Biology 130l Exam Review

1. Identification of Some Macromolecules
1) What are 2 types of treatment controls, and explain each.

Positive control: gives positive result if experimental conditions were followed correctly
Negative control: gives negative result if experimental conditions were followed correctly

2) What are the most abundant elements in living material?

Carbon, Hydrogen, Oxygen, Nitrogen, Sulfur and Phosphorus

3) What are the 4 major biological macromolecules?

Carbohydrates (monosaccharides & polysaccharides), lipids, proteins, & nucleic acids.

4) What is the original colour of iodine, and what colour does it turn in presence of starch and glycogen?

Starch: yellow --> blue-black (amylase in starch reacts with iodine)
Glycogen: yellow --> reddish-brown (due to the multi-branched component)

5) How do plants and animals store simple sugar glucose?

In the form of polysaccharides. In plants, starch is the polysaccharide with glucose units linked by glycosidic bonds. Glycogen, in animals, is a larger polymer.

6) How do starch and glycogen differ?

Molecular weight, overall shape and degree of branching in the final polysaccharide structure. Amylose is unbranched and helical, where glucose units are joined by a(1-->4) linkages. Amylopectin has straight, branched sections from a(1-->6) linkages. Glycogen is similar to amylopectin, but more highly branched.

7) What makes sugar a reducing sugar?

The terminal aldehyde group makes it an aldose sugar, which reacts in Benedict's test to make glucose a reducing sugar. Blue solution will develop precipitate ranging from yellow, green, red or brown (positive).

8) What happens when benedict's solution mixes with solution with reducing sugar?

The blue cupric ions (Cu++) in benedict's solution get reduced to Cu+ by the terminal aldehyde group. The amount of Cu2O formed is proportional to the concentration of free aldehyde groups.
4Cu+ + 2OH +2e --> 2Cu2O + 2H+ +2e

9) What gives a false positive reaction?

Presence of other substances that could be oxidized (i.e. ketose sugar --> fructose)

10) What test is done for proteins? And how does the colour change occur?

Biuret test; The peptide bonds in the proteins with Cu++ ions and alkali give a violet peptide complex.

2. Isolation of Some Macromolecules

1) Why should the yeast be grinded with water and sand?

It will rupture the cell walls and cell membrane; the glucan (cellulose) in the cell walls, and glycogen, proteins and NA in cytoplasm will be released.

2) What is TCA used for?

Polysaccharides (starch and glycogen) are soluble in TCA. Proteins and NA aren't, so they'll remain in suspension.

3) What is centrifugation?

Supernatant is liquid above the pellet. The pellet is also precipitate. Controlling the speed and time of a centrifugal run will allow particles of different sizes/properties to be separated from the same suspension.
A fractionation technique where centrifugal force sediments suspended particles at the bottom of the tube.

4) What is in the supernatant, and what is in the pellets?

Supernatant: polysaccharides
Pellet: proteins and NA

5) What does NaCl do to the pellets?

Nucleic acids are soluble in strong NaCl, but protein isn't so it will remain in suspension.

6) What will boiling do to the protein? What will centrifugation do?

I will thicken (coagulate) the suspended protein molecules. Centrifugation will pack the proteins into a pellet, and nucleic acids will be the supernatant.

7) Why is ethanol added to supernatant?

NA are insoluble in ethanol, so it will precipitate out of the solution to form a white suspension.

8) After centrifugation, H2SO4 is added to the pellets. Why?

NA are soluble, so it will go into the solution.

9) What will boiling the acid do?

It's a hydrolyzing process, so it will break up NA into component nucleotide subunits, and then eventually into base and sugar and phosphoric acid subunits.

10) What's the difference between the boiled and unboiled NA solutions?

Boiled: Hydrolyzed NA
Unboiled: Unhydrolyzed NA

11) What will barium hydroxide do?

