Molecular + cellular Flashcards
(428 cards)
0
Q
Checkpoints control transition between phase of cell cycle. Is regulated by:
A
Cyclins
Cycle - dependent kinases (CDK)
Tumor suppressor
1
Q
- Which is the shortest phase of cell cycle?.
2. which phases are variable?
A
- Mitosis
2. G1 and G0
2
Q
What is G and what is S in cll cycle
A
Gap
Synthesis
3
Q
M phase includes
A
- mitosis (Prophase, Metaphase, Anaphase, Telophase)
2. cytokinesis (cytoplasm splits in 2)
4
Q
Which cell cycle regulator is CONSTITUTIVE AND INACTIVE
A
Cyclin dependent kinases (cdk)
5
Q
Which cell cycle regulators are phase specific?
Role?
A
Cyclins….activate cyclin dependent kinases (CDKs)
6
Q
Phases of cell cycle:
What is interphase?
A
- G1 (and G0)
- S phase
- G2
- M phases
Interphase: G1-S-G2
7
Q
- Tumor suppressors in cell cycle regulation (mechanism)
2. If mutated:
A
- P53 induce p21 which HYPOphosphorylates Rb –> binds to and inactivate E2F –> Inhibit G1 to S progression
- Unrestrained cell cycle division (eg Li-fraumeni)
8
Q
Which cell type is affected by chemotherapy
A
Labile
9
Q
Cell types according their proliferative ability: (and definition)
A
- Permanent - remain in G0, regenerate from stem cells
- Stable (quiescent) - enter G1 from G0 when stimulated
- Labile: never go to G0, divide rapidly with shirt G1. Most affected by chemotherapy
10
Q
Cell type that is also called QUIESCENT
A
Stable
11
Q
Permanent cell examples
A
Neurons, skeletal and cardiac muscle, red blood cells
12
Q
Stable (quiescent) cells examples
A
Hepatocytes, lymphocytes
13
Q
Labile cells examples
A
Bone marrow, gut epithelium, skin, hair follicles, germ cells
14
Q
Smooth vs rough
Endoplasmic reticulum according their structure:
A
Smooth endoplasmic reticulum LACKS SURFACE RIBOSOMES
15
Q
Cell type with short G1
A
Labile
16
Q
Smooth endoplasmic reticulum role:
A
- Steroids synthesis
2. Detoxification of drugs and poisons
17
Q
Cells rich in smooth endoplasmic reticulum:
A
- Liver hepatocytes
- Steroid hormones-producing cells of the adrenal cortex
- Steroid hormones-producing cells of the gonads
18
Q
Cells rich in rough endoplasmic reticulum
A
- Mucus-secreting goblet cells of the small intestine
2. Antibody-secreting plasma cells
19
Q
Rough endoplasmic reticulum role:
A
- Synthesis of secretory (exporter) proteins
2. N-linked oligosaccharides addition to many proteins
20
Q
What are Nissl bodies and what is their function
A
Nissl bodies are rough endoplasmic reticulum in neurons
Synthesize peptide neurotransmitters for secretion
21
Q
Free ribosomes (structure and function):
A
Unattached to any membrane
Site of synthesis of cytosolic and organelle proteins
22
Q
Proteasome stracture and function
A
Barrel-shaped protein complex that degrades damaged or UBIQUITIN-tagged proteins
23
Q
Ubiquitin - Proteasome system defects:
A
It have been implicated in some cases of PARKINSON disease + Alzheimer
Genes (parkinin, PINK1, DJ-2)
24
Peroxisome structure
Membrane enclosed organelle
25
What is Golgi apparatus?
Is the distribution center for proteins and lipids from the endoplasmic reticulum to the vesicles and plasma membrane
26
Peroxisome function:
Catolism of:
1. Very-long-chain fatty acids
2. Branched chain fatty acids
3. Amino acids
4. ethanol
27
Golgi apparatus function:
1. Modifies N-oligosaccharides on ASPARAGINE
2. Adds O-oligosaccharides on SERINE and THREONINE
3. Adds MANNOSE-6-PHOSPHATE to proteins from trafficking to lysosomes
28
Endosomes?
Sorting centers for materials from OUTSIDE the cell or from the GOLGI sending it to lysosomes for destruction or back to membrane/Golgi for further use
29
I-cell disease (inclusion cell disease also referred to as:
Mucolipidosis II
30
which enzyme is defective in inclusion cell disease and and what is the problem? What is low?
Phosphotransferase
Failure of Golgi to phosphorylate mannose residues
LOW LEVELS OF MANNOSE RESIDUES
31
I cell disease (inclusion cell disease) pathophysiology:
Inherited lysosomal storage disorder- defect in N-acetylglucosamil-1-phosphotranferase - failure of the Golgi to phosphorylate mannose residues (LOW MANNOSE-6-PHOSPHATE) on glycoproteins - proteins are secreted extracellularly rather than delivered to lysosomes
32
I cell disease (inclusion disease) results
1. Coarse facial features
2. Clouded corneas
3. Restricted joint movements
4. High levels of lysosomal enzymes
33
Is i-cell disease - course
I - cell disease is OFTE FATAL IN CHILDHOOD
34
Signal recognition particles (SRP):
SRP are abundant, cytosolic ribonucleoproteins that traffic proteins FROM THE RIBOSOME TO THE ROUGH ENDOPLASMIC RETICULUM
35
Absent or dysfunctional Signal recognition particles (SRPs)
Proteins accumulate in the cytosol
36
Golgi phases:
1. Endoplasmic reticulum phase - cis phase
| 2. Plasma membrane phase - trans phase
37
Vesicular trafficking proteins:
1. COPI
2. COPII
3. Clathrin
39
COPI function:
Vasicular trafficking protein:
1. Golgi to cis Golgi (retrograde)
2. cis Golgi to Endoplasmic reticulum
40
COPII function:
Vasicular trafficking protein:
| Endoplasmic reticulum to cis Golgi (anterograde)
40
Clathrin associated plasma membrane to endosomes:
| Example:
Receptor mediated endocytosis
| Example: LDL receptor activity
41
Clathrin function:
Vasicular trafficking protein:
1. Trans-Golgi to lysosomes
2. Plasma membrane to endosomes (receptor mediated endocytosis)
42
what is the proportion for each ATP molecule:
For each ATP, 3 Na go out and 2 K come in
43
Where is sodium potasium pump located and where is its ATP site?
Sodium - Potasium pump is located in the plasma membrane with ATP site on cytosolic side
44
Sodium potassium pump function:
1. 3 Na bind on the cytosolic side
2. 3 Na are released on the extracellular side and the cytosolic site hydrolyzes ATP to ADP ( Pi is linked to the pump)
3. 2 K bind on the extracellular site of the pump and Pi is released from the pump
4. 2 K are released in the cell
45
3 Drugs that inhibit sodium-potassium pump:
1. Quabain
2. Digoxin
3. Digitoxin
46
Quabain mechanism
Quabain inhibits sodium potasium pump by binding to K site
47
Digoxin and Digitoxin mechanism of action:
| Which is their indirect effect?
