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MCBM Exam 5 > Review > Flashcards

Review Flashcards

1
Q

What did Meselson & Stahl discover?

A

That DNA replication is semi-conservative using E. coli and “heavy” nitrogen

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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)

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3
Q

What is the composition of a 3rd generation N labeled DNA?

A

25% mid density

75% light density

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4
Q

What are the two requirements for DNA replication by DNA polymerases?

A

Template

Primer

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5
Q

What is a template?

A

A DNA strand used to specify the nucleotides to be incorporated into the daughter strand

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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

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7
Q

What provides the chemical basis for semi-conservative replication?

A

The need for a template for DNA synthesis

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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

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9
Q

What is the accuracy of replication due to DNA polymerase I?

A

10^6 - 10^8

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10
Q

Where does DNA replication begin?

A

At the ORI

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11
Q

How many proteins are involved in the replication of E. coli DNA?

A

Over 20; plus DNA polymerase III and control proteins

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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

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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

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14
Q

What are SSBs/what do they do?

A

Single stranded binding proteins

Stabilize single stranded DNA

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15
Q

Which steps in the initiation of replication need energy?

A

All of them

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16
Q

What repairs the Okazaki fragments?

A

DNA polymerase

DNA ligase

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17
Q

What is the 3d enzyme activity of DNA polymerase I?

A

5’ to 3’ exonuclease

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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

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19
Q

What are telomeres?

A

Buffer zones on the end of eukaryotic chromosomes

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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

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21
Q

What structures to telomeres form?

A

Unusual, non-helical structures

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22
Q

Do telomeres need a template to be synthesized?

A

No

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23
Q

How are telomeres synthesized?

A

By telomerase

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24
Q

What is telomerase?

A

A specialized reverse transcriptase

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25
Q

What component is found in telomerase that serves as a template for synthesis?

A

An RNA component

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26
Q

What do telomeres prevent?

A

Undesirable fusion of chromosomes

Aberrant recombination

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27
Q

What attaches chromosomes to the nuclear envelope?

A

Telomeres

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28
Q

What serves as a “mitotic clock”?

A

Telomeres - Short telomeres induces senescence and/or apoptosis

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29
Q

Where is there no telomerase activity found?

A

In differentiated cells

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30
Q

Where is telomerase active?

A

In:
Germ cells
Stem cells
Tumors

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31
Q

How many RNA polymerases does E. coli use for all transcript synthesis?

A

1

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32
Q

How many subunits is found in the core enzyme of E. coli?

A

5

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33
Q

What are the 5 subunits of E. coli core enzymes?

A

2 alpha
1 beta
1 beta’
1 omega

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34
Q

E. coli core enzymes are very inefficient at what?

A

Initiating RNA synthesis

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35
Q

What is the additional subunit needed to add to the E. coli core enzyme for efficient initiation?

A

Sigma factor - its addition completes the RNA polymerase holoenzyme

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36
Q

What are the three stages of RNA synthesis?

A

Initiation
Elongation
Termination

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37
Q

In which direction does the DNA coding (sense) strand run?

A

5’ to 3’

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38
Q

In which direction does the DNA template (anti-sense) strand run?

A

3’ to 5’

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39
Q

In which direction does the RNA transcript run?

A

5’ to 3’

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40
Q

What are the steps of elongation?

A

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

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41
Q

How fast does RNA polymerase move along the template?

A

About 50 bases per second

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42
Q

What facilitates unwinding during elongation?

A

-ve supercoiling

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43
Q

At any one point, about how many base pairs are unwound?

A

17

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44
Q

How many nuclear RNA polymerases do eukaryotes have?

A

3

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45
Q

Where is RNA polymerase I found?

A

Nucleolus

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46
Q

Where are DNA polymerases II and III found?

A

Nucleus

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47
Q

What does RNA polymerase II core enzyme protein require for efficient initiation?

A

Numerous additional proteins (it is a multisubunit protein)

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48
Q

What genes are transcribed by RNA polymerase I?

A

rRNA (18S, 28S, 5.8S)

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49
Q

What genes are transcribed by RNA polymerase II?

