PCR, DNA sequencing, genes (Benzer), Central Dogma, and Prokaryotic Gene Regulation

Lac-I Terminology

*Cis*: 2 mutations (genes/sequence) are on the same copy of the chromosome *Trans*: 2 mutations (genes/sequence) are on different copies of chromosome

Basics of amino acid -> protein

*DNA* 5' AACTTATTATCA 3' 3' TTGAATAATGT 5' --> *Transcription* *RNA* 5' AACUUAUUAU 3' --> *Translation& *Protein& Leu (L) - Tyr (Y) - Tyr (Y) - His (H) --------------------------- Nucleotide sequence (code) -> amino acid sequence Questions: 1) How is the code organized (is it overlapping?) 2) How long is a code word? (codon) 3) What is a code (what amino acids are encoded in which codons)

How Did We Work This All Out?

*Lac Operon Regulation (Jacob and Monod, 1961)* Use bacterial mating to introduce a small circular piece of DNA (F' plasmid, F') - carried copy of Lac operon - created partial diploid genotype Their experiment allow: - Complementation test to identify different genes - Determine dominance of different mututions (alleles) - Could map the locations of different mutations ---------------------------------------------------------Their experiment found 3 complementation groups corresponding to different genes 1) *Lac-Z region*: coded for enzyme Beta-galactosidase/Beta-gal to break down lactose into sugar glucose and galactose 2) *Lac-Z mutants*: can transport lactose across cell membranes but could not use sugar 3) *Lac-Y region*: coded for enzyme permease that transport lactose into the cell from outside. -------- *Lac-Z mutations (LacZ-): mutants could transport but not use lactose *Lac-Y mutations (LacY-) Permease, mutants could not transport lactose *Lac-I mutants (lacI-) - mutants produce B=gal and Permease even w/o lactose - constitutive/continuously expression of operon Note: - LacZ- and LacY- produce at high levels only if lactose was available - LacZ- and LacY- are close together on chromsome - LacI- is mapped further away --------------------------------------------------------- *Jacob and Manod proposed that the protein coded for by the lac-I gene regulating the expression of lac-Z and lac-Y*

How Changing Expression Levels Can Affect Health

*Melanoma patient* - had a family history of disease - researcher mapped the location of DNA associated w/ increased risk (chromosome 5) *Mutations* - CC*T*GAA -> CC*G*GAA (T>G) - mutations associated w/ Telomerase (TERT) gene - mutations was *not* in coding region Found in the upstream of transcriptional start site, and not affect the amino acid sequence of protein Other identifed mutations in this area, the telomerase gene* associated w/ increased in risk of cancer How could mutations that don't even affect protein that are being made affect a phenotype?

Types of RNA

*Messenger RNA (mRNA) - carries info on how to make protein from DNA in nucleus to the cytoplasm - transcribed from protein-coding genes - short lived (seconds-hour) - Processed before translation (eukaryotes) *Transfer RNA* - help translate info stored in nucleotide code into a protein sequence - used during translation - deliver amino acid to growing polypeptie chain -many modified bases *Ribosomal (rRNA)* - present in ribosome Less Well Known RNA *Small nuclear RNA (snRNA) - help process other mRNA molecules *MicroRNA (miRNA)* - post transcriptional regulation of mRNA *Small Interfering RNA (siRNA) and piwi (piRNA)* - defense against virus or transposons *Long non-coding RNA (IncRNA)* - regulate gene expression and chromatin structure

Mutation Vocab

*University* if a sequence of nucleotide in E. coli specified the amino acid X, the same sequence of nucleotide in any organism would specify the same amino acid X *Codon*: The # of nucleotide in mRNA required to specify an amino acid *Degeneracy*: The code is said to be degenerate if a given amino acid may be specified by 2 or more codons *Nonsense*: If no amino acid is specified by codon, then that codon is a nonsense codon *Missense*: A codon that has mutated such that it encodes a different amino acid *Non-Overlapping*: The genetic code is none-overlapping, which means each nucleotide is part of only a single codon. Contrasts overlapping code in which a single nucleotide could be part of more than 1 codon *Reading Frame*: Refers to how a string of nucleotide is divided up into consecutive triplet codons and determine whether a nucleotide would be 1st, 2nd, or 3rd position of a codon. Each nucleotide sequence has 3 possible reading frame through. In most cases, only 1 reading frame is actually used in translation. The reading frame used during Translation is set by locations of the start codon.

