1
Q
Shotgun Strategy
A
- DNA extraction
- DNA fragmentation (broken up into tiny pieces)
- Cloned into vectors
- Transform Bacteria, hybridize and grow, isolate the vector DNA
- Sequence genome library
- Assemble continuous fragments using computer software
2
Q
Primer Walking Strategy
A
- Start sequencing from specific site in genomic DNA
- Design pimer at a site based on sequence information
- Start sequnecing with designed primer
- Repeat until formed sequence
- Often used to “close the gaps” in shotgun sequencing, or obtain small sequence
3
Q
Enzymatic Method
A
- Single stranded molecules are sequenced
- All strands of different lengths end with ddA - causes it to stop
- Strands are separated by length in gel electrophoresis
- Anneal primer to one dna strand
- Split into 4 reactions: dATP, dTTP, dCTP, dGTP
- DNA polymerase is added to all of the rxns
- longest DNA strand is furthest from primer
- shortest strand is shortest from primer
- causes chain termination
4
Q
DNTP
A
OH group allows strand extension at 3’ end
5
Q
ddNTP
A
does not allow extension at 3’ end
6
Q
Standard Vector Primers for sequencing
A
- need new primer for every new DNA template sequenced
- cannot design a primer if no previous sequence information is known
- insertion site are binding sites for vector primers
7
Q
Automated Sanger DNA sequencing
A
- allows for large scale DNA projects
- Use a different colour fluorescent dye to tag each ddNTP
- The DNA primer is unlabeled
ADVANTAGES: - Can read up to 900 nucleotides per Rx
- Cost effective —› <$4 per sequence determination with a standard plasmid vector primer
- Can perform all 4 ddNTPs Rx in one sample and load in same lane of gel
8
Q
Next Gen Sequencing
A
Examples
- High-throughput sequencing (HTSeq)
- Massively Parallel Sequencing
- Sequencing By Synthesis - SBS (Illumina)
- Pyrosequencing (454 Life Sciences)
- Ion semiconductor sequencing (Ion Torrent Systems)
- SOLiD - Sequencing by Ligation (Applied Biosystems)
ADVANTAGES:
- Can obtain 800 - 5000x more DNA sequence per day
- 6 - 25x cheaper than “old” technology
- Disadvantage over Sanger method
ELIMINATES:
- insertion and cloning of DNA
- transformation of vector to BAC
- isolation of plasmid
ADDED:
- ligate “adaptor sequences” to each end of DNA fragment and PCR amplify
Prep of genomic library
- isolate genomic DNA
- fragment genomic DNA
- Ligate DNA primers (adaptors) to each end of genomic fragments
- attach tagged DNA fragments to slide
- PCR amplify to abtain large #
- perform DNA sequencing reaction directly through the slide
9
Q
Third Gen Sequencing
A
- Single molecule real-time sequencing (SMRT) – Pacific Biosciences (PacBio)
- Nanopore sequencing – MinION Portable Gene Sequencer (Oxford Nanopore)
-Pool of dNTPs, each labeled with a different colour fluorescent tag attached to g-phosphate is added to well. - Due to location of laser and detector -> only the labeled dNTP transiently held by DNA pol could be detected -> recorded as a pulse of coloured light.
- When the dNTP is incorporated into DNA being synthesized -> the a-phosphate is cleaved off -> pulse of light is lost.
- Pulses of different colour light correspond to different dNTP incorporated by single DNA polymerase in PacBio (SMRT) sequencing
- Each type of dNTP is labeled with different colour fluorescent tag. In this example: yellow -> C, red -> G, blue -> A, green -> T.
DISADVANTAGES in contrast to illumina - HIGHER ERROR RATE
- INCREASED COST
- NOT AS WELL ACCESSED
10
Q
Illumina sequencing
A
- DNA sequencing by synthesis but does not involve permanent chain termination
- All 4 colours can be viewed after each cycle
- Each modified nucleotide has a different coloured fluorescent tag
T - green
A - yellow
C - red
G - blue - a solution is added to remove the block off the 3’ end
11
Q
3 examples of large scale genome projects
A
Personal genome project
–> Correlate genotypes with health info
Cancer genome project
–>Correlate drug sensitivity
Pediatric Cancer Genome
–>Find genetic causes of childhood cancers
12
Q
Reading Frames
A
- There are 6 potential protein sequences that can be derived from any double-stranded DNA fragment
13
Q
What is the rate of stop codons
A
1/21.3
14
Q
What is the correct reading frame
A
The longest strand with no stop codon
15
Q
What is the central dogma
A
DNA–>RNA–> protein
- DNA is for permanent storage of genetic information
- RNA is for transient storage of genetic information
- Protein is the final gene product
16
Q
RNA polymerase
A
- major enzyme involved in transcription
- RNA is synthesized from 5’ —› 3’ from DNA template
- Catalyzes formation of phosphodiester bond from linkage of ribonucleotides (rNTPs)
- Does not require primer to initiate synthesis
- Modest proofreading mechanism → - Error rate of 1/104 rNTPs incorporated
17
Q
DNA polymerase
A
- major enzyme involved in DNA replication
- DNA is synthesized from 5’ —› 3’ from DNA template
- Catalyzes formation of phosphodiester bond from linkage of deoxyribonucleotides dNTPs)
- Require pre-existing primer (either DNA or RNA) to initiate synthesis
- Good proofreading mechanism → - Error rate of 1/107 dNTPs incorporated
- can stop or stall causing hairpins
18
Q
What are mRNAs
A
Messanger RNAs
- code for proteins
Percent?
