Week 1 Flashcards
(154 cards)
1
Q
Where are most lipids synthesized in the cell?
A
Endoplasmic Reticulum (Smooth more specifically)
2
Q
Through what mechanism do proteins made in the endoplasmic reticulum reach the Golgi apparatus?
A
Vesicular Transport
3
Q
Where are proteins translated?
A
At ribosomes in the cytosol
4
Q
What are the characteristics of signal sequences bound for the:
A) ER (ER signal sequence)
B) Mitochondria
C)Nucleus (NLS)
A
A) Large Hydrophobic Region
B) Interspersed Positively charged amino acids (basic AA)
C) Short contiguous positively charged domain (basic)
Exact amino acids does not matter, only that the sequence has a similar physical characteristic
5
Q
Describe the process through which proteins pass into the nucleus.
A
Proteins with nuclear localization sequence (short positively charged) are recognized by the nuclear import receptor. Nuclear import receptor then moves down cytosolic fibrils to pass through fibril filled nuclear pore. Once inside, the nuclear import receptor releases the protein.
RNA passes out through a similar mechanism.
6
Q
Some proteins are made in the mitochondria, but some are imported from the cytosol. Describe the process through which a protein enters the mitochondria.
A
An inter membrane import receptor protein recognizes the signal sequence on a protein (interspersed basic amino acids) and threads it into the adjacent trans locator protein in the outer membrane. The protein is unfolded as it is threaded through the translocator proteins on both the inner and outer membrane. Once inside the protein is remolded and the signal sequence is cleaved.
7
Q
Describe the difference between free ribosomes and bound ribosomes.
A
There is no difference. Ribosomes will bind to the Er when the translate a protein with the ER signal sequence.
8
Q
Proteins bound for translation in the ER can end up in which organelles?
A
ER, Golgi apparatus, plasma membrane, lysosomes, endosomes.
They may also be exported from the cell entirely
9
Q
Describe the process through which proteins enter the ER.
As a bonus describe the difference between soluble and membrane proteins.
A
During translation the ER signal sequence is created, a signal recognition particle (SRP) recognizes the signal and leads the ribosome to the ER where the SRP attaches to an SRP receptor. The protein is then fed into a protein translocator in the ER membrane while a signal peptide seems cleaves off the signal.
Side note: soluble proteins are are translated completely into the ER, while membrane proteins have a large hydrophobic region that causes the translocator protein to eject the hydrophobic region into the membrane.
Soluble proteins will reside in the lumen of some organelle while membrane proteins will reside in the membrane
10
Q
Describe the cause and clinical features of swyer syndrome.
A
Swyer syndrome, also known as XY gonadal dysgenesis, is caused by a mutated SRY gene on the Y chromosome. The mutation disables the SRY transcription factor that initiate male sex determination during development.
A common mutation is in the nuclear localization sequence of the protein.
Patients have female external sex characteristics without ovaries.
11
Q
What type of localization sequence remains part of the protein after transport into an organelle?
A
Nuclear localization sequence
12
Q
What type of RNA is the most abundant in cells?
A
Ribosomal RNA
13
Q
What is the function of miRNA
A
Inhibition of translation or targeting of mRNA for degradation
14
Q
What separates RNA structure from DNA structure?
A
RNA is single stranded which allows pairing with itself at palindromic sites. THis leads to secondary and tertiary structure while DNA only has secondary structure.
Side Note: This is how tRNA forms is characteristic clover and L shapes
15
Q
What is required for non-Watson-Crick base pairing in RNA?
A
1) An H acceptor (typ N:) and an H donor (typ NH)
2) the acceptor and donor come together in close proximity
3) the acceptor and donor interact at the right orientation
16
Q
Name the regions upstream and down stream of the open reading frame in a gene.
A
The 5’ and 3’ untranslated regions respectively
17
Q
What two protective elements are added to mRNA in eukaryotes?
A
The 5’ CAP and the 3’ poly A tail
18
Q
WHat is the difference between promoters and enhancers?
A
Promoters is the site of binding for RNA polymerase, on/off switch, immediately upstream of associated gene
Enhancer/silencer is a segment that can increase/decrease transcription by promoting the binding of a transcription factor. Can be in any orientation, and may be tens of thousands of base pairs away from gene upstream or downstream
19
Q
How many eukaryotic RNA polymerases are their?
