Exam 1 Flashcards
(195 cards)
1
Q
what are the 3 parts that make up folate?
A
- pteridine ring (site of redox)
- PABA (used to make sulfa antibiotics)
- poly glutamate tail (cleaved to n=1 in brush border)
2
Q
list 3 conditions than can inhibit folate absorption
A
- crohn’s disease
- alcohol abuse
- celiac disease
3
Q
what is most of the folate in the body made from?
A
folate is the reduced form of tetrahydrofolate (FH4)
4
Q
what is the most oxidized form of FH4? most reduced form?
A
Oxidized: N^10-formyl-FH4
Reduced: N^5-methyl-FH4 (its hard to return to the oxidized state which is used for more reactions)
5
Q
what substrate is the major source of the one carbon pool?
A
serine (which can be made form glycolysis intermediates)
6
Q
how does FH4 relate to cell division?
A
it’s essential for purine synthesis which is needed for cell division.
7
Q
name 3 drugs that interfere with folate enzymes and what they do (in general).
A
- fluorouracil - (cancer drug) prevents conversion of dUMP to dTMP
- methotrexate - (cancer drug) prevents conversion of FH2 to FH4
- trimethoprim - (antimicrobial) inhibits dihydrofolate reductase (same as methotrexate?)
8
Q
what is the main dietary source of folate? B12?
A
folate - leafy greens
B12 - animal products
9
Q
what two major reactions require vitamin B12?
A
- homocysteine to methionine (requires B12 AND folate)
2. methylmalonyl-CoA to succinyl-CoA (just B12)
10
Q
what atom is found in the center of a vitamin B12 molecule
A
cobalt
11
Q
what causes pernicious anemia?
A
a deficiency in intrinsic factor (which binds B12)
12
Q
what is intrinsic factor used for?
A
it binds B12 and then binds receptors in the small intestine for absorption
13
Q
what is the schilling test used for? what are the steps
A
to test if a patient has pernicious anemia
- two doses of B12, on labeled, one not then collect urine
- dose of labeled B12 with intrinsic factor
- two weeks of antibiotics then test levels again
- take pancreatic enzymes for several days then labeled B12
(if one test is abnormal then move on to the next)
14
Q
what 2 molecules are used to make S-adenosylmethionine (SAM)?
A
- methionine (B12 used to make this)
2. ATP
15
Q
what does SAM do?
A
it adds a methyl group to nitrogen or oxygen atoms in many different reactions.
it becomes SAH after it does so.
16
Q
what is the methyl trap hypothesis?
A
when folate gets trapped in the reduced n^5-methyl-FH4 form which isn’t as useful as the oxidized form
17
Q
what are some potential causes of hyperhomocysteinemia?
A
problems with B12, folic acid or PLP (therefore high levels of homocysteine can be indicators of a deficiency in one of these)
18
Q
list 2 reasons why a folate deficiency is especially bad for pregnant women
A
- it can lead to neural tube defects in the developing fetus
- a folate deficiency can lead to inhibition of DNA synthesis
19
Q
what is megaloblastic anemia? what causes it?
A
enlargement of RBCs due to a decrease in synthesis of thymine and purine bases (folate needed) which leads to decreased DNA synthesis. the large RBCs don’t’ function properly leading to anemia
caused by a deficiency of folate and/or B12
20
Q
how many enzymes are needed for make IMP from ribose 5-phosphate? how many ATP are used?
A
11 enzymes
6 ATP
21
Q
what 2 molecules is IMP used to synthesize? what molecules are the nitrogen donors to their respective purine ring?
A
- AMP - aspartate is nitrogen donor
2. GMP - glutamine is nitrogen donor
22
Q
what 5 molecules are used to make purine? what does each contribute?
A
- formate - 2 carbons
- amide N from glutamine - 2 nitrogens
- aspertate - 1 nitrogen
- CO2 - 1 carbon
- glycine - 2 carbons, 1 nitrogen (whole molecule)
23
Q
what regulates the production of the purines AMP and GMP? what other purine biosynthesis enzymes are regulated and by what?
A
AMP and GMP inhibit their own synthesis
- PRPP synthetase - inhibited by (G/A)DP
- glutamine phosphoribosyl aminotransferase - inhibited by (G/A)MP through (G/A)TP
24
Q
what types of molecules can be salvaged for purines?
A
free bases, nucleosides and nucleotides
25
what is defective in lesch-nyhan syndrom? what is the result?
hypoxanthineguanine phosphoribosyltransferase is defective
the result is that purines are not salvaged and insted converted into uric acid (can lead to gout?)
26
what is the ultimate result of adenosine deaminase deficiency?
lymphocytes use the purine salvage pathway. if it can't get purines then it will result then the body won't be able to produce T or B cells (SCID?)
patient must be placed in a sterile environment
27
what type of molecule is IMP used for make? UMP?
IMP = purines
UMP = pyrimidines
28
how many enzymes are needed for make UMP from ribose 5-phosphate? how many ATP are used?
6 enzymes
2 ATP
29
what 3 molecules are used to make pyrimidine? what does each contribute?
