Review Flashcards
(216 cards)
1
Q
What did Meselson & Stahl discover?
A
That DNA replication is semi-conservative using E. coli and “heavy” nitrogen
2
Q
After one generation, what was the density of the N found in the DNA of the Meselson & Stahl experiment?
A
Half heavy (15) and half light (14)
3
Q
What is the composition of a 3rd generation N labeled DNA?
A
25% mid density
75% light density
4
Q
What are the two requirements for DNA replication by DNA polymerases?
A
Template
Primer
5
Q
What is a template?
A
A DNA strand used to specify the nucleotides to be incorporated into the daughter strand
6
Q
What is a primer?
A
A segment of nucleic acid (RNA or DNA) base paired to the template, with a free 3’-OH
7
Q
What provides the chemical basis for semi-conservative replication?
A
The need for a template for DNA synthesis
8
Q
After adding a base, what is done by DNA polymerase I?
A
It pauses with the base still in the active site
If the base is incorrect, proofreading nuclease moves back and cuts it out
Polymerase resumes
9
Q
What is the accuracy of replication due to DNA polymerase I?
A
10^6 - 10^8
10
Q
Where does DNA replication begin?
A
At the ORI
11
Q
How many proteins are involved in the replication of E. coli DNA?
A
Over 20; plus DNA polymerase III and control proteins
12
Q
What is the prepriming complex?
A
dnaB (a helicase)/dnaC dimers bind to open complex and unwind origin DNA to expose single strand template
13
Q
How does replication get initiated?
A
dnaA binds to oriC and forms the initial complex
Open complex formed by denaturation
Prepriming complex is formed
SSBs bind to single stranded DNA
14
Q
What are SSBs/what do they do?
A
Single stranded binding proteins
Stabilize single stranded DNA
15
Q
Which steps in the initiation of replication need energy?
A
All of them
16
Q
What repairs the Okazaki fragments?
A
DNA polymerase
DNA ligase
17
Q
What is the 3d enzyme activity of DNA polymerase I?
A
5’ to 3’ exonuclease
18
Q
What does DNApolymerase I’s exonuclease do?
A
Removes RNA from 5’ end of Okazaki fragment in front of DNA polymerase I molecule
-DNA ligase covalently seals the gaps in the backbone
19
Q
What are telomeres?
A
Buffer zones on the end of eukaryotic chromosomes
20
Q
How do telomeres act as buffer zones?
A
Have hundreds of tandem repeated copies of GT-rich sequence (usually 6-8 bases long) - if one is cleaved off, it doesn’t matter
21
Q
What structures to telomeres form?
A
Unusual, non-helical structures
22
Q
Do telomeres need a template to be synthesized?
A
No
23
Q
How are telomeres synthesized?
A
By telomerase
24
Q
What is telomerase?
A
A specialized reverse transcriptase
25
What component is found in telomerase that serves as a template for synthesis?
An RNA component
26
What do telomeres prevent?
Undesirable fusion of chromosomes
| Aberrant recombination
27
What attaches chromosomes to the nuclear envelope?
Telomeres
28
What serves as a "mitotic clock"?
Telomeres - Short telomeres induces senescence and/or apoptosis
29
Where is there no telomerase activity found?
In differentiated cells
30
Where is telomerase active?
In:
Germ cells
Stem cells
Tumors
31
How many RNA polymerases does E. coli use for all transcript synthesis?
1
32
How many subunits is found in the core enzyme of E. coli?
5
33
What are the 5 subunits of E. coli core enzymes?
2 alpha
1 beta
1 beta'
1 omega
34
E. coli core enzymes are very inefficient at what?
Initiating RNA synthesis
35
What is the additional subunit needed to add to the E. coli core enzyme for efficient initiation?
Sigma factor - its addition completes the RNA polymerase holoenzyme
36
What are the three stages of RNA synthesis?
Initiation
Elongation
Termination
37
In which direction does the DNA coding (sense) strand run?
5' to 3'
38
In which direction does the DNA template (anti-sense) strand run?
