Gene expression and recombinant DNA

Question 1

Gene expression describes the end product, what is that end product?

Answer 1

*Usually a protein
**May be RNA
rRNA
tRNA
micro RNA (miRNA)
mRNA, depending on context

Question 2

What controls prokaryotic gene regulation? (3)

Answer 2

1. Mainly by initiation of txn
2. Regulation of operons
   - repressors
   - activators
3. σ factors - helps RNA polymerase bind promoter
   (locate the start site)
4. Attenuation - premature termination of transcription
   -ultimately decreases gene expression because trx/translation occur at same time
   (depends on secondary structure)

Question 3

What controls eukaryotic gene regulation? (7)

Answer 3

Chromatin remodeling
Gene-specific TF and regulatory sequences
RNA processing - *splicing or RNA editing (replace a nucleotide on the RNA, nOT the DNA)
RNA transport
mRNA stability/degradation
Translation
Protein activity
Protein stability/degradation

Question 4

An operon is one gene or two?

Answer 4

• functional unit of DNA
• cluster of genes
• under control of a single PROMOTER
• all on or all off

Question 5

explain the lac operon example

Answer 5

-lactose is the inducer, which binds to and dissociates the repressor so that RNA polymerase may synthesize the enzymes for lactose break down

Question 6

What is a co-repressor?

Answer 6

• binds repressor
• Negative feedback
• helps to repress
• an example would be if the enzymes that digest lactose are over produce, they can bind the repressor, which causes the repressor to bind DNA
• another example high tryptophan concentration leads to represson of enzymes that synthesize tryptophan

Question 7

What about positive control?

Answer 7

ACTIVATOR (lac operon has both an activator and a repressor)

Question 8

what is an example of 2 ways gene expression is altered without altering DNA sequence?

Answer 8

histone modification
DNA methylation
(studying these is called Epigenetics)

Question 9

Chromatin remodeling

note: DNA wound around histone = chromatin
histones are unique to eukaryotes
each gene has its own promoter (euk)

Answer 9

changing heterochromatin to euchromatin
(tightly wound DNA to less tightly wound DNA)
-makes the genes accessible and they have a chance to be expressed

Question 10

how can we achieve chromatin remodeling?

note: histones are normally positively charged

Answer 10

histone modification!

• add an acetyl group to the histone and this negative charge repels the DNA (because DNA is also negatively charged)
• loosening the DNA around the histone: thus transforming heterochromatin into euchromatin

Question 11

How can we convert euchromatin to heterochromatin?

Answer 11

HDAC (removes the acetyl group)

Question 12

Result of DNA methylation?

Answer 12

silences gene expression

fragile X syndrome? - inherited metal retardation

Question 13

gene specific transcription factors, what are they?

Answer 13

they can inhibit or enhance transcription
(could be a hormone that binds DNA)
-AH-ha! would need to be a steroid hormone to get into the nucleus - cortisol!

Question 14

RNA editing

Answer 14

RNA processing - that replaces a nucleotide on the RNA

NOT on the DNA!

Question 15

mRNA stability

Answer 15

stability
5’ cap
polyA tail

degradation
miRNA - incorporated into RISC complex - roams cytoplasm looking for its complement so it can degrade it

Question 16

gene expression - translation level regulation?

Answer 16

take advantage of

- translation initiation factors

Question 17

What to transferrin and ferritin do?

Answer 17

transferrin - binds iron in blood, transporting it, and allowing it to enter cells
ferritin - found inside cells, stores the iron

Question 18

synthesis of transferrin

Answer 18

• when Iron is low there is a protein that binds the mRNA to stabilize it (must bind to 3’ end or we couldn’t initiate translation)
• when Iron is high, Iron will bind to the this same protein causing it to unbind from the mRNA; destabilizing the mRNA because we dont need to synthesize transferrin

Question 19

synthesis of ferritin

Answer 19

here the IR-BP (iron binding protein) has the opposite effect - it prevents translation! (binds 5’ end!)

so Iron binding allows translation because if there is a lot of Iron we need to store it

Question 20

In bacteria, genes X, Y, and Z are under the control of the same promoter. This is referred to as:

A coding seq
Monocistronic
Controlled expression
Operon (promoter + gene)
Gene expressoin

Answer 20

Operon (promoter + gene)

Question 21

The genes for A, B, and C are expressed only when the molecule Leu is present. Leu binds to a molecule found at the promoter region, thereby releasing the molecule from the promoter. This type of regulation of gene expression involves:

Activation by activator
Repression by an inducer
Constitiutive activation 
Repression by a corepressor 
Attenuation of transcription

Answer 21

Repression by an inducer

Question 22

Determine if the regulation of gene expression is found only in eukaryotes (E), only in prokaryotes (P), or in both (B).

