1
Q
What are key traits that define life?
A
Complexity
2
Q
Why does life require a continual input of energy?
A
To maintain order and avoid decay toward equilibrium with the environment.
3
Q
What are macromolecules?
A
Large organic molecules essential for life’s functions.
4
Q
What is the function of the plasma membrane?
A
It acts as a selective barrier
5
Q
Why are cells considered open systems?
A
They exchange energy and materials with their environment.
6
Q
What is the main function of proteins in the cell?
A
They perform most cellular functions including structure
7
Q
What are enzymes?
A
Proteins that catalyze biochemical reactions.
8
Q
Where is genetic information stored?
A
In DNA.
9
Q
How is DNA chemically simpler than proteins?
A
DNA has 4 nucleotide building blocks; proteins have 20 amino acids.
10
Q
Why was the structure of DNA important?
A
It helped prove that DNA is the genetic material.
11
Q
What links nucleotides in DNA and RNA?
A
Phosphodiester bonds between 5’ and 3’ carbons of ribose or deoxyribose.
12
Q
What forms the backbone of nucleic acids?
A
Phosphate and (deoxy)ribose sugars.
13
Q
What is the orientation of a nucleic acid strand?
A
5’ end (phosphate) to 3’ end (hydroxyl).
14
Q
Why is RNA less stable than DNA?
A
RNA has a 2’ OH group that can cause spontaneous cleavage.
15
Q
Why is DNA more suitable for long-term information storage?
A
DNA lacks the 2’ OH
16
Q
What are the purines in DNA?
A
Adenine (A) and Guanine (G).
17
Q
What are the pyrimidines in DNA?
A
Thymine (T) and Cytosine (C).
18
Q
Which bases pair together in DNA?
A
A-T (2 hydrogen bonds)
19
Q
What is meant by antiparallel DNA strands?
A
One strand runs 5’→3’
20
Q
What is the structure of double-stranded DNA?
A
A right-handed double helix with base pairs inside and backbone outside.
21
Q
What are major and minor grooves in DNA?
A
External grooves that allow proteins to access base information.
22
Q
Why is the major groove important?
A
It exposes unique chemical features for protein-DNA binding.
23
Q
What provides evidence of common ancestry among life forms?
A
Shared use of amino acids and nucleotides.
24
Q
Why is the conserved genetic code significant?
A
It allows gene transfer between species (e.g.
25
What part of the ribosome catalyzes protein synthesis?
Ribosomal RNA (rRNA)
26
What are different ways to visualize tertiary structure?
By shape
27
What are domains in protein tertiary structure?
Distinct structural/functional regions of a protein.
28
What can protein domains represent?
A function
29
Can domains function independently?
Yes
30
What are C-terminal and N-terminal domains?
Protein regions near the carboxyl (C) or amino (N) end.
31
What does the modular nature of proteins mean?
Many proteins share similar domains
32
Give an example of domain reuse in proteins
Epidermal Growth Factor (EGF) domains appear in diverse proteins like Neu oncogene.
33
What is the structure of GFP?
A barrel of 11 beta sheets surrounding a central alpha helix with the chromophore.
34
How is the GFP chromophore formed?
Through post-translational modification and cyclization of the peptide backbone.
35
What stabilizes GFP structure?
Alpha-helices at the ends of the protein and crowded side-chains inside the barrel.
36
What is quaternary structure?
Assembly of multiple polypeptide subunits into a functional protein complex.
37
Example of quaternary structure?
Influenza hemagglutinin: a trimer of identical HA1 and HA2 subunit pairs.
38
What are supramolecular complexes?
Large protein assemblies ("molecular machines") with multiple subunits
39
How is protein structure determined experimentally?
Through X-ray crystallography or cryo-electron microscopy.
40
What is a limitation of structure determination methods?
They require purified proteins and take months to years to complete.
41
Can protein structure be predicted from sequence?
Yes
42
How is AI used in protein structure prediction?
AI can convert sequences into structures rapidly and predict protein interactions.
43
What are the benefits of AI-driven structure prediction?
Fast
44
What is a limitation of AI-based structure prediction?
It’s often a black box—how predictions are made isn’t always clear.
45
What is the ubiquitin conjugating system?
"A system that covalently links ubiquitin to other proteins using multiple steps and specialized machinery.")
46
("What is the first step in ubiquitin conjugation?"
"Activation of the carboxyl terminus of ubiquitin.")
47
("What enzyme is involved in transferring ubiquitin to target proteins?"
"E3 ubiquitin ligase.")
48
("How many E3 ligases are encoded in the human genome?"
"Approximately 600
49
("What is polyubiquitinylation?"
"Addition of multiple ubiquitins to a protein
50
("What residues do ubiquitin ligases recognize?"
"Exposed hydrophobic residues on misfolded or damaged proteins.")
51
("What recognizes polyubiquitinylated proteins?"
"Ub receptors in the proteasome.")
52
("What do deubiquitinases (DUBs) do?"
"They hydrolyze bonds between ubiquitins to recycle them.")
53
("What role do ATPases play in proteasomal degradation?"
"They unfold proteins and transport them into the proteasome's core.")
