Molecular And Cell Biology Flashcards

Question

What is bacteriophage T2?

Answer 1

The host is Escherichia coli. It destroys the chromosomes and replicates its genone.

Answer 2

1) label bacteriophage DNA or protein with radioactive isotope 2) Infect a bacteria (only DNA enters) 3) Separate phage ghosts and bacteria (use blender and centrifuge) 4) test for radioactivity

Answer 3

32P and 35S are unstable isotopes - can be detected by Geiger counter. Growing the bacteriophage in media with them will produce radioactively labelled protein/DNA.

Answer 4

Supernatant had phage ghosts, pellet had bacteria. They were tested for radioactivity - It showed that DNA was injected, not proteins.

Answer 5

Purines - A,G (2 carbon rings) Pyrimidines - C,T,U (1 carbon rings)

Answer 6

Sugar + base

Answer 7

Used chromatography to seperate and isolate nucleobases. Chargaff's rules %A = %T, %C = %G %AT does not = %GC

Answer 8

A-T and G-C hydrogen bonded pairs Antiparallel Right handed double helix Major (big gap) and minor grooves (little gap) One helical turn every 10.5 bp

Answer 9

AT = 2 CG = 3 5' to 3' polarity

Answer 10

The binding of one purine and one pyrimidine

Answer 11

3.4 nm, 10.5 bp

Answer 12

Specialised region where microtubules attach - doesn't have to be in the centre

Answer 13

Repetitive DNA at end of chromosomes Protect the ends of chromosomes

Answer 14

Single, circle chromosome Plasmids also found Passed between cells by conjugation Few million bp

Answer 15

Have domains which can regulate gene expression, cut DNA and protect DNA e.g. restriction endonucleases, transcription regulators, histones

Answer 16

Proteins which bind to reg sequences near promotors to stimulate or block transcription. Bend DNA in favourable or unfavourable ways e.g. lac operon - lac repressor binds to DNA to block transcription

Answer 17

Enzyme which cuts DNA at specific sequences. Restricts action of viruses. Bacteria is protected as methylated

Answer 18

Proteins which chromatin is wrapped around.

Answer 19

Uracil - uridine Adenine - adenosine Guanine - guanosine Cytosine - cytidine

Answer 20

Short helices in RNA caused by intramolecular base-pairing. Secondary structural elements. e.g. tRNA

Answer 21

Minor groove - major too narrow

Answer 22

G-U, A-C - wobble base pair These can stabilise RNA

Answer 23

Lacks a OH on second carbon The OH makes ribose more reactive and prone to hydrolysis

Answer 24

Secondary - 2D map defined through intramolecular base-pairing. Tertiary - interactions that connect regions separated in secondary structure - can be canonical.

Answer 25

Two adjacent A bases interacting with the edge of a G-C base pair.

Answer 26

From RNA polymerases - transcription. RNAP active site contains a short RNA/DNA heteroduplex

Answer 27

Promotor regions until reaches terminator region.

Answer 28

Protein complex containing 5 subunits: 2 alpha - binds transcription factors 2 beta - catalytic w - assembly and stability

Answer 29

Provide specificity to RNAP for the gene promotor. In prokaryotes Released from RNAP

Answer 30

I = rRNA II = mRNA, noncoding RNA III = tRNA, 5s RNA Have conserved core structure

Answer 31

General transcription factor Required to assemble RNAP II onto promotors in eukaryotic cells. A preinitiation complex involves a multi-step pathway. e.g. TFIIA, TFIIB

Answer 32

X-ray crystallography - saw x pattern (helix), regular pattern, distance between spots (one turn) = 3.5 nm

Answer 33

Used nitrogen isotopes to experiment semi-conservative DNA. 14 and 15 - nitrogen 1) grew the bacteria in media to make heavy (15) and light (14) DNA 2) separate by ultracentrifugation 3) mix with caesium chloride 4) look at DNA using UV light 5) saw different bands after every generation 1st - 1 band 2nd - 2 bands 3/4/5 - 2 bands but hybrid getting lighter and lighter

Answer 34

1 in E.coli Tens of thousand in humans - bidirectional replication forks

Answer 35

Adds nucleotides one at a time 5'-3' using template strand

Answer 36

Primase generates the primer Ligase joins the new DNA together (loose ends into a single strand)

Answer 37

Topoisomerase relieves pressure from overwinding by breaking and resealing DNA. Helicase breaks H bonds between two strands

Answer 38

Prevents the two DNA strands from reannealing in DNA replication

Answer 39

Leading strand - DNA points towards replication fork - continuous Lagging - DNA points away so must be discontinued and primed multiple times BOTH 5'-3'

Answer 40

Pieces of DNA - they are stuck together to make lagging strand

Answer 41

Primer is removed - leaves a gap so lagging strand is incomplete - telomeres solve this - telomerase can replenish telomeres

Answer 42

Large (rRNA) - in agarose gels Small - acrylamide gels mRNA is not clearly seen

Answer 43

mRNA - 5% rRNA - 75% tRNA - 10%

Answer 44

- capped at 5' end - pre-mRNA splicing - 3' end processed (cleavage and polyadenylation - adding a stretch of adenine nucleotides) - happens in nucleus - the cap and poly(A) protects and promotes translation

Answer 45

A guanosine nucleotide is added to the 5' end of RNA pol II transcripts - linked by 5'5' triphosphate linkage. Then methylated

Answer 46

Where an mRNA can code for multiple proteins - in prokaryotic cells

Answer 47

Genes are interrupted by introns Prokaryotic cells DO NOT HAVE THIS

Answer 48

Introns and exons have splice site sequences 5' - GU (start of intron) 3' AG (end of intron) Introns also have a branchpoint 'A'

Answer 49

Carried out by a ribonucleoprotein (RNP) called spliceosome These are made from smaller RNA/proteins called snurps

Answer 50

Involves 2 transesterification steps - 1) 5' exon removed and the intron forms a lariat structure involving branchpoint adenosine 2) exons joined and intron lariat released

Answer 51

Yes! Nuclear splicing is said to have evolved from this Enzymes with RNA catalytic subunit - ribozyme

Answer 52

- infer protein sequences from DNA and its sequence - infer protein function by comparison - design tools to study protein function e.g. drugs

