They add carbonyls onto guanine which makes it as likely to pair with adenine as with cytosine which then results in DNA mutations during replication
8 oxo guanine
What common mistakes occur during replication that can lead to mutations?
1 Slippages occur during microsatellites leading to deletions or duplications of genes
2 Tandem repeats can lead to unequal crossing over in meiosis leading to deletions and duplications of major portions of DNA
What is haploinsufficiency?
When 50% of a protein is not enough to be functional. This causes heterozygous individuals with a null mutation to have a disease phenotype
What is a dominant negative?
A mutant protein that loses its normal function and also interferes with the normal protein product such as through competitive inhibition in transcription factors and multimeric proteins
What types of errors will base excision repair fix?
Purine loss and 8-oxo-guanine
What is the function of MUTYH protein and what happens when it is mutated?
It is involved in 8-oxo-guanine DNA repair mutations cause a recessive phenotype leading to many colon polyps and increased risk for colorectal cancer called MUTYH associated polyposis
What types of errors can nucleotide excision repair fix? What kind of phenotypes result from mutations to this machinery?
Repairs bulky DNA lesions such as thymine dimers. Mutations to machinery lead to recessive phenotypes increasing sensitivity to the sun and risk of skin cancer
What 4 genes are responsible for mismatch repair? Which of them dimerize?
MLH1, MSH2, MSH6, and PMS2
MLH1 and PMS2 dimerize as do MSH6 and MSH2
Outline the steps of the double stranded DNA damage signal and repair system including what protein (s) are involved and what results from mutation to that protein.
1 Maintenance of structure and integrity of DNA - BLM (helicase) - Bloom Syndrome (recessive)
2 Signal the repair process - ATM - ataxia telangiectasia
3 Locate damage and trigger repair- FANC genes - Fanconi anemia
4 Repair of DNA - BRCA1 and BRCA2- Hereditary breast and ovarian cancer (dominant)
5 End joining of dsDNA - NBN, Rad50 and MRE11A - Nijmegen breakage syndrome
What protein is involved in single stranded DNA repair?
PARP1
Why could PARP1 potentially be used as a therapeutic agent for cancers related to a mutated BRCA1 or BRCA2?
PARP 1 is involved in single stranded DNA repair and BRCA1 and 2 are related to double stranded repair. These processes have some overlap and if both are compromised, cell death is triggered. Cancers where BRCA1 or 2 is mutated would then trigger cell death if PARP1 is compromised
What is the difference between a nucleotide and a nucleoside?
Nucleosides do not contain phosphate groups
How many rings do purines have? How about Pyrimidines?
Purines: 2
Pyrimidines: 1
What is the major source of nucleotides in humans?
De novo synthesis of nucleotides
Outline the Pentose Phosphate pathway and explain its importance. Include the rate limiting enzyme
Glucose 6 phosphate from glycolysis is converted to Ribose 5 phosphate by the reduction of 2 NADP to NADPH. Then Ribose 5-phosphate is converted to 5 Phosphoribosyl-1-pyrophosphate PRPP which is important in nucleotide synthesis. NADPH is also an important antioxidant.
The rate limiting enzyme is glucose 6 phosphate dehydrogenase
What occurs as a result of glucose 6 phosphate dehydrogenase deficiency and why? What is the inheritance pattern?
Results in hemolytic anemia in response to infection, certain drugs and fava beans which all increase levels of reactive oxygen species. this occurs because glucose 6 phosphate dehydrogenase catalyzes the rate limiting step of the pentose phosphate pathway which produces NADPH an important antioxidant
X-linked recessive
Outline the major steps of purine biosynthesis from Ribose 5-phosphate. Include the key enzyme and major substrates.
Ribose 5P—> PRPP—> 10 steps —> IMP—> 2 steps—> AMP or GMP
Key enzyme: glutamine PRPP aminotransferase
Important substrates: Amino acids (glutamine x2, glycine, aspartate), Carbon sources (HCO3- and THF)
Outline the pathway of adenosine catabolism. Include 2 key enzymes
AMP-> Adenosine->Inosine->Hypoxanthine->Xanthine->Uriel acid
Key enzymes: Adenosine deaminase - catalyzes adenosine to inosine
Xanthine oxidase- catalyzes hypoxanthine to Xanthine and Xanthine to Uric acid
OUtline the pathway for catabolism of Guanine.
Include 1 key enzyme
GMP->Guanosine->Guanine->Xanthine->Uric acid
Key enzyme Xanthine oxidase- catalyzes Xanthine to Uric acid
What symptoms would occur as a result of a deficiency of adenosine deaminase and why?
Severe combined immunodeficiency called (scid) occurs because ADA leads to the buildup of ATP which inhibits RNR which catalyzes dNTPS. Without dNTP’s cells can’t divide and this kills T lymphocytes and B lymphocytes which constantly divide
Describe how gout and its treatment relates to purine metabolism.
Gout occurs when Uric acid builds up and forms crystals leading to chronic inflammatory arthritis. This occurs as a result overactivity of purine catabolism. It can be treated with allopurinol which resembles hypoxanthine and inhibits Xanthine oxidase lowering the production of Uric acid
Describe the major and minor pathways of purine salvaging and the enzymes for each.
Minor
Adenine + PRPP-> AMP + ppi
Enzyme: Adenine phosphoribosyltransferase APRT
Major
Hypoxanthine + PRPP->IMP +PPI->AMP or GMP
Guanine+PRPP->GMP+ppi
Enzyme: Hypoxanthine-guanine phosphoribosyltransferase HPRT
What disorder results from HPRT deficiency and why?
Lesch Nyhan syndrome- severe hyperuricemia and compulsive self-mutilation.