1. glutamine - 1 nitrogen
2. HCO3 - 1 carbon
3. Aspartate - 3 carbons, 1 nitrogen
30
what is the 2 step process of pyrimidine salvage?
1. free bases converted to nucleosides via pyrimidine nucleoside phosphorylase
2. nucleosides converted to nucleotides via nucleoside kinases
31
describe the process for deoxyribonucleotide formation
1. ribose (NDP) converted to deoxyribose (dNDP) via ribonucleotide reductase (conversion of -OH on NDP to -H on dNDP)
2. nucleoside diphosphate is then converted to triphosphate
32
for the following molecules list whether they are made de novo or not.
1. pyrimidine
2. purine
3. deoxyribonucleotides
1. yup
2. yup
3. nope, made from ribonucleotides
33
what is the result of a dNTP deficiency? in excess?
deficiency - lethal due to inability to make DNA
excess - mutagenic, especially if one is a higher concentration than the others
34
what molecule are purines broken down to? what problems can it cause?
what about pyrimidines?
uric acid and then excreted in urine. can cause gout if uric acid is excessive
pyrimidine degradation does not cause problems
35
what are the 2 main causes of gout? which is more common? what is used to treat gout?
1. lack of excretion of uric acid [90% of the time]
2. overproduction of uric acid (purine degradation) [10%]
treated with allopurinol
36
what two enyzmes are defective in orotic aciduria? how do you treat it?
1. orotate phosphoribosyl transferase
2. orotidine 5'-decarboxylase
treated with oral uridine
37
how does the synthesis of purines differ from pyrimidines?
purines are build onto PRPP whereas with pyrimidines the base is made first and then PRPP is added
38
how many rings do the following molecules have?
1. purines
2. pyrmidines
which bases are which?
purines - 2 rings (A and G)
pyrimidines - 1 ring (C and T)
39
what tautomeric form of nitrogenous bases is the dominant form?
lactam (keto)
40
what type of bond would you find between a nitrogenous base and a sugar? would this bond normally be in the "syn" or "anti" position?
N-glycosidic bond
normally in the anti position (due to less steric hindrance?)
41
what substituent is attached to the 5' end of a phosphate backbone? what about the 3' end?
5' - phosphate
| 3' - free OH
42
what bond holds the phosphate backbone of DNA together?
phosphodiester bonds
43
which nitrogenous bases pairs with which? how many hydrogen bonds does each pair form?
A and T(U) = 2 H-bonds
G and C = 3 H-bonds
44
is DNA a right or left handed helix?
right handed (clockwise)
45
which base pair more stable? how does this relate to the melting (denaturation) temperature?
G:C
the more G:C content in DNA, the higher the denaturation point
46
how do the following molecule react to basic solutions?
1. DNA
2. RNA
1. DNA - mostly stable (at high levels is can cause denaturation)
2. RNA - breaks apart (basic solution attacks 2' OH and breaks the phosphodiester bond)
47
how many svedberg units are prokaryotic and eukaryotic ribosomes and their subunits?
prokaryotic - 70S (50S and a 30S)
eukaryotic - 80S (60S and a 40S)
(prokaryotes have odd first numbers and eukaryotes have even first numbers)
48
where would you find an anti-codon? what does it do?
on tRNA. its the part responsible for bringing in the amino acids being added to the polypeptide
49
what is Tm(subscript)? how do you calculate it? how does salt concentration affect Tm?
Tm is the point where 50% of the DNA is denatured.
Tm (in celcius) = 69.3 + 0.41(%G:C content)
salt concentration helps stabilize the solution and therefore raises Tm
50
what amino acids are most common in histones? how are histones charged?
lysine and arginine
positively charged
51
what is the function of the following histone types?
1. H1
2. H2A
3. H2B
4. H3
5. H5
H1 joins the nucleosomes (beads) together
all the others work together to form the nucleosomes
52
what is zidovudine (ZDV)? how does it work?
an HIV drug show to slow progression.
it acts as an inhibitor of HIV's reverse transcriptase preventing it from being able to add nucleotides to a 3' end
53
what is 5-fluorouracil? how does it work?
an anti cancer drug
it inhibits the enzyme thymidylate synthase which is responsabe for the production of deoxythymidine. without that calls can't make DNA
54
what is azithromycin? how does it work? are there any side effects?
an antibacterial drug.
it binds to the 50S ribosome subunit and prevents protein synthesis
however it can also sometimes bind to mitochondiral ribosomes
55
what is ciprofloxacin? how does it work?
an antibiotic
it binds/inhibits bacterial DNA gyrase
56
list the function of the following enzymes:
1. helicase
2. topoisomerase I
3. topoisemerase II
4. single strand binding proteins
5. DNA polymerase
6. Primase
7. Ligase
8. telomerase
1. helicase - unzips DNA strand
2. topoisomerase I - cuts one strand of DNA
3. topoisemerase II (gyrase) - cuts both strands to relieve supercoiling
4. single strand binding proteins - binds to the DNA as it unwinds to prevent re-association and enzyme degradation
5. DNA polymerase - synthesized DNA (also some have proofreading activity)
6. Primase - adds RNA primers upstream (polymerase needs 3' OH to function)
7. Ligase - joins strands together
8. telomerase - adds nucleotides (TTAGGG) to the 3' end
57
what is the conservation of DNA replication?
