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484 Cards in this Set
- Front
- Back
Functional unit of genetic information is the _____. This is composed of _____.
|
1. Gene
2. DNA |
|
What are the 3 informational macromolecules in the cell?
|
1. DNA
2. RNA 3. Protein |
|
List in the order in which they are produced:
RNA DNA Protein |
1. DNA
2. RNA 3. Protein |
|
Each gene is transcribed individually in
|
Eukaryotes
|
|
Introns and exons are not found in _____.
|
Bacteria (nothing is ‘cut out’ like in eukarya)
|
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The sequence of nucleotides is aka a ____.
|
Gene
|
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What codes for RNA?
|
Gene
|
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What codes for the Amino Acid sequence?
|
mRNA
|
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What codes for polypeptides?
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Amino Acid sequence (which folds into protein
|
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What are the 4 nucleotides found in DNA?
|
1. Adenine (to T)
2. Guanine (to C) 3. Cytosine (to G) 4. Thymine (to A) |
|
Which bases melt at a higher temperature?
|
Guanine and Cytosine because they have 3 hydrogen bonds vs. 2
|
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Which type of RNA is coded for on DNA?
|
All 3 types (mRNA, tRNA, rRNA)
|
|
What is transcription?
|
DNA to RNA
|
|
All cells and some viruses contain DNA in what type of structure?
|
Double stranded (double helix)
|
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How are the two strands of DNA arranged according to one another?
|
Anti-parallel
|
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The two strands of DNA form a what?
|
double helix
|
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What type of bond holds the 2 strands of DNA together?
|
Hydrogen bond
|
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This bond is strongest between what?
|
Guanine and Cytosine (3 hydrogen bonds=higher melting point)
|
|
The backbone of DNA is composed of:
|
Phosphates & deoxyribose (pentose sugar)
|
|
DNA’s backbone is held together by what?
|
Covalent bonds (phosphodiester bonds)
|
|
Compare DNA to RNA:
|
DNA:
- double stranded - contains deoxyribose (no OH at position #3) - contains thymine RNA: - single stranded - contains ribose - contains uracil |
|
How many strands of DNA are transcribed and in what direction?
|
Only ONE strand in 5’ to 3’
|
|
What does translation facilitate?
|
RNA to polypeptides
|
|
Where does translation occur in:
1. bacteria (prokaryotes) 2. fungi (eukaryotes) What about transcription: |
Bacteria:
- lack a nucleus so transcription and translation occur in the cytoplasm NOTE: bacteria has no nucleus therefore transcription and translation may occur simultaneously Fungi: - transcription in nucleus - translation in cytoplasm |
|
Replication always occurs ___ to ___.
|
5’ to 3’
|
|
PCR is an example of the _____.
|
Denaturation (caused by high heat which breaks hydrogen bonds b/w bases)
|
|
Which DNA melts at a higher temp?
GC rich OR AT rich? |
GC rich b/c 3 hydrogen bonds b/w bases.
|
|
What physical configuration does DNA undertake to be able to fit inside the space allocated?
|
Supercoiling
|
|
Double helix = underwound is an ex. of:
|
Negative supercoiling
|
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Double helix = overwound is an ex. of:
|
Positive supercoiling
|
|
Which type of supercoiling is most common in bacteria?
|
Negative supercoiling
|
|
Where can we find overwound supercoiling
|
Archaea (overwound double helix = positive supercoiling)
|
|
What enzyme is responsible for the supercoiling of DNA?
|
DNA Gyrase
|
|
T or F: histones in eukaryotes are the same as histones in prokaryotes.
|
False. These do not indicate the same thing.
|
|
What enzyme allows for the DNA to unwind at the replication fork?
|
Helicase
|
|
What holds the DNA apart so that it does not go back together
|
Stabilizing proteins
|
|
T or F: Viruses contain either RNA or DNA.
|
TRUE. (viruses NEVER have both; this is why they need a host to survive)
|
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How many genes does the chromosome contain?
|
All genes (= DNA = full genome of bacteria)
|
|
Are viruses linear or circular?
|
Can be both
|
|
Are viruses single or double stranded?
|
Can be both
|
|
Are plasmids linear or circular?
|
Most are circular
|
|
Are plasmids single or double stranded?
|
Most are double stranded
|
|
What do plasmids carry?
|
1. antibiotic resistance
2. gene for toxin 3. gene for capsule |
|
Are viruses intracellular or extracellular?
|
Extracellular
|
|
Are plasmids intracellular or extracellular?
|
Intracellular
|
|
Which genetic element has “housekeeping” genes?
|
Chromosome (ie: glycolysis, etc = things the cell has to do again and again)
|
|
T or F: A plasmid is self-replicating?
|
FALSE. Plasmids are not self-replicating and only carry a couple of genes
NOTE: Plasmids are NOT part of chromosome |
|
The leading strand replicates continuously from _____ to _____.
|
5’ to 3’
|
|
The lagging strand replicates discontinuously from _____ to _____.
|
3’ to 5’
|
|
What does primase do?
|
Makes RNA primers on both DNA strands
|
|
_____ polymerase can start on its own.
|
RNA
|
|
_____ polymerase needs an –OH group on end to start.
|
DNA
|
|
What does DNA polymerase do?
|
Copies DNA
|
|
Is DNA replication conservative or semiconservative?
|
Semiconservative = 1 old strand (parent) + 1 new strand
|
|
Why does replication always proceed from 5’ to 3’?
|
Because of free –OH group needed on 3’ end to add nucleotide.
NOTE: 3’ end is the “growing point” |
|
DNA synthesis begins at the what?
|
Origin of replication
|
|
The zone of unwound DNA where replication occurs is the what?
|
Replication fork
|
|
What enzyme unwinds the DNA?
|
DNA helicase
|
|
What is uniquely found on the lagging strand
|
Okazaki fragments = pieces of DNA created by discontinuous replication
(= approx 1000 nucleotides) |
|
What connects the DNA pieces together?
|
Ligase
|
|
What cuts the RNA primer out and why is this important?
|
RNAase; cuts RNA primer out to make DNA (imp. b/c RNA primer has uracil which may cause mutation if not cut out)
|
|
Is RNA primer needed for the leading or lagging strand during replication?
|
Both
|
|
What winds and unwinds supercoils of DNA?
|
DNA gyrase
|
|
What connects Okazaki fragments?
|
Ligase (each requires its own RNA primer)
|
|
What unwinds DNA for replication?
|
DNA helicase
|
|
What holds DNA apart for copying?
|
Single strand binding protein
|
|
Which strand requires RNA primer during replication (leading or lagging)?
|
Both
|
|
In which direction does the leading strand replicate? Lagging?
|
1. 5’ to 3’
2. 3’ to 5’ |
|
In bacteria, because they have _____ chromosomes, replication occurs in the _____ direction with _____ origin(s) of replication and _____ replication fork(s) which makes replication _____.
|
1. circular
2. bi-directional 3. one 4. two 5. faster |
|
A complex of multiple proteins involved in replication is called a _____.
|
Replisome
NOTE: DNA moves (pulled through replisome) = replisome does not move |
|
What is the theta structure and what is it unique to?
|
Theta structure = when DNA of bacteria is half-way through replication = unique to bacteria b/c DNA is circular forming this structure which resembles the Greek letter ‘theta’.