3-5% of RNA
19
Q
What are rRNAs
A
Ribosomal RNAs
- form the basic structure of the ribosome and catalyze protein synthesis
Percent?
Majority of RNA 75%
20
Q
what are tRNAs
A
Transfer RNAs
- central to protein synthesis as the adaptors between mRNA and amino acids
Percent
10-15%
21
Q
Telomerase RNA
A
Serves as a template for telomerase enzyme that extends ends of chromosomes
<5%
22
Q
snRNAs
A
Small nuclear RNAs
- function in a variety of nuckear processes, including splicing pre-mRNA
<5%
23
Q
snoRNAs
A
Small nucleolar RNA
- help to process and chemically modify rRNAs
<5%
24
Q
IncRNAs
A
Long non-coding RNAs, not all of which appear to have a function, some serve as scaffolds and regulate diverse cell processes, including X chromosome inactivation
<5%
25
miRNAs
MicroRNAs
regulate gene expression by blocking translation of specfic mRNAs and causing their degradation
<5%
26
siRNAs
Small-interfering RNAs
turn off gene expression by directing the degradation of selective mRNAs and helping to establish repressive chromatin structures
<5%
27
piRNAs
Piwi-interacting RNAs
bind to piwi proteins and protect the germ line from transposable elements
<5%
28
First Human Genome Sequencing and who was involved
- cost 2.7 billion
- took 15 years
- 2013
- Francis Collins
- Craig Ventor
29
Genome Library
All possible DNA fragments of genomes
30
What is a Contig
A series of overlapping DNA sequences used to make a physical map that reconstructs the original DNA sequence of a chromosome or a region of a chromosome
31
What would happen in Sanger sequencing if too much ddA is present
The odds of pulling a ddA are very high which would cause chain termination or a stoppage
Contrast, if there was too little ddA, chains will be longer
32
Tell me about gel electrophoresis
- bottom is the positive node
- from bottom to top; smallest to largest
- Bcuz negative and positives attracts(smaller the space smaller the DNA)
- 5' end is lined up at the positive node
33
Why are adaptors added in Next Gen sequencing
- you know the sequence of the adapter therefore you know what primer you need to use
34
Why is a PCR step added in Next Gen sequencing
- To build up an amount of copies to have a bigger signal
35
Would a signal with 2x the regular intensity be likely to 2x incorporation of the same nucleotide in the SAME cycle in Illumina sequencing
No
36
Which of the following statements are FALSE about comparison between RNA and DNA polymerase:
1. The proofreading of RNA pol is not as good as DNA pol
2. Both RNA and DNA pol catalyze the formation of phosphodiester bonds
3. While DNA pol synthesizes DNA from a DNA template, RNA pol synthesize RNA from RNA template
4. While DNA pol requires primers to initiate DNA synthesis, RNA pol can initiate RNA synthesis de nova
5. None of the above
While DNA pol synthesizes DNA from a DNA template, RNA pol synthesize RNA from RNA template
37
Which of the following is TRUE about DNA sequencing:
1. The DNA sequences for both strands of double-stranded DNA template are usually obtained to check for any technical errors in the sequencing procedure
2. In sanger dye terminator sequencing with fluorescent tags, the DNA sequence of both strands of double stranded dna template can be determined simultaneously in the same tube
3. In sanger dye terminator sequencing it is still necessary to split the reactions into 4 samples for each ddNTP
4. Nanopore is another form of sequencing involving chain termination by the incorporation of modified nucleotides
5. 2 of the above
The DNA sequences for both strands of double-stranded DNA template are usually obtained to check for any technical errors in the sequencing procedure
38
Error rate of DNA polymerase
1/107
39
Error rate of RNA polymerase
1/104
40
sigma factor
called holoenzyme
Completes the enzyme and binds to promoter sequence
41
Transcription cycle of RNA polymerase
Initiation starts and sigma factor and core subunit associate to form holoenzyme
holoenzyme slides along DNA
sigma factor recognizes promoter sequence on DNA
Transcription bubble forms: ~10 ntd of single stranded DNA
Scrunching occurs - short fragments of RNA, pulls DNA towards promoter
RNA polymerase breaks free of promoter, and releases sigma factor
Elongation starts, the jaw closes and increases rate of transcription
Termination starts, RNA pol reaches and transcribes termination sequence on template DNA
Hairpin structure formed in RNA corresponding to the DNA termination sequence causes the RNA pol to release its hold on RNA
42
Hairpin
Forms inverted repeats and only forms on RNA because RNA is single stranded so it snaps together.
