BMS Part II Flashcards
Define locus
site on chromosome where gene is located
Define allele
variants of a gene located on same locus
Define locus heterogeneity
Different mutations on different genes produce the same phenotype e.g. familial hypercholesterolemia
Define allelic heterogeneity
different mutations at same allele cause similar phenotype e.g. Duchenne, CF, B-thalassemia
Define phenotypic heterogeneity
different mutations in the same gene cause different phenotypes e.g. Hirschsprung, CF
severity of disease depends on how protein function is affected by mutation
Define hemizygous
Male patients only- abnormal gene on X
Define compound heterozygote
Mutations are at different locations in the gene e.g. CF
Define obligate heterozygote
Clinically unaffected but must carrier mutant allele based on pedigree (e.g. normal parents of affected child)
Autosomal recessive diseases: define features of pedigree factors that increase risk factors that affect clinical severity
1) Two mutant alleles (can be compound heterozygotes), rare
2) skips a generation (parents are asymptomatic carriers), prevalence among siblings (1/4 chance of disease), equal prevalence among M/F
3) Carrier frequency (consanguinity i.e. related, genetic isolates i.e. Tay Sachs, inbreeding i.e. same small pop)
4) Sex influenced phenotype (hemochromatosis- too much iron, disprop affects males)
Autosomal dominant: define features of pedigree what is incomplete dominance factors that affect clinical severity
1) can have 1 or 2 mutant alleles (though 2 is rare), majority of CURRENT known Mendelian disorders
2) disease is in every generation, equal prevalence among M/F, prevalence among siblings (1/2 chance of disease), normal siblings have all normal offspring
3) homozygous BB is more severely affected than heterozygous Bb e.g. familial hypercholesterolemia
4) reduced penetrance, variable expressivity, sex-limited phenotype (e.g. male-limited precocious puberty)
X-linked recessive (XLR)
define
features of pedigree
what is unbalanced X inactivation/dosage compensation
1) need two mutated X alleles to exhibit the phenotype
2) No M-M transmission (offspring M of affected M are carriers), prevalence M»>F, women are carriers, any women with phenotype due to unbalanced X-inactivation
3) if female is Xx but the rate of inactivation of the mutated X is out of prop–> female will exhibit phenotype
females are somatic mosaic
X-linked dominant (XLD)
define
features of pedigree
1) only need 1 mutated X allele to exhibit phenotype
2) NO M-M transmission (offspring M of affected M are normal, offspring F are all affected), prevalence F»>M
What is the difference between reduced penetrance and variable expressivity?
1) Penetrance- does disease show up? prop with mutation who express symptoms
Expressivity- how does disease show up? same mutation but severity of phenotype varies
*can have differential expressivity- minor clinical abnormalities vs definite clinical abnormalities
What are exceptions to Mendelian inheritance?
1) Mosaicism- somatic or germline (depending on when the mutation occurs)
pedigree clue– two normal parents who have multiple affected children with AD or XLR
2) New mutations- v rare
pedigree- if you see a new mutation for a genetic lethal disease e.g. Duchenne
3) Misattributed paternity
4) Genomic imprinting - methylation is off in gametes
5) Reduced penetrance/variable expressivity
6) mitochondrial inheritance
7) TNR expansion disorders
Difference between chromatin and chromatid?