This occurs because HPRT salvages purines and if they are not salvaged they will produce Uric acid in over abundance and create hyperuricemia
What is the committed step of purine biosynthesis?
Conversion of PRPP to 5-phosphoribosyl amine (2nd step)
List the substrates and key enzyme of Pyrimidines biosynthesis
Substrates: amino acids (glutamine and aspartate) and Carbon sources: CO2 and THF
Key enzyme: carbamoyl phosphate syntheses II
What is the committed step of pyrimidine biosynthesis?
Production of carbamoyl phosphate by carbamoyl phosphate synthetase II (CPSII)
Outline the major steps in synthesis of Cytosine.
CO2+Glutamine + ATP->4 Steps-> +PRPP-> 2 steps-> UMP->UDP->UTP->CTP->dCTP
Outline the major steps in Thymine biosynthesis
CO2 + Glutamine +ATP->4 Steps->+PRPP-> 2 Steps-> UMP-> dUMP-> dTMP
methylene tetrahydrofolate (THF) is required for final step
How are Pyrimidines salvaged?
Cytidine + ATP -> CMP +ADP
What are the end products of catabolism of Pyrimidines? HOw are they different from the end product of purine catabolism?
Cytidine and uridine -> alanine
Thymidine-> amino butyrate
Their rings are no longer intact making them water soluble so they don’t cause disorders unlike uric acid the product of purine biosynthesis.
What causes orotic aciduria and what are its symptoms?
Symptoms: megaloblastic anemia, crystalline sediment of orotic acid in urine, slow growth
Caused by a deficiency in UMP synthetase which prevents biosynthesis of Pyrimidines. Lack of Pyrimidines leads to the above symptoms
What is a major difference between the biosynthesis of purines and that of Pyrimidines.
In pyrimidine biosynthesis, heterocyclic rings are synthesized first and then bound to ribose phosphate. The opposite is true in purine biosynthesis.
How is the production of deoxyribose thymine different from that of other Pyrimidines?
All other Pyrimidines are converted to deoxyribonucleotides from their corresponding ribonucleotide. However, thymine is converted from deoxyribouracil.
What enzyme catalyzes the conversion of ribonucleotide of cytosine to deoxyribonucleotide?
Ribonucleotide reductase
Describe how ribonucleotide reductase is allosterically regulated. What disorder does this relate to?
ATP activates it and dATP inhibits it. This relates to SCID in which adenosine deaminase is deficient leading to accumulation of dATP’s which inhibit ribonucleotide reductase preventing the production of dNTPs
What is folate? How is it converted to its active form?
Folate is a B-vitamin that plays an important role in nucleotide synthesis by being a carbon source. It’s active form is tetrahydrofolate (THF) it is converted from folate by successive reductions with NADPH by the enzyme dihydrofolate reductase
Describe how each of the following function as chemotherapeutic agents:
5 fluorouracil
Methotrexate
Hydroxyurea
All function in some way by inhibitting pyrimidine biosynthesis preventing the production of nucleotides needed for replication
5-fluorouracil is a structural analog of thymine and blocks the conversion of dUMP to dTMP
Methotrexate is a structural analog of folate and competitively inhibits dihydrofolate reductase preventing the formation of THF which is necessary for nucleotide synthesis including conversion of dUMP to dTMP
Hydroxyurea inhibits ribonucleotide reductase preventing the formation of deoxyribonucleotides from their corresponding ribonucleotides
Briefly describe the structure of the ER import sequence.
It is found near the N-terminus and is made up of hydrophobic amino acids
Briefly describe the mitochondria import sequence.
It is found near the N-terminus and contains regularly distributed basic (Positively charged) amino acids
Briefly describe the structure of the nuclear import and nuclear export signal sequences.
Import: Short and made up of positively charged amino acids
Export: short and made up of hydrophobic amino acids
What is the ER retention signal? What is it like?
It signals that a protein is to be maintained within the lumen of the ER rather than to be transported to another organelle. It is 4 amino acids at the C terminus with varying characteristics
Define paternal and maternal imprinting.
In paternal imprinting, the father’s copy of a gene is methylated and turned off. IN maternal imprinting it is the mother’s copy that it methylated and turned off
Explain how the transferrin receptor is an example of RNA stability used to control gene expression
Transferrin gene is constantly transcribed but its mRNA is degraded and not translated unless iron is low in which case it is immediately translated. Allows the body to react immediately to low iron
What is the function of snRNA?
They regulate splicing
What is aplastic anemia?
A rare form of anemia caused by failure of the bone marrow to produce all blood cell types
Where do most achondroplasia mutations come from?
80 to 90% are de novo mutations and all de novo mutations come from the father
Explain the function of the two gene therapies to treat SMA.
Spinrata-causes inclusión of exon 7 to be more likely in smn2.
Zolgensma delivers the smn1 gene in adeno associated virus
What is the haldane hypothesis?
It states that a genetically inherited disease causing death before eproduction should decrease by 1/3 each generation. This shows us that 1/3 of Duchenne muscular dystrophy cases are de novo mutations because the frequency is remaining constant
What is heteroplasmy?
A specific form of mosaicism that results from differences in the number of normal and abnormal mitochondria within each cell
What is the natural, resting state of DNA?
30nm fiber
What are the possible causes of polyploidy?
1 Fertilization of ovum by 2 sperm
2 an error in meiosis 2 of oogenesis
Is the father or mother more likely to contribute a structurally abnormal chromosome?
The father
What is the likely phenotype for an individual with a chromosome translocation?
They may be phenotypically normal if they have a full compliment of genetic material but have problems with reproduction because of difficulties of lining up chromosomes in meiosis
What other sex chromosome abnormalities exist in addition to turner’s Syndrome and klinefelter’s syndrome. What are the symptoms?
