FOM Week 5 Flashcards
Why is DNA used to store information
It is stable
It is easy to copy/access
It can store ‘infinite’ amount of information
DNA Methylation
A long term covalent modification used to silent DNA from being transcribed
Very common in heterochromatin and repetitive DNA
DNA Acetylation
A short term modification used to open up DNA from the nucleosome
Occurs on lysine residues
Done by the enzymes HAT and HDAC
What makes up the Human Genome
Half is repetitive (LINEs, SINEs, Retroviral)
Half is unique (Introns, Exons, Non repititive DNA that is not involved in transcription)
Active Metabolite to Treat Actinic Keratosis
Actinic Keratosis is a pre malignant skin cancer
The drug gets incorporated into the DNA and causes strand breaks leading to cell death
What happens in FSHD
Their repeats in repetitive DNA get lost and therefore are no longer recognized as repetitive DNA. This leads to their transcription which causes the pathogenesis
Compare LINEs vs SINEs
LINEs are about 20% of genome. SINEs are 13%
LINEs have transposon capability
LINEs are around 6000 nt long. SINEs are around 280
Explain the Effects of Low Methylation in LINEs
People who have low methylations have a higher risk of dying early and getting some forms of cancer
Some LINEs are oncogenes if they become expressed
Mosaicism/Chimerism
Occurs during development and gives you multiple copies of genes
Occurs in about 20% of people
What can happen if DNA replication goes wrong
Cell catches it and repairs itself
Apoptosis
Cancerous
Rules of DNA Replication
It must only occur once per cell cycle
It must be regulated
It must be accurate
It is semi-conservative
Basic Steps to DNA Replication
Locate the origin and unwind from there
Initiate replication using a RNA primer
Elongate while preventing supercoils from forming
Reassemble into chromatin once done
Origins in Eukaryotes vs Prokaryotes
Prokaryotes only have one-two origins and are conserved
Eurkaryotes have 30k-50k origins and are not as conserved but still AT rich
Explain the Formation of the Pre-Replicative Complex
ORC binds to the origin
CDC6 binds to the ORC and recruits CDT1 which carries the MCM helicase along with it
Regulation of CDC6 and CDT1 in the Cell Cycle
CDC6 is only active during the G1 phase
CDT1 is only present during the G1 phase and then gets degraded
Geminin is a protein inhibitor of CDT1 as well and is highly present in the S and G2 phase
Enzymes Involved in DNA Replication
MCM Helicase DNA Primase DNA Polymerase (delta/epsilon) PCNA Topoisomerase DNA Ligase
Explain the Role of RPA during Replication
It is involved in binding the SSB’s to prevent reannealation of the DNA strands
Lagging Strand vs Leading Strand
Lagging stand goes away from the replication fork and requires discontinous replication
Leading strand goes towards the fork and is synthesized as one long fragment
Quinolone/Etoposide
Quinolone is an antibiotic that inhibits topoisomerase
Etoposide in an anti tumor drug that inhibits topoisomerase
Explain the Role of Chaperones in DNA Replication
They work to rearrange the nucleosomes so the DNA is accessible by the machinery and then reposition them once it is complete
Explain the Role of Telomerase
In the lagging strand, replication can never copy all of the DNA because of its need for lots of primers
Telomerases add nucleotides to the end of the chromosomes to prevent them from shortening after each replication
Cancer Cells have lots of them
Role of Blood
Transport O2 and nutrients to tissues
Transport CO2 and waste away from tissue
Works to maintain homeostasis
Composition of Blood
55% Plasma (90% water, lots of albumin)
Buffer region which contains WBCs
45% RBCS
Hematopoiesis During Development
Originally occurs in the yolk sac, then the liver/spleen
Once we are born it occurs in the bone marrow
At about age 20 it stops in long bone because the BM gets replaced by adipose
Erythrocytes
About 7 micros in diameter
Have no organelles
Are bioconcave in shape
EPO
A hormone produced by the kidneys
Gets released during times of hypoxia to stimulate more production of RBCs
HbA1C
A measurement used for the long term measure of glucose levels in the blood (2-3 months)
Glucose naturally binds to the n terminus of Hb and causes problems to the RBC
Neutrophils
12-15 micros in diameter
Multi lobed nuclei
60% of all the leukocytes
They are very short lived once they enter tissue
Eosinophils
12-15 micros in diameter
Bi lobed nuclei
The granules have two shades of staining in the EM
Involved in parasitic infection/modulation of inflammation
Basophils
12-15 micros in diameter
Bi lobed nuclei
The granules are large and stain very densely
Involved in releasing histamine
Lymphocytes
6-8 micros in diameter
Include B, T, and NK cells
30% of circulating leukocytes
Lifespan is hours to years
Monocytes
12-20 micros in diameter
Differentiate into macrophages in the tissue