Digoxin and digitoxin directly inhibit sodium-potassium pump. That leads to indirect inhibition of the sodium calcium exchanger- high intracellular calcium concentration- cardiac contractility
48
Most abundant protein in human body and its general function
Collagen - organizes and strengthens extracellular matrix
49
Which protein is responsible to organizes and strengthens extracellular matrix?
Collagen
50
How does collagen take its final conformation?
Extensively modification by post-translational modification
51
Most common type of collagen
Type 1. (90%)
52
Which cells product collagepn type 1 in bones?
Osteoblasts
53
Collagen type 1 is founded to:
1. Bone
2. Skin
3. Tendon
4. Dentin
5. Fascia
6. Cornea
7. Late wound repair
54
Disease of low production of collagen type 1
Ostogenesis imperfecta type 1
55
What is reticulin
Is a type of fiber in connective tissue composed of type III collagen secreted by reticular cells. Reticular fibers crosslink to form a fine meshwork (reticulin). This network acts as a supporting mesh in soft tissues such as liver, bone marrow, and the tissues and organs of the lymphatic system
56
Collagen type 2 is founded to:
1. Cartilage (including hyaline)
2. Vitreous body
3. Nucleus pulposus of intevertebrate discs
57
Where is type 3 collagen founded?
1. Skin
2. Blood vessels
3. Uterus
4. Fetal tissue
5. Granulation tissue
58
Type 3 collagen deficiency:
Vascular type of Ehlers - Danlos syndrome (uncommon)
59
2 disease associated with collagen type 4
1. Alport syndrome
| 2. Goodpasture syndrome
60
Defective of collagen type 4
| Clinical symptoms
Alport syndrome - isolated hematuria (glomerulonephritis), sensory hearing loss, ocular disturbances
61
Autoantibodies against type 4 collagen (basal membrane)
| Clinical symptoms
Goopasture syndrome
| Hematurua (rapidly progressive glomerulonephritis) and hemoptysis, classically in young adult males
62
Where is collagen type 4 founded
1. Basement membrane
2. Basal lamina
3. Lens
63
Plasma membrane structure
Assymetric lipid bilayer
64
Plasma membrane composition
Cholesterol, phospholipids, sphingolipids, glycolipids, proteins, ergosterol (fungal membrane)
65
Animal vs fungal plasma membrane
Fungal plasma membrane contains ergosterol
66
Bacterial vs eukaryotic plasma membranes
Bacterial plasma membrane lacks sterols (with some exceptions)
68
Immunohistochemical stains for intermediate filaments
1. Vimentin
2. Desmin
3. Cytokeratin
4. GFAP
5. Neurofilaments
69
Vimentin stain is specific for: (cell type and identifies)
cell type: mesenchymal tissue (fibroblast, endothelial cells, macrophages
identifies: mesenchymal tumors (sarcomas) but also many other (enometrial ca, renal cell ca, meningiomas)
70
Desmin stain is specific for: (cell type and identifies)
Muscle cells
| muscle tumors
71
Cytokeratin stain is specific for: (cell type and identifies)
Epithelial cells
| eg. SCC
72
GFAP Stain is specific for: (cell type and identifies)
Neuroglia
| Astrocytoma, GBM
72
Osteogenesis imperfecta is a genetic bone disorder also called:
Brittle bone disease
73
Neurofilaments stain is specific for: (cell type and identifies)
```
Neurons
neuronal tumors (eg. neuroblastoma
```
75
Ostegenenis imperfecta is caused by a variety pf gene defects. Most common? genes?
Most common is autosomal dominant with low production of otherwise NORMAL COLLAGEN 1
genes: COL1A1 and COL1A2
75
Causes of blue sclerae in osteogenesis imperfecta
Due translucency of the connective tissue over the CHOROIDAL VEINS
76
Clinical manifestations of osteogenesis imperfecta
1. Multiple fractures with minimal trauma (may occur during birth)
2. Blue sclerae
3. Hearing loss (abnormal ossicles)
4. Dental imperfections - opalescent teeth that wear easily due to lack of dentin (dentinogenesis imperfecta)
77
Causes of hearing loss in osteogenesis imperfecta
Abnormal ossicles - easily fracture
78
Which disease can mimic child abuse
Osteogenesis imperfecta can mimic child abuse, but bruising is absent
79
Causes of dental imperfections in osteogenesis imperfecta
opalescent teeth that wear easily due to lack of dentin (dentinogenesis imperfecta)
80
Osteogenesis imperfecta in imaging
1. Severe skeletal deformity
| 2. Limb shortening due to multiple fractures in a child
81
Ehlers-Danlos syndrome etiology
Faulty collagen synthesis
82
Most common clinical manifestations of Ehlers-Danlos syndrome
1. Hyperextensible skin
2. Tendency to bleed (easy bruising)
3. Hypermobile joints
83
How many types of Ehlers-Danlos syndrome are exist?
6+ types
84
Is Ehlers-Danlos syndrome an inherited disease?
| Is it severe
Inheritance and severity VARY
85
Is Ehlers-Danlos syndrome autosomal dominant or recessive?
Ehlers-Danlos syndrome can b autosomal dominant or recessive
86
Except hyoeextensible skin, tendency to blled and hypermobile joints, what else clinical manifestations can be associated with Ehlers-Danlos syndrome?
1. Joint dislocation
2. Berry aneurism
3. Aortic aneurism
4. Organ rupture
87
The most common type of Ehlers-Danlos syndrome
Hypermobility type (joint instability)
88
Which is the classical type of Ehlers-Danlos syndrome? Which collagen type of collagen is affected?
Joint and skin symptoms
| MUTATION in collagen type 5
89
What is vascular type of Ehlers-Danlos syndrome?
| Which type of collagen is affected in vascular type of Ehlers-Danlos syndrome?
Vascular and organ rupture
| DEFICIENT type 3 collagen
90
3 types of Ehlers-Danlos syndrome and clinical manifestations
1. Hypermobility type ( joint instability) most common
2. Classical type (joint and skin symptoms) (5 collagen)
3. Vascular type (vascular and organ rupture) (3 collagen)
92
What type of tissue does Menkes disease affect / mode of inheritance
Conective tissue
92
copper is necessary cofactor of which enzyme
Lysyl oxidase
93
Menkes disease - mechanism
Impaired COPPER absorption and transport due to defective Menkes protein (ATP7A) --> low activity of LYSYL OXIDASE (copper is necessary cofactor)
94
Clinical manifestations of Menkes disease:
1. Brittle hair
2. Kinky hair
3. Growth retardation
4. Hypotonia
95
What is lysyl oxidase?