Answer 53

By base pairing with anticodons within cognate tRNAs - the tRNAs are charged/aminoacylated (connected to appropriate amino acid) - mediated by aminoacyl-tRNA synthetases

Answer 54

Peptidyl group and aminoacyl groups bind - peptide bond

Answer 55

Nonpunctuated Some have same meaning - degenerate

Answer 56

Cell extracts were programmed to make proteins using artificial RNAs made with polynucleotide phosphorylase. e.g. poly(u) RNA made phenylalanine

Answer 57

64 - 61 are sense = code for amino acids - 3 are stop codons - UAA, UAG, UGA The start codon is AUG - methionine Met and Trp have unique codons - others degenerate

Answer 58

Code for same amino acid - usually differ by 3rd nucleotide Some recognised by isoacceptor tRNA's - charged with same amino acids Others can be recognised by the same tRNA - wobble base-pairing

Answer 59

Inosine - 1st position on anticodon Can pair with U,A,C G can pair with C or U

Answer 60

Cloverleaf - 5' and 3' drawn together Amino acid is bound to 3' hydroxyl on A nucleotide All tRNA have 3' terminal CCA Modification of 1st position of anticodon allows wobble

Answer 61

Increases thermodynamic ability in tRNA Anticodon on D arm, acceptor on TYC arm Base pairing between the arms

Answer 62

Mediated by aminoacyl-tRNA synthetases - needs ATP 20 different ones - causes ester link between carboxylic group of AA and 3' hydroxyl group

Answer 63

Large RNP particle - has two unequal subunits Codon/anticodon occus on small subunit Peptide bonding occurs on large subunit There are 3 tRNA binding sites (A,P,E) Has a peptidyltransferase centre (PTC) - RNA rich - makes peptide bonds - RNA catalysed

Answer 64

rRNA transcription and early steps in nucleoli Later steps in nucleosomes and cytoplasm

Answer 65

2 tRNAs are bound at a given time, either at A & P, or P & E. Contains cycles of: - aminoacyl-tRNA binding - peptide bond formation - translocation of the ribosome along mRNA When aminoacyl-tRNA binds to A - tRNA is released from E

Answer 66

P = peptidyl site A = aminoacyl site E = exit site

Answer 67

The aminoacyl-tRNA brought to ribosome by the elongation factor EF1A (EF-Tu in prokaryotes) Translocation needs EF2 (EFG in prokaryotes) Both are GTPases - 2 GTP molecules are hydrolysed per cycle

Answer 68

EF1a brings Methionyl-tRNAs to bind to AUG codons and associate with eukaryotic initiation factor 2, eIF2 (IF2 in prokaryotes)

Answer 69

Allows the start codon to start at P site - in prokaryotes - near the start codon - 3' end of 16s rRNA

Answer 70

tRNAmet (bound to eIF2) and 5' mRNA bind through interaction with cap-binding complex. The preinitiation complex scans along mRNA - finds AUG - kozak sequence The large subunit is then recruited

Answer 71

Eukaryotic release factor 1 (eRF1) or RF1/2 in prokaryotes trigger peptide hydrolysis . eRF3 (GTPase) releases peptide by releasing RF1/2 from ribosome

Answer 72

Genes that are constitutively expressed

Answer 73

cis - mutation within the same gene - DNA sequences that effect gene regulation trans - in a different gene - protein or RNA factors that regulate the expression of target gene

Answer 74

Transcriptional

Answer 75

Can activate (activators) - positive control - at weak promotors Can repress (repressors) - negative control Activators in E.coli interact with a subunit of RNA polymerase - promote binding

Answer 76

Inducers - bind to TF and stimulate activators or inhibit repressors Corepressors - bind to TF and stimulate repressors or inhibit activators

Answer 77

Codes for 3 genes - lacZ, lacY and lacA Controlled by transcription repressor gene - lacI LacI - binds to operator (lacO) and blocks RNA polymerase Lactose is the inducer - inhibit repressor Negative - inducible

Answer 78

lac operon also regulated by activator - catabolite activator protein Binds to lac promotor when associated with cAMP (inducer) - stims transcription cAMP is inhibited by glucose (ideal energy source) Lac operon - expressed by absence of glucose and presence of lactose

Answer 79

Pre-mRNA can occur in different patterns - different proteins

Answer 80

Translation is downregulated by integrated stress response Can also be regulated by specific mechanisms e.g. Ferritin expression is responsive to Fe2+

Answer 81

Most commonly - phosphorylation of serine, threonine or tyrosine by kinases

Answer 82

eIF2 hydrolyses GTP to GDP - regeneration requires eIF2B but eIF2a is phosphorylated by integrated stress response and binds very tightly to it so it cannot be recycled

Answer 83

1) insert DNA into a vector (usually a plasmid) - makes recombinant DNA 2) transfer to host 3) replicate

Answer 84

1) origin of replication - replication in host 2) selectable marker - survival of host cells e.g. antibiotic resistant gene 3) multiple cloning site - where we insert gene - has restriction enzyme sites

Answer 85

Use restriction enzymes - recognise short specific sequence There are 3/4 types: - types I and III cleave DNA at random places far from recognition sequence - type IV cleave modified DNA - type II cut DNA at a specific place Name them: Species, Strain, Enzyme e.g. EcoRV

Answer 86

Most commonly protein homodimers DNA sequence usually palindromic Recognise specific DNA sequence Can generate overhangs or blunt edges - overhangs are compatible

Answer 87

1) initial binding is non-specific 2) moves along until finds specific site 3) this binding causes structural changes 4) catalysis requires Mg2+ 5) generates 5' phosphate and 3' OH ends

Answer 88

Sticky ends have to interact - DNA ligase catalyses new phosphodiester bond

Answer 89

- insert doesn't join vector - little DNA - DNA may be mixed into lots of other molecules

Answer 90

Removal/addition of 5' phosphate - need to form phosphodiester so needs to be there Adding - T4 polynucleotide kinase Removing - Calf Intestinal Phosphatase (CIP) - this can prevent self-ligation

Answer 91

Fill in 5' overhang or remove 3' overhang - T4 DNA polymerase or DNA polymerase I Remove 5' overhang - Mung bean nuclease

Answer 92

1) electroporation = brief high voltage 2) chemical trasformation - heat shock - causes membrane changes that allow DNA uptake - not that efficient - use antibiotics to find cells with the DNA