Chromatin- fibers composed of DNA + histones
Chromatid- DNA after replication (Sister chromatids), made of condensed chromatin fibers
Define c-value
Amount of DNA in one gamete
*wide variety between C-value and organism complexity (also with chromosome number and complexity)
Compare RNA and DNA in the genome
RNA precedes DNA in evolution
RNA more complex/diverse in actions
DNA more chemically stable –> evolutionary advantages
Conclusions from the ENCODE project
1) Chromatin has 7 functional states
2) 60-75%of DNA transcribed into RNA
3) 80% is “functional”– reproducible biochemical signature
4) non-coding transcripts as abundant as coding ones–> will be important as determinant of health/disease
Describe repetitive sequences:
impact
types
example
More recent the repeat- less divergence from predecessor segment –> proteins encoded are more similar
repeats enhance probability of recombination/structural changes
1) Tandem repeats - adjacent repeating sequences, evolutionally recent repeats are more similar –> lead to recombination
2) Short repeats - satellite (100+ bp)/microsatellites (few bp) tandem repeats –> form small satellite when DNA is fractionated by density
3) Retrotransposons - encode reverse transcriptase acting on mRNA creates segmental duplication
LINE-encode reverse transcriptase
SINE- do not, e.g. Alu sequences
*reverse transcriptase comes from viruses
Examples of diseases due to repetitive sequences:
R/G color blindness
Continuous gene syndrome
1) R/G color blindness– recombination bw duplicated genes on the X chromosome;
misalignment in meiosis and improper segregation into sperm cells; one has only red receptor gene (colorblind) and the other has 1 red + 2 green receptors (normal)
2) DiGeorge/Prader Willi/Angelman- deletes large gene sequence repeats
use FISH to tell you region of DNA deleted
Define pseudogenes
Non functional, pieces of DNA back in the genome from action of reverse transcriptases; not transcribed bc they lack promoters
Describe the three techniques for karyotype staining:
G-banding
FISH
CGH (Comparative genomic hybridization)
1) G-banding- detects large changes
arrest chromosomes in metaphase, stain with Giemsa so bands form (dark G bands)
Chromosomes have unique banding pattern, size, also centromere position (meta, sub-meta, or acrocentric)
can only detect large deletions (one band = 45 genes)
2) FISH- detects smaller changes with known location
Interphase FISH is faster, metaphase FISH is better resolution
need specific probe- test only as good as the probe
green is the internal control, red is the test probe–if red is missing, there is a mutation
but if red is there, there could still be a mutation elsewhere
con: cannot detect single nucleotide changes, resolution decreases as # probes increases
3) CGH- detects changes even if location is not known
oligonucleotides on glass slide/ microarray
PCR test patient DNA (green) with reference DNA (red) and allow both to hybridize on glass slide to detect small changes in the genome
yellow- both bind equally
red- no patient DNA
green- too much patient DNA
can also plot hybridization ratios (L–>R is p–>q arm) to quantify the deletion/duplication
con: only for deletions/duplicates NOT inversions/translocations
Describe Robertsonian translocation
D (13-15) and G (21-22) group acrocentric chromosomes only; fusion of chromatids and loss of p arms –> so 3 chromosomes instead of 4
*not necessarily deleterious bc p arms of acrocentric chromosomes have multiple copies of genes for rRNA
Three ways to ID chromosomes
1) Size
2) Centromere index = length of p arm / total chromosome length
3) G banding
Steps of meiosis
Meiosis I
1) Interphase- chromosomes duplicate
2) Prophase I- chromosomes condense, synapsis- tetrads line up; crossing over (swapping homologous sections), nuclear breaks down + spindle forms
3) Metaphase I- tetrads line up in independent assortment, spindles attach to kinetochore
4) Anaphase I- spindle microtubules contract and pull chromosomes to opposite ends
5) Telophase- nuclear membrane reforms + cytokinesis
Meiosis II
1) Prophase II- spindle reforms, nuclear breaks down again
2) Metaphase II- lining up, spindles attach
3) Anaphase II- spindles contract
4) Telophase II- nuclear membrane reforms + cytokinesis
Difference between meiosis in M/F
M- spermatogenesis: meiosis I and II begins at puberty- 4 sperm produced- and continues throughout life
F- meiosis starts in utero- all oocytes formed before birth, arrested in metaphase II until fertilization (one oocyte during ovulation each month)
1 polar body extrudes at ovulation, 2nd at fertilization, they die when egg is fertilized
Nondisjunction
Either Meiosis I or II - failure of homologous chromosomes or sister chromatids to separate
leads to abnormal zygotes e.g. Down, Turner
Define uniparental disomy