Lysyl oxidase is an extracellular copper enzyme that catalyzes formation of aldehydes from lysine residues in collagen and elastin precursors.
96
What is elastin?
Elastin is a protein in connective tissue that is elastic and allows many tissues in the body to resume their shape after stretching or contracting.
97
Location of elastin
1. Skin
2. Lungs
3. Elastic ligaments
4. Vocal cords
5. Ligamenta flava (connects vertebrae)
6. Large arteries
98
Elastin is rich with which aminoacids and in which forms
PROLINE and GLYCINE and LYSINE in nonhydroxylated forms
99
Elastin is broken down by
Elastase
100
Elastase in normally inhibited by:
a1-antitrypsin
101
Elastin cross-linking takes place
| What is the purpose of cross-linking
Extracellularly
| It gives elastin its elastic properties
102
Elastin structure
Tropoelastin with fibrillin scaffolding
103
What is fibrillin
Glycoprotein that forms a sheath around elastin
104
What is ligamenta flava
Ligament that connects vertebrae-relaxed and stretched conformation
105
Disease caused by a defect in fibrillin
MARFAN
106
Diseased that can be caused by a1-antitrypsin deficiency
Emphysema (from excess elastase activity)
107
Pathophysiology of emphesema
A1-antitrypsin deficiency, resulting in excess elasetase activity
108
Wrinkles of aging are due to
Low collagen and elastin production
109
Microtubule shape
Cylindrical
110
Microtubules are cylindrical structure composed of:
A helical array of polymerrized heterodimeres of α and β tubulin
111
Polymerized heterodimers of microtubules are composed by
1. α-tubulin
| 2. β-tubulin
112
Microtubules Heterodimers - GTP association
Each dimer has 2 GTP bound
113
Microtubule has 2 ends
1. Positive end
| 2. Negative end
114
Microtubules are incorporated into:
1. Flagella
2. Cilia
3. Mitotic spindle
115
Microtubule is a dynamic structure
Grows slowly
| Collapses quickly
116
What is protofilament in microtubule
| How many protfilaments in each microtubule
A vertical line of heterodimers
| 13
117
Which is the role of microtubules in transport in neurons
They are involved in slow axoplasmic transport transport in neurons
118
What is the role of the molecular motor proteins?
Molecular proteins TRANSPORT CELLULAR CARGO toward opposite ends of microtubules
119
2 molecular motor proteins
1. Dynein
| 2. Kinesin
120
Dynein direction
Retrograde to microtubule (+ end to - end)
121
Kinesin direction
Anterograde to microtubule (- end to + end)
122
Dynein vs kinesin
Dynein: + to -
Kinesin: - to +
123
Drugs that act on microtubules and their clinical uses
1. Mebendazole (anti-helminthic)
2. Griseofulvin (anti-fungal)
3. Colchicine (anti-gout)
4. Vincristine (anti-cancer)
5. Vinvlastine (anti-cancer)
6. Paclitaxel (anti-cancer)
124
Anti-fungal drug that acts on microtubules
Griseofulvin
126
Cilia structure
- 9+2 arrangement of microtubules
the base of a cilium below the cell membrane, called the basal body condists of 9 microtubules TRIPLETS with no central micotubules
126
axonemal dynein - ATPase role:
It links peripheral 9 doublets and causes bending of cilium by differential sliding of tubules
127
Molecular motor protein of cilia
Axonemal dynein - ATPase
128
Energy of dynein and kinesin
ATP
129
Disease associated with cilia
Kartagener syndrome
130
Kartagener syndrome is also called
Primary ciliary dyskinesia
131
Pathophysiology of kartegener syndrome (primary ciliary dyskinesia)
Immotile cilia due to a dynein arm defect
132
Example of situs inversus
Dextrocardia (on CXR)
133
Kartagener syndrome clinical manifestations
1. Male infertility
2. Female infertility
3. Risk for ectopic pregnancy
4. Bronchiectasia
5. Recurrent sinusitis
6. Situs inversus
135
Cause of infertility in Katagener syndrome
male: Immotile sperm
female: Dysfunctional fallopian tube cilia
136
Cytoskeletal elements?
a netwrokd of protein fibers within the cytoplasm that sapports cell structure, cell and organelle moementt, and cell division
137
cytoskeletal elements - types
1. microfilaments
2. intermediate filaments
3. microtubules
138
microfilaments - predominate function and examples
muscle contraction, cytokinesis
| ex: actin, microvilli
139
intermediate filaments - predominate function and examples
maintain cell structure
| ex: vimentin, desmin, cytokerain, lamins, Glial fibrillary acid proteins (GFAP), neurofillaments
139
Cells tha produce collagen
Fibroblasts
140
Microtubule - predominate function and examples
Movement, cell division
| ex. cilia, flagella, mitotic spindle, axonal trafficking, centrioles
141
Synthesis of collagen
| Where
Translation of collagen α chains (preprocollagen)
| Rough endoplasmic reticulum
142
phases of collagen production and the site of them
1. Synthesis (RER)
2. Hydroxylation (RER)
3. Glycosylation (RER)
4. Exocytosis (from fibroblasts)
5. Proteolytic processing ( outside fibroblasts)
6. Cross linking ( outside fibroblasts)
143
Preprocollagen sequence
Usually Gly-X-Y (X and Y are proline or lysine)
144
Collagen is 1/3...
Glycine
145
......... content best reflects collagen synthesis?
Glycine
146
Hydroxylation as a part of collagen production
| Where
Hydroxylation of specific proline and lysine residues
| Rough endoplasmic reticulum
147
It is necessary for collagen hydroxylation:
Vitamine C
149
Vitamine C deficiency in collagen synthesis
Inhibits hydroxylation of collagen (Scurvy)
149
Procollagen?
