Unit 2 - Week 1 - Amberg 1, Amberg 2, Shrimpton, Chang Flashcards
(121 cards)
1
Q
What are the three types of Creutzfeld-Jacob disease?
A
- 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%
2
Q
What is one main difference between CJD and Alzheimer’s Disease?
A
Rapid deterioration in CJD, complete dementia by ~6 months, death by ~1 year (progression is slightly slower in vCJD)
3
Q
What are two cardinal manifestations of CJD?
A
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
4
Q
Prions are very dangerous because? Give 2 reasons.
A
- No immune response is raised against them 2. They are resistant to just about every degradation process
5
Q
What is the protein-only hypothesis?
A
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.
6
Q
Why do AD, Parkinson’s and HD pose a challenge to the protein-only hypothesis?
A
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
7
Q
What is the conformational change involved in the normal to pathogenic version of the PRP gene?
A
PRPc (alpha-helical) *normal* –> PRPsc (beta-sheet) *pathogenic* sc stands for “scrapie form”
8
Q
What are some general functions of the PRPc gene?
A
It is involved in maintaining the brain’s white matter, regulating innate immune cells, responses to oxidative stress, and neuron formation.
9
Q
What are two ways you can obtain PRPsc?
A
- Mutation of your PRPc 2. Exogenous source - meat, blood
10
Q
About how many types of human prions are there?
A
4, as proven by Western Blot analysis. Type 1 = sporadic CJD **PRPsc type 4 is seen uniquely in vCJD**
11
Q
What kind of brain disease is caused by CJD?
A
A spongiform encephalopathy
12
Q
Explain the model for prion self replication.
A
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.
13
Q
What is the only way to diagnose sCJD?
A
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).
14
Q
In what what age group is sCJD most common?
A
>65 years most common, slight majority of females noted
15
Q
What kind of CJD represents bovine to human transmission?
A
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.*
16
Q
What is one clinical difference between vCJD and sCJD?
A
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
17
Q
14-3-3 protein is a sensitive marker for which CJD?
A
sCJD, but not vCJD
18
Q
What kind of EEG pattern is associated with vCJD?
A
Slow wave pattern, not the same as sCJD (no PSWCs)
19
Q
Describe the microscopic findings of a brain biopsy in sCJD.
A
Diffuse staining with vacuoles
20
Q
Describe the microscopic findings of a brain biopsy in vCJD.
A
PrP plaques with rings of vacuoles surrounding an amyloid core, this core stains darker
21
Q
What is the mean age of onset of vCJD?
A
Range is 11-74, mean is 29
22
Q
Is sCJD transmittable from person to person?
A
No; but vCJD can be transmitted via blood transfusion or via contaminated meat.
23
Q
Has there been any medication successful in treating CJD?
A
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.
24
Q
What is really the only way to break down prions?
A
Soak in NaOH for at least an hour and autoclave for at least 1 hour.
25
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.
26
How would you denature dsDNA for a hybridization experiment?
High temp or high pH
27
In nucleic acid hybridization, a slightly lower temperature than it takes to reanneal ssDNA produces what?
Imperfectly paired dsDNA
28
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.
29
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.
30
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.
31
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.
32
How can you see the DNA on the gel after electrophoresis?
1. 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.
33
What kinds of gels are used in gel electrophoresis and for what size DNA are they intended?
1. 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.
34
A Southern blot is used for:
DNA
35
A Northern blot is used for:
RNA
36
A Western blot is used for:
protein
37
What are the steps in Southern blotting?
1. 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.
38
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.
39
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.
40
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.
41
How are F1 plasmids inserted into bacteria during cloning experiments?
Transformation, same process as with bacteriophage lambda cloning.
42
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.
43
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\*\*
44
What kind of virus is bacteriophage lambda?
45kb, dsDNA, linear, hijacks enzymes to replicate via \*rolling circle replication\*
45
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.
46
What is the most useful and stable way to maintain large pieces of human genomic DNA in cloning experiments?
BAC
47
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.
48
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.
49
What is a library?