54
("What happens to proteins inside the proteasome?"
"They are digested into short peptides of 2-24 amino acids.")
55
("What happens to peptides after proteasome degradation?"
"They are further degraded into single amino acids in the cytoplasm.")
56
("What happens when misfolded proteins accumulate?"
"They can form aggregates if not properly degraded.")
57
("Why do protein aggregates form?"
"Misfolded or incompletely degraded proteins interact to hide hydrophobic residues.")
58
("What conditions promote protein aggregation?"
"High protein concentration and environmental changes.")
59
("What forms can protein aggregates take?"
"They can be amorphous or structured
60
("Where can protein aggregation be observed?"
"In cells
61
("What are amyloid fibrils?"
"Arrays of β-sheets formed by short segments of misfolded proteins.")
62
("How are amyloid fibrils structured?"
"β-strands align perpendicularly to the filament axis
63
"Why are amyloids clinically significant?"
"They are associated with diseases like Alzheimer's
64
"What increases the risk of amyloid formation?"
"Aging and mutations in proteins."
65
What is trypsin and what does it do?
Trypsin is a serine protease that hydrolyzes peptide bonds adjacent to arginine and lysine, using a serine residue in its catalytic site.
66
How does trypsin achieve substrate specificity?
Trypsin has a negatively charged pocket that accommodates large, basic side chains like arginine and lysine.
67
What defines substrate specificity in proteases like elastase?
The substrate recognition pocket in elastase is obstructed by bulky valine side chains, allowing cleavage only adjacent to small side chains like alanine or glycine.
68
Which amino acids are involved in the catalytic site of trypsin?
Aspartate-102, Histidine-57, and Serine-195.
69
How does protein folding affect enzyme catalysis?
Folding brings distant amino acids into close proximity at the catalytic site, enabling the reaction.
70
Describe the two-step catalytic mechanism of trypsin.
Step 1: Peptide bond cleavage and formation of Ser195 acyl enzyme complex. Step 2: Hydrolysis of the acyl enzyme complex to release the peptide.
71
Why is trypsin activity pH-dependent?
Trypsin relies on His-57, which needs to donate and accept protons optimally at around pH 7, close to its pK value.
72
What is the pH optimum for chymotrypsin and why?
Chymotrypsin has a pH optimum around 8. Activity decreases below pH 9 due to protein misfolding.
73
What is the pH optimum for lysosomal hydrolases?
Around 4.5, matching the acidic environment of the lysosome.
74
What is allosteric regulation?
Binding of a ligand at one site induces conformational changes that affect activity at the active site.
75
Give an example of allosteric regulation in chaperones.
ATP binding induces conformational changes in chaperones to regulate activity.
76
How does calmodulin regulate proteins?
Calcium binding changes calmodulin’s shape, enabling it to bind and regulate target proteins.
77
What is the role of GTP in G-proteins?
G-proteins are active when bound to GTP ('on') and inactive with GDP ('off').
78
How is G-protein switching regulated?
GAPs promote GTP hydrolysis ('on' to 'off'); GEFs promote GDP-GTP exchange ('off' to 'on').
79
What are post-translational modifications?
Chemical changes to a protein after translation, such as phosphorylation or ubiquitinylation.
80
What do protein kinases do?
They catalyze phosphorylation of amino acid side chains, modifying protein activity.
81
What do protein phosphatases do?
They remove phosphate groups from proteins, reversing kinase action.
82
How many protein kinases are in the human genome?
More than 500 different protein kinases.
83
What is fluorescence in molecular biology?
Fluorescence is the emission of light by a molecule (fluorophore) after it absorbs light energy and returns from an excited state to its ground state.
84
What are fluorophores?
Molecules that absorb light (excitation) and emit it at a longer wavelength (emission).
85
What happens during the excitation phase of fluorescence?
A photon is absorbed, and an electron in the fluorophore moves to a higher energy orbital.
86
What happens during the emission phase of fluorescence?
The excited electron returns to the ground state, releasing light energy as fluorescence.
87
What is photobleaching?
The chemical modification (often oxidation) of fluorophores after many excitation-emission cycles, causing them to stop fluorescing.
88
What are the key components of a fluorescence microscope?
1) Light source, 2) Excitation filter, 3) Dichroic mirror, 4) Objective lens, 5) Emission filter, 6) Sensitive camera.
89
What does the dichroic mirror do in a fluorescence microscope?
It reflects the excitation wavelength and transmits the emission wavelength.
90
What is the purpose of excitation and emission filters?
Excitation filter selects the light that excites the fluorophore, and emission filter allows only emitted light to reach the detector.
91
Where was fluorescence first observed in animals?
In jellyfish (Aequorea victoria) and coral (Discoma sp.).
92
How is GFP used to label proteins in living cells?
By creating gene fusions of GFP to the gene of interest, either at the N- or C-terminal end.
93
What determines whether a GFP tag is placed at the N- or C-terminus?
The choice depends on the structure and function of the protein being studied.
94
How are gene fusions for GFP typically introduced into organisms?
Via molecular cloning using vectors or by endogenous tagging through homologous recombination.