|
|
In the process of DNA replication in bacteria, the _____ will unwind the DNA going in both directions, the _____ proteins will be there to prevent the DNA from re-bonding, the _____ _____ will come in and begin copying the leading and lagging strands. The leading strand is transcribed _____ and the lagging strand _____. The fragments formed by discontinuous replication are called _____ _____.
|
1. helicases
2. stabilizing 3. DNA polymerase 4. continuously 5. discontinuously 6. Okazaki fragments |
|
Why is bacteria DNA replication called semiconservative?
|
The parent acts as template and synthesizes new DNA for complimentary strand.
|
|
What must happen first for binary fission to take place?
|
Chromosomes must duplicate which takes about 40 minutes (60 minutes for entire binary fission process)
|
|
What do DNA Helicases do?
|
Separate the strands for replication
|
|
What do Primases do?
|
Catalyzes primer on each strand
|
|
Are mutation rates in cells common?
|
NO = only 10 to -8 or 10 to -11 errors per base inserted.
|
|
What performs the ‘proofreading’ activity in eukaryotes, prokaryotes, and viral DNA replication systems?
|
Polymerase
|
|
What’s happening during transcription?
|
Making RNA from DNA (mRNA, tRNA, and rRNA)
|
|
In what direction does transcription occur?
|
5’ to 3’
|
|
What makes transcription happen?
|
RNA Polymerase
NOTE: ‘POLYMERASE’ always = copy |
|
How many strands is transcribed
|
One strand
|
|
Where the RNA polymerase binds =
|
Promoter (begins transcription)
|
|
Where repressor protein binds =
|
Operator (stops transcription)
|
|
What is the job of the promoter?
|
To start transcription
|
|
What is the job of the operator?
|
To stop transcription
|
|
What is located after the promoter and the operator and what does it code for?
|
Structural Genes which codes for mRNA
|
|
The structural genes in bacteria are _____.
|
Polycistronic = mRNA will code for multiple genes
EX: Lac operon has 3 diff. structural genes for one mRNA (all 3 controlled by 1 promoter and 1 operator) |
|
What does the Lac Operon code for?
|
The enzymes used in lactose metabolism
|
|
What is the last thing that is found on a gene?
|
Terminator
|
|
Where is the stop codon located?
|
On mRNA strand to stop translation
|
|
What does the terminator do?
|
Stops transcription
|
|
What would happen if no terminator?
|
Transcribe entire chromosome
NOTE: not useful b/c don’t need everything all at one time; ie: cell does not always need enzymes for lactose fermentation b/c it really wants to use glucose (lactose = glucose + galactose), genes for glycolysis expressed all the time |
|
Where does the polymerase bind?
|
Promoter
|
|
What controls whether the transcription happens or not?
|
Operator
|
|
What will happen if the repressor protein is not there? What is this called?
|
It can keep going so transcription can happen. This is called induction = take the protein off = induce the gene = turn the gene on.
NOTE: turn the gene off = repressing it = stopping it |
|
Where does the polymerase bind?
|
Promoter
|
|
What binds to the promoter region to help the polymerase to get everything situated to be able to do transcription?
|
Sigma factors
|
|
What ends the transcription process?
|
Terminator
|
|
Transcription happens where there is no _____.
|
Repressor protein
NOTE: this is a PHYSICAL thing = the repressor protein binds to the DNA causes the polymerase to physically run into it effectively stopping transcription |
|
Transcription happens where there is no _____.
|
Repressor protein
NOTE: this is a PHYSICAL thing = the repressor protein binds to the DNA causes the polymerase to physically run into it effectively stopping transcription |
|
Where does the repressor protein bind?
|
Operator site
|
|
Where does mRNA go once created by transcription?
|
Ribosome
NOTE: 70s in bacteria (80s in eukaryotes) |
|
Information stored in DNA is copied to RNA during
|
Transcription (this info used to synthesize specific proteins)
|
|
What are the 3 types of RNA used to cells?
|
1. mRNA = carry info for making specific proteins from DNA to ribosomes
2. tRNA = deliver A.A. to ribosomes 3. rRNA = used to form ribosomes |
|
What are the 3 stages in the process of transcription?
|
1. initiation
- RNA polymerase binds to promoter site on 3’ end of DNA and breaks H-bonds holding DNA strands together on both sides - Binds to different promoter sites differently 2. elongation - RNA Polymerase moves along DNA opening up bubble in DNA - As it moves, RNA Polymerase adds bases to the 3’ end of the growing RNA transcript 3. termination - RNA Polymerase and newly synthesized RNA transcript are released from DNA - Can occur through 2 mechanisms - 1. self termination - 2. enzyme dependent termination |
|
Does RNA polymerase bind to all promoter sites the same way?
|
NO. It binds to some more strongly than others = one way that cells can control gene expression = more strongly is more likely gene is to be transcribed
NOTE: in general, higher levels of transcription leads to higher levels of translation which leads to higher levels of concentration of that polypeptide |
|
Which strand of DNA is the RNA transcribed from?
|
Template strand
|
|
In what ways can termination occur?
|
1. self termination = the RNA sequence transcribed to the terminator causes the RNA to hydrogen bond to itself pulling the RNA Polymerase off the DNA
2. enzyme dependent termination = a termination protein binds to the terminator and pushes RNA Polymerase off the DNA |
|
The sigma factors bind with the _____ at the _____ and recognize 2 sites on the promoter called the _____ and _____. These sites are considered _____.
|
1. Polymerase (helps to hold Polymerase to DNA)
2. promoter 3. Pribnow box (10 bases b/4 transcription = -10 region) 4. -35 region (approx. 35 bases upstream of transcription) 5. conserved = the same over and over again throughout diff. generations = sequences stay the same NOTE: “-“ = to left and “+” = to right |
|
The Pribnow box is aka the what?
|
TATAAT box
|
|
When does the sigma factor get released during the process of transcription?
|
Stays on the whole time until terminator reached.
|
|
What will end the gene for transcription?
|
Terminator
|
|
Why is polycistronic important in bacteria and what is it called when they are grouped together?
|
An mRNA bacteria, very seldom code for just one protein but will have multiple ones and when grouped together they are called operons.
|
|
Translation = _____ to _____.
|
mRNA to polypeptide
|
|
How many possible codons?
|
64
|
|
How many Amino Acids?
|
20
|
|
What is the start codon?
|
AUG (“ALL U GO!”) = codes for methionine = every protein starts with methionine but may not necessarily keep the methionine (may get chopped off)
|
|
What stops translation? What is another name for this?
|
Stop codons aka non sense codon = do not code for amino acid (nothing there = stop)
|
|
What does a sense codon code for?
|
Amino acid
|
|
Non sense codons code for what?
|
Stop codons = do not code for an A.A.