A large collection of recombinant DNA clones. There are two types of libraries: genomic and cDNA
50
The abundance of a clone in a cDNA library reflects:
The expression level in the original tissue. If the gene you are looking for is expressed at low levels in the tissue used to isolate the RNA, it will be underrepresented in the library and difficult to isolate.
51
What are the steps in creating a cDNA library?
1. Lyse cells and purify mRNA from tissue. 2. Hybridize with poly-T primer (to join with poly-A primer in original strand) 3. Use reverse transcriptase to make complementary strand of DNA attached the the mRNA. 4. Dissolve mRNA with alkali, NB remaining DNA will form a 3' hairpin loop 5. Hairpin loop acts as primer, use DNA polymerase to make complementary strand 6. Treat with ssDNA nuclease to cleave hairpin loop, end with ds cDNA!
52
What are the common steps in screening a library with a radioactive probe?
1. Add nitrocellulose paper to copy the colonies and fuse them for denaturing. 2. Lyse bacteria and denture DNA with alkali 3. Add radioactive DNA probe 4. Probe hybridizes to DNA of interest 5. Expose with X-ray film
53
What kind of DNA polymerase is best for PCR reactions?
DNA Polymerase from thermophilic micro-organisms that can withstand repeated treatments at 95C (just below boiling), ie Taq polymerase
54
Why is PCR so awesome?
Only the region between the primers is amplified each cycle. If the reaction was 100% efficient, it would create 2^# of cycles of DNA of interest.
55
What is the purpose of a "real time" PCR machine?
A real time PCR machine uses fluorescence to quantify product production at each step of the reaction. The resulting curve (Amt vs. # cycles) allows you to find the amount of original template, and the efficiency of the process.
56
What is multi-plex PCR? Provide one clinical application.
Multi-plex PCR is when you fo PCR with a number of different primers, for example to identify and hybridize to particular alleles, and run the reaction all at once. As long as these primers can all be separated by gel electrophoresis, the experiment can test for multiple alleles of a disease gene. Example is looking for many different exons that can be deleted in the dystrophin gene in diagnosing versions of MD.
57
What is the function of dystrophin?
Dystrophin helps to stabilize the sarcolemma of muscle cells.
58
True or False: PCR can be used to detect pathogen genes in a host.
True
59
How would you test for HIV using PCR?
1. Take blood sample from pt 2. Remove cells by centrifugation 3. Extract viral genome from centrifugated stuff 4. Use reverse transcriptase because HIV is an RNA virus (retrovirus), make DNA 5. PCR amplification 6. Run gel \*\*called RT-PCR (reverse transcriptase PCR)
60
In a Western Blot, indicate what the purposes of SDS and mercapoethanol are.
SDS denatures the protein and provides a uniform negative charge for running on the gel. Mercaptoethanol dissolves disulfide bridges.
61
What questions can you answer with a Northern blot?
1. Is the mRNA for a particular gene present? 2. What is the expression level of the mRNA? 3. Is the mRNA the correct size?
62
When would you want to perform non-denaturing gel electrophoresis on a protein?
When you are interested in seeing if there is a change in charge from WT/expected, for example in sickle cell anemia. Skip the heating, SDS and mercaptoethanol. Protein separation is then based on size AND charge.
63
What is isoelectric focusing?
A technique used to separate proteins by charge only. Set up a pH gradient and run voltage, and proteins will settle at their pI, or isoelectric point, where they have a net charge of 0.
64
What is two-dimensional gel electrophoresis?
2D-gel-e is when you start with isoelectric focusing to separate the proteins by charge, and then you run the gel to separate by size.
65
The antibody specific to a protein you wish to work with is called the \_\_1\_\_. Most Western assays require a \_\_2\_\_ that will \_\_3\_\_.
1. primary antibody 2. secondary antibody 3. bind to the primary antibody's constant region and fluoresce.
66
What is a common enzyme that is bound to a secondary antibody for fluorescence?
HRP, horseradish peroxidase. Bathe in substrate that causes HRP to fluoresce, then expose to film to observe.