95
What is a chromophore in fluorescent proteins?
The part of the molecule responsible for fluorescence, formed by a post-translational modification.
96
What is special about the chromophore of GFP?
It forms inside a beta-barrel structure through cyclization of the peptide backbone.
97
How many fluorescent protein colors are available?
There are fluorescent proteins emitting from blue to far-red, allowing multiplexing in experiments.
98
Why is GFP useful for live-cell imaging?
Unlike immunofluorescence, which requires fixed (dead) cells, GFP allows real-time tracking in living cells.
99
What does your lab study using fluorescence in live cells?
DNA replication and genome integrity in live S. cerevisiae and human cell lines.
100
What serves as the template for new daughter DNA strand synthesis?
The parental strand serves as the template, with the new strand being complementary to it.
101
In which direction is the new DNA strand synthesized?
New strands are synthesized in the 5’ to 3’ direction, adding nucleotides to the 3’ end.
102
What chemical reaction occurs during DNA polymerization?
The phosphate group of the incoming deoxynucleotide triphosphate (dNTP) reacts with the 3’ hydroxyl group of the growing DNA chain, forming a phosphodiester bond.
103
What mechanism does DNA replication follow?
DNA replication proceeds through a semiconservative mechanism.
104
What structure forms as DNA unwinds during replication?
The replication fork forms as the DNA duplex opens progressively.
105
Which enzyme separates the DNA strands at the replication fork?
DNA helicase separates the duplex strands for replication.
106
What catalyzes the addition of nucleotides during DNA replication?
DNA polymerase catalyzes the polymerization of nucleotides.
107
What are the substrates required by DNA polymerase?
Deoxynucleoside 5’ triphosphates (dNTPs) and a primer (DNA or RNA).
108
Why is a primer necessary for DNA polymerase?
DNA polymerase can only add nucleotides to an existing strand; it requires a primer to provide a 3’ hydroxyl group to extend.
109
What is the role of topoisomerase in DNA replication?
Topoisomerase relieves supercoiling tension ahead of the replication fork.
110
What happens if the primer is RNA?
DNA polymerase extends the primer, resulting in a molecule with RNA at the 5’ end and DNA at the 3’ end.
111
What enzyme synthesizes the RNA primer?
Primase, a specialized RNA polymerase, synthesizes short RNA primers complementary to single-stranded DNA.
112
Why is DNA replication described as antiparallel synthesis?
Because the two strands of DNA are antiparallel, one strand (leading) is synthesized continuously 5’ to 3’, while the other (lagging) is synthesized discontinuously in fragments.
113
What problem arises from DNA's antiparallel nature during replication?
The lagging strand must be synthesized in the opposite direction to the replication fork movement, producing Okazaki fragments.
114
What are Okazaki fragments?
Short discontinuous DNA fragments made of RNA and DNA on the lagging strand.
115
How are Okazaki fragments processed?
RNA primers are removed by FEN-1 and ribonuclease H, replaced by DNA by DNA polymerase delta, and ligated by DNA ligase.
116
What is the replisome?
A multiprotein complex (molecular machinery) that carries out DNA replication.
117
What is the composition and function of the CMG helicase?
CMG helicase is a hexamer of MCM proteins with Cdc45 and GINS; it binds the leading strand and unwinds DNA.
118
What is the role of Replication Protein A (RPA)?
RPA binds single-stranded DNA to stabilize it and prevent secondary structure formation.
119
Which polymerase carries out leading strand synthesis?
DNA polymerase epsilon (Pol ε).
120
What is PCNA and its function?
PCNA is a homotrimeric sliding clamp that holds DNA polymerase on the DNA template, increasing processivity.
121
What does the primase/Pol alpha complex do?
Primase synthesizes RNA primers; Pol alpha extends primers with DNA, creating RNA-DNA hybrid primers.
122
Which complex completes Okazaki fragment synthesis?
DNA polymerase delta (Pol δ)/PCNA complex replaces Pol alpha to complete Okazaki fragment synthesis.
123
What is the function of RFC in DNA replication?
RFC is the clamp loader that opens PCNA and loads it onto DNA at the primer site.
124
How are RNA primers removed during lagging strand synthesis?
Ribonuclease H and FEN-1 displace RNA primers from Okazaki fragments.
125
How are gaps filled after RNA primer removal?
Pol delta fills the gaps with DNA.
126
How are DNA fragments joined?
DNA ligase seals the nicks between DNA fragments.
127
How does DNA replication initiate at origins?
Replication origins, often AT-rich regions, are marked by the Origin Recognition Complex (ORC) which loads helicase with help from initiation factors.
128
When is the MCM helicase loaded and activated?
MCM helicase is loaded in G1 phase in an inactive form and activated in S phase by phosphorylation and interactions.
129
How many helicases are loaded at each origin and in what direction do they move?
Two helicases are loaded at each origin, moving in opposite directions for bidirectional replication.
130
Summarize the replication steps after helicase unwinding."
RPA binds single strands → primase/Pol alpha synthesizes primers → Pol delta/RFC/PCNA extends primers → primers removed by FEN-1 and RNase H → DNA replaced and ligated.