Both chromosomes come from one parent e.g. Prader Willi, Angelman (imprinted genes)
How do you use polymorphic markers to determine where meiosis II occurs? Stage of meiosis and parental origin
If the alleles near the centromere are NOT identical –> nondisjunction in meiosis I
If alleles near centromere ARE identical –> nondisjunction in meiosis II
For parental origin - need to analyze polymorphic DNA markers
Explain types of rearrangements
Balanced
Unbalanced
Balanced
1) Reciprocal translocation- exchange of segments between non-homologous chromosomes; usually harmless
2) Inversion- single chromosome undergoes breakage and rearrangement
Unbalanced
3) Deletion
4) Duplication
Difference between paracentric and pericentric inversions
Paracentric- inversion on one arm of the chromosome (either p or 1)
Pericentric- inversion includes the centromere
Requires two chromosome breaks
Balanced rearrangement in the carrier parent but high instability in gamete formation- miscarriages or affected children
Define isochromosome
Chromosome has lost one of its arms and replaced it with a copy of the remaining arm
e.g. Turner’s syndrome, Down’s
Gene density of human DNA
12-15 genes in 1 M bp
22,000 genes over 23 chromosome pairs
Difference between ataxia and apraxia
Ataxia- loss of coordination of motor control (e.g. in HD)- muscle/movement disorder
Apraxia- inability to execute movement despite having both desire AND capability - motor nerve disorder
Describe the mt inner and outer membrane and mitochondrial matrix
Inner- impermeable to molecules, rich in protein, where electron transport chain/ox phos takes place, contains cristae (convoluted folds which increase surface area)–> # of cristae depend on energy needs of the cell
Outer- permeable to small molecules/ions, contains signaling receptors
Matrix- proteins for TCA, etc. mt RNA and DNA, protein synthesis machinery
Differences between mitochondrial and nuclear genome
1) Mt DNA is much smaller–> 2-50 mtDNA in matrix of each mt, ~10-200 mt per cell (20-10000 mtDNA molecules per cell)
2) mt DNA circular
3) no introns
T/F:
1) Protein transport is unidirectional
2) Mt have fixed copy number, shape, size
1) True - protein from cytosol imported into mt
2) False- through fission and fusion, mt shape is changing; size depends on energy demand conditions
Explain mt DNA replication
Can occur independently of the cell cycle
Replication and transcription take place in the D-loop
DNA polymerase gamma- 3 activities (polymerase, exonuclease/proofreading, lyase for DNA repair), nuclearly coded protein
Twinkle- helicase
afterwards, mtDNA packaged 1 or 2 at a time into nucleoids
mtDNA repair less efficient bc there is high copy number buffer
Explain mt DNA transcription
Takes place in the D-loop, no cap added
polycistronic, encodes 13 mRNA, 2 rRNA, and 22 tRNA
requires mt RNA pol, regulated by TFAM (transcription factor activator), and mt transcription factors B1 and B2
Explain mt translation
-Encodes 13 proteins
-1 tRNA can pair with any of 4 codons- need only 22 tRNA (all are mt encoded)
-Proteins nuclearly coded and imported:
Initiation- IF2 and IF3
Elongation- mtEFTu, mtEFTs, mtEFG1
Termination- only 2 stop codons
*most mitochondrial proteins come from cytosol (not RER and Golgi)
Explain the 6 distinguishing features of mtDNA genetics
1) High mtDNA mutation rate
- close to source of ROS, DNA pol gamma is not great at proofreading, no introns so mutations happen in coding exons
2) Maternal inheritance - paternal mitochondria destroyed when sperm fertilizes the egg
3) Genetic bottleneck - during oogenesis, reduction in primary oocyte then amplification –> redistributes mt DNA among daughter cells–> can have different levels of mutated mtDNA
4) Random segregation of mt and mtDNA between daughter cells
- heteroplasmy- mixture of mutant and normal mt in the same cell
5) this is associated with the threshold effect–> cross threshold of mutant mtDNA for phenotypic expression
- daughter cells have wide variation in prop of mutant/normal mt
- threshold level varies among mutation and tissue
6) Changes with age
- number of mtDNA mutations increases with age due to ROS and defective DNA pol gamma, also levels of ox phos decline
Common features of mt diseases
1) Lactic acidosis - ox phos impaired –> systems that need the most energy are most impaired (heart, brain, muscles, lungs)
2) Ragged red fibers- Appear due to proliferation of large, diseased mt in myofibers/muscle fibers (in 1/3 diseases)
- in diseases with RRFs- ataxia, retinopathy, and dementia
3) Absence of cytochrome c oxidase (partly encoded by mtDNA) - needed for electron transport chain
4) Affect multiple systems (ones that need a lot of oxygen) e.g. heart, lungs, muscles, brain
What are the 4 assumptions for a population in equilibrium?
