FOM Week 5
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. What would you like to do?
What does the idea of genetic determinism mean?
- That we are nothing more than our genes
- This is too simplistic; doesn't allow for effect of environment
What is the basic flow of information?
- DNA-->RNA via transcription
- RNA-->Proteins via translation
- RNA-->DNA via reverse transcription
What is the typical conformation of chromosomes during interphase?
When would you be most likely to find chromosomes in the linear fashion they are typically depicted in?
How many chromosomes are in the body?
22 autosomes (44 chromosomes) and 2 sex chromosomes
What is the primary function of a centromere?
To connect 2 sister chromatids (one from mom, one from dad)
What is the basic structure of a chromsome?
2 sister chromatids connected by a centromere; each chromatid has telomeres, a p short arm, and a q long arm
What are the defining characteristics of a DNA helix?
- anti-parallel strands (5'-->3' and 3'-->5')
- 3.4 A betewen base pairs
- 34 A per 10 bp turn
- 10.5 bp per turn
- phosphodiester backbone on outside and bases on inside interacting via h-bonds
- Major and minor groove
- Phosphorylated on the 5'carbon sugar
A DNA double helix is most compact during which phase of mitosis?
Most compact during metaphase; much looser during interphase
What is the difference between euchromatin and heterochromatin?
- Euchromatin is light staining on an EM; chromatin is generally looser; consists of fertile, gene-rich areas of DNA
- Heterochromatin is dark staining on an EM; chromatin is generally more compact; consists of sterile, gene-free deserts; contains many repeats, telomeres, and centromeres
What are the key functions of chromatin organization?
Packages DNA and is critical in regulating transcription
~200 bps wrapped around a histone octamer core (2X4 histone proteins)
Definition: Histone 1
linker histone thata assists in higher order packing
Nucleosome fibers (beads on a string) compact further to form this
Definition: Interphase nucleus
- looped solenoids; each loop contains ~100,000 bps
- Must be unpacked and loosed to replicate
What causes SEPSIS?
Free histone proteins in endothelial cells; these are toxic to the cell; causes a global hyperinflammatory response that leads to multiple organ failure
Order the following compounds from most organized to least organized:
histone octamer core, solenoid, interphase nucleus, nucleosome, histone
- Interphase nucleus
- Histone octamer core
Rich in lysine; highly basic; interacts electrostatically with phosphates on DNA backbone
What is the site of histone modifications?
The exposed N-terminus tail
What are the 3 most common modifications to chromatin structure?
Acetylation, methylation, and phosphorylation
What is the function of modifying the chromatin structure?
- To make the structure tighter or looser to regulate gene transcription (this alters the access to the DNA)
- Some modifciations mark active or inactive genes
- There are more than 100 different modifications
What does acetylation of the chromatin structure do?
- Acetylation of lysine uncompacts the DNA; target of many drugs; requires acetyl transferase and aceytl CaA (donates the aceytl group)
- Taking it from an essentially positive charge to an essentially negative charge
What does methylation of the chromatin structure do?
- Methylation of lyine and arginine make the chromatin structure lipid friendly;
- methylation on the cytosine of CpG results in irreversible tightening to transcriptionally silent DNA
- Repeat DNA is highly methylated
- Common in many normal developmental processes (silencing tissue specific genes to grow the right kind of tissue in the right places; X chromosome in females, one is silent because of methylation)
What does phosphorylation of the chromatin structure do?
Gives it a negative charge
What is epigenetics?
- DNA information that is not based on the nucleotide sequence but is inherited
- Histone modifications and DNA methylation are examples
Where are the 2 locations you can find the human genome in a cell?
- Nucleus (3.6E9 bps and ~24,000 genes)
- Mitochondria (1.7E4 bps and ~40 genes)
What is the average inter-individual variations?
- 3-3.5E4 nucleotide differences
- ~1000 copy number variations (CNVs) although the size of the variable blocks ranges from 500 bp to 1.3 mbp
What are the critical requirements of the human genome?
It must be compacted but accessible for expression, copying, and repair
What are the different classes of repetitive DNA?
- Interspersed repeats (LINES, SINES, retroviral-like elements, and DNA transposons)(GCTGAGG...GCTGAGG...etc)
- Tandem interspersed elements (GCGCGC....GCGCGC...etc)
- Tandem repeats (Satellite DNA, Minisatellite DNA, and Microsatellite DNA) (TTAGGGTTAGGGetc.) no spaces in between
What does LINES stand for and what does it mean?
- Long interspersed elements
- A type of interspersed element
An interspersed repeat is derived from what?
A transposable element (Function is to replicate and jump around the genome)
Which interspersed element is the most common repeat?
SINES (Short interspersed elements)
What is satellite DNA?
- found at mainly at centromeres, this is a type of tandem repeat (TTAGGTTAGG)
- between 5 and 200 bps per repeat
- in humans there are at least 4 different repeat types
What is minisatellite DNA?
Clusters of tandem repeats up to 20kb in length; repeat units are up to 25 bp
What is microsatellite DNA?
- shorter clusters than minisatellite; these are tandem repeats that are usually <150 kb in length and <13 bp
- These are found at telomeres and can be used for DNA fingerprints and identifying individuals
Which type of repetitive DNA can be used to fingerprint individuals and to which class does it belong?
Microsatellite DNA, belongs to the tandem repeat class
Which type of DNA facilitates recombination events?
