bio 207 6 and 7
what are the challenges of molecular genetics
- challenge of scale (very thin and no physical features that mark genes)
-challenge of numbers (1 gene- 1/millionth genome, some genes ecpressed in small fraction of cells)
what are people able to do now in molecular genetics (5)
locate a gene/ DNA sequence, remove/copy DNA sequence, Visualize DNA, store new DNA sequences, Edit any genome
what does recombinant DNA allow you to do
locate a gene/ DNA sequence, remove/Copy DNA sequence, Visualize DNA, store new DNA sequences.
what is recombinant DNA (rDNA)
technique in molecular genetics where scientists combine DNA from two sources to a create new DNA. this is usually done using a vector like a plasmid.
what is recombinant also reffered to as
genetic engineering
what is a plasmid
small circula double stranded DNA that can naturally exist in bacterial cells along side the bacterial genome . have the ability to replicate indepedent of bacterial cell genome using an (ori) encodded in a dna sequence inside plasma.
what are linkers
synthetic DNA fragments containing restriction sites
an ideal cloning vector has what 3 things
an ori, one or more selectable markers, recognition sites for one or more restriction enzymes
why is selectable marker in a plasmid necesarry
because it allows cells containing the vector to be selected or identified
why are recognition sites important for a vector
plasmids need these sites for cleavage because this is where the restriction enzymes (restriction endonucleases) can cut the DNA at specific sequences. allows insertion/ removal of specific genes/ DNA fragments within the plasmid.
what happens when restriction enzymes cut at these sites
creates sticky ends (overhangs/blunt ends) that can be used to join other pieces of DNA. allowing precision when inserting new foreign DNA.
what is common when cleaving the foreign DNA and the plasmid
typically cleaved with the same restriction enzyme
what is notable about plasmids used today
they have highly engineered sequences to optimize various functions (high copy number, inducible, high protein expression).
how did fred griffiths exp demonstrate transformation in bacteria.
activated by high stress enviornment, bacterium takes up foreign DNA from dead bacterium and incorporates it within its genome, survival mechanism, provides anatiobiotic resistance
does DNA have to be bacterial in source to experience transformation
no (hence recombinant DNA)
what does it mean to make bacteria competent
make it able to take up foreign DNA
how do we co opt bacteria transformation ability
after creating a recombinant plasmid with the gene of interest, the plasmid is introduced into bacteria under special conditons allowing them to take it up. once it is taken up the bacteria can replicate it and make copies of the plasmid.
do all bacteria sucessfully take up the plasmid
no, that is why scientists use antibiotic selection when experimenting, since the plasmid generally has antiobiotic resitance, only bacteria that succesffully taken up the plasmid will survive when grown on agar plant with antiobiotic. allows for selection and isolation
what is easy expansion
bacterial cells with their resident plasmids can be quickly cheaply expanded
what is indefinite storage
these transformed bacteria can be put in glycerol and frozen and stored at -80 celsisus with little to no effect on cell health of DNA degradation (ability to store new genetic seuqences)
is circular dna stable
yes circular DNA is very stable
how do we obtain/ purify ONLY transformants
using selectable markers on the plasmid (genes that encode a marker) which are typically visual or growth selective.
what is an example of a selectable marker
ampicillin resistance gene. when bacteria are spread on a lawn of media containing ampicillin only transfromants will grow into colonies
what is then done with the transfromant colonies
a single colony is picked and cultures into large isogenic (same genotype) bacterial cultures. plasmid DNA can be purified from these cultures
what do plasmids and transformations allow
allow us to multiply and maintain DNA sequences indefinitely
what are endonucleases?
enzymes that cleave the phosphodiester bonds within a polynucleotide chain. They cut DNA or RNA at specific internal sites, unlike exonucleases, which remove nucleotides from the ends of the chains.
what are restriction enzymes
DNA endonuclease enzymes
where were restriction enzymes found
they were discovered in bacteria as a defense mechanism agaiant invading viruses by functioning as a part of a bacterial innante immune system by identifying and cutting foregin DNA. stop viral DNA from integrating into the bacterial genome
why is it extermely useful that different enzymes have different recognition sequences?
allows researchers to target and cut DNA at very specific sites. This enables precise genetic manipulation, such as cloning, gene editing, and the construction of recombinant DNA
what are the 3 ways of cutting that enzymes can do ?
5' overhang, 3' overhag, blunt end
5' overhang? 3' overhang? blunt end?
5' leaves a cut in a way that leaves a single stranded 5' end, 3' leaves a single stranded 3' end. blunt end cuts straight through both strands leaving no overhangs
what needs to happen for two dna sources to hybridize
they need to be cut/digested with same enzyme and mixed for the overhang ends (sticky ends) to hybridize due to the correct homology
what is the role of T4 DNA ligase
covalently seal (ligates) 'nicks' in sugar phosphate bonds on two ends. Thus, creates stable double stranded piece of DNA.
Where do recognition sequences occur in plasmids
Recognition sequences occur at specific locations in the plasmid where the restriction enzyme cuts. also called restriction sites and only occur at this one location in plasmid.
how are restriction enzymes named?
Each restriction enzyme is named based on the species of bacteria in which it was discovered, with the first portion of the name (in italics) representing the bacterial genus or species.
what is a common method of visualizing DNA
through gel electrophoresis
what is the gel electrophoresis medium
gels (agarose or polacrylamide) where one side has small wells
how does Gel electrophoresis work pertaining to its current
gels are covered in ionic buffer and electric field is applied, DNA is made of acids and acids are negatively charged so they slowly migrate to th eother end of the gel (postive pole)
what moves faster in the gel smaller or larger fragments
larger DNA fragments move slower through the gel matrix thus run slower than small DNA fragments (stay closer to the negative side)
how do we see the DNA after it travels through the gel
DNA is stained with soemthing that binds to the nucleic acids (ex. ethidium bromide), this can be imaged with a cemra and a an appropriate light source (ex. UV light). certian DNA bands can be cut out of the gel and purified
how do we copy and then multiply/amplify speciic DNA sequences in a very efficent manner
polymerase chain reaction (PCR)
who developed PCR
kary mullis 1983
breifly in 3 steps how do cell replicate
1. single stranded DNA template (unwound by Helicases)
2. replication machinery (DNA polymerase)
3. RNA primers (created by primase)
4. free dNTPS
briefly explain PCR. what is used to seperate DNA, how does it receieve dna polymerase, what primers are used how are dNTPs supplied.