Involved in APC process
How does diapedesis occur
Macrophages in the tissue relase ILs that bind to the endothelial
The endothelial will begin to express selectin receptors
Neutrophils have selctin ligands and will interact with the receptors causing slow rolling
Neutrophils begin to express ICAM and integrin which leads to a tight bind
Neutrophils will then exit into the tissue
What is a mutation
It is when the DNA has been damage and unrepaired. They are permanent
Can be somatic or germline
What type of damage does UV light cause in DNA
It forms thymidine dimers. Also causes ROS
Around 4000 per hour in sunlight
What type of damage does smoking cause in DNA
It forms aromatic DNA adducts
What type of damage does medical imaging cause to the DNA
It forms DSBs
At a very low rate though
Effects of DNA Adducts
They chemically modify the DNA
This leads to improper base pairing or DSBs
Cisplatin
A cancer drug that forms intrastrand cross links in DNA making them inseperable
This means not replication or transcription can happen
Steps to Repairing Simple Lesions
Recognize the mutation
Remove the mutation by nuclease
Replace with DNA pol
Ligate the nicks
Steps to Repairing Thymidine Dimers and Cross Links
Nucelotide Excision Repair
Same as the simple lesions except more base pairs are removed (more complex)
Fanconis Anemia
A genetic disease when the ICL repair does not work
Leads to a build up of genetic mutations
Two Ways to Repair DSBs
Homologous Recombination:
- Error free
- Can only occur during S or G2 phase
Non Homologous End Joining
- Occurs when homologous recombination cannot
- Two ends of the chromosomes are joined together without properly filling in the break
What happens in cells with a buildup of mutations
Cell death
Neoplasia
PARP1
An enzyme that recognizes mutations and flags down the machinery needed to repair
In cancer cells we inhibit this and it prevents DNA repair leading to so many mutations that eventually the cell will die
Importance of Regulating Gene Transcription
It leads to the differentiation of cell lineages
Determine the morphology and function of cells
Promoter
A region of DNA next to the gene and is where RNA pol 2 binds
Defines the transcription start site
Enhancers and Silencers
Can be located all throughout the genome and be even be in between genes
They are specific to a certain gene
This is where the transcription factors bind
Effects of Mutations in TFs
It will lead to improper expression of the gene leading to malfunction of the cell
Lineage TFs for Cardiocytes
GATA4
TBX2
TBX5
3 Domains of TF
DNA binding domain
Transcriptional Activation Domain
Dimerization domain
How do domains bind to the DNA
They recognize specific bases based on bonding and also shape of the DNA based on major/minor groove
How do TFs alter transcription
- They recruit proteins to alter chromatin structure (CRC/HAT/HDAC)
- They recruit other TFs
- They recruit kinases to activate RNA pol
Lineage TFs for pluripotent induced stem cells
Oct3
Oct4
Sox2
Expose fibroblasts to these
How does environment influence transcription
They environment of cells determine which TFs are present
Ex is infection, inflammation, cell cycle stage, etc
NF-kB
Inhibits production of inflammatory genes by blocking the receptor from binding glucocorticoids
Null Hypothesis
There is difference/association between exposure and disease
RR=1
Alternative Hypothesis
There is a difference/association between exposure and disease
RR does not equal 1
What do each of these tests compare
Chi Square
T Test
ANOVA
Chi square compares categorical data
T test compare numerical/mean data
ANOVA compares numerical/mean data as well but is better
What does a high and low p value mean
A high p value means that your data is consistent with the null hypothesis
A very low p value means that your data is inconsistent with the null hypothesis
Type 1 Error vs Type 2 Error
Type 1 error is false positive
Alpha is the probability of committing a type 1 error (want less than .05)
Type 2 error is a false negative
Beta is the probability of committing a type 2 error (want less than .20)
What is the most reliable test to base data off
Confidence Interval
You want these to be small and narrow
Informed Consent
The process of communication between a physician and patient that results in the patients authorization to undergo a specific medical intervention
Negligence
Deviation of the standard of care
Leads to malpractice
Battery
When consent was not given from the patient but you went ahead with the intervention anyways
What came from the patient self determination act
Patients have the right to:
- participate in their healthcare decisions
- accept or refuse treatment
- prepare an advance directive
- receive clear information
Implied Consent
In times of emergency where they patient cannot respond and has no one to speak on his behalf
3 Elements of Informed Consent
Preconditions