131
How many hydrolytic depurination events occur daily per cell?
Approximately 2,000 to 10,000 events.
132
How often does cytosine deamination occur per cell?
About once every 5 days.
133
How often does guanine oxidation occur per cell?
About once every 5 days.
134
How many adenine methylation events occur per day per cell?
Around 600 events.
135
What types of DNA modifications can alter base pairing or block DNA polymerase?
Deamination, oxidation, and alkylation produce many modified bases that can alter base pairing or block polymerase.
136
Define mutation.
"Permanent
137
What causes mutations?
Mutations can occur spontaneously, from transposable elements, or replication errors.
138
What is a mutagen?
Chemical compounds or radiation that increase mutation frequency.
139
How are mutagens related to carcinogens?
Many mutagens are carcinogens that cause cancer.
140
What causes mismatches in DNA?
Mistakes during DNA replication.
141
How often does DNA polymerase make a mistake without proofreading?
About 1 mistake per 10,000 nucleotides.
142
How much does proofreading exonuclease activity improve DNA polymerase fidelity?
About 100-fold improvement, reducing errors to 1 in a million.
143
How much does mismatch repair (MMR) improve replication fidelity?
Approximately 1000-fold improvement, reducing mutations to 1 in 10^9-10.
144
How does paternal age affect mutation rates in offspring?
Mutations inherited from fathers increase with age due to ongoing germ cell production.
145
What human disorders are linked to increased paternal age?
Autism and schizophrenia.
146
Why do viruses have higher mutation rates?
Higher mutation rates increase the chance of evolving successful variants.
147
Which eukaryotic DNA polymerases have 3’ to 5’ exonuclease proofreading activity?
DNA polymerases delta and epsilon; polymerase alpha does not.
148
What is the role of proofreading exonuclease activity?
It removes incorrectly incorporated nucleotides by excising them.
149
How does mismatch repair (MMR) function in humans?
MSH2/MSH6 detect mismatch on daughter strand, recruit MLH1/PMS2 endonuclease to cut near mismatch, helicase and exonuclease remove segment, then Pol delta and ligase repair the gap.
150
What role does PCNA play in mismatch repair?
PCNA remains on DNA after polymerase detaches and interacts with MMR proteins.
151
What is base excision repair (BER)?
Repair of small base modifications like deamination or oxidation.
152
What happens when cytosine deaminates spontaneously?
Forms uracil, which is abnormal in DNA and can cause mutations if unrepaired.
153
How does BER repair uracil in DNA?
UNG (uracil DNA glycosylase) removes uracil base, APE1 cuts backbone, DNA Pol beta fills gap, AP lyase removes sugar phosphate, and ligase seals nick.
154
What type of DNA damage does nucleotide excision repair (NER) fix?
Bulky lesions like thymine-thymine dimers caused by UV light or chemical adducts from aflatoxin and smoke.
155
What proteins initiate damage recognition in NER?
XPC and 23B form a complex to recognize DNA lesions.
156
What role does TFIIH play in NER?
TFIIH unwinds DNA around the lesion.
157
Which proteins cut out the damaged DNA during NER?
XP-F and XP-G endonucleases cut 24-32 base fragments containing the lesion.
158
How is the DNA gap filled after NER excision?
DNA polymerase fills the gap and DNA ligase seals the strand.
159
What diseases are linked to defects in mismatch repair?
Increased susceptibility to colon and ovarian cancer.
160
What diseases are linked to defects in nucleotide excision repair?
Increased susceptibility to skin cancer.
161
What is translesion synthesis (TLS)?
A DNA damage tolerance process where special TLS polymerases bypass lesions during replication.
162
Why are TLS polymerases error-prone?
They lack proofreading activity and often insert incorrect bases opposite lesions.
163
When does TLS occur?
When the replisome encounters unrepaired DNA lesions and normal polymerases stall.
164
What is a Holliday junction?
A DNA structure formed during homologous recombination where two double-stranded DNA molecules exchange strands.
165
How does the resolution of Holliday junctions affect cells?
It determines whether parental or recombinant chromosomes are produced, impacting genetic variability.
166
What is the primary purpose of double-strand breaks (DSBs) during meiosis?
To generate genetic variability through recombination between maternal and paternal chromosomes.
167
What is Non-Homologous End Joining (NHEJ)?
An error-prone DSB repair pathway active in G1 phase that joins DNA ends without sequence homology.
168
Why is NHEJ considered error-prone?
It processes DNA ends with exonuclease and polymerase activity to create blunt ends, leading to nucleotide loss or addition and possible chromosomal translocations.
169
Which proteins form the initial binding complex in NHEJ?
DNA-dependent protein kinase (DNA-PK) and the Ku70/Ku80 heterodimer.
170
During which cell cycle phases is homologous recombination (HR) the primary DSB repair pathway?
During S, G2, and M phases when sister chromatids are available.
171
What is the key advantage of homologous recombination over NHEJ?
HR uses a homologous template for accurate DNA repair, minimizing mutations.
172
What DNA repair mechanisms mainly operate during the S phase besides HR?
Mismatch repair (MMR) and translesion synthesis (TLS).