semiconcervative - one old strand and one new
58
what direction is the leading strand synthesized in? what about the lagging strand?
are they continous?
which eukaryotic polymerase is used at which strand?
leading - 5' to 3' (continuous) - Pol. epsilon
lagging - 3' to 5' (not continuous) - Pol. delta
59
list 3 differences between prokaryotic and eukaryotic DNA replication
1. eukaryotic genomes are much bigger
2. euakryotes have histones/nucleosomes
3. prokaryotes have circular DNA
60
what are the general functions of the following processes?
1. base excision repair (BER)
2. nucleotide excision repair (NER)
3. mismatch repair
1. BER - removal of a damaged base that cannot be directly repaired
2. NER - correction of large segments (pyrimidine dimers of bulky substituents
3. mismatch repair - fixes errors in replication that were missed by proofreading
61
what is translocation? what's the difference between balanced and unbalanced?
the exchange of large segments of DNA between two non-homologous chromosomes.
balanced - no loss of genetic function
unbalanced - results in extra or missing genes
62
what is the difference between DNA and cDNA?
cDNA is made from an RNA template (via reverse transcriptase) therefore it will not contain any introns
63
what direction is RNA synthesized?
5' to 3' (complementary to DNA template strand)
new bases added to 3' end
64
which way does DNA dependent RNA polymerase synthesize RNA and read DNA?
synthesizes: 5' to 3'
reads DNA: 3' to 5"
65
how many subunits does DNA dependent RNA polymerase have? what are they?
4 subunits
- 2 alpha
- 1 beta
- 1 beta prime
66
what is the sigma factor? how does it relate to RNA polymerase?
the sigma factor is a protein than scans the DNA for promoters. once it finds one, it allows RNA polymerase to bind to it.
RNA polymerase w/o sigma = apoenzyme
RNA polymerase w/ sigma = holoenzyme
67
for the following RNA polymerases, list which RNA type they make:
- I
- II
- III
- I = rRNA (except the small 5S rRNA)
- II = mRNA
- III = tRNA and 5S rRNA
68
differentiate the DNA template strand and coding strand in terms of RNA
template strand - used by RNA polymerase to make RNA
coding strand - resembles the RNA produced but NOT used by RNA polymerase
69
what does polycistronic mean?
it's a mechanism used by some prokaryotes where a single RNA produces codes for multiple genes that are all part of the same metabolic pathway. (ex. lac operon)
70
which end of a gene would you find a promoter?
5' (starting end)
71
list 2 prokaryotic promoter elements and where they are usually found
1. pribnow box (TATAAT) at -10
| 2. TTGAGA at -35
72
list 2 eukaryotic promoter elements and where they are usually found
1. hogness (TATA box) at -25
| 2. CAAT and GC rich regions found between -40 and -100
73
differentiate cis vs. trans DNA factors
cis - DNA sequence than can bind to a protein factor
trans - protein than can potentially bind DNA sequence
74
for the following, list what they are and how they affect transcription:
1. enhancer element
2. repressor
3. silencer element
1. enhancer element - cis element that binds a transcriptional activator (increases transcription)
2. repressor - protein that binds to a silencer element (slows transcription)
3. silencer element - cis element that binds to repressors (slows transcription)
75
what is hnRNA?
pre-mRNA, RNA that has not received any processing yet
76
what is done to RNA to make a fully mature mRNA?
1. 5' cap added (methylated GTP)
2. poly A tail added (binding site for proteins and stabilizes RNA)
3. introns spliced out
4. transported out of the nucleus to cytoplasm
77
what molecule preforms RNA intron splicing?
snRNPs
78
what is thalassemia?
an autosomal recessive blood disorder (found in the Mediterranean region) where a variant or missing gene in the hemoglobin gene affects for its' made
79
list 2 altered bases you would find in tRNA
what is added to tRNA during processing? where?
1. thymine
2. pseudouracil
3' CCA added (mnemonic: Can Carry Amino acids)
80
1. what is actinomycin D do?
2. what does rifampicin (or rifamycin) do?
3. what does streptolydigin do?
4. streptomycin?
5. tetracycline?
6. chloramphenicol?