3' to 5'
39
In which direction does the RNA transcript run?
5' to 3'
40
What are the steps of elongation?
Next nucleotide aligned w/template; first phosphodiester bond formed
RNA polymerase moves along template 1 base at a time
DNA unwound in front of enzyme; rewound behind
41
How fast does RNA polymerase move along the template?
About 50 bases per second
42
What facilitates unwinding during elongation?
-ve supercoiling
43
At any one point, about how many base pairs are unwound?
17
44
How many nuclear RNA polymerases do eukaryotes have?
3
45
Where is RNA polymerase I found?
Nucleolus
46
Where are DNA polymerases II and III found?
Nucleus
47
What does RNA polymerase II core enzyme protein require for efficient initiation?
Numerous additional proteins (it is a multisubunit protein)
48
What genes are transcribed by RNA polymerase I?
rRNA (18S, 28S, 5.8S)
49
What genes are transcribed by RNA polymerase II?
mRNA
50
What genes are transcribed by RNA polymerase III?
tRNA
| 5S rRNA
51
What is a primary transcript?
A newly synthesized RNA molecule
52
What has to be done to prokaryote mRNAs before they are ready to be used in translation?
Nothing
53
What modification does RNA undergo to become tRNA?
Base modifications
54
What must occur to RNA to become rRNA?
Cleavage of large primary transcript into functional fragments
55
What is found on the 3' end of mature tRNA?
CCA
56
What two enzymes are at work to modify a primary transcript into mature tRNA?
RNase P
| RNase D
57
What things are done in mRNA processing?
Addition of 5' cap
Addition of 3' polyA tail
Splicing of introns
58
What is function of the 3' polyA tail on mRNA?
Enhance mRNA stability
59
What is the 5' cap?
7-methylguanosine, added via 5',5'-triphosphate linkage
60
What is it thought that the 5' cap does for mRNA?
Enhance translation
61
How many codons are there?
64
62
How many stop codons?
3
63
What are the stop codons?
UAA
UGA
UAG
64
What is the initiation codon?
AUG
65
What is UUU?
Phe
66
What is CCC?
Pro
67
What is GGG?
Gly
68
What is AAA
Lys
69
What is stage 1 of protein synthesis?
"Charging" tRNA with proper amino acid
70
How are amino acids attached to tRNA during charging?
Via ester bonds (carboxylic acid to sugar OH) to the 3' OH
71
What catalyzed the aminoacylation of the 3' OH (charging) of the tRNA?
Amino acyl-tRNA synthetases
72
What is stage 2 of protein synthesis?
Initiation
73
How is initiation started?
By recognition of the initiation codon
74
What is the "regular" tRNA in bacteria?
tRNA^Met
75
What is the initiation tRNA in bacteria?
tRNA^fMet
76
What is the starting amino acid in bacteria?
N-formylmethionine
77
What donates the formyl group to tRNA^fMet?
N610-formyltetrahydrofolate
78
What are the 7 components required to initiate protein synthesis?
1. 30S ribosomal subunit (contains 16S rRNA)
2. mRNA
3. Initiation fMet-tRNA^fMet
4. Three initiation factors (IF-1, IF-2, IF-3)
5. GTP
6. 50 S ribosomal subunit
7. Mg^2+
79
How many steps does it take the 7 initiation components of bacteria to assemble into the initiation complex?
3 steps
80
What is step 1 of assembling the initiation complex (bacteria)?
Binding positions the initiation codon on the 30S subunit
| -Codon bound to P site of 30S
81
How is the initiation codon distinguished in step 1 of initiation complex assembly (bacteria)?
By proximity to the Shine-Dalgarno sequence
82
What blocks the A site in the assembly of the initiation complex (bacteria)?
IF-1
83
What is step 2 of assembling the initiation complex (bacteria)?
Initiator tRNA binds to P site
| -30S subunit, mRNA, IF-1, IF-3 complex is joined by IF-2 and fMet-tRNA^fMet
84
What is step 3 of assembling the initiation complex (bacteria)?
Large 50S subunit binds to complex
85
What is the 1st step of elongation?
