Exam II Flashcards
Pedigrees Replication II etc. (214 cards)
1
Q
change that affects the number of chromosomes in a cell
A
Numerical mutation
–aneuploidy: error duirng meirosis/mitosis
2
Q
change in the structure of an individual chromosome
A
structural mutation
ex: partial duplication, triplication, deletion, inversion, translocation
* copy number variation
3
Q
change that alters an individual gene
A
change in DNA sequence
4
Q
specific sequence of DNA present in at least 1% of the general population regardless of clinical phenotype
A
polymorphism
5
Q
Polymorphisms include
A
LINES/SINES short tandem repeats segmental duplications (large repeats)
*regions of squence homologies at edges
6
Q
Factor VIII and Hemophilia are both results of
A
Gene inversion
–intrachromosomal mispairing (exons 1-22)
*bleeding in joints, soft tissue and CNS
7
Q
The following describes what category of copy number variation (CNV)?
- common in general population (1%)
- < 10 kb in length
- enriched for genes involved in drug detox and immunity
(psoriasis, Crohn’s, glomerulonephritis)
A
Copy Number Polymorphism
8
Q
Which of the following best describes copy number variation in the form of microdeletions and microduplications?
A. relatively common in the population
B. always seen in genomes
C. disproportionately ID’d in patients with mental retardaton, developmental delay, autism, etc.
A
Answer: C
- rare in the population
- hundreds to > 1 mil bp
- recently appeared in individual genomes
9
Q
Regions within a chromosome that have high sequence homology to another region within a chromosome
A
segmental duplications
10
Q
In normal meiosis, chromosomes align precisely at the recombination site and the crossovers do not change in the number of genes in the recombinant chromosomes.
However, presence of duplicated sequences on a chromosome can increase the frequency of chromosome misalignment and lead to copy number variation. What are the products of unequal crossover?
A
Increased copy number of genes on one recombinant chromosome (gain), while decreasing the copy number on the other chromosome (loss)
**copy number variation
11
Q
Identical genetic information on homologous chromosomes
A
Homozygous
12
Q
Non-identical information on homologous chromosmes
A
Heterozygous
Aa
13
Q
Wild-type allele
A
- shared by 50% or more of the population
* common
14
Q
Variant allele
A
DIfferent from 50% of population sharing WT
15
Q
Mutations are generated by what events?
A
Replication
Repair
Recombination
Chromosome Segregation
16
Q
Mutations are very rare (1 error/1 billion nucleotides). There are fewer than on mutation per cell division.
Mutations rates can vary from gene to gene. What can cause these errors?
A
Nucleotides Proofreading Mismatch repair Template Nucleotide selection
NOTE: 75 new mutations in genome from one or other parent
17
Q
Which of the following has a higher mutation rate?
A. achondroplasia (FGFR3)
B. Retinoblastoma (RB1)
A
Achondroplasia
18
Q
An arbitrary designation that uses the most common sequence or arrangement in a population at any one position in the genome
A
Reference genome sequence
19
Q
change that alters an individual gene
*change in DNA sequence
A
Gene mutation
20
Q
change in codon such that one amino acid is changed
nucleotide change that results in a different amino acid
A
missense mutation
ex: change from glutamate (GAA) to glutamine (CAA)
21
Q
amino acid changed to stop codon
A
Nonsense mutation
ex: UAC (Tyr) to UAA (stop)
22
Q
changes reading frame for translation
A
Frameshift mutation
23
Q
addition of one or more nucleotide bases
A
Insertions
24
Q
Loss of one or more nucleotide bases
A
Deletions
25
The genetic code is degenerate. What does this mean?
specific amino acids are coded by more than one codon
* 3rd position wobble (50% of cdons)
* STOP: UAA, UAG, UGA
26
Change of a single nucleotide
Point mutation
*if the DNA sequence changed, but the aa sequence is the same = SILENT mutation
27
A point mutation in which a single nucleotide change results in a codon that codes for a different amino acid.
Missense mutation
* sense of reading frame changes
ex: AT for GC
28
A missense mutation that results in an amino acid change, but the properties of the amino acid remain the same (e.g. charged, hydophobic, etc.)
Conservative
*may not affect protein function
29
A missense mutation that can lead to the loss of protein function
(esp. in highly conserved regions)
Non-conservative
| -change aa to a different aa with different properties
arg for threonine
30
What are the possible consequences of a missense mutation
1. gain of function
--more protein/inc. enzyme activity
(hypermorphic)
2. loss of function
- -reduces/eliminates protein
3. dominant negative mutation
- -abnormal protein that interferes with normal protein
31
Gain of function:
1. Hypermorphic mutations cause an _____ in normal gene function.
2. Neomorphic mutations cause a dominant gain of gene function that is ______ from normal function.
* Wilson Genetic Mutations I
1. Increase in normal gene function
- -results from inc. gene dose, inc. protein, etc.
x: RET; Y kinase receptor
2. Different from normal
- -cause new protein functions
ex: Decarboxylation of isocitrate to alpha-ketoglutarate via isocitrate dehydrogenase
32
________ mutant protein acts as a "sponge" that soaks up the WT proteins preventing them from interacting with other normal proteins
*loss of function
Dominant negative
33
_______ mutation describes a mutation that causes a partial loss of gene function.
Hypomorphic mutation
* *CFTR mutant = does not move Cl- ions out
- -mucous build up
*less, inactive, defective
34
_____ mutation occurs when a codon that codes for an amino acid is converted to a stop codon that stops translation
Nonsense mutation
- -loss of function
- -shortened protein
35
The presence of an exon-exon junction within 50-55 nucleotides downstreatm of the premature stop codon signals abnormality and recruits the machinery for degradation of the message. This is known as
Nonsense-mediated mRNA decay
* Premature termination (truncated protein)
* No translation occurs *No protein is formed
*LOF
36
Treatment of Nonsense mutation involves drugs that allow ribosomes to read through the termination codon.
This is known as
Aminoglycoside-mediated suppression of premature stop codon
-antibiotics inhibit bacterial protein synthesis at high doses
37
What is the effect of aminoglycoside-mediated suppression of premature stop codons in eukaryotes?
--less efficient binding
- -translational read through by the insertion of a random amino acid at the stop codon
- -restores function w/ low level of functional proteins
38
What happens when you lose a termination codon?
Abnormal protein is translated
- -introns included at 3' end
- -translation continues until next STOP
39
What is expected in a mutation in exon-intron splice sites?
--Sequence may or may not be spliced
--Spliceosome splices at the next available splice site (cryptic)
* at end: good
* in middle: bad
40
Why are Single nucleotide polymorphisms important?
SNP can mark a gene or can alter gene expression
*regions of chromosomes w/ multitude of SNP's
41
HLA protein -- transplants on chromosome 6 is an example of
SNP
42
Aneuploidies are due to _______ events while gene mutations are due to ______
1. maternal non-disjunction
2. paternal; spermatogenesis
paternal: achondroplasia, marfan, etc.
43
A mutation event may either have a
1. positive effect
2. neutral effect
3. negative effect
Differentiate
1. Positive
- -fixed in population
ex: sickle cell (hetero)
2. Neutral
- -fixed in population as polymorphism
3. Negative
- -rare
- -ex: sickle cell (homo)
44
ABO blood types are classified by the presence or absence of the antigens A and B which are carried on the surface of the RBC's.