47,XXX and 47,XXY- these usually show no significant physical phenotype and only a mild learning disability
What is the usual cause of Turner’s syndrome?
Lack of a paternally inherited X chromosome
What is a cystic hydroma? What condition is it associated with?
A sac of fluid that forms during development near the back of the neck associated with Turner’s syndrome
Give the cause and major symptoms of Williams disease.
Caused by a deletion on chromosome 7
Symptoms: delayed speech and development, unique face, mild to moderate mental retardation, feeding issues, and cocktail personality
For which if the genetic testing methods can SNPs be a problem?
Any of them that use probes or primers such as PCR, Sanger sequencing and multiple probe ligations MLPA
Where does erythrophoiesis occur?
In the bone marrow, specifically the medullary space of long bones and especially in the hip
What is erythropoietin?
A hormone that is released in response to low levels of oxygen and causes increased erythropoiesis
What is extramedullary erythropoiesis? What is it an indication of?
Production of red blood cells occurring outside of the bone marrow. Most often in the liver, spleen and lymph nodes. Occurs normally in embryos but in adults it is a sign of RBC turnover or bone marrow failure. It is also associated with hereditary spherocytosis
What structural changes occur to forming red blood cells across each stage of erythropoiesis (generally)?
They get smaller, more hemoglobin and more chromatin condensation with each step
What is a holoprotein? Describe how both hemoglobin and myoglobin can be classified as holoproteins?
A holoprotein is a protein that contains at least one non-protein prosthetic group. The heme group is a non-protein prosthetic group
Describe the structure of the heme group.
It is a porphyrin ring with 4 nitrogen containing rings each of with interacts with Fe which is at the center
What protein structures are most prevalent in myoglobin and the hemoglobin subunits?
Alpha hélices
What is the Bohr effect?
The effect of low pH on hemoglobin binding curve. Namely, at low pH protons bind to hemoglobin stabilizing the T state and dramatically lowering the affinity for oxygen
How are all of the different hemoglobin genes arranged in the genome?
The various hemoglobin genes are arranged so that the genes for like subunits are grouped together.
Chromosome 16 contains 1 zeta gene and 2 alpha genes close to each other
Chromosome 11 contains 1 delta, 1 epsilon, 2 gamma and 1 beta gene all close together
Suppose 1 mutant hemoglobin variant lowers the affinity for oxygen and another raises the affinity for oxygen. Which will likely be more damaging to the individual?
The one that raises affinity
Describe hemoglobin C gene.
Caused by a mutation at the exact same locus as sickle cell disease that changes to a positively charged amino acid instead of a hydrophobic amino acid (like in sickle cell). It is homozygous recessive and causes a milder anemia than sickle cell. Individuals who are heterozygous for the hemoglobin C allele and sickle cell allele have sickle cell anemia but it is usually milder than those who have homozygous for sickle cell trait
What other factors besides a mutation could lead to a gain of function disease of protein folding?
Abnormal protein concentration and protein infections
What acronyms correspond with each type of systemic amyloidosis?
Primary = AL Secondary = A Familial = ATTR
What is the difference between synonymous and non-synonymous SNPs?
Synonymous do not cause amino acid changes while non-synonymous do
What is another name for a minisatellite? What is another name for a microsatellite?
Minisatellite = variable number tandem repeat Microsatellite = short tandem repeat
Explain the term compound heterozygote.
An individual who has inherited two partially functional genes. Phenotype depends on the severity of the reduced function of each gene. An example would be someone who inherited one sickle cell allele and one hemoglobin C allele. In this case they would display a phenotype more severe than homozygous for hemoglobin C but less severe than homozygous for sickle cell
What is the role of mismatch repair in DNA repair?
It fixes replication errors, mismatched nucleotides, and problems with microsatellites
Where does metabolism of purines take place?
Liver
What enzyme catalyzes the formation of deoxyribonucleotide thymine?
Thymidine synthetase
What are the 3 main functions of the plasma membrane?
1 Receiving information
2 Import and export
3 Movement and change of shape
What is meant by the fluid mosaic model of the plasma membrane?
Describes the way the membrane acts which is like a fluid in 2 dimensions
Describe the general components that make up the plasma membrane and how they are arranged.
The major component is phospholipids which contain a hydrophilic head and 2 hydrophobic tails. These form a lipid bilayer with hydrophilic heads on the outside and hydrophobic tails in the center. Another major part of membranes are proteins. Cholesterol is present inside of the membrane and fills in the gaps between hydrophobic tails as well as making the membrane less permeable. Finally sugars are found on the outside of the membrane covalently attached to either proteins or lipids.
Diagram the structure of a general phospholipid in the plasma membrane.
A hydrophilic head attached to phosphate which is attached to a glycerol molecule with 2 hydrophobic tails coming off of it. On one of the fatty acid tails, a cis double bond causes a kink
What is meant by the term amphipathic and how does it relate to molecules in the plasma membrane?
Amphipathic means having both a hydrophobic and hydrophillic region in the same molecule. Proteins and lipids in the membrane are amphipathic allowing them to form a bilayer
What are the four major types of phospholipids? Briefly describe how they differ. What is the minor 5th? And what is its importance?
1 Phosphatidyl ethanolimine
2 Phosphatidylserine
3 Phosphatidylcholine
4 Sphingomyelin
1-3 are exactly the same other than the hydrophilic head group which is reflected in their name.
Phosphatidyl serine is - charged while the rest are neutral
4 has a choline head group but is attached to the hydrophobic portion by a different molecule other than glycerol
5 (minor) Phosphatidyl inositol- important for recruiting signaling molecules
Describe the ways that lipids move within the plasma membrane.