Triple helix of 3 collagen α chains bind by hydrogen and disulfide bonds
150
Glycosylation as as a part of collagen production
| Where
Glycosylation of pro-α-chain hydroxylysine residues and formation of procollagen via hydrogen and disulfide bonds (triple helix of 3 collagen a chain
RER
151
Procollagen bonds
Hydrogen and disulfide bonds
152
Problems forming triple helix (procollagen)
Osteogenesis imperfecta
153
Exocytosis as a part of collagen production
Exocytosis of procollagen into extracellular space
154
Proteolytic processing as a part of colllagen production
| Where
Cleavage of disulfide - rich terminal regions of procollagen, transforming it into insoluble tropocollagen
Outside fibroblasts
156
Cross linking as a part of collagen production
| Where
Covalent lysine - hydroxylysine ( cross linkage) by lysyl oxidase (copper) to make collagen fibrils. (Reinforcement of many staggerd tropocollagen molecules)
156
Collagen production pathway
Preprocollagen - procollagen - tropocollagen - collagen fibrils
157
Problem with cross linking of tropocollagen
1. Elhers-Danlos
| 2. Menkes disease
159
DNA charge
| Histone octamer charge
DNA --> Negative
| Histone octamer charge --> Positive
160
Chromatin structure
DNA loops twice around histone octamer to form nucleosome bead
161
Nucleosome bead
Negatively charged DNA around positively charged histone octamer
162
Amino acids of histones
reach lysin and arginine
163
Types of histones
H1 H2A H2B H3 H4
164
Nucleosome core histones
H2A H2B H3 H4 (each 2 times)
165
The only histone that is not in the nucleosome core
H1
166
H1 location and role
H1 binds to nucleosome and to linker DNA thereby STABILIZING the chromatin fiber
167
DNA IN MITOSIS
In mitosis DNA condenses to form chromosomes
168
Cell cycle phase of chromosomes
Mitosis
169
Cell cycle phase of DNA and histone synthesis
S PHASE
170
CELL CYCLE OF HISTONE SYNTHESIS
S PHASE
171
Heterochromatin
Condensed, transcriptionally inactive, sterically inaccessible DNA
172
Euchromatin
Less condensed, Transcriptionally active, sterically accessible
173
Transcriptionally active and inactive DNA
active --> Euchromatin
| inactive --> Heterochromatin
174
Sterically accessible and inaccessible DNA
accessible --> Euchromatin
| inaccessible --> Heterochromatin
175
Chromatin is like
Beads on a string
176
DNA methylation at......represses transcription
CpG islands
177
The role of methylation at CpG islands
Repress trancription
178
Which nucleotides are methylated during DNA replication and in which strand
Cytosine and Adenine
| Template strand
179
What is the purpose of template strand cytosine and adenine methylation during DNA replication
Mismatch repair enzyme can then distinguish between old and new strands in prokaryotes
180
How does histone chemical modification influence DNA
1. Histone methylation repress DNA transcription (activate it in some cases)
2. Histone acetylation relax DNA, allowing for trancription
181
Purines vs Pyrimidines according to types and structures
Purines (A, G) - 2 rings
| Pyrimidines (C, T, U) - 1 ring
182
Thymine difference in chemical structure
Thymine has a METHYL
183
Cytosine and uracil chemical relationship
Deamination of cytosine makes uracil
184
Uracil found in
RNA
185
Thymine found in
DNA
186
Nucleotides bounds
Hydrogen bounds
187
Nucleotides pairs and number of bonds / stronger?
G-C (3 H bonds)
A-T (2 H bonds)
G-C is stronger
188
High G-C content -->
High melting temperature of DNA
189
Amino acids necessary for purine synthesis
GAG
1. Glycine
2. Aspartare
3. Glutamate
190
NoucleoSide - structure
Base + (deoxy)ribose (sugar)
191
NucleoTide - - structure
Base + (deoxy)ribose (sugar) + PHOSPHATE
192
Phosphate is linked to nucleotide by
3-5 phosphodiester bond
193
Besides glycine, aspartate, glutamate, which substance participate in purine synthesis
N10-Formyl tetrahydrofolate
194
Purine base production de novo requires
Aspartate
Glycine
Glutamine
THF
195
Purine production process
1. Start with sugar (ribose 5-P) + phosphate = PRPP (phosphorybosil pyrophosphate (enzyme: PRPP synthetase)
2. Add GAG ( to make the base) = IMP (inosinic acid)
3. IMP - AMP or GMP (enzyme for IMP to GMP IMP dehydrogenase)
196
The initial sugar for purine synthesis
Ribose 5 - P
197
Main enzyme in purine synthesis
PRPP synthetase (phosphoribosyl pyrophosphate synthetase)
198
Pyrimidine base production requires
Aspartate
199
Pyrimidines synthesis process (generally)
1. Make temporary base (orotic base)
2. Add PRPP (sugar and phosphate)
3. Base modification
200
temporary base for pyrimidine synthesis
Orotic base
201
Orotic base production
1. Glutamate + CO2 + 2 ATP = carbamoyl phosphate 2 + glutamamine + 2 ADP + 2P (enzyme:carbamoyl phosphate synthetase 2)
2. Carbamoyl phosphate + aspartate = orotic acid (enzyme:dihydroorare dehydrogenase)
202
Orotic acid. Next step for pyrimidines synthesis?
Orotic acid+PRPP=UMP (uridine monophosphate)
UMP to UDP
UDP to dUDP (enzyme: ribonucleotide reductase)
203
UDP. 2 possible next steps
1. CTP
| 2. dUDP - dUMP - dTMP (thymidylate synthetase)
204
DUMP TO DTMP
dUMP + N5N10 Methylene THF=dTMP +DHF (enzyme: thymidylate synthetase)
205
THF cycle
N5N10 Methylene THF - DHF (thymidylate synthase) - THF (dihydrofolate reductase) - THF - N5N10 Methylene THF
206
Deoxyribonucleotides synthesis
Ribonucleotides are synthesized first and are convertes to deoxyribonucleotides by ribonucleotide reductase
207
Enzyme: ribonucleotide reductase
Convert ribonucleotides to deoxyribonucleotides
208
Dihydroorate dehydrogenase inhibitor (explain)
Leflunomide (Carbamoyl phosphate to orotic acid)
209
Ribonucleotide reductase inhibitor (explain)
Hydroxyuria (UDP to dUDP)
210
IMP dehydrogenase inhibitor (explain)
Mycophenolate
Ribavirin
(IMP to UMP)
211
Thymidylate synthase inhibitor (explain)
5-fluorouracil (5-FU) --> forms 5-F-dUMP
| dUMP to dTMP
212
PRPP to IMP inhibitor
6-mercaptopurine (6-MP)
| Azathioprine (prodrug of 6-MP)
213
Dehydrofolate reductase inhibitor in humans, bacteria and protozoa
Human - methotrexate
Bacteria - trimethoprim
Protozoa - pyrimethamine
214
Genetic code is unambiguous
Each codon specifies 1 amino acid
215
Genetic code is degenerate/redundant
Most amino acids are coded by multiple codons
| Exceptions: methionine, tryptophan
216
Amino acids that coded by one codon (and which codon)
Methionine - AUG
| Tryptophan - UGG
217
Genetic code is universal
It conserved throughout evolution
| Exception in humans: mitochondria
218
Exceptions of universal genetic code in human
Mitochondria
219
Genetic code is comma-less, non overlapping
Aread from fixed starting point as a continuous sequence of bases
Exceptions:some virus
220
Exceptions of comma-less, nonoverlapping gemetic code
Some virus
221
Genetic codes features
1. Unambiguous
2. Degenerate/redundant
3. Comma-less, nonoverlapping
4. Universal
222
Carbamoyl phosphate is involved in 2 metabolic pathways
1. De novo pyrimidine synthesis
| 2. Urea cycle
223
IMP to urine - pathways and enzymes (and combinations with other pathways)
- IMP --> Inosine --> Hypoxanthine (XO) --> Xanthine (XO) --> Uric acid --> urine
- IMP AMP
- Adenosine --> Inosine (ADA)
- Guanine --> xanthine
- Hypoxanthine + PRPP --> IMP (HGRTT
224
HGPRT? APRT? meaning and action
```
Hypoxanthine guanine phosphoribosyltransferase:
- Hypoxanthine + PRPP to IMP
- Guanine + PRPP to GMP
Adenine phosphoribosyltransferase:
- adenine + PRPP to AMP
```
225
Purine salvage deficiencies - Adenine
Nucleoic acid --> AMP
| Adenine + PRPP --> AMP (APRT)
226
Purine salvage deficiencies - guanine
Nucleic acid --> GMP --> Gianosine --> Guanine --> Xanthine
| Guanine + PRPP (HGPRT) --> GMP
227
ways of IMP synthesis
1. de novo (Ribose-5-P via PRPP synthetase)
2. Hypoxanthine + PRPP to IMP (HGPRT_
3. - IMP AMP
228
Adenosine deaminase deficiency pathophysiology
ADA is required for degration of adnononisne and deoxyoadenosine (nucleosides) (to make them inosine) --> increased dATP --> toxicity in lymphocytes --> Severe combined immunodeficiency (SCID)
229
Is adenosine deaminase deficiency inheritable?