43
Which of the following statements are false:
Initiation of transcription occurs at the ATG start codon
Promoters are regulatory DNA sequences found infront of genes
Initiation of transcription occurs at the ATG start codon
- This is translation start
44
UTR
Transcribed not translated
45
Consensus Sequences
are located within the promoter region
46
High expression Gene
Large amount of protein and RNA
47
Low expression gene
small amount of protein and DNA
48
No expression gene
No protein
49
How many strands of DNA are used as a template for RNA synthesis
One strand of DNA
50
Promoter Sequences
Are asymmetrical so RNA polymerase can only bind in one direction
Bottom strand is tempate strand if RNA pol is proceeding 5'--> 3' , left to right
Top strand is template strand if RNA pol is proceeding 3'-->5' right to left
51
What RNA is transcribed by RNA pol I
5.8S, 18S, 28S rRNA
alpha amanitin - insensitive
52
What RNA is transcribed by RNA pol II
mRNA, snoRNA, miRNA, siRNA, LncRNA, snRNA
alpha amanitin - very sensitive
53
What RNA is transcribed by RNA pol III
tRNA, 5S RNA, some snRNA and other small RNA
alpha amanitin - moderately sensitive
54
What does eukaryotic RNA pol require the presence of to bind to promoters
Requires the presence of additional initiation proteins (transcription factors) before they can bind selectively to different promoter sequences.
55
Order of transcription binders
TBP, TFIID, TFIB, TFIIA, TFIIF, TFIIH, TFIIE
56
What does a typical eukaryotic RNA polymerase II core promoter consist of
TATA box
Initiator element (INR)
57
Transcriptional start site is marked with what
+1 arrow
58
TBP
TATA binding protein
usually the first transcription factor to bind
59
Formation of the PIC
Transcription pre-initiation Complex
INR is start of transcription
TBP binds to TFIID
TFIIB then binds to TFIIA
Both bind together
The kink in the promoter is the physical location
TFIIF then binds to RNA polymerase
TFIIH then TFIIE then all bind together
This begins initiation of transcription (UTP, ATP, CTP, GTP,) which begins elongation
60
Phosphorylation of Ser in Hepapeptide
*Still have to write this* skip
61
Which direction is protein synthesis in
N terminus to the C terminus
62
N terminus
Amino end - H3N+
63
C terminus
carboxyl end - C-O=O-
64
Peptide bond
O=C-N-H
N and C terminus correlated
65
Role of tRNA in translation
tRNAs are RNA molecules between 70-90 nucleotides long
transferrnas that transfer genetic information into protein sequence
66
rate of tRNAs in amino acids
1/20
67
Clover structure
leaf 1 - D end - has D codons
leaf 2 - anticodon loop - GAA anticodon
leaf 3 - T loop - Psi located
3' end - attached to Phe
68
69
Where is an activated amino acid located
3' end of each tRNA
70
3 binding sites
E site, P site, A site
71
E site
Exit site
tRNA exits ribosome
72
P site
Peptide site
Peptide binding
73
A site
Aminoacyl tRNA
74
how many binding sites for mRNA and tRNA
1 binding site for mRNA and 3 sites for tRNA
75
How many tRNAs can be bound on the ribosome at the same time
2 tRNAS
76
Where is the tRNA that is connected to the polypeptide chain
P-site
77
Step 1 in translation of mRNA
Incoming Aminoacyl-tRNA binds to a site
tRNA linked to growing Peptide chain isat Psite
Spent tRNA Exits from E site
78
Step 2 in translation of mRNA
Peptide bond forms between amino acids 3 and 4
peptide chain is now linked to tRNA
79
Step 3 in translation of mRNA
Large ribosome subunit moves relative to small subunit so sites are unaligned creating hybrid sites
tRnA 3 is at E/P hybrid site
tRNA 4 is at P/A hybrid site
80
Step 4 in translation of mRNA
Small ribosome subunit shifts 3 nucleotides down mRNA - resets
A site is empty and available for incoming tRNA
Spent tRNA can dissociate from E site
81
Translation Initiation
The AUG/ATG codon specifies the start of translation and the amino acid methionine
there can be several in one reading frame
82
How does the reading frame affect amino acids encoded?