Describe these
1. Type A
- -A antigen
- -Anti-B abs
2. Group B
- -B antigen
- -Anti-A abs
3. Group AB
- -no abs
- -A and B antigens
4. Type O
- -anti-A and B abs
- -no Ags
45
H antigen
Located on the surface of RBC's (precursor to A, B, O)
46
Fucosyltransferase
Enzyme formed by H gene
--adds fucose to oligossaccharide chain attached to glycosphingolipid (RBC) or protein (secretions)
47
True/False: Individuals either will express H antigen (HH or Hh) or WONT (hh, Bombay). They will have antibodies against H, A and B antigens
True
48
alpha 1-3 N acetylgalactosaminyltransferase (A type; AB type)
or a 1-3 galactosyltransferase (B type) convert _____ to ______
H antigen to A, B or O
49
The O blood allele is the result of a loss of function mutation caused by
1 SNP
--deletion = frameshift mutation
--no funtional protein
*Only express H antigen
50
A change from A allele to B allele occurs by
Neomorphic mutation
A: 4 SNP's
B: 2 aa changes
51
Where are ABO antigens expressed?
surface of RBC's, epithelial cells, sensory neurons, platelets, vascular, tears, milk, saliva, amniotic fluid, semen
*expression dependent on FUT1 (cell surface) or FUT 2 (secretory)
52
Type O individuals are more susceptible to severe cholera. There is no increased risk of infection, but they are more likely to develop severe illness if infected.
Why?
Cholera toxin has higher affinity to type O antigen
* stimulates intracellular signalling
* reduce # individuals in endemic populations
53
Type _____ individuals are more at risk of infection by malaria (P. falciparum)
Type A
* A antigen = co-receptor for P. falciparum
* Type O is protective
54
Antibodies against A and B types are IgM. IgM abs cannot cross the placenta. What is the significance of this during pregnancy?
Fetal ABO incompatibility
Maternal IgG abs against A/B cross placenta and bind RBC's
55
Individuals expressing A1 allele that are infected w/ E. coli or Clostridium epxress
B-like antigen
56
HLA antigens in humans are the most polymorphic gene systems known. They result in serological differences in protein structure leading to different immune responses. They are a result of SNP (kink/bend) What methods are used to identify them?
1. Serology
| 2. DNA chip hybridization
57
HLA antigens in humans are the most polymorphic gene systems known. They result in serological differences in protein structure leading to different immune responses. They are a result of SNP (kink/bend) What methods are used to identify them?
1. Serology
2. DNA chip hybridization
3. Restriction Fragment Length Polymorphism
- -restriction enzyme
58
_______ diosrders are a group of inherited conditions caused by increased numbers of specific trinucleotide repeats within the genome. These are repetitive sequences within genes that increase from a normal range into a pathogenic number of repeats
Trinucleotide repeats
*tandem repeat of 3 nucleotides
* *ex: CAG -- glutamine
* *Fragile X
59
Features of Trinucleotide repeats
* dynamic - expand and contract
* prone to errors during divsion
* inc. w/ age during gametogenesis
*anticipation of clinical phenotype
60
Anticipation is associated with trinucleotide repeats. DNA segments with an abnormal number of these repeats can change as the gene is passed from parent to child. If this number increases, it is known as expansion.
What is anticipation?
If the # of trinucleotide repeats > premutation size, then offspring will inherit more repeats and be more likely to have a clinical disorder.
*early onset and more severe with each generation
61
Huntington Disease is repeat expansion that occurs through
spermatogenesis
*passed on by male
62
Fragile X is a disease causing failure of chromatin condensation during mitosis. It is associated with repeat expansion that occurs only through
oogenesis
* X-linked (maternal)
* 5' UTR (CGG methylation)
* repeats: 60-200 = premutation
63
Myotonic dystrophy is a form of repeat expansion that can be cause by maternal or paternal transmission.
If < 100 repeats:
If larger:
1. paternal:spermatogenesis ( <100)
2. maternal: oogenesis (>100 repeats)
3' UTR
64
How do repeats expand?
Formation of hairpin loops during base excision repair by strand displacement
*toxic oxidation cycle
65
Methods for IDing genetic variation include
1. PCR -- amplify tandem repeats
(gel electrophoresis -- view product)
2. Southern blotting
- -probe hybridization
3. Next Gen sequencing
- -high througpout
66
List potential origins of spontaneous mutations
1. Spontaneous DNA damage
2. Replication errors
3. Endogenous mutagens (Reactive O2)
Mutation hotspots (induce nucleotide mutations):
* nitrosamines tobacco
* benzo(pyrene) cigarettes
* benzene industrial solvent
* formaldehyde
* sachharin
* *Ionizing radiation (X-rays)
* *Thalidomide (treats leprosy, myeloma, lupus, HIV-related cancer, blood/bone marrow cancers)
67
UV light is a mutagen. Discuss the effects of UV light on DNA
* Thymine dimers
- -base pair substitution during replication occurs if not repaired
- -insertion
68
Deamination of 5-mehtylcytosine results in the Cytosine turning into _____.
Thymine
*C methylation to form 5 methylcytosine -- Thymine
12X higher at dinucleotide sequences (mutation hot spot)
69
Xeroderma pigmentaosum is a disorder that results from a defect in Nucleotide Excision Repiar.
What are the features?
- Skin tumors
- lethal malignancies by 20
- dry, scaly skin
- abnormal pigmentation
70
# Define the following terms:
1. Genotype
2. Phenotype
3. Autosomal
1. Genotype: sequence of DNA
2. Phenotype: What is seen visually, lab results
3. Autosomal: Chromosomes 1-22
(not X and Y)
71
# Define the following terms:
1. Dominant
2. Co-dominant
3. Incompletely dominant
4. Recessive
1. Dominant: phenotype expressed in homo and hetero
- -regardless of if normal
2. Co-dominant: expression of two different allele loci
(ex: ABO)
3. Incompletely dominant: Phenotype of a dominant disorder differs in severity
- -homozygotes: more severe (achondroplasia)
4. Recessive: phenotype expressed only in homozygotes
72
# Define the following terms:
1. Penetrance
2. Expressivity
3. Haplotype
1. Penetrance: Percentage of people with a certain genotype that display a clinical phenotype
* *all or none
* *do they display it or dont they?
2. Expressivity: severity of expression
* same disease causing genotype (ex; NF1)
3. Haplotype: set or allele at a locus or cluster of loci
73
_______ describes a single, abnormal gene or gene pair that can have multiple different phenotypic effects in different organ system
Pleiotropy
*different functions in different tissues
74
What are the characteristic of autosomal dominant disorders?
1. Affected parent: Aa; Unaffected parent: aa
2. Equal # of females and males affected
3. Both sexes transmit the affected allele to offspring
4. Vertical transmission
((doesn't skip generations (except reduced penetrance or denovo))
Lethal in homozygotes**
Direct father to son **
75
In autosomal recessive disorders, affected progeny arise from mating of two heterozygous parents.
What are the features of autosomal recessive disorders?
1. equal number of males/females affected
2. Both sexes (parents) transmit an allele to offspring
3. Horizontal transmission (skips generations)
- -seen in one or more siblings w/ unaffected parents
4. Direct father to son transmission
5. Frequently associated with consanguinity
* *recurrence risk: 25% (both heterozygous parents)
* *recurrence risk if one parent is heterozygous (Aa) and one is homozygous affected (aa): 50%
76
Achondroplasia is an incompletely dominant disorder that results from a mutation in what gene? What are its featuers?