it is one of the MCC AR SCIDs
230
Adenosine deaminase (ADA) role
Adenosine to inosine (both nucleosides)
231
Lesch-Nyhan syndrome problem
Defective purine salvage due to HGRPT) deficiency or absent which convert Hypoxanthine to IMP and guanine to GMP
232
Lesch-nyhan syndrome results in:
| Inheritable?
Excess uric acid production and de novo purine synthesis
| X linked recessive
233
Lesch-nyhan syndrome treatment
Allopurinol
| Febuxostat (2nd line)
234
Lech-nyhan syndrome clinical manifestations:
```
Mnemonic HGPRT
Hyperuricemia
Gout
Pissed off (aggression, self-mutilation)
Retardation
DysTonia
```
235
Which is more complex, eukaryotic or prokaryotic DNA replication?
Eukaryotic replication is more compex but uses many analogous enzymes
236
In both, prokaryotes and eukaryotes, DNA replication is ..... and involves both ......and .....synthesis
In both eukaryotes and prokaryotes, DNA replication is SEMICONSERVATIVE and involves both CONTINUOUS and DISCONTINUOUS (OKAZAKI FRAGMENT) synthesis
237
Okazaki synthesis
Discontinious synthesis
238
Origin of replication
Particular consensus sequence of base pairs in genome where DNA replication begins
Single in prokaryotes
Multiple in eukaryotes
239
Number of replication origin
Single in prokaryotes
| Multiple in eukaryotes
240
Replication fork
Y-shaped region along DNA template where leading and lagging strands are synthesized
241
Helicase
Unwinds DNA template at replication fork
242
Enzyme responsible for DNA template unwinding at replication fork
DNA helicase
243
Single-stranded binding proteins (DNA) - function
Prevents strands from reannealing
244
topoisomerase inhibitors (and action)
fluoroquinolones: gyrase (prokaryotic topoisomerase 2) and topoisomerase 4
etoposide, tenoposide --> topoisomerase 2
Irinotecan, topotecan --> topoisomerase 1
245
Prokaryotic topoisomerase type 2 is also called
Gyrase
246
DNA topoisomerases
Create a single or double stranded break in the helix to add or remove supercoils
247
Primase (dna)
Makes an RNA primer on which DNA polymerase 3 can initiate replication
248
Where is DNA polymerase 3 founded
Only in prokaryotes
249
Polymerase type 3 function
1. Elongates leading strand by adding deoxynucleotides to the 3 end
2. Elongates lagging strand until it reaches primer of preceding fragment
3. 3 to 5 exonuclease activity proofreads each added nucleotide
250
Enzyme with 5 to 3 synthesis and profreads with 3 to 5 exonuclease
DNA Polymerase type 3
251
DNA polymerase type 1 - action / found
Degrades RNA primer (5 to 3 exonuclease)replace it with DNA (5 to 3 elongation and 3 to 5 exonuclease activity proofreading)
Only prokaryotes
252
DNA polymerase 1 vs DNA polymerase 3
They have the same function but DNA polymerase 1 also excise RNA primer with 5 to 3 exonuclease
253
DNA ligase
Formation of phosphodiester bond with a strand of double -stranded DNA (ex joints Okazaki fragments)
254
Telomerase
RNA dependent DNA polymerase that adds DNA to 3 ends of chromosome to avoid loss of genetic material with every duplication
255
Enzyme that participate in DNA replication
1. Helicase
2. Single -stranded binding proteins
3. DNA topoisomerase
4. Primase
5. DNA polymerase 3 (prokaryotes)
6. DNA polymerase 1 (prokaryotes)
7. DNA ligase
8. Telomerase (eukariotes)
256
type of mutations in DNA
1. silent 2. Missense 3. Nonsense
| 4. Frameshift 5. Splice site
257
Silent mutation
Nucleotide substitution but codes for same amino acid
| Often 3rd position of codon (tRNA) wobble
258
Missense mutation (and example)
Nucleotide substitution resultin in chamged aminacid
If new amino acid is similar to chemical structure---> conservative
example Sicke cell disease
259
Conservative missense mutation
If new amino acid is similar in chemical structure
| example: Sicke cell disease
260
Nonsense mutation
Nucleotide substitution resulting in early stop codon --> nonfunctional protein
261
Frameshift mutation
Deletion or insertion of a number of nucleotides not divisible by 3 resulting in misreading of all nucleotides downstream, usually resulting in a trancated, NONFUNCTIONAL PROTEIN
262
Frameshoft mutation - example
| splice site mutation - example
Frame: 1. Duchenne muscular dystrophy 2. Tay Sachs disease
Splice: cancers, dementia, epilepsy, some types of β-thalassemia
263
splice site mutation?
mutation at a splice site --> retained intron tn the mRNA --> protein with imparaid or altered function
264
Severity of mutations (in order) (from most severe to less
Frameshift --> nonsense --> mssense --> silent
265
Transition? (For silent, missense, nonsesne mutation)
Purine to purine or pyrimidine to pyrimidine
266
Transversion? (For silent, missense, nonsesne mutation)
Purine to pyrimidine
| Or pyrimidine to purine
267
DNA repair - ways
1. Nucleotide excision repair (single strand)
2. Base excision repair (single strand)
3. Mismatch repair (single strand)
4. Nonhomologous and joining (double strand)
268
Nucleotide excision repair?