Repetitive DNA (it is also highly unstable)
Huntington's disease and myotonic dystrophy are caused by what?
Trinucleotide repeat expansions caused by slippage during replication or recombination; the strands reanneal incorrectly and create an outward loop and new non-coding regions
What are 3 types of double stranded breaks?
- Chromosomal translocations (exchange of fragments between 2 chromosomes; associated with cancer)
- Gross deletions
- Chromosomal inversion (piece breaks off and is reattached backwards)
DSBs are more common with certain repeats; very devastating effects
Familial hypercholesterolemia is caused by what?
- an LDL receptor deficiency
- There is unequal crossing over of DNA strands leads to deletion of exon 5; not enough LDL transporter is created, this is critical for cholesterol levels
What is the key difference between RNA and DNA?
- RNA has an extra -OH on 2' carbon of rung
- ribose vs. deoxyribose
Which two base pairs form 3 hydrogen bonds when they pair?
- A-->T forms 2 h-bonds
How is uracil created?
- Through the deanimation of cytosine
- This small structural change leads to big conformational changes
Which is larger, purines or pyrimidines?
What are the purines?
- PURe As Gold
- Adenine and Guanine
What are the pyrimidines?
- CUT the PY
- cytosine, uracil, and thymine
Which nitrogenous base fits the description of ketone purine?
Which nitrogenous base has an extra methyl group?
During what stage of replication does the DNA undergo dramatic condensation?
during mitosis in preparation for the separation
True or false?
Histone deacetylation serves to strengthen the interaction between histones and the phosphate backbone.
TRUE; Deaceytlation causes histones to become more positively charged
The modification that occurs at the 5' cystine and is often associated with gene inactivation is?
DNA methylation; fragile X syndrome is associated with this
The majority of the human genome is made up of what?
If K>>>>1, the mixture in the container will be mostly what?
If K<<<1, what can be said about the solution at equilibrium?
It will be mostly reactant
The primary biochemical lesion in homozygotes with familial hypercholestermia is what?
The functional deficiency of plasma membranes for low-density lipoproteins
What is the process called by which DNA is doubled?
What are the goals and challenges of DNA replication?
- Fidelity (matching origingal as closely as possibnle)
- Timing (duplicate only when necessary and only once)
- Quantity (each daughter cell gets only one copy of entire genome)
- Dysregulation of DNA replication is often associated with cancer
What does it mean to say that DNA replication is semi-cosnervative?
each daughter DNA gets one copy of parental DNA and one new copy
In which direction are DNA strands read during replication?
- New strands are synthesized in 5'-->3'
- 3' end is the hydroxyl group; it creates a phosphodiester bond via nucleophilic attack
Why do telomeres shorten as the cell ages?
Because nucleotides can not be added to the 5' end
What are the functions of the G1 and G2 phases?
cell growth and doubling of mass
What is the function of the S phase?
DNA replication; lasts approximately 10-12 hours
What is the function of the M phase?
chromosome segregation and cell division; lasts about 1 hour
What are the steps of DNA synthesis and replication?
- 1. Identify origins of replication (Ori)
- 2. Unwind the double-stranded DNA (dsDNA) via helicase
- 3. Initiate and recruit necessary machinery
- 4.Ligate newly syntehsized DNAs on lagging strand
- 5. Reassemble into chromatin
How many Oris do prokaryotes have?
1; protein factors recognize this sequence
How many Oris do eukaryotes have?
multiple; conserved origins do exist, but they are generally not specific nucleotide sequences as in prokaryotes
When does the origin of recognition complex (Orc) bind to the Ori DNA?
late M phase/early G1
What do the cdc6 and cdt1 of the Orc recruit?
- They recruit and load the 6 subunit MCM helicase complex onto Ori during G1
- This process is known as licensing replication
What is the function of licensing replication?
- It is how the cell ensures that replication will only occur once per cell cycle;
- after this step, cdc6 is removed from nucleus and degraded; cdt1 is degraded by proteasome after S phase; it is also inhibited in S and G2 phase by germinin
- the timing is orchestrated by cyclin-dependent kinases (CDKs)
- there are multiple, redundant mechanisms to regulate the cell cycle
What is the function of germinin?
inhibits cdt1 during the S and G2 phase so that replication only occurs once
What is the function of replication protein A (RPA)?
It is a single stranded DNA binding protein that keeps denatured (unwound) DNA from reannealing
What "machinery" is necessary to initiate and elongate DNA?
- Primase enzyme (DNA priamse/pol alpha)
- DNA Polymerase enzyme (DNA pol delta)
- PCNA (proliferating cell nuclear antigen)
What is the function of DNA primase/pol alpha?
This synthesizes RNA primer so that enzymes can't directly combine nucleotides to form DNA (makes a 10-15 nt RNA primer, but is necesssary for the synthesis of the lagging strand)
What is the function of the DNA pol delta?
This DNA polymerase enzyme copies DNA; it synthesizes the new DNA
What is the function of PCNA?
It clamps the DNA polymerase delta onto the DNA; this is a marker used in tests to check for proliferating cells; slides along the DNA as it is synthesized
What is the function of topoisomerase?
- prevents overwinding of DNA ahead of the replication fork; it creates a single-stranded nick and allows the DNA to spin and create a negative supercoil
- This is a target for cytotoxic drugs because blocking this stalls replications (antibiotics and antitumor)
What are Okazaki fragments and why do they occur?