NOTE:
*50% offspring affected
*25%: lethal
25%: no phenotype
Mutation in FGFR3 (GOF)
- FGFR 3 (neg. regulator of bone growth)
- receptor Y kinase stays activated
- inhibits chondrocyte prolif. and bone formation
Features:
* more severe phenotype in homozygous affected
* short stature, large head
77
FGFR 3 mutations are seen in individuals with achondroplasia. What is the function of normal FGFR3? What is the most mutable nucleotide seen?
tumor suppressor (prevents excessive cll division)
* *most mutable: Guanine @1138 in FGFR3
* *>80% have denovo mutation -- paternal germline
78
What are current drugs being tested for treatment of achondroplasia?
*listen to lecture
Treatment:
- BMN 111 (acts at CNP)
- anti-nausea drug Meclozine (acts at
79
Factors that influence expression and maintenance of disease causing alleles include:
1. de novo mutations
2. reduced penetrance
3. age-dependent penetrance
4. expressivity
Explain
1. De-Novo:
- recurrence risk remains same in gen. population
- 50% if dominant; 25% in recessive
2. Reduced penetrance:
- parents w/ clinical genotype do not express phenotype
3. age-dependend penetrance
- delay in age of onset (Huntington)
4. Expressivity
- parents: mild phenotype; offsping: severe
80
Split hand/foot malformation is an autosomal dominant disease with reduced penetrance. What are the features of inheritance?
- incomplete penetrance
- 6th-7th week of development
- appears to skip a generation (but parents are carriers)
81
Neurofibromatosis type I (NF1) is an autosomal dominant disorder of the nervous system, eyes, and skin.
What are the clinical features?
- Lisch nodules (iris)
- cafe au lait spots on trunk (15mm)
- neurofibromas
**Complete penetrance; Variable Expressivity
82
NF1 encodes neurofibromin (ras GAP). ras is a small G-protein, that is inactive when bound to GDP. It is activated by the binding to GTP which is facilitated by ______ and signals cell growth, proliferation and differentiation.
Inactive: GDP bound by GAPs
Active: Guanine nucleotide exchange factor (GEF)
83
Huntington disease is a disease that causes progressive loss of motor control, dementia and psychiatric disorders. Clinical features include caudate atrophy, enlarged lateral ventricles, and gliosis.
What is the mode of inheritance?
Autosomal Dominant
1. Delayed age of onset
2. Vertical; Father to son transmission
3. Equal affected females and males
4. Repeat expansion (middle of exon)
5. Pre-mutation
6. Anticipation
7. Variable penetrance (some express phenotype)
*CAG - glutamine
84
Tay Sachs disease is a lysosomal storage disease (GM2 gangliosidosis). It can lead to neurological degenerative disorders which are fatal in early childhood. How is it inherited?
```
NOTE:
Increased frequency in genetic isolates:
-Ashkenazi jew (100%)
-Canada (consanguinity)
-Amish
```
Autosomal recessive
Features:
- onset 6 mos
- blindness
- mental/physical regression
* no treatment - ERT can't cross BBB
* premarital genetic test: 1 carrier only
85
True/False: Hexosaminidase A plays a critical role in the brain and spinal cord. This enzyme is found in lysosomes and functions in breaking down GM2 ganglioside. A mutation in this enzyme can result in buildup of GM2 and neuron toxicity.
*Tay Sachs
True
86
Cystic Fibrosis is an autosomal recessive disorder that results in mutation in the CFTR gene.
It is associated with allelic heterogeneity. What does this mean?
Heterogeneity: different mutations at the same locus which lead to the same or similar phenotypes.
* compound heterozygotes: autosomal recessive w/ allelic heterogeneity
* newborn screening: high levels of trypsin in serum
87
There are over 1400 different CFTR mutations. What are potential affects of such?
1. Inactive (Class III) Normal CFTR channel does not move Cl- ions causing buildup of mucous on outside of cell
2. Less (Class V): Normal channel, but doesn't make it to membrane
3. Defective (Type IV)
Symptoms:
* airways obstructed with mucous
* abnormal glucose tolerance (require insulin)
* congenital bilateral absence of vas deferens (azoospermia)
* CF lung
88
Differentiate between chromosomal and gonadal sex
Chromosomal: X or Y
Gonadal: phenotypic appearance
89
The Y chromosome is smaller than the X and contains less genetic material. It contains genes that specify male sexual development known as
1. SRY genes (sex determining region)
* short window of expression
* TDF: testis determining factor
2. AZFc genes
*mutation = can't make sperm
(azoospermia, oligospermia)
3. Pseudoautosomal regions
- homologous sequences at either end of X and Y chromosomes (p arms)
- -can undergo homologous recombination
90
True/False: Recombination can occur between X and Y chromosomes (outside of pseudoautosomal regions) resulting in XX males and/or XY females
True
91
True/False: X-degenerate regions of the Y chromosome are regions on the Y chromosome that are identifiably similar/homologous to the X chromosome. These regions contain single copy genes and are able to escape X inactivation
True
92
X inactivation Center (XIC) is located where? What gene does it carry?
Xq in band Xq 13
XIST:
- -non-coding RNA (>200nt)
- -master regulatory locus for X inactivation
- -expressed on inactive X chromosome
- -silent on active X
- -form heterochromatin (barr body)
- -random inactivation
93
True/False: The amount of XIST RNA produced is dependent on the number of X chromosomes within a cell
True
* all but 1 are inactivated
* XITE is downregulated
94
How is XIST inhibited on the active X chromosome?
1. Express/upregulate XITE
2. activates TSIX
- -antisense of Xist
3. TSIX binds XIST = inhibits XIST
95
Describe X-inactivation during development (embryogenesis)
1. Initially, XIST not expressed in sperm/egg
2. Fertilzation
3. Upregulate XIST of paternal X during 2-cell stage
- -maternal X epxressed
4. Inner cell mass: dec. XIST
- -both X's active
5. Differentation of epiblast: random X inactivation
6. Primordial germ cells:
- -migrate to genital ridge
- -downreg XIST
- -reactivation of X chromosomes
96
During X-inactivation, XIST bind the X chromosome and _____ in the nuclear envelope. Binding enables the X chromosome to be pulled to the nuclear periphery to become heterochromatic.
Lamin B
97
Which of the following describes the characteristics of the Inactivated X chromosome?
A. most genes silenced. Only 15% expressed
B. Facultative heterochromatin: Barr body
C. Enriched for macroH2A histone modifications
D. Enriched for heterochromatin marks
E> All of the above
Answer: All of the above
98
The pseudoautosomal region largely escape activation, however, not all of it escapes. What regions are most likely to be inactivated?
longer length
99
Non-random X-inactivation occurs when the X chromosome is structurally abnormal or in X:autosome translocation. What is important about non-random inactivation?
Prefer to maintain autosomal information (autosomal disomy) rather than X information
100
What are the characteristics of X-linked recessive disorders?
1. only in males (homozygous females are rare)
- -can have mosaic in females
2. No father to son transmission
3. Heterozygous females = carriers
ex: muscular dystrophy, hemophilia
NOTE: XHXH mothers will have unaffected sons; carrier daughters; XHXh mothers -- affected sons and carriers
101
An X-linked recessive disorder that is characterized by excessive bleeding due to a deficiency in factor VIII (no coagulation). The amount of bleeding is inversely related to the amt. of factor 8.