A

mRNA

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50
Q

What genes are transcribed by RNA polymerase III?

A

tRNA

5S rRNA

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51
Q

What is a primary transcript?

A

A newly synthesized RNA molecule

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52
Q

What has to be done to prokaryote mRNAs before they are ready to be used in translation?

A

Nothing

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53
Q

What modification does RNA undergo to become tRNA?

A

Base modifications

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54
Q

What must occur to RNA to become rRNA?

A

Cleavage of large primary transcript into functional fragments

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55
Q

What is found on the 3’ end of mature tRNA?

A

CCA

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56
Q

What two enzymes are at work to modify a primary transcript into mature tRNA?

A

RNase P

RNase D

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57
Q

What things are done in mRNA processing?

A

Addition of 5’ cap
Addition of 3’ polyA tail
Splicing of introns

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58
Q

What is function of the 3’ polyA tail on mRNA?

A

Enhance mRNA stability

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59
Q

What is the 5’ cap?

A

7-methylguanosine, added via 5’,5’-triphosphate linkage

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60
Q

What is it thought that the 5’ cap does for mRNA?

A

Enhance translation

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61
Q

How many codons are there?

A

64

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62
Q

How many stop codons?

A

3

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63
Q

What are the stop codons?

A

UAA
UGA
UAG

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64
Q

What is the initiation codon?

A

AUG

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65
Q

What is UUU?

A

Phe

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66
Q

What is CCC?

A

Pro

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67
Q

What is GGG?

A

Gly

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68
Q

What is AAA

A

Lys

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69
Q

What is stage 1 of protein synthesis?

A

“Charging” tRNA with proper amino acid

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70
Q

How are amino acids attached to tRNA during charging?

A

Via ester bonds (carboxylic acid to sugar OH) to the 3’ OH

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71
Q

What catalyzed the aminoacylation of the 3’ OH (charging) of the tRNA?

A

Amino acyl-tRNA synthetases

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72
Q

What is stage 2 of protein synthesis?

A

Initiation

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73
Q

How is initiation started?

A

By recognition of the initiation codon

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74
Q

What is the “regular” tRNA in bacteria?

A

tRNA^Met

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75
Q

What is the initiation tRNA in bacteria?

A

tRNA^fMet

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76
Q

What is the starting amino acid in bacteria?

A

N-formylmethionine

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77
Q

What donates the formyl group to tRNA^fMet?

A

N610-formyltetrahydrofolate

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78
Q

What are the 7 components required to initiate protein synthesis?

A
  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+
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79
Q

How many steps does it take the 7 initiation components of bacteria to assemble into the initiation complex?

A

3 steps

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80
Q

What is step 1 of assembling the initiation complex (bacteria)?

A

Binding positions the initiation codon on the 30S subunit

-Codon bound to P site of 30S

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81
Q

How is the initiation codon distinguished in step 1 of initiation complex assembly (bacteria)?

A

By proximity to the Shine-Dalgarno sequence

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82
Q

What blocks the A site in the assembly of the initiation complex (bacteria)?

A

IF-1

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83
Q

What is step 2 of assembling the initiation complex (bacteria)?

A

Initiator tRNA binds to P site

-30S subunit, mRNA, IF-1, IF-3 complex is joined by IF-2 and fMet-tRNA^fMet

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84
Q

What is step 3 of assembling the initiation complex (bacteria)?

A

Large 50S subunit binds to complex

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85
Q

What is the 1st step of elongation?

A

Bind the next aminoacyl-tRNA (to the A site)

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86
Q

What is the 2nd step of elongation?

A

Peptide bond formation

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87
Q

What are the 5 stages of protein synthesis?

A
  1. Activation of Amino Acids
  2. Initiation
  3. Elongation
  4. Termination and ribosome recycling
  5. Folding
88
Q

During elongation, where does the peptide bond form?

A

Between the 2 amino acids

89
Q

What enzyme catalyzes the peptide bond formation during elongation?

A

Peptidyl transferase

90
Q

What type of enzyme is peptidyl transferase?