They move freely in: lateral diffusion (side to side), flexión (movement of tails), and rotation (spinning in place)
Flip flop or changing from one side of the bilayer to the other is rare and requires flipase or flopase enzymes. This type of movement is important in membrane synthesis
Describe the asymmetry of the lipid bilayer.
There are differences between the outer and inner layers of the bilayer. Mainly glycolipids and proteins are found only on the outer layer and the inner layer is more negatively charged due to the presence of Phosphatidyl serine and Phosphatidyl inositol
What types of things will and won’t pass freely through the plasma membrane.
Will: hydrophobic molecules small or large such as O2 or steroids
Rarely will: Very small hydrophilic molecules
Won’t: large hydrophilic molecules or charged molecules of any size
What are lipid rafts?
Regions of the membrane with distinctive structure and function. They contain a lot of cholesterol and glycosphingolipids which makes them thicker and less fluid and this determines what proteins will be present here. They are often involved in cell signaling
What are the 2 major categories of membrane proteins and the subgroups within each category.
1 Integral- directly attached to the membrane and require detergents to be separated
A Transmembrane- passes through the entire membrane
B Membrane associated
C lipid linked - B and C are covalently bound to membrane components
2 Peripheral- interact non-covalently with membrane proteins and can be separated without detergent
A Protein attached
What are the 4 main functions of membrane proteins?
1 Transporters
2 Anchors
3 Receptors
4 Enzymes
Describe the primary structure of transmembrane proteins.
They are amphipathic with regions of hydrophobicity which pass through the center of membranes and hydrophilic regions that remain on the outside of the bilayer
Describe the common secondary structures of transmembrane proteins.
They often form alpha helices in their hydrophobic regions which allow them to show hydrophobic amino acids on the outside of the helix. Sometimes multiple amphipathic alpha helices will combine together to form a hydrophilic pore where hydrophilic molecules can pass through the membrane
Less often, beta sheets can be found which can form beta barrels which appear barrel like and function similarly to hydrophilic pores formed by alpha helices
In what ways can the lateral diffusion of membrane proteins be restricted? (4)
1 They can be bound to cytosolic proteins
2 they can be bound to extracellular proteins
3 they can be bound to proteins on other cells
4 The cell can form barriers to diffusion
What is the cell cortex?
A framework of fibrous proteins that links proteins in the membrane together to strengthen the otherwise weak membrane
What are glycoproteins and proteoglycans?
Glycoproteins are proteins linked to sugars that are common in the outer side of the plasma membrane. Proteoglycans are a specific kind of glycoprotein that is heavily glycosylated with long, repeating, unbranched amino containing sugars unlike the shorter non-repeating and branched sugars on most glycoproteins. Proteoglycans are especially important in forming the extracellular matrix and connective tissues
Describe the glycocalyx. What are its functions? (3)
The layer of sugars on the outside of the plasma membrane made up of glycolipids and glycoproteins. It functions to protect the membrane from mechanical damage, cell-cell signaling and lubrication of extracellular space in vascular system
Describe radiography in terms of energy , how it shows tissue differenc, benefits and limitations.
Energy: X-rays
How it shows differences: different electron densities
Benefits: Fast, great resolution
Limitations: Poor tissue contrast, uses radiation
Describe Computed tomography (CT) in terms of energy , how it shows tissue differenc, benefits and limitations.
Energy: X-rays
HOw it shows differences: Different attenuation of x-rays relative to water
Benefits: Very good contrast at all tissue densities
Limitations: TOns of radiation
Describe magnetic resonance imaging (MRI) in terms of energy , how it shows tissue differenc, benefits and limitations.
Energy: Radio waves
How it shows differences: different rates of nuclear relaxation
Benefits: Best tissue contrast, no radiation
Limitations: Slow and expensive, magnet dangers
Describe Ultrasound in terms of energy , how it shows tissue differenc, benefits and limitations.
Energy: Sound waves
How it shows difference: Different acoustic impedance
Benefits: Real time imaging, no radiation
Limitations: User dependent, many artifacts
Describe nuclear medicine in terms of energy , how it shows tissue differenc, benefits and limitations.
Energy: Gamma rays
How it shows difference: different levels of radioactivity
Benefits: See physiology and pathophysiology in action
Limitations: Worst resolution, Radiation
Compare and contrast simple diffusion and protein-assisted diffusion.
Diffusion always refers to passive movement of molecules down their concentration gradient. In simple diffusion they cross the membrane directly- this is only possible for small, hydrophobic molecules. In protein assisted diffusion, proteins embedded in the membrane allow movement of larger or hydrogphilic compounds
What is flux? What is net flux?
Flux is the amount of molecules crossing a membrane at any given time. Molecules are always crossing in both directions so net flux is the difference between the two one-way fluxes
What is Fick’s law?
It explains how much of a given molecule will diffuse across a membrane.
Diffusion is directly proportional to:
K- permeability of solute
A- Surface area of membrane
C = solute concentration gradient
Diffusion is inversely proportional to :
delta x - distance the solute must travel
What factors influence permeability constant (K)?
Lipid solubility- directly proportional
Solute size- indirectly proportional
What are the two main types of protein-assisted diffusion?
Ion channels
Facilitated diffusion
Compare and contrast ion channels and facilitated diffusion in terms of how they work, how quickly they work, what they can transport and how they select for solutes.
They both function on passive diffusion that is they do not require energy and transport only in the direction of the concentration gradient. Ion channels simply work by creating a very small pore in the membrane that will allow ions through, they work very quickly, can only transport ions and use size and charge of amino acids within the pore to select solutes.