A
3, RNAPolyII transcribes mRNA, tRNA, and rRNA
20
Q
True or false: Transcription factors are needed for the transcription of all genes.
A
True: General transcription factors form the initiation complex or basal transcription machinery needed to transcribe a gene
21
Q
True or False: Sequence specific transcription factors always interact directly with basal transcription machinery.
A
False: sometimes they interact with the basal transcription machinery through a cofactors.
22
Q
What is the TATA Box.
A
A conserved sequence found in the promoters of genes that allows binding of transcription machinery.
Bound by TataBinding Protein in TFIID
23
Q
Is ther a primer required in de novo synthesis of RNA?
A
No
24
Q
What direction is RNA synthesized?
A
5’ to 3’
DNA is read 3’ to 5’
25
What is found at the first nucleotide of exon 1?
The transcriptional start site (TSS)
26
Describe the combinatorial mechanism
Promoters are made of many binding sites. The different combination of these cis binding sites determine the activity of the gene in a given cell or environment.
27
Describe the concept of modularity.
Proteins and transcription factors are made up of modular and interchangeable functional domains that are found in many genes across the genome.
SOrt of a mix and match of functions to produce different proteins.
28
How are families of transcription factors classified?
On the basis of the DNA domains that they bind to.
29
What are two methods of making multiple products from one gene?
Alternative splicing and alternative polyadenylation
30
Define codon degeneracy
The ability of multiple codons to code for the same amino acid. Third base in the codon is typically degenerate.
31
What two portions of the tRNA determine its identity?
The 3’ end carries the amino acid, and the anti codon hybridizes with the mRNA codon
32
Insertion or deletion of nucleotides can result in what sorts of mutations?
Non-sense if an early stop codon is introduced
Protein level insertion if nucleotide insertion is divisible by three
Protein level deletion if nucleotide deletion is divisible by three
Frameshift if insertion or deletion is not divisible by three
33
Silent mutation definition
Nucleotide change that does not result in a change of amino acid.
Does not refer to an amino acid change that does not result in altered phenotype.
34
What direction are proteins synthesized in
N to C
| MRNA read 5’ to 3’
35
What is the sequence that marks polyaenylation in mRNA processing?
AAUAAA
36
MRNA is not proofread as closely. why?
Short lived and many are made at one time. Any given mistake is likely to have little if any consequence.
37
What is the chemical structure of the 5’ cap?
7-methyl guanosine with a triphosphate linkage
38
What type of ribosomes do mitochondria have?
70s ribosomes (prokaryotic)
Cytosolic ribosomes are 80s
39
What is the function of snRNA
Goes on to form SNRNP which are critical in the splicing out of introns during mRNA processing
40
Through what mechanism is RNA degraded
Nucleophilic attack of 2’-OH group splits phosphodiester bond
41
Distinguish between polyploidy and aneuploidy.
Polyploidy- more than one set of chromosomes (3n triploidy most common, e.g. 69,XXX)
Live births rare
Aneuploidy- a chromosome number that is not a multiple of the haploid number n.
Monosomy- 2n-1
Trisomy- 2n+1
Tetrasomy- 2n+2
42
What are the three trisomies that can result in term births?
13, 18, 21
43
What is the most common trisomy?
16, but it typically results in a 1st term miscarriage.
44
Describe the cause and clinical features of Down syndrome.
Trisomy 21
Characteristic facial appearance, cognitive delays with a mild to moderate intellectual disability, commonly associated with:
heart defects
Digestive abnormalities
Autism
obsessive compulsive behavior
Affected speech
Increased risk for early onset Alzheimer’s
45
Describe the signs of trisomy 18 (Edwards syndrome)
Small facial features with a relatively large head.
Overlap of 2nd and 5th digits
Small posterior lay rotated ears
Retarded intrauterine growth and low birth weight
Severe intellectual disability
Typically die before birth, 5-10% survive to first year.
46
Describe clinical features of trisomy 13
Many midline abnormalities such as heart, brain, facial and genitourinal defects.
Characteristic signs are cleft lip/palate, small eyes, and hypotonia
Sometimes polydactyly
Most die within days or weeks, only 5-10% survive to first year
47
Describe the clinical features of Turner syndrome (45,X)
95% result in miscarriaage with 85% missing the paternal X (Y only not survivable)
Cystic hygroma- failure of thoracic duct to connect to internal jugular vein, results in congestive heart failure. If this closes infant may survive.