67
Western assay tells you: \_\_1\_\_ and \_\_2\_\_
1. Is a protein present? IE in DMD, no dystrophin being made 2. Is a protein the right size?
68
Immuno-fluorescence microscopy tells you:
Does a certain protein have proper cellular expression and localization? Is the protein there at all?
69
What are the steps in performing immuno-fluorescence microscopy?
1. Obtain tissue section 2. Incubate sample with primary Ab 3. Wash with secondary Ab (w/ tag) 4. Put sample in microscope and view at excitation wavelength for tag, location of protein is visualized
70
DNA micro-arrays are used to \_\_1\_\_ analyze the \_\_2\_\_ of many thousands of \_\_3\_\_ in a collection of \_\_4\_\_ or \_\_5\_\_.
1. Simultaneously 2. expression level 3 genes 4. cells 5. tissue
71
Cluster analysis of DNA micro-array experiments is particularly helpful in understanding:
Cancer gene activity, related genes to cancers (ie lung cancer), grouping by gene type and consequent severity
72
RNAi and morpholinos can be used to \_\_1\_\_ the expression of genes in human cells.
1. down-regulate
73
Gel electrophoresis for DNA tells you:
1. Presence 2. Size 3. Quantity of DNA
74
What is accomplished by GFP fluorescence?
GFP is fused to a protein of interest, in situ, which can be observed in cellular processes.
75
True or False: PCR can be used to clone genes.
True!
76
What would provide the greatest sensitivity for detecting specific nucleic acid sequences?
PCR
77
True or False: Indirect immunofluorescence is the best way to determine protein expression levels.
False.
78
What is the cause of Fragile X Syndrome?
Repeat expansion of CGG, when it exceeds ~200 repeats, the whole area gets methylated, including the FMR1 promoter and the FMR1 gene is silenced as a result
79
What is the MOI of Fragile X Syndrome?
Roughly, a X-linked dominant disorder with reduced penetrance. Girls have it but not enough to make it fully dominant.
80
The risk of having a child with FRAXA is in the children of premutation \_\_1\_\_ and the \_\_2\_\_ of premuation \_\_3\_\_.
1. carrier females 2. grandchildren 3. males
81
Fragile X-related Tremor/Ataxia Syndrome (FXTAS) and premature ovarian failure (POF) occur in \_\_1\_\_ due to \_\_2\_\_ of the FMR1 mRNA.
1. premuation males and females 2. RNA toxic gain-of-function (excess)
82
Fragile X syndrome is the most common cause of \_\_1\_\_ mental retardation and is an example of a disease resulting from \_\_2\_\_.
1. inherited 2. dynamic mutations
83
Cystic fibrosis is a common autosomal \_\_1\_\_ disease with very significant \_\_2\_\_.
1. recessive 2. allelic heterogeneity.
84
Name some characteristics of Fragile X syndrome:
1. Severe mental retardation 2. Long face (mostly seen as child ages), large ears and head 3. Large testicles
85
Why does Haldane's rule not apply to Fragile X syndrome?
The mother has to be a premutation carrier.
86
How do you text for Fragile X syndrome?
1. PCR 2. Southern Blot Both have greater sensitivity than cytogenetic methods, but you cannot routinely do PCR across the full mutation because it will not replicate properly--it's too big
87
What is the Sherman paradox?
Aka anticipation or dynamic mutation, shows that the incidence of Fragile X syndrome increases with each passing generation.
88
At birth the \_\_1\_\_ globin gene replaces the \_\_2\_\_ globin gene, so that some diseases do not become apparent until after birth.
1. Beta 2. Gamma
89
What are Heinz bodies?
In the absence of beta globin chains, alpha globins for homotetramers which precipitate and cause destruction of the RBC, and eventually also destruction of the bone marrow.
90
Most beta globin gene mutations are:
Point mutations
91
Fetal Hgb has the formula:
a2g2
92
Embryonic Hbg has the formula:
a2e2, and 2 others
93
What is a symptom consequence of sickle cell anemia?
Hemolytic anemia, caused by rigid structure of HbS.
94
When is sickling most likely to occur?
Sickling occurs when there is low O2 concentration.
95
What is the clinical presentation of HbSHbC?
