2-Biochemistry - Dna

Front 1

What are the purine and pyrimidine bases?
What's a nucleoside vs. a nucleotide?

Back 1

Purine: A & G (Pure-AG)
Pyrimidine: C & T (cut)
Nside = base + sugar
Ntide = base + sugar + phosph

Front 2

Double Helix:
- what hold bp's together?
- width of helix?
- rotation repeats every?

Back 2

- H bonding
- 2 nm
- q10 base pairs it repeats the helical structure

Front 3

How do proteins control the expression of certain genes?

Back 3

By binding to the major and the minor grooves of the helix

Front 4

What are the effects of stringency conditions on hybridization?

Back 4

High stringency (elevated temp and low ionic/salt solution) = promotes hybridization only if there is an exact match
Low stringency conditions allow hybridization if strands not exactly complementary

Front 5

Mitochondrial DNA:
-role
-mutation rate
-inheritance

Back 5

- synthesis of mitochondrial proteins
- high mutation rate
- maternal inheritance

Front 6

What do the RNA Pol I, II & III synthesize?
What inhibits them?

Back 6

RNA Pol I: rRNARNA Pol II: mRNA
RNA Pol III: tRNA
a-amanitin inhibits Pol II and III (only at high concentrations). Will NOT inhibit mitochondrial pol.

Front 7

What 6 things does a mature mRNA strand contain? What has it lost?

Back 7

- 5' cap
- Regulatory region
- Promoter region (TATA box)
- Start codon
- Exons
- Poly-A tail

** Introns are lost in processing

Front 8

List the steps involved in initiation of transcription.
What happens if there are multiple or mutated promoters?

Back 8

1. TF binds to a TATAA, CG, or CCAAT box.
2. The Transcription Factor, attracts RNA Pol II which then binds
3. In the presence of ATP, the TF will P-late the Pol II, thus activating it and initiating transcription.
Multiple promoters = multiple start sites = multiple proteins produced
Mutated = less transcription of coding regions.

Front 9

Where are enhancers found and what do they do?

Back 9

- much further down or upstream. They bind proteins that may help TF find the promoter/initiation sites. May only be active in certain cell types = thus causing tissue-specific expression of genes.

Front 10

What is the role/function of the following regulatory proteins:
1. Chromatin openers
2. Response elements
3. Silencers

Back 10

1. Allow a certain gene to be accessible to RNA polymerase
2. RE = specific DNA segments that facilitate the binding of other regulatory molecules.
3. Silencers = similar to enhancers only they decrease transcription

Front 11

Define Cis and Trans acting effects. (with regards to transcription)

Back 11

1. Cis action locus regulates (via Trans Factors) genes of the same chromosome
2. Tans-acting: a gene codes for a protein that has regulatory effects on another gene

Front 12

Describe the process of splicing.
(how do splicesomes know where to cut? and what keeps exons in line?)
What happens if you get mutations?

Back 12

spliceosome splices introns containing GU at the 5' splice site and AG at the 3' splice site.
During splicing a "lariat" is formed that allows sequential exons to come together in the same order as they occurred in the parent DNA.
Mutations will either cause the removal of splice junctions or the insertion of new splice junctions.

Front 13

What are the 2 principal routes for the amplification of DNA?

Back 13

1. Molecular cloning: insert human DNA into E.Coli and have them make a bunch of DNA
2. Polymerase Chain Reaction: use biochemical reactions to replicate DNA, w/o any cells. Can add reverse transcriptase and made dsDNA from RNA

Front 14

What are 2 methods for selecting specific pieces of the genome?

Back 14

1. Restriction Enzymes: recognize specific sequences in the the DNA and cute both strands producing Xtic fragments of dsDNA.
2. Probes: complimentary pieces of DNA that bind specific segments of DNA.

Front 15

What are the 2 main types of genetic analysis? What are the 3 subtypes?

Back 15

1. Gel Electrophoresis - separates RE-digested DNA fragments based on size
2. Blotting: transfers GE bands onto nitrocellulose (better medium for blotting).
- Southern = DNA
- Northern = RNA
- Western = Proteins (with Ab probes!)

Front 16

What 3 things can reverse transcriptase do?

Back 16

1. Generate complimentary DNA (i.e. cDNA) from mRNA.
2. Hydrolyze the mRNA
3. Produce a DNA strand that is complimentary to the cDNA

Front 17

Why does ssDNA form a hairpin loop?

Back 17

For DNA polymerase to synthesize a complementary strand a free 3'-OH end is needed. This is provided by the sscDNA itself by generating a hair pin loop at the 3' end by coiling on itself. The polymerase extends the 3'-OH end and later the loop at 3' end is opened by the scissoring action of S1 nuclease.

Front 18

What is meant by probe degeneracy?

Back 18

The number of possible probes for a given amino acid sequence.

Front 19

What is the
"Functional Approach" to cloning disease genes?

Back 19

Works from the actual biochemical defect back to the DNA source:
defect --> protein --> amino acid --> gene

Front 20

What is the "Cadidate Gene Approach" to cloning disease genes?

Back 20

Applies to disease of specific proteins whose function is known.

Front 21

What is the "Positional cloning" approach to disease gene cloning?

Back 21

Do a linkage analysis of several families to find the location of the suspected gene, then sequence it and look for mutations.

Front 22

How does gene therapy work? What are 2 problems?

Back 22

Attempts to cure a disease by establishing a functional gene in a diseased genome, to compensate for the mutant gene (replacement, correction or augmentation)
problems: random insertion could create a new disease, carcinogenesis of vectors