There are 3 potential protein sequences that can be derived from any mRNA molecule because it is single stranded only 3 not 6
If the AUG codon sownstream does not have Shine dalgarno upstream, then it will not be recognized as a start codon
83
Shine-Dalgarno motif
located 10 ntd upstream of AUG start codon (to the left)
in prokaryotes
84
What allows translation of Polycistronic mRNAs
Ribosomes can assemble directly on AUG codon within interior of mRNA
85
What is added to mRNAs in eukaryotes for translation
A "cap" and a poly A tail are added to mRNAs post-transcriptionally
86
Translation in eukaryotes
The small subunit ribosome complex binds to cap structure at 5' end of mRNA
Starts scanning 5'-->3' along the mRNA until it reaches the 1st AUG
Translation begins at the 1st AUG encountered 90% of eukaryotic mRNA
87
What is the preferred translation start site in Eukaryotes
The Kozak sequence (ACCAUGG) surrounding the 1st AUG
*If sequence is missing "leaking scanning" of ribosome may occur such that an AUG codon further downstream would be chosen for translation
88
Translation Initiation in Prokaryotes (4)
Small ribosome subunit binds directly to AUG on mRNA
No scanning occurs
Translation initiation depends on presence of sequence upstream of AUG -- > shine dalgarno
Translation of different proteins from same mRNA commonly occurs --> results of ribosome binding at internal AUG codons within polycistronic mRNAs
89
Translation initiation in Eukaryotes (4)
Small ribosome subunit binds to cap structure at 5' end of mRNA
Scanning of ribosome along mRNA from 5'-->3'
Translation initiation usually occurs at 1st AUG encountered by ribosome and presence of Kozak Sequence
Translation of different proteins from same mRNA rarely occurs --> "leaky scanning" of the ribosome occurs when the 1st AUG does not have "good" kozak sequence
90
What are the functions of tRNA dependent on
its folded 3D structure
91
10% of nucleotides within a tRNA molecule are modified by what modifications
Covalent modifications
92
Promoting intramolecular base pairings does what to the structure of tRNA
Stabilizes tRNA structure
93
Isosine
deamination of A
Important in wobble base pairing
94
Modified Uridine Nucleotides
Uridine
-Normal nucleotide (ntd) in all cellular RNA
Pseudouridine
-Modified ntd(psi) found in cellular tRNA (also rRNA, snRNA, snoRNA)
N1-Methylpseuouridine
-Chemically modified pseudouridine in mRNA made for COVID vaccine
95
What did Katalin Kariko and Drew Weissman do to win the 2023 Noble Prize
stabilize and reduce cellular toxicity of in-vitro transcribed mRNA
96
Genetic code Numbers
18 amino acids with >1 codon
2 AMino acids with only 1 codon
3 stop codons
64 different codons in total
97
True or False: Stop codon does not have a complementary tRNA or anticodon
TRUE
98
where does wobble base pairing occur
occurs between anticodon on tRNA and codon on mRNA in 3rd position
99
How many tRNAs are needed to recognize the 4 glycine codons
3
100
Wobble Base pairing
A mismatch tolerated at the third position
1 tRNA with "I" at wobble position can recognize both codons
101
True or false: Codons can specify more than one amino acid
FALsE
102
True or false: An amino acid can be specified by more than 1 codon
TRUE
103
True or False: Although there are 3 binding sites for tRNA, on a ribosome, only 2 tRNAs can bind to a ribosome at the same time
TRUE
104
True or false: The first site that an incoming tRNA binds to on a ribosome is the "E" or Entry site
FALSE
105
What does the cell cycle do
Produces two genetically identical daughter cells so that is genetic information is passed onto the next generation of cells
106
Steps in the cell cycle
Interphase
Prophase
Prometaphase
Metaphase
Anaphase
telophase
cytokinesis
107
What does interphase consist of
G1+S+G2 phase
108
When does cell growth occur
In all phases of the cell cycle except mitosis
109
What is G0 phase
Resting phase
110
Flanking
the nucleotide sequences adjacent to a specific DNA sequence of interest.
111
3 major checkpoints in the cell cycle
Enter Mitosis
-G2/M checkpoint
-Is all DNA replicated
-is the environment favourable
Metaphase/Anaphase checkpoint
- are all chromosomes attached to the spindle
Start checkpoint
- In late G1
- Is the environment favourable
112
Fission Yeast - Name?
Schizosaccharomyces pombe
At low temperatures, can carry out the cell cycle
At high temperatures, is restrictive
-Unstable therefore, no longer functions after G1
113
Budding Yeast - Name?