Bind the next aminoacyl-tRNA (to the A site)
86
What is the 2nd step of elongation?
Peptide bond formation
87
What are the 5 stages of protein synthesis?
1. Activation of Amino Acids
2. Initiation
3. Elongation
4. Termination and ribosome recycling
5. Folding
88
During elongation, where does the peptide bond form?
Between the 2 amino acids
89
What enzyme catalyzes the peptide bond formation during elongation?
Peptidyl transferase
90
What type of enzyme is peptidyl transferase?
Ribozyme - it is the large subunit 23S rRNA
91
What is stage 4 of protein synthesis?
Termination
92
What begins termination?
Ribosome encounters an in-frame stop codon in the A site
93
What are the 3 termination factors that may bind to the termination codon?
RF1, RF2, or RF3
94
RF1 binds to which termination codons?
UAG
| UAA
95
RF2 binds to which termination codons?
UGA
| UAA
96
What effect do the termination factors have on protein synthesis?
Induce peptidyl transferase to transfer peptide chain to water, releasing the peptide chain from the ribosome
- Uncharged tRNA in P site released
- 30S and 50S subunits dissociate
97
How many termination factors are found in eukaryotes?
1 - eRF
98
What determines whether ribosomes are free or membrane-bound?
The protein being synthesized
99
What proteins are made by free ribosomes?
Nuclear proteins
Peroxisomal proteins
Mitochondrial proteins
100
Where are nuclear, peroxisomal and mitochondrial proteins made?
In the cytoplasm
101
What signal sequence brings proteins into the nucleus?
Internal nuclear localization sequences
102
What signal sequence brings proteins into peroxisomes?
C-terminal sequence SKF
103
What signal sequence brings proteins into the mitochondrial matrix?
N-termina sequence, rich in positively charged amino acids and ser & thr
104
What proteins are made in the RER?
Secretory proteins
Lysosomal proteins
Integral membrane proteins
105
Ribosomes bound to the endoplasmic reticulum synthesize what 3 classes of protein?
Secretory proteins
Lysosomal proteins
Integral membrane proteins
106
How is the signal sequence removed from secreted proteins?
By cleavage via signal peptidase
107
What occurs on the cytosol side of the ER?
Nucleotide activated sugar precursors are added stepwise to the phosphate group forming a dolichol pyrophosphate intermediate
108
What happens to incomplete core-dolichol pyrophosphate?
It is translocated across ER membrane into lumen
109
What are the 1st 7 sugars in the synthesis of core oligosaccharides?
2 GlcNAc
| 5 mannose
110
Where are nucleotide activated precursors found (oligosaccharides)?
Cytosolic side
111
Where are dolichol-phosphate activated precursors found (oligosaccharides)?
Lumen side
112
What sugars are added in the ER lumen (oligosaccharides)?
4 mannose
| 3 glucose
113
When the oligosaccharide is transferred to a specific Asn residue in protein, what is released?
Dolichol pyrophophate
114
How is dolichol phosphate regenerated?
By action of a phosphatase
115
How does tunicamycin affect the addition of sugars?
Blocks the 1st step in oligosaccharide synthesis
116
How does Bacitracin affect addition of sugars?
Blocks phosphatase from recycling dolichol phosphate
117
Why is Bacitracin a useful antibiotic?
The bacterial enzyme is very sensitive to it
118
What is an important signaler for lysosome targeting?
Mannose-6-phosphate
119
What signals for protein to return to the ER?
KDEL
120
What does KDEL stand for?
Lys-Asp-Glu-Leu
121
Where is KDEL found?
Carboxy-terminal on ER proteins
122
Most cell surface receptors are what?
Transmembrane glycoproteins
123
Where are many receptors located?
In specialized regions of the plasma membrane - coated pits
124
What does the cytosolic side of the "pits" have?
Thick coat of clathrin
125
With what does clathrin help?
Endocytosis
126
What does clathrin do in endocytosis?
Forms a lattice around the coated pit, excising it from the membrane, and forming a coated vesicle
127
Eukaryotic RNA polymerases have very low affinity for ___ without help from DNA binding proteins.