Chromatin remodeling
Initiation of transcription
Gene-specific TF
Repression of an operon
Attenuation of transcription
mRNA processing
Phosphorylation of eIF2α

Answer 22

Chromatin remodeling (E)
Initiation of transcription (B)
Gene-specific TF (E)
Repression of an operon (P)
Attenuation of transcription (P)
mRNA processing (E)
Phosphorylation of eIF2α (E)

Question 23

A patient presents with fatigue, pale skin color, and shortness of breath. A blood test reveals that this patient has a low RBC count and low iron. In terms of gene expression, what would we expect to find:

in terms of ferritin?

in terms of transferrin?

Answer 23

Low ferritin expression (no iron to store)
– at transcript level: no translation of ferritin (binding protein at 5’ end blocks translation)

High transferrin receptor expression (to bring iron into cell) – translation of transferrin receptor (binding protein at 3’ end bound and stabilizing mRNA)

Question 24

Molecular technique that could be used to identify disease/genetic counseling?*

Answer 24

southern blot (DNA analysis)
ex - do they have the sickle cell gene or not?

Question 25

what technique could we use for forensic analysis or paternity test?

Answer 25

DNA PCR of STR

• note: most the the DNA between any two people is identical (so how would this work? - STR (there are some difference - called short tandem repeats)
• so we’d have to use DNA recombination to isolate these STR portion of the DNA

Question 26

determining DNA fragment size on the gel

Answer 26

DNA migrates toward + electrode (smaller size products can move through faster so they are closer to the + end)

Question 27

what is Recombinant DNA and what can we use it for?

Answer 27

Joining DNA sequences into new combinations

-use it to
identify
amplify (PCR)
analyze

Question 28

What do you need for PCR?

Answer 28

Template (section that you would like to copy): DNA
Primers: oligonucleotides
Nucleotide Mix: dNTPs
Adenine
Guanine
Thymine
Cytosine
Enzyme: DNA polymerase (Taq)

Needs double-stranded start site
You provide the start with oligonucleotide primers!
You decide what is amplified!
Buffer: specific for enzyme

Question 29

Northern blot?

Answer 29

could analyze mRNA expression

Question 30

Western blot?

Answer 30

could analyze protein expression

probe is an antibody

Question 31

Identifying polymorphisms to diagnose disease

Answer 31

Polymorphisms = single nucleotide change in DNA

1. Use a disease specific probe + normal probe
   - child 1 has both normal, child 2 has both F508
2. Restriction enzymes to cut DNA (has to be a restriction enzyme site present in the region that contains the polymorphism and if the gene is normal the restriction enzyme will cut there. But if polymorphism is present the enzyme will not cut the DNA

Question 32

What is the purpose of a 2D gel?

Answer 32

Discovery technique to analyze protein expression
but its like trying to find a needle in one hay stack that isn’t in the other hay stack)

Separate proteins by isoelectric point (separating proteins by charge)
Then placing the tube horizontally on the gen (then top to bottom they separate by size)

Question 33

how could we look at mRNA expression without using a northern blot? (because that involves a radio active label - fuck that we don’t want that)

Answer 33

Use reverse transcriptase to convert the mRNA to DNA, you can tell how much mRNA is present based on the amount of DNA you get
*only create cDNA library (RT transcribes coding regions only!)

Question 34

*Can you use RT-PCR to determine viral infection?

Answer 34

YEP. just need one copy of the virus

-so get a blood sample to determine if someone is infected with *HIV

Question 35

*what are microarrays used for?

Answer 35

*great for analyzing GENE EXPRESSION

she loves microarrarys
any question involving what to use for analyzing gene expression this will surely be the answer

Question 36

*Are recombinant DNA techniques used to produce therapeutic compounds?

Answer 36

ah YEAH. today we use e. coli to make insulin

Question 37

other gene therapy techniques, that she neglected.

Answer 37

-Delivering a defective gene to drive down something that is overexpressed
Use of retroviruses for gene therapy

Question 38

In determining paternity by DNA testing, the most commonly used molecular technique is:

Restriction digest, DNA gel
PCR of STR, DNA gel
Genome sequencing 
DNA gel Southern blot
2D gel elctrophoresis

Answer 38

PCR of STR, DNA gel

Question 39

A pathologist takes a sample of a lung tumor as well as a sample of healthy lung tissue from the same patient. She determines changes in gene expression in order to make an informed decision about the proper course of drug therapy for this patient. Which molecular technique does she most likely use?

Western blot 
Microarray analysis
Genome sequencing 
PCR of STR, DNA gel
2D gel electrophoresis

Answer 39

Western blot – (looking for a specific type of protein, we are looking for global changes)

Microarray analysis (we can specify sequences and look at genes involved in what we want to look at)

Genome sequencing (this takes too long, expensive)

PCR of STR, DNA gel (more for forensic analysis)

2D gel electrophoresis (wont give you sutle differences in expression levels, messey looking, needle in hay stack