Saccharomyces cerevisiae
At low temperatres, the cell cycle continues
G1--> S-->S/M-->M
At high temperatures, restricted
so no longer functions past M phase
"budding" is not complete
114
Why are temperature sensitive mutants required for these studies of the cell cycle?
Cultures of the cells containing these mutations could not be maintained if the effects fi these mutations are present at all temps
115
Two key components of the cell cycle
Cyclins and Cdks
116
What is a cyclin and what does it do
Cyclin forms a complex with its cyclin-dependent kinase (Cdk) partner and activates protein kinase activity
117
G1-cyclins
Help regulate activities of G1/S cyclins
Cyclin partner in vertebrates
-Cyclin d
-Cdk 4 & 6
118
G1/S cyclins
Binds Cdks in late G1;commits cell to "START" and DNA replication
Cyclin partner in vertebrates
-Cyclin E
-Cdk2
119
S-cyclins
Binds cdks soon after "start" stimulates chromosome duplication; controls early mitotic events
Cyclin partner in vertebrates
-Cyclin A
-Cdk 2 &1
120
M-cyclins
Promotes entry of cell into mitosis
Cyclin partner in vertebrates
-Cyclin B
-Cdk 1
121
3 cyclins that are required in all eukaryotic cells
G1/S cyclins
S-cyclins
M cyclins
122
True or false; In yeast a single type of Cdk protein (Cdk1) controls all the stages of the cell cycle by changing different cyclin partners at the different stages
TRUE
123
Activation of S-CDK
1) active site is blocked by t-loop, inactive
2) Active site binds to cyclin, t loop still blocking active site, partially active
3) Active site and cyclin bind and activate T loop by adding activating phosphate with CAK
124
Inhibition of M-CDK
Phosphorylation of inhibitory site in the M-cyclin/cdk complex by Wee1 kinase inhibits cdk activity
Cdc25 phosphotase removes phosphate reactivating M-CDK
125
What does binding of CKI p27 to human cyclin A/Cdk2 do?
Adding p27 to an active site, inactivates enzyme activity by blocking ATP binding site and distorts active site of Cdk
126
What is APC/C and how is its acitivity controlled?
Anaphase-promoting complex or cyclosome
Activity is controlled by changes in its association with Cdc20
127
Activation of APC/C
Inactive APC/C or ubiquitin is binded in mitosis to cdc20 which activates it.
128
Initiation of Mitosis with CDK
Active cdk binds to M-cyclin - this initiates mitosis
It is then binded to active ubiquitin along with E1 and E2(enzymes) which creates a polyubiquitin chain
Degradation of M-cyclin in proteasome which then causes inactivation of M-cdk kinase activity
Then mitosis is completed
129
Mechanisms for controlling the cell cycle (6)
Cyclin binding
Phosphorylation by kinases
(phosphorylation state)
Dephosphorylation by phosphatases
(Phosphorylation state)
Binding of CDk inhibitors (CKIs
Controlled proteolysis
Transcriptional regulation
(Differential expression of cyclin genes)
130
The control of DNA replication during S-phase control of entry into mitosis using the activation of M-Cdk
Cdk1 and accumulation of M-cyclin bind together (inactive)
Cdk-activating kinase and CDK inhibitory kinase phosphorylate complex so it is primed for activation (Inactive)
Since Wee1 is inactive while phosphorylated, Cdc25 inactive phosphatase removes the phosphate
Cdc is activated when phosphorylated so this creates a positive feedback loop
(Active)
131
What happens in this situation:
Wee1 mutation--> no wee1 kinase
Cannot inhibit Mcyclin/cdk--> premature mitosis --> small "wee" cells
132
What happens in this situation:
Cdc25 mutation --> no cdc25 phosphotase
cannot activate Mcyclin/cdk--> no mitosis--> large cells
133
What would happen if the same cell had both cdc25 mutation and wee1 mutation
premature mitosis and small cells because the wee1 activation happens first
134
Explain the role of Myc in the Retinoblastoma protein in controlling the entry into G1 and S phase in animal cells
Myc increases expression of G1 cyclin genes and genes hat promote cell growth
Myc binds to active G1-cdk
Unphosphorylated Rb is an inhibitor of G1, an active Rb protein is binded to an inactivated E2F protein
E2F protein is a TF that inactivates entry into G1 and S
G1-cdk phosphorylates Rb to inactivate it and force to dissociate from E2F
Then the E2F protein is active and can continue on through G1 and S without Rb protein
135
What is the expected result of a mutation that would INCREASE the activity of Myc?
Unregulated progression into cell cycle--> uncontrolled cell growth and division
136
What is the expected result of a mutation that would inactivate the activity of Rb?