Surviving infants have excellent outcomes
48
Describe the clinical features of Kleinfelter syndrome (47,XXY)
A Male phenotype with female secondary sex characteristics such as enlarged breasts and hips, female pubic patterns, etc.
49
Which sex chromosome aneuploidies display no significant phenotype>
47,XXX and 47,XYY
These are typically found by chance and accompanied by mild learning disabilities
50
What are common causes of structural chromosome abnormalities?
DsDNA breaks within chromatids, telomere instability, recombination between nonhomologous chromosomes, centromere pair separates at wrong location in meiosis
51
Name the two type of chromosome translocations.
Reciprocal: exchange between two nonhomologous chromosomes
Robertsonian: translocation between two acrocentric chromosomes that fuse at their centromeres. Satellites are lost in this process.
52
Translocations are balanced chromosome abnormalities. These typically result in normal phenotypes, but may cause problems during what process?
Translocations may cause abnormal genetic content in gametes leading to miscarriages during reproduction.
53
Define mosaicism
Two or mor genetically distinct cell lines that crop up after conception.
Can be widespread if it happens during development, or localized to certain tissues if it occurs later.
Hallmark of cancer formation
54
When does cohesion dissociate during mitosis?
The majority releases during prophase, the cohesion at the centromere releases at anaphase.
55
Which stages of mitosis do low and high resolution karyotypes come from?
Metaphase and prometaphase respectively.
Prometaphase gets better resolution because the chromatin is less condensed.
56
What are the three chromosome subtypes based on centromere position?
Meta centric (middle)
Sub meta centric (middle adjacent)
Acrocentric (closer to end)
57
Loss of telomere triggers what process?
Apoptosis
58
Describe the equation and assumptions of hardy Weinberg equilibrium
P+q=1
P^2 +2pq +q^2=1
Random mating, no selection, no migration, mutational equilibrium
59
Describe the cause and clinical features of osteogenesis imperfecta type I and type II.
Type 1: caused by 1 null allele of proalpha1 chains. Half number of normal collagen triple helix.
Frequent fracture of long bones due to insufficient collagen. Typically heal normally without deformity.
Type 2: caused by mutant proalpha1 chain that does not bind into triple helix as tightly (glycine replacements) Causes 1/4 of normal collagen fibers.
Innumerable fractures at birth typically fatal within first few weeks.
60
Why are lethal disease more commonly survived by those with chimerism.
The mixed genotype of someone with chimerism allows the severity of a typically lethal disease to be diminished.
61
What is the cause and clinical features of achondroplasia?
Cause: FGFR3 fibroblast growth factor receptor gene has a GOF in which it is a constitutively active inhibitor of bone growth.
Autosomal dominant and recessive lethal (homozygous lethal). P.G380R mutation
Clinical features: Diminished height, dwarfism
62
Define anticipation.
THe tendency for triplet expansion diseases to worsen in severity and age of onset in successive generations.
Examples: myotonic dystrophy, Fragile X, Huntington’s
63
Describe the cause and clinical features of Huntington’s disease.
Cause: abnormally high number CAG (Q) repeats (>36) in the gene for Huntington’s protein which is expressed in a Wide range of cells.
Clinical features: average onset of 37 years with a high penetrate, wide range of symptoms that begin similar to those associated with normal aging such as forgetfulness, impaired coordination, and personality changes.
These symptoms progress into cognitive non-function, immobilization, and mute patients followed by death 10-20 years after onset.
64
Describe the cause and clinical features of spina muscular atrophy.
Cause: recessive mutation resulting LOF of survival motor neuron 1 (SMN1) protein.
Clinical features: degeneration of motor neurons resulting in muscle trophy.
Varying severity based on copy number of partially compensating SMN 2 gene.
Treatments include exon skipping to incorporate skipped exon 7 in SMN2 gene and produce more viable SMN2 protein (constitutive treatment), and adenovirus gene therapy of SMN 1 protein (1time)
65
In what cases can x-linked recessive disease affect females?
X/autosome translocations- X genes attach to autosome that is not inactivated.
Skewed x-inactivation- not 50:50 inactivation as expected
66
Describe the cause and clinical features of duchenne muscular dystrophy.