- Because DNA is synthesized in the 5'-->3' direction; one of the strands is in the wrong orientation because of anti-parallel conformation
- These fragments are about 1000 bps long
- Each fragment has an RNA primer
How are Okazaki fragments ligated together?
- DNA pol delta initiates at primer and elongates DNA to next RNA primer; this displaces the RNA primer and endonuclease cuts off the flap; leaves a nick (absence of a phosphodiester bond)
- DNA ligase seals nick using ATP
What is the Hayflick limit?
it is the amount of times a cell can replicate before the telomeres become too short; 50-60 cell divisions before senescence
What is the correlation between cancer and telomeres?
Somatic cells have regained telomerase activity
What is the mutation in Xeroderma pigmentosum?
- Cells are incapable of repairing damaged DNA
- Exposure to the sun causes massive amount of tumorous cells
What is the difference between a somatic mutation and a germline mutation?
- Somatic mutations affect only the individual
- Germline mutations produce gametes or zygotes that are mutated; offspring are affected but these mutations are likely benign to the host
What are some common causes of DNA mutations?
mistakes in copying, recombination, genome, mishandling, or physical/chemical damage
Missing bases, altered bases, incorrect bases, deletion/insertion, dimer formation, strand breaks, interstrand cross-links, and tautomer formation are all examples of what?
What are the 2 types of bp substitutions?
- Transitions (pyr-->pyr and pur-->pur)
- and Transversions (pyr-->pur and pur-->pyr)
What are the 2 types of base pair mutations?
bp substitutions and deletions
When DNA pol detects that an incorrect nucleotide has been added, what happens?
- DNA pol back up (3'-->5' exonuclease), correct nucleotide is inserted, continues on
- Low failure rate of proofreading
Chemical mutagens can produce what with respect to DNA replication?
- Altered base pairing
- Double stranded breaks in DNA (blocks replication fork)
- DNA adducts (addition of a chemical moiety to DNA)
What happens when cisplating cross-links nucleotides?
failure of strand separation; stalls replication or produces double stranded breaks; both outcomes are cytotoxic
How does UV light (and other physical mutagens) affect transcription?
It produces thymidine dimers, which creates a cyclobutane ring; this can either stall replication (cytotoxic) or replication will occur without base pairing (mutagenic)
What is urelamin?
An adaptation of the skin that helps absorb UV light
How is a mismatched bp fixed?
- single strand incision
- requires nuclease, polymerase, and ligase
- One strand is methylated and non-methylated strand is repaired
- happens shortly after replication
What is base excision repair?
- fixes damaged bases that are recognized by DNA glycolase
- Need nuclease, polymerase, and ligase to repair
- No genetic effects observed with defects in this pathway (incompatible with life)
What is nucleotide excision repair?
- Recognizes helix distortion lesions
- need polymerase and ligase to repair
- can not fix thymidine problems without this pathway
- Can either be transcription coupled or global NER
What are the 2 types of double strand breaks repairs and which is thes safer of the 2?
- non-homologous end joining
- and homologous recombinationi
- The latter is the safer
What is non-homologous end joining double stranded break repair?
- ends are recognizes, and protein kinases are recruited and activated; this leads to the recruitment and activation of end processing enzymes
- Ends join randomly which is very dangerous
What is homologous recombination of double stranded break repair?
- this is restricted to the S and G2 phases because alignment of sister chromatids is critical
- ssDNA invades undamaged homologous template and using many, many proteins the damaged strand uses intact strand as a template to synthesize the removed DNA
- Allows for correct hybridization and rejoining of broken ends
What are the general functions of blood?
- transport of nutrients, oxygen, wastes, carbon dioxide, hormones, humoral agents, and immune cells
- Maintenance of homeostasis and buffer coagulation
What are the 3 primary components of blood?
- Plasma (water, albumin, globulins, electrolytes, etc.)
- Buffy coat (WBCs)
- Red blood cells
What is the composition of plasma?
- 90% water
- 8% proteins (albumin, globulins, fibrinogins)
- 2% other solutes (electrolytes, nitrogens, nutrients, and regulatory substances)
What is the function of albumin?
Maintain osmotic pressure and act as a carrier protein
What is the function of gamma globulin?
- Humoral immunity and antibodies
- Synthesized by plasma cells
What are the functions of alpha and beta globulins?
- maintain osmotic pressure and act as carrier proteins
- synthesized in the liver
What is the function of fibrinogin?
How are blood stains typically prepared?
Blood or bone marrow smear allowed to dry; stained with Reis stain
What organelles are present in an RBC?
- no nucleus; only plasma membrane, cytoskeleton, hemoglobin, and glycolytic enzymes; no other organelles
- Characteristic donut shape
- 7.5 um in diameter
- 4-6 million RBCs per microliter of blood
What is anemia and what causes it?
- pathologic conditioin characterized by low blood concentration of hemoglobin
- Caused by decreases in number of erythrocytes, amount of hemoglobin, or function of hemoglobin
What is the hematocrit?
- Volume of RBCs per unit of volume blood; the percent total volume occupied by RBCs
- normal is 38-48%
- Females typically a little lower than males
What is the mean corpuscular hemoglobin concentration (MCHC)?
- the ratio of hemoglobin to hematocrit
- normal is 32-36%
What creates the characteristic donut (biconcave) shape of the erythrocyte?
membrane proteins and interactions between them (glycophorin, actin, tropomyosin, adducts)
What are spherocytosis and ellipocytosis?
