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Study Notes 3rd Year > BIOL 3080 pt 1 > Flashcards

BIOL 3080 pt 1 Flashcards

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
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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
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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
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4
Q

DNTP

A

OH group allows strand extension at 3’ end

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5
Q

ddNTP

A

does not allow extension at 3’ end

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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
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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
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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

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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
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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
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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

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12
Q

Reading Frames

A
  • There are 6 potential protein sequences that can be derived from any double-stranded DNA fragment
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13
Q

What is the rate of stop codons

A

1/21.3

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14
Q

What is the correct reading frame

A

The longest strand with no stop codon

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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

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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
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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
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18
Q

What are mRNAs

A

Messanger RNAs
- code for proteins
Percent?
3-5% of RNA

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19
Q

What are rRNAs

A

Ribosomal RNAs
- form the basic structure of the ribosome and catalyze protein synthesis
Percent?
Majority of RNA 75%

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20
Q

what are tRNAs

A

Transfer RNAs
- central to protein synthesis as the adaptors between mRNA and amino acids
Percent
10-15%

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21
Q

Telomerase RNA

A

Serves as a template for telomerase enzyme that extends ends of chromosomes
<5%

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22
Q

snRNAs

A

Small nuclear RNAs
- function in a variety of nuckear processes, including splicing pre-mRNA
<5%

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23
Q

snoRNAs

A

Small nucleolar RNA
- help to process and chemically modify rRNAs
<5%

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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%

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25
Q

miRNAs

A

MicroRNAs
regulate gene expression by blocking translation of specfic mRNAs and causing their degradation
<5%

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26
Q

siRNAs

A

Small-interfering RNAs
turn off gene expression by directing the degradation of selective mRNAs and helping to establish repressive chromatin structures
<5%

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27
Q

piRNAs

A

Piwi-interacting RNAs
bind to piwi proteins and protect the germ line from transposable elements
<5%

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28
Q

First Human Genome Sequencing and who was involved

A
  • cost 2.7 billion
  • took 15 years
  • 2013
  • Francis Collins
  • Craig Ventor
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29
Q

Genome Library

A

All possible DNA fragments of genomes

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30
Q

What is a Contig

A

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

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31
Q

What would happen in Sanger sequencing if too much ddA is present

A

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

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32
Q

Tell me about gel electrophoresis

A
  • 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
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33
Q

Why are adaptors added in Next Gen sequencing

A
  • you know the sequence of the adapter therefore you know what primer you need to use
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34
Q

Why is a PCR step added in Next Gen sequencing

A
  • To build up an amount of copies to have a bigger signal
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35
Q

Would a signal with 2x the regular intensity be likely to 2x incorporation of the same nucleotide in the SAME cycle in Illumina sequencing

A

No

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36
Q

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
A

While DNA pol synthesizes DNA from a DNA template, RNA pol synthesize RNA from RNA template

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37
Q

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
A

The DNA sequences for both strands of double-stranded DNA template are usually obtained to check for any technical errors in the sequencing procedure

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38
Q

Error rate of DNA polymerase

A

1/107

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39
Q

Error rate of RNA polymerase

A

1/104

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40
Q

sigma factor

A

called holoenzyme
Completes the enzyme and binds to promoter sequence

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41
Q

Transcription cycle of RNA polymerase

A

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

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42
Q

Hairpin

A

Forms inverted repeats and only forms on RNA because RNA is single stranded so it snaps together.

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43
Q

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

A

Initiation of transcription occurs at the ATG start codon

  • This is translation start
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44
Q

UTR

A

Transcribed not translated

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45
Q

Consensus Sequences

A

are located within the promoter region

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46
Q

High expression Gene

A

Large amount of protein and RNA

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47
Q

Low expression gene

A

small amount of protein and DNA

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48
Q

No expression gene

A

No protein

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49
Q

How many strands of DNA are used as a template for RNA synthesis

A

One strand of DNA

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50
Q

Promoter Sequences

A

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

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51
Q

What RNA is transcribed by RNA pol I

A

5.8S, 18S, 28S rRNA

alpha amanitin - insensitive

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52
Q

What RNA is transcribed by RNA pol II

A

mRNA, snoRNA, miRNA, siRNA, LncRNA, snRNA

alpha amanitin - very sensitive

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53
Q

What RNA is transcribed by RNA pol III

A

tRNA, 5S RNA, some snRNA and other small RNA

alpha amanitin - moderately sensitive

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54
Q

What does eukaryotic RNA pol require the presence of to bind to promoters

A

Requires the presence of additional initiation proteins (transcription factors) before they can bind selectively to different promoter sequences.

