Quiz

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• 5 points

• 10 minutes (starting on the hour)

• #2 pencils

1. DNA was identified as the genetic material in experiments done byA. Mendel.B. Beadle and Tatum.C. Avery, MacLeod, and McCarty.D. Watson and Crick.

2. During formation of haploid eggs and sperm, one member of each chromosome pair is separated toeach cell in a type of cell division calledA. mitosis.B. meiosis.C. cytokinesis.D. gametogenesis.

3. When thymidine and adenosine interact within the DNA molecule they formA. two hydrogen bonds.B. three hydrogen bonds.C. nucleotide triphosphates.D. sphodiester bonds.

4. An experiment in which cells were grown in 15N after growth in 14N and their DNA then separatedby density gradient centrifugation demonstrated thatA. DNA is replicated by DNA polymerase.B. DNA replication is conservative.C. DNA replication is semiconservative.D. A forms double helices by hydrogen bonding between base pairs.

5. In the base pairing within a DNA double helix,A. purines pair with purines.B. A pairs with U and G pairs with C.C. A pairs with G and C pairs with T.D. A pairs with T and G pairs with C.

6. DNA contains the base uracil whereas RNA contains the base thymine.A. TrueB. False

7. RNA molecules that serve as templates for protein synthesis are calledA. transfer RNAs.B. messenger RNAs.C. ribosomal RNAs.D. cytoplasmic RNAs.

8. Which of the following deletions will not change the reading frame of a gene?A. One nucleotideB. Two nucleotidesC. Three nucleotidesD. Four nucleotides

9. RNA is copied from DNA by a process called __________.A. transformationB. translationC. replicationD. transcriptionE. transfection

10. A codon is aA. region of DNA coding for one protein.B. sequence of three nucleotides on a tRNA that binds to an mRNA.C. sequence of three nucleotides on an mRNA that binds to specific tRNAs.D. sequence of three nucleotides on the coding strand of DNA.

11. Restriction nucleases are enzymes thatA. act only in a single species of bacteria.B. act only on the ends of DNA strands.C. cleave DNA only at specific sequences.D. cleave only nuclear DNA.

Energy Metabolism

• Metabolism (meta=after; Bol=to throw)– Greek : Metabole = change)

• Catabolism (kata=down; Bol=to throw)

• Anabolism (ana=upward; Bol=to throw)

Thermodynamics• First Law: Energy cannot be created or destroyed.• Second Law:

– Heat will flow only from hot to cold– Entropy of a closed system always increases – The second law, in its most general form, states that the world acts

spontaneously to minimize potentials

– All reactions proceed in an “energetically favorable” direction until they reach equilibrium

• Intrinsic properties of reactants and products

• Relative concentrations of reactants and products

• Temperature

Gibbs Free Energy

• G (Gibbs free energy) H(heat)+S(entropy)

• ∆G=∆H-T∆S (releasing heat or increasing entropy makes ∆G negative)

• Change(∆) in (Gibbs)free energy= ∆G = ∆Gº +RTlnK

• Convention– If ∆G is negative A and B continues to get made into C and D

– If ∆G is positive C and D continues to get made into A and B

– Therefore if nothing is happening any more (ie equilibrium is reached), then ∆G is ZERO

– When ∆G =0 then ∆Gº=-RTlnK

• The standard free energy ∆Gº is different that the free energy ∆G

• The standard free energy at 37ºC in water is ∆Gº’

A+B C+D

Reaction Equilibrium

K =[A][B][C][D]

Thought Experiment:Mix 100 As with 100 Bs

A+B C+D50 50 50 50

A+B C+D99 199 1

A+B C+D1 99 991

No reaction

Complete Reaction

Partial Reaction

K =0.0001

K =10000

K =1

-15

-10

-5

0

5

10

15

10-9 10-7 10-5 10-3 10-1 101 103 105 107 109

K = Ratio products to reactants ([C][D]/[A][B])

Relationship of Equilibrium Position and Standard Free Energy at 37ºC

∆Gº= -RTlnK ∆Gº'= -0.62kcal/mol lnK

K=1/1

∆Gº =0

K=10000/1

K=1/10000

∆Gº = - 6

∆Gº =+ 6

Which way do reactions go?Two paradoxes

• Reactions that break bonds are generally thermodynamically favorable (catabolism)– They release heat– They increase entropy