NOTE: recall sense mutations = changes the A.A. and non sense mutations = causes a stop codon = stops transcription |
|
What is the ‘wobble’?
|
What is the ‘wobble’?
|
|
What does the anti-codon bond to?
|
mRNA
|
|
Is RNA double or single stranded?
|
Single
NOTE: tRNA shaped like a ‘T’ b/c of complimentary base pairing |
|
Stop codons include:
|
UGA
UAG UAA |
|
The order that the codons are in is called the what?
|
Reading frame
NOTE: look at this with mutations |
|
What, on mRNA, base pairs with rRNA helping to hold mRNA in place?
|
Shine-Dalgarno sequence = keeps reading frame in place b/c if reading frame changes = mutation (ie: sickle cell anemia is caused by a 1-base mutation)
|
|
What has the anti-codon and carries the amino acid?
|
tRNA
|
|
What does the anti-codon base pair with?
|
Codon which is on mRNA
|
|
Where does protein synthesis happen?
|
Ribosomes
|
|
How many ribosomes does bacteria have?
|
70s (eukaryotes have 80s)
NOTE: “S” = Svedberg units which is measured by use of a centrifuge |
|
E.Coli has ___ dinstict ribosomal proteins.
|
52
|
|
What are the 3 main stages of translation?
|
1. initiation = start
2. elongation = peptide getting longer 3. termination |
|
What is happening during elongation?
|
Polypeptides being formed by the addition of peptide bonds b/w A.A.
NOTE: Amino group on one end and carboxyl group on other = dehydration rxn to form peptide bond = happening during elongation |
|
What kind of a bond is a peptide bond?
|
Covalent bond
|
|
Once 2 A.A. are bonded together, what happens to the tRNA?
|
It goes away and can be used again.
|
|
Why does transcription stop when it reaches a stop codon?
|
b/c stop codon does not have A.A. attached so there’s nothing to make peptide bond.
|
|
Many antibiotics kill the bacteria because they interfere with _____ at the _____.
|
1. translation
2. ribosome |
|
Why are translation and transcription coupled in bacteria?
|
b/c no nucleus
NOTE: translation can begin before transcription is completed |
|
Multiple ribosomes on one mRNA is what?
|
Polysome
NOTE: this helps them grow faster |
|
What is polycistronic?
|
Multiple genes on one mRNA
|
|
A polypeptide (protein) sometimes needs help from what to reshape or refold into its correct shape?
|
Chaperonin
|
|
T or F: protein folding is very important to its function.
|
TRUE
|
|
What are found on proteins that allow transport out of the cell?
|
Signal sequences
NOTE: bacteria secrete enzymes (lipases) to break down lipids into fatty acids and glycerol so that it can transport them in…but to be able to secrete the proteins, it’s gotta be able to go through the PM and also the outer Cell Wall. |
|
What are we really talking about when we talk about gene expression? (GIVE EXAMPLE OF LAC OPERON)
|
EX: lac operon expressed = enzymes for lactose fermentation made and lactose gets fermented
|
|
An operon that can be turned on and off is called a what?
|
Inducible operon
|
|
The operons for the genes in glycolysis are called what?
|
Constitutive enzymes = constitutive genes = constitutive proteins b/c made all at the same time (constantly made at a basic level the cell needs to survive)
|
|
Why would we want to turn some genes on or off?
|
b/c we don’t need everything all the time
EX: glucose available = do not need enzymes to break down other sugars |
|
What are the 2 major levels of regulation in the cell?
|
1. control the activity of pre-existing enzymes
2. control the amount of an enzyme |
|
Where is the gene found?
|
Chromosome
|
|
What causes transcription to happen?
|
RNA Polymerase
|
|
What causes transcription to happen?
|
RNA Polymerase
|
|
What does this do?
|
DNA to mRNA
|
|
DNA to mRNA is called what
|
transcription
|
|
Where does translation happen?
|
At ribosome
|
|
What is the allosteric effect?
|
When something binds to an enzymes changing the shape (recall feedback inhibition) preventing proper binding of substrate = not able to make end product we are looking for = controlled by enzyme activity
|
|
Where does RNA Polymerase bind?
What happens after this? What binds here? |
Promoter sequence on DNA
Operator Repressor protein |
|
What makes or blocks transcription from happening?
|
What makes or blocks transcription from happening?
|
|
What can happen when protein binds to DNA?
|
1. may catalyze a specific rxn (ie: transcription by RNA Polymerase)
2. may block transcription = negative regulation (control) 3. may activate transcription = positive regulation (control) |
|
Protein binding to DNA and blocking transcription = _____ control.
|
Negative
|
|
Protein binding to DNA and activating transcription = _____ control.
|
Positive
|
|
Negative control is talking about _____.
|
Repression (= repressor protein binds to the operator and PHYSICALLY blocks RNA P-ase from passing/transcribing the gene)
|
|
In what stage do the cells grow?
|
Log (or exponential) phase
|
|
“Cell makes it when it needs it but when it doesn’t, it turns those genes off “ is an example of what?
|
repression
|
|
What is an operon made of?
|
1. promoter
2. operator (regulatory element) 3. multiple structural genes |
|
All of the structural genes of an operon are transcribed into what?
|
Single mRNA molecule which is then translated into proteins
|
|
What type of operon is not transcribed unless they are activated? What are they activated by?
|
1. Inducible
2. activated by an inducer |
|
What type of operon is always transcribed? How do these get turned off?
|
1. Repressible
2. when deactivated by a repressor |
|
Multiple genes controlled by one promoter is called what?
|
Polycistronic
|
|
Repression =
|
Blocking
|
|
Induction =
|
Making
|
|
Many bacterial genes are regulated in groups called what? What are the elements of this?
|
1. operon
2. promoter, operator, and a string of structural genes (3 in Lac operon) |
|
What type of operon is the lac operon? Why is this important?
|
1. inducible (meaning the genes are usually not transcribed)
2. b/c it wouldn’t be efficient to have enzymes made all the time to catabolize lactose when lactose is not available |
|
The inducer acts by doing what?
|
By turning off the repressor…not by actively stimulating the genes to be transcribed.
|
|
What promoter and operator together form the what?
|
Control region
|
|
When is the lac operon induced or stimulated to produce proteins of lactose catabolism? How does this happen?
|
When is the lac operon induced or stimulated to produce proteins of lactose catabolism? How does this happen?
|
|
Since allolactose stimulates transcription of the lac operon, it is called what? This acts by turning off the ____ and not by actively stimulating the genes to be transcribed.
|
1. inducer
2. repressor |
|
What does E.Coli chromosome have near the lac operon that helps to control the production of enzymes to catabolize lactose?
|
A regulatory gene that is constantly transcribed and translated to produce repressor protein which binds to the operator DNA at the operator physically preventing RNA Polymerase from moving beyond the promoter = no enzymes to catabolize lactose
|
|
What does regulatory gene code for?
|
Repressor protein
|
|
TQ repressor proteins are made by the regulatory gene of a repressible operon, once made, are active or inactive?
|
Inactive (unless – for ex. – activated by binding of excess tryptophan to them which then binds to the trp operator to stop transcription of structural genes coding for tryptophan biosynthesis.
|
|
What is an example of a repressible operon found on E.Coli.
|
Trp operon = promoter, operator, and 5 structural genes = codes for biosynthesis of tryptophan
|
|
In the case of the trp repressor protein, what does it need attached to it?
|
Tryptophan and then can bind to the operator (attached to repressor when excess tryptophan in cell = blocks gene for tryptophan biosynthesis = not blocking genes for tryptophan but blocking genes for the enzymes to make tryptophan)
|
|
What will happen to the repressor protein in E.Coli when there is not excess tryptophan available to the cell?