Facilitated diffusion works by binding the solute at a specific site causing a conformational change which allows the solute to be released within the cell, they work much slower, they can transport ions or larger molecules, and they select based on the specific molecule’s ability to bind at the active site.
In what 3 ways can ion channels be gated?
1 Ligand gated - activated by binding of a signaling agent
2 Voltage gated- activated by changes in membrane potential
3 Mechanically gated- activated by physical deformation of the surrounding membrane
What is an electrochemical gradient?
Diffusion of ions depends not only on the concentrations of that specific ion but also concentrations of charge on either side of the membrane
What is membrane potential? What is the resting potential of cells like?
Membrane potential is the difference in charge on either side of the membrane. Generally, cells have more negative charge inside than out (-70mv)
What are the 2 types of active transport how are they different?
Primary- uses ATP for energy
Secondary uses ion gradient for energy
Diagram how primary active transport works.
Solute binds to pump protein-> ATP is hydrolyzed -> Pi binds to pump-> conformational change of pump allows solute to enter/exit the cell
Describe how the sodium potassium pump works. What type of transport is it and why?
It transports 3 sodium out of the cell and 2 potassium into the cell against their gradients with the use of 1 ATP.
This makes them primary active transport
What are the two types of secondary active transport? What is the usual energy source in the body?
The usual energy source is the sodium gradient.
1 Co-transport = solute moves in the same direction as sodium
2 Counter-transport/antiport = solute moves in the opposite direction of sodium
Why can water move across the membrane even though it is polar?
It can’t unless there are channels called aquaporins which are present in different concentrations in most cells. Amount of aquaporins determines speed of osmosis.
What is the typical osmolarity of body fluids?
300 mOsm
What is the difference between osmolarity and tonicity?
Osmolarity is the concentration of all solutes
Tonicity is the concentration of only non-permeable solutes
Most solutes in the body function as if they were non-permeable so the two are usually very similar
How does the cell respond to a hypertonic, isotonic and hypotonic solution?
Hypertonic- water leaves, cell shrinks
Isotonic- no net movement of water, no change in cell size
Hypotonic- Water enters cell, cell swells
Why do sodium and potassium behave as non-penetrating solutes even though they can pass through channels in the membrane?
Because they are quickly pumped by the sodium potassium pump to maintain their gradients.
What is the role of the Smooth endoplasmic reticulum? What is its structure?
It is made up of roles rolls of membrane which may be contiguous with the rough ER but does not contain ribosomes.
It’s function is to synthesize fatty acids and phospholipids
What types of cells would you expect to have abundant smooth endoplasmic reticulum?
Cells which produce a large quantity of lipids such as steroid producing cells for example the adrenal cortex or the leydig cells in the testes
What is smooth endoplasmic reticulum called in skeletal cells? What is its specialized function?
Sarcoplasmic reticulum, it sequesters calcium ions which play an important role in the contractile impulse
Describe the specialized role of the smooth endoplasmic reticulum in liver cells.
It helps to detoxify some hydrophobic compounds turning them into water soluble compounds which can be excreted in the urine. These include toxins such as carcinogens and pesticides and drugs like ethanol and the barbiturates thiopentanal and phenobarbital
What are the 3 main functions of vesicular transport?
1 Deliver newly made molecules to destinations
2 Communicate with extracellular environment
3 Ingest extracellular particles and solutes
Distinguish between the 2 major pathways of vesicular transport
Secretory pathway- in to outmoves from the ER to the golgi and from there it can move to the plasma membrane or lysosomes
Endocytic pathway - out to in
Moves from outside of the cell to components within the cell
What is budding? What is fusion?
Budding is when the vehicle breaks off from the membrane at its origin
Fusion is when the vesicle joins the membrane at its target
What is vesicle coating? What are its 2 functions?
Proteins that cover the outside of vesicles during budding and will be removed prior to fusion
They function to help vesicle bud off and to aid in sorting of proteins
Describe the structure of clathrin. What is its function?
It is the most important vesicle coating protein
It has 3 light and 3 heavy chains that form a 3 armed spiral structure called a triskelion. When coating vesicles it creates a honeycomb like lattice structure around the outside of the forming vesicle forcing the membrane into a circular shape
Diagram and describe how clathrin and other proteins are involved in protein sorting.
Clathrin binds to adaptin which binds to a receptor that is specific for a type of cargo. Different forms of adaptin exist in different areas which allow different receptor/cargo pairs to enter vesicles. After budding, clathrin and adaptin are released from the vesicle but the receptor and cargo remain
What protein pinches budding vesicles off from the membrane?
Dynamin
Describe the process and proteins involved in docking and fusion of vesicles.
First, Rab protein on the vesicle binds to a tethering protein on the target which pulls the vesicle closer to the membrane. Here v-Snare on the vesicle binds to T-snare on the target and the two pull each other so close together that fusion occurs
What are the 3 most common post-transcriptional modifications that take place in the ER?
1 Disulfide bonds- both intrachain and interchain
2 Lipid membrane anchors are added covalently
3 Glycosylation- usually to the N-terminus of asparagine residues but occasionally also to serine or threonine
Detail the process of glycosylation in the ER?
Dolichol protein embedded in the ER membrane holds the oligosaccharide until it will be added to the asparagine residue by the enzyme oligosaccharyl transferase
Describe the protein quality control that occurs in the ER.
ER does not allow proteins that either misfolded or did not join with the proper subunits to leave the ER. This is accomplished by chaperone proteins which bind to the unfolded protein and prevent other interactions until it is properly folded. If the protein never folds properly it is held by chaperones until it is eventually exported to the cytosol for degradation
Explain the connection between cystic fibrosis and ER quality control of proteins.