1) Random mating
2) No changes in population due to migration
3) No random fluctuations in allele frequency due to natural selection, genetic drift, etc.
4) No positive or negative selection- all genotypes reproduce equally well
What are the 5 factors that influence genetic variation (/shift allele frequency)?
1) Natural selection- heterozygotes have the advantage *
2) Genetic drift- allele frequencies change by chance alone, esp in small pop, due to sampling error - increases homozygosity since some alleles are wiped *
3) Founder effect- one rare allele at high frequency in a particular pop derived from 1 ancestor *
4) Pop bottleneck- some alleles at high frequency because of population constrictions *
* changes allele frequencies
5) Inbreeding- increases homozygosity frequency, can also happen bc of arranged marriage
What is the Hardy-Weinberg equation
For 2 alleles, A and B
p+q=1
Total number of A= p^2 + 2pq
Total number of B=q^2 + 2pq
What is the coefficient of inbreeding f
probability that a person with 2 identical genes received both from the same ancestor (e.g. grandmother with CF mutation passing on the disease to great-grandchild)
for first cousins- f=1/8, for their offspring, f=1/16
Define Bayesian terminology prior prob conditional prop joint prob posterior prob
Prior prob= Mendelian prob of carrier
Conditional prob=other information in family
Joint prob= prior x conditional
Posterior prob=ratio of joint prob of one outcome/sum of all joint probs
Define polymorphism and types of polymorphisms.
Define population stratification
Define heterozygote advantage
1) Differences in DNA sequence between individual
- mutation (e.g. end paragraph early), silent (eg spelling change), functional (eg replace . with ! punctuation)
Also defined as multiple phenotypes in same population (pop genetics)
2) nonrandom mating between groups causes different allele frequencies between subpopulations
eg. different HW probabilities of CF based on race/ethnicity
3) heterozygote has higher fitness than homozygotes e.g. sickle cell anemia
* maintain polymorphism when selecting for heterozygotes
Define heritability
=variance in dizygotic twins - variance in monozygotic twins / variance in dizygotic twins
1= trait due completely to heritability
Describe genetic risks for common multifactorial diseases and how you ID
Common variant- less individual, more population risk (higher RR)
ID through GWAS –> isolate DNA from thousands of individuals with disease—> ID SNP alleles co-inherited with disease
rare variant= mutation, less population, more individual
ID through deep genome sequencing
What was the ENCODE study and major findings?
1) Looking to see if the 97% of the genome that doesnt encode for protein had functionality
found that 80% of genome is involved in at least 1 biochemical event –> can be associated with disease
What is the epigenetic hypothesis?
What is the importance of proteomics/metabolomics in looking at disease?
1) Risk of common disease due to epimutations- changes in epigenetic signature that affects gene regulation and can lead to disease
-provide basis for variance in phenotypes among common diseases
-interface between environment and gene expression
-can look at methylation status of DNA through hybridization
2) Proteomics- looking at proteins
Metabolomics- different disease cells have different metabolic profiles (e.g. Warburg effect- cancer cells use high rate of glycolysis instead of ox phos)
can use these -omics analyses to target drugs
Causes of pathological hypoxia
1) Reduced 02 supply- high altitude or lung disease
2) localized ischemia (reduction in blood flow)
3) Abnormality of tumor microvessels
How does high altitude lead to hypoxia? What are symptoms of hypoxia at high altitude?