7. erythromycin
1. binds to DNA and sterically hinders replication and transcription (super toxic)
2. binds to beta-subunit of bacterial RNA polymerase and precents transcription initiation (not elongation)
3. prevents message elongation (not initiation)
4. binds 16S rRNA thus preventing translation initiation
5. binds 30S ribosomal rubunit, blocks binding of aminoacyl-tRNA to the A site
6. binds 50S subunit blocks binding of AA to tRNA (can also inhibit mitochondrial activity)
7. binds 50S subunit, prevents translocation
81
what is alpha-amanitin? what does it do?
its a poison found in death cap mushrooms (Amanita phalloides) that inhibits eukaryotic RNA polymerase II (which is responsible for making mRNA)
- initially causes mild GI symptoms
- 48 hours later causes massive liver failure (transplant needed)
82
what is the start codon?
what are the 3 stop codons?
start - AUG (sets reading frame)
stop - UAG, UAA, UGA
83
describe the following types of mutations
1. silent
2. missense
3. nonsense
1. silent - base change doesn't change AA
2. missense - base change changes AA
3. nonsense - base change forms stop codon
84
what enzyme attaches amino acids to tRNAs? how many are there? where does the AA attach on the tRNA?
aminoacyl-tRNA synthetase
one for each tRNA
AA attaches on the terminal adenine of CCA
85
what are the 3 stages of protein synthesis? what happens at each step?
1. initiation (components of translation machinery are assembled and the first AA is added?
2. elongation (AAs are added to the carboxyl end of the growing polypeptide)
2. 5. (translocation)
3. termination (release factors bind, translation stops [not tRNA with complementary anticodon])
86
what molecule catalyzes the peptide bond formation during elongation of protein elongation?
what is this molecule?
peptidyl transferase
it's a ribozyme, not a protein (it's the enzymatic activity associated with the large ribosomal subunit [50S or 60S])
87
what is translocation? what molecules are required for this? how does it affect tRNA?
the movement of the ribosome along the mRNA molecule
requires EF-G (eEF2) and GTP
- moves peptidyl tRNA from A site to P
- moves uncharged tRNA from P site to E (where it's released)
88
in what organism would you find an fMET?
in prokaryotes
89
what is osteogenesis imperfecta?
a defect in collagen assembly or stability due to a missense mutation and/or defects in post-translational modifications (hydroxylation of proline and lysine, it gets glycosylated)
90
what is the signal recognition particle? what does it do?
it's a protein-RNA complex that binds new proteins and targets them to the ER for post-transcription modifications
91
what is I-call disease?
a disease characterized by the lack of the phosphotransferase activity needed to transfer mannose-6-phosphate to enzymes.
M6P is added to protein in the golgi and it acts as a targeting molecule for the lysosome. without M6P, the protein will be secreted out of the cell and the lysosome become non-functional and become filled with debris they can't digest (inclusion bodies)
92
define aneuploidy
irregular number of chromosomes in a cell
93
what type of cancer will most likely result due to a mutation in the BRCA-1, 2 gene? what does the protein from this gene normally do?
Breast cancer as well as ovarian and prostate cancer (mostly breast?)
BRCA is a tumor suppressor protein that's responsible for repair of the DNA via homologous recombination
94
list 6 differences between normal cells and cancerous malignant cells
1. malignant cells have an infinite proliferative capacity (resistant to senescence)
2. they are anchorage independent
3. they are resistant to growth inhibition (also not inhibited by cell-cell contact)
4. resistant to apoptosis
5. malignant cells show signs of DE-differentiation
6. they can be metastatic (now where they're suppose to be?)
95
what is an oncogene?
onco genes result from gain-of-function mutations of growth promoting genes.
the normal form of the gene is called a proto-oncogene
96
what is a tumor suppressor gene?
normal genes that encode growth-inhibiting products that become inactivated in cancer cells (loss-of-function mutations)
97
what are are the 6 classes of oncogenes?
1. cellular growth factors
2. hormone and growth factor receptors
3. signal transduction proteins
4. GTP-binding proteins
5. nonreceptor protein kinases
6. transcription factors
98
list 3 things that could cause the conversion of a proto-oncogene to an oncogene
1. radiation/chemical mutagen
2. gene rearrangement (where the proto-oncogene is placed under the control of a strong transcriptional activator OR it fuses with another gene making it hyper reactive)
3. gene amplification
99
what kind of protein is Ras? what molecular pathway does it participate in?
what activates it?
what does it activate?
a monomeric G-protein that participates in the cascade mediating cell growth/mitosis control by growth factors
the binding of a growth factor by a receptor relays the signal to RAS (a GTPase) causing it to bind GTP and become active.
it stimulates the MAP Kinase phosphorylation cascade ultimately resulting the the transcription of nuclear proteins that move the cell cycle along
P.S. RAS gene is a proro-oncogene
100
what is NF-1?
what condition is caused if it gets mutated?
a tumor supressor protein that activates the GTPase activity of Ras (thus inactivating it)
mutations in NF-1 result in neurofibromatosis
101
what does the retinoblastoma protein do? what transcription factor does it bind to? what does the TF do?
its a tumor suppressor protein that negatively regulates the cell cycle. it arrests the cell in the G1 phase thus halting any inappropriate proliferation.
it binds to many transcription factors but E2F1 and inactivates it. (R2F is responsible for pushing the cell through the G1/S transition)
102
a dysfunction in at least one of four cell cycle regulator are present in nearly all cancers. what are those regulators?
1. p16/INK4
2. cyclin D
3. CDK4
4. Rb
103
what is p53? generally what does it do? what does it do if it senses DNA damage or hypoxia (3)?
