Unit 2 - Week 1 - Amberg 1, Amberg 2, Shrimpton, Chang Flashcards
What are the three types of Creutzfeld-Jacob disease?
- sCJD - sporadic CJD, no known cause, 85-95% 2. Familial CJD, fCJD, inherited genetic risk, 7-10% 3. Iatrogenic - iCJD, exposure during medical procedures, <1%
What is one main difference between CJD and Alzheimer’s Disease?
Rapid deterioration in CJD, complete dementia by ~6 months, death by ~1 year (progression is slightly slower in vCJD)
What are two cardinal manifestations of CJD?
Myoclonus (twitching), provoked by startling Rapid and progressive mental deterioration *but patient does not have to have both sets of symptoms at the same time
Prions are very dangerous because? Give 2 reasons.
- No immune response is raised against them 2. They are resistant to just about every degradation process
What is the protein-only hypothesis?
In the ‘protein only’ hypothesis, prion diseases are thought to result from the conformational change of a normal isoform of a prion protein (PrPC) to a protease-resistant, pathogenic form called PrPSc.
Why do AD, Parkinson’s and HD pose a challenge to the protein-only hypothesis?
Those diseases also result in misfolded proteins that cause aggregates/result in neurodegeneration. Skeptics argue that these diseases are not infectious, therefore misfolded protein alone is not enough to transmit disease
What is the conformational change involved in the normal to pathogenic version of the PRP gene?
PRPc (alpha-helical) *normal* –> PRPsc (beta-sheet) *pathogenic* sc stands for “scrapie form”
What are some general functions of the PRPc gene?
It is involved in maintaining the brain’s white matter, regulating innate immune cells, responses to oxidative stress, and neuron formation.
What are two ways you can obtain PRPsc?
- Mutation of your PRPc 2. Exogenous source - meat, blood
About how many types of human prions are there?
4, as proven by Western Blot analysis. Type 1 = sporadic CJD **PRPsc type 4 is seen uniquely in vCJD**
What kind of brain disease is caused by CJD?
A spongiform encephalopathy
Explain the model for prion self replication.
The PRPsc protein seed attaches itself to a normal protein PRPc, and converts it to a PRPsc, the nucleus of the proteins grows and becomes an amyloid fiber, then the fiber breaks and liberates new bits which allows for the dissemination of infectious material.
What is the only way to diagnose sCJD?
Brain biopsy, can also use detection of 14-3-3 protein in CSF, abnormal MRI or characteristic EEG pattern of periodic synchronous bi-or triphasic sharp wave complexes (PSWCs).
In what what age group is sCJD most common?
>65 years most common, slight majority of females noted
What kind of CJD represents bovine to human transmission?
vCJD, variant CJD, typically transmitted through CNS, retina, bone marrow, distal ileum and trigeminal and paraspinal ganglia. *Muscle and milk do not contain BSE *if* the cow was butchered properly.*
What is one clinical difference between vCJD and sCJD?
vCJD has a slightly slower progression than sCJD, but still very fast as compared with AD. Also, unique to vCJD has “prominent involvement of lymphoreticular tissues” aka peripheral pathogenesis, ie tropism in tonsils
14-3-3 protein is a sensitive marker for which CJD?
sCJD, but not vCJD
What kind of EEG pattern is associated with vCJD?
Slow wave pattern, not the same as sCJD (no PSWCs)
Describe the microscopic findings of a brain biopsy in sCJD.
Diffuse staining with vacuoles
Describe the microscopic findings of a brain biopsy in vCJD.
PrP plaques with rings of vacuoles surrounding an amyloid core, this core stains darker
What is the mean age of onset of vCJD?
Range is 11-74, mean is 29
Is sCJD transmittable from person to person?
No; but vCJD can be transmitted via blood transfusion or via contaminated meat.
Has there been any medication successful in treating CJD?
Prp13 is a peptide that can break beta sheet formation, and has been shown to delay symptom onset in mice, but so far we have no tx for this. Anti-PRP monoclonal antibodies have been shown to reduce PRPsc levels and prion infectivity in scrapie models. Viral vector with anti-PRP antibody encoded in it was shown to delay pathogenesis in mice.
What is really the only way to break down prions?
Soak in NaOH for at least an hour and autoclave for at least 1 hour.
Why do some experts suggest that AD is an infectious disease?
Mice with PRPc deletion do not develop AD, however mice injected from brain tissue of AD patients will develop AD, suggesting that on some level, the misfolded amyloid-B a prion.