|
No tryptophan will bind to repressor protein preventing it from binding to the operator which allows for RNA Polymerase to bind to the promoter and synthesize all genes to synthesize tryptophan (5 total) = this is polycistronic b/c 5 genes (multiple genes) which are all needed for same thing (tryptophan biosynthesis) controlled by one promoter
|
|
repression =
|
Blocking
|
|
Induction =
|
Making it happen (ex. Lac operon)
|
|
Enzymes are synthesized only when they are needed in what? What type of control is this?
|
1. induction
2. negative control |
|
What is a regulatory mechanism the stops transcription called?
|
Negative control (ie: repression and induction)
|
|
What is a regulatory mechanism the stops transcription called?
|
Negative control (ie: repression and induction)
|
|
What does allolactose do?
|
Binds to repressor protein turning it off which blocks its ability to bind to the operator which induces (by allowing RNA Polymerase to physically pass this point on chromosome) the transcription of the 3 genes to produce lactose for the cell.
|
|
The collective term for inducers and repressors is what?
|
effectors
|
|
TQ The regulatory gene is aka the _____ and codes for the _____ protein. It is located _____ from the promoter.
|
1. I-gene
2. repressor protein 3. upstream (before) |
|
What is the inducer for the lac operon?
|
Allolactose (binds to repressor protein)
|
|
What is lactose broken down into? What enzyme helps to do this?
|
1. glucose + galactose
2. B-galactosidase (this will be referred to in mutation lecture also) |
|
The binding of allolactose to the repressor protein causes a what?
|
Allosteric change in the repressor protein (shape change) which prevents the repressor protein from binding to the operator enabling the transcription of genes necessary to form enzymes to break down lactose.
|
|
A substance that represses enzyme synthesis is what?
|
Corepressor (refer tryptophan video)
|
|
A substance that induces enzyme synthesis is what?
|
Inducer
|
|
What is an operon?
|
Cluster of genes arranged in a linear fashion whose expression is under control of a single operator (ie: lac operon = 3 genes for 1 mRNA)
|
|
TQ What does negative control do?
|
Stops / blocks transcription
|
|
TQ What does positive control do?
|
Proteins will bind (usually along w/ polymerase) to allow transcription to happen (MALTOSE CATABOLISM AN EXAMPLE OF +)
|
|
What is an example of negative control?
|
Lac operon / tryptophan
|
|
What is an example of positive control?
|
Maltose
|
|
TQ In positive control, the activator protein binds to the what?
|
Activator binding site (not operator)
|
|
TQ In positive control, the activator protein binds to the what?
|
Regulon
|
|
T or F: Different signals help cells to know what it needs?
|
TRUE. For example: temp, pH, nutrients, etc)
|
|
TQ A mechanism by which bacteria assess their population density is what?
|
Quorum sensing = helps determine if enough bacteria around to do something (ie: 1 cell making a toxin vs. 1 million cells making toxin)
|
|
Where can luciferase be found?
|
In lightning bugs (this enzyme allows them to light up = bioluminescence)
|
|
P. aeruginosa switches from free living to growing as a biofilm is an example of what?
|
Quorum sensing
|
|
What is a biofilm?
|
When bacteria grow together and can grow all the way across a surface (ie: mop bucket or teeth day after)
|
|
What is chemotaxis?
|
Moving from pt. A to pt. B (toward or away from something)
Due to Chemicals |
|
TQX What is used to describe the phenomenon that cells use glucose first?
|
Catabolite repression (represses other things to use glucose 1st)
|
|
TQX What kind of curve is observed when a cell uses all glucose available then starts to break down different sugar?
|
Diauxic growth curve (NOTE: flat area in curve = when glucose is exhausted and cell producing enzymes to catabolize other)
|
|
What do heat shock proteins produced by cells help with?
|
Repair after being exposed to high temps.
|
|
TQX What is an example of differentiation?
|
Making of endospores when cell runs out of food, water, etc.
|
|
TQX What are 2 examples of this found in Caulobacteria?
|
1. Swarmer cells = disperser role
2. Stalked cells = reproductive role |
|
TQX When RNA folds back onto itself to block transcription (instead of repressor protein) is called what?
|
Attenuation
|
|
How do bacteria control what protein is getting expressed?
|
1. activity of enyme
2. controlling the amount = blocking transcription(+ or -) |
|
Why would a cell want to control what protein is expressed?
|
To conserve NRG = only make what needs
|
|
what binds at an operator site
|
Repressor protein
|
|
What makes the repressor protein
|
I-gene or regulatory gene
|
|
What does the repressor bind to? What does it do?
|
1. operator
2. blocks polymerase from going forward |
|
What happens what lactose is present?
|
Allolactose binds to repressor protein causing shape change = falling off of operator = transcription possible = induction
|
|
What happens what lactose is present?
|
Allolactose binds to repressor protein causing shape change = falling off of operator = transcription possible = induction
|
|
TQ What is catabolic repression?
|
Block genes for anything else but glucose utilization and once used up make genes to use other things = way cells use glucose 1st
|
|
TQ Where does the inducer bind?
|
Repressor protein
|
|
TQ What is the inducer for the lac operon?
|
Allolactose
|
|
TQ Where do repressor proteins bind?
|
Operator
|
|
TQ What is the difference b/c + and – control? Give an example.
|
+ : starts transcription (example is gene for maltose)
|
|
What causes bacteria to fluoresce?
|
Luciferase
|
|
TQ What is an diauxic growth curve?
|
Two growth curves in one (ie: glucose used up then lactose used up)
|
|
TQ What is an diauxic growth curve?
|
Two growth curves in one (ie: glucose used up then lactose used up
|
|
TQ How does attenuation work?
|
RNA folds up onto itself to block transcription (repressor not used) (ie: in case of excess tryptophan)
|
|
Why are RNA viruses more dangerous?
|
They mutate very easily
|
|
What causes SARS?
Where did it come from? What made it possible to be transmitted? |
Coranavirus (RNA virus)
Savant cats (birds to savant cats to people) Close proximity of animals and humans in markets, etc. |
|
Why can viruses be used to treat bacterial infections and/or cancer?
|
1. bacteriaphage
2. virus looks for protein receptor that’s different on cancer cells that’s not on normal cells so it will infect cancer cells and lyse them and not normal cells. |
|
What is a bacteriaphage? Why does it infect the bacteria and not other cells?
|
1. Virus that infects a bacteria.
2. Viruses are very specific. They have receptors that only fit certain cells (ie: bacteriaphage will lyse anthrax cells and not affect other cells = able to target specific cells like using a virus instead of drug) |
|
Why are viruses not considered living cells?
|
Because they can NOT replicate on their own
|
|
Virology is the …
|
Study of viruses
|
|
The protein coat around a virus…
|
Capsid
|
|
Inside the protein coat is…
|
DNA or RNA
|
|
How is a virus host specific?
|
It has specific receptors
|
|
What is the difference between viruses that infect bacteria and animals
|
Viruses that infect bacteria do not enter the cell, they only inject genetic material. Viruses that infect animal cells the whole virus goes into the cell.
|
|
What are the two ways a virus can exit the cell?
|
Lyse cell or buds out
|
|
What does HIV infect
|
T Cells
|
|
Does a bacteriophage have an envelope?