It carries out acid-base neutralization. A drop of each NA solution is placed on litmus paper. If unchanged, it's still acidic. So, barium hydroxide is added drop by drop. Titration is reached when litmus turns slightly blue--> slightly alkaline, and past a pH of 7. At the end of titration, barium sulfate will form as white precipitate.

12) Why is pancreatic enzyme added to protein?

It will suspend the pellet, hydrolyzing the protein into amino acid subunits. In living cells, hydrolysis is carried out by enzymes. Thymol is added to prevent bacterial growth.

13) What will phosphate buffer do to protein?

It will suspend the pellet, and it will be unhydrolyzed protein.

3. Characterization of Some Macromolecules

1) What is chromatography?

Technique that separates mixtures into their individual components. The solution is absorbed by the cellulose fibers on the paper. The degree of absorption depends on the structure; the one that sticks the most will be slowed down the most.

2) What is the stationary phase and mobile phase?
Stationary: the matrix (motionless substance such as cellulose)
Mobile: the solvent (influenced by solubility, molecular weight and polarity)
*If substance completely soluble --> moves fast, but no separation
*If insoluble --> no migration at all

3) When is chromatography terminated?
When the solvent has almost reached the opposite end of the matrix.
Rf = d travelled by substance/ d travelled by solvent (constant for any given substance in a particular solvent system and matrix)

4) The proteins have gone through chromatography. What does ninhydrin-acetone do?
Reacts with amino acids to turn purplish-pink colour.

5) What do you see after chromatography of NA?
Under U.V. lamp, compounds with nitrogenous bases absorb the U.V. light to appear as a dark spot.

6) What categories do alanine, histidine, aspartic acid, lysine and methionine fall? Adenine, cytosine and uracil?

Those that have highest Rf values are hydrolyzed protein/NA; they're non-polar with hydrophobic interactions (they have the greatest affinity to the mobile phase).

4. Spectroscopy

1) What is a spectrophotometer?

A white light source that is focused on a prism that separates white light into its distinct narrow portions of the spectrum-bands of radiant energy. It measures the entire visible spectrum.

2) What is the relationship between the concentration light-absorbing solute and their absorbance?

They're proportional, according to Beer's Law (linear).

3) How do you create an absorption spectrum?

Read the absorbance of substance at different wavelengths to determine the maximum absorption. Concentration curve is then made (% transmittance vs. concentration); Determine concentration of unknown. 4) Why are pigments deeply coloured?

Because it is unable to absorb wavelengths of that particular colour.

5) What are the major pigments?

Chlorophylls (a & b) and carotenoids (carotenes and xanthophylls).

6) Why are leaves of most plants green?

Chlorophyll molecules absorb all colours, except green; the green wavelengths are reflected back to us.

7) What regions in visible spectrum are most strongly absorbed by chlorophyll a & b?

a: 420 and 665nm b: 455 and 640nm

8) How did the chromatogram look?

Top to bottom: carotenoids (orange), xanthophyll (yellow), chlorophyll a (bluish-green), chlorophyll b (yellowish-green)
5. Enzymes

1) What are enzymes?
Biological catalysts and proteins.

2) What is their specificity determined by?

The number and order of their component amino acids and 3D configuration of protein.

3) Describe process of enzymes.

Substrate + Enzyme --> Substrate-enzyme complex --> product + enzyme

4) All enzyme-mediated reactions are reversible, BUT ....

direction of reaction depends on conditions, such as substrate & enzyme concentration, reaction time. 5) What is salivary amylase?

Digestive enzyme in saliva that acts on starch. It breaks off maltose from ends of starch chain and consumes water. To digest substrate, it requires hydrolysis (hydrolytic reaction).

6) What is phosphorylase?

Enzyme that acts on starch by removing glucose molecules, rupturing glucose-glucose bonds, and consuming phosphoric acid. (Phosphorylase -->Phosphorolysis). Phosphoric acid dissociates and energy released creates glucose-phosphate bond in glucose-1-phosphate.

7) What is the direction of reaction mediated by phosphorylase determined by?
Relative concentrations of reactants and products.

