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Describe the four types of mutations causing activities. |
1. tautomeric shift (TH: keto -> enol) (AD: amino -> imino)
2. ionizing radiation (xray, gamma ray -> one or two strand break)
3. uv radiation (pyrimidine dimers)
4. chemicals
- base analogs (5BRU)
- free radicals (O^3)
- deamination (ADENINE -> hypoxanthine)
- alkylation (methylbromide, ethylene oxide)
- intercalating agents (proflavin) |
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Describe some of the DNA repair mechanisms. |
1. BER (incorrect base at a specific bp)
2. NER (lesions in the genome - could be small regions or entire chromosomes)
3. AP endonuclease (baseless / apurinic sites)
4. Post replication mismatch repair
5. 3' -> 5' exonculease activity (transcription - gene repair)
6. Repair of double-strand breaks
- homologous (good)
- non-homologous (bad, adds more mutations) |
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DNA glycosylase is specific for cleaving this mutation. |
C -> U deamination -- specifically cleaves the uracil base but leaves the glycosidic bond in place but this too must be removed |
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If a base has been removed during the repair mechanism phase, what enzyme comes along and cleaves the glycosidic link? |
AP endonuclease |
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How do the repair mechanisms distinguish an old strand from a new one? |
Bacteria: The old strand has either pyrimidine dimers or has certain bases methylated
Humans: The new strand is the one with the nick and therefore mismatched base |
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This base is a mutational hotspot. |
5-methyl-cytosine --- it is deaminated to thymine which causes an incorrect bp to form
C -> G [This no longer happens]
T -> A [This now happens after several replications] |
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What are four DNA repair defects and their diseases? |
1. Xeroderma Pigmentosum
- autosomal recessive
- defect of genome-wide NER
2. Cockayne Syndrome
- autosomal recessive
- defect of transcription-coupled NER
3. HNPCC (non-polyposis colon cancer)
- autosomal dominant
- defect post-replication mismatch repair
4. Ataxia-telangiectasia
- autosomal recessive
- defect in PK involved in (non)-homologous end joining |
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Most DNA is 5 - 7% _______________.
Why is this important? |
Negatively supertwised
This favors unwinding via Type I (swivelase) as it doesn't require ATP |
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Topoisomerase Type II is the target of _____________ and _______________. |
Antibiotics and chemotherapy |
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What are some of the inhibitors of Type II Topoisomerase?
How do they work? |
Ciprofloxin (prokaryotes)
Doxorubicin (eukaryotes)
The inhibitors cause strand breakage in the chromosome |
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What is the major functional difference between DNA polymerase I and III?
Why is this? |
DNA polymerase III starts from where RNA primer left off and finishes to the end
DNA polymerase I remove the RNA primer and lays down DNA in its place
- It is the only one to have 5' -> 3' exonuclease activity |
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UV damage and repair is initiated by this complex. |
uvrABC
- Make nicks before and after the area of damage |
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What are snRNA and what is their function? |
snRAN are small RNA molecules found in the nucleus.
They are important in RNA splicing and telomere maintenance.
They are referred to as SNRNP (snurps) = spliceosome complex |
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What are the components of the RNA polymerase holoenzyme? |
alpha, beta, beta prime, and sigma |
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Which component of the RNA polymerase first bind to the Shine-Dalgarno sequence? |
Sigma subunit |
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What are the two types of transcription terminal signals?
Which one is used in emergency situations (e.g. cell low on energy)? |
1. GC stem loop
2. Rho
- This is used in emergency situations as Rho can race down the mRNA strand and create a hairpin loop |
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Name two elongation factors tin both prokaryotes and eukaryotes where energy is spent. |
EF-Tu (pro) & eEF-1A (euk) = bind all aminoacylated tRNAs, GTPase
EF-G (pro) & eEF-2 = translocation, GTPase |
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Achondroplasia (ACH) is a form of dwarifism. The mutation which causes this disease is a ______ which in turn causes this change to happen? |
Single nucleotide polymorphism
- The codon for glycine (GLY) becomes changed and instead codes for arginine (ARG) |
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Restriction enzymes can leave two different types of ends. What are they and which one is better for integrating with a genome? |
Blunt and sticky ends
- Sticky ends aid in genome integration because they have 1 or more bases protruding from each end |
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What are the steps and raw supplies needed for PCR?