Information
Consent
How much information needs to be given to a patient
Enough information that a typical physician would give and enough information that the average patient would need to know in order to make an informed decision
Trends of US Health Care
We spend the most on medical treatment and research yet do not have the best outcomes
Ranked 30th in infant mortality
Of all developed countries, we have the lowest percentage of people without insurance
How do health disparities/inequalities arise
Differences in quality of care
Differences in access to care
Differences in lifestyles
Barriers to Access of Care
Number of PCPs Financial Geographical Cultural Language barriers
What is the Gap
The gap is people who do not have health insurance. They live in states where medicaid was not expanded after the ACA was signed and do not make enough to apply for insurance through the exchange
Which group of people are most likely to not have health insurance
Young working people
4 Rules to Equity Based Care
Make SDH number 1 priority
Ensure a respectful, empowering clinic
Tailor care to the population served
Deal with cultural competence
Apoptosis
Programmed cell death that involves a series of biochemical rxns leading to chromatin condensation and blebbing
Things that cause apoptosis
Natural causes
Mutation in DNA
Loss of organelle function
Necrosis
Cell death that is less orderly and due to stress from infection/pathogens
The cell leaks and causes inflammation
Examples of irreversible cell injury
Pathogens Membrane defects Calcification of mitochondria ROS Lots of DNA mutations
Examples of reversible cell injury
All the -trophy's hydropic swelling Mitochondria swelling Lack of ATP Etc
Eosinophila
Common in necrotic cells
They stain very red because of their denatured proteins
Karyorrhexis vs Karyolysis
Destruction of the nucleus
Dissolution of the DNA
Coagulative Necrosis
The most common
Occurs from hypoxia
Liquefactive Necrosis
When the rate of destruction is higher than the rate of repair
Forms abscesses which are balls of puss/bacteria
Caseous (Cheesy) Necrosis
Necrosis associated with mycobacterium (Tb)
Can spread very rapidly if left untreated
Fat Necrosis
Occurs in adipose tissue and is often associated with pancreatitis
Forms calcium deposits from saponification of the lipids
3 Steps to RNA Processing
5’ Cap
3’ Poly A Tail
Splicing
What function does the 7 methyl Guanosine cap have
It helps to stabilize
It is the binding side of the ribosome
It helps recognize mRNA as self
What function does the poly A tail have
It helps to stabilize
It helps to increase translation efficiency
What contributes to the reason that 1 gene can produce many different forms of a protein
Alternative splicing
Produces multiple forms of the same protein but they each have a particular function
How does mutated SMN1 lead to SMA in infants
SMN1 interacts with snRNPs and is needed for their assembly
This leads to lower snRNP levels so splicing gets all messed up leading to malfunctioning proteins
snRNPs
Critical for splicing
Have 5 parts to them:
-U1 and U2 bind to the splicing sites
-U4, U5, and U6 then join to form the spliceosome
KLF6 Splice Variant
KLF is a tumor supressor gene
This SV binds to the same DNA region as normal KLF but does not carry out any function. It just blocks KLF from binding. Called a dominant negative SV
List the Types of RNA
mRNA- used to create proteins rRNA- used to make up the ribosomes tRNA- used to bring aa to ribosomes snRNA- used to assemble the snRNP miRNA- used to regulate translation
What is the cap binding complex
eIF4F. Has three different parts to it
It recruits the ribosomes and make sure only mRNA is translated
It gets elevated in cancers
eIF4F
eIF4E- The limiting factor. Actually binds to the cap
eIF4A- has helicase activity to straighten the RNA
eIF4G- a large scaffold protein that interact with poly A tail
Strong vs Weak mRNAs
Strong mRNAs do not need a high level of eIF4E to be translated (housekeeping proteins)
Weak mRNAs do need high levels of eIF4E to be translated (sensitive proteins)
Most mRNAs are strong
Steps to the Initiation of Translation
- eIF4F binds to the cap
- eIF2 loads met-tRNA onto the small sub unit
- eIF1 and 3 interact with cap binding complex to bring in the ribosome
- Small ribosome scans until it locates AUG
- Large subunit binds and elongation occurs
PAPB
Binds to the poly A tail and interacts with eIF4G to improve translation
eIF2 Role in Elongation
It is responsible for translocating the ribosome
Requires GTP
Diptheria toxin inhibits this process
eIF1 Role in Elongation
It is responsible for bringing the tRNA to the ribosome
Requires GTP
PERK Kinase
Becomes activated when there are lots of unfolded proteins present
It phosphorylates eIF2 making it inactive which means the ribosome cannot move
Walcott Rallison Syndrome