Cause: X-linked disorder in which the dystrophin gene is mutated. It is believed that 1/3 of mutations are de novo (constant rate, loss of 1/3 to childhood lethality). Dystrophin is absent from cells and no longer protects muscle fiber membranes.
Clinical features: delays in motor development, proximal muscle weakness, calfpseudohypertrophy, fibrosis of myofibers (muscle fibers), high serum of creative kinase (due to breakdown), progressive skeletal muscular atrophy.
dilated cardiomyopathy later in life, mild mental retardation, wheelchair bound by early teens, death in late teens.
67
Becker muscular dystrophy
Similar to DMD, but symptoms are milder with a later onset due to an incomplete LOF in dystrophin gene.
Dystrophin will be reduced/truncated.
68
Dystrophin
Protein with largest gene known to date, protects membranes of striated muscle by linking the F-actin cytoskeleton with the extra cellular matrix via the dystrophin associated complex.
69
Genetic heterogeneity
The idea that one disorder may have multiple mutations that can cause it.
Allelic heterogeneity- a variety possible mutations in the same gene
Locus heterogeneity- mutation possible in different genes, typically in the same biochemical pathway
Clinical heterogeneity- different phenotypes from mutations in same gene.
70
Mitochondrial inheritance
From maternal ovum, heteroplasmy- many different mitochondrial genomes in any given cell
71
What types of tissues are most affected by mitochondrial diseases?
Highly aerobic tissue including eyes, muscles, nerves, and cardiac
72
Proband
Arrow pointing to person of interest in pedigree
73
WHat shortcut can be used to represent large numbers of siblings in a pedigree chart?
A number inside a circle or square to represent the number of females/males in a sit ship.
74
What are two exceptions to the central dogma?
Reverse transcriptase and functional RNAs such as tRNA and miRNA
75
Chromosome amplification is often seen in what type of disease?
Cancer
although gene amplification is often correlated with cancer it is not necessarily the cause and more experimentation is needed to determine the cause.
76
Which nucleotide can be methylated?
Cytosine, typically in cpg islands. Methylation does not obstruct base pairing.
Globin gene has many cpg islands so that it can be inactivated in most cell types.
Barr body
77
How is methylation preserved during replication?
After replication, methylation on parental strand triggers methylation on daughter strand.
78
Where are steroid hormone receptors typically found
Inside the nucleus acting as transcription factors
79
How does the cell respond to low iron levels?
A protein binds to the 3’ end of the mRNA for transferrin receptor protein.
The more transferrin is then synthesized and placed into the membrane so that iron can be brought into the cell.
80
How does iron deficiency result in less hemoglobin?
In iron deficiency anemia, low iron levels lead to low heme levels. GLobin production is subsequently down regulated at the translational level by the low heme levels.
81
Describe red blood cell gene regulation in the following:
A)Non-RBC
B)Immature RBC
C)Mature RBC
A) methylation of genes
B)Chromatin condensation
C) gene loss and translational control for mRNA still in circulation.
82
How are Barr bodies created?
Extensive methylation of non-active X chromosome in females.
83
What benefit does an inaccurate reverse transcriptase confer to the HIV?
Fast mutation rate that prevents vaccines from being effective.
84
WHich mutation confers immunity to HIV?
10% of Europeans have a mutation to the ccr5 receptor in their cd4 t-lymphocytes that prevents entry by HIV.
85
Prokaryote only antibiotics
```
Rifamycin
Gentamycin
Tetracycline
Neomycin
Erythromycin
Chloramphenicol
Kanamycin
Streptomycin
```
Mnemonic: Racing GT’s can injure NECKS
86
Eukaryotic Only antibiotics
Cycloheximide
Alpha-amanitin
Ricin
Diptheria toxin
Mnemonic: I’m a CARD carrying eukaryote
87
Prokaryote and Eukaryote antibiotics
Actinomycin D
Puromycin
Mnemonic: AP bio cover both proks and euks
88
Systemic lupus erythematosus causes and clinical features
AUto-immune disorder caused by antibodies against many self antigens including snRNP’s (disrupt splicing)
Symptoms vary widely, but common ones are joint pain and swelling, and a butterfly rash on cheeks.
89
Testicular feminization
Males with inactive androgen receptors fail to respond to steroidal androgens
90
Chronic myelogenous leukemia
Genetic cancer caused by translocation between chromosomes 9 and 22 “Philadelphia chromosome”.