Answer 93

DNA replication in a tube - DNA doubles each cycle can solve cloning problems - not having enough DNA and DNA might be mixed with other DNA

Answer 94

1) denaturing - DNA dissociates into single strands at high temperatures (95) 2) primer annealing - primers bind to complementary sequence (55-65) 3) primer extension - DNA polymerase synthesises new strands from 3' end of primer (68-72)

Answer 95

Taq - thermostable and has high processivity and extension rate (2-4 kb per minute) however - low accuracy and adds adenine overhang - no proofreading Pfu - better thermostability, slower extension rate (1kb per minutew), products are blunt ended, more accurate - proofreads

Answer 96

- has to be minimum 17 base pairs - be specific to template - come in pairs - appropriate melting temp

Answer 97

Template DNA polymerase Primers Deoxyribonucleoside triphosphate (dNTPs) Buffer Thermocycler

Answer 98

Temperature that primer dissociates from DNA - 60/64 degrees - determines what the annealing temp should be (5 less than Tm) To work out: add 4 degrees for G/C or 2 for A/T If too low - primers may bind to other parts of sequence If too high - may not bind at all

Answer 99

Yes! - there's no phosphate at the end - add T4 PNK, taq has overhang which needs to be removed - T4 DNA pol We can add sequences at 5' end which is complementary to restriction enzymes.

Answer 100

RNA is reverse transcribed into DNA (cDNA - copy/complementary) - this is then amplified with PCR

Answer 101

reverse transcriptase synthesises the first strand of cDNA: 1) poly(dT) primers bind poly(A) tail of mRNA 2) mRNA removed This makes first strand 1) synthesised by klenow fragment of DNA polymerase I 2) hairpin formed by RT acts as primer 3) the ssDNA loop can be digested by nuclease This makes the second Then do normal PCR

Answer 102

PCR but we can see how much DNA at any time PCR is exponential at the beginning - until things run out We can measure product by: - fluorescent dye - SYBR green fluoresces when binds to dsDNA - they are proportional - fluorescent probe - sequence specific - is bound to DNA until replaced by strand - then fluoresence - we can use several

Answer 103

Cycle threshold - where fluorescence exceeds background levels Difference in Ct = relative measure of what sample had most sample. we can do 2^-difference to find difference Lower CT = more template at start We can also minus test from sample for A , then B then minus A and B then do 2^-this number

Answer 104

Mini prep: Grow lots of bacteria - then break them open - plasmids are small - we will have purified recombinant DNA

Answer 105

We can use restriction enzymes to digest the DNA and see if we get the correct sized fragments We can also use PCR - see if we get the correct product We can use electrophoresis to separate fragments - add dye (midori green/ethidium bromide)

Answer 106

If we add restriction sites to the gene and vector, but have different enzymes that will effect them, we can see different patterns if the DNA is not present/inserted wrong - restriction fragment length polymorphism - been mutated or gotten longer due to disease - won't bind

Answer 107

Use 2 primers: 1 for DNA, 1 for vector If we get PCR products - the DNA and vector are correctly combined, no product - no DNA or incorrect DNA - allows us to identify diseases, cancer ect. & genetic fingerprinting

Answer 108

Allows us to see that there's no PCR mutations 1) DNA is amplified using one primer 2) ddNTP (dideoxynucleoside triphosphate) is added - stops sequencing as there's a missing OH (there's also normal dNTP) 3) If we add one type e.g. ddCTP then we know that's the last nucleoside 4) we can then use electrophoresis to see how big the fragments are 5) repeat with ddATP, ddGTP and ddTTP

Answer 109

One reaction has all ddNTP - all have different fluorescent labels - use capillary gel electrophoresis which is read by computer

Answer 110

- A taq DNA polymerase - dNTPs - ddNTPs - Buffer - Template DNA - Primer

Answer 111

RT-PCR - we can use different isoforms of mRNA and look at the different types. Hybridisation-based technique - can use RNA to make a probe (DNA and RNA together) To see RNA expression we can use northern blots and microarray To see localisation use fluorescence in situ hybridisation

Answer 112

5' to 3' so if something was complimentary - make sure we reverse

Answer 113

1) RNA is separated by electrophoresis (by size) 2) transfer RNA to membrane to allow for blotting 3) add a probe (radioactive) then wash membrane - unprobed will wash away This is good for comparing mutated genes ect. - can only use one molecule at a time

Answer 114

Oligonucleotides are attached to a spot on a chip - each oligonucleotide corresponds to different gene - we add cDNA (made from RNA) which is fluorescent - we can see where it binds more mRNA = more fluorescence They give relative levels e.g. equal cDNA = combination of colours

Answer 115

Same as northern blot - but probe is fluorescent and visualised by microscopy - we can see these within cells RNA localisation

Answer 116

Easy to visualise (fluorescent) or assay (enzyme) e.g. enzymatic reporter genes: luciferase and beta-galactosidase - we can use these to see how promotor is regulated and organised - we put it next to the promotor and see how much it is expressed in different conditions increased expression = increased promoter activity

Answer 117

Usually by primary antibodies - we also use secondary antibodies (usually have a conjugate) as they recognise the primary antibody e.g. primary = rabbit, secondary = antirabbit

Answer 118

1) proteins separated by electrophoresis 2) transfer to membrane 3) add antibody - binds to specific protein also add secondary antibody which usually has a conjugate which produces light

Answer 119

Add primary antibody to cell - and a secondary which has a conjugate which makes light - can see different molecules

Answer 120

Binds to target protein and allows us to track and see them. Green fluorescent protein was the first

Answer 121

Use cloning - fuse coding sequence of the protein to a fluorescent protein (GFP) - it will be transcribed and we can track the protein.