Promoters
128
Which type of chromatin is "turned off"?
Heterochromatin
129
DNA is ___ in heterochromatin.
Hypermethylated (at CpG dinucleotides)
130
Histones are ___ in heterochomatin.
Deacetylated
131
In euchromatin, DNA is ___.
Hypomethylated
132
In euchromatin, histones are ___.
Acetylated
133
What is the difference between Prader-WIlli and Angelman?
The 15q11-13 region contains genes that are differentially silenced in male vs female gametes
134
If mutant chromosome 15 is inherited from the father, what genes are expressed?
Maternal genes
135
If only the paternal genes of chromosome 15 are expressed, what syndrome is present?
Angelman
136
In Angelman syndrome, which genes are mutant?
Maternal chromosome 15
137
If only the maternal genes of chromosome 15 are expressed, what syndrome is present?
Prader-Willi
138
Which genes are mutant in Prader-Willi syndrome?
Paternal chromosome 15
139
What is the TATA box?
Core promoter found at -25-30
140
What are some eukaryotic enhancer elements?
CCAAT box
GC box
Hormone response elements (HREs)
141
What binds the CCAAT box?
CTF1, C/EBP
142
What binds the GC box?
Sp1
143
Specific DNA sequences are recognized by specific ___ ___.
Transcription factors
144
What DNA binding protein recognizes the sequence GGGCGG?
Sp1
145
What DNA sequence is recognized by Oct-1?
ATGCAAAT
146
What DNA sequence is recognized by NFkB?
GGGGACTTTCCC
147
What are the 4 transcription factor structural domains?
Helix-Turn-Helix
Zinc Finger
Leucine zipper
Activation domains
148
Where are helix-turn-helix structural domains found?
Homeodomain proteins
-Hox
-Msx-1
Barx-1
149
Where are zinc finger structural domains found?
Steroid hormone receptors
- ER
- PR
- AR
- GCR
150
Where are leucine zipper structural domains found?
Protein/proteins interactions
| -AP-1
151
Where are activation structural domains found?
Interacting with RNA polymerase
| Highly acidic or glutamine-rich regions
152
In electrophoretic mobility shift assays (EMSAs), what is being separated?
Unbound DNA
| DNA+Protein
153
What are the 8 steps for binding of activation complexes and RNA polymerase (Pol II)?
1. Activators
2. Chromatin modifiers
3. Coactivator complex (mediator)
4. TATA binding protein (TBP) and TFIIB
5. Individual basal transcription factors
6. Pol II binds to Inr region
7. Carboxyl terminus of Pol II complexes with mediator
8. TFIIH phosphorylates Pol II and initiates transcription
154
How was Dolly cloned?
Nucleus from mammary cell injected into unfertilized oocyte
| Zygote transferred to pseudopregnant female
155
What is the basis for most recombinant DNA techniques?
Complementary base pairing
156
What plasmid is usually used in recombinant DNA?
pBR322
157
What are the important sites on plasmid pBR322?
Pstl
BamHI
SalI
158
What do type II restriction endonucleases recognize?
4-8 base pair palindromes
159
Type II restriction endonucleases may produce what?
Sticky ends
160
What is used to fragment plasmid and human DNA so that they have sticky ends?
Restriction enzymes
161
What is the Sanger dideoxy method (ddNTP) of DNA sequencing
ddNTP analogs inhibit DNA polymerase as it synthesizes the complementary strand
162
What is used to separate the mixtures in ddNTP?
PAGE
163
What is used to determine how different bodies metabolize drugs?
Amplichip CYP450
164
What is ARMS PCR/Allele-specific PCR used for?
To detect point mutations using allele-specific primers
165
How are PCR products fractionated in ARMS PCR?
By gel electrophoresis
166
What is ex vivo gene therapy?
Performed on cells that are removed from the patient, manipulated and returned
167
Ashanti de Silva has SCID and is able to function because of what?
Ex vivo gene therapy
168
What are two important current targets for gene therapy?
