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BIOC1009 CH > Practical Stuff > Flashcards

Practical Stuff Flashcards

1
Q

Which organism has the most base pairs of DNA?

A

Frog - 2.8 x 10^13

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2
Q

Roughly how many base pairs are found in a human gene?

A

10^4 bases

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3
Q

How is animal DNA arranged?

A

DNA arranged in chromosomes in nucleus

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4
Q

What occurs when bacterial DNA is packaged for use in a lab?

A

Two different conformations of DNA arise, one supercoiled (helix) one coiled (circular)

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5
Q

How will a supercoiled and coiled DNA vary on an electrophoresis trace?

A

Supercoiled is more compact so will travel further than the equivalent coiled DNA

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6
Q

What is used to cut DNA into smaller pieces?

A

Restriction endonucleases, made naturally by bacteria

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7
Q

What are restriction endonucleases used for by bacteria?

A

Used to protect from pathogenic invasion (e.g. virus), breaks down DNA

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8
Q

How does a bacterial cell protect it’s own DNA from breakdown by restriction endonucleases?

A

Methylates the target sequence so the RE cannot bind

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9
Q

What is an important feature of all restriction sites?

A

They are palindromic

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10
Q

What does EcoR1 cut between?

A

G/AATTC and CTTAA/G

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11
Q

What does Taq1 cut between?

A

T/CGA and AGC/T

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12
Q

How would you work out the probability of a specific sequence n bases long occurring?

A

1/(4^n) e.g 6 bases = 1/(4^6)

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13
Q

Are shorter or longer sequences more likely to occur in DNA?

A

Shorter sequences, higher probability, you fucking dick

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14
Q

After DNA is cut with restriction enzymes, how are they separated?

A

Use agarose gel electrophoresis and compare to fragments of known size

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15
Q

Which part of the DNA chain has a negative charge?

A

The phosphate in the DNA phosphate backbone (at pH of above 1)

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16
Q

What type of molecule is agarose?

A

A polysaccharide, which forms open gels

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17
Q

Which way will DNA strands travel through electrophoresis plate?

A

Travel from anode to cathode

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18
Q

What is Ethidium Bromide used for in electrophoresis?

A

Used to visualise DNA, intercalates between bases and fluoresces at 300nm (UV light)

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19
Q

Why does an uncut plasmid and a plasmid cut at one point run at different positions on the gel?

A

Even though they have the same no. of bases, the uncut plasmid would be more compact so travel further along gel

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20
Q

When drawing a calibration curve what should you plot?

A

log10 number of base pairs (x) against distance migrated (y)

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21
Q

Do animals store amino acids?

A

No you fucking cretin, excess AA are deaminated, carbon skeletons used as fuel

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22
Q

What are amino acids interconverted between to remove nitrogen?

A

Interconverted between amino acids and ketoacids (oxo acids)

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23
Q

What enzyme moves amino groups between alpha-amino acids and 2-oxoacids?

A

Transaminases/Aminotransferases, transfer from an alpha-amino acid to an alpha-keto acid (2-oxoacid)

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24
Q

What catalyses the oxidative deamination of glutamate?

A

Glutamate Dehydrogenase

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25
Q

What is another word for transaminases?

A

Aminotransferases

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26
Q

What is the cofactor for transaminases?

A

Pyridoxal Phosphate (derived from Vitamin D)

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27
Q

What is the Keq for transaminases?

A

~1

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28
Q

Pyruvate (2-oxoacid) + Glutamate —> ?

A

Alanine + 2-Oxoglutarate

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29
Q

What coenzyme does glutamate dehydrogenase use?

A

NAD+ and NADP+

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30
Q

How was the heart extract prepared?

A

Heart was minced, homogenised in ice-cold buffer, centrifuged at 20,000g for 20mins, supernatant dialysed exhaustively against ice cold buffer

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31
Q

What colour are the 2,4 dinitrophenyl-hydrazones of oxoacids?

A

Yellow

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32
Q

How are amino acids detected on TLC?

A

Spraying with ninhydrin, appear purple

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33
Q

What is the average size of a eukaryotic cell?

A

1-2micrometers

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34
Q

How do you convert eyepiece units into regular units?

A

Use eyepiece graticule scale and calibration slide, match up one end of the eyepiece graticule/calibration slide and find how long it is

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35
Q

How is movement generated by cells?

A

Using motor proteins (kinesin, myosin) to convert chemical ATP energy into kinetic energy

36
Q

What type of filament does myosin travel along?

A

Actin filaments

37
Q

What type of filament does kinesin/dynein travel along?

A

microtubules

38
Q

Which protozoa travels faster, paramecium or amoeba?

A

Paramecium travels faster

39
Q

What contains a pseudopodia (false foot)?

A

Amoeba

40
Q

How do you determine the optimum pH of an enzyme?

A

Vary pH and measure initial rate of reaction of enzyme

41
Q

What is it important to do when investigating effect of enzyme concentration on reaction rate?

A

Important to use a shot incubation time to ensure [Substrate] is not affecting results e.g, initial rate

42
Q

What do you plot on a line weaver burke?

A

1/v (Y) against 1/[S] (x) (v=reaction rate, [S]=substrate concentration)

43
Q

What is the equation for V?

A

V = Vmax.[S]/(Km+[S])

44
Q

What is the equation for Kcat?