Unregulated progression into cell cycle--> uncontrolled cell growth and division
137
What is p53
a transciption factor for the expression of p21 - an inhibitor of G1/S and S-cdk complexes
unstable transcription factor found at low levels
Mutations in p53 occur in ~50% of cancers
138
Explain the Dna damage checkpoint in late G1
Mdm2 and p53 bind
Damaged DNA due to xrays damage p21 gene
mdm2 is removed and p53 is phosphorylated to be stable and active
Active p53 binds to the regulatory region of p21 gene
transcription of p21 mRNA
Translation of p21 mRNA
If p21 does not bind to active G1/S-cdk then there is entry into S --> p53 low
If p21 binds to G1/S-cdk, it inactivates and there is no entry into S because it has damaged DNA and allows cancer cells to accumulate
139
What is mdm2
an ubiquitin ligase
140
2 types of cancer critical genes
Proto-oncogenes/oncogenes
tumor supressor genes
141
proto-oncogenes/oncogenes (3)
Promotes cell proliferation
indentified by gain of function mutations - increased or unregulated activity
Dominant trait
142
Tumor supresser genes (3)
Inhibits cell proliferation
identified by loss of function mutations
Recessive trait
143
Two main categories of cancer critical genes
Overactivity mutation
underactivity mutation
144
Overactivity mutation
A normal cell has a single mutation event which creates an oncogene
Activating the mutation enacbles oncogene to promote cell transformation which causes cancer cells
145
Under-activity mutation
a normal cell has a mutation event which inactivates tumor supressor gene
there is no effect of the mutation if one copy is present
A second mutation occurs and inactivates the second copy of the gene
Two inactivating mutations functionally eliminate the tumor supressor gene promoting cell transformation causing cancer cells
146
Which of the following are true about cancer critical genes:
The mutation of any one of the cancer critical genes in a cell would result immediately in the cell becoming cancerous
The normal (un-mutated) function of an oncogene is to promote cell proliferation
The gene for a tumor repressor becomes activated in a cancerous cell
The activation of an oncogene is characterized as a recessive trait
none of the above
The normal (un-mutated) function of an oncogene is to promote cell proliferation
147
4 mechanisms that can activate a proto-oncogene into an oncogene
Deletion or point mutation in coding sequence
- hyperactive protein made in normal amounts
Regulatory mutation
-normal protein greatly overproduced
Gene amplification
-normal protein greatly overproduced
Chromosome rearrangement
-nearby regulatory DNA sequence causes normal protein to be overproduced
-fusion to actively transcribed gene produced hyperactive fusion protein
148
Product of proto-oncogene
Usually made in small amounts and or have low activity
149
product of oncogene
usually made in greater amounts and or have higher or unregulated activity
150
EGFR
Epidermal Growth Factor Receptor
151
Inactive state EGFR
Extracellular domain of receptor, outside cell
cytoplasmic domain of receptor
152
Active only when binds to EGF
binding of growth factor triggers intrcellular signaling
signal transduction cascade --> stimulates cell cycle
153
Always Active EGFR
Since regulatory region has been deleted, missing extracellular region
trucated receptor triggers intracellular signalling in absence of growth factor
154
What is the EGFR most common in
oncogenic events in glioblastoma (brain cancer)
155
Retinoblastoma in a normal healthy individual
active form of Rb protein binds the transcription factor E2F and prevents it from activating transcription of G1/S and S-cyclin
Regulates cells entry into S-phase
NO TUMOR
156
Hereditary Retinoblastoma
Occasional cell inactivates its only good Rb gene copy
Excessive cell proliferation leading to retinoblastoma
All cells in body lack one of the functional copies of the Rb gene --> multiple tumors affecting b oth eyes
MOST PEOPLE WITH INHERITED MUTATION DEVELOP TUMORS
also have increased risk of other cancer later in life
157
Nonhereditary retinoblastoma
Occasional cell inactivates one of its two good Rb genes
the second copy of Rb is very rarely inactivated in the same line of cells
Excessive cell proliferation leading to retinoblastomas
Defec in the Rb gvene originates in a single retina cell
A single tumor in one eye
ONLY ABOUT 1 in 30000 NORMAL PEOPLE DEVELOP TUMOR
158
True or False: a single mutation is enough to cause cancer
FALSE
development of cancer results in the accumulation of several mutations
159
True or False: The incidence of cancer increases with the age of the individual
TRUE
But there is a decrease in cancer after 90 years because slower cell turnover/proliferation
160
True or false: There is a lag time from initial exposure to carcinogen to cancer development
TRUE
Long incubation period due to the multiple changes/mutations that needs to occur within a cell before it becomes cancerous
161
Which of the following is true about Rb?
