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Flashcards in Molecular + cellular Deck (428):
0

1.Which is the shortest phase of cell cycle?.
2.which phases are variable?

1. Mitosis
2. G1 and G0

1

Checkpoints control transition between phase of cell cycle. Is regulated by:

Cyclins
Cycle - dependent kinases (CDK)
Tumor suppressor

2

What is G and what is S in cll cycle

Gap
Synthesis

3

M phase includes

1. mitosis (Prophase, Metaphase, Anaphase, Telophase)
2. cytokinesis (cytoplasm splits in 2)

4

Which cell cycle regulator is CONSTITUTIVE AND INACTIVE

Cyclin dependent kinases (cdk)

5

Which cell cycle regulators are phase specific?
Role?

Cyclins....activate cyclin dependent kinases (CDKs)

6

Phases of cell cycle:
What is interphase?

1. G1 (and G0)
2. S phase
3. G2
4. M phases

Interphase: G1-S-G2

7

1. Tumor suppressors in cell cycle regulation (mechanism)
2. If mutated:

1.P53 induce p21 which HYPOphosphorylates Rb --> binds to and inactivate E2F --> Inhibit G1 to S progression
3. Unrestrained cell cycle division (eg Li-fraumeni)

8

Which cell type is affected by chemotherapy

Labile

9

Cell types according their proliferative ability: (and definition)

1. Permanent - remain in G0, regenerate from stem cells
2. Stable (quiescent) - enter G1 from G0 when stimulated
3. Labile: never go to G0, divide rapidly with shirt G1. Most affected by chemotherapy

10

Cell type that is also called QUIESCENT

Stable

11

Permanent cell examples

Neurons, skeletal and cardiac muscle, red blood cells

12

Stable (quiescent) cells examples

Hepatocytes, lymphocytes

13

Labile cells examples

Bone marrow, gut epithelium, skin, hair follicles, germ cells

14

Smooth vs rough
Endoplasmic reticulum according their structure:

Smooth endoplasmic reticulum LACKS SURFACE RIBOSOMES

15

Cell type with short G1

Labile

16

Smooth endoplasmic reticulum role:

1. Steroids synthesis
2. Detoxification of drugs and poisons

17

Cells rich in smooth endoplasmic reticulum:

1. Liver hepatocytes
2. Steroid hormones-producing cells of the adrenal cortex
3. Steroid hormones-producing cells of the gonads

18

Cells rich in rough endoplasmic reticulum

1. Mucus-secreting goblet cells of the small intestine
2. Antibody-secreting plasma cells

19

Rough endoplasmic reticulum role:

1. Synthesis of secretory (exporter) proteins
2. N-linked oligosaccharides addition to many proteins

20

What are Nissl bodies and what is their function

Nissl bodies are rough endoplasmic reticulum in neurons
Synthesize peptide neurotransmitters for secretion

21

Free ribosomes (structure and function):

Unattached to any membrane
Site of synthesis of cytosolic and organelle proteins

22

Proteasome stracture and function

Barrel-shaped protein complex that degrades damaged or UBIQUITIN-tagged proteins

23

Ubiquitin - Proteasome system defects:

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)