Cancer
| Blindness
169
Gene therapy has been used to treat what ocular condition?
Choroidemia - to prevent blindness
170
What does a pluripotent cell come from?
Blastocyte
171
What can a pluripotent cell form?
Cell types from all 3 germ layers
172
Where does a multipotent cell come from?
An adult
173
What can a multipotent cell form?
Closely related cell types
174
Why isn't iPS successful yet?
It is tumorigenic
175
What happens to cells when there is no external signal to stimulate them?
They go into a prolonged G1 due to a lack of G1 cyclins, which are destroyed during mitosis
176
What is required to push the cell cycle from G0 to G1?
Cyclin D
177
Where is cyclin E found?
G1S checkpoint
178
What is cyclin A used for?
DNA synthesis
179
What is cyclin B for?
Entry into mitosis
180
What must occur to cyclin B during metaphase and anaphase?
Be degraded via ubiquitin pathway
181
How are Cdks/Cyclins regulated?
```
Via:
Cyclis proteolysis
Transcriptional regulation
Inhibitor proteins
Covalent modification (phosphorylation/dephosphorylation)
```
182
When is cyclin B-CDK1 active?
Mitosis
183
When is cyclin A-CDK2 active?
S-G2
184
What proteins are required for inhibitor proteins?
p21
185
What are the 2 important tumor suppressor proteins?
p53, pRb
186
What are the 4 important transcription factor proteins?
Jun
Fos
Myc
E2F
187
What are the important proteins in cyclic proteolysis via ubiquitin ligases?
SCF
| APC
188
Which cyclin is an important checkpoint in cancer development?
Cyclin E
189
What 2 things can activate cyclin D?
Growth factors
| Inflammation
190
What is the most important checkpoint of the cell cycle?
G1-S
191
What proteins are at work in the G1-S checkpoint?
p53
| Retinablastoma protein
192
What are the 5 critical DNA damage checkpoints?
```
G1/S
Mid S
G2/M
M
Post-M
```
193
What occurs at the G1/S checkpoint?
DNA damage assessment
194
What occurs at the Mid S checkpoint?
DNA replication checkpoint I
195
What occurs at the G2/M checkpoint?
DNA replication checkpoint II
196
What may occur if the Mid S checkpoint doesn't function?
Telomere dysfunction
Rearrangements
Amplifications
197
What occurs at the M checkpoint?
Spindle assembly checkpoint
198
What occurs at the Post-M checkpoint?
Polyploidy checkpoint
199
What does APC target?
Cyclin B, securin and separase
200
What does APC allow to occur?
The onset of anaphase
201
What does the flipping of phosphotydil serine in the cell do?
Targets things for phagocytosis
202
HPV is highly associated with which type of cancer?
Cervical cancer
203
What do HPV proteins E6 & E7 do?
Bind and inactivate p53 and pRb tumor suppressor proteins
204
BCl2 is targeted by HPV E6 which causes what?
Maintains cells that should undergo apoptosis
205
What a euploidy?
Having a extra full set of chromosomes, usually due to polyspermy
206
What is aneuploidy?
Having an extra single chromosome due to a nondisjunction
207
What are bivalents or tetrads?
Pair of duplicated homologues - 2 pairs of sister chromatids
208
What is the region where bivalents crossover?
Chiasmata
209
What is a dictyotene?
prolonged prophase 1 of oocyte
210
The primary oocyte sits in what zone?
Zona pellucida
211
What does LH trigger?
Proteolytic breakdown of ovarian wall by granulosa-secreted enzymes
212
What cells produce inhibitors to limit enzymatic breakdown (ovulation)?
Theca cells
213
What does an LH surge cause?
PKC activation and an increase in calcium
214
PKC targets c-mos, which increases MPF activity. What does MPF do?
Targets nuclear lamins and triggers germinal vesicle breakdown and the completion of meiosis I
215
In fertilization, what does sperm bind to?
ZP3
216
After sperm binds to ZP3, Ca increases causing what?
The release of acrosomal enzymes which allow penetration through ZP
