Amino Acids lectures

Question 1

non-polar/aliphatic amino acids

Answer 1

G, A, V, L, M, I

Question 2

aromatic amino acids

Answer 2

Y, W, F

Question 3

Polar and uncharged amino acids

Answer 3

T, S, C, P, N, Q

Question 4

polar and charged amino acids

Answer 4

H, K, R

Question 5

polar and negatively charged amino acids

Answer 5

D, E

Question 6

Add _____ group to proline to make ______ . Need vitamin C to do this, so if you have scurvy you also have collagen problems because collagen is rich in _______.

Answer 6

Hydroxyl

hydroxylproline
hydroxylproline

Question 7

________ is important in proteins that help with blood clotting. Vitamin K is needed for___________, so a deficiency in Vitamin K causes trouble clotting.

Answer 7

Carboxyglutmate

the enzyme that carboxylates glutamate

Question 8

O-linked Glycosylation

Answer 8

sugar added to serine or threonine.

Question 9

N-linked Glycosylation

Answer 9

sugar added to asparagine.

Question 10

Why is glycosylation important to proteins?

Answer 10

Addition of the sugar molecule can help proteins become more soluble, can also plays a role in adhesion between cells- cell cell glycoproteins.

Question 11

Lack of the ability to properlyt glycosylate proteins is called ________; ______ is the most common.

Answer 11

congenital disorder of glycosylation (CDG).

CDG1a

Question 12

What was the most fundamental conclusion drawn from the Ribonuclease refolding experiment -

Answer 12

• the primary sequence of a protein determines its structure.
  Dr. Chris Anfinson showed that denatured ribonuclease A can completely refold to its native state on its own, suggesting that all the information required to fold a protein into its native state is embedded in the primary amino acid sequences of the protein.

Question 13

Chaperone proteins

Answer 13

Not all proteins can refold on their own. In many cases, protein folding is helped by a special class of proteins called chaperones. The two major classes of chaperones are Hsp70 and GroEL.

Question 14

Hsp70

Answer 14

induced by heat. Binds to hydrophobic areas to prevent them from aggregating and thus giving the proteins another chance to refold.

Question 15

GroEL

Answer 15

7 different subunits in eukaryotes. Binds hydrophobic surface of unfolded protein and puts it in its own cavity so that the protein can refold properly. Much more generic- not induced by heat shock.

Question 16

Explain why sometimes protein disulfide isomerase or protein prolyl isomerases are
required for protein folding.

Answer 16

Sometimes, proteins require disulfide isomerase and prolyl isomerase for proper folding.
Protein disulfide isomerase catalyzes the exchange between free Cysteines and disulfide bonds which facilitate the formation of correct disulfide bonds in protein.
Note: disulfide bonded proteins are usually secreted or exposed on the plasma membrane
Prolyl isomerase catalyzes the conversion between trans and cis Proline which is required for the proper folding of some proteins.

Question 17

Give an example of a disease that can be diagnosed using a restriction fragment length polymorphism (RFLP) and a use of DNA fingerprinting. Describe at least three experimental stages required in each of these procedures.

Answer 17

Screening for disease alleles characterized by restriction fragment length polymorphisms (RFLP) –Disease allele has to lead to a loss or gain of a cleavage site for a restriction endonuclease. The classic example is screening for the HbS mutation of sickle cell anemia that destroys a restriction site for the restriction endonuclease MstII. Digest patients DNA with “diagnostic” restriction enzyme first, followed by southern or PCR analysis, then electrophoresis and detection of altered size of restriction fragment.

Question 18

Southern Blotting:

Answer 18

allows detection of specific DNA fragments from complex mixtures via hybridization

Question 19

Northern blotting

Answer 19

– Used to identify RNA fragments of known sequence from a complex mixture of DNA fragments, using a method very similar to southern blotting.

Question 20

Describe at least 1 distinct use for PCR amplification in the diagnosis of a genetic condition in your patients.

Answer 20

Screening for disease alleles using allele-specific PCR. The allele(s) must have been previously characterized. Detects small mutations, such as point mutations or small insertions/deletions. The PCR primers are designed so that they will hybridize with the mutant allele but not with the normal allele. The presence of a PCR product indicates the presence of the disease allele. Can test for presence of any one of multiple disease alleles at the same time by designing the reaction such that the PCR product from each mutant allele is a different size. Used for diagnosing 100ʼs of genetic alterations: Cystic fibrosis, Tay-Sachs disease, Beta-thalassaemia, HLA typing.

Question 21

Describe the three main stages that are repeated multiple times during PCR amplification. State the approximate temperature of each step, and relate this temperature to the state of the DNA molecules in the PCR reaction.

Answer 21

1 - Primers are synthesized that are complementary to both strands of a specific sequence of DNA.
2 - A thermally stable DNA polymerase (such as Taq) the four nucleotide triphosphates are added to the reaction containing primers and DNA template.
3 - DNA strands are denatured by heating to ~95 ̊C and the solution cooled to ~55 ̊C to allow the primers to hybridize to the template.
4 - The polymerase uses each strand as a template and makes a copy by extending the 3’ end of the primer, at around ~72 ̊C.
5 - If you heat again and then cool, the process repeats.
6 - Repeating this cycle 35 times gives 23 or 10 billion copies of the gene.