173
Why are DNA-damaging chemotherapy drugs effective against tumors?
Because tumor cells divide rapidly and rely on DNA replication, making them vulnerable to DNA damage.
174
What characterizes mismatch repair (MMR) in humans?
MSH2 and MSH6 detect mismatches on daughter strands, triggering MLH1/PMS2 to cut and remove error-containing DNA, which is then resynthesized.
175
What is translesion synthesis (TLS)?
A DNA damage tolerance mechanism where specialized polymerases bypass lesions, often inserting incorrect bases and lacking proofreading.
176
Name two organisms known for exceptional DNA repair and radiation resistance.
Tardigrades and Deinococcus.
177
What is the main challenge in repairing double-strand breaks in G1 phase?
Only one genome copy is present, so error-prone NHEJ is used instead of homologous recombination.
178
Fill in the blank: NHEJ requires _____ ends for ligation, which are generated by exonuclease and polymerase activity.
blunt
179
What are chromosomal translocations?
Abnormal chromosome regions formed by joining fragments from two non-homologous chromosomes, often due to NHEJ.
180
What is the main function of the Ku70/Ku80 complex?
To bind DNA ends and recruit DNA-PK during NHEJ.
181
How does DNA replication affect mutation rates in germ cells?
Mutation rates increase with paternal age due to continuous germ cell production.
182
Which polymerases have proofreading 3’ to 5’ exonuclease activity in eukaryotes?
DNA polymerases delta and epsilon (not alpha).
183
Name the protein complex that loads PCNA onto DNA.
RFC (Replication Factor C).
184
185
Question
Answer
186
Visual summary: Repair Type - NHEJ; Cell Cycle Phase; Accuracy; Key Proteins; Outcome
NHEJ; G1; Error-prone; Ku70/Ku80, DNA-PK, Ligase; Small insertions/deletions, chromosomal translocations possible
187
Visual summary: Repair Type - Homologous Recombination; Cell Cycle Phase; Accuracy; Key Proteins; Outcome
Homologous Recombination; S, G2, M; High fidelity; RAD51, BRCA1/2; Accurate repair, genetic recombination
188
Visual summary: Repair Type - Mismatch Repair (MMR); Cell Cycle Phase; Accuracy; Key Proteins; Outcome
Mismatch Repair; S; High fidelity; MSH2, MSH6, MLH1/PMS2; Removal of replication mismatches
189
Visual summary: Repair Type - Translesion Synthesis (TLS); Cell Cycle Phase; Accuracy; Key Proteins; Outcome
Translesion Synthesis; S; Error-prone; TLS polymerases; Bypass DNA lesions with mutagenesis
190
What are the two main levels of DNA organization?
Chromatin and Chromosomes.
191
Why is DNA more compacted during metaphase?
To facilitate easier division of DNA during cell division.
192
What stages of the cell cycle make up interphase?
G1, S, and G2 phases.
193
What is chromatin during interphase?
Less compacted DNA that allows transcription and replication.
194
What are nucleosomes composed of?
Histone proteins and DNA, separated by linker DNA.
195
How is DNA organized around nucleosomes?
DNA is wound around a histone octamer (~2 turns).
196
What histones form the nucleosome core?
Two copies each of H2A, H2B, H3, and H4.
197
What role does histone H1 play?
Stabilizes and organizes the 30-nm chromatin fiber.
198
Describe the solenoid and 2-start helix models of 30-nm fiber formation.
Solenoid: stacked circles; 2-start helix: two helices wrapped around each other.
199
How do histone tail modifications regulate chromatin?
Modifications like acetylation neutralize positive charges, affecting chromatin condensation and transcription.
200
What is euchromatin?
Decondensed, gene-rich chromatin that is transcriptionally active.
201
What is heterochromatin?
Highly condensed chromatin, rich in repetitive DNA and transcriptionally inactive.
202
What are polytene chromosomes?
Giant interphase chromosomes with many parallel chromatids (e.g., from fly salivary glands).
203
What do chromatin puffs represent?
Regions of chromatin decondensation with active transcription.
204
What proteins mediate DNA looping in chromatin?
Structural Maintenance of Chromosomes (SMC) proteins.
205
What specialized SMC proteins condense chromosomes during metaphase?
Condensins (Condensin I and II).
206
What are the three essential chromosome elements for replication and inheritance?
Origin of replication, centromere, and telomeres.
207
What is the function of the centromere?
Links chromosomes to spindle fibers via the kinetochore for segregation.
208
What unique histone is found at centromeres?
CENP-A, a centromere-specific histone variant.
209
What is the telomere problem during DNA replication?
Lagging strand cannot fully replicate chromosome ends, causing shortening.
210
How does telomerase solve the telomere problem?
It extends telomeres by adding repeats using its RNA template.
211
What type of enzyme is telomerase?
A reverse transcriptase (DNA polymerase using RNA as a template).
212
In which cells is telomerase active?
Germ cells and stem cells; often reactivated in cancer cells.
213
What is the significance of telomerase activity in cancer?
It allows unlimited cell division, making telomerase a target for cancer therapy.
214
How do telomeres protect chromosome ends?