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55
Q

Order of transcription binders

A

TBP, TFIID, TFIB, TFIIA, TFIIF, TFIIH, TFIIE

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56
Q

What does a typical eukaryotic RNA polymerase II core promoter consist of

A

TATA box
Initiator element (INR)

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57
Q

Transcriptional start site is marked with what

A

+1 arrow

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58
Q

TBP

A

TATA binding protein

usually the first transcription factor to bind

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59
Q

Formation of the PIC

A

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

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60
Q

Phosphorylation of Ser in Hepapeptide

A

Still have to write this skip

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61
Q

Which direction is protein synthesis in

A

N terminus to the C terminus

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62
Q

N terminus

A

Amino end - H3N+

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63
Q

C terminus

A

carboxyl end - C-O=O-

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64
Q

Peptide bond

A

O=C-N-H

N and C terminus correlated

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65
Q

Role of tRNA in translation

A

tRNAs are RNA molecules between 70-90 nucleotides long

transferrnas that transfer genetic information into protein sequence

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66
Q

rate of tRNAs in amino acids

A

1/20

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67
Q

Clover structure

A

leaf 1 - D end - has D codons

leaf 2 - anticodon loop - GAA anticodon

leaf 3 - T loop - Psi located

3’ end - attached to Phe

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68
Q
A
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69
Q

Where is an activated amino acid located

A

3’ end of each tRNA

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70
Q

3 binding sites

A

E site, P site, A site

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71
Q

E site

A

Exit site
tRNA exits ribosome

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72
Q

P site

A

Peptide site
Peptide binding

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73
Q

A site

A

Aminoacyl tRNA

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74
Q

how many binding sites for mRNA and tRNA

A

1 binding site for mRNA and 3 sites for tRNA

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75
Q

How many tRNAs can be bound on the ribosome at the same time

A

2 tRNAS

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76
Q

Where is the tRNA that is connected to the polypeptide chain

A

P-site

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77
Q

Step 1 in translation of mRNA

A

Incoming Aminoacyl-tRNA binds to a site
tRNA linked to growing Peptide chain isat Psite
Spent tRNA Exits from E site

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78
Q

Step 2 in translation of mRNA

A

Peptide bond forms between amino acids 3 and 4
peptide chain is now linked to tRNA

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79
Q

Step 3 in translation of mRNA

A

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

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80
Q

Step 4 in translation of mRNA

A

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

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81
Q

Translation Initiation

A

The AUG/ATG codon specifies the start of translation and the amino acid methionine

there can be several in one reading frame

82
Q

How does the reading frame affect amino acids encoded?

A

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
Q

Shine-Dalgarno motif

A

located 10 ntd upstream of AUG start codon (to the left)

in prokaryotes

84
Q

What allows translation of Polycistronic mRNAs

A

Ribosomes can assemble directly on AUG codon within interior of mRNA

85
Q

What is added to mRNAs in eukaryotes for translation

A

A “cap” and a poly A tail are added to mRNAs post-transcriptionally

86
Q

Translation in eukaryotes

A

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
Q

What is the preferred translation start site in Eukaryotes

A

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
Q

Translation Initiation in Prokaryotes (4)

A

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
Q

Translation initiation in Eukaryotes (4)