Hemophilia
No treatment:
- -transfusions: contaminated
- -recombinant: antibodies, inhibitors
- -gene therapy: antibodies
102
Fragile X (X-linked disorder) is a trinucleotide expansion disorder that is caused by methylation of CGG/CpG repeats.
What are the clinical features of Fragile X?
- macrocephaly
- developmental and language delay
NO cure: Treat symptoms
103
FMR1 (fragile X mental retardation) is a gene that encodes for the protein FMRP. Mutations of this gene can lead to Fragile X syndrome, and autism in boys. What is the role of FMRP?
FMRP (RNA binding protein) binds pre/post-synaptic proteins and acts as a translational brake on synthesis of synaptic proteins.
It reversibly stalls ribosomes on target mRNA
104
Premutation carriers of Fragile X are at risk of developing:
1. Fragile X-associated tremor/ataxia
- -cerebellar dysfunction
2. Premature ovarian failure by 40
3. Parkinson or Alzheimer's and ataxias
1. adult onset
2. Females
3. Grandfathers w/premutation
*inc. signal in cerebellar peduncles
105
DNA analysis for fragile X syndrome: The patient's DNA is cut with 2 enzymes
1. cuts DNA (methylated or un-methylated)
2. Only cuts UN-methylated DNA
Run gel electrophoresis and probe for FMR1
**See ppt.
- -If methylated: larger band of DNA; top of gel
- -If unmethylated: 2 cuts
*compare to normal
106
True/False: Mitochondria originated among extant bacterial phyla. The alpha-proteobacteria are the closes identified relative
True
2/3 of mitochondrial proteins are bacterial in origin
*Circular DNA
107
The number of mitochondria in a cell is based upon the energy needs of said cell.
What are the functions of mitochondria?
- ATP production (ox phos)
- Lipid and aa metabolism
- Apoptosis
- Sequester Ca2+
- ROS production
Genome:
- 13 proteins
- 22 tRNAs
- 2 rRNA
108
True/False: the properties of mitochondria vary depending on the tissue
True
| localization, fusion/fission, mitophagy, biogenesis
109
What are the properties of the mitochondrial genome?
- 16,000 bp
- thousands of copies (polyploid)
- no introns
- 93% coding DNA (only 3% in nuclear genome)
- 37 genes encoded (20,000 in nuclear)
- maternal inheritance
110
True/False: Mitochondrial DNA replication is relatively accurate (2x10-6 errors/nt) over short repeat sequences. However, longer homeopolymeric tracts (>4bp) can lead to slippage and imbalance in dnTPs.
True
* >300 mutations
* G-T mismatches** (POLG enzyme issues)
111
True/False: Under normal conditions, mitochondrial DNA is homoplasmic (uniform; single type of DNA). However, many pathogenic mutations can lead to Heteroplasmy (non uniform; different DNA sequences).
True
NOTE: Heteroplasmy can reult in mosaic phenotypes
(random sorting during cell division; daughter cells may end up with different proprotions of normal and mutant)
*inc. meiotic disjunction events
112
True/False: The proportion of mutant DNA within mitochondria determines penetrance and the level of expression
True
**Pleiotropy (multiple tissues affected)
113
_______ describes selective degradation of mitochondria by autophagy. This occurs when mitochondria has been damaged (e.g. ROS) and involves PINK and Parkin proteins.
Mitophagy
PINK: moves to outer membrane; recruits Parkin
Parkin: ubiquitinates for degradation by proteosome
114
What are some symptoms of mitochondrial diseases?
- hypertrophic cardiomyopathy
- Leigh syndrome lesions
- sideroblastic anemia
- Hereditary optic neuropathy
- bowel distension
115
Nuclear DNA is inherited from _______, while mitochondrial DNA is inhertied from _____
Nuclear: all ancestors
Mitochondrial: single lineage; mother
116
In sperm, mitochondria are localized to ____, while in oocytes, mitochondria are localized ______
1. sperm: midpiece
- -active
2. oocytes: oocyte plasma membrane
- --silent
- -dec. ROS
117
Models of Mitochondrial maternal inheritance
1. mtDNA is eliminated in mature spermatozoa
2. mtDNA is restricted to 1 blastomere -- forms placenta
3. mtDNA is ubiquinated and degraded in paternal
118
True/False: Females with mutant mitochondrail DNA can produce a healthy embryo via use of a donor with normal mitochondria. The mutant female donates her nuclear DNA, but uses the mitochondria from the unaffected mother.
True
119
In order for a eukaryotic cell to reproduce, it must replicate the DNA in each chromosome and accurately distribute these replicated chromsome into 2 daughter cells. This is carried out by the cell cycle, a dedicated system. What are the stages?
1. G1 phase: resting
2. S phase: chromosomal duplication
- -initiated at ORI
3. G2: rest; edit
4. M phase: duplicated chromosomes are segregated into daughter cells and cytokinese occurs
120
The cell cycle is controlled by two key components:
1. ________ undergo a cycle of synthesis and degradation in each round of division. The most important regulator of Cdk activity.
2. _______ when active, phosphorylates intracellular proteins that initate or regulate major events of the cell cycle.
1. Cyclins
- -regulate Cdk's
2. Cyclin dependent kinase
- -phosphorylate -- activate
- -only active when a cyclin is bound
121
There are 4 different classes of cyclin that are expressed at different (specific) phases of the cell cycle.
When is S cyclin expressed?
- longest time expressed
- expressed in S phase
- overexpressed in G2 (error checking) and M
122
True/False: Initiation of DNA replication is dependent upon an ORI. In humans, ORI are distributed over the entire length of each chromosome, and replication proceeds bi-directionally.
True
*proceeds until adjacent replication forks meet each other/or until reach end
123
What are the steps involved in initiation of DNA replication (formation of the pre-replication complex)
1. ORC recognizes ORI
2. CDc6 and Cdt1 associate with ORC
- -assemble helicase MCM 2-7
3. S-Cdk simulates assembly of other proteins at ORI
4. DNA pol and other proteins recruited at MCM helicase (activation)
5. Begin replication
124
Mutations in ORC 1, ORC 4, ORC 6, CDT1, and CDC6 (pre-replication complex) can lead to this syndrome, which features microtia, absent patellae, lobar congenital emphysema, pachygyria, and ventricular enlargement.
Meier-Gorlin Syndrome
*primordial dwarfism
125
________ polymerases are able to bypass areas of DNA damage and continue DNA replication. Some are error free and insert the correct bases. Others are error prone and insert incorrect bases.
Bypass polymerases
126
List the polymerases involved in eukaryotic DNA replication
1. Pol alpha: RNA/DNA primers
2. Pol beta: Base-excision repair
3. Pol gamma: Mito DNA replication and repair
4. Pol delta: lagging strand synthesis and repair
5. pol epsilon: leading strand synthesis
*pol alpha: only one w/out 3' to 5' exonuclease
127
List the steps in DNA replication
1. ______ opens the template strand and forms a replication bubble at the ORI.
2. _____ synthesizes a primer which is elongated by DNA polymerase
3. The first two primers establish the ______ strands for both L and R. of replication fork. Additional primers will be synthesized along the _____ strand template for synthesis of okazaki fragments.