91
describe Methotrexate and how cancer cells develop resistance to it
Methotrexate is a competitive inhibitor for dihydrofolate reductase (dihydrofolate analog). It reduces production thymine to prevent cell proliferation.
Cells with cancer may amplify their DHFR gene to amplify the reductase enzyme and overcome the inhibitor.
92
Sickle cell anemia
Mis-sense mutation glu-> Val at position 6 causes hydrophobic region on outside of hemoglobin resulting in a sticky hemoglobin that has the tendency to aggregate.
Functions properly at high oxygen and phenotype is only seen at low oxygen conditions. Confers resistance to malaria so gene is more common in areas where malaria is present due to selective pressure.
93
Thalassemia
Any genetic mutation that causes a dysfunctional or reduced amount of globin proteins (either alpha or beta)
Single most common genetic disorder in the world
94
A) Iron deficiency anemia
B) Vitamin deficiency anemia
C) Hemolytic anemia
D) Aplastic anemia
A) lack of iron in diet blocks translation of globin mRNA in last stage of RBC maturation
B) lack of B12 necessary for hemoglobin production
C) can be caused by various reasons, all result in a destruction of RBC
D) rare form, failure of bone warrior to produce all types of blood cells
95
Genomic imprinting
When a gene from one parent is methylated or inactivated and the inactivation is passed on to the offspring allowing the other parents allelic contribution to dominate.
96
Karyotyping
What is it? What are the strengths/weaknesses?
Examining the overall chromosomal structure of an individual.
Arrest cells in metaphase (low-res) or pro-metaphase(high res)
Non-specific, low resolution test that can identify large scale chromosomal abnormalities such as translocations and deletions, rearrangements in cancer, mosaicism, etc.
test can be indicated for recurrent miscarriages, infertility, confirmation of anueploidies (13, 18, 21), developmental and sexual differentiation abnormalities, dysmorphia, mental retardation, developmental delays, neoplasticism conditions.
Cannot detect uniparental disomy, methylation, small structural changes (<5Mb)
97
Fluorescence in situ hybridization (FISH)
What is it? What are the strengths/weaknesses?
Fluorescent probes are hybridized to specific sequences on metaphase cells overnight.
Indications are similar to karyotyping (infertility, suspected aneuploidy, neoplasticism conditions, developmental abnormalities/delays, mental retardation, etc.) but is specific for certain sequences. Need to know what you are testing for before hand.
Can be used to identify deletions, duplications, translocations, mosaicism, non-random rearrangements in cancer,etc.
Will not detect uniparental disomy or methylation. Quicker and cheaper than karyotyping. Limited by available probes.
98
Copy number variation (CNV)
Gain or loss of genetic material that may result in developmental abnormalities, tumor initiation/progression, or no alteration in phenotype.
Can be assessed using Karyotype, FISH, microarrays depending on necessary resolution.
99
Array CGH
Comprehensive evaluation of CNV in single analysis.h
Detects only deletions, duplications, chromosome gain/loss.
Non-targeted, better resolution than karyotype or FISH. metaphase spread not needed. Probes are from clinical samples of DNA.
Patient DNA and control DNA compete for probes in micro-array, light up as different colors depending on relative number.
Does not detect balanced rearrangements, UPD, imbalances between probe regions, alterations below 20 kB, methylation changes.
100
PCR
Polymerase chain reaction, copies a region of DNA using a heat resistant DNA polymerase, target specific DNA primers, and Thermo cycling to continually denature helixes and anneal primers.
Also neeed dNTPs to build DNA daughter strands
Used to amplify DNA for micro satellite analysis, Sanger sequencing
101
Microsatellite analysis
Use 13 locations of repeat sequences for human identity testing, paternity testing, transplant monitoring, forensic analysis.
102
Sanger sequencing
Examine a region base by base.
PCR to amplify then sequence using polymerase, and dNTP mixed with fluorescently tagged ddNTPs.
Record color of different sized strands that have been excited with a laser after separation in a capillary gel.
Targeted to certain areas and can detect smalll alterations such as SNP with high accuracy
103
Multiple probe ligation amplification
Used to detect small deletions and duplications on the order of an exon to the entire gene. No smaller, no larger.
Typical application is to detect abnormalities in dystrophin gene in DMD and BMD.
104
Whole exome sequencing
Genomic DNA is fragmented, adapters are lighted to the ends, and fragments are amplified on a flow cell and sequenced.