Answer 122

To see protein-protein: - pull down assay - immunoprecipitation - yeast two-hybrid Protein-DNA - chromatin immunoprecipitation

Answer 123

We analyse protein interaction in vitro Uses fusion proteins which have an affinity to a certain ligand, GST is commonly used which binds to glutathione 1) make recombinant DNA (made of protein and the other protein e.g. GST) by placing in e.coli 2) make cell lysate - break them open 3) bind GST to affinity ligand on a bead 4) wash away any unwanted stuff 5) We then have a purified sample then we can identify what can bind to protein X

Answer 124

Identical to pull down assay but uses antibodies of beads

Answer 125

Uses fusion proteins Transcription factors have separate domains for different functions DNA binding domain is fused to bait, transcription activation is fused to prey DNA BD binds to promotor If reporter gene is transcribed = bait and prey interact

Answer 126

Used to see protein interacting with DNA Use antibody to purify protein - assay DNA associated with protein 1) crosslink DNA to protein 2) chromatin fragmentation 3) immunoprecipitation 4) DNA purification - get rid of DNA 5) analyse DNA

Answer 127

cholesterol and sphingolipids form microdomains called rafts - slightly thicker

Answer 128

Phosphatidyl - ethanolamine - serine (negative) - choline Sphingo-myelin

Answer 129

Show us the tensile strength of membranes

Answer 130

Hyper - higher solute outside - cell shrinks Hypo - lower solute outside - cell bursts Isotonic - same

Answer 131

NO! - lipids composition is always different in each half

Answer 132

Blood groups - ABO Blood group is determined by structure of oligosaccharides attached to sphingomyelin Terminal sugar of oligosaccharides determines groups Also needed for coagulation (clot) - phosphatidylserine Needed for cell recognition - macrophage can identify phosphatidylserine or phosphatidylethanolamine

Answer 133

End of oligosaccharides O - none A - GalNAc = N-acetylgalactosamine B - Galactose

Answer 134

Established by ionic conc Combination of membrane potential and conc gradient

Answer 135

Voltage gates sodium channel - SCN9A

Answer 136

Glucose/sodium symporter at apical Sodium/potassium pump at basal Glucose carrier at basal

Answer 137

Hypothesis 1 - membrane formed around DNA Hypothesis2 - endosymbiosis (entered a eukaryotic cell)

Answer 138

Wrapped around histones - packaged in chromosomes Before cell division - called chromatin

Answer 139

In electron microscope: Heterochromatin - dense staining interphase DNA Euchromatin (genes more oftenly transcribed) - less dense staining interphase DNA Nucleolus - highly dense stained RNA We can also see chromosomes using FISH

Answer 140

Yes! Chromosomes occupy specific territories within the nucleus - can be identified by chromosomal painting

Answer 141

Yes! - they can move around in an ATP-dependant manner Nucleolus Speckles - pre-mRNA processing Cajal bodies - splicing PML bodies - storage

Answer 142

Not membrane bound - processes rRNA to produce ribsosomes - collection of rRNA genes, precursor rRNA, mature rRNA, rRNA processing enzymes, snoRNP's - also processes other types of RNA - tRNA & mRNA

Answer 143

Double membrane with pores - supported by a meshwork called the lamina The lamina ensures that the membrane is asymmetric - may also play a role in gene regulation

Answer 144

Genetic disorders of the lamina in nucleus e.g. Hutchinson-Gilford progeria syndrome

Answer 145

Seen by SEM - controls what enters in a size-dependant manner - particles with mw>50000 cannot enter by diffusion but can by active signal-dependant transport - signal for entry is by a specific peptide sequence - can open to 26nm in diameter

Answer 146

Equilibrates lipids It catalyses the flipping of phospholipids

Answer 147

Ensures membrane asymmetry Facilitates flipping to cytoplasmic monolayer

Answer 148

Negative charged phospholipids (phosphatidylserine) mainly in cytosolic leaflet

Answer 149

Translocase transfers to inner leaflet Scramblase abolishes asymmetry Equilibrium favours translocase

Answer 150

Endoplasmic reticulum (SER and RER) Form hollow tubes and flatterned sacs - chambers are called cisternae

Answer 151

1) Quality control 2) Synthesis 3) Storage 4) Detoxification

Answer 152

Phospholipid and cholesterol synthesis Steroid hormone synthesis Synthesis of storage of glycogen Calcium store

Answer 153

Zymogen granules have enzymes important for digestion Stimulation -> Ca release -> Vesicle fusion -> enzyme release

Answer 154

In the form of vesicles and tubules Vesicles bud off ER and are received by Golgi The vesicles are coated to help formation, either: - clathrin - COPI - COPII Coating needs to be discarded before it can fuse with the membrane

Answer 155

Through SNARE Two types: - v-SNAREs: found in vesicle membrane - t-SNARE: found in membrane of target In nerves: SNARE binds to a helical bundle with 3 components

Answer 156

- modifying and packaging proteins - renewing plasma membrane - delivery of material to other organelles

Answer 157

Bind at cis face - leave at trans face

Answer 158

From golgi to membrane - secretory pathway From golgi to lysosomes From golgi to secretory vesicles (storage)

Answer 159

Nutrients Antibodies Enzymes Signals Viruses Bacteria

Answer 160

Can be: - recycled - degraded - transcytosis Membrane is recycled

Answer 161

Small scale e.g. clathrin Macropinocytosis Phagocytosis

Answer 162

Uptake of large particles - bacteria/apoptotic cells Pathogens usually coated by antibodies - opsonization Can be frustrated - when target is too large and two macrophages are trying to engulf - ligand coated particle binds to phagocyte surface receptor, cell extends pseudopods to engulf particle

Answer 163

Cells form actin driven ruffles which can fuse to form macropinosomes Similar to phagocytosis Not selective Used by cancer cells to take up nutrients

Answer 164

Target binds to receptor in clathrin coated pit - enters as coated vesicle - becomes uncoated and fuses with endosome. Vesicles bud off endosome to return receptors to membrane

Answer 165

Consists of 3 heavy and 3 light chains - polymerise into lattice which forms the pits

Answer 166

Needed to pinch off clathrin coated vesicles

Answer 167

The gradient of pH

Answer 168

Intraluminal vesicles (found in the multivesicular body)

Answer 169

By dictyostelium

Answer 170

Define intracellular organelles - in the endocytic pathway

Answer 171

Hydrolases and lipases

Answer 172

We wash then with two buffers: TFB1 and TFB2 which contain rubidium and calcium - this induces permeabilisation We then transform by hear shock We then do antibiotic selection

Answer 173

Tube with water - negative control Tube with vector and ligase - controls for self-ligation Tube with vector and insert Tube with vector - positive control