Discuss what is needed at each step of the way. |
* Require double stranded DNA
* Require two different RNA primers, one for each strand
Step 1: Heat solution to denature/de-anneal DNA strands
Step 2: Add RNA primer to the solution
Step 3: Cool down the solution to allow annealing of primer to DNA
Step 4: Increase temperature to allow thermo-stable polymerase to operate efficiently & optimally; don't forget the dNTPs
[REPEAT] |
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Which of the electrophoresis gels has a loose mesh and which one has a tight mesh? |
Loose mesh: Agarose
Tight mesh: Polyacrylamide/bis-acrylamide |
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What is the major difference between gel electrophoresis and pulsed field electrophoresis? |
Pulsed field electrophoresis can resolve a much larger size molecule.
This has definite uses in a hospital where it can be uses to track down different strains of illnesses. |
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High stringency of hybridization is made possibly by |
* Increased temperature
* Decreased salt
For short probes:
* Decreased GC content
* Decreased length |
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What are the different kind of blots and what do they target? |
SNOW
DROP
Southern: DNA
Northern: RNA
Western: Protein
Dot Blot: DNA or RNA |
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True or false.
A Northern blot can use DNA or an oligo as a probe. |
True |
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A cancer cell is reacting differently to a newly designed drug. You want to measure the level of transcription of a gene encoding a multidrug resistance transporter.
Which blotting method would you choose? |
Northern |
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A cloning a glycoprotein will require the use of this cell line. |
Eukaryotic |
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What is the difference between polygenic and multifactorial genetic disorders? |
Multifactorial genetic disorders also include environmental causes into their understanding of the disease |
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General transcription factors are the sites of ____________ |
Promoters (CAAT, TATA, ...) |
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What are three ways that genomics aids in medical practice? |
1. Identifying optimal patient-centered therapeutic dosages
2. Identifying optimal-patient centered therapies
3. Improvement of patient-centered long-term risk assessment |
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Are ALU repeats SINEs or LINEs? |
SINEs |
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How are minisatellites and microsatellites used in the world? |
Minisatellites: DNA fingerpriting
Microsatellites: Tracking individual polymorphisms in individuals of a family -- establish kinship/parentage |
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Chromatin is made active by?
Chromatin is made inactive by? |
* HAT (histone acetyl transferase) adds acetyl groups to lysine and thus 'opening up' DNA
* Histones can have methyl groups added to lysine and arginine
* Histones can phosphorylate serine, threonine, and lysine
- This increases rates of transcription and replication
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Histones can be deactivated by HDAC (histone de-acetylation complex)
- This decreases transcription and replication rates |
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What are four types of DNA binding proteins?
Where do they bind to on the chromosome? |
1. Helix Turn Helix
2. Helix Loop Helix
3. Leucine Zippers
4. Zinc Fingers
*** They all bind to the major groove of the chromosome |
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The toll-like receptor found on human immune cells senses this on bacterial cells. |
LPS |
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What are three ways by which prokaryotes transfer genetic information? |
1. Conjugation (F Plasmid)
2. Transduction (Viral infection)
3. Transformation (Pick up DNA from environment) |
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What is transcriptional silencing? |
The hypermethylation of promoter CG islands by some cancers. |
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Which of the following mutations occurs at the promoter region and which acts farther away?
Cis-acting
Trans-acting |
Cis-acting mutations occur at the promoter region |
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On a eukaryotic mRNA strand, what is the sequence of protein bindings and ribosome activation? |
* TFIID binds to TATA
- TBD is a subunit of TFIID
* IIA, IIB, and IIH bind to TFIID
* IIH phosphorylates RNA Polymerase II
* RNA Polymerase II begins transcription |
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Translation initiation is dependent on these factors. |
elongation factors (eEF-2, eEF-1a) |
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What toxin directly effects elongation factor 2 and disables protein translation? |
Diphtheria toxin |
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