- Result of defects in the proteins ankyrin and spectrin; the RBCs can't move through the capillaries as well; body recognizes them as bad and demolishes them; leads to anemia
- spherocytosis (circular)
- ellipocytosis (football shaped)
What do sickle cell anemia and thalassemia have in common?
Both have defects in hemoglobin (the oxygne carrying structure)
How does diabetes lead to elevated hemoglobin A1C levels?
- excess glucose interacts with amino acids to form reactive intermediates that eract with circulating proteins to form advanced glycated end products (AGE) which slow conductioni speed and can lead to endothelial leakage and thicker BM
- also AGE leads to non-enzymatic glycation of hemoglobin; glycohemoglobin (HbA1C)
Why is glycohemoglobin a measurement of average blood sugar over the past 2-3 months?
- erythrocytes are freely permeable to glucose, which binds the N-terminal of valine irreversibly
- 120 days is average lifespan of erythrocyte
What are leukocytes?
Another name for WBCs; function to defend body against foreign material and infectious disease
What are neutrophils?
- 10-15 um; most abundant WBC in circulation
- Function: migrate to site of infection to phagocytize bacteria
- Histologically: multi-lobed nucleus with densely clumped chromatin; strand of nuclear material connects the lobes
What are the 3 types of cytoplasmic granules in neutrophils?
- Specific (Small and numerous; contain phospholipases; target bacteria)
- Azurophilic (large and less numberous; lysosomes)
- Tertiary (Phosphatases, metalloproteinases)
What are the 3 types of neutrophils?
- Band neutrophils (U-shaped nucleus; immature neutrophil; gives idea of proliferation)
- Barr body (only in females)
What are eosinophils?
- 12-15 um; less abundant in circulation than neutrophils
- Function: might into connective tissue; first line of defense against parasites
- Histologically: bilobed nucleus; large cytoplasmic granules
What are basophils?
- 12-15 um; least abundant WBC in circulation
- Function: migrate into connective tissue and play a role in hypersensitivity reactions
- Histologically: key feature is large, intensely staining granules; nuclues is not very evident but biolobed if you can see it)
What are lymphocytes?
- 8-10 um; smallest nucleating cell in circulation
- Histologically: roughly same size as RBC; large nucelus with pale cytoplasm surrounding; no granules
- Differentiate to T-cells (recognize antigens), B-cells (antibody protection), and NK cells (kills virus-infected cells and timor cells)
T-cells, B-cells, and NK cells are examples of what type of WBC?
What are monocytes?
- 12-20 um
- Function: migrate from circulation where they differentiate to macrophages and are involved in bacterial phagocytosis, antigen presentation, and removal of cellular debris (garbage collector)
- Histologically: no granules; largest nucleating cell in circulationg; nucleus is abnormally shaped; chromatin stains in rope-like pattern
What are platelets and where do they come from?
- Thrombocytes; 2-3 um
- Function: adhere to endothelium to promote vessel repair, blood clotting, and vasoconstriction
- Cellular fragments of megakerocytes
What is diapedesis?
- Transendothelial migration
- Occurs at post-capillary venules in response to a seignal
- Includes capture and rolling (upregulation of selectins that WBCs loosely associate with), firm adhesion (cell adhesion molecules; CAMs; integrins), spreading, diapedesis (histamines functionally reduce cell-cell interactions between endothelial cells), and attraction to site of infection by cytokines
What is hematopoeisis?
- The process of producing mature RBCs
- Site of this shifts through development: yolk sac, spleen/liver, bone marrow by late teens
- Includes myeloid series and erythroid series
How can you tell if a cell is in the myeloid series?
- the nucleus is oddly shaped
- These stem cells produce neutrophils, eosinophils, and basophils
How can you tell if a cell is in the erythroid series?
- Nucleus is round
- Produces RBCs when it looses its nucleus and other organelles
Which structure is more likely to be single-stranded: RNA or DNA?
- RNA though it can form higher order structures
- Also, RNA has additional 2' hydroxyl which reduces its stability
What is the function of tRNA?
adaptor between AA and mRNA sequence
What is the function of mRNA?
contains protein coding sequence from gene; it is made from transcription
What is the function of rRNA?
structural and catalytic componenet of ribosomse; shows that RNA can also catalyze reactions, not only proteins
What is the function of snRNA?
What is the function of miRNA?
inhibits translation and degrades mRNA
Most simply, what is transcription?
- Highly regulated (Primarily at initiation steps)
- First step in genotype-->phenotype
Why did we spend so much time examining transcription in E.coli?
Because the general principles of bacterial transcription apply to eukaryotes as well
RNA polymerization begins with what common molecule?
ATP or GTP
In which direction is RNA synthesized?
5'-->3', just like DNA replication
Which of the following does not require a primer: transcription or replication?
Transcription does not
What is the TSS?
- Transcriptional start site
- RNA polymerase begins transcribing from this point
What is the promoter region?
- Sequence info that directs transcription
- Usually in the 5' (or upstream) direction of the TSS
- Found in essentially all genes
- Necessary for recruitment of general transcription factors
How do enhancers/silencers alter transcription?
- In one of 2 ways: post-initiation events (eukaryotes only) and recruitment of transcription factors and RNA polymerase to core promoter
- Can be near of far, upstream or downstream
- Combination of various elemtns makes the regulation complex
What is teh core promoter in prokaryotes?