A

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
Q

What are the functions of tRNA dependent on

A

its folded 3D structure

91
Q

10% of nucleotides within a tRNA molecule are modified by what modifications

A

Covalent modifications

92
Q

Promoting intramolecular base pairings does what to the structure of tRNA

A

Stabilizes tRNA structure

93
Q

Isosine

A

deamination of A

Important in wobble base pairing

94
Q

Modified Uridine Nucleotides

A

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
Q

What did Katalin Kariko and Drew Weissman do to win the 2023 Noble Prize

A

stabilize and reduce cellular toxicity of in-vitro transcribed mRNA

96
Q

Genetic code Numbers

A

18 amino acids with >1 codon
2 AMino acids with only 1 codon
3 stop codons
64 different codons in total

97
Q

True or False: Stop codon does not have a complementary tRNA or anticodon

A

TRUE

98
Q

where does wobble base pairing occur

A

occurs between anticodon on tRNA and codon on mRNA in 3rd position

99
Q

How many tRNAs are needed to recognize the 4 glycine codons

A

3

100
Q

Wobble Base pairing

A

A mismatch tolerated at the third position

1 tRNA with “I” at wobble position can recognize both codons

101
Q

True or false: Codons can specify more than one amino acid

A

FALsE

102
Q

True or false: An amino acid can be specified by more than 1 codon

A

TRUE

103
Q

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

A

TRUE

104
Q

True or false: The first site that an incoming tRNA binds to on a ribosome is the “E” or Entry site

A

FALSE

105
Q

What does the cell cycle do

A

Produces two genetically identical daughter cells so that is genetic information is passed onto the next generation of cells

106
Q

Steps in the cell cycle

A

Interphase
Prophase
Prometaphase
Metaphase
Anaphase
telophase
cytokinesis

107
Q

What does interphase consist of

A

G1+S+G2 phase

108
Q

When does cell growth occur

A

In all phases of the cell cycle except mitosis

109
Q

What is G0 phase

A

Resting phase

110
Q

Flanking

A

the nucleotide sequences adjacent to a specific DNA sequence of interest.

111
Q

3 major checkpoints in the cell cycle

A

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
Q

Fission Yeast - Name?

A

Schizosaccharomyces pombe
At low temperatures, can carry out the cell cycle

At high temperatures, is restrictive
-Unstable therefore, no longer functions after G1

113
Q

Budding Yeast - Name?

A

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
Q

Why are temperature sensitive mutants required for these studies of the cell cycle?

A

Cultures of the cells containing these mutations could not be maintained if the effects fi these mutations are present at all temps

115
Q

Two key components of the cell cycle

A

Cyclins and Cdks

116
Q

What is a cyclin and what does it do

A

Cyclin forms a complex with its cyclin-dependent kinase (Cdk) partner and activates protein kinase activity

117
Q

G1-cyclins

A

Help regulate activities of G1/S cyclins

Cyclin partner in vertebrates
-Cyclin d
-Cdk 4 & 6

118
Q

G1/S cyclins

A

Binds Cdks in late G1;commits cell to “START” and DNA replication
Cyclin partner in vertebrates
-Cyclin E
-Cdk2

119
Q

S-cyclins

A

Binds cdks soon after “start” stimulates chromosome duplication; controls early mitotic events

Cyclin partner in vertebrates
-Cyclin A
-Cdk 2 &1

120
Q

M-cyclins

A

Promotes entry of cell into mitosis

Cyclin partner in vertebrates
-Cyclin B
-Cdk 1

121
Q

3 cyclins that are required in all eukaryotic cells

A

G1/S cyclins
S-cyclins
M cyclins

122
Q

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

A

TRUE

123
Q

Activation of S-CDK

A

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
Q

Inhibition of M-CDK

A

Phosphorylation of inhibitory site in the M-cyclin/cdk complex by Wee1 kinase inhibits cdk activity

Cdc25 phosphotase removes phosphate reactivating M-CDK

125
Q

What does binding of CKI p27 to human cyclin A/Cdk2 do?

A

Adding p27 to an active site, inactivates enzyme activity by blocking ATP binding site and distorts active site of Cdk

126
Q

What is APC/C and how is its acitivity controlled?