Answer 174

It reduces self-ligation of the vector to itself

Answer 175

18-28 nucleotides in length GC composition should be 50-60% The melting temp should be 60-65% (4 x(G+C) + 2x(A+T))

Answer 176

Slowest in gel electro: open circular Linear Fastest in gel electro: closed circular(supercoiled) Small plasmids are usually supercoiled

Answer 177

To isolate recombinant DNA from E.coli Most commonly alkaline lysis - causes denaturing of DNA which reanneal after neutralisation - plasmids reanneal - genomic DNA is too big and gets tangled - easy to remove

Answer 178

Makes ATP and has a key role in apoptosis

Answer 179

1-2 micrometres long Double membrane Inner membrane highly folded - cristae - contains REDOX proteins Inner matrix has enzymes responsible for energy production, tRNAs, enzymes, DNA, ribosomes Outer membrane has porins allowing entry of molecules <5000 kDa and contains enzymes involved in mitochondrial lipid synthesis Intermembrane space has enzymes that use ATP to phosphorylate other nucleotides - H+ pumped in here

Answer 180

Circular double stranded - ~ 15-17 kbps Encodes for 37 genes Inherited from mother

Answer 181

In a dynamic flux between fission and fusion Fission - splits into 2 - 1 is good, 1 has debris so goes through mitophagy Fusion - 2 into 1 Mitophagy - digested by a lysosome by encasing it in an autophagosome

Answer 182

Proteins N-terminal signal sequence is recognised by proteins embedded in membrane called TOMs - translocators of the outer membrane The protein translocates through TOM then TIM23 (translocator of inner membrane) into the matrix Signal is then cleaved off

Answer 183

Bind interacting proteins to chain - chaperones Needs energy to dissociate The signal sequence is +ve so the H+ gradient drives it through the IM

Answer 184

Major protein is porin - beta barrels TOM can't insert them - SAM (sorting and assembly machinery) inserts them and folds the protein

Answer 185

1) TOM then TIM23 2) Protein completely enters matrix - signal sequence cleavage unmasks a 2nd signal causing insertion into OXA complex if multipass - snake through TOM as a loop - allows chaperones to bind to stop folding - guide to TIM22

Answer 186

Single membrane - no DNA or ribosomes Found in all eukaryotic cells - carry out oxidative reactions - have catalase and urate oxidase Remove hydrogen atoms and make hydrogen peroxide: RH2 + O2 --> R + H2O2 - peroxidases use the peroxide Used in alcohol and fatty acids metabolism

Answer 187

Most made in cytosol and inserted in the membrane of preexisting peroxisomes - by peroxins - proteins don't need to be unfolded Pex5 recognises signal sequences (ser-lys-leu) and accompanies cargo into peroxisome - ubiquilated then recycled back New peroxisomes arise from preexisting ones - fission

Answer 188

Outer mitochondrial membrane: - TOM (Translocator of Outer Membrane) - SAM (Sorting and Assembly Machinery) Inner mitochondrial membrane: - TIM23/22 (Translocator of inner membrane) - OXA (Cytochrome OXidase Activity)

Answer 189

Hollow tubes of alpha + beta tubulin Has 13 protofilaments Make cilia and flagella used to move sperm and respiratory fluids

Answer 190

Same in both just different lengths - flagella longer Has a axoneme MADE OF MICROTUBULES: - 9 doublets +2 microtubule assembly (9 doublets on the outside, 2 in the middle) - has radical spokes (holds outer and inner together) - has dynein arms (outer and inner of the 9 doublets) - motor protein which allows sliding - causes polarity as they touch one doublet but there isn't any on the other side of doublet - the rings look like they overlap - the full ring is complete (A) fibres - 13 protofilaments, the half ring is incomplete (B) fibres - 10 protofilaments

Answer 191

Inner - waveform Outer - power Cilia and flagella have different waveforms

Answer 192

Anchor doublets so don't slide past eachother - dynein will cause bending - not elongation

Answer 193

At beginning of axoneme 9 x 3 (triplet) microtubule array 0.2 micrometre (diameter) x 0.4 micrometres (height) There is no inner pair

Answer 194

Has a plus and minus end (polarity) - plus end will be polymerised, minus is degraded Globular with energy source in middle (ATP)

Answer 195

Microtubules have a bigger diameter (24nm), actin is 5-9nm Microtubules are hollow (actin solid) Microtubules are 50kDa, actin is 42kDa Microtubule has a/b heterodimer Microtubule has 450 amino acids, actin has 375 Microtubules have GTP (GTP in alpha, GDP in beta), actin has ATP (ADP in filament)

Answer 196

Monomer nucleating Monomer sequestering Capping Cross-linking Bundling Depolymerizing Membrane binding

Answer 197

Cilia/flagella - filament = microtubule, motor = dynein Cytoskeleton/muscle - filament = actin, motor = myosin

Answer 198

Coil of 2 alpha helices

Answer 199

Filopodium - small projections so cell can test environment Lamellipodium - meshwork of filaments - extends cell membrane Stress fibres - strong fibres which pull cell Cortical actin

Answer 200

Migration Tissue integrity and cell shape Proliferation Differentiation

Answer 201

Extracellular meshwork of proteins and hydrated macromolecules Different types: Basal lamina Collagen and Elastic fibres Proteoglycans and Glycoproteins

Answer 202

Fibrous proteins - collagens and elastin Adhesion proteins - fibronectin and laminin Hydrated macromolecules - glycosaminoglycans and proteoglycans

Answer 203

Glycine, proline, hydroxyproline repeats Triple helix - 3 alpha chains Produced by fibroblasts and epithelial cells 1.5 nm wide

Answer 204

Ehlers-danlos Syndrome Can pull skin as there is less collagen - less strength - bad in the heart

Answer 205

1) synthesis of pro-alpha chain 2) hydroxylation of some prolines - allows crosslinking 3) glycosylation of some hydroxyprolines 4) 3 pro-alpha chains assemble 5) procollagen triple helix formation 6) secretion 7) propeptides are cleaved 8) self-assembly into fibril 9) clusters of collagen fibrils form a collagen fibre

Answer 206

Energy, guidance, mechanical interaction with something outside, swimming/crawling, microtubules and microfilaments