The mutated protein in the cystic fibrosis gene could be at least partially functional if it reached the plasma membrane but instead it is prevented by the quality control mechanisms of the ER leading to its degradation and a severe phenotype
Describe the structure of the golgi.
The golgi is made up of several long, flattened membranes piled on top of each other kind of like a stack of pancakes. One end is called the cis face which is where vesicles enter and the other is the trans face where vesicles exit. Cargo moves by vesicles from one membrane bound layer to another throughout the golgi until reaching the trans face
What actions occur to proteins as they move through the golgi?
They are sorted and oligosaccharides are added or removed
Differentiate between the two types of exocytosis.
Constitutive secretion- occurs at all times unregulated in all cells. It carries new proteins and lipids to the plasma membrane and transports other cargo outside of the cell
Regulated secretion occurs only in specialized secretory cells such as those that release hormones. In this case vesicles storing tightly packed cargo are produced and wait near the membrane until a signal is received that causes them to fuse to the membrane releasing their contents.
What are acid hydrolases?
The proteins in the lysosome that degrade different types of molecules and function under the lower pH of the lysosome relative to the rest of the cell. They are highly glycosylated to allow them to withstand the low pH.
Describe how lysosomal enzymes are targeted to the lysosome from the ER.
These proteins contain a short amino acid sequence that targets them to lysosomes. Once in the golgi, a mannose-6 phosphate is added to these proteins. In the trans face of the golgi, the mannose-6phosphate binds to mannose-6phosphate receptors in clathrin coated pits with then form vesicles bound for early or late endosomes which will mature into lysosomes
Describe the causes and symptoms of lysosomal storage diseases. Give a few of the most common and the most severe.
Diseases caused by a defected hydrolase or cofactor that functions in the lysosome to degrade a specific molecule. This causes that molecule to build up and become toxic. Babies with these diseases appear normal at birth but show symptoms very early. Life expectancy is 15 years. Most common are : Gaucher disease, Hurler syndrome and Hunter syndrome.
The most severe is I-cell disease which causes is a defect in the enzyme that adds mannose-6-phosphate to hydrolases so none of them reach the lysosome.
What are the 3 types of endocytosis?
1 Phagocytosis- injestion of large particles only in specialized immune cells
2 Pinocytosis- non-specific constitutive injestion of fluid and solutes that occurs in all cells
3 Receptor mediated endocytosis- very specific uptake of molecules which bind to receptors in the membrane
How are constitutive exocytosis and pinocytosis related to each other?
Both are occurring constantly and non-specifically and they compliment each other so that one process doesn’t significantly change the amount of membrane in a cell
Briefly describe how phagocytosis works.
Membrane extends projections called pseudopods out to engulf whatever large particle it is trying to injest until it covers the entire particle and brings it in
What is the fate of endocytosed molecules?
They first go to early endosomes near the plasma membrane. This causes most receptors to release their substrate because of the slightly acidic environment. Endosomes sort cargo and most of the cargo released from receptors ends up in the lysosomes for degradation
Explain how LDL cholesterol gets into cells.
LDL cholesterol is a complex containing cholesterol surrounded in a lipid monolayer which is surrounded by an organizing protein. This allows transport of cholesterol in blood although it is hydrophobic. This entire complex binds to receptors and is endocytosed. In the early endosomes, the cargo is released from the receptor and in the lysosome, it is degraded, releasing cholesterol which diffuses out through the membrane and into the cytosol of the cell
Describe the cause and symptoms of familial hypercholesterolemia.
Caused by a mutation in the LDL receptor that would normally allow for receptor-mediated endocytosis that causes it to be either missing or non-functional. Thus LDL doesn’t enter cells and accumulates in the blood stream which can lead to atherosclerosis and early cardiovascular disease. Patients are treated by controlling their diets and using cholesterol lowering drugs such as statins
Why does signal transduction usual occur over many steps instead of a few?
This allows:
1 Signals to be regulated at each step
2 Signals to be amplified
3 Signals to be distributed to several processes at the same time
What 3 actions/molecules often form part of a signal cascade?
1 Enzyme activity- ex kinases
2 Protein-protein interactions
3 Second messenger molecules
How can different cells respond differently to the same signal?
Different receptors could be present on each cell that start differing cascades or the signaling pathways could be different downstream
Why is withdrawal of a signal important? What actions can be taken to withdraw the signal?
Proper withdrawal is as important as signaling itself because it if is withdrawn too quickly it can’t have a large enough effect and if it is withdrawn too slowly it could have too dramatic of effects or the receptor could be downregulated as a result. Withdrawal can occur by removing the signal molecule via diffusion, degradation or endocytosis by neighboring cells or it could occur by downregulation of the receptors either by reduced synthesis or internalization
Describe the cause, symptoms and treatment for myasthenia gravis
Myasthenia Gravis is an autoimmune disease in which antibodies are formed against acetylcholine receptors in skeletal muscle preventing reactivity to acetylcholine. Symptoms include severe fatigue, a droopy eyelid, unsteady walking and difficulty swallowing. It can be treated by acetylcholinesterase inhibitors which prevent degradation of acetylcholine and increase its concentration
What type of signals might cause immediate modifications? What signals might cause slower, long-term changes?
Immediate changes could be caused by changes in protein activity for example in changing the shape of the cell.
Slower long-term results take place from signals which affect gene expression because time is needed to transcribe, translate, modify and transport protein that is expressed
Compare and contrast the structure of G-protein coupled receptors and Receptor tyrosine kinases.
Both contain an extracellular domain which binds to the signal molecule and both contain intracellular domains which bind to proteins. The main difference is that GPCR’s pass through the membrane 7 times while RTK’s pass through only once.