4. The leading strand will be synthesized by ______, associated with a sliding clamp.
5. The PCNA trimer is assembled around the primed DNA by the clamp-loader __________.
6. The lagging strand is synthesized by _______
1. Helicase -- opens template strand
- -form replication bubble at ORI
2. Pol alpha/primase -- synthesizes a primer
- -elongated by DNA pol
- -2nd primer at opposite template
3. Leading strands: First 2 primers
- -Lagging strands: Additional
4. DNA polymerase epsilon: Leading strand
- -sliding clamp: PCNA (proliferating cell nuclear antigen subunits)
5. replication factor C (RFC).
6. DNA polymerase delta
128
DNA Replication Part II: Explain the process of DNA replication.
1. After separating the template strands, _______ binds to the ssDNA to prevent annealing
2. DNA polymerase alpha is complexed with primase
3. Primase synthesizes an RNA primer containing ~10nt using the template strand.
4. Primase activity ceases and DNA polymerase alpha elongates the RNA primer with ~15-30 _________.
5. Pola/primase dissociates and RFC binds to assemble the PCNA sliding clamp.
1. Replication protein A binds
2. DNA pol A/primase complex
3. Primer synthesis
4. DEOXYribonucleotides
5. Assemble PCNA sliding clamp
6. DNA pol delta (lagging strand) binds PCNA clamp and elongates Okazaki fragments and displaces RNA primer
129
In the lagging strand, the RNA primer can be removed by one of three mechanisms. Short primer flap cleavage is the most common mechanism.
In short primer flap cleavage, DNA pol delta pushes up a small flap (~2-10nt) and then ______ comes and cleaves at the angle of the flap removing the RNA primer.
FEN 1 (flap endonuclease 1)
* DNA pol delta synthesizes DNA as it displaces the RNA primer of previous fragment
* DNA pol delta dissociates leaving a nick
* Sealed by DNA ligase
130
True/False: During replication, the H3 and H4 histones remain bound to the DNA. Gaps are filled in by newly assembled H3-H4.
True
*H2A and H2B join later
131
The ends of linear chromosomes are composed of repetitive DNA sequences (telomeres). Telomeres are important in preventing the loss of gene-coding chromosomal DNA.
Telomeres are 6-nucleotide repeats of TTAGGG at 3' end of DNA. What is the enzyme that catalyzes telomere addition to ends of DNA? What are its features?
Telomerase
* contains a short RNA strand -- template for telomere synthesis
* proteins w/ reverse transcriptase activity
NOTE: Premature aging = shorter than normal telomeres
132
________ is a ribonucleoprotein which has reverse transcriptase capacity. It carries tightly bound non-coding telomerase RNA (TR) which varies between 150-1300nt. It is different from DNA polymerase in that it carries its own template and synthesizes ssDNA.
Telomerase
(a.k.a. telomere terminal transferase)
1. reverse transcrip. = TERT
2. non-coding RNA = TR
3. synthesizes ssDNA
* *Adds G-T repeats
NOTE: activation in somatic cells = inc. tumors
133
In DNA replication, the single stranded 3' end of telomeres tucks into the duplex DNA of the telomeric repeat sequences. This forms
t-loop
134
This is a complex of proteins that binds to telomeres and protects the ends
Shelterin complex (x7)
*no 3' OH at end
135
________ is a disease characterized by prematurely shortened telomeres and progressive bone marrow failure. It results from having only one copy of funtional telomerase RNA gene.
Dyskeratosis congenita
*skin pigmentation, nail dystrophy, mucosal leukplakia
136
Sickle Cell anemia occurs from a transversion that produces an amino acid change in hemoglobin. What is the change?
GAG (glutamate) to Valine (GTG)
*CRISPR gene editing
137
True/False: Werner syndrome causes premature aging due to genetic instability
True
138
List examples of 3 nucleotide insertion mutations
1. Hungtington - CAG (Gln)
- -threshold: >35
2. Fragile X - CGG (Arg)
>230
3. Myotonic dytrophy: CTG (Leu)
- ->50
* if in important motif = bad
* beyond threshold = bad
139
There are three general classes of DNA damage:
1. Single base changes
2. Structural distortions
3. Strand breaks
Strand breaks are caused by high energy radiation (X-rays) and generation of reactive oxygen species that lead to a break in one or both strands. How are the anti-neoplastic drugs Bleomycin and Doxorubicin associated with strand breaks?
*Both cause double stranded breaks
1. Bleomycin:
- binds DNA; forms ROS
2. Doxorubicin:
- intercalates into DNA
- inhibits topoisomerase II
140
DNA damage: Single base changes
Deamination involves removal of an amino group. There are three possible types:
1. Cytosine deamination
2. Guanine deamination
3. Adenine deamination
Describe each
1. Cytosine:
- produces U
- U pairs w/ A
2. Guanine:
- produces Xanthine
- X pairs w/ C
3. Adenine
- produces HypoXanthine
- HX pairs w/ C
141
DNA Damage: Single base changes:
Hydrolysis of the N-glycosyl bond between a purine and deoxyribose, resulting in an abasic site
Depurination
142
DNA Damage: Single base changes
| Addition of a methyl or other alkyl group to bases
e.g. O6-methylguanine
Alkylation
143
DNA Damage: Single Base Changes
The combination of a substance with oxygen. G pairs with C, but 8-oxoguanine pairs with A.
Oxidative damage
144
DNA Damage: Structural Distortions may cause mutation or physically impede replication or transcription.
Pyrimidine dimers (thymine-thymine dimer) is an exmaple. How?
Form Covalent bonds between adjacent Thymines in the presence of UV light
*distorts double helix geometry
145
DNA Damage: Structural Distortions
How do Benzo-alpha pyrene (tobacco smoke, coal tar, charbroiled food) and Cisplatin (anti-neoplastic) affect DNA structure?
1. Benzo-a-pyrene:
- intercalates in DNA
- binds Gs at N2
- distorts
2. Cisplatin
- crosslinks DNA
- binds purines at N7
- prevent DNA replication
146
DNA Repair: Excision Repair accurately repairs DNA damage. There are two types:
1. Base Excision Repair
2. Nuclear Excision Repair
Base Excision repair fixes what kinds of structures? What enzymes does it use?
Structures:
- alkylated bases
- deaminated bases
- abasic sites
DNA glycosylase - recognizes altered base and catalyzes removal
(ex: Uracil DNA glycosylase)
147
DNA glycosylase is an enzyme involved in base excision repair. It recognizes DNA damage, and removes the base by cleaving the N-Beta glycosyl bond, leaving an abasic site. How is this site repaired?
1. 5' end is cleaved by AP endonuclease (APE1)
2. 3' side is cleaved by DNA pol Beta (AP lyase activity)
- -also fills gap
3. Nick sealed by DNA ligase
* prokaryotes: Pol I
* euk: long patches: Flap endonuclease
148
DNA Repair: Excision Repair accurately repairs DNA damage. There are two types:
1. Base Excision Repair
2. Nuclear Excision Repair
In Nuclear Excision Repair, the damage (e.g. thymine dimer) is recognized by a DNA complex (enzymes), and nicks are made on either side of the damage by what enzyme?
After the oligonucleotide is released, DNA pol fills the gap and it is sealed by ligase.
Excinuclease (2 endonuclease - 5' and 3' nicks)
--betwen 12 and 15nt later on each side
149
In humans, there are 20-30 proteins that participate in NER. Specific defects in proteins that participate can lead to Xeroderma Pigmentosum. What are features?
Dry skin and freckles
* phostosensitivity
* 1000-fold risk of skin cancer
* progressive neurodegeneration
150
There are several genes, that, when mutated give rise to Xeroderma Pigmentosum. These genes and their protein products are designated by XP.