Answer 207

Disaccharide chains - 70-200 units long Holds water Highly charged Proteoglycan - 95% sugar (80 saccharides) Glycoprotein - 60% sugar (15 saccharides)

Answer 208

Big protein with aggrecans attached - effective for holding water

Answer 209

An adhesion glycoprotein Self assembles It has binding sites for integrins - allows to adhere to cells

Answer 210

An adhesion glycoprotein Self associates Can bind to collagen and the cell

Answer 211

Bind matrix through divalent cations Removal of cations - cells detach Binds ECM to cells

Answer 212

Is a transmembrane receptor - connects to cytoskeleton and integrins Used for signalling Contains FAK, Paxillin, Talin and Vinculin

Answer 213

B1 integrin - embryonic lethal day 5 a5 integrin - embryonic lethal day 10 fibronectin - embryonic lethal day 9 talin - embryonic lethal day 6-8

Answer 214

Tropoelastin ----> elastin Need lysyl oxidase

Answer 215

Moves organisms or cells

Answer 216

ATP powers the stroke Calcium binds to troponin/tropomyosin so myosin can bind to actin

Answer 217

causes bleeding gums/nose bleeds

Answer 218

Have cadherins - links to another cadherin - binds in cell to actin cytoskeleton Calcium dependant Homophilic interaction - cadherin binds to cadherin

Answer 219

Adherens junctions Focal adhesion

Answer 220

Cadherin -> catenin -> actin -> myosin

Answer 221

Hemidesmosomes - attach cells to basal lamina between integrins and intermediate filaments Desmosomes - cell-cell junction between cadherins and intermediate filaments

Answer 222

Autoimmune disease where immune system produces antibodies which attack desmoglein (cadherin which hold keratinocytes in the epidermis) - causes blistering

Answer 223

Known as occluding junctions or zonulae occludens Found in epithelia Prevents fluid, ion and membrane flow, allows transcellular and paracellular transport, specialises membrane regions - apical (glycolipid and cholesterol), basal (phosphatidylcholine) Sealing strands of claudin and occludin

Answer 224

Is a connexon made of connexins (6 subunits can be hetero/homotypic) - form open channel 100-500nm long, 2-4nm gap between cells, 1.5 nm pore Allows cell-cell communication Regulates pH, Ca, membrane potential and cell signals In connective tissue, heart, neurones ect.

Answer 225

Slows down leukocytes when there is injury

Answer 226

So we can isolate our specific sequence Add primers - forward attaches to start of gene, reverse to end of our gene We can add sequences to the 5' end of our primers - restriction site sequences This allows for us to have sticky ends - no treatment needed

Answer 227

Extrinsic factors are needed - some other signals can override this and stop division There is a master governor making the major decision regarding cell fate = cell cycle clock in nucleus

Answer 228

M phase - mitosis: PPMAT and cytokinesis Gap 1 - cells increase in size, ribosomes and RNA are produced and the cell is prepared for cell division S phase - DNA replication Gap 2 - cell checks DNA and is preparing for division

Answer 229

G1 - checks for DNA damage and favourable environmental conditions S - check DNA for damage G2 - check for damaged or unduplicated DNA M - check for chromosome attachment

Answer 230

G1 - 10 hours S - 7.5 hours G2 - 3.5 hours M - 1 hour - 22 hours

Answer 231

Cell will test the environment from G1 to an hour or two before S - has until the R point (restriction) E.g. cell has serum and growth factors removed 80% into G1 - went back to G0, when removed one hour before - went to S/G2/M phase

Answer 232

Deregulation of the R-point

Answer 233

Genetic approach - requires cells that have a mutation in a potential cell cycle gene Biochemical approach - requires large amount of cells in same transition state

Answer 234

Yeast This is because: - rapid division rate - cell cycle control genes are highly conserved - can be group as haploids or diploids - easy to grow

Answer 235

We can grow diploid cells to maintain lethal mutation then study them haploid There are temp sensitive mutations - allow growth in permissive temps (Cdc genes)

Answer 236

Yes! We sucked out the cytoplasm from a cell in M phase and put it in a cell arrested in G2 - caused cell to go to M phase. Something catalysed this transition. It was called maturation promoting factor

Answer 237

A protein kinase-based machine (AA sequence of CDK - cyclase dependant kinase) - regulated by cyclins and Tyr phosphorylation

Answer 238

Selective extraction of kinase - incubate with protein substrate and ATP - electrophoresis of substrate and imaging

Answer 239

Cell cycle transition involves the irreversible destruction of cyclins Cyclins activate kinases

Answer 240

Cdk1 - mammals have multiple as well as multiple cyclins

Answer 241

Cyclin E - low levels in G1 until R point - rapid increase Cyclin A - levels increase as S phase Cyclin B - levels increase in M Collapse of cyclin levels - degration (ubiquitination dependant) LOOK AT GRAPHS - lecture 25, slides 28/29

Answer 242

G1 - Cyclin D - Cdk4, Cdk6 G1/S - Cyclin E - Cdk 2 S - Cyclin A - Cdk2 Cdk 1 M - Cyclin B - Cdk 1

Answer 243

We can use cell-free mitosis - can use a cytoplasm and remove it at different stages to study changes

Answer 244

Chromosome condensation Sister-chromatid resolution

Answer 245

Have the same genes arranged in the same order - one from mum, one from dad

Answer 246

Newly copied DNA strands joined by a centromere

Answer 247

M-Cdk - cyclin B Cdc25 phophatase removes an inhibitory cyclase from M-Cdk then activates a positive feedback loop - activated by S-Cdk - triggers assembly of mitotic spindle - chromosome condensation - breakdown of nuclear envelope - rearrangement of actin cytoskeleton and golgi Wee1 inhibits M-Cdk but S-Cdk inhibits it

Answer 248

Progression through metaphase and anaphase is driven by protein destruction - cyclin levels are zero The APC is a ubiquitin ligase activated by Cdc20 1) Targets S+M cyclins - destroyed which unactivates most CDKs and CDK targets are dephosphorylated APC/C is kept on in G1 - turned off as G1/S-CDK activated so cyclins can accumulate 2) Targets securin - protects protein linkages keeping sister chromatids together - destroyed so sister chromatids separate - anaphase

Answer 249

Loss of heterozygosity - genes need mutations on both alleles to cause phenotypic change - 2 hit hypothesis e.g. 2 sporadic or one familial and one sporadic Hemizygosity - loss of an allele