heat used to seperate double stranded DNA instead of helicase
dna polymerase supplied a test tube
dna primers (synthetically created)- small chains of nucleotides (17-25) that are complementary to the template and provide 3'-OH end for the polymerase to work with
free dNTPs supply in a test tube
what does PCR stand for
polymerase chain reaction
what is in the PCR reaction mixture (tube) - 5
1. DNA template ( double stranded)
2. primers (forward and reverse)
3. dNTPs
4. DNA polymerase (heat resistant)
5. buffer (ions)
what is step 1 of pcr
heat to 90-100 celsisus for a few minutes
how do primers anneal to single strands in the second step of PCR
cooled 30-60 celsius rapidly for less than a minute. primers are in high conc, rapid cooling give advantage for small primer to bind template before larger single stranded DNA comes back together.
what is used to flank a sequence of interest in pcr in step 2
both forward and reverse primer
what happens during step 3 of PCR
heat to 72 celsisu, polymerase tolerates the rapid heating in step 1
how was PCR revolutinized
by the discovery of thermal resistant DNA polymerase in bacteria, thermus aquaticus
what is a thermocycler
machine that allows repeated cycling of 3 primary steps (heating and cooling) 25-35 times
in pcr what does each cycle of robust amplification do to the number of target dna
ideally doubles the number of copies of the target DNA.
copies of DNA= 2^N X inital copies of DNA. N is the number of PCR cycles.
due to specificty imparted by the forward and reverse primers, we are amplifying only a specific sequence... what does this mean?
Forward Primer: Binds to the beginning of the target sequence on the sense strand of DNA.
Reverse Primer: Binds to the end of the target sequence on the antisense strand of DNA.
By choosing primers that are complementary to specific regions flanking the target sequence, you ensure that only the desired segment of DNA is amplified
how long of primers do you want for PCR
20nt long
how should you design the forward and reverse primers
Forward Primer: This should be designed to bind just upstream of the start codon (usually in the 5' untranslated region or UTR).
Reverse Primer: This should be designed to bind just downstream of the stop codon, in the 3' UTR or exon.
what does DNA sequencing and DNA libraries allow us to do
locate a gene/DNA sequence
true or false, every cell in our bodies have the same genetic code
true
are all the genes in a genome expressed in a cell
no, on average, only 1/10th the genes in the genome is expressed in a cell
how does the same genetic code without our cells give rise to very diverse tissues, all with their own different and unique functions
cells of different tissues express only a subset of genes
what is the first step of how cDNA is created
the first step is isolation of total RNA. first you collect cells or tissues of interest than extract the total RNA from the sample.
after isolating total RNA from your sample the next step is to generate complementary DNA from mRNA... how is this done?
with oligo (dT) primers which are short sequences od deoxythumidine which bind to the poly-A tail of eukaryotic mRNA molecules. the poly A tail is unique to mRNA so the oligo primer ensure that only mRNA is reverse transcribed. (this is the starting point for the reverse transcription reaction)
what does reverse transcriptase do after the oligo (DT) primers
RT is an enzyme that synthesizes cDNA from mRNA, it synthesizes the first strand by copying the mRNA template. it had both RNA- dependent DNA polymerase activity (mRNA--> cDNA) and RNase H activty (degrade RNA template after cDNA is made)
in what direction does RT synthesize
synthesizes cDNA from the 3' end of the mRNA and works toward the 5' end. results in a single stranded cDNA copy of the mRNA. RNase nicks the mRNA strand.
where does DNA polymerase I come in when creating cDNA
after RT and the first strand of cDNA being made, DNA polymerase I which comes from E.coli and has both polymerase and exonuclease activies is used to syntehsize the second strand of cDNA using dNTPS. adding complementary bases to the first cDNA strand.
what else does DNA polymerase do after synthesizing the second strand
since it also has RNase H activty, which removes the RNA strand from the original template, so it can be degraded after reverse transcription
what is the end product after oligo, RT and DNA poly I
double stranded cDNA molecules where one strand is complementary to the mRNA and the other strand is the reverse complement
what happens after you have the double stranded cDNA
it can be inserted into a cloning vector.
how is the cDNA cloning vector prepared
you will cut the vector (plasmid) with appropriate restriction enzymes to generate sticky or blunt ends that will allow for insertion of the cDNA.
whar happens after the vector for cDNA is prepared
the double stranded cDNA is ligated into the vector using DNA ligase which forms covalent bonds between the cDNA insert and the vector. now the cDNA is part of the plasmid vector and can be introduced into bacterial cells
what is transformation (cDNA)
how the ligated is introduced into competent E.coli, the bacteria that actually take up the plasmid will begin to replicate creating clones of the cDNA insert. the colonies can then be screened to identify the ones containing the desired cDNA insert
what is specific about cDNA library
it is specific to a particular tissue, cell type, state or time. they can be created to capture gene expression profile of specific conditon or stage in the life cycle of an organism
why do we use DNA sequencing
helps us understand the protein sequence a gene product will make (gene structure/function) as well as for designing primers for downstream molecular cloning of these gene sequences into plasmids
who was sanger sequeuncing made by and what is also called
sanger sequencing is the standard used for seuqencing DNA fragments in laboratories. created by frederick sanger and colleagues in 1970s, it is also called dideoxy sequencing.
how is dideoxy (sanger) sequencing similar to PCR (3)
1. DNA replication with DNA polymerase
2. DNA template
3. primers
what are the 5 differences between PCR and sanger sequencing
1. number of primers
2. use of ddNTPS
3. concentration of ddNTPS
4.labeling for detection
5. amount of DNA required
pcr vs ss: number of primers
pcr- 2 primers (F and R)
ss- 1 primer
pcr vs ss: use of ddNTPS
PCR- uses dNTPS (deoxyribonucleotide triphosphate) to extend the DNA strand
ss: uses ddNTPS (dideoxyribonucleotide triphosphates), which lack the 3' hydroxyl group, causing DNA synthesis to terminate when incorportaed.
pcr vs ss: concentration of ddNTPS
PCR: dNTPS are present in equal amounts
ss: ddNTPS are present at a 100 fold lower conc than dNTPS to ensure controlled termination of DNA synthesis
pcr vs ss: labelling for detection
pcr: no special labelling is required
ss: primer or ddNTPS are labelled (radioactive or fluorescent) to visualize and read the sequence after electrophoresis
pcr vs ss: amount of DNA required
pcr: small amount of template DNA as the target region gets amplified
ss: large amount of DNA becuase PCR amplification or cloning comes before sequencing to produce a large amount of target DNA
which version of ss labels the primer
the original version the primer is radioactively on the 5' end. the automated version the primer is not labelled
what is labelled in the automated version of ss
each ddNTP (A,C,G,T) is labelled with a different fluorescent probe (illustrated by green, purple, black, red etc.)