|
No, they
|
|
Where are enveloped viruses seen primarily?
|
Animals
|
|
Where does the envelope come from?
|
Host cells phospholipid bilayer
|
|
TQ What are the three shapes of viruses?
|
Rod Shaped – (Helical Symmetry)
Spherical – (Icosahedral symmetry) Complex – (Mix of both) |
|
Why does a bacteriophage need lysozyme?
|
To get through the cell wall when entering and exiting the cell.
|
|
TQX What are neuraminadases?
|
Enzymes that cleave glycosidic bonds in sugars; allows viruses to exit host,
usually associated with the spread of viruses throughout host tissue. |
|
What polymerase does viruses carry
|
Reverse transcriptase
|
|
TQ To grow a virus in the lab you must…
|
grow the host (bacterium first)
Infect with virus Look for clear areas = placques plaques = # of bacteria in original sample |
|
What is a lawn of bacteria?
|
When bacteria grow across the entire medium
|
|
Why would this process not work for identifying lysogenic viruses?
|
Because lysogenic viruses won’t form placques
|
|
Animal viruses must be grown in ______ cultures
|
Tissue
|
|
How diagnose a viral infection?
|
Indirect diagnosis: Look for specific antibodies instead of growing tissue cultures (difficult)
|
|
TQ Titer is
|
Number of infectous units per sample
|
|
Placque Assay
|
Analogous to the bacterial colony; one of the most accurate ways to measure virus infection.
|
|
What are the steps to the replication cycle of a bacterial virus?
|
1. Attachment
2. Penetration (Injection) 3. Synthesis of Nucleic acid and protein (transcription & Translation) 4. Assembly & packaging (Cytoplasm) 5. Release (Lysis & budding) |
|
Burst Size = ?
|
Number of virions released after one cycle (NOTE: “virion” = virus INSIDE host = infectious whereas virus OUTSIDE host = “virus particle” = usually not infectious)
|
|
Bacteriophage T4 is what kind of virus?
|
lytic virus
|
|
What does a restriction enzyme do?
|
Cuts DNA at a specific seuence
|
|
How can a host cell stop infection from a virus?
|
By having restriction enzymes in its cytoplasm (which would chop up the DNA injected by virus)
|
|
What does the virus do to resist the hosts Restriction enzymes/
|
Methylation of Viral DNA
Restriction Inhibitory Proteins |
|
The __________ system is used to name viruses.
|
Baltimore System
|
|
What is a +RNA and a –RNA virus
|
Positive Strand RNA virus: single strand RNA used directly as mRNA
Negative Strand RNA virus: Single strand RNA that must transcribe – strand into + strand of mRNA. |
|
How does the naked virus get in
|
Inject DNA
|
|
Envelope virus gets in by
|
phagocytosis (Cell eating)
|
|
How does ssDNA and dsDNA infect its host?
AKA? |
ssDNA (+): synthesis of (-) strand, then transcription of (-) strand into mRNA (+)
dsDNA (+): (-)strand transcribed into mRNA (+) |
|
What is the temperate mode of the viral life cycle.
|
The virus DNA has been inserted into the host DNA and both are replicated, does not kill host (Method of multiplying virus)
AKA – Lysogenic or Latent Stage |
|
What is the temperate mode of the viral life cycle.
|
The virus DNA has been inserted into the host DNA and both are replicated, does not kill host (Method of multiplying virus)
AKA – Lysogenic or Latent Stage |
|
What is an example of a temperate virus?
|
Lambda
|
|
T4 is ___________ that infects _____________.
|
Bacteriophage E-Coli
|
|
What is the portion that holds the DNA and what is it made out of?
|
Capsid (Proteins)
|
|
What are the extensions that look like legs called? What is their importance?
|
Tail fibers – Have receptors that match up with the host cell wall.
|
|
When the cell lyses that is called what stage?
|
The release stage AKA Lysis Stage (hello)
|
|
What happens when it lysis the host?
|
Kills the cell
|
|
Whats that clear space on the culture.
|
Placque
|
|
What is the difference between a virus that infects bacteria and one that infects a human?
|
Animal virus has to enter the cell, the capsid has to be uncoated, releasing the DNA into the cell.
|
|
What does the virus have that allowed it to inject its DNA into the host?
All this is part of what phase of the lifecycle |
It had lysozymes that broke the beta 1,4 linkages of the cell wall.
Eclipse |
|
DNA Polymerase will
|
Copy the DNA (Including the Phages DNA)
|
|
It will go through _______ & _________ to make more proteins.
|
Transcriptions / Translation
|
|
Why will it make more lysozyme?
|
To break out of host cell
|
|
What is this a lytic virus?
|
B/c it lysis the cell.
|
|
__________ is the number of viruses released after one cycle of infection?
|
Burst Size
|
|
What is a temperate virus?
|
They don’t kill the cell b/c they incorporate the viral DNA into the host DNA (ie Lambda)
|
|
What is a prophage?
|
when the Lambda DNA goes into the host chromosome
|
|
Temperate viruses are also called ______________ ___________ ____________.
|
AKA Lysogenic, dormant, latent
|
|
How does the viral DNA of temperate viruses get copied?
|
When the cell goes through binary fission, it will duplicate the viral DNA.
|
|
Once the viral DNA is incorporated into the E-Coli bacteria it is now called _________________.
|
Lysogenized Bacteria.
|
|
What does a restriction endonuclease do?
|
Cut DNA at very specific sequences/
|
|
What is the importance of the sticky ends of DNA.
|
Allows for the incorporation of the cut plasmid into the host DNA
|
|
What does ligase do?
|
Seal the gap where sticky ends come together
|
|
Why does a prophage stay in the lysogenic stage?
|
The reason that a prophage stays in the lysogenic stage is that there are repressor proteins that inhibit lytic stage
|
|
What could we eventually use phages for?
What kind of problem could this cause? |
Fight infection
If it’s a gram negative bacteria, Lipid A in the LPS is a toxin. Too many cells lyse at once could poison the host. |
|
How do we know most of the animal viruses are enveloped?
|
The animal cell does not have cell wall
|
|
Papoloma virus causes
|
Warts
|
|
Adenovirus
|
Infects adenoids, causing upper respiratory infections
|
|
Parvovirus
|
Infects Dogs
|
|
What is a Reovirus
|
Respiritory Enteric Orphan
Associated with upper respiratory or colon/gut infection that did not fit into other categories. |
|
Rhabdovirus causes…
|
Rabies
|
|
Coronavirus causes …
|
SARS
|
|
What is unique about a Retroviruses?
|
Has Reverse Transcriptase…RNA DNA
|
|
_____ viruses tend to mutate more often.
|
RNA
|
|
When you get infected with any of these viruses you build up what?
|
Antibodies
|
|
How do you build up antibodies?
|
Body recognizes protein as foreign, makes antibodies, next exposure antibodies bind to virus and blocks attachment.
|
|
How long does it take to make antibodies?
|
1 week to 10 days
|
|
How does a vaccine work?
|
It introduces your body to the virus, and you make a secretory antibody (IGA), that I in your secretions and it never comes into body.
|
|
How do viruses normally get out of host cell?
|
Bud out
|
|
Transformation means to do what?
|
Change
|
|
What is contact inhibition? And why is it important in tumors.