Quicker, more extensive, and cheaper than Sanger, but more prone to errors
105
UVB and UVC irradiation can cause what types of mutations and how are those mutations repaired?
Thymine diners in which adjacent thymines are fused together thus distorting the helix.
These mutations re repaired via nucleotide excision repair
106
How do platinum compounds fight cancer?
By cross linking bases (preferentially guanine) in DNA. This DNA damage initiates apoptosis.
107
What happens if mutations such as depurination and deamination of cytosine are not repaired?
Replication will lock in the mutation.
108
Haploinsufficenicy
When one mutated allele is enough to produce disease phenotype “dominant”
109
Dominant LOF mutation
Mutant protein loses function and interferes with function of normal allele.
Typically a protein which has normal binding function and mutant enzymatic function. Binds and blocks
Osteogenesis imperfecta. Abnormal collagen from one allele can screw up entire collagen triple helix
110
Basic excision repair
Excise damaged base and repair with other strand.
Repairs purine loss and guanine oxidation
Can be targeted for cancer treatment. Slow down BER to induce apoptosis
111
Nucleotide excision repair
Remove entire nucleotides to fix.
All LOF mutations with proteins involved in this process result in diseases that exhibit sensitivity to sunlight.
112
DNA Mismatch repair
Protein complexes in cell that repair errors in dna replication
113
BRCA1 and BRCA2
Genes involved in dsDNA damage repair. Often associated with the LOF mutation that causes breast cancer.
114
PARP1 inhibition
PARP1 is an enzyme involved in ssDNA break repair. In BRCA1/2 cancer patients inhabiting PARP1 can trigger apoptosis by closing down ssDNA repair system on top of the already damaged dsDNA repair system from the BRCA mutations.
115
How does Hemoglobin bind with oxygen?
There are 4 nitrogen atoms at the center of a porphyrin ring that bind Fe2+.
In hemoglobin there are histidines on either side of the heme plane. The fe2+ binds the proximal histidine to pull it out of plane. O2 binding to distal histidine pulls the fe2+ back into plane and reacts at a favorable bonding angle for oxygen (bent)
116
How does hemoglobin binding oxygen cause coopertivity?
The binding of O2 pulls the iron back into the heme plane which subsequently pulls on the proximal histidine and flexes the globin chain.
117
CO is a normal metabolite that is bound to approximately 1.5% of hemoglobin (3-4% in smokers) Although CO binding of hemoglobin is 300 times more favorable, models had predicted CO being 10,000 times more favorable. What accounts for this difference?
CO prefers a linear binding angle, but the distal histidine and iron placement force a bent angle bond.
118
As o2 pressure decreases, hemoglobin displays a ________affintity for oxygen
Decreased. This allows it to deliver oxygen to tissue even at decreased oxygen levels.
119
Define Kd
The dissociation constant. The concentration of oxygen that results in half saturation of myoglobin or hemoglobin.
Kd=[Mb]*[O2]/[Mb*O2]
Saturation =
120
The Hill coefficient
Outdated measure of hemoglobin cooperativity. Not accurate for two reasons:
Assumed trimer
Assumed straight line saturation curve
121
Which bonds flex when hemoglobin moves from its low affinity (T) state to its high affinity (R) state.
Alpha 1/2
Alpha1/beta2
Alpha2/beta1
122
What are the three common allosteric effectors of hemoglobin that shift the hemoglobin saturation curve to the right (lower affinity) and how do they work?
1) pH, H+ attracts to and stabilizes ionic interactions between monomers in T state (exercise)
2) BPG is a negatively charged atone the fits into the positively charge pocket between the two beta subunits stabilizing the T state. (Short term adjustment to high altitude)(BPG requires inosine as a carrier to enter RBC)
3) CO2, carbamoylation of Ntermini on all monomers and stabilizes T state. (Accounts for 20% of CO2 carried out of tissue)
123
What other globin monomers are present besides alpha and beta?
Gamma- fetal hemoglobin, beta analog, high affinity
Delta- beta analog, minor adult form
Epsilon- embryonic hemoglobin, beta analog
Zeta- pre-embryonic, alpha analog
124
What chromosomes carry the globin proteins?
16 carries 2 alphas and 1 zeta
11carries 2 gamma 1 epsilon and 1 beta
125
In thalassemia, what compensation mechanism is commonly seen?