A

Ribozyme - it is the large subunit 23S rRNA

91
Q

What is stage 4 of protein synthesis?

A

Termination

92
Q

What begins termination?

A

Ribosome encounters an in-frame stop codon in the A site

93
Q

What are the 3 termination factors that may bind to the termination codon?

A

RF1, RF2, or RF3

94
Q

RF1 binds to which termination codons?

95
Q

RF2 binds to which termination codons?

96
Q

What effect do the termination factors have on protein synthesis?

A

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
Q

How many termination factors are found in eukaryotes?

98
Q

What determines whether ribosomes are free or membrane-bound?

A

The protein being synthesized

99
Q

What proteins are made by free ribosomes?

A

Nuclear proteins
Peroxisomal proteins
Mitochondrial proteins

100
Q

Where are nuclear, peroxisomal and mitochondrial proteins made?

A

In the cytoplasm

101
Q

What signal sequence brings proteins into the nucleus?

A

Internal nuclear localization sequences

102
Q

What signal sequence brings proteins into peroxisomes?

A

C-terminal sequence SKF

103
Q

What signal sequence brings proteins into the mitochondrial matrix?

A

N-termina sequence, rich in positively charged amino acids and ser & thr

104
Q

What proteins are made in the RER?

A

Secretory proteins
Lysosomal proteins
Integral membrane proteins

105
Q

Ribosomes bound to the endoplasmic reticulum synthesize what 3 classes of protein?

A

Secretory proteins
Lysosomal proteins
Integral membrane proteins

106
Q

How is the signal sequence removed from secreted proteins?

A

By cleavage via signal peptidase

107
Q

What occurs on the cytosol side of the ER?

A

Nucleotide activated sugar precursors are added stepwise to the phosphate group forming a dolichol pyrophosphate intermediate

108
Q

What happens to incomplete core-dolichol pyrophosphate?

A

It is translocated across ER membrane into lumen

109
Q

What are the 1st 7 sugars in the synthesis of core oligosaccharides?

A

2 GlcNAc

5 mannose

110
Q

Where are nucleotide activated precursors found (oligosaccharides)?

A

Cytosolic side

111
Q

Where are dolichol-phosphate activated precursors found (oligosaccharides)?

A

Lumen side

112
Q

What sugars are added in the ER lumen (oligosaccharides)?

A

4 mannose

3 glucose

113
Q

When the oligosaccharide is transferred to a specific Asn residue in protein, what is released?

A

Dolichol pyrophophate

114
Q

How is dolichol phosphate regenerated?

A

By action of a phosphatase

115
Q

How does tunicamycin affect the addition of sugars?

A

Blocks the 1st step in oligosaccharide synthesis

116
Q

How does Bacitracin affect addition of sugars?

A

Blocks phosphatase from recycling dolichol phosphate

117
Q

Why is Bacitracin a useful antibiotic?

A

The bacterial enzyme is very sensitive to it

118
Q

What is an important signaler for lysosome targeting?

A

Mannose-6-phosphate

119
Q

What signals for protein to return to the ER?

120
Q

What does KDEL stand for?

A

Lys-Asp-Glu-Leu

121
Q

Where is KDEL found?

A

Carboxy-terminal on ER proteins

122
Q

Most cell surface receptors are what?

A

Transmembrane glycoproteins

123
Q

Where are many receptors located?

A

In specialized regions of the plasma membrane - coated pits

124
Q

What does the cytosolic side of the “pits” have?

A

Thick coat of clathrin

125
Q

With what does clathrin help?

A

Endocytosis

126
Q

What does clathrin do in endocytosis?

A

Forms a lattice around the coated pit, excising it from the membrane, and forming a coated vesicle

127
Q

Eukaryotic RNA polymerases have very low affinity for ___ without help from DNA binding proteins.

128
Q

Which type of chromatin is “turned off”?

A

Heterochromatin

129
Q

DNA is ___ in heterochromatin.

A

Hypermethylated (at CpG dinucleotides)

130
Q

Histones are ___ in heterochomatin.

A

Deacetylated

131
Q

In euchromatin, DNA is ___.