- -10 box and -35 box
- Consistent distance from start site
- Contact points for RNA polymerase proteins
- Region between -10 box and TSS is unwound
What are 2 enhancers found in prokaryotes?
- UP element and FIS site (much farther upstream than UP)
- Conserved; something specific always binds to these
What 5 subunits make up RNA polymerase?
- beta (catalytic subunit)
- beta' (clamps DNA during transcription)
- Sigma (interacts with -10 and -35 boxes; targets polymerase to promoter; initiation factor; open complex is more stable than a closed complex in the sigma cycle)
- and 2 alpha subunits
When transcription begins, what happens to the sigma subunit?
it dissociates from the holoenzyme RNA polymerase so that it polymerase can go somewhere; critical for promoter clearance and elongation
What are the 2 components of the alpha subunit of RNA polymerase?
- alpha CTD: the c-terminus; interacts with enhancers and silencers
- alpha NTD: N-terminus; binds to core promoter
With transcription, what is the difference between the holoenzyme and the core polymerase?
Core polymerase is the holoenzyme minus the sigma subunit (elongation)
What is the general rule of thumb reagardinf rate of transcription?
The more contacts you have between the DNA and the holoenzyme, the more translation you'll have
What are the different transcriptional states of genes?
- Repression: foot on the brake; some translation because you can never completely stop it
- De-repression: foot on the brake, proceeds slowly
- Activation: hit the gas and go
- Constitutive expression: cruise control; for the general housekeeping genes; always on, but can be regulated
What is the best way to regulate transcription?
- Regulate the recruitment of RNA polymerase to the promoter by: chaning promoter strength (depends on 10/35 elements), additional Poly RNA to DNA contacts (includes UP element with sigma)
- Proteins that bind to enhancers/silencers affect RNA pol recruitment too
- Post-recruitment regulation is also important
What are the transcriptional states of lac operon in E. coli?
- Repression: when no lactose is present; lac repressor binds specific DNA sequence in operator and prevents RNA pol from binding promoter
- De-repression: lactose is there, but not the only sugar present; allactose (intermediate metabolite of lactose breakdown) binds lac repressor can causes conformational change so that it can't bind DNA anymore
- Activation: lactose is only sugar presentp; cAMP binds CAP which interacts with sequence upstream of promoter; increases recruitment of RNA pol to promoter
With regards to transcription, what is allosterism?
The idea that binding of small molecules away from the active site alters function
What are the different kinds of RNA polyemrase in eukaryotes and whihc is most common?
- I (transcribes gene for rRNA)
- II (transcribes gene for mRNA and miRNA; sensitive to alpha amanitin which shuts off transcription completely; most common)
- III (trancribes genes for tRNA, 5s rRNA, and snRNA)
- Mitochondrial (transcribes genes for mitochondrial genome)
What are the core promoter elements of transcription in eukaryotes?
TATA, Inr (initiator), and DPE; function similarly to 10/35 box in bacteria
Which type of regulatory element in eukaryotes affects the transcriptional baseline?
Proximal promoter elements
How do distal regualtory elements in eukaryotes aleter transcription?
- Recruitment of trancription factors to core promoter
- Altering chromatin structure
- Intronic regulatory elements (exclusively on the introns)
- Can be used upstream of downstream (1-50kbp distance)
What are locus control regions?
Regulatory elemnts that govern transcription of a group of genes at a single chromosomal locus; can be thousands to millions of bps
What are the steps of transcription by RNAPII in eukaryotes?
- loosen chromatin structure
- Binding of general transcriptioin factors toc ore promorter
- Binding of RNA pol II to DNA
- Phosphorylation of RNAPII, promoter clearance, and product elongation
What are HDACs and what are they involved in?
- Histone de-acetylases
- acetylate lysines of chromatin structure to loosen it (along with HATs)
- Inhibitors of HDACs are currently being tested as cancer treatments because they stop transcription
What are the general transcription factors that bind to the core promoter in eukaryotes?
- TFIIA, B, D, E and H
- TFIID binds the core promoter (TATA box) and all others are just involved in the assembly and recognition of core promoter
What makes up the pre-initiation complex in eukaryotic transcription?
- Mediator complex (guides RNAPII to TFII)
- RNAPII (huge 12 subunit complex)
What is the rate limiting step of transcription in eukaryotes?
formation of the pre-initiation complex
Which part of the TFII complex is responsible for phosphorylating RNAPII?
- TFIIH and a second kinase, Ptef-B phosphorylate RNAPII on its c-terminus
- Once this happens, RANPII leaves TFIIs and Med and moves down strand; now called the initiation complex
- To have productive elongation (>50 bps), Ptef-B must add some more phosphates to RNAPII
How does the positive regulatory element affect transcription in eukaryotes?
enhances in 2 ways: binds to HAT to increase loosening of chromatin and helps to recruit TFII and Med proteins to promoter
How does the chromatin remodeling complex (CRC) affect transcription in eukaryotes?
enhances transcription by peeling DNA from nucleosome
How do transcriptional repressor proteins work in eukaryotes?
- block the binding of general transcription factors and
- recruit HDAC enzymes that deacetylate the nucleosomes; this results in compact, inaccessible chromatin
What are the 4 tasks of adolescents?