what is the difference in tube # in the automated and original version of ss
original: many copies of target DNA and primer are added to 4 tubes
automated: everything goes into one tube and one well on gel
in the original version of ss what goes in each tube (4)
1. lots of dNTPS
2.DNA polymerase
3. a small amount (1/100) of one of the 4 ddNTPS (A,C,G,T)- low conc to ensure random incorporation/chain termination at various points
how are dna bands read in the orignal ss and whats it con
gel electrophoresis to seperate by DNA length, very laborious, expensive
how does automated ss read dna sequences
carried out by automated machines using laser scanners, imagers and software to read out DNA sequence
what is insulin
a peptide hormone, used in the treatment of diabetes
what must we do for to use these techniques to produce insulin (3)
1.identify human tissues that expresses insluin (pancreas)
2. purify RNA from pancreatic tissue
3. use purified RNA to create cDNA from pancreatic mRNA
after making the insulin cDNA what needs to be done
using known genetic sequence of insulin,design a forward (3' end) and reverse (complentary 5' end) DNA primer for inulin cDNA
then to the 5' ends of the forward and reverse primer add an extenstion of a few nucleotides which contain a restriction enzyme recognition site
what do the primers do for the insulin cDNA
in a PCR reaction they amplify the insulin cDNA
after insulin cDNA is amplified then what happens
- load you PCR product on an agarose gel, then use gel electrophoresis to make sure your DNA band is the size you expect
after bands are made from the insulin DNA then what happens
cut your insulin cDNA band out of the gel and purify the DNA from the agarose gel
after insulin cDNA band is cut out and purified then what should you do
add restriction enzyme to your DNA fragment to cut at the restriction sites that you put on the ends, use the same enzyme to digest a plasmid optimized for expressing a protein in E.coli
why do we use the same RE for the dna fragment cutting and to digest the plasmid? what happens after this step?
we use the same RE to digest both becuase it will leave complentary DNA ends that will fit together. then ligate the insulin cDNA into the expression plasmid
what happens with the newly synthesized plasmid with the insulin gene
it gets transformed into competent e.coli cells, then we allow it to grow then spread on a culture plate to obtain single colony isolates
after the insulin colonies grow then what do we do
we perform a PCR on anumber of different bacterial colonies (1-4) to confirm they contain the insert and not just a religated plasmid
what do we have to not just have a religated plasmid
designing one primer in the plasmid and one primer within the insulin cDNA would produce a product
how do we know we have a postive PCR product
use gel electrophoresis to visualize wheter you have a postive PCR product or not
after we get our positve PCR product results what do we do
After selecting colonies or cultures with the plasmid containing the insert, we purify the plasmid DNA. We then design primers that span both the insulin cDNA and the surrounding plasmid regions (flanking parts). The plasmid is sent for sequencing, and the resulting DNA sequence is analyzed to confirm the correct insertion of the insulin cDNA and ensure there are no mutations.
so now the cDNA has been properly sequenced now what?
we grow a larger bacterial culture, and because we cloned the insulin cDNA into an expression plasmid we can add a chemical (IPTG) to induce the bacteria to transcribe and translate the human insulin cDNA on the plasmid. This allows the bacteria to produce insulin. We can then purify the insulin from the bacterial culture for further use.
what are the "weird" sequences in genomes of bacteria and archae (1993)
CRISP: clustered regularly interspaced short palindromic repeats
what is unique about the spacers within the CRISPR sequences
CRISPR was initially thought to be a form of bacterial immunity but then it was found the spacers cointaied DNA that come from foreign sources such as bacteriophages (viruses that infect bacteria), archaeal viruses, and plasmids (small, circular DNA molecules in bacteria). These foreign DNA sequences are inserted into the bacterial genome as a sort of genetic "memory" of past infections, allowing the bacteria to recognize and defend against those specific invaders if they come back.
what is significant about cas proteins
porteins associated with CRISPR, which were initially thought to be involved in general DNA repair, actually played a crucial role in this immune system. they recognize and cut foreign DNA sequences/ defend bacteria and repair dna
what are the 3 functions of CRISPR/Cas system
1. evolved in bacteria to combat invading DNA elements (ex. viruses), similar to an adaptive immune system
2. functions as a type of oragnismal memory of past invaders
3.leads to the targeted destruction of invading DNA
how do bacteria remember past invaders
they create crispr arrays in the bacterial genome (acquisiton phase)
what are the two parts of CRISPR arrays
Protospacers: These are foreign DNA sequences (from viruses or plasmids) that match the unique spacer sequences in the CRISPR system. Essentially, they are the exact targets for CRISPR's defense mechanism.
Palindromic repeats: These are short DNA sequences that flank the spacers. They read the same forward and backward, which helps the CRISPR system recognize and target the corresponding foreign DNA when it reappears.
how do Bacteria use this "memory" to target/destroy future invaders?
they express complementary RNA to similar invaders (expression phase)
what are the first 2 steps of the expression phase
The CRISPR array (the sequence of spacers and repeats) is transcribed into a long precursor RNA called pre-crRNA.
The pre-crRNA is then cut by Cas nuclease proteins (or RNase II) into shorter crRNAs, each containing one of the unique protospacer sequences that match the foreign DNA previously encountered. These crRNAs are crucial for recognizing and targeting the invading DNA.
what are the 3 steps following the cleaved crRNA
crRNA + tracrRNA + Cas9 form complex
cas9/crRNA/tracrRNA complex binds to complementry DNA of new (but similar) invaders
complex cleaves causing double stranded breaks
what are the 2 molecular aspects of targeting/cleavage
1.crRNA contains 20-50nt of invader homology--> CRISPR-Cas can target very unique DNA sites (like PCR primers)
2. protospacer adjacent motif (PAM) site is required downstream of spacer- enables cas9-mediated recognition and cleavage of target DNA.
what is the pam sequence in the crispr-cas9 system
(for the crispr-cas9 system, this PAM sequences is: 5'-NGG-3' <-- short 3nt sequence, occurs very frequently throughout a genome (every 8bp in humans).
