1 - Intro Lecture Flashcards
What are chromosomes composed of?
a centromere from which four arms protrude, and a telomere at each end
_______ helps to confer stability to the ends of the chromosome
telomere
____ pairs of autosomal chromosomes
22
___ sex determining chromosomes
2
XX (female)
XY (male)
Chromsomes are composed of ______ which is compactly folded mixtures of DNA and basic DNA-binding proteins
chromatin
Chromatin is composed of individually packaged units called ________
nucleosomes
How do nucleosomes appear as by electron microscopy ?
as beads on a thin string
Describe what a nucleosome consists of
octamer of basic DNA binding proteins called histones with a constant length of DNA wound around it
Each octamer consists of ?
two copies of histones- H2A, H2B, H3, and H4
Each nucleosome is separated from its neighbours by a length of DNA that is bound to _________
histone H1
What do nucleosomes do?
package DNA but also help to regulate gene expression
Compare prokaryotic and eukaryotic cells
Prokaryotic:
- no nucleus
- peptidoglycan
Eukaryotic:
- nucleus
- Golgi vesicles
- mitochondria
slide 4
Describe the basic prokaryotic gene structure
- no splice sites so no splicing
- promoters are much less complex
- a consensus sequence for binding RNA pol
- sometimes a simple regulatory mechanism
- no poly A signal so no poly A tail
- half life of RNA controlled by other mechanisms
- no 5’ methyl-guanosine cap
- ribosomes bind to an RNA sequence called the shine-dalgarno sequence
Describe DNA replication
- Synthesis is 5’ to 3’, the incoming nucleotide triphosphate is added to the 3’OH of the existing DNA strand
- Diphosphate (PPi) isa good leaving group this along with hydrolysis of PPi to phosphate drives the reaction forward.
During DNA synthesis, the DNA polymerase makes a mistake about every _____ base pairs
10,000
The DNA polymerases have ______-________ activity which means they can replace improperly paired bases with the correct ones.
proof-reading
These DNa polymerases also have exonuclease activity so they can do what?
remove the mismatched base in addition to adding the right one
__________ cuts one strand of DNA, allowing it to twist around by one turn and then re-ligate. It does this repeatedly to relieve the torsional strain in the DNA created by the helices.
topoisomerase 1
The leading strand is synthesized continuously in the _______ direction.
5’ to 3’
The lagging strand is also synthesized in what direction?
5’ to 3’ (but in small discontinuous stretches known as Okazaki fragments)
A small RNA primer is made by ____ _______ to initiate synthesis. This is later degraded by RNase H followed by filling in DNA by DNA pol
RNA to primase
The fragments are joined by ______
ligase
What unwinds DNA?
What does unwinding cause?
- helicase
- torsional strain on the DNA
Helicase unwinds DNA so ___ _________ can gain access to the DNA.
DNA polymerase
Replication starts at a specific sequence called the _____ of replication some times abbreviated ori
origin
The Lac operon is an example of a ___________
prokaryote
Lac operon:
What is the preferred carbon source?
glucose
Lac operon:
If glucose isn’t available, then ______ can be digested into galactose and glucose as a carbon source
lactose
Prokyarotic gene regulation:
When would there be no need to make genes for digesting lactose?
if lactose is not present or if sufficient glucose is
Prokyarotic gene regulation:
What does the lac operon control?
the expression of genes lac Z, Y, and A
Prokyarotic gene regulation:
What dose Lac I do?
Produces the lac repressor which binds to the operator in the absence of lactose. If lactose is present it binds to the repressor and prevents it from binding to the operator
Prokyarotic gene regulation:
What does RNA polymerase do?
Binds to the promoter. If the repressor is bound to the operator RNA polymerase cannot transcribe lac Z, Y and A
Prokyarotic gene regulation:
What does CAP do?
Binds the cAMP receptor protein which helps RNA polymerase bind to the promoter
Prokyarotic gene regulation:
What does the cAMP receptor protein do?
Binds cAMP and only then will it bind to the CAP site
Prokyarotic gene regulation:
The [cAMP] increases as [glucose] _______
decreases
**inverse relationship
make sense of slide 13
OKAY
Gene regulation in _______ is more complicated than in bacteria.
eukaryotes
Eukaryotic gene regulation:
Does RNA polymerase bind to the promoter by itself?
it does not
Eukaryotic gene regulation:
A group of proteins must also bind to the promotor (along with RNA polymerase), what are they?
- transcription factors
- co-activators
- co-repressors
- depending on the proteins that are bound expression can be higher or lower
Eukaryotic gene regulation:
A Cis acting element is a DNA sequence that a DNA binding protein (_____ acting element) will bind it
Trans
What are the promoters region of a eukaryotic gene?
the TATA box
the GC box
the CCAAT box
What is the consensus sequence of the TATA box?
TATAAAA
What binds to the TATA box?
Transcription factor IID (TFIID)
What does TATA-binding on the TATA box do?
bends the DNA and forces the helix to open slightly, probably allowing better access to RNA polymerase
What is the consensus sequence of the GC box?
GGGCGG
GC box:
The transcriptional activator ____ binds to this region
Sp1
What is the consensus sequence of the CCAAT box?