They prevent exonuclease degradation, end-to-end fusion, and signal chromosome ends.
215
Why do somatic cells have limited division capacity?
Because telomerase is inactive and telomeres shorten with each division.
216
Describe the nucleosome structure (visual summary).
DNA wraps ~2 turns around a histone octamer (H2A, H2B, H3, H4), with linker DNA and histone H1 stabilizing the 30-nm fiber.
217
Describe chromatin compaction from nucleosome to metaphase chromosome (visual summary).
Nucleosomes ('beads on a string') → 30-nm fibers (solenoid or 2-start helix) → loops formed by SMC proteins → metaphase chromosomes condensed by condensins.
218
What is the genome?
The entirety of an organism’s hereditary information, usually DNA (some viruses have RNA).
219
What mainly causes differences in genome size among eukaryotes?
The amount of non-coding DNA.
220
What percent of the human genome codes for proteins?
About 3%.
221
What is a gene?
A nucleic acid sequence necessary for synthesis of a functional product (polypeptide or RNA).
222
What do eukaryotic gene exons contain?
The coding region or Open Reading Frame (ORF).
223
What separates exons in eukaryotic genes?
Introns, which are spliced out during mRNA processing.
224
What are control regions in a gene?
Promoters and cis-regulatory factors that help regulate transcription.
225
What is a transcription unit?
A DNA region between initiation and termination sites transcribed into a single primary transcript.
226
What is alternative splicing?
Generating multiple transcripts and protein isoforms from one gene by varying exon inclusion.
227
What distinguishes solitary and gene family protein-coding genes?
Solitary genes occur once; gene families are duplicates with similar sequences/functions.
228
What are orthologs?
Same protein in different species, often with conserved function.
229
What are paralogs?
Related proteins in the same species arising from gene duplication.
230
What percent of the genome is simple sequence repeats (SSRs)?
About 6%.
231
What are minisatellites?
Repeats of ~14–100 bp, 20-50 tandem units, often in centromeres and telomeres.
232
What are microsatellites?
Repeats of 1-4 bp, up to 600 bp long, often inside transcription units.
233
What is backward slippage?
DNA polymerase slips during repeat replication, causing expansion linked to diseases like Huntington’s.
234
How are SSRs used in forensic science?
DNA fingerprinting, paternity testing, criminal identification.
235
What percentage of the genome is made of transposons?
Approximately 3%.
236
How do DNA transposons move?
Cut and paste mechanism using DNA transposase, causing 9 bp duplications.
237
What percentage of the genome do retrotransposons represent?
About 40%.
238
How do retrotransposons replicate?
Copy themselves via an RNA intermediate using reverse transcriptase.
239
What are LTR retrotransposons?
Have long terminal repeats, similar to retroviruses but lack envelope proteins.
240
What are LINEs and SINEs?
Nonviral retrotransposons; LINEs encode proteins for retrotransposition; SINEs use LINEs proteins.
241
Describe the mechanism of LINE insertion.
RNA exported, proteins ORF1 and ORF2 bind RNA, complex re-enters nucleus, DNA cut at AT-rich sites, reverse transcription primes insertion.
242
How do transposable elements impact genomes?
They influence evolution and can cause mutations leading to disease.
243
Visual summary: What are SSRs and their role?
SSRs are tandem repeats (mini- and microsatellites); they are hypervariable and used in DNA fingerprinting and linked to diseases via repeat expansions.
244
Visual summary: How do retrotransposons replicate?
LTR retrotransposons transcribe RNA → reverse transcribed to DNA → integrated into genome by integrase; LINEs/SINEs use similar RNA intermediate mechanisms without integrase.
245
What happens to the genomes of obligate endosymbiont bacteria?
They undergo genome reduction over time.
246
How do solar-powered sea slugs obtain photosynthesis capability?
By stealing chloroplasts from algae (kleptoplasty).
247
What kind of symbiotic relationship do corals have?
Endosymbiotic relationship with phototrophic dinoflagellates.
248
Where are most proteins for mitochondria and chloroplasts encoded?
In the nucleus.
249
What evolutionary process has affected gene location between organelles and nucleus?
Gene transfer from organelles (mitochondria, chloroplasts) to the nucleus.
250
What is unique about mitochondrial ribosomes?
They have sequence similarity to bacterial ribosomes.
251
Do proteins coded in mitochondria leave the organelle?
No, all proteins coded in mitochondria stay within the organelle.
252
How are mitochondria inherited?
Through cytoplasmic inheritance, independent of nuclear DNA inheritance.
253
How are mitochondria distributed during cell division?
They are distributed evenly among daughter cells.
254
What effect do mutations in mitochondrial DNA have in mammals?
They may be related to aging.
255
What is observed in mice with defective mitochondrial DNA polymerase proofreading?
They exhibit premature aging.
256
Visual summary: Describe the gene transfer between organelles and nucleus.
Over evolution, many genes originally in mitochondria and chloroplasts have moved to the nucleus, but mitochondria still retain their own DNA and ribosomes.
257
Visual summary: How is mitochondrial inheritance different from nuclear inheritance?