what is the most widely used system in molecular genetics for genome editing
crispr-cas9
what is needed to make the effector complex (crispr-cas9)? what is different with how researchers do it
natural system required crRNA and tracrRNA to pair and bind with Cas9 to make effector complex. reasearchers combine crRNA and tracrRNA into one singlue guide RNA (sgRNA)
what is the structure of sgRNA
contains ~20nt "seed" sequence that can direct the effector complex to any specific DNA sequence
what happens to cleaved DNA
DNA repair, two DNA repair mechanisms exist to fix double stranded breaks: nonhomologous end joining (NHEJ) and Homology directed repair (HDR)
explain nonhomoglous end joining (NHEJ) , when used and pros and cons.
when: homologous template is NOT present
(Used when cells are in G1 and no sister
chromatid available as a template)
pro: (good for making random disruptive mutations to
your gene of interest)- good for when you want to disrupt a gene (like in gene editing)
con: Error prone often leading to deletions, insertions,
and translocations because it doesn't have a template to guide it
explain homology directed repair ? how is it different in transgenics (genetic eng)?
when: homologous template IS present – Normally, it
is the sister chromatid after DNA replication.
con: Disadvantage is HDR is not very efficient – success
rate for this strategy of gene editing is one in 100-
1000.
transgenics: researchers provide a donor DNA (a synthetic piece of DNA with the desired sequence/homolgous template) to guide the repair process. This allows them to insert desired new sequences into a gene, such as adding a fluorescent protein tag to track or study a protein.
somatic mutations were the ones in the soma and aren't heritable where as germ line muations were in the germ cells which are heritable. which is more ethical for gene therapy?
• Somatic gene therapy – ethically more permissible
• Germ-line gene editing – Within the traditional research community, this
possibility is not currently on the table (for ethical reasons)
what is the first challenge of CRISP-cas9 system for gene therapy
1. ethical challenges
what is the second challenge of crispr-cas9 system for gene therapy
delivery of materials into target cells
what are the two delievery methods
Viral Vector Delivery: Engineered viruses are used to deliver the CRISPR components (DNA, Cas9, sgRNA) into cells. Viruses are effective at entering cells, but they have risks, including immune reactions and potential unintended genetic changes.
Non-Viral Delivery Vectors: Alternatives like nanoparticles can carry the CRISPR materials into cells without using viruses. These can be safer, but often face challenges in efficiently getting the materials into cells.
will the delievery reach Target Tissues?
The question of whether CRISPR components will effectively reach and enter the correct tissues remains a significant challenge. Delivery methods, like viral vectors and nanoparticles, can sometimes struggle with targeting the right tissues and cells efficiently. Even if the delivery method works, there’s no guarantee that the CRISPR components will reach enough cells in the target tissue to make a meaningful impact on gene therapy.
efficeny of gene editing
Homology Directed Repair (HDR), which is crucial for precise gene editing, is not very efficient (only successful in about 1 in 100 to 1,000 attempts).
For gene therapy to work, a significant number of cells in the target tissue need to be edited. If only a small fraction of cells are successfully edited, the therapy may not be effective enough to restore function.
what is the third Challenge of CRISPR-Cas9 System for Gene Therapy
immunological challenges
what are the immunological challenges
* immune response against cas9 protein (since it is a bacterial protein) which destroys the protein function, there is also a immune response to delivery mechanisms (delivery of CRISPR-Cas9 constructs)
this leads to autoimmune-like disease/symptoms
where is there the most progress for gene therapy (not necessarily crispy-cas9 gene therapy)
Immune-privileged organs are parts of the body where the immune system has limited access or activity such as the eyes (treatment of inherited retinal diseases)
what are the two broad genetic approaches
forward and reverse genetics
explain forward genetics
• Forward genetics: begins with a phenotype due to random
mutations. Then researcher uncovers what gene is responsible for phenotype.
explain reverse genetics
Reverse genetics: begins with interest in a gene of unknown
function. Then, induce mutations specifically in gene and check if phenotype occurs.
what is forward genetics better for
Forward genetics better
for discovering unknown
genes important for a
function/phenotype.
what is reverse genetics better for
Reverse genetics good if
you have a strong
hypothesis that a gene is
important for a trait of
interest.
DNA fingerprinting (DNA profiling): what are microsatelities?
• Microsatellites: short tandem repeats (STRs), variable
number of copies of repeat sequences possessed by
many organisms
• Detected by PCR
• Fragments represented as peaks on a graph
how do the peaks of homo and hetero on graphs appear differntly from microsattetiles
• Homozygotes: single tall peak
• Heterozygotes: two shorter peaks
what can dna figerprinting be used for
to identify people. take an individual dna, subject to pcr, the length of the dna fragment from the pcr depends on the # of copies of the microsatellite sequences. the fragments are seperated by gel electrophoresis, different sized fragments appear as different bands. these patterns of fragments produced by individuals differ
what does a dna profile show
represents the pattern of DNA fragments produced by performing PCT on the STR loci. number below each peak= number of STRs in that DNA fragment
what else can dna fingerprinting be used for in relation to the law
used to determine the presence of a suspect at a crime scene, ex. the dna profile of suspect 2 matches dna evidence collected at the crime scence, results from 4 str loci.
how else can DNA fingerprinting identify people
usual means of victim id were of little use with remains, but used dna fingerprinting and also carried out on mitochondrial DNA
what is DNA barcoding
Technique to identify different species using
DNA
• Use genes that have high interspecies
variability (between species) and low
intraspecies variability (within species)
what is the most common gene used for animals for dna barcoding
Most common gene used for animals is the
mitochondrial gene cytochrome c oxidase I
(COI aka COX1)
what can dna barcoding be used for (5)
Can and has been used to identify:
• The species of meats sold at markets/in restaurants, to make sure food labels are accurate
Species of plants, pollen, insects (esp. larval stage)
• The diet of an organism (DNA testing of stomach contents)
• Human matrilineal lineages
• Animal remains, fecal samples, monitoring for invasive species
hpw dp crpytozoologists use dna barcoding
for evidence of yeti,bigfoot, sasquatch etc.