What are they?
1. XPC – damage recognition
2. XPB – has helicase activity to open double-stranded DNA.
3. XPD – has helicase activity to open double-stranded DNA
4. XPF – a subunit of the 5’ endonuclease.
5. XPG – 3’ endonuclease.
6. XPA – involved in the assembly of the preincision complex.
151
There are several genes, that, when mutated give rise to Xeroderma Pigmentosum. These genes and their protein products are designated by XP.
What are they?
1. XPC – damage recognition
2. XPB – has helicase activity to open double-stranded DNA.
3. XPD – has helicase activity to open double-stranded DNA
4. XPF – a subunit of the 5’ endonuclease.
5. XPG – 3’ endonuclease.
6. XPA – involved in the assembly of the preincision complex.
152
Nucleotide Excision Repair is also capable of RNA polymerase rescuing, also known as Transcription-coupled DNA repair.
This repair involves recruitment of NER proteins to the stalled RNA polymerase. How does it differ from NER?
RNA pol II is the damage sensing protein in the cell
--stalls and backtracks to degrade the messed up RNA
*TFIIH plays a crucial role
153
Mismatch (strand-directed) Repair corrects Replication errors. In the bacterial system, mismatch repair can easily scan hemi-methylated nascent DNA from methylated parental DNA. Mismatched base pairs create a distortion in the DNA which is recognized by what protein? Describe the steps in mismatch repair.
Prokaryotes:
-Dam methylase -- N6 of Adenine (5' GATC)
1. Mut S find mismatch
2. Recruit Mut L
(scanning bi-directional; ATP)
3. Finds GATC (hemi) site
4. Recruits MutH
- -endonuclease (site specific)
- -cleaves unmethylated GATC (daughter)
5. Muts-Mut L recruit helicase II
- -unwind DNA
6. Stop shortly after mismatch is excised
7. SSb coating -- filled in by Pol III and Ligase
154
Hereditary nonpolyposis colorectal cancer (HNPCC, or Warthin-Lynch syndrome) is a mutation in mismatch repair enzymes resulting in the inability to remove single nucleotide mismatch. The genes affected are: MLS1, MSH2, MSH6 and PMS2 which are homologues of E. coli
mutS and mutL genes
155
Accurate DNA polymerases (i.e., DNA polymerase delta and epsilon,) will not insert bases opposite lesions (e.g., thymine-thymine dimers).
However, eukaryptes do have error-prone DNA polymerases that insert nucleotides opposite lesions in a process referred to as ____________
Translesion synthesis
- no proofreading ability
- all fall off
1. DNA polymerase eta (n): incorporates two A's opposite thymine dimers.
2. DNA polymerase iota (i): incorporates a nucleotide opposite abasic sites and adducted bases.
3. DNA polymerase xi (squiggly E): incorporate nucleotide randomly across non-coding lesions.
NOTE: Pol delta and epsilon can take over once lesion is spanned
156
DNA Repair: Double Strand Breaks
There are two mechanisms for repairing double stranded breaks (due to ionizing radiation):
1. ________________: broken ends joined by ligases
- -loss of nucleotides at joining
- -somatic cells and old age
2. _______________: DNA repaired using sister chromatid as template (S and G2 phases). No loss of info.
1. Non-homolgous joining
2. Homologous joining
- -accurate repair
157
Homologous Recombination Defects: True/False
Mutations in Brca 1 and 2 genes compromise homologous recombination DNA repiar leading to breast and ovarian cancer.
True
158
_________________describes a rare neurodegenerative disease characterized by:
1. Ataxia = poor coordination
2. Telangiectasia = small dilated blood vessels
3. Progressive cerebellar degeneration, telangiectasia, immunodeficiency, premature aging and predisposition to cancer development
Ataxia Telangiectasia (AT)
- mutation in DNA recombination repair enzymes (homologous recombination)
* hypersensitivity to ionizing radiation
159
DNA Repair:
Nonhomologous end joining (NHEJ) is an important pathway for repairing double strand breaks. Describe the steps
1. Ku70-Ku80 binds to the DNA ends.
2. DNA-PKcs (DNA-dependent protein kinase catalytic subunit), complexed with Artemis, bind Ku70-Ku80.
3. DNA-PKcs autophosphorylate and phosphorylate Artemis.
4. Phosphorylated Artemis acts as an endonuclease and generates overhangs on each side of the break.
5. The DNA ends anneal and Artemis cleaves any unpaired DNA segments.
6. Small DNA gaps are filled in by DNA pol µ or λ (shown as red lines in the previous figure).
7. Nicks are sealed by a complex containing a DNA ligase (XLF-XRCC4-DNA ligase IV).
160
List the enzymes involved in non-homologous end-joining
1. Ku70-Ku80 - bind DNA ends
2. DNA PKcs - protein kinase catalytic subunit
3. Artemis - nuclease
4. µ or λ - fills in gaps
5. XRCC4, XLF, ligase IV - seals nicks
161
_________ is a process by which an RNA strand (primary transcript) is made from a DNA template strand using RNA polymerase. The gene is the basic functional unit of this process.
Transcription
162
Transcription: The primary transcript (RNA strand) is identical in sequence to the ___1____ strand, and is complementary to the ____2____ strand.
1. Identical to Coding Strand
- -except U instead of T
2. Complementary to Template
* top strand usually coding strand, bottom usually template
163
A sequence of DNA that encodes an RNA (encoding region). It also contains DNA sequences that regulate the production of the encoded RNA (transcription)
examples: (e.g. 5' promoter and 3' regulatory sequences/enhancers)
Gene
* unit of transcription
* The RNA may be translated to polypeptide
164
Transcription:
1. RNA synthesis is catalyzed by _________.
2. Transcription begins when RNA polymerase binds to the _____ at the start of the gene (Initiation). It is normally upstream of the start point (1st bp) and forms a transcription bubble.
3. _______, or the sequential addition of ribonucleotides can then occur.
1. RNA polymerase
2. Promoter (DNA)
3. Elongation
*RNA pol moves along the template until it reaches a terminator sequence and then releases from the DNA strands (Termination)
NOTE:
closed complex: pre-initiation
Open complex: Initiation
165
The transcription start stie is known as the
+1 position
Promoter and untranscribed areas: negative (upstream)
Coding sequence: positive (downstream)
166
List some of the similiarities between DNA replication and transcription
1. Substrates: 4 NTP's
2. Use enzymes to select NTP (template-directed manner)
3. 2 DNA strands must be transiently separated (bubbles) to gain access to genetic info
4. Linkages formed between nucleotides are 3' to 5' phosphodiester bonds
5. Chain growth occurs in 5' to 3' (Watson Crick)
167
How is DNA transcription different from Replication?
1. asymmetric (one strand is the template)
2. RNA molecules are discrete in size
- -specified by Start and Stop
3. As RNA chain is synthesized, it is not H-bonded to DNA template
- -ss molecule
4. RNA pol can initiate synthesis of RNA de novo: no primer
5. small region (10-12bp) of DNA is unwound at a time
- -replication bubble: 100 nt
6. The RNA product is extensively modified after transcrip.
7. Many copies are made
(replication: only 1 copy)
8. RNA pol do NOT contain proofreading activity
- -higher error rate
168
RNA polymerase catalyzes the addition of nucleotides to a growing RNA chain in the 5' to 3' direction. It uses DNA as a template and adds ribonucleotides. It does NOT need a primer to initiate RNA synthesis.