Specific endonucleases release the oligonucleptide-containing damaged bases --> DNA polymerase and ligase fills and reseal the gap -->
269
Defective in xeroderma pigmentosum
Nucleotide excision repair (single strand) --> UV causes pyrimidine dimers
270
What is the problem in dna after ultraviolet exposure
Pyrimidine dimers
271
What type of lessions does nucleotide excision repair system repair
It repairs BYLKY helix - distorting lesioms
272
nucleotide excision repair system - acts during
G1
273
Important in repair of spontaneous/toxic deamination
| Acts during
Bade excision system
| All cycle
274
Base excision repair system steps
1. Glycosylase recognized altered base and creates apurinic/ apyrimidinic site (AP site)
2. One or more nucleotides are removed by AP-endonuclease, wchich cleaves 5 end.
3. Lyase cleaves 3 end
4. DNA polymerase β fills the gap
5. DNA ligase seals the gap
4. DNA polymerase fills the gap
275
Base excision repair system enzymes
1. Glycosylase
2. AP exonclease
3. Lyase
4. DNA polymerase β
5. DNA ligase
276
Defective in hereditary nonpolyposis colorectal cancer (HNPCC)
mismatch repair system
277
Mismatch repair system
| Acts during
Newly synthesized strand is recognized, ,ismatched nucleotides are removed, and the gap is filled and realised
G2
278
The first step of mismatch repair system
Recognizes newly synthesized strand
279
defective in Nonhomologous and joining repair system - example
1. ataxia telengiectasis
| 2. fanconi anemia
280
Nonhomologous end joining repair system
Brings together 2 ends of DNA fragments to repair. Double stranded breaks. No requirement for homologous
281
Nonhomologous end joining repair system - disadvantage
some DNA may be lost
282
Double stranded DNA repair system
Nonhomologous end joining
283
Single strand DNA repair system
1. Nucleotide excision repair
2. Base excision repair
3. Mismatch repair
284
DNA synthesis direction
RNA synthesis direction
protein synthesis direction
RNA/DNA 5 to 3
| PROTEIN N-terminus to C-terminus
285
Energy source for bond in RNA/DNA synthesis
The 5 end of incoming nucleotide bears the triphosphate
286
mRNA read direction
5 to 3
287
DNA chain termination drugs mechanism of action
They have a modified 3 OH, preventing addition to the next nucleotide
288
The triphosphate bond is the target of which enzyme
3hydroxyl attack
289
Start codon
AUG
| Rarely GUG
290
Start codon - eukaryotes - amino acids
Methionine, which may be removed before translation is completed
291
Start codon - prokaryotes - amino acids
Codes for formylmethionine (f-met)
292
mRNA stop codons
UGA (U Go Away)
UAA (U Are Away)
UAG (U Are Gone)
293
2 strands of a gene
1. Template strand
| 2. Sense/coding strand
294
Promoter - definition
Site of RNA polymerase II and multiple other transcription factors bind to DNA upstream from gene locus
295
Promoter sequence
AT - rich upstream sequence with TATA and CAAT boxes
296
TATA box
| CAAT box
Template strand:ATATTA, GTTA
| Sense/coding strand: TATAAT, CAAT
297
Promoter mutation
Dramatic decreasing in level of gene trancription
298
Silencer
Site where negative regulators (repressors) bind
299
Enhancer
Stretch of DNA that alters gene expression by binding transcription factors
300
Enhancers and silencers location
Close to, far from or even within (in an intron) the gene whose expression it regulates
301
Regulation of gene expression DNA sites
1. Promoter
2. Enhancer
3. Silencer
302
Eukaryotes RNA polymerase
1. RNA polymerase type 1 - rRNA
2. RNA polymerase type 2 - mRNA
3. RNA polymerase type 3 - tRNA (5S rRNA)
NUMBERED IN THE SAME ORDER THAT THEIR PRODUCTS ARE USED IN PROTEIN SYNTESIS
303
Prokaryotes RNA polymerase
1 RNA polymerase makes all 3 kinds of RNA (Multisubunit complex)
304
Most numerous RNA
largest RNA
smallest RNA
most numerous: rRNA
largest: mRNA
smallest: tRNA
305
a-amantin (where is founded, mechanism of action, clinical manifestations)
```
Amanita phalloides (death cap mashrooms),
It inhibits RNA polymerase type 2
Causes severe hepatotoxicitu if ingested
```
306
Amanita phalloides (death cap mushrooms) contain
a-amantin
307
Rifampin - mechanism of action
inhibits RNA polymerase in prokaryotes
308
Actinomycin D mechanism of action
inhibits RNA plymerase in both prokaryotikes and eukariotes
309
Enzyme that opens DNA at promoter site
RNA polymerase type 2
310
RNA polymerase vs DNA polymerase according proofreading function and chain initiation
RNA POLYMERASE HAS NO PROOFREADING FUNCTION BUT IT can initiate chains
311
hnRNA
Heterogenous nuclear RNA
312
Heterogenous nuclear RNA (hnRNA). (eukaryotes)???