A

Kcat = Vmax/[E0], turnover number = number of substrate molecules each enzyme converts per unit time

45
Q

What points occur at the Y and X intercept?

A

X=0=1/Vmax Y=0=-1/Km

46
Q

What is Km?

A

Michaelas constant, substrate concentration when reaction rate is half of Vmax, measure of how strongly something binds, small Km = strong binding

47
Q

What is a competitive inhibitor?

A

Inhibitor binds reversibly to active site, enzyme can still process substrate (Vmax same), but cannot bind S (Km changes), so gradient increase, rotating around 1/Vmax point (X=0)

48
Q

What is a non-competitive inhibitor?

A

Inhibitor binds to a remote site, affects activity not ability to bind, so Vmax changes, Km same, line rotates around Y=0 (-1/Km)

49
Q

What is an irreversible inhibitor?

A

Binds to active site and shuts down activity, so [E] decreases

50
Q

Where is the electron transport chain?

A

On the inner mitochondrial membrane

51
Q

What is oxidative phosphorylation?

A

Process in mitochondria by which ADP is phosphorylated to ATP

52
Q

Where do the electrons arise from to reduce water?

A

NADH or FADH2 provide the electron flow in the ETC

53
Q

What happens if there is no H+?

A

No proton gradient, ATP cannot be synthesised by rotation of ATP synthase

54
Q

What is said of phosphorylation of ADP and electron transport?

A

They are coupled

55
Q

What does complex I do to what coenzyme?

A

Reoxidises NADH to NAD+

56
Q

What complexes are also hydrogen pumps?

A

Complex I, III, IV (1, 3, 4)

57
Q

What does complex II do to what coenzyme?

A

Reoxidises FADH2 to FAD+

58
Q

Does only FADH2 have to be used by coenzyme II?

A

No, FAD containing enzymes can oxidise substrates such as succinate (bound FAD->FADH2)

59
Q

What else can complex II oxidise?

A

Succinate

60
Q

Which carrier carries electrons from complex I/II to III?

A

Coenzyme Q (Ubiquinone)

61
Q

What is the mobile electron carrier that carries electrons between complex III and IV?

A

Cytochrome C

62
Q

What is the final electron acceptor?

A

Oxygen

63
Q

How many ATPs are generated by one NADH/FADH2

A

2.5 or 1.5

64
Q

What ion is present at cytochrome c’s core?

A

Fe2+/Fe3+

65
Q

What two forms does ubiquinone cycle between?

A

CoQ and CoQH2

66
Q

Is Coenzyme Q membrane soluble?

A

Yes

67
Q

Where is cytochrome c present on the membrane?

A

Membrane associated, not soluble so travels up and down inter membrane space membrane

68
Q

Which complex is present only in the membrane and matrix side?

A

Complex II - it is not a proton pump

69
Q

What is used to measure the rate of ATP production?

A

Oxygen electrode, isolated mitochondria given oxidisable substrate and known amount of ADP

70
Q

What is the overall equation of the oxygen electrode?

A

O2 + 4Ag+ + 4Cl- + 4H+ —> 4AgCl + 2H2O

71
Q

What are the conditions of the oxygen electrode?

A

Platinum cathode, silver-silver chloride anode, bathed in saturated KCl, separated by PTFE membrane (known as the Clarke electrode)

72
Q

What happens in state 2 respiration?

A

When mitochondria have substrate but no ADP to phosphorylate

73
Q

What happens in stage 3 respiration?

A

ADP and substrate present (Added to state 2), initial rate of oxygen use is proportional to amount of ADP added

74
Q

What happens in state 4 respiration?

A

ADP exhausted, rate of oxygen consumption drops to 0 (if you add more ADP the return to state 3)

75
Q

Define uncoupler and give examples

A

Compound which can dissolve in the membrane and carry hydrogen ions back across the membrane, dinitrophenol

76
Q

What complex is malate oxidised through?

A

Complex I, NADH pathway, using NAD+ dependant enzyme malate dehydrogenase

77
Q

What complex is succinate oxidised through?

A

Complex II, FADH2 pathway, using Flavoprotein succinate dehydrogenase

78
Q

How do we calculate P:O ratio?

A

On % oxygen against time trace: rule straight lines across state 2 and 4, then rule down state 3 line, measure height of whole table, measure height between state2/3 and state 3/4 crossing over points, (height of state/height of whole table)x oxygen at 100%, then: moles of ADPadded/moles of oxygen used

79
Q

Why is dinitrophenol proved to be an uncoupler?

A

O2 uptake increases considerably, P:O ratio decreased, this is because ADP cannot be phosphorylated as no proton gradient but ETC still carries on so O2 still produced

80
Q

What are the effects of amytal?

A

oxygen uptake ceases using malate (w/ or w/out dinitrophenol), denatures complex I, but complex II, III and IV are fine (succinate)

81
Q

What are the effects of antimycin A?

A

Affects complex III and IV - so no oxygen uptake w/ malate or succinate

82
Q

What are the effects of oligomycin?

A

Affects ATP synthase, oxygen consumption when DNP present

83
Q

What are the effects of potassium cyanide?

A

Affects complex III and IV - so no oxygen uptake with malate or succinate

84
Q

What are the effects of rotenone?

A

Affects complex I, but not complex II

85
Q

What is the role of 2,4 Dinitrophenylhydrazine solution in practical B?

A

Used to label oxyacids yellow and stop reaction

BIOC1009 CH (1 decks)

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