How would you denature dsDNA for a hybridization experiment?
High temp or high pH
In nucleic acid hybridization, a slightly lower temperature than it takes to reanneal ssDNA produces what?
Imperfectly paired dsDNA
5’-GGATCC-3’ might be recognized by what?
A restriction enzyme (bacteria use to cut up foreign/unmethlyated DNA). They frequently recognize palindromic sequences, meaning sequences that read the same in both 5-3 directions/strands.
What is a restriction map?
A restriction map is a when the genome of a particular organism is “landmarked” by specific restriction sites. This feature is also used (in forensics) to identify differences in lengths of RFLP’s in humans.
What is a common result of restriction enzyme cutting?
5’ or 3’ overhangs in the DNA, deemed “sticky ends” are common results of restriction enzyme cutting, which can be useful in DNA cloning. NB: Blunt ends work poorly, and sticky-end ligations are best when the overhangs are complementary.
In what direction does DNA try to move in gel electrophoresis?
The phosphodiester bond makes the DNA very negatively charged, thus it migrates toward the anode in the gel when the voltage is applied. The smaller DNA fragments move faster and thus separate from the larger fragments.
How can you see the DNA on the gel after electrophoresis?
- Use Ethidium bromide to bind to DNA and then it will fluoresce under UV light, or 2. The DNA has already been labeled with a radioisotope and will be exposed on X-ray film or phospho-imager.
What kinds of gels are used in gel electrophoresis and for what size DNA are they intended?
- Agarose - from seaweed - for large DNA molecules (500 bp - a few mega bp’s) 2. Acrylamide - forms tight matrix - for small DNA molecules (25-1000 nucleotides) *Bear in mind that DNA can be ds or ss.
A Southern blot is used for:
DNA
A Northern blot is used for:
RNA
A Western blot is used for:
protein
What are the steps in Southern blotting?
- Gel electrophoresis 2. Add strong base to denature DNA 3. Transfer to nitrocellulose paper –DNA is now immobilized on the paper and read to hybridize 4. Add probes with sequence of interest. NB probes usually contain radioactive 32-P. 5. Wash off excess stuff 6. Visualize on X-ray film or phospho-imager screen.
What is the enzyme used to join DNA overhanging sequences created by restriction enzymes?
Typically T4 (bacteriophage) DNA ligase, especially for bacterial cloning of human DNA.
What is the smallest type of vector used in molecular cloning?
Plasmid, holds up to 15kb, used for genomic or cDNA, insert into bacteria or yeast. If cloning human stuff, cDNA is the way to go because of the size limit you work with.
What are the largest types of vectors used in molecular cloning?
BAC’s and YAC’s. Bacterial artificial chromosomes, used in bacteria, are genomic chromosomes that can hold 100-300 kb’s. Yeast artificial chromosomes, used in yeast, are genomic chromosomes that can hold 100-2000 kb’s.
How are F1 plasmids inserted into bacteria during cloning experiments?
Transformation, same process as with bacteriophage lambda cloning.
In bacterial cloning experiments, why do researchers keep the antibiotic-R gene?
By plating the bacteria onto the agar with the antibiotic, the non-transformed bacteria will be killed, leaving only the bacteria that picked up the DNA you want to clone.
The virus bacteriophage lamba naturally carries genes for what?
Amplification of its genome Synthesis of coat proteins DNA packaging molecules **Non-essential regions can be removed and replaced by DNA fragment of interest**
What kind of virus is bacteriophage lambda?
45kb, dsDNA, linear, hijacks enzymes to replicate via *rolling circle replication*
What is a cosmid?
A cosmid is a gutted version of bacteriophage lambda, leaving only sequences required to replicate and package DNA. In this way, you can insert a bit more DNA (genomic) into the cosmid than you can for a regular bacteriophage lambda for cloning experiments.
What is the most useful and stable way to maintain large pieces of human genomic DNA in cloning experiments?
BAC
What is one major drawback of using YAC’s for cloning?
Yeast have very high rates of homologous recombination, which can lead to the scrambling of the DNA of interest. A lot of human DNA has Alu and LINE sequqnces, and those are apt to recombine.
How do you construct a YAC library?
By taking the two YAC arms, and keeping them separated (telomere and markers on each end), insert lots of human genomic DNA), and then ligate to the YAC arms, and transform into yeast. Each yeast will have a different piece of human DNA, thus creating the library.