A

Anaphase-promoting complex or cyclosome

Activity is controlled by changes in its association with Cdc20

127
Q

Activation of APC/C

A

Inactive APC/C or ubiquitin is binded in mitosis to cdc20 which activates it.

128
Q

Initiation of Mitosis with CDK

A

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
Q

Mechanisms for controlling the cell cycle (6)

A

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
Q

The control of DNA replication during S-phase control of entry into mitosis using the activation of M-Cdk

A

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
Q

What happens in this situation:

Wee1 mutation–> no wee1 kinase

A

Cannot inhibit Mcyclin/cdk–> premature mitosis –> small “wee” cells

132
Q

What happens in this situation:

Cdc25 mutation –> no cdc25 phosphotase

A

cannot activate Mcyclin/cdk–> no mitosis–> large cells

133
Q

What would happen if the same cell had both cdc25 mutation and wee1 mutation

A

premature mitosis and small cells because the wee1 activation happens first

134
Q

Explain the role of Myc in the Retinoblastoma protein in controlling the entry into G1 and S phase in animal cells

A

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
Q

What is the expected result of a mutation that would INCREASE the activity of Myc?

A

Unregulated progression into cell cycle–> uncontrolled cell growth and division

136
Q

What is the expected result of a mutation that would inactivate the activity of Rb?

A

Unregulated progression into cell cycle–> uncontrolled cell growth and division

137
Q

What is p53

A

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
Q

Explain the Dna damage checkpoint in late G1

A

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
Q

What is mdm2

A

an ubiquitin ligase

140
Q

2 types of cancer critical genes

A

Proto-oncogenes/oncogenes

tumor supressor genes

141
Q

proto-oncogenes/oncogenes (3)

A

Promotes cell proliferation

indentified by gain of function mutations - increased or unregulated activity

Dominant trait

142
Q

Tumor supresser genes (3)

A

Inhibits cell proliferation

identified by loss of function mutations

Recessive trait

143
Q

Two main categories of cancer critical genes

A

Overactivity mutation

underactivity mutation

144
Q

Overactivity mutation

A

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
Q

Under-activity mutation

A

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
Q

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

A

The normal (un-mutated) function of an oncogene is to promote cell proliferation

147
Q

4 mechanisms that can activate a proto-oncogene into an oncogene

A

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
Q

Product of proto-oncogene

A

Usually made in small amounts and or have low activity

149
Q

product of oncogene

A

usually made in greater amounts and or have higher or unregulated activity

150
Q

EGFR

A

Epidermal Growth Factor Receptor

151
Q

Inactive state EGFR

A

Extracellular domain of receptor, outside cell

cytoplasmic domain of receptor

152
Q

Active only when binds to EGF

A

binding of growth factor triggers intrcellular signaling

signal transduction cascade –> stimulates cell cycle

153
Q

Always Active EGFR

A

Since regulatory region has been deleted, missing extracellular region

trucated receptor triggers intracellular signalling in absence of growth factor

154
Q

What is the EGFR most common in

A

oncogenic events in glioblastoma (brain cancer)

155
Q

Retinoblastoma in a normal healthy individual

A

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
Q

Hereditary Retinoblastoma

A

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
Q

Nonhereditary retinoblastoma

A

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
Q

True or False: a single mutation is enough to cause cancer

A

FALSE

development of cancer results in the accumulation of several mutations

159
Q

True or False: The incidence of cancer increases with the age of the individual

A

TRUE

But there is a decrease in cancer after 90 years because slower cell turnover/proliferation

160
Q

True or false: There is a lag time from initial exposure to carcinogen to cancer development

A

TRUE

Long incubation period due to the multiple changes/mutations that needs to occur within a cell before it becomes cancerous

161
Q

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

A

The normal function of the product of the Rb Gene is to stop the entry of a cell into S-phase

162
Q

DNA viruses associated with human cancers

A

Papillomavirus

Hepatitis B virus

Epstein-Barr virus

human herpesvirus 8

163
Q

RNA Viruses associated with Cancer

A

Human T cell leukemia virus type I

Human immunodeficiency virus (AIDs, HIV)