What are the three nuclear RNA polymerases and one mitochondrial polymerase involved in transcription of eukaryotic cells?
1. RNA polymerase I: Pre-rRNA (5.8s, 18s, 28s)
2. RNA polymerase II: pre-mRNA (protein-coding and snRNA's)
3. RNA pol III: tRNA, 5srRNA, snRNA's, small RNA's
169
True/False: RNA polymerase plays a role in maintaining the transcription bubble (site of RNA synthesis). In addition, it has distinct channels for DNA to enter and exit, for rNTP's to enter, and for the growing RNA transcript to exit.
True
170
________ is produced by Amanita phalloides mushrooms. It acts as a differential inhibitor of eukaryotic RNA polymerases. At low concentrations, it inhibits RNA pol II, at high concentrations it inhibits pol III.
alpha-amantin
* low conc. 1mg/ml
* high conc. 30mg/ml
171
_____________ are needed to initiate transcription in eukaryotes. These factors recognize and bind specific DNA sequences within a promoter. Once they bind to the promoter, they participate in recruiting DNA polymerase.
Transcription factors
* RNA pol I and III: small # of tf's
* RNA pol II: highly variable promoters bound by many different tf's (no universal promoter)
NOTE: prokaryotes have delta 70: (-10) and (-35)
172
True/False: Promoters for RNA pol II are made up of different combinations of different consensus sequences that are recognized by different transcription factors.
True
173
There are different elements within an RNA pol II promoter:
1. __________: consensus sequences in the immediate vicinity of the transcription start point. (where we find tf's)
2. _________: consensus sequences that bind tf's within 100-200 bp upstream (in front of) the start point
3. _________: consensus sequences that can be on either side of the start point, in any orientation and at great distances away.
1. Core (basal) elements
* **TATA
2. Proximal elements
3. Enhancer elements
174
Core elements for RNA Pol II are bound by General (basal) transcription factors. These are the minimum factors needed to transcribe a gene using RNA pol II.
What are their functions? What are examples?
* low level transcription
* position RNA pol II near start point
* aid in opening DNA (open complex)
* promote release of Pol II from promoter
ex:
1. TFIID: TATA and recruits TFIIB
- --used by all 3 polymerases
2. RPII: catalytic
3. TFIIH: helicase activity and protein kinase acitivty (phosphorylates CTD of RPII - release from promoter)
175
Before transcription can begin, a pre-initiation complex must be formed. What are the general transcription factors involved?
1. TFIID - assembly
- -TBP on TFIID binds TATA
- -conformational change of helix
- -other TFIIX factors join
2. TFIIH - elongation
--hydrolyzes ATP
--opens double helix and phosphorylates RNA pol II
(C-terminal domain)
176
Proximal and Enhancer elements are consensus sequences that, when bound to the transcription factors, increase the efficieny of RNA pol II and transcription. How do they differ?
1. Proximal:
- 100-200 bp upstream of start
- inc. tf assembly
2. Enhancer elements
- either upstream or downstream
- distance away (50 kb from promoter)
- inc. tf assembly and promoter transcrip regardless of orientation (loop)
- yeast: upstream activator sequences
177
Describe the process of transcription by RNA pol II
1. Pol II recruited to DNA by tf's
- -prereplication complex
2. Formation of transcription bubble
a. Initiation complex
b. Elongation complex
c. Phosphorylation of CTD during initiation
3. . Elongation
4. Transcription terminates and CTD is phosphorylated
178
True/False: initiation is primer independent and produces short, abortive transcripts, while transcription elongation is continuous until termination.
True
179
What are methods used by polymerase during elongation to improve accuracy?
1. Pyrophosphorylosis
2. Nucleolytic proofreading
1. Pyrophosphorylosis (kinetic proofreading):
- -polymerase stalls after mismatches and removes incorrect base
- -catalytic reaction runs in reverse
2. Nucleolytic proofreading:
- polymerase reverses direction by one or few nt
- breaks RNA phosphodiester bond upstream from mismatched base
*error rate: 1 in 10^5
180
True/False: Transcription: Eukaryotic RNA undergoes simultaneous processing to produce mature and functional RNA molecules after synthesis.
RNA processing may include:
1. Nucleotide removal/addition
2. Base modifications
3. Separation of RNA sequences
True
181
Transcription: Pre-mRNA processing is highly coordinated and occurs in the nucleus.
It involves:
1. 5' capping
2. Intron Splicing (snRNPs)
3. Poly A binding proteins
4. Poly A tail (3')
What are the enzymes involved in 5' capping of the pre-mRNA?
1. C-terminal domain phosphorylation (RNA pol II)
- -serine 5' = initiates 5' capping
- -serine 2 = initiates splicing
2. Gaunylyltransferase: Adds 5' cap
- -once capping is complete, cap binding complex (CPB) binds
NOTE:
* 5' to 5' phosphate linkage via Guanine 7 methyltransferase
* partial methylation: 2' O mehtyltransferase
182
Transcription: Pre-mRNA processing is highly coordinated and occurs in the nucleus.
It involves:
1. 5' capping
2. Intron Splicing (snRNPs)
3. Poly A binding proteins
4. Poly A tail (3')
RNA polymerase transcribes past the site corresponding to the mature 3' end. How does the 3' Poly A Tail get added?
1. Endonuclease cleaves transcript ~20nt of AAUAA (3')
(polyadenylation consensus sequence)
--CTD of pol II
2. Poly A polymerase (PAP) makes tail
3. Poly A binding proteins (PABP) bind to tail
*add 20-200 adenine at 3' site
183
Transcription: Pre-mRNA processing is highly coordinated and occurs in the nucleus.
It involves:
1. 5' capping
2. Intron Splicing (snRNPs)
3. Poly A binding proteins
4. Poly A tail (3')
Pre-mRNA possess exons (joined together = mature mRNA) and introns (intervening sequences) that need to be removed. How are these introns removed?
Spliceosome
(snRNA's and proteins - 3' and 5' ends)
Introns: always begin with GU and end with AG
NOTE: 2 transesterification reactions:
- -branch point: 2' OH adenine attacks 5' splice site
- -2'-5' lariat structures
- -second reaction (5' attacks 3') joins the exons
184
Alternative splicing can give rise to multiple mature mRNA's from a common primary transcript. These splicing patterns can be tissue specific.
What are examples of 2 different proteisn that are formed from the same gene via splicing?
Clacitonin (1-4) and Calcitonin-related gene transcript (1-6)
185
Epigenetic modifications of histones
1. phosphorylation
2. acetylation
3. methylation (me, me2, me3)
4. ubiquitination
5. sumoylation
1. phosphorylation
chromosome condensation
2. acetylation
promotes gene expression; separates histone tails
3. methylation (me, me2, me3)
inhibit gene expression
4. ubiquitination
targets histone for
methylation
5. sumoylation
inhibit gene expression
6. biotinylation
- enriched at transcriptionally silent chromatin
7. ADP-ribosylation
H1 ribosylation associated with relaxed chromatin
structure; associated with protection of genomic DNA
methylation
186
Epigenetic factos are hereditary factors that are independent of DNA sequence. Imprinting involves inhibition of gene xpression that is inherited. Imprinted genes are not expressed and are inactive. What causes imprinting?