Answer 250

Chromosomes end in wrong daughter cell

Answer 251

Interpolar microtubules - overlap Kinetocore microtubules - attach to kinetochores (centromeres) - trial and error and the appropriate attachment is sensed by tension from chromatids Astral microtubules - contact cell cortex to position spindle Centrosome - centriole surrounded by pericentriolar matrix - nucleate microtubules

Answer 252

Bound to an inactive separase - no phosphorylation to it as cyclins are destroyed and Cdks are inactivated - these would inhibit it but the APC stops this - the APC/C destroys securin This activates separase - breaks sister chromatids apart

Answer 253

Chromosome in wrong cell - elimination by apoptosis. Sometimes the cell can eliminate a chromosome - sometimes it's normal, sometimes it will cause loss of heterozygosity

Answer 254

In G2/M - crossing over could occur - a chance that one cell gets both mitotic copies of a mutation.

Answer 255

DNA polymerase begins replication on a template strand but then jumps to a different chromosome then back to normal. This may pass a mutation to a different chromosome

Answer 256

Levels: Western blot Localisation: Immunofluorescence fusion proteins Interactions: Pulldown assay, immunoprecipitation, yeast two-hybrid, ChIP (DNA-protein) Methodology: Antibody, fusion protein

Answer 257

Levels: quantitative RT-PCR, northern blot, microarray, luciferase assay Localisation: FISH Methodology: PCR, hybridisation, reporter genes

Answer 258

Localisation: FISH Interactions: ChIP (DNA-protein) Methodology: PCR, hybridisation

Answer 259

Forms haploids - gametes One homologue for each chromosome is in the gamete 2 steps: meiosis I and II Ovary: oogonium --> primary oocyte --> secondary oocyte --> mature egg Testes: spermatogonium --> primary spermatocytes --> secondary spermatocytes --> spermatids

Answer 260

Crossing over and independant assortment

Answer 261

Centrioles and chromosomes are replicated (like mitosis) Maternal and paternal homologs pair up One complete chromosome (2 chromatids) pulled to separate poles Crossing over occurs

Answer 262

like mitosis just makes haploid cells

Answer 263

Pairing - facilitated by synaptonemal complex It aligns then for anaphase Allows genetic recombination between paternal and maternal DNA Can takes years to complete (mitosis is less that 30 minutes)

Answer 264

Chromosome homolog pairing - brought 400nm apart - recombination complex binds them together Axial core (binds chromatin via cohesion) are crossed liked by transverse filaments to form synaptonemal complex This aligns the two chromosomes and helps crossing over (recombination)

Answer 265

- both kinetichores attach to spindle pole - by protein complex which is removed after meiosis I - crossing over - cohesin is only removed from arms - keeps chromosomes together

Answer 266

At least one crossing over - no more than 4 Regulation makes sure there's at least one and inhibits others close by - crossover interference

Answer 267

i) abnormalities in chromosome number ii) chromosome structural rearrangements

Answer 268

Different numbers of chromosomes - non-dysfunction Monosomy - 1 copy of a chromosome - lethal Trisomy - 3 copies - usually lethal Polyploidy - extra sets e.g. triploid - lethal Can have an extra sex chromosome - normal lifespan One sex chromosome - X - normal but infertile, Y - not viable Caused by failed separation in Meiosis I/II or mitosis - either from sister chromatids or homologous chromosomes

Answer 269

Severe intellectual disability Low birth weight A small, abnormally shaped head A small jaw and mouth Clenched fists with overlapping fingers Congenital heart defects Various abnormalities of other organs Most die before 1 month - edward's syndrome

Answer 270

Undeveloped midface Malformed ears Wide-spaced eyes Microcephaly Congenital heart disease Usually die shortly after birth

Answer 271

Complete or partial absence of a second sex chromosome in females - poor growth - short - delayed puberty - congenital heart defects - skeletal abnormalities 1% survival rate as there is haplodeficiency (pseudoautosomal genes need to be expressed in both alleles) or imprinted genes on X

Answer 272

Usually in homologous recombination or DNA damage - represent 4.7% of 1st trimester abnormalities Can view them via spectral karyotyping (SKY)

Answer 273

Mostly through necrosis or apoptosis

Answer 274

Physical damage - trauma (cuts/burns) and extreme temperature Toxins - external (snake venom), internal (bacteria) Acute hypoxia/ischaemia - stroke

Answer 275

Physiological: - tissue size maintenance - development - removal of immune cells - hormone-dependant involution - inappropriate interactions e.g. anoikis Pathological: - DNA damage - viral infection 50 billion cells daily

Answer 276

Reversible: membrane collapses, organelle and cell swelling Irreversible: increased intracellular calcium, autolysis, cell bursting, makes an inflammatory response Causes blebbing - cells leak into extracellular fluid No ATP required

Answer 277

Shrinkage Nuclear breakdown Apoptotic bodies - budding Phagocytosis No inflammatory response Requires energy Controlled cell death Capsase is mediator

Answer 278

Cells in middle die - necrosis Cells at edge die - apoptosis This restricts spread of cell death

Answer 279

Metamorphosis: e.g. frog Thyroid hormone in blood causes apoptosis in tails Digit formation in mice cause by local signal proteins Neuronal connections are refined by the competition for survival factors - not enough factors = apoptosis

Answer 280

C-elegans - good model to study apoptosis Some Ced genes can recognise the apoptotic signal and some cause phagocytosis of the cells Many of these genes are conserved: EGL-1 - BH3-only proteins Ced 9 - Bcl 2 Ced 4 - APAF-1 Ced 3 - capsases Reduced Ced 3+4 - gives excess adult cells Reduced Ced 9 - massive cell death EGL-1 stop CED 9 CED 9 stops CED 4 CED 4 promotes CED 3 - then cell death

Answer 281

Causes apoptosis C = cysteine in active site asp = aspartic acid cleave target proteins Irreversible Over 10 Ced3 homologues

Answer 282

Initiator: activated by apoptosis signals and activate executioner capsases Executioner: cleave over 1000 proteins Amplifying proteolytic cascade: one initiator can activate multiple executioner