what are the 6 steps of dna barcoding
1. extract mystery dna
2. amplify COI region with PCR
3. check on gel/ column purify
4. DNA (sanger) sequence product from column purify
5. obtain dna sequence
6. identify likely species using DNA sequence database
why put the cDNA in plasmids
because circular DNA
is stable, whereas
linear DNA is not
(will be degraded
over time)
what is gene transcription
gene (DNA)--> RNA
what do cells undergoing transcription look like under the microscope
under the electron microscope, dna moleculues undergoing trasncription exhibit christmas tree like structures
RNA vs DNA subunits
RNA: rNTPS (ribonucleoside triphosphates) contains ribose sugar, 2' carbon of sugar has a OH group makes RNA more unstable than DNA
DNA: dNTPS (deoxyribonucleoside triphosphates)
what base does rna have instead of thymine (in DNA)
rna- uracil
how does rna and dna differ in secondary structure and flexibility
DNA typically forms a double helix with a stable, predictable structure due to its double-stranded nature/more rigid/less flexible
RNA is usually single-stranded and can fold into more complex 3D shapes (like loops, hairpins, and bulges) due to its ability to base-pair within itself. more flexibile/less rigid- can adopt more shapes
since RNA has a flexible secondary shape what does that allow
allow some RNA to acts as ribozymes (proteins) or catalytic RNA
- storing genetic info for protein translation
what can ribozymes do
ribozymes:
-cut/edit out own sequence
-connect RNA molecules together
-replicate other RNA
-catalyze peptide bond formation between amino acids
what are nucleotides joined by in RNA and DNA
phosphodiester bonds
what is the early RNA world theory
4 bil years ago only RNA, ribozymes
then RNA ribozymes--> protein enzymes
now: dna--> rna,ribozymes--> protein enzymes
what are the 4 main classes of RNA
ribosmal RNA (rRNA), Messenger RNA (mRNA), Transfer RNA (tRNA), CRISPR RNA (crRNA)
give cell type/location of function in eukaryotic cells/function: ribosmal rna
prokaryotic and eukaryotic
cytoplasm
structural and functional components of the ribosome
give cell type/location of function in eukaryotic cells/function: messenger RNA
prokaryotic and eukaryotic
nucleus and cytoplasm
carries genetic code for proteins
give cell type/location of function in eukaryotic cells/function: transfer RNA
prokaryotic and eukaryotic
cytoplasm
helps incorporate amino acids into polypeptide chain
give cell type/location of function in eukaryotic cells/function: CRISPR RNA
prokarytoic
---
assists destruction of foreign DNA
what 3 major components are needed for transcription (DNA--> RNA)
- a DNA template
-the raw materials (ribonucleotide triphosphates) needed to build a new RNA molecule
- The transcription apparatus, consisting of the proteins necessary for catalyzing the synthesis of RNA
how does trancription differ from replication
transcription is highly selective in what DNA sequences are transcribed. genes are transcribed from only one of the DNA strands (the template strand) either strand of DNA can be the template strand (depends on the gene)
template strand is also called
the transcribed strand, the dna strand that is used as template to syntehsize the RNA molecule. RNA is complementary to this strand
the non template strand is also called the
coding strand. it has the same sequence as the RNA except RNA uses uracil instead of thymine, it is not used as the template for transcription
what happens during transcription
the RNA molecule is synthesized 5' to 3' direction and is complementary and antiparallel to DNA template strand
what is a transcriptional unit
stretch of DNA encoding RNA molecule and sequences
necessary for its transcription (Promoter, RNA-coding region, Terminator).
what is a promoter (2)
1. Contains sequences that Transcriptional Apparatus recognize and bind
2. Dictates which DNA strand is template strand and Transcription Start Site
what is the terminator (2)
1.Contains sequences that signal to end transcription.
2. Usually part of the RNA coding sequence and resulting transcript
how are nucleotides numbered during transcription
First nucleotide transcribed is numbered +1 and numbering increases downstream.
Nucleotides upstream of start site are numbered negative moving upstream (promoter and up)
what is the transcriptional appartus
RNA Polymerase and array of accessory proteins
what is RNA polymerase
RNA Polymerase is large multimeric enzyme (several
polypeptide chains)
what is the core enzyme for
Core enzyme catalyzes addition of rNTPs , thus
elongation of RNA molecule.
what are the parts of the cor enzyme (5), which one is no important
Core enzyme has 5 subunits:
a - alpha (2)
b - beta (1)
b’ - beta prime (1)
w - omega (1)
a, b, b’ are essential, while w is not but helps
stabilize enzyme.
what is the role of accesory proteins during transcription
Accessory proteins join and leave polymerase at
different phases of transcription.
Thus, different accessory proteins regulate special
transcriptional functions.
what are the 3 stages of transcription
initation, elongation, termination
intiation (4)
Transcriptional apparatus that assembles on promoter and begins RNA synthesis
1. Promoter recognition/binding
2. Formation of transcription bubble
3. Creation of first bonds between rNTPs
4. Escape of transcriptional apparatus from promoter
what in unique about promoters
Promoters have different affinities for RNA polymerase
what is so important about the promoter recognition and binding
Critical step for regulating selective gene expression (when and how frequent)
Promoter sequence encodes information (that RNA Polymerase “reads”)
1. Where to start
2. Which strand is template strand
3. Which direction to move
consensus sequence
Consensus Sequence – set of most commonly encountered nucleotides among sequences with considerable similarity, or consensus.
Similar location with respect to start site.
most bacterial promoters have two consensus sequeunces where? what about the sequences between/around consensus
one located at -10 bp - Pribnow box TATAAT
another at -35 bp - TTGACA
Sequence between/around consensus vary greatly from promoter to promoter
(mutations there have little impact on transcription).
what is the sigma factor
Sigma factor (s) is a bacterial accessory protein that binds to core RNA polymerase
(forming the RNA polymerase “holoenzyme”).
what does the sigma factor do to the polymerase
Sigma stabilizes polymerase binding to proper promoter start sites ensuring initiation of gene transcription.
when is sigma required? is there only one
Sigma required only for promoter binding and initiation and detaches after a few RNA nucleotides have been added.
Multiple types of Sigma exist that are specific for particular sets of promoters
what does the RNA polymerase holoenzyme do
RNA Polymerase Holoenzyme bind consensus sequences in promoter (initially weakly)
during the formation of the trancription bubble during intiation what happens
Holoenzyme changes structure leading to tighter binding and unwinding of DNA to form transcription bubble (nucleotides -12 to +2).
what happens during the creation of first bonds between rNTPS
-RNA polymerase starts by adding the first ribonucleoside triphosphate (rNTP) to the DNA template.
-The first base added doesn’t use the rNTP's triphosphate bonds for energy.
-As a result, the RNA transcript has 3 phosphates on the 5' end.
-For subsequent rNTPs, two phosphates are cleaved off during the addition of each new base.
