Exam 1: Recombinant DNA, Amino Acids, and Proteins

Question 1

Recombinant DNA refers to:

Answer 1

laboratory techniques to manipulate DNA, and includes topics such as cloning vectors, cDNAs, protein expression vectors, and PCR (polymerase chain reaction).

Question 2

1. Cloning is the:
2. Cloning vectors are:
3. They include:

Answer 2

1. production of an exact copy of a DNA segment.
2. DNA molecules into which “foreign” DNA is inserted for cloning.
3. plasmids, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs).

Question 3

1. What is cDNA?
2. How is the first DNA strand of cDNA synthesized?
3. How is the second DNA strand of cDNA synthesized?

Answer 3

1. cDNAs are DNA copies of mRNA molecules.
2. Reverse transcriptase, which copies RNA into DNA, is used to synthesize the first DNA strand.
3. After the mRNA strand is degraded by ribonuclease treatment, the second strand is synthesized with DNA polymerase.

Question 4

1. What are protein expression vectors used for?
2. How are foreign proteins expressed in E. coli?

Answer 4

1. Protein expression vectors are used to synthesize proteins, including human proteins, in E. coli bacteria and other host cells.
2. To express a foreign protein in E. coli, a cDNA molecule representing the gene of interest is inserted next to a promoter and a ribosome- binding site in the vector.

Question 5

1. What is PCR, and what does it involve?

Answer 5

1. The polymerase chain reaction (PCR) is an in vitro technique to greatly amplify the amount of a DNA segment.
2. It involves multiple cycles of heating and cooling a reaction mixture that includes the DNA template, oligonucleotide primers, deoxynucleotides, and a heat-stable DNA polymerase.

Question 6

are useful in recombinant DNA experiments because they cut DNA at specific sequences typically nucleotides in length. An example is the restriction enzyme EcoRI, which recognizes the sequence GAATTC.

Answer 6

• Restriction endonucleases
• 4-8

Question 7

Blotting and hybridization methods allow specific DNA fragments ( blotting), RNA molecules ( blotting), or proteins ( blotting) to be detected in mixtures of these molecules.

Answer 7

1. Southern
2. Northern
3. Western

Question 8

In , functional versions of defective genes are introduced into humans by virus-mediated gene delivery or other techniques to correct damage caused by gene mutations.

Answer 8

human gene therapy

Question 9

Amino acids consist of:

1. a carbon atom (the α-carbon) covalently linked to
   
   1. 2. 3. 4. • At physiological pH, the amino group is and the carboxyl group is .

Answer 9

1. an amino group (-NH2),
2. a carboxyl group (-COOH),
3. a variable side chain (R-group),
4. and a hydrogen (-H).

• protonated (-NH3+)
• unprotonated (-COO-)

Question 10

The nonpolar or hydrophobic amino acids are:

1.

2.

3.

4.

5.

6.

7.

8.

Answer 10

Hydrophobic: A Very Likable Isabelle Planted My Purple Tree

1. alanine (Ala, A) -alkyl chain R groups
2. valine (Val, V), “ “
3. leucine (Leu, L), “ “
4. isoleucine (Ile, I), “ “
5. proline (Pro, P) - unusual cyclic structure
6. methionine (Met, M) - sulfur-containing
7. phenylalanine (Phe, F) - aromatic
8. tryptophan (Trp, W). “ “

Question 11

The polar amino acids are:

1.

2.

3.

4.

5.

6.

7.

8.

Answer 11

Polar: Girls Say That They Care About Guys.

1. glycine (Gly, G) -simplest R group (H)
2. serine (Ser, S) -hydroxyl in R group
3. threonine (Thr, T), “ “
4. tyrosine (Tyr, Y), “ “
5. cysteine (Cys, C) -sulfur-containing
6. asparagine (Asn, N) -amide in R group
7. glutamine (Gln, Q) “ “

Question 12

Acidic Amino Acids (which have a net negative charge at pH 7.0):

1. 2.

Answer 12

Acidic: Awesome Architects Gossiped Angrily.

1. aspartic acid (Asp, D) - carboxyl in R group
2. glutamic acid (Glu, E) “ “

Question 13

The basic amino acids are:

1.

2.

Answer 13

Basic: Los Angeles Hat

1. lysine (Lys, K) - positively-charged N group
2. arginine (Arg, R). “ “
3. Histidine (His, H) “ “

Question 14

Amino acids have two possible configurations, D and L, that are mirror image isomers or enantiomers. Amino acids found in proteins are all acids.

Answer 14

L-amino

Question 15

1. which link amino acids together, are formed by the reaction of the group of one amino acid and the group of another
2. The atoms on both sides of the C—N peptide bond are in .

Answer 15

1. Peptide bonds, carboxyl group (-COO-), amino group (-NH3+)
2. the same plane.

Question 16

Proteins are typically several hundred amino acids in length, ranging from about .

Answer 16

100 to over 1000

Question 17

The polypeptide chains are folded into precise three-dimensional structures by . Disulfide bonds between can also be important.