Up regulation of expression for zeta (in alpha thalassemia) or gamma (in beta thalassemia) globin proteins.
126
How many copies of the alpha globin gene are there?
4, 2 copies on each chromosome 16
In thalassemia, severity depends on how many dysfunctional copies there are.
127
Heinz body anemia’s
A hemolytic anemia in which point mutations, typically in the beat subunit, lead to an unstable hemoglobin molecule.
128
GLycosylation of hemoglobin
A spontaneous non-enzymatic event that is directly proportional to glucose concentration in blood.
A good indicator of sustained hyperglycemia in diabetics
129
What is primarily responsible for genetic variability from person to person?
Repeated elements.
130
SNP
Single nucleotide polymorphism. An allele that is one nucleotide off from the wild type and occurs in at least 1% of the population. 90% of genes contain one and they are responsible ~85% of variation in gene expression.
131
Synonymous mutation vs. non-synonymous.
Has nothing to do with phenotype. Simply refers to whether or not a change in amino acids happens with a nucleotide mutation.
132
Copy number variations
Variations in the number of copies of a section of DNA.
Deletions, duplications
17% of variation, typically stable and heritable.
133
Where are tandem repeats typically found in the genome?
In non-coding regions
134
Satellite DNA
Large sections of tandemly repeating non-coding DNA
Telomeres are a mini-satellite (TTAGGG)
There are also micro satellites throughout the entire genome (coding and non-coding) that require frequent DNA repair
135
Explain the following nomenclatures for genetic variants
1) C.187delAG
2) c.682+1G>A
3) p.Glu6Val
4) p.Gly796*
5) c.402delC (p.Phe134Leufs*51)
C denotes that sequence was obtained from cDNA, g denotes genomic DNA. P denotes a change in protein structure.
1) deletion of AG at position 187
2) +denotes a change in the splice site at position 682
3) Glu changed to Val at position 6
4) *denotes a termination codon (nonsense mut. )
5) a del of c at position 402 resulted in a frame shift (fs)
136
In Anfinsen’s study What was the order of steps needed to correctly unfold and refold the protein?
Reduce disulfide bridges->denature with urea-> remove urea->oxidize cysteines
137
What is the main force that drives protein folding?
What drives the formation of secondary structures?
Hydrophobicity does most of the work. The inside of a globular protein is typically made up of a cluster of hydrophobic residues that clump together.
Unsatisfied H bonds in the clumped hydrophobic region drive the protein into more stable secondary structures such as helixes and sheets so that other amino acids may satisfy these Hbonds in the apolar environment.
138
Levinthal’s paradox of protein folding
Protein folding can’t occur via trial and error. Too many possible configurations.
There must be a predetermined pathway.
139
What in vivo conditions can lead to protein aggregation?
Cellular crowding- raises apparent concentration and lowers apparent solubility of protein leading to a greater rate of aggregation. (Polysomes, proteins in the nucleus, proteins denatured due to stress)
140
What types of proteins can improve folding in vitro?
peptidyl-prolyl cis/trans isomerase (PPI)- speeds up the usually slow process of cis trans isomerization of proline to reduce time to proper folding
Protein disulfide isomerase- catalyzes disulfide bridge formation and shuffling, localized to ER
Molecular chaperones- prevent and reverse inappropriate interactions, often through sequestration, do not provide folding help
141
What are the three main types of chaperone proteins
Small heat shock proteins.
Low molecular weight (bind hydrophobic sequences until buried) ATP dependent binding
Chaperoning- cage proteins to prevent interactions. ATP dependent shutting of gate
142
Systemic amyloidosis
Densely pack beta amyloid sheets are deposited systemically
Beta sheets identified through apple green color on a Congo red stain
Primary accounts for 75%, produced by plasma cells in bone marrow
Secondary ~7% secondary to chronic inflammation
Familial ~10% mutations in transthyretin
143
Cystic Fibrosis
Mutation of cystic fibrosis transmembrane conductance regulator (CFTR) that causes misfolding. A lack of cl- transport across membrane causes a sticky mucus build up outside cells.
A number of different severities based on type of misfolding. Can happen at many stages of protein synthesis/implantation/function
```
No protein made
Less protein made
No implantation in membrane
No function
Less function
Less stability
```
144
Alpha-1-antitrypsin deficiency (AATD)
NOrmally alpha 1 antitrypsin is made in liver and is sent to coat lungs to protect from neutrophil elastase.