The normal function of the product of the Rb Gene is to stop the entry of a cell into S-phase
The phosphorylated form of the Rb protein is the active form
The Rb protein binds to and inhibits function of Myc
The inherited form of cancer resulting from mutations in the Rb gene is the appearance of tumors in both eyes of the patient
2 of the above
The normal function of the product of the Rb Gene is to stop the entry of a cell into S-phase
162
DNA viruses associated with human cancers
Papillomavirus
Hepatitis B virus
Epstein-Barr virus
human herpesvirus 8
163
RNA Viruses associated with Cancer
Human T cell leukemia virus type I
Human immunodeficiency virus (AIDs, HIV)
Hepatitis C
164
Papillomavirus
Associated cancer
-warts
-Carcinoma of uterine cervix
Areas of high incidence
-worldwide
Vaccine available
165
Hepatitis B virus
Associated cancer
-liver cancer
Areas of high incidence
-southeast asia, tropical africa
Associated with chronic hep B
166
Epstein-Barr virus
Associated cancer
-Burkitts lymphoma - B lymphocytes
- Nosopharyngeal carcinoma
Areas of high incidence
-West africa, Papua New Guinea
-Southern china, greenland
167
Human herpesvirus 8
Associated cancer
-Kaposi's sarcoma (skin cancer)
Areas of high incidence
-central and southern africa
168
Human T cell leukemia virus type I
Associated cancer
- Adult T cell leukemia/lymphoma
Areas of high incidence
-Japan, west indies
169
Human immunodeficiency virus (AIDs, HIV)
Associated cancer
-kaposi's sarcoma via human herpesvirus 8
Areas of high incidence
-central and southern africa
170
Hepatitis C
Associated cancer
-Liver cancer
Areas of high incidence
-worldwide
171
Progressive stages of colon cancer
Disease is responsible for 11% of all cancer deaths in the US and canada
Originates in a small benign tumor or adenoma - visible as protruding mass of tissue called a polyp
Larger polyps frequently can be divided into two subsections: one that have almost normal appearance of epithelial cells and the other have undifferentiated cells in abnormal organizations
uses FIT test - Fecal immunochemical test
Cells keep diving and accumulating cancer cells
172
How are genetic abnormalities detected in colorectal cancer cells?
1. Screen the tumors for mutation in genes known and suspected to be involved in other cancers
2. exacmine many different colorectal tumors to look for common genetic alterations --> large chromosomal deletions
3. Examine families with genetic predisposition to colorectal cancers
173
FAP
Familial Adenomatous Polyposis Coli
FAP is due to deletion or inactivation of both alleles of the APC (Adenomatous polyposis coli) gene located on chromosome 5
The normal APC protein is an inhibitory component of the Wnt signalling pathway
if the APC protein is missing or nonfunctional, an overgrowth of the colonic epithelium will occur--> polyps develop
174
Development of a Colorectal carcinoma Pathway
1. Normal epithelium
2. Loss of APC or early mutation
3. hyperplastic epithelium
4. early adenoma
5. Activation of K-Ras
6. intemediate adenoma
7. loss of Smad4 and other tumor suppressors
8. Late adenoma
9. Loss of p53, later mutation
10. Carcinoma
11. other unknown alteration, activation of VEGF - growth of blood vessels
12. invasion and metastasis
175
True or False: Mutations found in both polyps and malignant tumors are likely to occur early
TRUE
176
True or False: Mutations found only in malignant tumors are likely to occur later
TRUE
177
Metastasis of Cancer cells
Normal epithelium
A local invasion happens; cells grow a benign tumour
cells become invasive and enter capillary
they adhere to the blood vessel wall in liver
escape from lood vessel to form micrometastasis - exit circulatory system
colonize liver forming full-blown metastasis
178
Which of the following is/are FALSE about colorectal cancer?
The majority of cases are not identified after the age of 55
The lost of the APC gene/activity occurs early in the development of this disease
There are inherited and non-inherited forms of this disease
A loss in p53 activity is commonly found in cells during their development from benign to malignant tumors
none of the above
A loss in p53 activity is commonly found in cells during their development from benign to malignant tumors
179
Angiogenesis
is controlled by factors released by surrounding tissues
In well oxygenated areas, HIF is kept at low levels but its continual degradation- no blood vessel growth, hypoxia inducible factor
In low oxygenated areas, HIF stimulates transcription of the gene encoding the Vascular Endothelical Growth factor - VEGF stimulatges endothelial cells to sprout
180
VHL
von hippel lindau tumor suppressor
ubiquitin ligase
involved in degradation of HIF
Acts as a tumor supressor
181
Chronic Myelogenous leukemia
BCR gene encodes a protein with serine/threonine kinase activity but its function is unclear (chromie 22)
Abl gene encodes a protein tyrosine kinase involved in cell signalling, activates the production of T cells (chromie 9)
Translocation occurs and Bcr and Abl are fused
transcription occurs and a poly a tail is added
translation occurs and it is a Bcr Abl protein which is a unique gene targetted in chemotherapy
the Abl kinase domain becomes hyperactive
Stimulates excessive proliferation of the white blood cells made in bone marrow
182
Which of the following is/are TRUE about chronic myelogenous leukemia (CML)?