AKA "the guardian of the genome"
its a transcription factor regulating the cell cycle, DNA repair and apoptosis.
1. halts the cell cycle so new DNA synthesis will not replicate the damaged DNA
2. up-regulated DNA repair genes
3. is DNA damage is too severe, it triggers apoptosis
104
what is Li Fraumeni syndrom?
its a mutation in p53. this causes a 25-fold increased chance of getting cancer.
mutations in p53 is the most frequent mutation leading to cancer,
105
what does p27 do? how does it relate to breast cancer?
it inhibits cyclin and Cdk, thus blocking the entry of the cell into the S phase
the prognosis of breast cancer is determined using p27 levels. (reduced p27 predicts a poor outcome)
106
what is the protein "patched"? how does it relate to HH (hedgehog) and "smoothened"?
Patched is a receptor for HH. in its unbound form it is actively inhibiting the protein smoothened which is a transcription factor. binding of a HH to patched releases the inhibition on smoothened allowing it to work
107
what are cadherins? how do they relate to catenins?
what is the result of a mutation in CDH1 (E-cadherin)?
proteins than bind cells together. they are anchored by catenins
mutation in CDH1 result in diffuse gastric cancer
108
list two functions of catenins
what is APC (adenomatous polyposis coli)? what happens if it gets mutated (2)?
1. anchor cadherins
2. act as transcription factors (activate transcription of Myc gene and Cyclin D1 gene)
APC is a molecule (protein?) that inhibits beta-catenin. if it get mutated then beta-catenin will cause the cell to over proliferate and it can result in sporadic colon cancer
inherited mutations cause familial adenomatous polyposis (FAP)
109
what is tumor necrosis factor (TNF)? what does it do?
its a ligand that binds the death receptor (this activates caspases) triggering apoptosis via the extrinsic pathway
110
list 2 ways caspases can trigger apoptosis
1. TNF binds the death receptor (extrinsic)
| 2. cytochrome C in mitochondria (intrinsic)
111
list 2 initiator caspases and 3 executors
initiators: 8, 10
executors: 3, 6, 7
112
how do procaspases go from their procaspase form in the mitochondria to triggering apoptosis (intrinsic pathway)? what other proteins are involved?
procaspases are activated by an apoptosome, which is the combination of cytochrome C and Apaf-1 (pro-apoptotic protease activating factor-1) which forms an asterisk shaped molecule. it then goes and cleaves other procaspases which go on to cleave others resulting in an amplified photeolytic response resulting in apoptosis
113
what is a piliadelphia chromosome? what condition is is closely associated with? what is the mechanism of the condition?
is a chromosome 9:22 rearangement
it is associated with 95% of chronic myelogenous leukemia (CML)
the condition is cause via a fusion of the ABL gene (a proto-onco gene) and the BCR gene. when these two bind together, it causes ABL tobe consitutatively active thus converting it into an oncogene.
114
what is the molecular cause of burkitt's lymphoma?
caused by a translocation of the c-myc gene so that its under the control of the promoter for immunoglobin heavy chain (which is very active).
normally c-myc is a transcription factor that is induced by growth factor signaling cascade. therefore over expression of this caused overproliferation of WBCs
115
what are the molecular characteristics of neuroblastoma?
the condition occures as a result of N-Myc gene amplification. this amplification manifests either as (1) double minutes, which are small extra-chromosomal DNA that encodes the gene [also other genes?] or (2) integrated homogeneous staining regions (HSR) in the DNA
116
list 4 nonsurgical cancer treatments that target DNA
1. anti-folates
2. base analogues
3. alkylating agents
4. ionizing radiation
if p53 is inactive then the triggering of apoptosis is inhibited thus limiting the use of #3 and 4
117
lise 4 nonsurgical cancer treatments that do not target DNA
1. interference of the mitotic spindle
2. choke off blood supply (blocking angiogenesis)
3. monoclonal antibodies for a protein that may be the chief cause
4. active site inhibitors that attack specific areas of DNA
118
differentiate positive and negative regulation of transcription
positive - required a protein for transcription to occur
negative - a protein is required to block transcription
119
in the lac operon, what does the repressor and inducer do?
the repressor binds the operator thus preventing transcription.
the inducer binds to the repressor causing it to detach from the operator thus allowing transcription
(this model is true for many other genes/operons)
120
what is a co-repressor?
a protein that must bind a repressor before that repressor can be active
121
list 7 levels in which eukaryotic genes expression is regulated.
1. DNA and the chromosome
2. transcription regulated (via TFs)
3. processing of transcripts (RNA)
4. RNA transport and localization
5. initiation of translation
6. stability of mRNA
7. stability of the protein
122
what can be done to histones to change their charge?
acetylation
123
where would you normally find methylation of DNA in the DNA? what is the purpose of methlyation? how specific is methlyation of DNA?
on cystines in GC-rich areas (CpG islands) which is often near or in a promoter region for a gene.
methylated promoters can either increase or decrease transcription
Methylation patterns are specific to each cell type (it may have played a role in their differentiation). this means certain cells can repress or favor specific proteins that fit their needs
124
chat chromosome is affected in prader-willi syndrome and angelmen syndrome
chromosome 15
125
list 2 diseases that are the result of triple repeat expansion, what the repeats are and where they're found.