How are Diploids Helpful

1) *Allow you to ask different type of questions* Ex. If a cell contained DNA that promote diff. traits, like large or small, which trait is dominant? Wild type (wt) -> small plaques Mutation (r1 & r2) -> large plaques Result: Benzer made diploids in wild type and mutant phage, but always saw small so... *Mutants are recessive to wildtype (wt) alleles - Recessive mutation are believed to be due to the loss of function converted by a protein or gene - Also suggest that production of small plaque require functional; gene or protein to be present -------------------------------------------------------------- 2) Examining the Gene Ex. Benzer isolated thousands of different mutations that all cause the same phenotype, the production of large plaques Phage DNA: Mutations -> large plaques - 1 model explains this result is b/c mutations cause the same phenotype b/c they all affect the same gene Benzer tested the model by using diploids w/ diff combinations of diff mutants 1) Uses mutations r1 and r2 2) See if both mutations were recessive to wide type, which they were since they produced small plaques 3) Made diploids using DNA from each mutants (r1 and r2) 4) Results in all small plaques = functional protein *Proves that mutation affect the same gene is incorrect* ------------------------------------------------------------- New model: Mutations in 2 different genes have the same phenotype r1 wt / wt r2 -> Small size b/c each DNA mutant would provide functional copy. Example of *Complementation Test*

What Did Benzer's Finding Mean for Bead Theory?

1) Genes are the smallest functional unit of heredity - *Supported b/c data show mutations in diff. locations of gene tend to cause the same phenotype meaning the whole gene is responsible for controlling phenotype* 2) Genes are the smallest structural unit of heredity - *Wrong, data indicate the smallest structural components of gene is a nucleotide and that a recombination of nucleotide together give rise of a complete structure of a gene*

Immune Response

1) Infection (bacteria or pathogens) 2) Recognition (triggers responses to destroy) 3) Mount a defense (e.g. produces antibodies against the pathogen) - Can take a few days - Infection can cause symptoms before immune system responds - Immune system can remember pathogen and destroy it, which prevent making you sick.

DNA vs RNA

1) RNA as Hydroxyl OH on sugar while DNA has Hydrogen (H) - the extra oxgyen (from OH) makes RNA more unstable and breaks down more quickly than DNA 2) RNA uses Uracil (U) instead of Thymine (T) - uracil an thymine are both *pyrimindes* and identical except for the presence of a methyl group in thymine that is absent in uracil

Amplifying DNA with PCR

1) Sample of DNA collected w/ many possible target sequence 2) Mixed with polymerase enzyme, DNA primers, nucleotide triphosphate, and buffer solution to maintain correct environment for enzyme 3) Heated (near boiling) to break hydrogen bond to create single stranded template strands 4) If done well, DNA primers attaches to speocfic target sequence by forming complimentary base pair b/w primers & target template DNA molecule 5) DNA polymerase adds nucleotide to the 3' primer end to create 2 copies of target DNA fargment 6) Sample is heated again to separate all DNA into single strand again. 7) Repeated process to create 4 total copies of target DNA fragment --> millions *Backward primers*: 5' -> 3' *Forward primers": 3' -> 5' - DNA polymerase adds onto primer and move in the direction of the template

Gel Electrophoresis Notes

1) Smaller fragment migrates further down than larger fragment 2) Gel separate molecules by size, not composition 3) DNA and RNA are charged linear molecule, so they can easily be separated by size. However, in gel, proteins are treated with detergents that coat the proteins and cause them to unfold and develop a uniform charge. Results in that protein can also be separated by size in gel 4) Samples can form the same starting material, but treated in different ways because DNA molecule cut with different enzymes that recognize different sequences, and cut at different places and different numbers of times producing different sized fragments. 5) In order to see molecule on gel, even with stains and other labeling, 1000s of copies of molecule must be present in order for those molecules to be visible on gel