- Independence from parents
- Being like their peers
- Accepting body image
- Establishing sexual, vocational and moral identities
What are the 3 stages of adolescence?
- Early adolescence (10-13)
- Middle adolescence (14-16)
- Late adolescence (16-2....)
What are the typically characteristics of early adolescence?
Onset of puberty, concern with body, desire to be separate from family, peer importance, and concrete thinking
The following characteristics are indicative of which phase of adolescence: completion of puberty, peers set standards, some desire to be separate from family, increased independence and some abstract thinking?
The following characteristics are indicative of which stage of adolescence: complete physical maturation, secure body image and gender role, emancipation from parents, nearly complete cognitive development, creation of life goals?
What are the stages of puberty in a typical girl?
- thelarche (with asymmetrical breast development)
- pubarche (6 mo later)
- menarche (2 years later)
In a typical boy, what is the first stage of puberty?
testicular enlargment (98% of the time)
Who is more likely to enter pubtery first, males or females?
Females (completed in 4 years)
Males start later but complete in approximately 3 years
What does the pneumonic device HEADSS tell you to ask about in the adolescent interview?
- Education (insight to learning disabilities)
- Activities (counsel on exercising)
- Drugs (ask if their friends do it)
- Sex (what to expect and do friends have sex?)
What can you legally refuse to tell a parent about their adolescent?
Everything except whether the teen has thoughts of harming themself or others or if you suspect abuse (mandated reporter)
Definition: Healthy behavior
- One that helps prevent illness and promotes health
- Unhealthy behaviors play at least some role in each of the 12 leading causes of death; most common contributers to mortality are alcohol, tobacco, firearms, motor vehicle, diet, activity, and sexual behavior
- Nationally, health habits tend to cluster in geographic regions
Defnition: Fundamental attribution error
- When making attributions about actions or behavior, people tend to over-emphasize dispositional factors and under-emphasize situational factors
- This is a possible reason for the persistence of bad health behaviors despite the knowledge that they're bad
What are the 3 most prominent influencers of an individual's universal behaviors?
- Psychopathology (engage in different behaviors if you're depressed, bipolar, etc.)
- SES (important to distinguish between situational and generational poverty here)
- Cultural perspectives
What must be known to alter or intervene with an unhealthy behavior?
Why they engage in behavior and what would motivate them to change
In terms of behavior and psychology, what is a construct?
- variable or factor that influences whether a behavior will be changed
- Includes habits, intent/willingness to act, cues, social influence, emotions, and environment
The following order defines which psychological model of behavioral modification:
individual-->interpersonal-->community-->institutions-->structures, policies and systems?
What are the three levels at which behaviors can be modified?
- Individual (medications and counseling)
- Situational (family, social groups, job, etc.)
- Environmental (taxation, laws, ad campaigns, etc.)
What is the health belief model?
- Individual's beliefs about their health and what will improve their health
- Influenced by self-efficacy (confidence that specific task can be completed), perceived susceptibility, severity, benefits, and barriers
- Not really a very good predictor of behavior changes
What are the essential characteristics of a stage theory?
- Small number of distinct stages
- Temporal sequence
- Similar barriers facing all people in same stage
- Different barriers facing people in different stages
Precontemplation, contemplation, preparation, action, and mainenance best describe which model of behavioral change?
Transtheoretical Model (TTM)
What does the social learning theory say about behaviors?
- Behaviors are learned throguh modeling or the observation of oterh engaged in said behavior
- You are also more likely to imitate the behaviors of those you have the most contact with
- massive, wide-spread pre-mature cell death
- Generally in response to injury; always considered pathological
- Caused by leaking of lysosome enzymes into the cytoplasm and their consequent digestioin of the cell
Definition: Reversible cell injury
characterized by reduced oxidative phosphorylation of the cell and consequent depletion of energy stores; also causes cellular swelling because of changes in ion concentrations (Na+)
What are 2 types of cell death?
Necrosis and Apoptosis
- programmed cell death of a single cell
- Can be caused by cell injury or be part of a normal process
Hypoxia and free radical, physical agents, chemical agents/drugs, infectious organisms, immunologic reactions, genetics derangements, and nutritional imbalances can all cause what?
At which point does a reversible cell injury become irreversible and lead to cell death?
Not possible to pinpoint the time, they are on a continuum
What are the 3 types of radicals associated with hypoxia and consequent cell injury?
- Superoxide anion (O2.) formed by cytochrome p450; can be removed by superoxide dismutase
- Hydrogen peroxide (H2O2) removed by catalase or glutatione peroxidase
- Hydroxyl radical (.OH) which initiates lipid peroxidation (ruins membranes) and DNA damage
Defects in volume regulation, increased permeability to Na+, cellular (hydropic) and mitochondrial swelling, plasma membrane blebs, dissociation of ribosomes, and aggregation of nucleolus are all characteristic of what?
Reversible cell death
Under a light microscope, how can you tell necrosis and apoptosis apart?
Can not be differentiated under a microscope
In necrosis, there is a dramatic change in the concentration of which intracellular ion?
Calcium; leads to calcification and formation of dense bodies that cause mitochondrial damage; also increases oxidative stress and lipid peroxidation which causes CytC to leak out of mitochondria and signal cell to die
What happens as a direct result of membrane defects in necrosis?
cells aggregate, coagulate, and change color (makes it easy to identify cell death location)
What is the morphology of a cell undergoing necrosis?