A

Hypomethylated

132
Q

In euchromatin, histones are ___.

A

Acetylated

133
Q

What is the difference between Prader-WIlli and Angelman?

A

The 15q11-13 region contains genes that are differentially silenced in male vs female gametes

134
Q

If mutant chromosome 15 is inherited from the father, what genes are expressed?

A

Maternal genes

135
Q

If only the paternal genes of chromosome 15 are expressed, what syndrome is present?

136
Q

In Angelman syndrome, which genes are mutant?

A

Maternal chromosome 15

137
Q

If only the maternal genes of chromosome 15 are expressed, what syndrome is present?

A

Prader-Willi

138
Q

Which genes are mutant in Prader-Willi syndrome?

A

Paternal chromosome 15

139
Q

What is the TATA box?

A

Core promoter found at -25-30

140
Q

What are some eukaryotic enhancer elements?

A

CCAAT box
GC box
Hormone response elements (HREs)

141
Q

What binds the CCAAT box?

A

CTF1, C/EBP

142
Q

What binds the GC box?

143
Q

Specific DNA sequences are recognized by specific ___ ___.

A

Transcription factors

144
Q

What DNA binding protein recognizes the sequence GGGCGG?

145
Q

What DNA sequence is recognized by Oct-1?

146
Q

What DNA sequence is recognized by NFkB?

A

GGGGACTTTCCC

147
Q

What are the 4 transcription factor structural domains?

A

Helix-Turn-Helix
Zinc Finger
Leucine zipper
Activation domains

148
Q

Where are helix-turn-helix structural domains found?

A

Homeodomain proteins
-Hox
-Msx-1
Barx-1

149
Q

Where are zinc finger structural domains found?

A

Steroid hormone receptors

  • ER
  • PR
  • AR
  • GCR
150
Q

Where are leucine zipper structural domains found?

A

Protein/proteins interactions

-AP-1

151
Q

Where are activation structural domains found?

A

Interacting with RNA polymerase

Highly acidic or glutamine-rich regions

152
Q

In electrophoretic mobility shift assays (EMSAs), what is being separated?

A

Unbound DNA

DNA+Protein

153
Q

What are the 8 steps for binding of activation complexes and RNA polymerase (Pol II)?

A
  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
Q

How was Dolly cloned?

A

Nucleus from mammary cell injected into unfertilized oocyte

Zygote transferred to pseudopregnant female

155
Q

What is the basis for most recombinant DNA techniques?

A

Complementary base pairing

156
Q

What plasmid is usually used in recombinant DNA?

157
Q

What are the important sites on plasmid pBR322?

A

Pstl
BamHI
SalI

158
Q

What do type II restriction endonucleases recognize?

A

4-8 base pair palindromes

159
Q

Type II restriction endonucleases may produce what?

A

Sticky ends

160
Q

What is used to fragment plasmid and human DNA so that they have sticky ends?

A

Restriction enzymes

161
Q

What is the Sanger dideoxy method (ddNTP) of DNA sequencing

A

ddNTP analogs inhibit DNA polymerase as it synthesizes the complementary strand

162
Q

What is used to separate the mixtures in ddNTP?

163
Q

What is used to determine how different bodies metabolize drugs?

A

Amplichip CYP450

164
Q

What is ARMS PCR/Allele-specific PCR used for?

A

To detect point mutations using allele-specific primers

165
Q

How are PCR products fractionated in ARMS PCR?

A

By gel electrophoresis

166
Q

What is ex vivo gene therapy?

A

Performed on cells that are removed from the patient, manipulated and returned

167
Q

Ashanti de Silva has SCID and is able to function because of what?

A

Ex vivo gene therapy

168
Q

What are two important current targets for gene therapy?

A

Cancer

Blindness

169
Q

Gene therapy has been used to treat what ocular condition?

A

Choroidemia - to prevent blindness

170
Q

What does a pluripotent cell come from?

A

Blastocyte

171
Q

What can a pluripotent cell form?

A

Cell types from all 3 germ layers

172
Q

Where does a multipotent cell come from?

173
Q

What can a multipotent cell form?