|
Cells grow to certain density then stop, tumors acquire a mutation that renders contact inhibition unproductive
|
|
Retroviruses
|
Enveloped, RNA virus, contains reverse transcriptase, also have integrase and protease.
|
|
What does + orientation mean when speaking of RNA viruses
|
Everything is the same as mRNA, which means translation of protein and copied to make more of RNA
|
|
Viroids are…
|
only RNA that infects plants.
|
|
Prions are…
|
infectious proteins that cause normal proteins to change shape.
|
|
Prions
|
Infectious Protein: Normal protein changes into mis-shaped protein, causing plaques in brain
|
|
Prions disease in generally called:
|
Spongiform Encephalopathy
|
|
Diseases caused by Prions and what animal it affects…
|
1. Scrappies (Sheep)
2. Cruchfeld-Jacobsen Disease (Humans) 3. Kuru (Humans – Papua New Guinea Tribe) 4. Mad Cow Disease (Cows Duh) 5. Chronic Wasting Disease (Deer) |
|
First prion found…
|
First prion found… Sheep, called scrappies b/c they would scrape off their coats, transmitted to cows b/c sheep brain was fed to cows in feed.
|
|
Primary workhorse for bacterial genetics
|
E-coli
|
|
What do you see when you look at the E-coli Chromosome?
|
LAC Operon
TRP Operon Histodine |
|
Origin of Replication
|
Where replication starts
|
|
What is a Plasmid?
|
Made out of DNA, Circular, with few genes, Can be passed between bacteria.
|
|
How can you cut a plasmid?
|
With restriction enzyme (Endonuclease)
|
|
Plasmids are used to…
|
Transfer genes into other bacteria
|
|
To clone something one would…
|
Cut plasmid with restriction endonuclease, insert gene of interest, insert plasmid into bacteri, allow bacteria to transcribe & translate cloned product
|
|
Plasmids have origin of replication so they can…
|
make multiple copies within the cell
|
|
Taking the plasmids out is called…
|
Curing
|
|
What was it about the temperate phages, what did they?
|
They incorporated their DNA via a plasmid into the host cell or chromosome.
|
|
Is the plasmid essential for cell function?
|
No, but it does enhance the bacteria in some way. (May be a toxin, capsule or resistance gene)
|
|
_______________ is used in bioremediation b/c it can break down octane & camphor.
|
Psuedomonias
|
|
What are the things that bacteria produce via plasmid that can make them more virulent?
|
Resistance
Tumor Formation Bacteriosin Hemolysin Toxins and Capsules |
|
______________ means the antibiotic won’t kill the organism.
|
Resistance
|
|
Bacteriosins are…
|
Proteins produced by bacteria that inhibit or kill closely related species or even different strains of the same species (similar to antibodies)
|
|
Cloning is …
|
when you take a gene from one place, put it in a bacteria and get the gene product back.
|
|
Mutation is …
|
a heritable change in the genetic sequence, can be positive, negative or neutral.
|
|
Wild type is the organism found in ________.
|
Nature
|
|
Two types of radiation that can cause damage to DNA
|
Ionizing and Non Ionizing
|
|
Examples of Ionizing radiation
|
x-rays & Gamma rays
|
|
How do Ionizing radiation cause damage?
|
Cause free radicals that break DNA because of the short wavelength which penetrate causing cancer.
|
|
Examples of non-ionizing radiation.
|
UV Light and Microwaves
|
|
Non-ionizing radiation causes the DNA strand to form ______.
|
Thymine–Thymine Dimers, (more exposure causes more dimmers and the cell can’t repair.)
|
|
Thymine–Thymine Dimers, (more exposure causes more dimmers and the cell can’t repair.)
|
Skin Cancer
|
|
Skin Cancer
|
Amino Acid ; protein
|
|
What are nucleoside analogs?
|
Chemical mutagens that look like a regular nucleotide base but have a different side chain. When incorporated into the DNA strand it will cause the base pairing to be wrong.
|
|
What are nucleotide analogs?
|
Chemical mutagens that look like a regular nucleotide base but have a different side chain. When incorporated into the DNA strand it will cause the base pairing to be wrong. exampleAZT – (aids treatment)
|
|
Example of frameshift mutations are…
|
Ethidium bromide – (stain that incorporates itself into the DNA used for Gel Electrophoresis)
|
|
Why would you want to know that ethidium bromide could cause a frame shift?
|
Duh, wear your gloves when handling…can cause cancer
|
|
A ________________is one that you can look for.
|
selectable mutation
|
|
Antibiotic resistance is a selectable mutation and if a bacteria is resistant to penecilin it will________ on a penecilin media.
|
GROW
|
|
______________ are things that you have to screen for by looking at a multitude of things to find certain characteristics
|
Nonselectable Mutation
|
|
What are some examples of nonselectible mutations?
|
Pigment Production, Loss of pigment
|
|
What are some examples of nonselectible mutations?
|
Pigment Production, Loss of pigment
|
|
Replica Plating looks for mutants that are _____________.
|
Auxotroph
|
|
________ is a mutant with a nutritional requirement (example; Histodine)
|
Auxotroph
|
|
____________ media has all of its ingredients and ____________ media lacks one ingredient.
|
Complete Minimal
|
|
____________ media has all of its ingredients and ____________ media lacks one ingredient.
Why does everything grow on the complete media? |
Complete Minimal
B/c all of the nutrients are there, |
|
Why are there some colonies that do not grow on the incomplete media?
|
B/c those are the auxotrophs that required the missing ingredient from the complete media.
|
|
How do you determine which are the mutants?
|
Compare the two colony growths from the complete and incomplete medias and see which did not grow on the incomplete media. Those are the mutants.
|
|
If you induce something you …
|
make it happen or force it to happen
|
|
Induced mutation…
|
One that we force to happen by exposing the bacteria to a mutagen
|
|
Spontaneous mutation
|
Happen in nature as a result errors in replication or the exposure to natural radiation.
|
|
Spontaneous mutation
|
Happen in nature as a result errors in replication or the exposure to natural radiation.
|
|
DNA to mRNA is what?
|
Transcription
|
|
mRNA to amino acid sequence is?
|
Translation
|
|
Where does the mutation happen?
|
on DNA strand
|
|
Silent mutation
|
When base pair substitution occurs and resulting mutation does not change the amino acid
(Example: AAA AAG both code for Phe) Change in Genotype NOT Phenotype |
|
Missence Mutation
|
The base pair substitution created a different codon that coded for a different amino acid. Results in a protein with a different conformation (shape).
|
|
The codons that code for amino acids are called _____________?
|
Sense codons
|
|
Nonsence Mutation
|
A base pair substitution that causes a stop codon to occur, instead of an amino acid, forming an incomplete protein. Likely to be most lethal.
|
|
Frameshift Mutation
|
When nucleotides are inserted or deleted which causes a change in the reading frame. This changes all of the codons that are downstream.
|
|
Reversion
|
Change that occurs when the mutation goes back to the wild type. (reverses of previous mutation)
|
|
Non-Ionizing radiation causes ______________ to occur.
|
Thymine – Thymine Dimers
|
|
Ionizing radiation causes …
|
free radicals which break DNA causing serious mutations
|
|
What is the SOS system?
|
What is the SOS system?
|
|
What is the SOS system?
|
Take salmonella bacteria that is an auxotrophic mutant, expose it to potential carcinogen, if back mutation occurs, the chemic may be carcinogen.
|
|
Transformation by definition is…
|
the uptake of free DNA
|
|
Where did free DNA come from?
|
A Plasmid of a cell that lysed
|
|
Cells that take up DNA are called ____________.
|
Competent
|
|
How could you make that were competent?
|
By growing in calcium
|
|
Explain Griffiths Experiment
|
Two strains of Streptococcus pneumonia,
Smooth (S) and Rough (R) strains R = not virulent S = Virulent (due to capsule) Heat killed S strain = not virulent Mix Heat Killed S and Live R = Virulent Live S cells found in blood of this rat. Tells us that the R strain picked up plasmid from S strain and develop capsule. |
|
What’s the difference between repression and induction?
|
Repression = when you have repressor protein bond to operator that stops transcription.