8) What is starch synthase? main enzyme that produces starch in an intact plant cell.

9) When will phosphorylase synthesize starch?
When enzyme attaches glucose molecule to starch primer molecule; makes it longer, giving a positive iodine test (forms blue colour-complex).

10) Why would a phosphorylase test be negative?

Lack of glucose-1-phosphate, no primer, inactive enzyme, +/- phosphorolysis

11) When would amylase be positive in benedict's test?
Mixed with starch suspension. (Just amylase, would give negative test)

6. Osmosis

1) What is osmosis?
The movement of water in and out of a cell, through the cell membrane.

2) What is diffusion?

Net movement is from high to low concentration. When equilibrium is reached molecules still move randomly, but no net movement (Brownian motion).

3) What is the function of cell membrane?

It is semi-permeable, and allows water to pass in both directions.

4) Describe concentrations at isotonic, hypertonic and hypotonic.

Isotonic: same concentration in cytoplasm; no net movement.
Hypertonic: Higher concentration of solute than cytoplasm (more water in cytoplasm); net movement is out of cytoplasm
Hypotonic: Lower concentration of solute than cytoplasm; net movement is into the cytoplasm.

5) What is dialysis tubing used for?

Cellophane resembles cell membrane; allows water to pass freely but not the solute molecules.

6) Explain what happened to solutions in water? Sucrose?

Water: Higher concentration lead to increased mass (water diffuses into bag)
Sucrose: Decreased weight (no water to diffuse into the bag)

7) What is lysis?

When too much water enters the cell, cell membrane will rupture, causing burst of animal cell. Bursting of red blood cell is haemolysis.

8) *Note diagrams of blood in each environment, and the Elodea sp.*
No solution: Vacuole filled with sap
In water: Turgor pressure
In sucrose: plasmolysis

9) What factors affect the hematocrit?

Bodily activity, anemia, and altitude.

10) Isotonic: valuole is filled with sap.
Hypertonic: Water leaves vacuole, causing it to shrink (plasmolyzed)
Hypotonic: cell wall prevents extra water from entering the vacuole (turgid).

7. Mitosis and meiosis

1) What is the purpose of mitosis?

Cell cycle in which parental nucleus divides into 2 new daughter nuclei, each identical to the parental nucleus. In eukaryotic cell, cytoplasmic division (cytokinesis) accompanies nuclear division (mitosis).

2) What is the major component of nuclei?

Chromosomes; each has a long, linear DNA molecule and has 1 centromere.

3) What is the centromere?

A specific DNA region where protein complex (kinetochore) is assembled to link chromosome to the mitotic spindle. Ends of chromosome has specific DNA sequences (telomeres).

4) What are the 3 major periods of interphase?

*they all look identical*
G1: cell grows, organelles duplicate, all chromosomes are unipartite
S: DNA synthesis (semi-conservative replication), chromosomes also duplicate, but # stays the same.
End of S phase, all chromosomes are bipartite (2 DNA molecules)
G2: Cell grows and prepares for mitosis, chromosomes are bipartite (2 sister chromatids -future chromosomes).

5) Prophase | Prometaphase | Metaphase | Anaphase | Telophase | -chromosome tightens coils (spaghetti-like)-threads get shorter and thicker | -Nuclear membrane and nucleoli will be gone -dark bodies-spindle formed and move to centre | -in a line-each centromere of chromosome attaches to separate spindle fiber | -centromeres separate-2 unipartite daughter chromosomes-sister chromosomes pulled to opposite poles | -uncoiling (loses rod-like appearance)-New nucleus looks like interphase nucleus |

6) Explain each zone of the onion root tip.

A: root cap (no mitosis)
B: Cell division (lots of mitosis)
C: Cell elongation (no significant mitosis)
D: Cell differentiation (no significant mitosis)

7) What is meiosis?

Formation of gametes (sex cells) from germ cell; forms nuclei with half the number of chromosomes of parental nucleus ("reduction division").

8) Difference between diploid and haploid. Homologous chromosomes?