Transcription and Translation in Prokaryotes and Eukaryotes

1) Takes place in different cellular compartmnts in eukaryotes, but not prokaryotes - This allows transcription and translation of an mRNA to take place simultaneously in prokaryotes 2) Transcription and translation of an mRNA can occur at the same time in prokaryotes 3) Eukaryotic mRNA and monocistronic while prokaryotic mRNA are often polycistrionic *Monocistronic RNA* - when the codes or a single type of protein even if that mRNA can be spliced in different ways to produce multiple similar protein *Polycistronic mRNA* - codes for multiple, completely different proteins that have different functions - These different proteins are involved in the same biological process - Polycistrnic mRNA are produced by operons and gens correspond to the separate coding region within the operon

Initiation of Transcription of E. Coli.

1) The RNA polymerase core enzyme and sigma subunit bind to ~10 and ~35 promoter consensus sequences 2) DNA unwinds near the start of transcription to form the open promoter complex *RNA Polymerase* - Template-dependent - Reads 3' -> 5' - Synthesizes 5' -> 3' CC: "The transcription of a gene is initiated when a group or complex of proteins, including RNA polymerase binds to the promoter region of DNA. When the DNA strands at the promoter are together, it is referred to as a *closed promoter*. The complex of proteins attaching to the promoter separate the DNA strands to create what is referred to as an *open promoter* where the template DNA is now exposed. The RNA polymerase enzyme is then able to use one of the two strands of DNA as a template to begin making a complimentary RNA molecule beginning at the plus one position and reading the template in the 3' to 5' direction and producing RNA in the 5' to 3' direction. Which of the two strands will serve as a template is determined by the spacing and orientation of the conserved sequences found in the promoter."

Massive Parallel (NextGen or "Shotgun" Sequencing

1) Use fragment DNA 2) Add adapter sequences 3) Attach to slide using adapters 4) Amplify fragment by PCR using primer 5) Sequence all fragment simultaneously and scan 6) Reads fragment and their colors 7) Uses computer to a align fragment 8) Reads sequence from fragment -------------------------------------------------------------- - Primers that recognized the adapter are used to make copies of each DNA fragment using PCR, which cause specific DNA sequence to become contentrated at discrete regions on slides - Sequencing reaction then performed on all the fragment on the slide at once. - Each nucleotide added is scanned by a laser to see which was added at each location based on color that fragment now fluoresces - Record is kept in which nucleotide were added to each separate DNA fragment in order - Computer uses to align each of ther sequence w/ each found on diff. fragfment to identify the complete sequence of chromsome present in the sample ------------------------------------------------------------- Human genome project (finished in 2003) - 6x10^9 base pairs - 13 years and 3 billion $ Current technology - 1 hour - $1,000 time and cost depend on accuracy needed

Vaccination

1) Vaccination (dead/weaken pathogen particles) 2) Recognition - dead virus 3) Secretion of antibodies to fight the infection - Virus is dead so can't cause illness - Immune system remembers the pathogen -> Immune - Developing vaccines can be slow (years) and expensive

REVIEW 3/19/21

1) What type of regulation does Brg Operon display? *Positive, Repressible* 2) Expression of operon A is controlled a regulatory protein and a cellular signal. Based on the experimental evidence below, what type of regulation does this operon display? *Negative, Repressible 3) Operon Z produces an enzyme needed to produce molecule X. Expression is regulated by the presence of X. You have found mutations (A-D) that disrupt a regulatory protein, operator, and structural gene for operon Z. The table shows X production (+) under different conditions. How the regulatory act: 4) Which mutation disrupts the regulatory protein *C* 5) The Lac-I region and lac operon from three different lac+ strains (strains 1, 2 and 3) were used to make partial diploids two different lac- strains (strains 4 and 5). ß-galactosidase activity was assayed in the presence and absence of inducer in each of the haploid and diploid strains. What are the genotypes of the lacI and lacO regions for each of the strain *Strain