Induction = when inducer binds to repressor protein so that it falls off and allows transcription to happen. |
|
Do bacteria have histones?
|
No. Histones are found in eukaryotic cells and hold DNA together.
|
|
What is the uptake of free DNA called?
|
transformation
|
|
Cells have to be what for transformation? What does this mean? How is this accomplished?
|
1. competent
2. able to uptake free DNA 3. some naturally occurring competent like streptococcus but to make most competent = - grow in high levels of calcium OR - electroporation (hitting bacteria w/ electrical pulses that makes them uptake some DNA) |
|
What is diff b/w transfection and transduction?
|
Transfection = transformation with DNA naked by itself taken from bacteriophage Transduction = transfer of DNA using a replicating virus (aka phage) itself
|
|
A virus that infects bacteria is called a ?
|
Bacteriophage OR phage
|
|
What are the 2 types of transduction?
|
1. generalized transduction
- initiated during lytic cycle of virulent bacteriophage - donor DNA can be any fragment of bacterial chromosome 2. specialized transduction - initiated during lysogenic cycle of temperate bacteriophage - donor DNA is very specific part of chromosome adjacent to prophage |
|
When is generalized transduction initiated?
|
During the lytic cycle of virulent bacteriophage
|
|
When is specialized transduction initiated?
|
During lysogenic cycle of temperate bacteriophage
|
|
What is a transducing phage?
|
Composed of phage proteins filled with bacterial DNA (carrying DNA from bacteria that was infected)
|
|
When is specialized transduction initiated?
|
During lysogenic cycle of temperate bacteriophages.
|
|
What happens
During lysogenic cycle of temperate bacteriophages. |
Phage DNA becomes incorporated into bacterial chromosome
|
|
What is the phage DNA called once it becomes incorporated into a bacterial chromosome?
|
prophage
|
|
A recipient bacterial cell incorporates new injected DNA into its own chromosome through what?
|
Recombination (bacterial cell is now recombinant)
|
|
The prophage is cut out from the host chromosome during what?
|
Induction (this may take with it some of the host bacterial genes therefore new recipient will have prophage + new bacteria genes)
|
|
What can specialized transduction be used for?
|
Transferring antibiotic resistant from one bacterium to another
|
|
What does the phage end up with during generalized transduction?
|
Bacterial DNA = new recipient cell able to make proteins that this codes for
|
|
What is transduction?
|
Transfer of DNA by phages
|
|
What is transduction?
|
Transfer of DNA by phages
|
|
In conjugation, the cells must do what?
|
Touch each other to transfer DNA
|
|
Touch each other to transfer DNA
What are the short ones called? |
Pilli
fimbrae |
|
What is the F factor?
|
Fertility factor aka F plasmid (E.Coli that have this = F+ = able to have conjugation pilus and serve as DNA donors during conjugation
|
|
What does a cell do once it has pilus?
|
Rolling replication = copy one strand of DNA which goes through pilus to F- which turns F- into F+
|
|
What’s special about F+ cells?
What is coded for on the plasmid? |
Has F factor (aka plasmid which can be self replicating)
Ability to make pilus |
|
What is a cell called when the F factor goes into the chromosome?
|
High frequency recombinant
|
|
What basically happens during Hfr factor?
|
Adding to diversity. Recipient cell remains F- because it does not get full copy of F+ genes but has new genes added to its chromosome.
|
|
It is called the _____ ______ replication when a bacterial cell goes from F+ to F-.
|
Rolling circle replication
|
|
When the F plasmid goes into the chromosome, the cell is called what?
|
Hfr cell (high frequency of recombination cell)
|
|
What are transposons and insertions sequences used to do? In what are they most commonly found in use?
|
1. another way to move DNA around
2. in eukaryotes |
|
What is the simplest transposable element?
|
Insertion sequence
|
|
What is an insertion sequence?
|
Series of nucleotides in a certain order that go into chromosome
|
|
What moves DNA b/w inverted repeats?
|
Transposons (these are larger than I.seq.)
|
|
The process of conjugation can be used to help geneticists do what?
|
Map the genome of bacteria
|
|
What is a nutritional mutant called?
|
auxotroph
|
|
The technique used to look for nutritional mutants is called what?
|
Replica plating (this involves the minimal (=has minimal) and the complete media (=has everything)
|
|
What is a segment of DNA that can moved from one location to another called? How does this happen?
|
1. transposon
- simple = contain only elements necessary for transposition - complex = also carry other elements (ie: gene for antibiotic resistance) 2. the segment of DNA also has the gene coding for transposase (enzyme that cuts out of one location and inserts in another and also forms covalent bond b/w itself and new host DNA) |
|
What is the simplest transposon called?
|
Insertion sequence = tranposase gene flanked by inverted repeat
|
|
What is the simplest transposon called?
|
Insertion sequence = tranposase gene flanked by inverted repeat
|
|
What do complex transposons consist of?
|
Consists of 2 simple transposons with another sequence of DNA b/w them
NOTE: this DNA is often a gene that gives some selective advantage to the organism containing transposon (ie: antibiotic resistance) |
|
What are the simplest transposable element?
|
The insertion sequence
|
|
Transposae moves any DNA between what?
|
Inverted repeats
|
|
“in-vitro” means what?
|
In a test tube
|
|
Following are the basic techniques at alter genetic material. What does each one do?
1. restriction enzymes 2. gel electrophoresis 3. nucleic acid hybridization 4. nucleic acid probes 5. molecular cloning 6. cloning vectors |
1. enzymes that cut DNA (blunt ends and sticky ends)
2. get different bands (use + and – poles to pull DNA through gel) 3. hybrid = combine from 2 sources (complimentary base pairing) 4. single stranded used to look for complimentary strands (usually put radioactive label on that probe) 5. cut gene from source (with restriction genes) and then cut and paste into plasmid (cut plasmid and gene with same restriction enzymes) and they hybridize. Transformation technique is used to put plasmid in bacteria which will undergo transcription / translation for the gene (ie: insulin) 6. carry the gene (DNA) = plasmid or phage |
|
Does DNA/RNA need to be in single strand or double stranded form to hybridize?
|
Single stranded form
|
|
What is the reason DNA/RNA hybridize?
|
Complimentary base pairing
|
|
What do vectors do?
|
Carry things
|
|
What is the vector for:
1. lyme disease? 2. malaria? |
1. tick
2. mosquito |
|
What are the two primary vectors used in recombinant DNA technology?
|
1. plasmids
2. phages |
|
What do restriction enzymes do?
|
Cut DNA
|
|
What do you end up with after restriction enzymes are used?
|
either
1. sticky ends = single stranded DNA at each end = can easily recombine / base pair with another single strand which end up with blunt ends 2. blunt ends |
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What is the 1st restriction enzyme found?