1) Air pressure drops as altitude increases
Air holds less molecules per area- lowers gas pressure
Lower number of 02 molecules per area–> lower 02 pressure
2) slower rxn time
learning/spacial memory impaired
MRI changes, hallucinations
What are the types of hypoxia inducible factors and where are they located?
Hif1, Hif2, Hif3
Hif1 and Hif2 are active as heterodimer- alpha is unstable cytosolic protein, beta is stable nuclear protein
need to go to nucleus and bind to beta subunit and then can activate transcription of hypoxia-inducible genes
Explain prolyl hydroxylation of Hif-1
Post-translational regulation
Prolyl hydroxylases (PDH 1-3) attach OH to Hif-1 –> need 02 and alpha-ketoglutarate
PDH active with Fe2+ cofactor, inactivated when oxidized to FE3+ (e.g. mt damage due to ROS)
How is Hif-1 degraded?
After hydroxylation, recognized by VHL (component of complex E3 ligase) –> ubiquinated and degraded
3 ways that Hif-1 can escape degradation?
1) Siah1/2 adds multi-UB chain to PDHs 1-3 and causes them to be degraded
2) VHL can also be ubiquinated and degraded
*also have mutations in VHL
3) VDU deconjugating enzyme removes the Ub chain from Hif-1
*VHL can ubiquinate Hif-1 OR VDU
Fastest way to activate hypoxia response is to prevent Hif-1 from degradation (rather than waiting for it to be transcribed)
How does hypoxia response intersect with inflammatory response pathways?
Hif-1 can activate the IkB kinase- which then phosphorylates the IkBalpha (which is then degraded)–> NFk can go into nucleus and activate transcription of inflammatory response (including making Hif-1)
*IkB kinase is needed for both inflammatory and hypoxia response –> need to coordinate mechanisms to achieve high levels of Hif-1 protein
What is the purpose of mir-210 in hypoxia response?
Transcriptional regulation
microRNAs that inhibit translation of normoxia genes during the hypoxia response –inhibits global protein synthesis
How is more Hif-1 protein synthesized when it induces the shutdown of global protein synthesis during hypoxia?
mTOR and S6-kinase promote preferential translation of mRNA encoding Hif1alpha
through PI3K MAPK pathway
What are the major effects mediated by Hif-1 during hypoxia?
1) Increased glycogen synthesis
2) Switch from ox phos–> glycolysis
3) Activates angiogenesis (new blood vessel devlpt)
4) Activates inflammatory response (by activating kinase)
5) alpha ketoglutarate produced, converted to citrate
6) mir-210 activated- inhibits normoxia expression
How does hypoxia induce increase in Hif-1 protein?
Reduced degradation (1) + increased gene expression (2)
1) Reduces function of prolyl hydroxylases (PHDs) which add OHs to proline of Hif-1alpha to target for degradation
2) prevents PHD inhibition of IkB kinase –> NFk factor activates transcription of HIf-1
What are the major post translational modifications of Hif1alpha?
1) proline hydroxylation –> required to be recognized by VHL for degradation
2) lysine acetylation–> promotes interaction with VHL–> degradation
3) Asparagine hydroxylation–> inhibits HIf1 interaction with transcription coactivator
What are the key features of trinucleotide repeat disorders?
1) Characteristics/consequences of expansion differ
2) Transmitting parent influences tendency for repeat expansion
3) if you have abnormal number of TNRs but no symptoms= pre-mutation
4) Display genetic anticipation– phenotype occurs earlier and/or phenotype is more severe from generation to generation
Huntington’s Disease
- mode of inheritance- parental preference for TNR?
- location of TNR expansion - how it correlates with phenotype
- clinical features of disease
1) Autosomal dominant
high tendency for repeat expansion during spermatogenesis– high likelihood a man in premutation stage will pass on HD to his children (paternal expansion)
2) Expansion in first exon of Huntington protein –> Causes aggregation of glutamines –> misfolded protein –> neuron degeneration and toxicity
>39 CAG
3) chorea, dystonia, ataxia, bipolar disorders, cognitive impairment/dementia
(Alzheimers + Parkinsons + Bipolar)
How do you detect TNR disorders?
