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BCH5413 TEST 2 QUESTIONS AND ANSWERS ALL REVISED AND UPDATED

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BCH5413 TEST 2 QUESTIONS AND ANSWERS ALL REVISED AND UPDATED Methylation: a primitive immune system for bacteria - hds-R - Answer-- plasmid transforms into cells are protected (restriction enzyme not expressed) Methylation: a primitive immune system for bacteria - mcrA/mcrB/mrr - Answer-degr...

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  • August 31, 2024
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BCH5413 TEST 2 QUESTIONS AND
ANSWERS ALL REVISED AND
UPDATED
Methylation: a primitive immune system for bacteria
- hds-R - Answer-- plasmid transforms into cells are protected (restriction enzyme not
expressed)

Methylation: a primitive immune system for bacteria
- mcrA/mcrB/mrr - Answer-degrades foreign DNA that is not properly methylated
- can occur to DNA obtained from mouse/human cells containing CpG methylated DNA
(can interfere with cloning of mammalian genomic DNA)

pCR 2.1 - TOPO - Answer-Tac Pol adds A-overhangs to PCR products

TOPO cloning
- what it stands for - Answer-TOPO = topoisomerase
- contains lacZa (can use blue/white color screening)
- f1 Ori (can make ssDNA)
- amp-r
- pUC Ori

drawback: PCR product doesnt get directionally cloned

PCR cloning: test digest - Answer-- restriction enzyme site in pCR 2.1 TOPO + PCR
product
- 5' -> 3': will yield fragment of 300 and 4600 band
- 3' -> 5': 700 and 4200 band

Protein expression in E. coli - Answer-- clone DNA out of TOPO vector into downstream
plasmid capable of expressing protein
- expression plasmid encodes lacZ gene (IPTG in environment will induce protein
expression); will product beta-galactosidase protein

*can also be done by having a plasmid expression vector, lacZ gene to transformed E.
coli -> protein expressed

Shuttle vector: replication in E. coli and yeast - Answer-- cloning steps can be
performed in E. coli, protein expression takes place in eukaryotic cell (yeast)

yeast shuttle vector
- contains... - Answer-- polylinker

,- Ori
- amp-r
- CEN: ensures segregation
- ARS (ori to be copied within yeast)
- URA3 (uracil synthesis; selection)

Yeast shuttle vector: pESC-URA - Answer-URA3 for yeast selection required for growth
on media without uracil

Mammalian shuttle vector
- pcDNA3.1 - Answer-- contains f1 ori (ssDNA)
- amp-r
- pucOri (copying in E. coli)
- pCMV (promoter to express in mammalian cell)
- has epitope tag
- neomycin resistance (selectivity)

*promoter will always be active, does not need to be induced

cDNA library construction
- standard approach steps in isolating a cDNA clone - Answer-1. isolate mRNA
2. convert to ds cDNA
3. choose vector and insert cDNA
4. create phage or bacterial library
5. screen
6. verify identity

Making a cDNA Library - Answer-- isolate mRNA
- make cDNA
- perform second strand synthesis for ds cDNA and add C overhangs (sticky ends)
- clone into vector with complementary G overhangs
- anheal and ligate

Packaging cDNA library into bacteriophage lambda
- problems and solution - Answer-Problem:
- transformation of plasmids (DNA) into bacterial cells is inefficient

Solution
- use bacterial virus (phage) vector, not a plasmid

Packaging cDNA library into bacteriophage lambda
- steps - Answer-- mRNA transcribed into ds cDNA
- ligate ds linker to all ends of cDNA
- can be cleaved with EcoR1 for sticky ends
- complementary overhang vector added
- in vitro packaging to create recombinant virus particles

,- library then used to infect E. coli

*when E. coli is infected with lambda clones you get a lawn of E. coli; where infection
has occured you will see a plaque

Cloning in Charon 4
- useful for? - Answer-- this type of vector accomodates large pieces of DNA

Steps
- after obtaining recombinant DNA combine with lambda packaging system
- infectious phages will infect E. coli and show plaques

Screening a library
- plaque hybridization - Answer-- lay membrane on petri dish and lift off a replica of
plaques
- DNA on filter corresponds to that of the plaques
- block filter with nonspecific DNA or protein and hybridize to labeled probe
- detect by autoradiography

DNA structure - Answer-- nucleic acid bases (purine (A,G) and pyrimidine (C,U,T))
- sugars (ribose, deoxyribose)
- sugars linked by phosphate groups (sugar-phosphate backbone)

*form antiparallel strands

In mammilian DNA, which base has various forms - Answer-cytosine

Chargaff's rule - Answer-A=T and C=G

Is DNA conservative or semi-conservative? - Answer-semi-conservative
- parental sequence and daughter strand bind together

Alternative Double stranded DNA conformation
- A and Z DNA - Answer-A DNA
- dehydrated form
- right handed helix
- 11 bp per helical
- bp tilted 19 degrees
- major groove narrow and deep
- found with dsRNA and RNA-DNA duplex

z-DNA
- >1% of cellular DNA
- favored by G-C repeats and alternating purines-pyrimidines
- left handed helix
- 12 bp per turn

, - bp tilted 9 degrees
- major groove flattened, nearly gone
- bound by poxvirus E3L virulence factor and down regulates apoptosis genes

Non-duplex DNA structures
- cruciform DNA - Answer-- inverted repeat sequence
- favored by excessive negative supercoiling
- AT-rich cruciforms associated with 'fragile' DNA

Alternative modes of base pairing - Answer-Watson Crick:
A--T and C---G
- adenine is at anti-conformation

Hoogsteen:
- adenine or guanine can be flipped to sin-conformation (different H-bonding)

Hoogsteen base pairing
- when can it occur - Answer-- in triplex and quadruplex DNA structures

Triplex DNA - Answer-- pyrimidine rich strand
- negative supercoiling

Chair DNA - Answer-- two G-rich strands
- down regulation of c-myc transcription

Intrinsic Bends in DNA - Answer-- distortion of the ideal B-DNA conformation resulting
from base stacking in the nucleotide sequence
Eg. Adenine-tract DNA results in 20 degree bends

- also can be found in duplex-oligonucleotide model DNA
- causes 23 degree bend and mis-stacking of one GC
- DNA is not a uniform structure

Does B-DNA have a perfect structure? - Answer-No
- sequence specific local variation in twist between paired bases, sugat conformation, tilt
of base pairs, rise distance, etc.

Supercoiled DNA
- negative v. positive - Answer-- left handed under-twisted DNA is in a negative
supercoil

- right handed over-twisted DNA is in a positive supercoil

*most is negatively supercoiled (topoisomerase releaves supercoiling)

Denaturation/Melting of DNA - Answer-- separation of DNA strands

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