-After a few bases are added, the sigma factor detaches from the RNA polymerase and transcription continues.
what happens during the escape of trancriptional appartus from promoter
Polymerase changes shape again such that it “lets loose” of consensus sequences in promoter and is free to move and transcribe downstream.
what are the 3 steps in elongation
1.Unwinding DNA at downstream edge of bubble
2. Adding nucleotides to RNA molecule
3. Rewinding DNA at upstream edge
trancriptional pausing happens during elogation what is it
RNA polymerase pauses due to backtracking, where it moves backward along the DNA template.
This pausing is important for proofreading the RNA transcript.
DNA supercoiling happens during elogation, what is it
DNA Supercoiling:
Supercoiling occurs ahead of the bubble (positive supercoiling) and behind it (negative supercoiling).
Topoisomerases are enzymes that help relieve supercoiling and "fix" the negative supercoiling behind the transcription bubble.
what are the 3 steps of termination of transcription
1.RNA Polymerase stops RNA synthesis
2. RNA molecule released from Polymerase and dissociates from DNA
3. Polymerase detaches from DNA
bacterial genomes posses one of two major types of termiantors that drive two distinct termination mechanisms: what two?
Rho-dependent or Rho-independent
rho dependent termination only causes termination when what is present
These terminators only cause termination
when an ancillary protein, rho (r) is present.
genes with the Rho-dependent termination has what two features
1. RNA sequence where rho binds called Rut
(rho utilization) site
2. Terminator sequence causes Polymerase
to pause. Polymerase pause allows rho time to
advance towards paused transcription sit
what activity does the Rho protein have
Rho protein has helicase activity that unwinds the RNA:DNA hybrid thus terminating transcription.
what are rho independent terminators called
Terminator sequence sufficient to cause termination
(called “intrinsic terminators”)
what are the two features of Rho-independent termination
Features:
1. Inverted repeats – two sections of terminator
have complementary (inverted) sequences.
2. String of ‘A’s in the DNA template follows last
inverted repeat (leading to string of ’U’s in RNA). The string of Uracil (U) cause Polymerase to pause, allowing time for the inverted repeat hairpin to form. Hairpin breaks binding of DNA to RNA and terminates transcription.
what is wrong with the location of eukaryotic DNA
Eukaryotic DNA is condensed into chromatin
structure and often inaccessible
how can we access eukaryotic dna
Nucleosome modifications regulate access to
DNA
what is chromatin remodeling
Chromatin-remodeling complexes: bind directly to DNA sites and reposition nucleosomes
Acetylation of histone proteins
Destabilize nucleosomes, making DNA accessible
how do chromatin remodeling complexes work
chromatin remodeling complexes reposition the nucleosomes, allowing transcription facors and RNA polymerase to bind to promoters and intiate transcription
how does adding acetly groups to tails of histone proteins affect it
The acetylation of histone proteins alters
chromatin structure and permits some
transcription factors to bind to DNA.
what is an example of histone modifications allowing for gene expression
Acetylation of histones controls flowering in
Arabidopsis
− Flowering locus C (FLC) gene
− Flowering locus D (FLD) gene
FLC and FLD example explain:
Flowering Locus C (FLC) gene: This gene represses flowering and happens when acetyl roups on histone proteins destablize chromatin strcture .
Flowering Locus D (FLD) gene: This gene promotes flowering (encodes a deacetylase enzyme) affecting histone modification by removing acetyl groups which then restore chromatid structure so FLC is not transcribed, flowering is not supressed and so flowering takes place
eukaryotes have 3 types of RNA polymerase (I, II, III) why?
Each RNA Polymerase type recognize different promoter
sequences.
type II RNA polymerase is the protein coding (pre-mRNA), the promoters of genes trancribed by RNA poly II consist of two primary parts
Core Promoter – just upstream of gene, similar to Bacterial promoter
Regulatory Promoter – upstream of core promoter, more varied consensus seq
Eukaryotic transcription requires accessory proteins that bind
directly to DNA and recruit RNA Polymerases (I,II,III). what is one type of accesory protein?
• Transcription factors (TF) - any DNA binding protein that affects the levels
of transcription (estimated >1000 TFs in humans)
where do TF's bind and what do they do
TFs bind regulatory promoter sequences and affect transcription by directly or indirectly contacting the basal transcriptional apparatus.
what forms the basal transcription apparatus
General transcription factors + RNA polymerase II forms the basal
transcription apparatus, which is sufficient to initiate minimal levels of
transcription (“basal transcription”
Transcription is frequently controlled by DNA binding proteins. how have they evolved? (hint d and m)
Many types of DNA binding proteins have evolved with functional parts (60-90amino acids), referred to as domains, that bind the DNA.
Many types have evolved that share characteristic domains called motifs (e.g. zing finger motif)
how does basal transcription work in eukaryotes (step 1 and 2)
1. TFIID binds to TATA box in the core promoter, 2. the trancription factors and RNA poly II bind to the core promoter
how does the TATA-binding protein (TBP)
The TATA-binding protein (TBP) binds to
the minor groove of DNA, straddling the
double helix of DNA like a saddle.
eukaryotic transcription has regulatory promoters and enhancers, what are enhancers?
Enhancers – more distant from gene, But the DNA can loop over allowing interaction with DNA binding proteins and Polymerase
what happens after the transcription factors and RNA poly II bind to the core promoter?
3. transcriptional activator proteins bind to sequences in enhancers
4. DNA loops out, allowing the proteins bound to the enhancer to interact with the basal transcription apparatus
5. transcriptional activator proteins bind to sequences in the regulatory promoter and interact with the basal transcription apparatus through the mediator
once RNA polymerase II and general transcription factors are
assembled on the core promoter… (3)
* 11-15 bp of DNA unwinds around the transcription start site
• Open complex - template DNA strand positioned in RNA
polymerase’s active site
• RNA synthesis begins
when does RNA poly II move off the promoter and proceed downstream
After the first ~30nt are polymerized, RNA polymerase II
moves off the promoter and proceeds downstream to
elongate the RNA molecule
how much of the RNA remains paired with the DNA template strand as transcription goes downstream
~8nts of RNA remain paired with the DNA template strand as
transcription progresses downstream
what is upstream and downstream, entering or exiting DNA
exiting DNA out of rna poly II: upstream
entering DNA: downstream
who his roger kornberg
Molecular structure of RNA
polymerase II and how it functions
during elongation have been
revealed through the work of Roger
Kornberg and colleagues.
how does the DNA enter the RNA poly II
The DNA double helix enters
RNA polymerase II through a
cleft in the enzyme and unwinds.
what is notable about the DNA-RNA duplex in the RNA poly II
The DNA–RNA duplex is bent at
a right angle, which positions
the 3' end of the RNA at the
active site of the enzyme.
what happens at the active site of the RNA poly II
At the active site, new
nucleotides are added to the 3'
end of the growing RNA
molecule.
what are RNA poly I and III simialr too
RNA Polymerase I, III similar mechanisms to
Bacteria
What does RNA Polymerase II do after completing the coding sequence?