A

Closely related cell types

174
Q

Why isn’t iPS successful yet?

A

It is tumorigenic

175
Q

What happens to cells when there is no external signal to stimulate them?

A

They go into a prolonged G1 due to a lack of G1 cyclins, which are destroyed during mitosis

176
Q

What is required to push the cell cycle from G0 to G1?

177
Q

Where is cyclin E found?

A

G1S checkpoint

178
Q

What is cyclin A used for?

A

DNA synthesis

179
Q

What is cyclin B for?

A

Entry into mitosis

180
Q

What must occur to cyclin B during metaphase and anaphase?

A

Be degraded via ubiquitin pathway

181
Q

How are Cdks/Cyclins regulated?

A 
Via:
Cyclis proteolysis
Transcriptional regulation
Inhibitor proteins
Covalent modification (phosphorylation/dephosphorylation)
182
Q

When is cyclin B-CDK1 active?

183
Q

When is cyclin A-CDK2 active?

184
Q

What proteins are required for inhibitor proteins?

185
Q

What are the 2 important tumor suppressor proteins?

186
Q

What are the 4 important transcription factor proteins?

A

Jun
Fos
Myc
E2F

187
Q

What are the important proteins in cyclic proteolysis via ubiquitin ligases?

188
Q

Which cyclin is an important checkpoint in cancer development?

189
Q

What 2 things can activate cyclin D?

A

Growth factors

Inflammation

190
Q

What is the most important checkpoint of the cell cycle?

191
Q

What proteins are at work in the G1-S checkpoint?

A

p53

Retinablastoma protein

192
Q

What are the 5 critical DNA damage checkpoints?

A 
G1/S
Mid S
G2/M
M
Post-M
193
Q

What occurs at the G1/S checkpoint?

A

DNA damage assessment

194
Q

What occurs at the Mid S checkpoint?

A

DNA replication checkpoint I

195
Q

What occurs at the G2/M checkpoint?

A

DNA replication checkpoint II

196
Q

What may occur if the Mid S checkpoint doesn’t function?

A

Telomere dysfunction
Rearrangements
Amplifications

197
Q

What occurs at the M checkpoint?

A

Spindle assembly checkpoint

198
Q

What occurs at the Post-M checkpoint?

A

Polyploidy checkpoint

199
Q

What does APC target?

A

Cyclin B, securin and separase

200
Q

What does APC allow to occur?

A

The onset of anaphase

201
Q

What does the flipping of phosphotydil serine in the cell do?

A

Targets things for phagocytosis

202
Q

HPV is highly associated with which type of cancer?

A

Cervical cancer

203
Q

What do HPV proteins E6 & E7 do?

A

Bind and inactivate p53 and pRb tumor suppressor proteins

204
Q

BCl2 is targeted by HPV E6 which causes what?

A

Maintains cells that should undergo apoptosis

205
Q

What a euploidy?

A

Having a extra full set of chromosomes, usually due to polyspermy

206
Q

What is aneuploidy?

A

Having an extra single chromosome due to a nondisjunction

207
Q

What are bivalents or tetrads?

A

Pair of duplicated homologues - 2 pairs of sister chromatids

208
Q

What is the region where bivalents crossover?

209
Q

What is a dictyotene?

A

prolonged prophase 1 of oocyte

210
Q

The primary oocyte sits in what zone?

A

Zona pellucida

211
Q

What does LH trigger?

A

Proteolytic breakdown of ovarian wall by granulosa-secreted enzymes

212
Q

What cells produce inhibitors to limit enzymatic breakdown (ovulation)?

A

Theca cells

213
Q

What does an LH surge cause?

A

PKC activation and an increase in calcium

214
Q

PKC targets c-mos, which increases MPF activity. What does MPF do?

A

Targets nuclear lamins and triggers germinal vesicle breakdown and the completion of meiosis I

215
Q

In fertilization, what does sperm bind to?

216
Q

After sperm binds to ZP3, Ca increases causing what?

A

The release of acrosomal enzymes which allow penetration through ZP

MCBM Exam 5 (11 decks)

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