A mutation in the PERK gene. Leads to lots of unfolded proteins causing cell death
Diabetes arises very early in these patients
miRNAs
Short nucelotide fragments (20-22) that bind to specific mRNAs preventing their translation
Changes to the Nuclear Envelope During Mitosis
At interphase the lamins and linker proteins are present
At prophase they begin to collapse
At telophase they begin to reform around daughter DNA
How do small and large molecules get into the Nucleus
Through the nuclear pores
Small ones can diffuse. Large ones require active transport using RAN proteins
In the cytosol there is lots of GDP. In the nucleus there is lots of GTP
It uses this gradient to transport cargo into and out
Ran GEF and Ran GAP
Ran GEF is in the nucleus and swaps out a GDP for GTP
Ran GAP is in the nucleus and hydrolizes the GTP to GDP
How are proteins shuttled into the ER
Co Translationally (most common) Post Translationally Both are done by the proteins having signal sequences
What modification to the proteins occur in the ER and Golgi
In the ER glycosylation first begins and there are also chaperones to help with folding
In the Golgi glycosylation continues along with sulfation
Clathrin Process
Used for endocytosis
When a ligand on the outside binds to its receptor the inside part binds AP2. This pulls down the area and recruits clathrin which surrounds and forms the vesicle
COPI and COPII Process
When a ligand binds its receptor a GEF protein called Sec12 places a GTP on Sar1 which activates it. This then recruits COPI or COPII which will form the vesicle
Explain How a Vesicle Gets to Its Right Location
The routing is controlled by a specific Rab protein. Has to have a GTP on it
On the vesicle there a V snares that interact with the t snares to form fusion
Tethering–> Docking–> Fusion
Where is clathrin, COPI, and COPII used in the cell
Clathrin is used for endocytosis of plasma membrane
COPI is used by golgi
COPII is used by the ER
Explain How a Protein Gets Into Mitochondrial Outer Membrane
Cytosolic chaperones present it to TOM
It then goes to SAM where it will get incorporated
Explain How a Protein Gets Into Mitochondrial Inner Membrane
Cytosolic chaperones present it to TOM
It then goes to TIM22 and then TIM23 where it will get incorporated
Explain How a Protein Gets Into Mitochondrial Matrix
Cytosolic chaperones present it to TOM
It then goes to TIM22 and then PAM where it will get incorporated into the matrix
What causes the release of the ligand from the receptor in the endosomes
Endosomes have lower pH so this lowers the affinity
Salmonella Endocytosis
They have evolved a surface protein that will bind to one of our receptors and become endocytosed by the cell where it can live/replicate/avoid the immune system
Listeria (monocytogenes) Endocytosis
They have evolved a surface protein that will bind to one of our receptors and become endocytosed by the cell where it can live/replicate/avoid the immune system
They also have tails that can hydrolize actin and use this to move from cell to cell
Anitgen Presentation
An Ag that has been bound by Abs will bind to macrophages. This will then get endocytosed and the Ag protein will get degraded
After this it will then move to the membrane where it will be presented to other immune cells
Exonucleases vs Endonucleases
Exonucleases attack from either the 5’ or 3’ and degrade one by one
Endonucleases attack at a specific sequence and and create two shorter fragments
Exosome
Exonuclease that degrades from the 3’ end
Scleroderma is an autoimmune disease that attacks them. It is lethal
5-fluorouracil is a drug that inhibits them
XRN1
Exonuclease that degrades from the 5’ end
How Cells Fix Stalled Translation
There is a hairpin that is blocking the ribosome from moving forward
An endonuclease cuts at the area creating two mRNAs and then XRN1 and exosomes degrade it
How Cells Fix Early Stop Codons
An endonuclease cuts at the area creating two mRNAs and then XRN1 and exosomes degrade it
This is common in T cell Receptors
How Cells Fix if There is No Stop Codon
The problem is at the 3’ end so only the exosome is used to degrade this mRNA
Explain How miRNA can be Used for Degradation of mRNA
A protein recognizes when miRNA binds to mRNA and this can lead to two things
It can be place in a p body or it can be degraded
How Cells Degrade Normal mRNAs
They first remove the poly a tail and 5’ cap
Then an XRN1 degrades from the 5’ end
How are short lived proteins degraded
How are long lived proteins degraded
Short are done by proteasome (have to be tagged with ubiquitin and requires ATP)
Long are done by lysosome (can be autophagy or chaperone guided)
How Cells Degrade Oligosaccharides
They are done in the lysosome
They first get taken off the protein and are degraded by glycosidases
How Cells Degrade Lipids
They get sent to the lysosome where there are lipases
The FAs can then be used for fuel/signaling/inflammation