Rho-indepedent

1)RNA being transcribed contains a region of RNA with a sequence that is complimentary to another nearby RNA sequence. 2)When these two regions are transcribed, the RNA molecule can fold back on itself to form a structure called a *stem-loop or hairpin.* 3)Formation of a stem loop causes RNA polymerase complex to pause transcription at that location. 4) If the stem-loop is followed immediately by a repeated stretch of adenine nucleotides, the transcriptional complex becomes very unstable and RNA polymerase will release the DNA and RNA molecules ending transcription Q) Why does termnation of transcription in prokaryotes often involve a string of adenine nucleotide? *A-U base pairs are weaker than G-C base pairs* - held together by 2H-bond and likely to break while holding transcriptional complex to the DNA

Codon-Anticodon Interaction

1st 2 nucleotides in codon most important - 3rd nucleotide bins weakly and imprecisely - a single tRNA can recognize from 1-3 codons 5' end of Anticodon 1) G 2) C 3) A 4) U 5) I (inosine) 3' end of codon 1) C or U 2)G only 3) U only 4) A or G 5) U, C, or A Fewer tRNAs needed b/c of wobble and degeneracy of the codon UUU & UUC -> Phe UUA & UUG -> Leu UCU, UCC, UCA, UCG -> Ser Note: - Some tRNA molecules may contain non-standard nucleotides on their anticodons, such as inosine which can form base pairs with up to three different nucleotide

Initiation

1st Step: Small ribomsome subunit near 5' end of mRNA - facilitated by 5' cap in eukaryotes - facilitated by Shine-Dalgarno sequence in prokaryotes (small subunit has complementary16S rRNA) 2nd Step: Charged initator tRNA (carrying methionine) binds to AUG in the P site of the ribosome (translation can begin with other codons but uncommon) Large subunit then binds - 2nd aminoacyl -tRNA binds to codon on A site

DNA Sequencing Gel Eletrcophoersis

4 Diff. sequencing reactions are performed for the same template DNA w/ diff ddNTP Results are examined by gel electrophoresis to separate fragment by sizes *Smallest fragment or lowest band*: the 1st nucleotide added by DNA polymerase *Larger fragment*: nucleotide added later in the sequencing reaction First nucleotide starts at the 5' end. -------------------------------------------------------------- To read, start from the smallest to the largest fragment. Identify the sequencing each band came from allow us to read DNA sequence

Single Base Pair Substitutions

5' GC AUG GAC UAU CUC AUC ACC 3' Which of the change describes below most likely affect the function of protein coded by the RNA sequence? a) 8C > U b) 14U -> G *c) 16U -> C* C b/c the third base is changed

Is the Code Overlapping?

A) Overlapping genetic code would change 3 consecutive codons w/ each base mutation B) A nonoverlapping code would change 1 codon with each base mutation -------------------------------- *Mutations and Protein* - Sickle Cell Anemia > genetic mutaton (change in DNA) --> abnormal hemoglobin protein (can distort red blood cells - Sickle cell mutation changes 1 nucleotide: 17T>A - Sickle cell protein differs from normal by 1 amino acid Normal: Val-His-Leu-Thr Sickle C: Val-His-Leu-*Val* *Result: Only happen in info that was stored in a non-overlapping protein*

Capillary Sanger Sequencing

All reaction in 1 tube Different fluorescent labels on ddNTPs Capillary gel electrophoresis (really small)

Comparing Prokaryotic mRNa to Genomic DNA*

Another differences b/w eukaryotic and prokaryotic RNA can be seen in the sequence of RNA compared to DNA In prokaryotes, an mRNA being used for transcription would MATCH CLOSELY with specific segment of DNA used to transcribe the RNA In contrast, an mRNA used for translation in eukaryote would match up w/ multiple separate section of DNA

mRNA Vaccine

Antibodies recognize the spike protein on virus Faster ands cheaper to develop For COIVID: Spike protein 1) *mRNA sequence* Scientists generated an mRNA sequence that codes for virus spike protein 2) *Lipid coating* - the RNA sequence, a blueprint for making the spike, is swathed in a lipid coating for delivery 3) *mRNA Vaccine - Once arrives, cells read the info in mRNA sequence to produce millions of copies of the spikes protein 4) *Spike proteins -> Antibodies - The protein fragments spur the immune system to produce antibodies that can protect when a real virus enters the body

How long is a Codon?