Describe the general process of activation of a G-protein coupled receptor.
The GPCR is bound to a trimer of G-proteins alpha, beta and gamma. The alpha is bound to a GDP but once the signal molecule is bound to the receptor, it causes the GDP to be replaced with GTP. This change causes the alpha and beta/gamma subunits to dissociate from the receptor and become active. These subunits bind to their targets to affect some change which they causes phosphorylation of the GTP back to GDP and the trimer reforms and binds to the GPCR once again.
What are some of the classes of GPCR functions?
They can regulate ion channel activity or regulate membrane bound enzymes such as adenylyl ciclases
What are the functions of Gi Gs and Gq GPCR’s?
Gi- inhibits adenylyl cyclase
Gs activates adenylyl cyclase
Gq activates phospholipase C
What pathways are enzyme linked receptors generally involved in?
Pathways related to growth, proliferation, differentiation and survival. Their substrates are often growth factors. For this reason dysregulation of these pathways often results in cancers
What is the most common type of enzyme linked receptor?
Receptor tyrosine kinases
Generally describe how receptor tyrosine kinases are activated.
When the signal molecule binds to the receptor, it causes them to dimerize. Each receptor cross phosphorylates the other. The negative charge from phosphorylation attracts new proteins and a large protein complex forms around the receptor which can begin signal transduction pathways
What are ion channel linked receptors? Where are they commonly found?
Receptors that when bound to signal molecule open or close ion channels changing the electrical charge across the membrane. These are common in neural signals.
What type of molecule might bind to an intracellular receptor? What will its function be?
A small hydrophobic molecule so that it can pass through the membrane
These are basically always involved in changing gene expression
Explain how the gas nitrous oxide functions as a signal molecule.
The enzyme NO synthase converts arginine to NO which diffuses across membranes into other cells. NO binds to gauntly cyclase which produces cyclic GMP a second messenger. The result of increased cGMP is relaxation of smooth muscles which causes dilation of blood vessels.
How far reaching are the effects of NO synthesis and why?
They are very localized due to the instability of NO.
Explain why nitroglycerin is an effective treatment for patients with angina. Why would it be problematic to take nitroglycerin with other drugs such as ED medications?
Angina is caused by inadequate blood flow to the heart. Nitroglycerin is converted to NO which causes dilation of blood vessels by creating cGMP. This lowers the blood pressure so if taken with other drugs that work in the same pathway, blood pressure could be lowered to dangerous or even fatal levels.
What is cAMP? How is it produced and degraded?
Cyclic AMP is a second messenger molecule common to signal transduction pathways. It is produced by adenylyl cyclase from ATP and degraded by phosphodiesterase to AMP.
What does cAMP do once created?
It activates protein kinase A which phosphorylates enzymes in the cytosol or can travel into the nucleus and activate other proteins which regulate gene expression
Explain how cholera affects the body at the cellular level.
Creates a toxin which irreversibly modifies the alpha subunit of Gs proteins by adding ADP-ribose. This prevents hydrolysis of GTP to GDP so the pathway is always active. This results in high cAMP levels causing a massive flow of water into the intestine and severe, life threatening diarrhea.
How is cGMP created and degraded?
It is synthesized by guanylyl cyclase and degraded by cGMP phosphodiesterase
What is PIP2? Describe how it is produced and what its effects are.
Phosphotidyl inositol in the plasma membrane can be phosphorylated in a variety of ways by PI kinases. One of these forms is PIP2. It can be broken down by phospholipase C to produce DAG and IP3. DAG remains in the membrane and binds to protein kinase C. IP3 triggers the opening of calcium ion channels in the ER causing the cytosol to be flooded with calcium. Calcium also binds to Protein kinase C and along with DAG activates it so that it will go on to phosphorylation other proteins
What is Ras? How is it activated and what does it do?
It is a monomeric G-protein that is activated downstream of almost all RTK’s. It activates the Map-kinase cascade which results in protein activity and gene expression related to survival. For this reason it is often mutated in cancers and is an important oncogene.
Outline the PI-3 kinase pathway including how it is stimulated what it does etc.
It is stimulated by Ras and other G-proteins. As a result of stimulation, Phosphatidyl inositol is phosphorylated at the 3 position hence PI3. This stays in the membrane but becomes active and activates other membrane proteins mostly related to growth and survival
Describe the JAK STAT pathway.
It is a short pathway involved in inflammation and immunity
Describe the TGF beta-SMAD pathway.
A short pathway involved in development. It uses serine/threonine kinases
THF beta binds to its receptor which dimerizes and cross phosphorylates. Then it activates SMADs which travel to the nucleus to affect gene expression
What are 3 methods by which membrane receptors become desensitized? When might this occur?
1 Downregulation: Decrease in receptor synthesis or increase in degradation (occurs slowly)
2 sequestration: receptors internalized by endocytosis (occurs rapidly)
3 Inactivation of GPCR’s: GPCR’s are phosphorylated preventing interaction with G-proteins (occurs rapidly)
This would occur in response to constant activation because of prolonged high concentration of substrate
How do GPCR’s become inactivated by phosphorylation?
G-protein-coupled receptor kinases (GRK’s) phosphorylated them and once phosphorylated they bind to arrestin molecule rather than G-protein
How are Beta adrenergic receptors in the heart activated? What pathway do they follow after activation and what is the end result?
They are activated in response to epinephrine and norepinephrine. They are GPCR’s that activate a Gs protein which activates adenylyl cyclase pathway and protein kinase A. The end result is increasing contractility and heart rate.
Describe the relationship between heart failure and Beta adrenergic receptor signaling. How can it be treated?