- Eosinophilia (due to binding of eosin to denatured cytoplasm proteins)
- Pyknosis (DNA condenses into solid chunks)
- Karyohexis (DNAases and RNAases break up nucleus)
- Karyolysis (DNAases chop up DNA into finer pieces)
What are the 5 types of necrosis?
- Coagulative necrosis
- Liquifactive necrosis (includes abscess formation and coagulative necrosis of the brain)
- Fat necrosis
- Caseous necrosis
- Fibrinoid necrosis
Definition: Coagulative necrosis
- Typical in hypoxic or ischemic injury (blood supply has been completely cut off)
- Myocardial infarction (cells block arteries; soft atherosclerotic plaques are most dangerous) and gangrene are 2 most common examples
What is the earliest histological sign of myocardial infarction?
Hemorrhage followed by entrace of neutrophils (turns tissue yellow)
Definition: Liquifactive necrosis
- occurs when rate of cell destruction exceeds rate of repair
- Abscess formation (tissue dissolves away and area filled with puss) and Coagulative necrosis of the brain (liquifies tissue; glial cells are responsible for healing because there are no fibroblasts) are the only 2 times this really occurs
Would you treat an abscess with antibiotics? Why or why not?
No because abscess is avascular and pH is to low for efficient functioning of antibiotic
Definition: Fat necrosis
- occurs exclusively in adipose tissue
- Only way to get it is pancreatitis (lipids released by fatty acids become saponified to form calcium soaps; look like amorphous blue areas on H and E stain) or trauma
Definition: Caseous necrosis
found in associated with mycobacteria (TB is most common); due to the presence of mycolic acids in their cell membranes; TB does not kill tissue, it's the dramatization of the body's immune response that does it; first and most importantly, you must rule out TB!
Definition: Fibrinoid necrosis
Indication of sever damage to blood vessel walls; damage triggers thrombosis and clotting cascade; caused by malignant hypertension
List the primary initiation site, recognition factor, and secodnary inititation site of transcription
- 1 site: core promoter (10/35 in prokaryotes, TATA in eukaryotes)
- Recog. Factor: TFIID (and complex)
- 2 site: downstream of TFII
List the primary initiation site, recognition factor, and secondary initiation site of DNA replication
- 1 site: Origin of replication (ori)
- Recognition factor: ORC
- 2 site: DNA polymerase/ pol alpha adjacent to ORC binding site
List the primary initiation site, recognition factor, and secodnary initiation site of the poly-adenylation of RNA
- 1 site: Poly A signal (AAUAA)
- Recognition factor: Cleavage factors
- 2 site: ~30 nucleotides downsream of AAUAA
List the primary initiation site, recognition factor, and secondary initiation site of splicing
- 1 site: differs based on what the cell needs
- Recognition factors: snRNPs
- 2 site: "branch-point" adenosine in splicing
List the primary initiation site, recognition factors, and secondary intitiation site of translation
- 1 site: 7-methyl guanosine cap of mRNA
- Recognition factors: eIF4F complex (Composed of 3 subunits, eIFA, eIFG, and eIFE)
- 2 site: AUG start codon (Kozak/shine-dalgarno sequence)
What does UTR stand for?
- Untranslated regions; sequences in mRNA that are not translated; often cotnain conserved sequences that affect mRNA stability
- 5' UTR is between 7-methyl cap and AUG start codon
- 3' UTR is after UAG stop codon (often contains sequences that enhance or inhibit translation)
Definition: Cap binding complex
- eIF4F protein complex; in eukaryotes only
- In translation, this is an adaptor between the 7-methyl guanosine cap and ribosome; recruits small subunit of ribosome to mRNA; ensures taht only mRNAs are translated
- Consists of 3 subunits; eIFA, eIFG, eIFE
What do 4E binding proteins do? (4E-BPs)
- Inhibit translation by binding eIF4F complex so it won't bind cap of mRNA
- If these are phosphorylated (as in diabetes), it won't bind the complex and translation is uninhibited
- eIF4F is expressed at higher levels in cancerous cells because not properly regulated
How do ribosomes involved in translation differ in prokaryotes and eukaryotes?
- Prokaryotes: 50S subunit and 30S subunit form 70S ribosome
- Eukaryotes: 60S subunit and 40S subunit form an 80S ribosome
What are the p site and the a site of a ribosomal subunit?
- The p site (peptidyl tRNA site) is on the larger subunit of the ribosome and is where the chain lengthens and grows
- The a site is where tRNAs bring in the next AA in the sequence
- AA chain shifts from p site to a site, new AA is added, tRNA leaves, ribosome translocates to next codon, and the elongated chain shifts back to p site
What does the term polysomes indicate?
There are multiple ribosomes on a single mRNA; these dissociate and are translocated in the direction of translation (5'-->3' N-terminus-->C-terminus)
What is a polysome fraction?
- A subpopulation of highly translated mRNAs in the cell
- Can control protein synthesis by moving mRNAs in or out of polysome fraction (conceptual movement)
How do tRNAs "know" which AA to bring to the chain next?
Specific for 3 letter codon
What is the basic structure of tRNA?
- anti-codon region that base pairs with the codon
- Arms of t are base paired together to stabilize structure
- acceptor stem (part attached to AA) has nucleotides base paired in a double helix and a part that is signle-stranded
- An ester bond links the AA to the molecule; AA needs to be protected by proteins until it's loaded onto the growing chain
What does an aminoacyl tRNA synthetase do?