Mitochondria are inherited through the cytoplasm (typically maternal) and distributed evenly to daughter cells, unlike nuclear DNA which segregates during mitosis/meiosis.
258
What was the starting material for sequencing ancient specimens like mummies?
DNA extracted from mummy samples.
259
What did early sequencing methods require for DNA analysis?
Cloning DNA into plasmids.
260
What is recombinant DNA technology?
Combining a vector and DNA fragment to create recombinant DNA that replicates in host cells for sequencing and manipulation.
261
What is a plasmid?
A circular dsDNA molecule that replicates independently of the chromosome, commonly used as vectors in recombinant DNA tech.
262
What is the role of restriction enzymes?
They recognize specific palindromic sequences and cut DNA, creating double-strand breaks with sticky or blunt ends.
263
What is an example of a restriction enzyme and its recognition site?
EcoRI recognizes GAATTC and cuts to produce sticky ends.
264
What enzyme ligates DNA fragments together?
T4 DNA ligase, which requires ATP.
265
What key features must a plasmid have to be used as a cloning vector?
An origin of replication (ORI), selectable marker (e.g., ampicillin resistance), and a polylinker with restriction sites.
266
What is the general procedure for cloning DNA into plasmids?
Cut plasmid and DNA with restriction enzymes, ligate fragments, transform bacteria, and select with antibiotic.
267
What are DNA libraries?
Permanent collections of DNA clones: genomic libraries (whole genome) or cDNA libraries (mRNA-derived).
268
Why is cDNA useful for gene studies?
It represents mature RNA without introns, easier to clone and express in bacteria.
269
How is cDNA synthesized?
Using reverse transcriptase on mRNA primed with oligo-dT, followed by second strand synthesis.
270
What special features do protein expression vectors have?
Inducible promoters (like IPTG), purification tags (e.g., HisTag), resistance markers, and ORI.
271
Why use inducible promoters in expression vectors?
To control protein expression and reduce toxicity or burden on host cells.
272
What advantage does producing proteins in E. coli offer?
High protein yield, ease of purification.
273
Visual summary: What does a cloning plasmid contain?
ORI, selectable marker, polylinker with restriction sites for insertion of DNA fragments.
274
Visual summary: Describe the cloning workflow.
Digest plasmid and DNA, ligate fragments, transform bacteria, select antibiotic resistant colonies, isolate plasmids, sequence or express DNA.
275
Visual summary: How is cDNA made from mRNA?
mRNA is primed with oligo-dT, reverse transcribed into cDNA, second strand synthesized, then cloned.
276
Visual summary: What features aid protein expression in bacteria?
Inducible promoter, purification tag, antibiotic resistance marker, and replication origin.
277
What is the purpose of PCR?
To amplify specific DNA fragments from a small sample for sequencing, cloning, detection, and gene editing.
278
What are the key components required for PCR?
DNA template, primers (oligonucleotides), dNTPs, and Taq DNA polymerase.
279
What are the main steps in PCR?
1. Denaturation - melting the double-stranded DNA; 2. Annealing - primers bind to complementary sequences; 3. Extension - DNA polymerase synthesizes new DNA strand.
280
Why does the melting temperature (Tm) vary?
It depends on the length of the DNA and the GC content; more GC pairs increase Tm.
281
Why are DNA primers preferred over RNA primers in PCR?
DNA primers are more stable and harder to degrade.
282
What is special about Taq polymerase?
It is heat-stable and can withstand high denaturation temperatures but lacks proofreading activity.
283
How is PCR product analyzed?
By gel electrophoresis to check size and amount of amplified DNA.
284
How can PCR be used for cloning?
By adding restriction enzyme recognition sites to primers, allowing the amplified fragment to be cut and ligated into plasmids.
285
What type of DNA did Dr. Paabo sequence from extinct species like the Tasmanian tiger and giant sloth?
Mitochondrial DNA.
286
What are some common applications of DNA sequencing?
Studying phylogenetic relationships, ancestry, forensic identification, and disease diagnosis.
287
What is the principle of Sanger sequencing?
Incorporation of fluorescently labeled dideoxynucleotides (ddNTPs) terminates DNA synthesis at specific bases, producing DNA fragments of varying length.
288
Why do ddNTPs terminate DNA synthesis?
They lack a 3' hydroxyl group necessary for forming the phosphodiester bond.
289
How are sequencing products separated and detected?
By gel electrophoresis or capillary electrophoresis with fluorescence detection.
290
What limits the speed of whole genome sequencing by Sanger method?
The number of samples processed simultaneously is limited and the method is time-consuming.
291
Visual summary: Outline the PCR cycle.
Denaturation (heat to separate strands), Annealing (cool to allow primers to bind), Extension (DNA polymerase synthesizes new strands).
292
Visual summary: How are restriction sites used in PCR cloning?
Restriction sites are added to primers, so PCR products can be digested and ligated into plasmid vectors.
293
Visual summary: Describe the Sanger sequencing method.
DNA synthesis with fluorescent ddNTPs terminates strands at different lengths; fragments separated by electrophoresis reveal DNA sequence.
294
What are the main steps in the sequencing pipeline?