PCR- use primer in affected part of gene sequence
larger products implies more repeats- can estimate # repeats based on size
Fragile X syndrome
- mode of inheritance- parental preference for TNR?
- location of TNR expansion - how it correlates with phenotype
- clinical features of disease
1) X-linked
maternal expansions in oogenesis–> high likelihood a female in premutation stage will pass on to children (females more likely to be carriers, males more likely to be affected)
*most common inherited cause of intellectual disability
2) Expansion in promoter (5’ UTR non coding region) of FMR1 gene
FMR1 protein is found in neurons- chaperones mRNA down axons where protein is translated- associated with maturation of neurons
3)
Premutation phenotype- tremor-ataxia syndrome (neurodegenerative), primary ovarian insufficiency (menopause before 40) *premutation phenotype bc transcription still happens–> insertions–> Excess RNA which sequesters normal RNA binding proteins needed for OTHER mRNA transcripts
Phenotype in affected males: low IQ, large testes, long narrow face + large ears
Phenotype in affected females: 50% of females with full mutation in ONE allele have intellectual impairment (phenotype associated with unbalanced X inactivation)
*in full mutation range- gene becomes methylated
Friedreich’s Ataxia
- mode of inheritance- parental preference for TNR?
- location of TNR expansion - how it correlates with phenotype
- clinical features of disease
1) Autosomal recessive
2) Expansion in first intron (non-coding region) of frataxin/FRDA gene which encodes mitochondrial protein (degeneration of cells in myelinated neurons –> demyelination)
excess repeats –> long intron–> cant sit on exon and create spliceosome –> unspliced protein degraded
3) Frataxin high in heart/spinal cord–> Cardiomyopathy, ataxia (progressive limb weakenss/gait disturbance), loss of vibration senses and leg tendon reflexes
Myotonic dystrophy
- mode of inheritance- parental preference for TNR?
- location of TNR expansion - how it correlates with phenotype
- clinical features of disease
1) Autosomal dominant
both parents can transmit TNR expansion up to 1000 repeats
>1000 repeats- from mom ONLY (e.g. congenital MD)
2) expansion in 3’ UTR of DMPK gene
RNA-mediated toxicity- Expansions cause hairpin loop in 3’ UTR –> sequesters binding/splicing factors –> messes up transcription/translation for other nearby genes (this also happens in Fragile X syndrome)
3) Myotonia (inability to relax voluntary muscle after rigorous effort), muscular dystrophy (most common cause of adult onset), Type II diabetes, cardiomyopathy
Difference between pharmacogenetics and pharmacogenomics
Pharmacogenetics- how one particular drug interacts with the body e.g. inherited genetic differences that influence response to a drug
Pharmacogenomics- how genetic differences within populations explain different responses to a drug
Difference between pharmacokinetics and pharmacodynamics
Pharmacokinetics- the way your body processes a drug (clearance, metabolism, transport) –> what body does to drug
Pharmacodynamics- site of activity of drug and downstream effect –> what drug does to body
What are CYPs and role in drug metabolism?
CYP= cytochrome p450 oxidase, found in the liver and contain heme
have a flap which opens up- drug comes in and heme group chemically modifies it
break down metabolites/xenobiotics, activate drugs (e.g. codeine–> morphine), catalyze reaction (e.g. by accepting e-), increase solubility of drugs (e.g. adding hydroxyls)
substrates include xenobiotics, vitamins, fatty acids, sterols, signaling molecules (multiple possible drug/CYP interactions)
What is warfarin, how does it work, how does it interact with CYPs?