• Reactions that make bonds are generally thermodynamically unfavorable(anabolism)– they create more order, therefore lower entropy

Why doesn’t everything break down?Resolution to the catabolism paradox

• Thermodynamics vs. Kinetics

• Activation Energy

Why does anything build up?Resolution to anabolism paradox

• Coupling of reactions

A-P-P-P A-P-P

∆Gº’=-7.3kcal/mol

Glucose Glucose-6-P

∆Gº’=+3.3kcal/mol

Oxidation /ReductionA. Passing H- around

NAD+

Reduced Oxidized

Oxidized Reduced NAD-H

H+

2e-

CS1 O

H

H

H

CS2 O

H

H

HCS2 O

H

CS1 O

H

OxidizedReduced

Carbon Oxidation

Oxidation /ReductionB. Reducing Oxygen

NAD+

Reduced Oxidized

Oxidized Reduced NAD-H

H+

2e-

OH H O O

OxidizedReduced

C OO

+3ATP

CS1 O

O

H

Metabolism• Catabolism

– Breaks bonds to yield energy in ATP currency– Results in smaller carbon skeletons

-Cells prefer to use glucose for energy generation (catabolite repression)

• Anabolism– Uses ATP to make bonds and increase the size of

carbon skeletons

Where does the glucose come from?

• Sun emits photons of light• Photons excite chlorophyll• Excited chlorophyll converts water to

oxygen, protons, and electrons• Electrons are coupled to ATP generation

and NADP+ reduction• ATP and NADPH are used to generate

glucose (Calvin cycle Fig. 2.39) • Glucose is eaten by animals

Light reactions

Dark reactions

Where did the glucose come from?

• Sun emits photons of light• Reducing atmosphere and sparks created

amino acids• Amino acids were assembled into

glucose?

BiosynthesisCarbohydrates, Lipids

• Gluconeogenesis– lactate, amino acids, glycerol to pyruvate– pyruvate to glucose

• Polysacharide synthesis (Fig.2.40)– glucose to UDP-Glucose to chain

• Lipid synthesis– Pyruvate to Acetyl-CoA to chain

BiosynthesisAmino acids

BiosynthesisNucleotides

Catalytic Mechanisms

Active Sites

Feedback Regulation

• Thermostat• Toilet

Allosteric Control• Allo (other ,different); Stere (solid, three dimensional)• Non-covalent: O2 in hemoglobin, metabolites in feedback control• Covalent: Phosphorylation, methylation,acetylation, etc..

Genetics• Mendelian

– independent segregation of traits; 1865– Traits determined by pairs of inherited factors (alleles)

• Chromosomes– Exist as pairs

• Linkage– dependent segregation of traits; early 1900s

• Incomplete Linkage – Recombination during Meiosis

• One gene-one enzyme– multiple mutations in one gene– George Beadle; Edward Tatum; 1941

Discovery of the Genetic Substance• Chromosomes have both DNA and Protein• Activation of Inactive Pneumococcus

– Oswald Avery; ColinMcleod; Maclyn McCarty; 1944

• Ratios of A:T and G:C are 1– Erwin Chargaff

• DNA is helical– Maurice Wilkins; Rosalind Franklin; 1952

• Model Building– James Watson; Francis Crick; 1953

• Semiconservative Replication– Mathew Meselson; Frank Stahl; 1958

Readout of DNA• Colinearity of genes and proteins

– Charles Yanovsky– tryptophan synthetase

• Discovery of mRNA– Sidney Brenner, Francois Jacob; Mathew Meselson– E. Coli; T4 phage

• Genetic Code; triplet code– In vitro translation– tRNA adapters– Mathew Meselson; Frank Stahl; 1958

Recombinant DNA Technology• Restriction Enzymes

– Restriction Maps

• Gel Electrophoresis• Vectors• Libraries• cDNA vs Genomic• Sequencing• PCR• Southern; Northern• Antibodies• Western Blots• Immunoprecipitations

Recombinant DNA Technology• Reverse Genetics

– Use yeast to illustrate

• Gene Transfer– Selectable markers

• Controlled mutagenesis

Molecular Biology Methods