Damage to the heart -> sympathetic response-> prolonged response -> desensitization of beta-adrenergic receptors-> decreased contractility and heart rate -> further weakens the heart
Beta blockers can be used to treat heart failure because paradoxically they actually correct the beta adrenergic receptor number and GRK activity
Draw the structure of the insulin receptor.
2 transmembrane tyrosine kinase domains bound to two completely extracellular domains by disulfide bonds. The extracellular domains are bound together by disulfide bonds as well and together they form an active site for insulin binding
What pathways are activated by the insulin receptor?
MAPK and PI3K-AKT pathways to induce glucose uptake, cell survival and proliferation
Major effects on glucose regulation come through the PI3K-AKT pathway
Explain the role of calcium ions in signaling generally.
Calcium ions are at a lower concentration in the cytoplasm than in the extracellular environment and in the ER because of ion pumps. Calcium ion channels can be opened in either location as a result of signaling which floods the cytoplasm with ions and can activate or deactivate other proteins
What gene is responsible for cystic fibrosis? Where is the gene located? What is its basic function?
CFTR cystic fibrosis transmembrane conductance receptor
It is located on exon 7
Functions as a Cl- ion channel in the ABC transporter family
What are some of the symptoms of cystic fibrosis?
Salty sweat
Severe cough with thick mucus
Digestive problems including leading to malnutrition
Obstruction and infection of airways
Damage to respiratory system and pulmonary disease (90% of mortality)
Describe and/or diagram the general structure of the CFTR gene.
It has two membrane spanning domains with 6 transmembrane domains each. Each also bound to a nucleotide binding domain and one is attached to the cytoskeleton. The two subunits are attached by the R (regulatory) domain in between them. The R domain is unique and not similar to other ABC transporters.
Describe how the CFTR gene is gated?
Gating refers to the regulation that takes place to determine when it is open or closed.
1 cAMP activates PKA
2 PKA phosphorylates R domain
3 ATP is hydrolyzed in NBD1 to open channel
4 PKA phosphorylates more sites on R domain
5 NBD2 binds ATP which further opens channel
6 ATP hydrolysis at NBD2 results in release of ADP and channel closing
7 R domain dephosphorylation closes channel
What are the 6 classifications of CFTR mutations? Which is the most common? Which are the most severe? Relate them to some of the pancreatic symptoms.
I lack of CFTR
II defective, misfolded CFTR (does not reach membrane)
III defective gating
IV Restricted Cl- movement thru channel
V reduced protein (alternative splicing)
VI accelerated turnover
>85% are type II
I-III are the most severe leading to pancreas insufficient phenotype
V-VI are the most mild and lead to pancreas sufficient phenotype
Diagram/Explain on a molecular level how CFTR mutation leads to salty sweat.
Sweat ducts are divided into secretory coil and reabsorptive duct. Secretory coil does not contain CFTR and simply passes isotonic solution into the duct. This solution then passes through the reabsorptive duct where CFTR is found which normally causes uptake of chlorine leading to uptake of sodium but not water (because there are no aquaporins) this results in a hypotonic sweat solution. However, defected CFTR in CF patients limits reabsorption of salts leading to salty sweat
Describe/diagram on a molecular level how cystic fibrosis affects the lungs and how those affects lead to symptoms.
CFTR is located in bronchial ciliated cells. Goblet cells throughout the lungs secrete mucus into this area. Normally CFTR transports chlorine out of cell into the mucus where sodium remains and water is pulled out by osmosis to thin out the mucus. In CF patients, chlorine is not transported out and as a result sodium is reabsorbed into cells. These combine to cause water to move out of mucus into cells instead of the reverse and the resulting mucus is very thick. This thick mucus builds up obstructing airways and leading to inflammation and infection and ultimately scarring and lung disease.
Describe/diagram how cystic fibrosis affects the pancreatic duct on the molecular level and how those affects lead to symptoms.
Similar to the lungs, CF patients do not secrete chlorine leading to absorption of sodium and retention of water leading to viscous pancreatic juice. In addition, chlorine gradients created in part by CFTR are used for counter transport of bicarbonate out of the cells. In CF patients the chlorine gradient is not created and bicarbonate cannot be secreted. This leads to thick mucus blocking the release of enzymes into digestive tract and much of the food is not digested or absorbed which can lead to malnutrition. In addition, pancreatic beta cells can be damaged by mucus limiting insulin production and resulting in diabetes.
What percentage of CFTR function is required to show no symptoms of CF?
> 10% function
What is the most common mutation leading to cystic fibrosis.
Deletion of phenylalanine at position 508 of the gene. In the NBD1. Causes misfolding of protein which prevents it from reaching the plasma membrane
What are some potential future treatments for Cystic Fibrosis?
1 Gene therapy attempting to deliver functioning CFTR gene
2 Pharmacotherapy focused on trafficking protein with F508del mutation to the membrane that will be at least partially functional and may reduce or eliminate symptoms
Why has Cystic fibrosis gene become and remained so prevalent in European populations even though it is so harmful?
It is possible that it confers a heterozygous advantage against cholera. Heterozygotes don’t show symptoms of CF but show less fluid loss and dehydration than the homozygous dominant phenotype. Cholera toxin enters intestinal cells and perpetually activates cAMP which activates CFTR in addition to other ion channels causing massive secretions of ions and water into the lumen leading to severe often fatal diarrhea. CF heterozygotes potentially are less harmed by cholera.
What are the two main pathways of degradation of intracellular proteins? Explain how they differ in terms of what they degrade and when?
1 Ubiquitin proteasome- degrades short lived and damaged or misfolded proteins, it is selective
2 Lysosomal pathways- degrade long lived proteins and organelles, can be selective or random and is unregulated in response to cellular stresses.