- charges tRNA
- each AA has specific tRNA synthetases, but all isoacceptor tRNAs for an AA use the same synthetase; each synthetase binds AA, ATP (AMP attaches and 2 Pis go away, eventually loose AMP) and tRNA
What is the function if eIF2 in translation?
- Eukaryotic initiation factor 2
- escorts met-tRNA to ribosome
- once start codon is recognized, eIF2 leaves and large ribosomal subunit combine with the small one
What is the function of eIF1 and eIF3 in translation?
- bridges small subunit to cap complex
- It's a linake for recruitment activity; interacts with cap binding complex to bring ribosome to mRNA
What is the sequence of events in translation in eukaryotes?
- Intial recognition event (cap complex binds mRNA cap)
- eIF2 loads met-tRAN onto small ribosomal subunit
- Small ribo subunit recruited to mRNA and bridged with eIF1 and eIF3
- Small ribo scans along mRNA looking for start codon
- Recognition of start codon (release of initiation factors, eIF1 and eIF3)
- Termination (UAG; release factor binds to a site and causes teh release of everything)
What is the Kozak sequence?
The sequence in eukaryotes to which the 16S rRNA base pairs with; the more similar the sequence is to this, the stronger and better the binding will be; normally occurs at first AUG codon after the cap
What is the Shine-Delgarno sequence?
The defined start sequence in prokaryotes; can occur at internal AUG codons
What catalyzes the formation of a peptide bond between AA and the newly synthesized chain in translation?
Peptidyl transferase; energy required for peptide bond is from ATP used in tRNA charging
What is the function of EF2 (elongation factor 2) in translation?
- Translocates the ribosome; it's a GTPase that binds GTP, hydrolyzes it, and phuses tRNA along when p site is empty
- Servesa s a timer/switch
What is the function of EF1 (elongation factor 1) in translation?
escorts the next AA-tRNA to the vacant a site
Forming a peptide bond and subsequent translocation of the ribosome requires how many ATP/GTPs?
1 ATP (tRNA charging) and 1 GTP (translocation by EF2)
What does the release factor (RF) do in translation?
- causes the termination of translation at a stop codon and the release of all subunits
- ribo subunits are recycled
How does PERK regulate translation?
- PERK kinase phosphorylates eIF2 so that it cannot participate in initiation
- need to shut down translation when the ER is stressed and proteins are misfolded and begin accumulating; shut down translation so that these can be fixed
What is the defect in Wolcott-Rahison syndrome?
- mutation in PERK gene so that the protein has reduced or no kinase activity; can't shut off translation
- cells that can't shut off undergo apoptosis; leads to neonatal diabetes, epiphyseal dysplasia, osteoporosis, growth retardation
- very rare
What is the primary target of over 50% of all antibiotics?
Where are micro RNAs (miRNAs) most often found?
- in the 3' UTR region; have a hairpin structure; 21-22 nucleotide non-coding RNAs; regulate gene expression through repression of translation and/or destabalization of RNA; represses translation by interferring with translocation
- Transcribed in the nucleus, processed in the cytoplasm to become active; target RISC (RNA induced silencing complex); binds this and guides it to specific mRNA; deadenylates mRNA and essentially destroys it
What are the 3 levels of prevention?
- Primary (avoidance of a disease; not smoking)
- Secondary (detection of disease in early, more treatable stage)
- Tertiary (management of disease to minimize discomfort)
When should you utilize a screening test?
When the patient will benefit from the use of it; depends on how early disease can be detected, sensitivity and specificity of test, rate of false positives, harm caused by screening, and amount of motrality/increased survival brought about by test
When can a disease be detected, and when is it most likely to be detected?
- Preclinical phase: after biological onset of disease, but prior to manifestation of symptoms
- Most likely to be detected during clinical phase: after the manifestation of symptoms, often too late
What are the 2 types of bias associated with screening?
- Lead time bias (Identifying disease earlier in natural course does not increase survival time)
- Length time bias (tendency of screening test to preferentially identify individuals who have a longer preclinical phase of disease)
What are the leading sites of cancer-related deaths in men and women?
Cases: men (prostrate, lung, colon) women (breast, lung, colon)
Deaths: men (lung, prostrate, colon) women (lung, breast, colon)
Many of the secondary killers cannot be prevented through behavioral modification which s why screening is so important
What does "validity of a test" mean?
- The ability to distinguish between who has a disease and who does not
- No test can have perfect specificity and sensitivity
- ability of a test to identify those who HAVE disease (look at the oclumns)
- Probability of testing positive given the presence of a the disease
- to be perfect, no false negatives
- ability of a test to identify those who do NOT have disease (second row of columns)
- Probability of testing negative given the absence of a disease
- to be perfect, no false positives
How are specificity and sensitivity related to prevalence?
- They are independent of each other
- S and S do not change
Defintion: predictive value
- Posterior or post-test probability
- given certain test result, what is the probability that you actually do or do not have the disease
- deals with rows
- Dependent on the prevalence of disease in a population; higher prevalence increases PPV and decreases NPV
Definition: Likelihood ratios
- Similar to predictive value
- Positive LR: Sensitivity/(1-Specificity)
- Negative LR: (1-Sens/Spec)
Which type of white blood cell releases histamines and heparin?
What would you like to do?
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