Answer 283

They can cause nuclear breakdown including the nuclear lamina They can prevent DNA repair by cleaving PARP They can cause cytoskeleton changes e.g. breaking down actin They cleave proteins that inhibit apoptosis, DNA repair, cell cycle and nuclear structure

Answer 284

Extrinsic Intrinsic

Answer 285

By tumour necrosis factor family are ligands for death receptors 6 receptors: death receptors which indirectly activate initiator capsases by DISC e.g. Fas ligand on killer lymphocyte --> capsase 8 (initiator), DISC is made = death-induced signalling complex

Answer 286

Triggered by: stress & development signals e.g. cytochrome c is released by mitochondria, Apaf1 is activated --> apoptosome is assembled --> caspase 9 is recruited (initiator) --> activates executioner capsases LOOK AT SUMMARY ON LECTURE 28, slide 22

Answer 287

Bcl2 family proteins: EGL-1 - BH3-only protein is pro Ced 9 - Bcl2 protein is anti There is a balance of each

Answer 288

A disease of aberrant cell proliferation and differentiation e.g. tumours have no layers compared to normal cells which are arranged in layers

Answer 289

No - some countries have a high risk for certain cancers and some have a lower risk There was two migrations of japanese people to hawaii 50 years apart - we can look at the cancer rates. Japanese - high stomach, low prostate/breast/prostate Caucasion - higher for all cancers Hawaiian Japanese - just below caucasion Shows that environment can dictate the cancers

Answer 290

Infection - a chicken with breast cancer had it's tumour grinded up and passed through a filter - then injected the filtrate into a healthy chicken - got a tumour - shows cancer can be caused by infection DNA contains a gene called Src (Oncogene) which controls pathways that control cell adhesion, proliferation and cell mobility - viruses make a hyperactive version of this tyrosine kinase gene which turns on all the pathways e.g. Nasopharyngeal cancer - epstein-barr virus Cervical - HPV Kasposi's - human herpesvirus 8

Answer 291

Aspergillus oryzae (Koji mold - rice, peanuts) - increases risk of liver cancer This is because when cytochrome P-450 modifies aflatoxin to aflatoxin-2,3-epoxide - this can bind to a guanine in DNA - mutated base

Answer 292

E.g. asbestos (naturally occurring silicate) - this can cause pleura mesothelioma

Answer 293

Mostly due to smoking - 33% Diet/obesity - 25% Viruses - 5% UV - 2% Genetic factors are also causing

Answer 294

Retinoblastoma Li-Fraumeni syndrome Wilm's tumour Gorlin's syndrome Breast cancer syndrome

Answer 295

A gene with the potential to cause cancer by transforming cellular behaviour - they are dominant e.g. SCR Mostly arise from genes involved in regulated proliferation - Proto oncogenes These come about from chromosomal rearrangement (hyperactive), gene amplification (protein made loads), regulatory mutation (protein made loads) or a deletion/point mutation (hyperactive protein)

Answer 296

Chromosomal changes cause cancer e.g. CML patients had different chromosomes: 22 and 9 due to translocation of some chromosome 9 to 22 and vice versa This is due to a fusion of genes ABL (positive regulator of cell growth) and BCR - ABL cannot turn itself off now FISH allows us to identify bits of DNA complimentary to what we add - we can see that chromosome 9 parts are in the wrong place

Answer 297

Most likely as viruses benefit greater from dividing cells

Answer 298

Ras - GTPase turns it off, exchange protein turns on When bound to GTP = active When bound to GDP = inactive When a growth factor binds to a TK receptor - phosphorylated Grb2 and Sos - Sos will activate Ras by removing GDP allowing Ras to add GTP - growth factor induced growth Glu61, Gly12 are critical for turning on and off - mutations make it go from a proto-oncogene to an oncogene

Answer 299

Causes cell growth Causes gene expression Causes cell morphology and movement Mutated Ras - cause tumours as can't turn off

Answer 300

Cell fusion - a normal cell and cancer cell fusion The cell put into mouse - normal healthy mouse There is something dominant in normal cells which will suppress the oncogene This implied the existence of tumour suppressor genes

Answer 301

Retinoblastoma Arises sporadically (in one eye) or familial (both eyes) When you plotted one eye vs both eyes - both eyes was a straight line showing only one thing needs to change (one hit meaning they had one inherited mutated gene and another happened randomly), one eye was a curve showing two things need to change (2 random mutations) This one/two hit hypothesis provides evidence for tumour suppression, that cancer requires loss of both wild types and the basis of inherited cancers

Answer 302

Retinoblastoma - Rb Li-Fraumeni syndrome - P53 Wilm's tumour - WT-1 Gorlin's syndrome - Ptc Breast cancer - BRCA-1 FAP - APC

Answer 303

Oncogenes are activating, gain of function and are dominant. Only one allele mutation is needed Tumour suppressor genes are inactivating, loss of function and recessive. Need two mutated alleles

Answer 304

More than one Incidence rate and age is a curve - showing it cannot be one mutation as it would be a straight line Higher number - nastier cancer

Answer 305

Chromosome painting - we can see translocations Cancer genome sequencing - we can see where things have moved compared to normal

Answer 306

DNA repair pathway defects Defects in correction mechanisms in DNA replication Defects in correction mechanisms for DNA segregation

Answer 307

Increased cell division and decreased apoptosis

Answer 308

E.g. DNA damage, telomere shortening, hypoxia Stable p53 will cause cell cycle arrest, senescence and apoptosis - cell cycle checkpoint p53 is mutated in almost all cancers - disrupt intrinsic apoptosis Mutated cell cycle checkpoints usually cause cancer e.g. Rb which operates in the restriction point

Answer 309

Polar head group with nonpolar hydrocarbon tail

Answer 310

The structure of the oligosaccharides attached to sphingomyelin O - no terminal sugar A - N-acetylgalactosamine (GalNAc) B - galactose

Answer 311

Mammalian membrane - Na+ Bacteria/yeast - H+

Answer 312

chaperone proteins

Answer 313

Intraluminal vesicles Late endosome: multivesicular body

Answer 314

Cilia: 2-10 x 0.25 micrometres Flagella: 100-200 x 0.25 micrometres

Answer 315

D type - Extracellular signals All - CDK inhibitors (CDI)

Answer 316

Coupled carrier ATP driven pump light driven pump