It is the initial transcript. It is then modified and becomes mRNA
314
Capping of 5 end (eukaryotes)
Addition of 7-methylguanosine gap
315
Polyadenylation of 3 end
Enzyme. (eukaryotes)
SIGNAL
Addition of 200 at 3 end
Poly A- polymerase (does not require template)
AAUAAA
316
AAUAAA (eukaryotes)
Polyadenylation signal
317
Location of mRNA translation (eukaryotes)
Cytosol (mRNA is transported out of the nucleous into cytosol)
318
P bodies function and structure (eukaryotes)
- distinct foci within the cytoplasm of the eukaryotic cell
- contain Exonucleases, decapping enzymes, microRNA
- mRNA control
- mRNA is may be stored their for future translation
320
Splicing of pre-mRNA
1. Primary transcript combine with small nuclear ribonucleoproteins (SnRNPs) and other proteins to form spliceosome
2. Lariat - shaped (looped) intermediate is generated
3. Lariat is released to precisely remove intron and join 2 exons
331
In eukaryotes, tRNA / mRNA rRNA is synthesized by
tRNA --> RNA polymerase type 3
mRNA --> RNA polymerase type 2
rRNA --> RNA polymerase type 1
332
From DNA to protein (the name of the processes
DNA --> hnRNA (transcription) --> mRNA (splicing) --> proteins (translation)
335
Shape of secondary structure of tRNA
Cloverleaf: anticodon end is opposite 3-aminoacyl end
337
CCA of tRNA
- CCA at 3 end along with a high percentage of chemically modified bases
- both eukariotic and prokaryotic
- 3 - ACC
- contently bound aminoacid
340
T arm of tRNA
Contains ΤΨC (ribothymidine, pseudouridine, cytidine) sequence necessary for tRNA-ribosome binding
341
TΨC sequence of tRNA
ribothymidine, pseudouridine, cytidine
343
Acceptor stem of tRNA
3-ACC-5 - OH is the amino amino acid acceptor side + more nucleotides
344
Sequence of tRNA amino acid acceptor site (acceptor site)
3-ACC-5 - OH
346
Energy of aminoacyl tRNA (formation of tRNA binded with aminoacid)
ATP
347
Amino acid matchmaker of tRNA
Aminoacyl-tRNA synthetase (1 per amino acid). It also scrutinizes amino acid before and after it binds to tRNA. If incorrect, bond is hydrolized
355
Posttanslational modifications types
1. Trimming
| 2. Covalent alternations
356
Triming (type of posttranslational) modification
| example
Removal of N- or C- propeptides from zymogen (inactivate enzyme precursor) to generate mature proteins
example: trypsinogen --> tripsin
359
3 steps of protein synthesis
1. Initiation
2. Elongation
3. Termination
362
Protein synthesis initiation prosses
Initiated by GTP hydrolysis. Initiation factors (eukaryotic IF) help assemble the 40s ribosomal subunit with the initiator tRNA and are released when the mRNA and the ribosomal 60s subunit assemble with the complex (initiation complex)
363
3 site of 60s rRNA elongation
APE
A site = incoming aminoacyl-tRNA (except for initiator methinine)
P site = accommodates growing peptide
E site= holds empty tRNA as it exits
366
Elongation of protein synthesis
1. Aminoacyl - tRNA binds tomA site (except initiator methionine)
2. rRNA (ribozome) catalizes peptide bond formation, transfer growing polypeptide to amino acid in A site
2. Ribosomes advances 3 nucleotides toward 3 end of mRNA , moving peptidyl tRNA to P site (traslocation)
368
Polymerase chain reaction (PCR)? (definition, purpose)
Molecular biology laboratory procedure used to AMPLIFY A DESIRED FRAGMENT OF DNA. Useful as a diagnostic tool
369
In clinical practice, PCR is useful as a: (and examples)
Diagnostic tool (ex. Neonatal HIV, herpes encephalitis)
370
Steps of Polymerase chain reaction (PCR) and temperature
1. Denaturation --> 95
2. Annealing --> 55
3. Elongation --> 72
These steps are repeated multiple times for DNA sequence amplification
371
Denaturation (PCR)
DNA is denaturated by heating to generate 2 separate strands (95 C)
372
Annealing (PCR)
During cooling, excess DNA primers anneal to a specific sequence on each strand to be amplified
(55 C)
373
Elongation (PCR)
Heat stable DNA polymerase replicates the DHA sequence following each primer (72 C)
374
PCR - after the 3 steps
Agarose gel electrophoresis
376
Bloatting procedures
1. Southern blot
2. Nothern blot
3. Western blot
4. Southwestern blot
378
The following processes occur the nucleus following transcription:
(eukaryotes)
1. Capping of 5 end
2. Polyadenylation of 3 end
3. Splicing out of introns
CAPPES, TAILED AND SPLICED TRANSCRIPT IS CALLED mRNA
379
Nothern blot is useful for:
mRNA evels, reflective of gene expression
384
mRNA quality control occurs at (eukaryotes)
Cytoplasmatic P - bodies
386
Splicome
Primary transcript combined with snRNP and other proteins
387
Antibodies against spliceosomal snRNPs (anti Smith)
Highly specific for SLE
388
Anti-Smith
Antibodies against spliceosomal snRNP (highly specific for lupus)
389
SLE highly specific antibodies
Anti - Smith (antibodies against splisocoemal snRNP)
390
Mixed connective tissue antibodies
Anti - u1 antibodies
391
Major elisa variations
1. direct
2. sandwich (indirect)
3. competitive
392
Anti-U1 RNP antibodies
Mixed commective tisseu disease
393
Exons
Contain the actual genetic information coding for protein
394
Introns
Intervening noncoding segments of DNA
395
Alternative splicing
Different exons are frequently combined by alternative splicing to produce a larger number of unique proteins
396
Abnormal splicing are implicates in
1. Oncogenesis
| 2. Many genetic disorder (β-thalassemia)
397
Fluorescence in situ hybridization (FISH)
Fluorescent DNA or RNA probe binds to specific gene site of interest on chromosomes. Used for specific localization of genes and direct visualization of anomalies (e.g. Microdeletions) at molecular level (when deletion is too small to be visualized by karyotype)
399
FISH is used to detect (explain)
1. microdeletion: no florescence on a chromosome compared to florescence at the same locus on the 2nd copy of that chromosome
2. Tranocation: florescence outside the original chromosome
3. Duplication: extrasite of florescence on one chromosome relative to its homologous chromosome
400
Prokaryotic RNA polymerase
1 RNA polymerase is a multisubunit complex that makes all 3 kinds of RNA
401
Number of nucleotides in tRNA
75-90
403
Transgenic strategies of gene into mouse genome involve
1. Random insertion of gene into mouse
| 2. Targeted insertion or deletion of gene through homologous recombination with mouse gene
404
Where is tRNA anticodon end
It is opposite 3 aminoacyl end
406
Where is the aminoacid on the tRNA
Is covalently bound to the 3 end of tRNA
407
3 tRNA areas
1. T arm
2. D arm
3. Acceptor stem
410
Cre - lox system
Can inducibly manipulate genes at specific developmental points (e.g. to study a gene whose deletion cause embryonic death)
411
tRNA TΨC function
Necessary for tRNA - ribosome binding
414
D-arm of tRNA
It contains dihydrouracil residues necessary for tRNA recognitiom by the correct aminoacyl-tRNA synthetase
417
Introns sequence
P-GU-A-AG
| P binds with A to form the loop
418
How many different aminoacyl-tRNA does exist
One per amino acid
419
What if amino acid that binded to tRNA is incorrect
Bond is hydrolyzed. If not hydrolyzed, trna reads usual codon, vut inserts wrong amino acid
420
Peptide bind energy
The amino acid tRNA bond has energy for formation of peptide bond
421
Responsible for accuracy of amino acid selection
1. Aminoacyl-tRNA synthetase
| 2. Binding of charged tRNA to the codon
422
tRNA wobble
Accurate base pairing is required only in the first 2 nucleotide positions of an mRNA codon, so codons differing in the third position may code for the same tRNA/amino acid ( as a result of degeneracy of genetic codes
423
Chaperone proteins
Intracellular protein involved in facilitating and/or maintaining protein folding
424
Champeron proteins in yeast
Some are heat shock proteins (ex Hsp60) that are expressed at high temperatures to prevent protein denaturing/misfolding
427
Covalent alternations (type of posttranslational modification)
Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, ubiquitination
428
Ribosomes
Eukaryotes: 40s + 60s-->80s
| Prokaryotes 30s+50s-->70s
430
tRNA actives energy
1. Charging (activation) - ATP
2. Gripping (initiation of protein synthesis) - GTP
3. Ribosomes translocation - GTP
431
Protein synthesis is initiated by
GTP hydrolysis
434
Enzyme that catalizes peptide bond formation
| How
Ribozome (rRNA)
| It transfers growing polypeptide to amino acid A site
435
Ribosome translocation
Ribosomes advances 3 nucleotides toward 3 end of mRNA, moving peptidyl tRNA to P site
437
Termination of protein synthesis
Stop codon is recognized by release factor and completed polypeptide is released from ribosome
445
Agarose gel electrophoresis
Used for size separation of PCR products (smaller molecules travel further). Compared against DNA ladder
447
Southern blot steps
1. DNA sample is enzymatically cleaved into smaller pieces
2. Electrophoresed on a gel
3. Transferred to a filter
4. Soaked in a denaturant
5. Exposed to a radiolabeled DNA probe that recognizes and anneals to its complementary strand
6. Double stranded piece of DNA is visualized when the filter is exposed to film
448
Nothern blot
Similar to southern blot, except that an RNA sample is electrophoresed.