RNA Polymerase II continues to synthesize RNA past the end of the coding sequence, creating pre-mRNA.
What happens to the pre-mRNA after RNA Polymerase II finishes transcription?
The pre-mRNA is cleaved at a consensus sequence, producing mRNA that will be translated into a protein.
What happens to the RNA that is still being transcribed after the pre-mRNA cleavage?
The RNA continues to be transcribed with a 5' hanging end.
What is the role of the exonuclease protein Rat1 in transcription termination?
Rat1 binds to the 5' hanging end of the RNA, "eats" its way toward the RNA polymerase, causing termination of transcription.
what helps produce the large phenotypic differences between humans chimpanzees
Changes in a relatively small number of regulatory
sequences help produce the large phenotypic
differences between humans and chimpanzees.
true or false: genes dont have to be tightly regualted in time and space
false
genotype vs phenotype
genotype: DNA sequence information
phenotype: anatomical, behavioural, phsyiological
gene expression def:
- Process by which a gene creates a “gene products” (e.g.,
proteins, RNA) that have cellular/organismal functions (e.g. underlie a phenotype).
gene regulation def:
Gene regulation are mechanisms that have evolved to control gene expression
what does gene expression provide
Gene expression - Provides the molecular basis for the relationship between
genotype and phenotype
what does gene regulation control
Gene regulation – controls the flow
of information (fast, moderate, slow, stopped,etc.)
structural genes def:
• Structural genes: encoding proteins
regulatory genes:
Regulatory genes: encoding products that
interact with other sequences and affect the
transcription and translation of these sequences
regulatory elements
Regulatory elements: DNA sequences that
are not transcribed but play a role in regulating
other nucleotide sequences
constitutive expression
Constitutive expression: continuously
expressed under normal cellular conditions
in bacteria gene regulation maintains what
In bacteria, gene regulation maintains internal
flexibility, turning genes on and off in response to
environmental changes
in multicellular eukaryotic organisms, gene regulation brings about what
In multicellular eukaryotic organisms, gene
regulation also brings about cell differentiation
transcription in bacterial cells is regulated by what
Transcription in Bacterial Cells Is
Regulated by Operons
what is an operon
Operon: promoter + additional sequences that control transcription (operator) + structural genes
what is a regulator gene
Regulator gene: DNA sequence–encoding products that affect the operon function but are not part of the operon
negative and postive control: ?
Negative and positive control: inducible and
repressible operons
What is the trp operon in E. coli?
The trp operon is a cluster of genes in E. coli that are involved in the biosynthesis of tryptophan.
How is the trp operon regulated?
The trp operon is regulated by repression. When tryptophan levels are high, the repressor protein binds to the operator and prevents transcription of the operon.
What happens when tryptophan levels are low in E. coli?
When tryptophan levels are low, the repressor protein is inactive, allowing transcription of the trp operon to occur, leading to the production of enzymes needed for tryptophan synthesis.
What are the components of the trp operon?
The trp operon consists of the promoter, operator, and five genes (trpE, trpD, trpC, trpB, trpA) that code for enzymes involved in tryptophan biosynthesis.
How does attenuation further regulate the trp operon?
Attenuation is a second level of regulation that controls transcription based on the availability of tryptophan. It involves premature termination of transcription when tryptophan is abundant.
negative control
Negative control: A repressor protein binds to the operator (partially overlap the promoter or beginning of first gene) to prevent transcription/ blocks RNA polymerase, turning gene expression off (seen in repressible operons).
repressible operons
Repressible operons: Typically on, but can be turned off by a corepressor (e.g., trp operon for tryptophan biosynthesis).
postive control
Positive control: An activator protein enhances transcription, turning gene expression on (seen in inducible operons). Frequently bind promoter and facilitate RNA
Polymerase binding and transcription
inducible operons
Inducible operons: Typically off, but can be turned on by an inducer (e.g., lac operon for lactose metabolism).
who uncovered the operon model of how bacteria regulated genes
Francois Jacob and Jacques Monod used classical genetics to
uncover the “Operon model” of how Bacteria regulate genes
What is an operon?
An operon is a cluster of multiple structural genes controlled by a single promoter. These genes usually have a related function.
What is the advantage of an operon?
Operons allow the cell to coordinate the expression of multiple related genes (e.g., proteins) with one promoter, ensuring efficient regulation.
What do regulatory genes do in operon systems?
Regulatory genes encode proteins or RNA that regulate transcription, similar to transcription factors in eukaryotes.
What are regulatory proteins and how do they function?
Regulatory proteins are DNA-binding proteins that can bind to the operon DNA to regulate transcription. They can act negatively as repressors (inhibit transcription) or positively as activators (enhance transcription).
How do regulatory proteins function in operon regulation?
Regulatory proteins bind to small molecules, which alters their shape and changes how they interact with the operon DNA, either positively or negatively.
What are allosteric proteins?
Allosteric proteins are proteins that change shape and function when they bind to other molecules, influencing their ability to bind DNA or other proteins.
Negative inducible operons
The control at the operator
site is negative. Molecule binding is to the operator,
inhibiting transcription. Such operons are usually off and
need to be turned on, so the transcription is inducible.
inducer
Inducer: small molecule that turns on the transcription
Operon is negative because regulator protein inhibits transcription when bound. what can cause increased transcription?
the allosteric modulator/inducer, which inhibits the function of the repressor
corepressor
Corepressor: a small molecule that binds to the repressor
and makes it capable of binding to the operator to turn off
transcription
Operon is negative because regulator protein inhibits transcription when bound. what causes decreased transcription
Allosteric modulator (small molecule) causes decreased transcription and is
called a co-repressor. Co-repressor is required for repressor protein function
the inducer is a precursor for what
The inducer is a precursor for enzymes (D,E,F) produced by the operon, thus
transcription itself leads to more enzymes and thus effects levels of inducer.
what does this operon feedback system allow for bacteria
This feedback system allows bacteria to adapt to their environment and function
more economically
when are genes D-F (enzymes) made, what precursor has to be present
Genes D-F (enzymes) are only made if precursor (inducer) is present (less
wasteful production of enzymes).