Histone modification (deactylation -- chromatin condensation)
hypermethylation of CPG islands (5' UTR)
187
Cytosine methylation
1. CpG poor promoters are frequently _____ in gametes and early development
2. CpG rich promoters are ________ methylated during early development and adulthood
1. Frequently methylated
| 2. Infrequently methylated
188
Identity by descent
1. Twins (identical)
2. Fraternal twins
3. 1st degree relatives
4. 2nd degree relatives
5. 3rd degree relatives
1. 100%
2. 50%
3. 50%
parents, siblings, children
4. 25%
- -aunts, grandparents, half sibilings
5. 12.5%
- -first cousins
189
What is an example of environmental regulation on genes?
1. Agouti gene -- yellow and obese
- -can be deactivated by high methyl diet
2. In utero effects can be negated by childhood experiences
190
Malnutrition in utero is associatd with
1. adverse metabolic profile
- -suboptimal glucose handling
- -high BMI
2. elevated LDL cholesterol
3. inc. risk of shizophrenia
191
During early development (zygote), there is high methylation of maternal genes and low methylation of paternal genes. When does this change?
1. Morula stage
- -low methylation of both
2. Blastocyst
- -high methylation of both
192
True/False: Pluripotent stem cells are repressive/permissive for gene expression based upon their lineage (what they are going to become)
True
*different methylation patterns for different types of cells
193
Epigenetic regulation of the CIrcadian rhythm
Bmal 1 (master regulator)
--lights *ZT10) on vs. lights off (ZT12) methylation
194
True/False: There is epigenetic regulation of the menstrual cycle that contributes to the induction of various genes during different stages of the cycle
True
195
There are epigenomic changes that occur during aging. Explain what happens
global hypomethylation
-hypermethylate tumor suppressor genes -- no brake on cell cycle
--acquire mutations; cancer
196
Give an example of how chronic pathology affects the epigenome?
--ex: have prolonged expression of chemokines
--sensitization and neuropathic pain
197
There are epigenetic changes that can occur in the germline and can be passed on to offspring and demonstrate increased severity. This describes
Epigenetic anticipation
198
Examples of epigenetic diseases
1. fragile X syndrome
2. prader willi (15q11--13)
3. angelman
199
Small RNA's like miRNA control regulation of many genes. Eukaryotes also use short interfering RNA (siRNA) to prevent protein synthesis of mRNA,
How do they perform translational silencing?
DGCR8/Drosha: binds mRNA
--removes 5' cap and poly A tail
Exportin 5: carries precursor mRNA to cytoplasm
DIcer proteins: mature miRNA
RISC: binds 3' UTR of mRNA to silence
200
What are mechanisms by which RNA polymerases terminate transcription?
1. Pol I and III:
- -T-rich sequences from 3' end
2. Pol II:
- few bp to several kbp downstream from 3' end
- coupled w/ 3' end porcessing
201
Two models have been proposed for 3' end processing via Pol II (during transcription)
1. Allosteric (anti-terminator model) which induces a confromational change in Pol II elongation proteins, terminating transcription.
2. The torpedo model which is more widely accepted.
Explain the torpedo model
-cleavage after poly A site creates an entry site for 5' to 3' exonuclease to degrade remaining RNA
202
rRNA is transcribed in the nucleolus. The rRNA genes are located in specific chromosomal regions known as the "nucleolar organizer". Hundreds of these genes occur in tandem in these regions.
Where are these regions found? How does Pol I transcribe these?
Chromosomes: 13, 14, 15, 21, 22
1. Class I tf's bind promoter
- -recognition by Pol I
2. UBF (upstream binding factor) binds core sequence and control elements
3. SL1 w/ TBP binds to UBF and Pol I initiating transcription
- -TBP bends DNA = open complex
NOTE: each gene has its own promoter and is transcribed separately
203
RNA pol I synthesizes 3 of the 4 rRNA's in the nucleolus. Thes rRNA's are produced as a large 45S precursor which is then broken down into its respective subunits: 28s, 18s, 5.8 s by ______
snoRNPs
204
RNA polymerase III transcribes 5s RNA and tRNA's. The promoter for RNA pol III lies within the transcribed region and the tf's involved bind RNA pol III and DNA.
Explain how RNA pol III does its job
1. Pol III tRNA promoter
- -Box A and B sequence elements
- -bound by TFIIIB and TFIIIC
2. Pol III rRNA promoter
- -Box A and C
- -TFIIIA, B, C
*these factors recruit Pol III to the trasncription start site
205
THe primary transcript of tRNA's usually have extra nucleotides at both the 5' and the 3' ends as well as an intron. How is this extra info removed?
1. 5' end:
- RNAse P
2. 3' End:
- exonuclease
3. Intron:
- endonuclease
*mature tRNA
4. CCA3' sequence must be added
- -tRNA nucleotidyltransferase
206
Some bases are modified by specific enzymes to the following:
1. Thymine by methylating ______
2. Pseudouridine, by rotating ______ and forming a C-C bond
3. Dihydrouracil, by reducing the ______ bond in uracil.
1. uracil
2. uracil
3. double-double bond
207
Translation is the process in which genetic information present in an mRNA is translated to a sequence of amino acids during protein/synthesis.
What are the necessary machinery for translation?
1. Remove CPB and circularize mRNA
- ELF4G/4E = 4F
2. mRNA
- -transcript (codons)
- monoscistronic
(prok - poly; no cap)
3. tRNA
- transport aa's to ribosomes
- adaptor molecule w/ anticodons
- CCA 3' sequence: binds aa's
- aminoacyl tRNA synthetases
4. Ribosomes
208
________ provide an environment for the recognition of codons in the mRNA by tRNA's. THey help catalyze the formation of a peptide bond between every amino acid of a polypeptide change.
ribosomes
209
An Open Reading Frame begins with an ______ (start) and continues in the sequential triplets to a termination codon (STOP). 2 of the 3 reading frames are typically blocked by START codons.
Start: AUG (methionine)**
--ATG sometimes
STOP: NOT part of reading frame
210
mRNA consists of a non-overlapping linear series of codons (three-base sequence). There are 64 possible codons, but 61 code for amino acids. Three are STOP codons.
What are the codons for STOP, START (meth) and tryptophan?
Start: (met) AUG
Tryp: UGG
STOP: UAA, UAG, UGA
*degeneracy of genetic code: different codon triplets code for the same aa
NOTE: degeneracy, but each codon can only code for one amino acid
211
True/False: The anticodon of each tRNA base pairs with codons in mRNA in a complementary and anti-parallel manner.
True
212
Some tRNA's recognize more than one codon. How is this possible?
Wobble hypothesis
- less stringent base pairing between the 1st position (anticodon) and 3rd position (codon/degenerate)
* non-watson-crick
(ex: G-U)
213
Amino acids must be activated by linkage to an appropriate tRNA before being incorporated into polypeptides. What molecule does this? What is significant about this molecule?
aminoacyl tRNA synthetases
* specific for amino acid
* recognize 1 or more tRNA
* *proofreading abilities ensure correct aa is added
214
Aminoacyl tRNA's are based on the amino acid they carry
1. ______ is the tRNA that carries alanine (charged with alanine)
2. In eukaryotes, ______ is the tRNS that specifically recognizes the start codon (AUG).
3. _______ is tRNA that carries methionine, but does not recognize the start codon (Met)
1. tRNA^ALA
2. tRNAi^Met
* i = initiator
* MettRNAi ^Met when charged
3. tRNAm^Met