Hepatitis C

164
Q

Papillomavirus

A

Associated cancer
-warts
-Carcinoma of uterine cervix

Areas of high incidence
-worldwide

Vaccine available

165
Q

Hepatitis B virus

A

Associated cancer
-liver cancer

Areas of high incidence
-southeast asia, tropical africa

Associated with chronic hep B

166
Q

Epstein-Barr virus

A

Associated cancer
-Burkitts lymphoma - B lymphocytes
- Nosopharyngeal carcinoma

Areas of high incidence
-West africa, Papua New Guinea
-Southern china, greenland

167
Q

Human herpesvirus 8

A

Associated cancer
-Kaposi’s sarcoma (skin cancer)

Areas of high incidence
-central and southern africa

168
Q

Human T cell leukemia virus type I

A

Associated cancer
- Adult T cell leukemia/lymphoma

Areas of high incidence
-Japan, west indies

169
Q

Human immunodeficiency virus (AIDs, HIV)

A

Associated cancer
-kaposi’s sarcoma via human herpesvirus 8

Areas of high incidence
-central and southern africa

170
Q

Hepatitis C

A

Associated cancer
-Liver cancer

Areas of high incidence
-worldwide

171
Q

Progressive stages of colon cancer

A

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
Q

How are genetic abnormalities detected in colorectal cancer cells?

A
  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
Q

FAP

A

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
Q

Development of a Colorectal carcinoma Pathway

A
  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
Q

True or False: Mutations found in both polyps and malignant tumors are likely to occur early

A

TRUE

176
Q

True or False: Mutations found only in malignant tumors are likely to occur later

A

TRUE

177
Q

Metastasis of Cancer cells

A

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
Q

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

A loss in p53 activity is commonly found in cells during their development from benign to malignant tumors

179
Q

Angiogenesis

A

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
Q

VHL

A

von hippel lindau tumor suppressor
ubiquitin ligase
involved in degradation of HIF
Acts as a tumor supressor

181
Q

Chronic Myelogenous leukemia

A

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
Q

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

A

All of the above

183
Q

Effects of ionizing Radiation in a Normal cell

A

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
Q

Effects of ionizing Radiation in a Cancer cell

A

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
Q

Down falls of radiation(2)

A

Causes death in normal cells
Cancer cells may still survive

186
Q

Rational design of drugs to target proteins encoded by critical cancer genes

A

growth factor and RTK inhibitor binds to receptor tyrosine kinase which causes cascade to Raf, Mek and Erk inhibitors

187
Q

MAB

A

Monoconal Antibody

188
Q

NiB

A

small molecule inhibitor

189
Q

2 ways to prevent cancer

A

Removal and elimination of known carcinogens
Early detection and treatment

190
Q

Problems associated with current cancer treatments (4)

A

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
Q

2 ways to develop new cancer treatments

A

Molecular markers to allow for early detection

Customized medicine - obtain genome sequence to personalize treatment

192
Q

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

A

The cost of DNA sequencing had greatly decreased since 2001 mainly due to advances in technology and availability of good reference genomes

193
Q

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

A

The probability of the chain termination occurring early in the sequencing reaction will increase

194
Q

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

A

It reconstructs the DNA sequence of a larger region of DNA from the sequences of smaller DNA regions

195
Q

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

A

The formation of a structure on the DNA template caused the DNA polymerase to stop at this position

196
Q

What are standard vector primers

A

Complementary to sequences in plasmids that are commonly used to carry fragments of cloned DNA

197
Q

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

A

The correct reading frame fro each gene is independent of those of its neighbouring genes

198
Q

What is the function fo the sigma factor in E coli RNA

A

Helps recognize different promoter sequences

199
Q

Whats the main reason primer walking is not normally used to sequence a genome

A

It would take too long

200
Q

What are SSU rRNAs commonly used for

A

construction of phylogenic trees

201
Q

tRNA charging

A

linking an activated amino acid to a tRNA

Study Notes 3rd Year (11 decks)

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