What type of operon is the lac operon of E. coli?
The lac operon is a negative inducible operon involved in lactose metabolism.
What regulates the lac operon?
The lac operon is regulated by the lacI gene (which encodes a repressor), the lacP promoter, and the lacO operator.
What is the inducer of the lac operon?
The inducer of the lac operon is allolactose, which binds to the repressor and inactivates it, allowing transcription.
What are the structural genes in the lac operon and their functions?
lacZ: Encodes β-galactosidase, which breaks down lactose.
lacY: Encodes permease, which helps transport lactose into the cell.
lacA: Encodes transacetylase, which modifies lactose derivatives.
Does the lac operon completely shut down transcription?
No, the repression of the lac operon never completely shuts down transcription; there is always a low level of transcription even in the absence of lactose.
Why is glucose the preferred energy source for E. coli?
Glucose is the preferred energy source for E. coli because it requires no further processing to enter glycolysis, making it a more efficient energy source.
How does E. coli use lactose for energy?
E. coli breaks down lactose through catabolism, producing glucose as one of the products, which can then be used as an energy source.
How do bacterial cells use the lac operon to respond to changing glucose/lactose levels?
Bacterial cells use the lac operon to efficiently switch on and off lactose metabolism based on the availability of glucose and lactose in the environment.
How does β-galactosidase play a role in lactose metabolism in E. coli?
β-galactosidase breaks down lactose into galactose and glucose (catbolize), allowing the cell to use lactose as fuel. The cell makes β-galactosidase and permease in a coordinated manner (called coordinate induction) using the lac operon. β-galactosidase also converts lactose into allolactose, which regulates lactose metabolism.
What happens when lactose is not available in E. coli?
When lactose is not available, the lac operon is repressed. The repressor is constitutively made, preventing the transcription of enzymes. The lac operon is in its basal state with no enzyme production.
What happens when lactose levels increase in E. coli?
When lactose levels increase, β-galactosidase converts lactose into glucose (for fuel) and allolactose. Allolactose acts as an inducer and reflects lactose levels. This triggers the lac operon to be induced in a negative inducible operon system, allowing lac enzyme levels to respond to lactose availability.
What happens to cAMP levels when glucose levels are low in E. coli?
When glucose levels are low, cAMP levels are high. and vice versa
What role does CAP (Catabolite Activator Protein) play when glucose levels are low?
When glucose levels are low, CAP binds to cAMP, and together, they bind just upstream of the lac operon promoter, promoting transcription.
How does low glucose affect lactose metabolism in E. coli?
Low glucose levels result in high cAMP, which activates CAP to promote transcription of the lac operon, turning on lactose metabolism.
What happens when glucose levels are high in E. coli?
When glucose is high, cAMP levels are low, leading to catabolite repression, which represses transcription of the lac operon.
What is catabolite repression?
Catabolite repression occurs when glucose represses the transcription of the lac operon, even in the presence of lactose.
What type of control does CAP represent in the regulation of the lac operon?
CAP represents positive control of the lac operon because it enhances transcription in response to low glucose and high cAMP.
What experimental system did Jacob and Monod use to study the lac operon?
Jacob and Monod used partial-diploid strains of bacteria, which contained an F plasmid carrying a second copy of the lac operon.
What is the purpose of the F plasmid in Jacob and Monod's experiments?
The F plasmid carried a second copy of the lac operon, allowing Jacob and Monod to mutate the lac operon independently and study the effects.
How did Jacob and Monod study the effects of mutations on lactose metabolism?
By using different combinations of mutations in the bacterial genome and the F plasmid, Jacob and Monod could determine whether the mutations had dominant or recessive effects on lactose metabolism.
What was the key approach Jacob and Monod used to understand how mutations affect the lac operon?
they used mutational analysis to assess how mutations in the genome and plasmid affected lactose metabolism and tested for dominant vs. recessive effects.
What is trans-acting regulation in gene transcription?
Trans-acting regulation occurs when the product of Gene A (on a separate piece of DNA) affects the transcription of Gene C.
What is cis-regulation in gene transcription?
cis-regulation involves a DNA sequence (such as Region B) located on the same DNA molecule that affects the transcription of Gene C.
What does it mean if Gene A product affects Gene C transcription?
If Gene A product affects Gene C transcription, it is considered a trans-acting factor.
What does it mean if a DNA sequence (like Region B) affects Gene C transcription?
If a DNA sequence affects Gene C transcription, it is considered a cis-regulating element.
How do structural mutations in lacZ and lacY affect E. coli?
Mutations in lacZ affect the amount of β-galactosidase produced, and mutations in lacY affect the amount of permease produced. These mutations affect the two enzymes independently.
What is the effect of mutations in lacI on E. coli?
Mutations in lacI affect both β-galactosidase and permease levels, with some mutations causing lac proteins to be produced constantly, regardless of lactose levels. This is called a constitutive mutant.
What happens if mutations occur in only one copy of lacI (either plasmid or genome)?
If mutations occur in only one copy of lacI, both lac operons function normally, indicating that the lacI+ gene is dominant over the lacI- gene.
What does the lacI+ gene being dominant over lacI- suggest?
It suggests that lacI is a trans-acting factor that encodes the regulator protein for the lac operon.
What happens in constitutive mutations in the lac operon?
some constitutive mutations, lac proteins are produced regardless of lactose availability- evident only if the structural genes on the same DNA were not mutated. These mutations occur in the lacO operator, preventing the binding of the regulator protein.- cis regulating elements
How are lacO mutations different from mutations in lacI?
lacO mutations are cis-regulating elements because they affect only the lac operon on the same DNA molecule (plasmid or genome), unlike lacI, which is a trans-acting factor.
What is the outcome of a genotype with lacI+ lacO+ lacZ- and lacI+ lacOC lacZ+?
The operon is ON in both low and high lactose conditions, exhibiting constitutive expression.
What happens with the genotype lacI+ lacO+ lacZ+ and lacI+ lacOC lacZ-?
The operon is OFF when lactose is low, but ON when lactose is high, showing normal regulation of the lac operon.
How do lacO mutations affect the operon in relation to lactose levels?
lacO mutations cause the operon to be ON regardless of lactose availability, resulting in constitutive expression of lac proteins.