- Warfarin- blood thinner
- Inhibits Vitamin K reductase- enzyme in Vit K recycling that reduces oxidized Vitamin K –> reduces levels of active Vit K–> impairs synthesis of vit K dependent clotting factors
- CYPs hydroxylate warfarin and make it more soluble
- with warfarin overdose- administer Vit K to outcompete warfarin effect
What is the pharmacodynamic effect of warfarin? What two variants have highest effect on warfarin sensitivity
-Warfarin inhibits VKORC1 (Vit K reductase) and prevents it from reducing Vitamin K into active form
-variations in VKORC1 and CYP2C9 affect response- mutation can increase affinity of drug for VKORC1 target and make patient more sensitive (need lower doses)
OR mutations to CYP2C9 can make patient intermediate/poor metabolizer (need lower doses because drug is in the system longer before being broken down)
Define linkage disequilibrium
combinations of alleles/genetic markers that occur more or less frequently than expected at random
Plasma membrane
- purpose in the cell
- what is it composed of
- viewed with which microscope
-interface between cell and environment, permeability barrier
-PM= lipid bilayer
lipids are amphipathic, also have membrane proteins
-viewed on EM (LM shows both plasma membranes + extracellular matrix)
What is spectrin and what is its role/significance?
Spectrin- cytoskeletal protein that is attaches to membrane proteins, stabilizes biconcave shape of normal RBCs
with mutation–> RBCs more spherical, more sensitive to hypertonic–> degraded in spleen
What are the major functions of membrane proteins?
1) Linker/anchor molecules e.g. binding with spectrin
2) Transporter molecules
3) Receptors
4) Enzymes
Microvilli
- purpose in the cell
- what is it composed of
- viewed with which microscope
- evaginations of apical cell membrane- increase absorptive surface for absorption, secretion, adhesion
- composed of a core actin filaments as well as myosin Ia motor protein and calmodulin –> ends in a terminal web of actin minus ends + spectrin + myosin II
- have a glyocalyx at the surface
- viewed on EM, seen as a brush border under LM
What is the glycocalyx?
- glycocalyx- fuzzy coat on tops of microvilli
- consists of sugar side chains attached to glycoproteins, glycolipids, and proteoglycans (sugar added to membrane proteins/lipids during biosynthesis in the RER) –> can be used for binding, adhesion, protection
Junctional complexes
- purpose in the cell
- viewed with which microscope
Provide contact between neighboring cells- promote adhesion and prevent passage of molecules
IDed on LM by small reddish dots - terminal bars; also IDed on EM
What is the nuclear lamina?
network of intermediate filament proteins that provide support to the nuclear envelope
What is the nucleolus? What can you see under EM?
site of synthesis of rRNA
can see lighter fibrillar centers (DNA between rRNA genes, has RNA pol I for transcription)
granular region- ribosome assembly
What are the main components of cyotosol?
1) Cytoplasm
2) free ribosomes/polysomes (in active translation)
3) Inclusions e.g. glycogen, lipid (dont have membranes, nonliving components), secretory vesicles (do have membranes)
4) Cytoskeleton i.e. actin, intermediate, microtubules (cannot see under LM)
Smooth ER
- function in the cell
- viewed with which microscope
- lipid and steroid biosynthesis, detoxification, metabolism, calcium storage
- view with EM
- lipids have no staining- washed out
Rough ER
- function in the cell
- viewed with which microscope
- why is there basophilic staining
- studded with ribosomes for protein synthesis (membrane, lyosomal, secretory)
- view with EM, continuous with nuclear envelope
- lots of RER in secretory cells
- basophilic dark staining bc (-) rRNA on ribosomes binds (+) dyes
Golgi apparatus
- function in the cell
- viewed with which microscope
- why are vesicles needed
- flattened sacs- polarity because proteins enter at cis face (more RER there) and leave at trans face (more vesicles there)
- view on EM, can see on LM only through silver stain
- need vesicles to transport material b/w golgi cisternae
Difference between constitutive and regulated secretion?
Constitutive- continual secretion of proteins e.g. plasma cells
regulated secretion- initiated by external stimulus, lots of storage and secretory granules
What is receptor mediated endocytosis?
How ligand-receptor complex can enter the cell:
associate with coat proteins to form coated pit–> coated vesicle–> fuse with early endosome –> lysosome
Lysosome
- function in the cell
- viewed with which microscope
-site of intracellular digestion
-hard to ID even at EM (cant see on LM) but if you use trypan blue can see places where dye accumulated in lysosomes
ONE exception is eosinophil (white blood cell) - lysosomes have crystalline inclusions which can be seen on EM
Peroxisomes
- function in the cell
- viewed with which microscope
- hydrogen peroxide metabolism, high enzyme concentrations- also have crystalline inclusions (like lysosomes)
- hard to ID even at EM (like lysosomes)
How do cells interact with their environment?