Answer 17

• noncovalent interactions (hydrophobic, hydrogen bonding, ionic)
• cysteine residues

Question 18

are proteins that contain, in addition to the polypeptide chain, components that include carbohydrate, lipid, nucleic acid, phosphate groups, metal ions, heme, or flavin.

Answer 18

Conjugated proteins

Question 19

Secondary structure is short-range structure along a polypeptide chain. It is formed by .

Answer 19

hydrogen bonding between amino acid residues.

Question 20

There are two types of secondary structures: α-helix and β- strand.

Describe each formation.

Answer 20

1. The α-helix is a spiral structure formed by a single polypeptide chain where each C=O of the backbone is H-bonded to the N-H group four amino acids along the chain.
2. In β-strand or β-sheet structures, the H-bonds are between strands rather than within a strand. The strands can be parallel or antiparallel, and can form extensive β-pleated sheet structures. The β-turn is a sharp bend where a polypeptide chain folds back upon itself.

Question 21

Tertiary structure is:

Answer 21

the folding of a polypeptide chain in three-dimensions. It is due to the 3-D arrangement of segments of α-helix and β-strand, as well as linking segments.

Question 22

Examples of Fibrous proteins:

Answer 22

1. α-keratin, found in hair and fingernails, is an example that is formed by the coiling of α-helices.
2. Fibroin in silk fibers and β-keratin in bird feathers are composed of stacked antiparallel β-sheets.
3. Collagen is a principal constituent of connective tissue and consists of a special collagen helix with three intertwined polypeptide chains.

Question 23

How is a peptide bond formed between ?

Answer 23

A peptide bond is formed by the reaction of the carboxyl group (COO-) of one amino acid and the amino group (NH3+) of another.

The reaction of the two amino acids results in the loss of a water molecule.

This leads to formation of the peptide bond or amide bond: CO—NH.

Question 24

Regulate the ability of other proteins to carry out their physiological functions. Examples: insulin–the hormone regulating glucose metabolism in animals; transcription factors that regulate gene expression

Answer 24

Regulatory Proteins

Question 25

hemoglobin–transports oxygen from the lungs to the tissues; membrane transport proteins– transport essential nutrients and other molecules across cell membranes

Answer 25

Transport Proteins

Question 26

casein–the most abundant protein of milk and the major nitrogen source for infants; zeins–corn proteins that serve as a nitrogen source for developing seedlings

Answer 26

Storage Proteins

Question 27

Produce cell movement in processes such as cell division, muscle contraction, and cell motility. Examples: actin and myosin–muscle proteins; tubulin–major component of microtubule filaments of the mitotic spindle

Answer 27

Contractile and Motile Proteins

Question 28

Role in cell defense, protection, or exploitation. Examples: immunoglobulins or antibodies–neutralize foreign molecules; thrombin and fibrinogen–blood clotting proteins

Answer 28

Protective Proteins

Question 29

Example: glue proteins that attach marine organisms

Answer 29

Exotic Proteins

Question 30

Larger molecules are excluded from a column of gel beads and emerge from the column sooner than smaller molecules.

Answer 30

Size Exclusion Chromatography (also known as gel filtration chromatography or molecular sieve chromatography)

Question 31

Affinity purification makes use of the specific interactions of proteins such as enzymes with metabolites or ligands. A ligand is covalently linked to an insoluble matrix, a cell extract is passed through the matrix, and a specific protein is eluted by addition of high concentrations of free ligand.

Answer 31

(b) Affinity Chromatography

Question 32

Based on movement of ions in an electric field; Carried out in a porous support matrix such as polyacrylamide or agarose.

(a) Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
(b) Isoelectric Focusing
(c) Two-Dimensional Gel Electrophoresis

Answer 32

Electrophoresis

Question 33

Briefly describe each level of protein structure

Answer 33

1. Primary Structure–amino acid sequence of a protein
2. Secondary Structure–two structural motifs: alpha-helix and beta-strand Stability due to formation of hydrogen bonds between N-H and C=O groups along the polypeptide backbone.
3. Tertiary Structure–folding of a polypeptide chain into its three- dimensional shape
4. Quaternary structure–subunit organization of a protein Refers to the kinds of subunits within a protein molecule, the number of each, and the ways in which they interact with one another.

Question 34

Two basic types of secondary structure: alpha-helix and beta-pleated sheet

Answer 34

Alpha-helix–spiral structure formed by a single polypeptide chain

Beta-pleated sheet–sheet-like structure formed between different polypeptide chains (or distant portions of the same chain)

Question 35

Quaternary structure refers to the association of monomeric protein subunits to form muti-subunit complexes.

–the interacting surfaces are identical and the resulting structure is dimeric.

Answer 35

Isologous interactions

Question 36

–involve nonidentical interfaces; these surfaces must be complementary.

Answer 36

Heterologous associations

Question 37

Structural and Functional Advantages of Quaternary Association

1. Stability–subunit association reduces the protein’s surface-to-volume ratio, which usually increases its stability
2. Genetic Economy and Efficiency–less DNA is required to code for a monomer
3. Bringing Catalytic Sites Together–formation of the multi-subunit complex may be necessary to bring all the catalytic groups together
4. Cooperativity–subunit interactions may produce cooperativity Binding is cooperative if binding at one site increases binding at other sites