1. fragile x syndrom (CGG) - 5-54 repeats are normal, 60-230 are carriers, 230-4000 are affected
2. huntington disease (CAG) - 10-35 is normal, greater than 35 is the disease
126
what can occur if a patient lacks the ability to respond to androgens (steroids)
the development of ambiguous genitalia or testicular feminization
127
what structure is common on all DNA binding domains on proteins?
an alpha-helix that binds the major groove
128
list 4 DNA binding domains used by proteins
1. zinc fingers (used for estrogen receptors?)
2. leucine-zipper
3. helix-turn-helix (doesn't dimerize)
4. helic-loop-helix
129
what is the purpose of genes having multiple promoter areas
different sites may direct activity in different circumstances
130
describe how the globin protein is regulated by the presence (or lack of) heme
Heme works to inactivate the kinase that inactivates eIF-2 (which is a protein required for translation which mediates the binding of tRNA-Met to mRNA)
when heme levels are high, eIF2 is not phosphorylated and is therefore active
131
what are miRNAs? what do they do?
they are small DS RNAs that work to regulate expression at the TRANSLATIONAL level (induces degradation and represses translation)
132
how does iron regulate mRNA stability?
| maybeonly applies to ferririn [iron storage protein] protein? LOOK THIS UP MORE
- low Fe allows more translation
| - high Fe = less translation (protein)
133
what are restriction enzymes? what do they do?
enzymes isolated from bacteria that cut double stranded DNA at specific base sequences
134
what is DNA ligase used for in vivo? in vitro?
in vivo - repairs single-stranded breaks (fixes nicks in phosphodiester backbone)
in vitro - used to join fragments of double stranded DNA
135
what is "ligation" of DNA? what is required for the DNA for this to occur?
joining two DNA molecules together
the DNA must have "stick ends" (overhanging single-stranded portions at the end of their strand). if they have blunt ends then you have to add "Linkers" to make them sticky before you can ligate them.
136
What do most DNA polymerases need in order to begin working?
a primer (power point says DS but the picture says SS. the SS primer likely couples with the template strand thus making it DS)
137
what enzymes is required for the production on recombinant eukaryotic proteins in prokaryots?
reverse transcriptase (makes processed cDNA since the bacteria can't do that for eukaryotic proteins)
138
what is a hybridization technique? what is a probe?
a technique involving the annealing of two single strands (DNA or RNA) of complementary sequence
its commonly used as a probe to identify to select specific DNA fragments (the probe is usually labels in some way)
139
like 3 commonly used hybridization techniques
1. southern blotting - identification of DNA fragments using a complementary probe
2. northern blotting - identification of an RNA fragment using a probe
3. western blotting - identification of proteins through their ability to bind specific antibodies
140
what are the steps to southern blotting (5)?
1. extract DNA and digest it with restriction enzymes
2. DNA fragments separated by electrophoresis
3. blot onto membrane and add an radioactive probe (which hybridized to a specific DNA sequence)
4. expose to an x-ray film
5. analyze film for labeled restriction fragments
141
list 4 applications to southern blotting in medicine
1. identifying specific genes
2. diagnosis of genetic disease (RFLP analysis)
3. carrier detection
4. DNA fingerprinting/profiling
142
what are the 2 DNA elements that were originally used for DNA profiling/fingerprinting?
what do we currently use?
1. minisatellite DNA comprises VNTRs (variable number tandem repeats) - 10 to 60 bases repeated
2. microsatellite DNA comprises STRPs (short tandem repeat polymorphisms) - usually less than 10 bases repeated
currently we just use PCR to analyze STRPs or specific multiple polymorphic alleles (multiplex PCR)
143
how many VNTRs must match a DNA sample to have a probability of one in a million? what about STRPs?
VNTRs - 4
STRPs - 13
144
what is the result of DNA trying to us a dideoxy nucleotide while synthesizing DNA? what are ddNTs used for?
it prevents the addition of additional bases afterward since they need the 3' OH which isnt no longer there
they are used for DNA sequencing (sanger method). if use do 4 separate reactions with the same template strand, one with each ddNT you can see how long each terminal strand is and elucidate the sequence of the DNA
145
list 4 reagents needed needed for a PCR reaction
1. DNA template
2. dNTPs (dATP, dTTP, dCTP, dGTP)
3. heat stable DNA polymerase (Taq)
4. oligonucleotide primer complementary to ends of region to be amplified
146
list the steps of PCR (6).
1. initial melting
2. melting
3. annealing
4. extension
5. repeat steps 2-4 26 times
6. final extension
147
List 2 advantages to PCR and 3 disadvantages.
Advantages
1. very small amount of DNA needed
2. very fast
Disadvantages
1. some knowledge of the sequence is required (for primer formation)
2. there is a size limit for template DNA (up to around 4Kb)
3. contamination is a huge issue
148
list 5 vectors used to introduce DNA fragments into cells and the maximum insert size.