In this deficiency, incomplete folding causes aggregation in liver, damaging liver, and allowing neutrophil elastase to damage lungs.
145
Prion diseases
Prions are infectious protein agents that have misfolded , and may cause other proteins to misfolding as well.
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Erythropoiesis
PRoduction of RBC, occurs predominantly in the bone marrow (medullary) space of hip, sternum, vertebrae, ribs, and long bones
Maturation takes ~10 days, life cycle is approx 120 days.
Throughout maturation hemoglobin levels increase and chromatin is continually condensed until expulsion at normoblast/reticulocyte stage.
Can happen in liver, lymph nodes, or spleen of adults who have a marrow failure or an increased turnover of RBC
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Erythropoietin
Hormone released by kidneys in response to low oxygen levels. Stimulates stem cell maturation into RBC
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Reticulocytes
What are they? What stains are used? What do they indicate? What levels are normal?
Last stage of RBC maturation before erythrocyte. ~1% of total RBC (0.5-2% is normal)
COunt is used as an indicator of over all RBC production. Abnormal levels can indicate high turnover due to hemolysis, or decreased production because of vitamin deficiency.
So named because of reticular (mesh like) network of increased ribosomal RNA
Some can be seen as polychromatic RBCs on wright-giemsa stain.
All can be seen on methylene blue stain (stain ribosomal RNA)
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Shistocytes
Fragmented RBC, helmet cells often triangular or helmet like in shape.
Formed by high mechanical shear force in flow (high blood pressure, heart valve, clotted vessels)
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Spherocytes
Spherical RBC without central pallor (depression). Typically caused by autoimmune hemolytic anemia. My also be hereditary (ANK1 mutation in cytoskeleton)
Decreased surface area/volume ratio.
Smaller in size, higher concentration of Hb, increased osmotic fragility(lyses easily)
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Target cells
Excess lipid deposition in RBC cell membrane causes a bump in the central pallor where hemoglobin can pool.
Increase surface area to volume ratio.
Typically seen in liver disease after splenectomy, also seen with thalassemias
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Ovalocytes/elliptocytes
RBC’s that take on an elliptical shape with rounded edges.
Typically present in blood (<5%)
May be hereditary or due to nutritional deficiencies. FE def-> long pencil like
B12 def-> wider and larger.
Hereditary is autosomal dominant and produces incidence of >25% in normal blood.
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Basophilic stippling
Clusters of ribosomal RNA leftover.
Seen with lead and arsenic poisoning, alcoholism, thalassemias
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Sickle cell deep dive
1) What types of events cause polymeriztion of hemoglobin molecules?
2) How does fetal hemoglobin help treat sickle cell anemia?
3) How do sickle cells cause clotting?
4) What are some clinical signs of sickle cell anemia?
5) how is sickle cell typically diagnosed?
6) what are some short and long term treatments?
1) Valines in the 6 position on the beta subunits can cause polymerization in the deoxy(T) state. Anything that causes hemoglobin to shift into the deoxy state can cause a crisis (low ph, high CO2, high BPG, low oxygen). Also high concentrations, such as those inside RBCs can cause polymerization as well (dehydration, decrease in RBC volume like sickling)
2) fetal hemoglobin results in more hemoglobin molecules with gamma globin chains instead of beta. The fetal hemoglobin molecules also actively terminate polymerization and reduce HbA concentrations.
3) polymerization causes long Cain’s of proteins that change the shape of and stiffen RBC’s. The now rigid RBC’s can get caught in smaller vessels. While sickling is initially reversible, repeated sickling can become permanent.
4) anemia symptoms, frequent infections, severe joint/bone pain, Vaso-occlusive crises, shortness of breath, poor healing, increased serum iron and bilirubin (from shortened RBC lifespan, lysis in 10-20 days), low hemoglobin and hematocrit, increased reticulocyte count. Also sensitivity to altitude change.
5) hemoglobin electrophoresis (digested and undigested, undigested is most common).
6) short term treatments include oxygenation, blood transfusion, hydration, antibiotics for infection, analgesics for pain.
Long term solutions include bone marrow transfusion, fetal hemoglobin gene therapy, hydroxyurea (also used as a chemotherapeutic inhibitor of RNR), iron chelaters .