Secrete and respond to signals
reversible (motile/change shape)
irreversible (divide/differentiate/die)
What are ligands? What are key features?
ligand- triggers signal by binding to receptor
- agonist or antagonist to receptor
- lock/key fit
- receptor not active forever
- receptors typically inactive until bound
What are the 4 major classes of receptors?
1) Ion channels
2) Steroid hormone receptors
3) Protein kinase receptors
4) 7-alpha-helix receptors
Ion channels
- what are they
- where are they
- example: ligand-gated
- related diseases and example
- pore-forming proteins that allow flow of ions down electrochemical gradient (do not need energy)
- can be anywhere there is a lipid bilayer- cell surface, organelles
- ligand-gated: binding of ligand triggers flow of ions across membrane - ligands are neurotransmitters (for nerve cells and muscle contraction)
- mutations can cause loss or gain of function e.g. cystic fibrosis caused by loss of function mutation in CFTR chloride channel, tetrodotoxin (pufferfish) blocks Na+ channel
Nuclear steroid hormone receptors
- what are they
- where are they
- example: estrogen
- related diseases and example
- steroid hormones control gene expression by binding to and activating hormone receptors in nucleus or cytoplasm –> receptor binds to response element on DNA–> activation domain triggers transcription factors
- anywhere in cell- hydrophobic so can cross cell membrane
- estrogen: estrogen binds to receptor, it dissociates from chaperone and dimerizes, enters nucleus to bind response element to activate transcription
- estrogen receptors overactive in majority of breast cancer; tamoxifen is competitive antagonist to estrogen and represses transcription
Protein kinase receptors
- what are they
- where are they
- example: ras
- related diseases and example
- receptors that induce signal transduction cascade; dimerize when ligand binds- the cytosplasmic kinase domains phosphorylate and activate
- 3 domains: 1) extracellular (binds ligand); 2) transmembrane; 3) cytoplasmic (kinase activity or binds PK protein)
- Ras is a small G protein that targets gene transcription in the nucleus (proteins in cell division) by stimulating a MAPK phosphorylation cascade
- ras one of most mutated proteins in cancers- we know the 2 mutations, they destroy intrinsic GAP function of ras so it is always active
Define: Grb GEF SoS G-protein GAP
Grb- G protein receptor binding–> binds to phosphorylated protein kinase receptors
GEF- activates G proteins through GDP–> GTP
SoS- GEF that binds to Grb and activates ras
G-protein- binds guanine nucleotides (e.g. GTP,GDP) and acts as molecular switch during signaling
GAP- inactivates G protein through GTP–> GDP
7 alpha helix receptors
- what are they
- where are they
- example: beta adrenergic receptor
- related diseases and example
- most abundant- detect odor, light, taste, hormones, neurotransmitters
- coupled to trimeric large G proteins; when ligand binds, receptor is a GEF and activates G protein –> alpha and beta/gamma subunits dissociate
- 3 classes of Galpha: Gsalpha (activates adenylate cyclase–> PKA –> Ca2+), Gialpha (inhibits adenylate cyclase–> PKA), Gqalpha (activates PLC–> PKC–> Ca2+ influx from SER)
- beta adrenergic receptor desensitization- bound by epinephrine and norephinephrine –> this complex is phosphorylated by BARK–> bound by Beta-arrestin–> blocks interaction with trimeric G protein
What is calmodulin?
Ca2+ binding protein
Active when 4 Ca2+ bound, in turn activates CAMK by binding to inhibitory domain and allowing active site to be accessible for kinase activity
CAMK is important – linked to learning, memory, and Alzheimer’s
How does tamoxifen work?
competitive antagonist for estrogen- binds to estrogen receptor to prevent estrogen from binding
goes into nucleus and activates genes that inhibit target genes (estrogen activates them)