1. plasmids - 2.5Kb
2. bacterophage - 20Kb
3. cosmid - 40 to 50Kb
4. bacterial artificial c'some (BAC) - 150Kb
5. yeast artificial c'some (YAC) - 1000Kb
149
what vectors are typically used for gene therapy?
viruses
150
define transduction, transformation and transfection
transduction - phage mediated gene transfer into prokaryotic cell
transformation - non phage mediated. bacteria picking up naked DNA form environment
transfection - gene transfer into a eukaryotic cell
151
how would you typically clone a DNA fragment?
1. insert it into some sort of vector
2. put that vector inside a bacteria
3. allow to bacteria to replicate into colonies
152
list 2 reasons to clone DNA
1. to amplify the amount of that DNA
| 2. for production of the encoded protein (from cloned cDNA)
153
what is a DNA library? what are the sources of genomic and cDNA libraries?
a large collection of recombinant DNA clones in which the DNA has been inserted into a vector
genomic - DNA fragments from entire organism (has all genes)
cDNA - from RNA of a particular organ (only genes from the tissue/cell type)
154
how do you screen a genomic library? what about a cDNA library?
genomic - select a clone that contains the individual gene and hybridize it with a probe
cDNA - PCR is the fragment isn't too large then hybridization with a probe and immunological screening
155
what is allele-specific oligonucleotide (ASO) hybridization?
AKA "dot blot". its where you get a fragment of DNA and hybridize it to specific synthetic primers to see if the patient has that particular gene/mutation.
ex. DNA sample hybridized to normal CF gene and mutant CF gene to test whether they have it, are a carrier or are healthy
156
why will PCR not work to test for myotonic dystrophy?
the mutation is too large ( >4000bases) to amplify
157
what is a DNA microarray?
basically just a huge ASO analysis chip that contains 10^5 ASOs per slide
158
list 8 therapeutic proteins produced by recombinant DNA tech
1. insulin
2. factor VIII
3. factor IX
4. human growth hormone
5. tissue plasminogen activator
6. interferon
7. erythropoietin
8. adenoside deaminase
159
which vaccine was the first to be made using recombinant DNA technology? how?
hepatitis B
protein antigens are produces in abcteria and therefore completely free of the infectious agent
160
list 2 types of gene therapy
1. somatic
| 2. germline
161
list 3 types of somatic cell gene therapy and the idea behind them
1. gene replacement therapy - replacing a mutated gene that causes disease with a healthy one
2. gene therapy for non-inherited diseases - introducing new genes to help fight a disease
3. gene-blocking therapy - inactivating a mutated gene that is functioning improperly
162
list 3 advantages and 3 disadvantages for using a retrovirus for gene therapy
advantages
1. enters into cell efficiently
2. stable integration into host DNA
3. targets only dividing cells
disadvantages
1. limited insert size (8-12Kb)
2. integrates into host genome
3. targets only dividing cells
163
list 3 advantages and 2 disadvantages for using a adenovirus for gene therapy
advantages
1. enters into cell efficiently (both dividing or not)
2. high expression of insert
3. does not integrate into host genome
disadvantages
1. can elicit serious immune response
2. does not integrate into host genome
164
list 3 advantages and 2 disadvantages for using a adeno-associated virus for gene therapy
advantages
1. enters into cell efficiently (both dividing or not)
2. little to no immune responce
3. integrates into host genome at specific site (c'some 19)
disadvantages
1. limited insert size (5Kb)
2. difficult to produce
165
list 3 advantages and 1 disadvantage for using a herpes simplex virus for gene therapy
advantages
1. can carry up to 20Kb
2. prolonged activity
3. infects nerve cells very efficiently
disadvantage
1. causes immune responce
166
list 2 advantages and 3 disadvantages for using a liposome for gene therapy
advantages
1. can accept large inserts
2. no immune response
disadvantages
1. inefficient cell entry
2. no integrating into host DNA
3. can be toxic
167
list 2 advantages and 2 disadvantages for using naked DNA for gene therapy
advantages
1. can accept large inserts
2. no immune response
disadvantages
1. very inefficient entry into cell
2. no integration into host DNA
168
in general which type of gene therapy vector is best suited for ex vivo therapy and which for in vivo therapy?
ex vivo: non-viral vectors
in vivo: viral vectors
169
list the top 3 most used vectors for gene therapy in order
1. adenovirus
2. retrovirus
3. naked/plasmid DNA
170
briefly, what is the process of ex vivo gene therapy? list 5 conditions this type is used to treat
patients cells are exrtacted, manipulated outside the body and re-introduced
1. SCID
2. familial hypercholesterolemia
3. gaucher disease
4. malignant melanoma
5. leukemia
171
list 4 conditions that can be treated using in vivo gene therapy
1. cystic fibrosis
2. various cancers (brain, ovarian, neck)
3. hemophilia B
4. duchenne muscular dystrophy (maybe?)
172
list a type of gene therapy approved for use (in Europe) and what condition it treats. what vector does it use?