These individuals are compound heterozygotes and have one glut--\> val and one glut--\> lys, and have a slightly milder form of sickel cell anemia.
96
What is the most common source of alpha-globin mutations?
Unequal crossing over, resulting in misalignments and deletions of one or more of the alpha globin genes.
97
HbS and HbC both destroy what restriction site?
Mnl 1, which can distinguish between A and S/C, but not bewtween S and C.
98
What is the purpose of using Dde I digest in diagnosing sickle cell anemia?
Dde I digest distinguishes S from C and A
99
What are B4 (HbH) tetramers?
In alpha-thalassemia, where there is little or no alpha globin being made, beta globin chains create inclusion bodies.
100
Beta thalassemia is characterized by:
1. Reduced beta globin chain production
2. B+ is reduced
3. B0 is null
4. 100+ point mutations can cause this (allelic heterogeneity)
101
Gene expression in the alpha and beta globin gene clusters is controlled by common \_\_\_\_\_\_\_\_\_.
locus control regions. Mutations in the LCR can also cause thalassemias.
102
What is hereditary persistence of fetal hemoglobin (HPFH)?
HPFH can result from a deletion which brings downstream enhancers within the proximity of the fetal globin genes, or from a point mutation in the upstream regulatory region of the Agamma of Ggamma genes.
103
Where is alpha thalassemia particularly common?
Southeast Asia
104
Hydrops fetalis is the result of what condition?
Alpha thalassemia, where the fetus is --/--, and makes no alpha hemoglobin.
105
Individuals with alpha-thal trait can be what two genotypes:
a-/a- (Thalassemia type 2) or aa/-- (Thalassemia type 1)
106
What is Hb Constant Spring?
Hb Constant Spring is a hemoglobinopathy, and is the most common **non-deletional** alpha-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. It is the result of a point mutation (UAA to CAA) which changes a stop codon and produces an unstabe alpha chain mRNA is produced.
107
Fun fact!
Of the two (equal) alpha globin gene products, alpha 2 is expressed at twice the level as alpha 1.
108
What condition does a gamma-globin homotetramer cause?
Hb Bart's, formed early in on the fetus when the gamma globins have no alpha partners. Can be an indicator of any alpha-thalassemia
109
The persistence of \_\_1\_\_ globin expression can compensate for the lack of beta globin genes in patients who have beta thalassemia.
Gamma (alpha-gamma tetramers)
110
What is the most common mutation in CF?
delta F508
111
Name some symptoms of CF:
1. Pancreatic insufficiency
2. Chronic lung problems and infections (is pseudomonas)
3. Some males may be infertile
4. steatorrhea (excess fat in stool)
5. malnutrition
6. hepatic cirrhosis
7. intestinal obstruction
112
What is the most common current method of diagnosing CF?
PCR
Linkage analysis with intragenic markers are used when direct analysis is not enough
113
CF therapy depends on:
The class of the mutation!
1. Nonsense - no synthesis
2. Missense - block in processing
3. Missense - block in regulation
4. Missense - Altered conductance
5. Missense - reduced synthesis
114
What is Kalydeco?
It is a CF drug that increases the time the CFTR channel stays open in \*\*G551D\*\* mutated patients. Will it be helpful with other Class III mutations?
115
In dynamic mutations, genomic instability is mostly due to:
(mostly triplet) repeat expansions
116
Define "dynamic mutation."
The variation in copy number is in some way related to stability, such that the higher copy number, the more likely to expand, thus the mutability of a product of mutation is different from that of its precursor.
117
What kind of instabilities to dynamic mutations effect?
Meiotic and mitotic instabilities
118
What disease is associated with a triplet repeat, but shows no anticipation?
FRDA, Friedrich's Ataxia
Repeat is in an intron, which is thought to impact splicing.
119
When mutations occur in noncoding regions, name two pathologic outcomes:
1. Change gene dosing/disrupt regulatory elements
2. Alter splicing/stability of mRNA
120
How many beta genes does a normal person usually have?
2, 1 gene each on chromosome 11
121
True or False: Fragile X Syndrome is due to expansion of a CAG repeat.
False. It's actually a CGG repeat.