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EcoR1
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What are the following examples of?
EcoR1, Hind III, Hhal |
Names of restriction enzymes
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Where do restriction enzymes come from?
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Naturally from the cells and helps to protect cell from invasion from foreign DNA by cutting it up (NOTE: cell has to protect itself from its own restriction enzymes so it modifies the DNA to do that)
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What is gel electrophoresis?
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1. dye DNA
2. run charge through gel (DNA naturally negatively charged) (NOTE: gel has pores to physically allow passage of certain sized DNA) 3. stain with ethidium bromide |
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What do you use to stain a gel electrophoresis? What does this cause and how?
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1. ethidium bromide
2. cancer b/c gets b/w bases causing a mutation |
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What kind of light is needed to see the stain in gel electrophoresis?
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UV light
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What is the map of the location of restriction enzyme cuts on a segment of DNA called?
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Restriction map
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What is a nucleic acid probe used for and in what technique? Is it single stranded or double stranded?
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1. it is used in nucleic acid hybridization that is used to look for another complimentary piece of DNA
2. single stranded |
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“Southern blot” and “Northern blot” are two names for what kind of technique?
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Nucleic acid hybridization
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What are the 3 main steps of gene cloning?
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1. isolation and fragmentation of source DNA (EX: source = pancreas cells for insulin) (NOTE: fragmented / chopped up by restriction enzymes and gel electrophoresis used to isolate whatever size DNA they want)
2. insert DNA into cloning vector (vector most likely plasmid but also could be phage) (NOTE: plasmid needs to be cut w/same restriction enzymes and it becomes hybridized) 3. introduction of cloned DNA into host organism (ie: E Coli) |
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What is used to seal the DNA during the hybridization process?
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DNA Ligase
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What is the process by which scientists can modify the genome of an organism?
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Recombinant DNA technology (insert desirable or remove undesirable genes)
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A method that is used by scientists to screen for nutritionally defective mutants is called what?
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Replica plating
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Explain this process:Replica plating
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1. bacterial cells treated with X-rays or mutagen (NOTE can be radiation or chemical) to induce mutation in genome
2. cells grown on master plate (complete medium) w/ all necessary A.A., etc to form colonies on the plates 3. plates pressed onto blocks covered with sterile velvet (stamping) 4. imprint is stamped onto 2 other plates (one w/ complete medium and one w/ minimal medium) 5. replica plates are examined for differences in the growth of the colonies (NOTE: cells that grow on complete medium and not on minimal medium = mutants which can then be isolated from master colony for study) |
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What is replica plating used to look for?
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Auxotrophs = mutant that has a nutritional requirement
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What is used to cut a plasmid at the gene?
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Restriction enzymes
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Why do the plasma and the gene go back together?
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Complimentary base pairing at the sticky ends?
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What enzyme allows it to seal off?
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Ligase
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How do we get plasmid into host cell?
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Transformation = uptake of free DNA
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The plasmid with the gene in it is called?
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Recombinant
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What are the 2 possible vectors?
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Plamids or phages
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Why do you want to cut them with the same enzyme?
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End up w/ same cut = increase chance of them coming together
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What is the host likely to be?
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Bacteria (E.Coli mostly)
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Why is it important that plasmids have selectable markers?
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To know if the plasmid picked up the gene or not (marker will help you know)
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What is pUC19?
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Example of a plasmid vector
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What does pUC19 have?
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Amphicillin resistance and lacZ gene (part of the lac operon)
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What does lacZ produce?
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B-galactosidase
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What technique is used to determine whether or not a colony has a vector with foreign DNA inserted = test for recombinants / mutants?
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Blue/White screening (have to get plasmid in there and also get gene in plasmid)
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What media is used to test for lactose utilization?
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X-gal = substitute for lactose
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If pUC19 makes B-galactosidase, what color will this media be?
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Blue
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If pUC19 makes B-galactosidase, what color will this media be?
If not, what color? What color would the mutant be? |
Blue
White White |
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What are the markers for pUC19?
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Amphicillin and B-galactosidase
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What are the most common markers?
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Antibiotic resistance and the lacZ gene (because of X-gal)
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What is the most common method of sequencing?
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Sanger dideoxy method (invented by Sanger)
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What is sequencing?
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Order of nucleotides
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What does PCR do?
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Amplifies DNA (when need more copies of DNA)
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What polymerase is used for PCR?
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tAq polymerase
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What’s special about this Thermos aquaticus
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Doesn’t denature in high heat
Thermos aquaticus=thermophil =archaea |
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What does high temp break?
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Hydrogen bond b/w bases to be able to separate DNA strands to replicate it
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What replicates DNA?
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DNA Polymerase
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What is the piece of DNA called that is being amplified by PCR?
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Target DNA
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What are steps of PCR?
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1. denaturing (=high temp=94 C)
-separates DNA strands 2. priming (=low temp=60 C) 3. extension (=int. temp=72 C) |
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What is the machine called that changes the temperature during the PCR process?
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thermocycler
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Why does PCR require a primer?
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b/c DNA P.ase needs 3 prime end (-OH)
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What are the most common cloning hosts:
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1. E.Coli = Gram – rod
2. Bacillus subtilis = Gram + rod (has spores) 3. Saccharomyces = yeast (used to make bread) |
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What is the disadvantage of using E.Coli for cloning?
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It is potentially a pathogen
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What is transfection? What are 4 ways transfection is accomplished?
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1. introduction of DNA into mammalian cells
2. accomplished by: - phagocytosis of DNA by host cell - using microinjection (like in vitro fertilization) - by electroporation - by gene gun |
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What are the nucleic acid probes or antibodies labeled with?
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Radioactive dye (ie: P32)
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Xrays and Gamma rays are examples of what? What do they cause?
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1. ionizing reaction
2. highly reactive ions & free radicals which can then combine w/ bases on DNA resulting in errors (mutations) OR can react w/ sugar phosphate backbone causing breaks in chromosomes |
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What is UV light an example of? What can it cause?
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1. non ionizing radiation
2. thymine dimmers |
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What are the 2 main types of mutations?
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1. base substitution mutation
- silent = no change in A.A. seq. (affect genotype but not phenotype) - missense = changes 1 A.A. = could be harmful or helpful - nonsense = changes A.A. to stop codon = premature ending of protein = harmful 2. frameshift mutation (lead to nonfunctional protein) - insertion = nucleotide base(s) inserted - deletion = nucleotide base(s) removed |
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What type of DNA repair mechanisms do cells have?
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1. light-repair enzymes = break bond b/w adjoining thymine nucleotides
2. excision-repair enzymes = cut out damaged DNA (DNA P-ase fills in) 3. mismatch-repair enzymes (NOTE: cannot repair once mutation becomes methylated) |
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What is the most common form of mutation?
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Thymine dimmers caused by exposure to UV light
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A protein called a what binds to the A site when a ribosome reaches a stop codon during translation?
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Release factor
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