GLYBERA, used to treat lipoprotein lipase deficiency
uses adeno-associated virus vector
173
list 4 types of genetic disorders and give 2 examples each
1. chromosome disorders (down syndrome, turners syndrome)
2. single-gene disorders (hemophilia, cystic fibrosis)
3. multifactorial disorders (diabetes, heart disease)
4. mitochondrial disorders (LHON, MERRF)
174
where would you find a centromere in a metacentric c'some? submetacentric? acrocentric?
metacentric - center
submetacentric - more towards one side
acrocentric - near the end of one side
175
what percent of our DNA is single-copy, dispersed repetitive DNA and satellite DNA?
single-copy - 45%
dispersed repetitive - 45%
satellite - 10%
176
list 3 specific dispersed repetitive DNA elements
1. LINEs
2. SINEs
3. Alu elements
177
list 3 specific satellite DNA elements
what is one of the medical uses of satellite DNA?
1. alpha
2. minisatellites (14-500bp)
3. microsatellietes (1-13bp)
satellite DNA is very polymorphic between people (less so between related people) therefore it can be diagnostic for many purposes (paternity test, forensics, fingerprinting etc...)
178
what is a chiasma? where on the c'some are these less likely to occure?
the point of recombination between two c'somes
crossing-over is less common near the centromere and near the telomere
179
what determines the rate of recombination of genes? what units are used?
their distance from each other (the closer they are, the less likely they will be separated)
distance measured in centimorgans (cM) [more of a measure of relative distance than actual distance; ex. "it takes 5 minutes to get there"]
180
what are 2 possible consequences of mutation in non-coding DNA?
1. can affect mRNA expression
| 2. can affect mRNA processing
181
what nucleotide sequence is at the beginning of all introns? what about the end?
begining - GT
| end - AG
182
# define the following mutation types:
1. duplication
2. transposons
3. expanded repeates
1. duplication - large regions (whole genes) that get duplicated
2. transposons - mobile genetic elements (can insert in or near another gene and alter expression)
3. expanded repeats - can be trinucleotide repeats, runs of one AA, etc... too many repeats can disrupt function
183
differentiate loss of function mutations with gain of function
LoF - mutated protein has no function (ex. active site gets mutated)
GoF - mutated protein has new or amplified function (ex. it gets higher substrait affinity)
184
what mutation leads to the formation of sickle cells?
glutamate 6 to valine (single missence mutation)
185
what are the causes of thalassemias? in which population is the most common it?
a defect in production of either alpha or beta-globin subunits of hemaglobin. (normal hemoglobin has 2 alpha and 2 beta subunits)
most common in Mediterranean populations
186
list 3 molecular causes of mutation
1. ionizing radiation - creates reactive radicals that react with bases, causes double-strand breaks
2. nonionizing radiation - shifts electrons around and causes reactions. ex. thymine dimers from UV
3. chemicals - base analogs inserts or chemical changes to bases. ex. deamination of C to U
187
list 3 factors that determine that rate of mutation for a gene
1. size of the gene - the larger, the more likely
2. age at replication - the older you are the more likely
3. Hot spots in gene - certain areas more likely to be mutated like CpG dimers (which usually get methylated. if the C gets deaminated it becomes a T)
188
what causes xeroderma pigmentosum?
a defect in the nucleotide excision repair mechanism (NER). this prevents the cell from being able to fix thymine dimers. therefore affected people are more profoundly affected by UV radiation
189
what is the hardy-weinberg equation? what do the variables represent?
(p^2) + 2pq + (q^2) = 1
```
p = frequency of WT (normal) gene
q = frequency of mutated gene
(q^2) = frequency of affected people
```
190
what is the multiplication rule or probability?
what about the addition rule?
the probability of multiple events occurring together
the probability of either one or another multiple events occurring
191
for the following conditions. list whether they are autosomal dominant or autosomal recessive
1. retinoplastoma
2. postaxial polydactyly
3. albinism
4. achondroplasia
5. cystic fibrosis
6. neurofibromatosis
1. retinoplastoma - AD (tumors form in retina)
2. postaxial polydactyly - AD (more than 5 digits on a limb)
3. albinism - AR
4. achondroplasia - AD (short legs due to cartilage problem?)
5. cystic fibrosis - AR (disrupted chloride transport)
6. neurofibromatosis - AD (variable expression from developement of a few "cafe au lait" spots to developement of many large skin tumors)
192
list 4 symptoms of cystic fibrosis
1. disrupted chloride transport
2. pancreatic secretions are affected leading to a clogging of the ducts (therefore enzymes don't reach the intestines)
3. lungs have thickened secretions, can't clear material
4. excess chloride in sweat
193
define pleiotropy. what type of genes can cause it?
multiple effects from a single mutation
1. DNA transcription factors
2. extracellular matrix proteins
194
# define the following terms:
1. allelic heterogeneity
| 2. locus heterogeneity
1. allelic heterogeneity - multiple mutant forms with the same gene
2. locus heterogeneity - multiple genes affecting a single pathway
195
what is anticipation in terms of genetic disorders?
the phenomenon where some dominant disorders get more sever or occur earlier in life in later generations