DNA isolation, fragmentation, library preparation, amplification, sequencing, assembly
295
What is the purpose of PCR in Illumina sequencing?
To obtain many copies of the same DNA molecule in a very small space using DNA clusters
296
What is ligated to both ends of double-stranded DNA in Illumina sequencing?
Linkers that contain sequences recognized by primers
297
What happens to DNA after denaturation in Illumina sequencing?
Single strands bind to primers on a surface (e.g., microscope slide) and DNA polymerase generates complementary strands
298
What happens after the DNA polymerase step in Illumina sequencing?
One single strand is shorter, the other is bound to the surface, then denaturation and washing remove unbound DNA
299
What is formed by DNA clusters in Illumina sequencing?
Colonies of identical DNA sequences
300
What type of detection does Illumina sequencing use?
Fluorescence-based detection
301
How is sequencing done in Illumina after DNA clusters are formed?
Add fluorescently labeled dNTPs one at a time, image fluorescence, chemically remove fluorophores, and repeat
302
Who sequenced the Neanderthal genome using NGS techniques?
Dr. Paabo's group
303
What are the advantages of NGS?
High sensitivity, requires small sample amounts, and produces huge numbers of reads per experiment
304
What information can sequence analysis provide?
Common ancestry, shared protein function or structure, timing of evolutionary events, mutations linked to disease
305
What is sequence similarity?
The degree of match between two sequences, usually expressed as a percentage
306
What are homologous sequences?
Sequences derived from a common ancestral sequence
307
What is the goal when searching for sequence similarity?
To find maximal similarity or best alignment between sequences
308
Why are gaps introduced in sequence alignment?
To maximize the match between sequences of different lengths
309
What is a dot plot used for in sequence analysis?
Visualizing alignments, changes, inversions, and duplications between sequences
310
What does BLAST stand for?
Basic Local Alignment Search Tool
311
What are BLASTN and BLASTP used for?
BLASTN for nucleotide similarity, BLASTP for protein similarity
312
How does BLASTN work?
Looks for exact matches, extends matches locally, allows short gaps
313
What distinguishes nanopore sequencing from other NGS methods?
No PCR amplification, no DNA polymerase, dNTPs or primers; based on changes in electric current through a pore
314
What are advantages of nanopore sequencing?
Single molecule sequencing, very long reads, detection of DNA modifications, portable
315
Why are long reads useful in sequencing?
They help map repetitive sequences and reduce the need for complex genome assembly
316
How is DNA methylation related to sequencing?
Methylation patterns detected by sequencing are linked to cancer progression and can aid clinical decisions
317
What is RNA polymerase quaternary structure?
It assembles with other polypeptides and proteins to form supramolecular complexes such as the transcription initiation complex.
318
What is the difference between a gene, exon, and open reading frame (ORF)?
Gene: entire nucleic acid sequence for a functional product; Exon: coding part of the genome; ORF: DNA sequence from start to stop codon defined by translation.
319
What does mature mRNA contain?
Only exons (no introns) after post-transcriptional processing.
320
Do bacteria generally have introns?
No, bacteria generally do not have introns.
321
How do LTR retrotransposons differ from LINEs in their mechanism?
LTRs produce RNA in the cytoplasm and use reverse transcriptase; LINEs produce RNA that is reverse transcribed into double-stranded DNA with reverse transcriptase that also has endonuclease activity, but no transposase.
322
Do transposons code for transposase?
Yes, transposons encode transposase.
323
What protein coding regions do LTRs and LINEs contain?
Reverse transcriptase, integrase, and nuclease that catalyze their own mobility.
324
How is DNA synthesis done in retrotransposons?
By reverse transcriptase, which has low fidelity and sometimes falls off causing non-coding repetitive sequences.
325
What is the DNA amplification factor in PCR?
Exponential amplification: 2^n, where n is the number of PCR cycles.
326
What are the two types of DNA breaks involved in homologous recombination?
Single strand breaks and double strand breaks.
327
What happens to DNA ends during homologous recombination?
5' ends are digested by exonucleases leaving 3' single-stranded overhangs.
328
What proteins mediate strand invasion in homologous recombination?
Rad51 in eukaryotes and RecA in bacteria.
329
What is a Holliday structure?
A cross-shaped DNA intermediate formed during homologous recombination.
330
What enzymes extend the 3' ends during homologous recombination?
DNA polymerase extends the 3' ends using the homologous strand as a template.
331
How many DNA polymerases are primarily involved in eukaryotic DNA replication and what are they?
Three main polymerases: Pol epsilon (leading strand), Pol delta (lagging strand), and Primase/Pol alpha complex (primer synthesis).
332
What is the role of Pol epsilon?
Leading strand DNA synthesis; binds to PCNA.
333
What is the role of Pol delta?
Lagging strand DNA synthesis; binds to PCNA.
334
What does the Primase/Pol alpha complex do?
Primase synthesizes RNA primer; DNA Pol alpha extends primer with DNA but lacks proofreading activity.
335
What does ribonuclease H do?
Removes RNA primers during DNA replication.
336
What is Fen-1’s function?
Helps displace and cut the RNA primer flap during lagging strand synthesis.
Biol 112
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