Diploid: nucleus with 46 chromosomes
Haploid: nucleus with 23 chromosomes
Homologous chromosomes: same genes (46 chromosomes paired into 23 pairs), but versions of genes are different (alleles)

9) Interphase | Prophase | Metaphase | Anaphase | Telophase | -same as mitosis | -synapsis(HC pair up- tetrads)-4 chromatids of 2 HC exchange parts | -in a line-tetrad attaches to spindle fibre-centromeres don't separate | -1 bipartite chromosome is moved to each pole | -half # of chromosomes of parental nucleus |

Interphase 2 | Prophase 2 | Metaphase 2 | Anaphase 2 | Telophase 2 | -Only goes through G2 period | -chromosome tightens coils (spaghetti-like)-threads get shorter and thicker | -2 unipartite daughter chromosomes | -centromeres separate-2 unipartite daughter chromosomes-sister chromosomes pulled to opposite poles | -uncoiling (loses rod-like appearance)-New nucleus looks like interphase nucleus (haploid) |

10) Describe each stage: Uninucleate Stage | Binucleate stage | 4-Nucleate stage | -diploid megaspore mother cell-1 diploid nucleus in cytoplasm-premeiotic interphase of interphase-1 | -2 nuclear products of meiosis 1-interphase condition-no cytokinesis | -4 haploid nuclei of meiosis 2-interphase condition |

11) When do oocytes begin meiotic division?
After fertilization (when sperm enters the egg's cytoplasm)

12) Differences between plant and animal oogenesis?

Animal | Plant | -has polar bodies-cytokinesis occurs-requires fertilization for meiotic division | -no cytokinesis-can have more than 1 nuclei-ovules causes meiosis |

8. Agarose Gel Electrophoresis

1) What is electrophoresis?

Technique for separating and analyzing mixtures of charged molecules.

2) Why are nucleic acids negatively-charged?
Due to the phosphate group on the sugar-phosphate backbone.

3) What happens when the nucleic acids are placed in an electric field?

Negatively-charged molecules (anions) will migrate to the positive electrodes (anode)

4) Why electrophoresis called Agarose Gel Electrophoresis?
Agarose gel is the most commonly use matrix.

5) What does the speed and distance of migration of nucleic acid molecules depend on?
It depends on the gel pore size and size and conformation of nucleic acid molecules.

6) How is pore size determined?
By the concentration of dissolved agarose; High concentration makes it dense and forms a gel with smaller pore size.

7) How is the size of a nucleic acid expressed?
Expressed by the number of base pairs (for double stranded) or nucleotides (single stranded).
Linear and relaxed --> travels faster than --> Circular and relaxed
Circular and twisted --> Fastest!!!

8) What is the difference between high and low percentage agarose gels?
High % : separates small DNA molecules; gels solidify fast and do not set evenly (can easily crack)
Low % : for large molecules; gels are very soft and break easily

9) What is ethidium bromide used for? What happens when it is exposed to UV radiation?
It is used to stain the gel to visualize the nucleic acids. It emits in the red-orange region of visible spectrum (makes nucleic acid visible).

10) Why is it more difficult to find single stranded nucleic acids?
The bases are not stacked, so affinity of ethidium bromide is very low.

11) Why must the gel be submerged in running buffer?
- allows current to flow through the gel
- prevents damage of molecules by controlling pH -controls ionization state

12) What is the role of dyes?
Monitors the progress of the gel; they're negatively charged, so they move in the same direction as nucleic acids.

13) Why is glycerol or sucrose used?
Provides a density to the nucleic acids so that it doesn't diffuse out of the gel.

14) What is a DNA molecular weight marker?
It is a set of DNA fragments of known molecular sizes.

17) Why are there twice as many bubbles at one pole?
More bubbles at the black pole cathode (close to wells) than red pole anode (end of gel); bubbles are hydrogen (cathode) and oxygen (anode) resulting from electrolysis of water. DNA migrates to the positive electrode (anode).