GGCCAATCT
What do enhancers do?
bind activators and repressors
Where are enhancers located?
some distance from the gene
Describe enhancer sequence
can be relative to the promotor or flipped in the opposite orientation
Activators bound at an _______ can work together to switch on a gene
enhancer
What does the 5’ methyl-guanosine cap do in RNA processing ?
Composed of a methyl-guanosine nucleotide covalently attached to the 5’ phosphate of the first nucleotide of the mRNA
- recognition signal for ribosome
- signal to export out of nucleus
- increases half life of mRNA
What does the Poly A tail do ?
- It is a specific mRNA sequence AAUAAA is recognized by the enzyme polyadenylate polymerase
- Cleave the primary transcript 11-30 bases at 3’ end
- Add a stretch of 200-250 adenosines (increases mRNA half life and nuclear export)
What is RNA splicing?
The process of removing introns from pre-mRNA to form mRNA is called RNA splicing or just splicing
RNA splicing is a 2-step process: Describe it
- The cleavage of the 5’ intron/exon junction occurs simultaneously with the formation of a new phosphodiester bond between the phosphate at the 5’ end of the intron and an adenine residue just within the 3’ end of the intron
- Another trans-esterification reaction occurs, in which the phosphodiester bond at the 3’ intron/exon junction is cleaved and the two cleaved axons are joined.
One of the largest known human genes is Titin, which has more than 175 ____
exons
In mammalian cells, intron removal largely depends on specific features of individual introns, what are some?
- How well the splice signal sequences match the consensus splice site sequences
- Intron length
- Other regulatory sequences located within the pre-mRNA such as the polypyrimidine tract
Describe polycistronic mRNA
- mRNA encode more than one protein
- the information for each arranged in tandem
Describe the Shine-Dalgarno box in mRNA
- a specific ribosome-binding sequence
- adjacent to the start site for each protein
Briefly describe what you need for the making of protein drugs
- the gene
- a vector to carry the gene (usually a plasmid)
- a way to get the gene into the vector
- a way to get the vector with the gene into the microorganisms or cultured mammalian cells
- a way to select for the microorganisms or cultured mammalian cells that have the vector with the gene
- a way to control the expression of the gene once it is in the microorganisms or cultured mammalian cells
- a way to lyse the cells and purify the protein drug away from other contaminating proteins made by microorganisms or cultured mammalian cells
Get the gene from the human cell by making what is known as ____
cDNA
Amplify the gene from cDNA using ______ ______ _____
polymerase chain reaction (PCR)
What is a plasmid?
A vector for transferring gemes
Describe a plasmid
A long sequence of circular double stranded DNA
- Place to put gene
- Selection mechanism
- Method of self replication
- A promoter (place to initiate translation)
______ may be used to transfer genes to mammalian, yeast, insect and bacterial cells
Vectors
Plasmids are put into bacteria and yeast by a process known as __________, in insect and mammalian cells this is known as transfection
transformation
What is antibiotic selection?
This allows to select for cells that have the plasmid
Ex. if we are using bacterial cells we will use an antibiotic resistance gene such as B-lactamase. If we grow the bacteria in the presence penicillin only the bacteria that have the plasmid and hence express the gene can grow.
What is an affinity tag?
Not always present
- Expressed as part of the protein of interest
- Allows us to capture the protein we expressed to purify it
What is a protease cleavage site?
Not always present
-A specific proteolytic cleavage site within the expressed protein usually 4 to 5 amino acids long to cut off the affinity tag to produce a protein free of the affinity tag
Once the protein is expressed, it can be purified with ______ _____
affinity resin
What is a multiple cloning region?
a short sequence with multiple restriction sites
What are restriction sites?
specific sequences that are specifically cleaved by restriction enzymes
The restriction sites in the multiple cloning region only appear _____ in the whole plasmid
ONCE
What is sub cloning?
the process of putting the gene of interest into a plasmid, can be done with restriction enzymes
_______ expression system is the most popular system of expression recombinant proteins
bacterial
______ expression system is a good alternative when a bacterial expression system is not adequate
yeast
Proteins expressed in yeast will have most of the _______ post-translational modification
eukaryotic
______ cells recognize most mammalian protein-targeting sequence, thus they can express most mammalian proteins
Insect
Insect cell vectors are called ________ vectors
Baculovirus
Insect cells grow more _______ than bacterial and yeast cells
slowly
________ cells provide the best expression system for generating recombinant eukaryotic proteins
mammalian
Mammalian cells grow _____
slowly
Advantages of bacteria expression system
Easy, fast, can produce a large quantity of protein rapidly
Disadvantages of bacteria expression system
No post-translational modifications
Advantages of yeast expression system
Some post-translational modifications, faster than mammalian
Disadvantages of yeast expression system
handling may be difficult
Advantages of insect cell expression system
Large quantities of proteins, some post-translational modifications, secreted and tagged forms
Disadvantages of insect cell expression system
Difficult to handle large plasmids
Advantages of mammalian cell expression system
post-translational modifications
Disadvantages of mammalian cell expression system
Slow, more complex, expensive