The genetic basis of this disease is a chromosome translocation
A tyrosine kinase becomes constitutively active in the affected cells
The drug that targets this disease binds to a protein that is unique to the affected cells
The drug was identified from a high throughput screen of many compounds
All of the above
All of the above
183
Effects of ionizing Radiation in a Normal cell
DNA damage occurs which activates p53 which causes cell cycle arrest
The damage is repaired and normal cell division is done
OR
The damage is too extensive which results in apoptosis which is programmed cell death
184
Effects of ionizing Radiation in a Cancer cell
Radiation of cell lacking p53, DNA damage occurs, no cell cycle arrest occurs
Cell division with damaged chromosomes occurs
Massive mitosis failure which results in cell death - tumor regression
OR
Continued mutation and cancer continues
185
Down falls of radiation(2)
Causes death in normal cells
Cancer cells may still survive
186
Rational design of drugs to target proteins encoded by critical cancer genes
growth factor and RTK inhibitor binds to receptor tyrosine kinase which causes cascade to Raf, Mek and Erk inhibitors
187
MAB
Monoconal Antibody
188
NiB
small molecule inhibitor
189
2 ways to prevent cancer
Removal and elimination of known carcinogens
Early detection and treatment
190
Problems associated with current cancer treatments (4)
Surgery to remove tumors is not practical in metastasized cancers
Radiation and chemo are usually toxic to normal cells
Development of resistance to either a single or multiple anticancer drugs - cancer cells evolve
Survival of a single cancer cell can lead to resurgence
191
2 ways to develop new cancer treatments
Molecular markers to allow for early detection
Customized medicine - obtain genome sequence to personalize treatment
192
What is TRUE about the DNA sequencing of genomes?
The DNA ancestry kits uses technology from illumina to offer complete genome sequencing to the public
The first complete human genome sequenced was that of James Watson
The genomes that have been completely sequenced as of 2021 are biased towards those of the nuclear genomes of plants
The cost of DNA sequencing had greatly decreased since 2001 mainly due to advances in technology and availability of good reference genomes
The cost of DNA sequencing had greatly decreased since 2001 mainly due to advances in technology and availability of good reference genomes
193
In sanger sequencing, the presence of too MUCH ddNTPs would give too many short DNA strands. What is the reason for this.
DNA sequencing will proceed from both ends of the DNA template
The activity of the DNA polymerase would be inhibited by the vast amounts of dNTPs
The binding of the primer would be inhibited by vast amounts of ddNTP
The probability of chain termination occurring early in the sequencing reaction will decrease
The probability of the chain termination occurring early in the sequencing reaction will increase
The probability of the chain termination occurring early in the sequencing reaction will increase
194
What is the main purpose of contig assembly in sequencing
It reconstructs the DNA sequence of a larger region of DNA from the sequences of smaller DNA regions
It checks for DNA sequencing errors
It checks that both strands of DNA fragment are 100% complementary
It identifies potential contamination of the DNA sample
It reconstructs the DNA sequence of a larger region of DNA from the sequences of smaller DNA regions
195
In the manual method of sanger sequencing, a band in all 4 lanes (G, A, T, C) are observed for a single nucleotide position on the audio-graph. What is the most likely reason for this?
The DNA sample is degraded
The DNA sample is contaminated
The DNA polymerase incorporated 4 different ddNTPs in the same position
The formation of a structure on the DNA template caused the DNA polymerase to stop at this position
The formation of a structure on the DNA template caused the DNA polymerase to stop at this position
196
What are standard vector primers
Complementary to sequences in plasmids that are commonly used to carry fragments of cloned DNA
197
Which if the following is true about the organization fo genes in any large region of genomic DNA from a eukaryote such as drosophilia
All genes located on the same chromosome would have the same reading frame
The correct reading frame fro each gene is independent of those of its neighbouring genes
It is common that the reading frame of adjacent genes overlap
The correct reading frame is always the longest reading frame
The correct reading frame fro each gene is independent of those of its neighbouring genes
198
What is the function fo the sigma factor in E coli RNA
Helps recognize different promoter sequences
199
Whats the main reason primer walking is not normally used to sequence a genome
It would take too long
200
What are SSU rRNAs commonly used for
construction of phylogenic trees
201
tRNA charging
linking an activated amino acid to a tRNA
Study Notes 3rd Year
flashcards
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