450
Method for studying mRNA levels
Nothern blot
451
Southern vs nothern blot
In nothern blot, RNA sample is electrophoresed
| In southern blot, DNA sample is electrophoresed
452
Western blot steps
1. Sample protein is separated via gel electrophoresis
2. Transferred to a filter
3. Labeled antibody is used to bind to relevant protein
453
Western blot is used for:
Confirmatory test fir HIV after + ELISA
454
western vs southern vs nothern blot difference according samples
Southern=DNA
Nothern=RNA
Western=protein
455
Southwestern blot
Identifies DNA-binging proteins (e.g. transcription factors) using labeled oligonucleotide probes
456
Karyotiping (method)
A process in which METAPHASE CHROMOSOMES ARE STAINED, ORDERED AND NUMBERED
457
In karyotypes, we observe
1. Morphology
2. Size
3. Arm-length ratio
4. Banding pattern
458
Karyotypes can be perform on a sample of
1. Blood
2. Bone marrow
3. Amniotic fluid
4. Placental tissue
459
Karyotyping is used to diagnose
CHROMOSOMAL IMBALANCE (e.g. Autosomal trisomies, sex chromosomes disorder)
460
Enzyme - linked immunosorbent assay(ELISA) is used to detect
The presence of of either a specific antigen (direct) or a specific antibody (indirect) in a patient's blood sample
461
Linked immunosorbent assay - direct
Uses a test antibody to see if a specific antigen is present in the patient's blood. A secondary antibody coupled to a color- generating enzyme is added to detect the antigen
462
Linked immunosorbent assay(ELISA) - indirect
Uses a test antigen to see if a specific antibody is present in the patients blood. A secondary antibody coupled to a color- generating enzyme is added to detect the antigen
463
ELYSA + result
If the target substance is present in the sample, the test solution will have an intense color reaction
464
ELYSA is used in many laboratories to determine:
Whether a particular antibody (e.g. anti-HIV) is oresent in a patient's sample
465
ELISA sensitivity and specificity
Both approach 100%, but both false-postive and false-negative results occur
467
When used fluorescence in situ hybridization instead of karyotype for direct visualization of anomalies
When deletion is too small to be visualized by karyotype
468
FISH signal
Fluorescense=gene is present
| No fluorescence=gene has been deleted
469
Cloning
It is the production of a recombination DNA molecule that is self perpetuating
470
Cloning methods
1. Isolate eukaryotic mRNA (post-RNA processing steps) of interest
2. Expose mRNA to reverse transcriptase to produse cDNA (lacks introns)
3. Insert cDNA fragments into bacterial plasmids containing antibiotic resistance genes
4. Transform recombination plasmid into bacteria
5. Surviving bacteria on antibiotic medium produce cDNA
471
Knock out
Removing a gene
472
Knock in
Inserting a gene
473
RNAi
RNA interference
474
RNA interference
dsRNA is synthesized that is complementary to the mRNA sequence of interest. When transfected into human cells, dsRNA separates and promote degradation of target mRNA, "knocking down" gene exression
475
RNA interference structure before transfected into human cells
dsRNA complementary to the mRNA sequence of interest
476
RNA interference structure in human cell
dsRNA separates and promotes degradation of target mRNA
477
Microarrays materials
Thousands of nuclei acid sequences are arranged in grip on glass or silicon
478
Microarrays concept
DNA or RNA probes are hybridized to the chip, and a scanner detects the relative amounts of complementary binding
479
Microarrays used to:
Profile gene expression levels of thousands of gene simultaneously to study certain disease and treatment
480
Microarrays are able to detect:
Single nucleotide polymorphism (SNPs) and copy number variations (CMV) for a variety of applications including genotyping, clinical genetic testing, forensic analysis, cancer mutation, genetic linkage analysis
481
Microarrays are able to detect SNPs and CNVs for a variety of applications including
1. Genotyping
2. Clinical genetic testing
3. Forensic analysis
4. Cancer mutation
5. Genetic linkage analysis
482
Termination signal of an eukaryotic gene
5-AATAAA-3
3-TTATTT-5
It is the same with the adenylation signal
483
flow cytometry is laboratory technique to assess
size, granularity, and protein expression (immunophenotype) of individual cell in a sample
484
flow cytometry comonly used in
workup of hematologuc abnosmalities (eg. paroxysmal noctural hemoglobinuria, fetal RBCs in mother's blood) and immunodeficiencies (CD4 cell count in HIV)
485
flow cytometry - mechanism
cells are tagged with antibodies specific to surface or intracellular proteins --> antibodies are then tagged with a unigue fluorescent dye --> sample is analyzed one cell at a time by focusing a laser on the cell and measuring light scatter and intensity of fluorescense --> data are plotted either as histogram (one measure) or scatter plot (any 2 measures)
486
Lac operon is a classic example of
genetic response to an environmental change
487
Glucose is the preferred metabolic substrate in E.coli. When is absent -->
if lactose is available --> the lac operon is activated to lactose metabolism
488
lac operon - mechanism of shift
low glucose --> increased adenylate cyclase --> incresaed cAMP --> activation of catabolic activator protein (CAP --> increased transcription
high lactose --> unbinds repressor protein from repressor / operator site --> increased transcription (alolactose is the real binding)
489
genes of Lac operon
1. LacZ
2. LacY
3. LacA
490
lac operon - low glucose / lac available?
strongly expressed
491
lac operon - high glucose / lactose unavailable
not expressed
492
lac operon - low glucose / lactose unavailable
not expressed
493
lac operon - high glucose, lactose available
very low (basal) expression)