Best guesses: - Codon would all be the same length - Codons were 3 nucleotide in length Four Nucleotide code --> 20 amino acids If the length of code is: - 1 nucleotide: 4 (4^ 1) codon - 2 nucleotide: 16 (4^2) codon - 3 nucleotide: 64 (4^3) codon - 4 nucleotide: 356 (4^4) codon *Likely length was 3 nucleotide long b/c the it is the shortest length to have enough different combination to code 20 different amino acids* --------------------------- *Crick and Brenner (1961) Isolated rII mutations using chemical that would cause insertion (+) or deletion (-) OF 1 nucleotide If triplet code was used, insertion or deletion would cause the triplet to be read incorrectly -> non functional protein Normal: A B C | A B C | A B C | A B C --> small plaque Deletion (-) Mutation A B C | A *C A | B C A | B C A* --> nucleotide read wrong and large plaque Insertion (+) Mutation A B C | A B *B | C A B | C A B* --> large plaque ^^ -------------------------------- What if a mutant phage experienced a 2nd mutation nearby (double mutant>) Could potentially restore function: a suppressor mutation Result: *- If the 1st was (+), the suppressor was (-) - If the st was (-), the suppressor was (+)* -------------------------------- Growth phage w/ mutliple mutation result - Phage w/ mulitple mutation prodiced large plaque indicating functional protein not being made - mutation w posing affects produced functional protein - some, triple mutation produced functional protein, but only when all were the same type of mutation (ex. + + +) > only happen if DNA was stored in triplet code

The LacI repressor

Binds to operator (lacO), blocks sigma factor from binding to the promoter; always present (default is OFF) Binds to the right at transcription start site of the lacZ operons and twists the DNA into a lopp preventing transcription of gene Contains 2 different functional region: 1st domain recognize and binds to the operator DNA in the lac operons 2nd domain recognize and binds lactose, allowing the lac protein to be turned off. LacI mutations that cause lacZ to be constitutively expressed either eliminate production of the lacI protein, all are allocated in the *DNA binding region*. Mutations that lactose binding domain are called *lacI^s mutations.* --------------------------------------------------------- A mutation in the lactose binding domain would prevent the lacI protein from binding to lactose Genotype / Phenotype lacI- lacO+ lacZ+ / constitutive 1) lacI^s lacO+ lacZ / B-gal expression never - lacI protein never turned off and would always bind to operator, preventing the lacZ from ever being expressed even w/ lactose present 2) laci^s lacO^c lacZ+ / B-gal expression expressed - lacI protein always active b/c of lacI^s mutations. However, the lacO^c mutations could prevent the active repressor from ever binding to the operator. Result is lacZ gene always expressed

Antibody Testing

Body produce antibody proteins against (rexognize) the virus Test look for these antibodies 1) Isolate blood plasma/saliva 2) Mix plasma w/ labeled virus protein 3) Anti-virus antibody (if present) binds to the virus protein 4) Look for antibodies 5) Check if the test is working properly Positive: Mark on T and C Negative Mark on only C

Enhancer Sequences

CC: "Expression of eukaryotic genes at high levels requires interaction with additional regions of DNA located outside the promoter called enhancer regions. Enhancer regions can turn expression of eukaryotic genes up or down and can be found both upstream and downstream of the promoter. That can also be found very far away from the gene itself." -------------------------------- Sequence needed to promote high levels of expression Can be near or far from promoter, upstream or down Recognize by transcription factor proteins Can turn expression up or down

Basal Apparatus

CC: like in prokaryotes, the RNA polymerase complex recognizes and binds to specific sequences in the promoter of a gene with the help of DNA binding proteins. In prokaryotes, this is enough to allow gene to be expressed at high levels. However, in eukaryotes, this will usually only allow a gene to be expressed at low levels called basal levels of transcription. ------------------------------ Promoter is recognize by multiple DNA binding proteins Recognize consensus DNA sequences in *cor promoter* Recruit RNA polymerase Coordinates interaction w/ other transcription factors - Required for expression - Only low levels of expression by itself

RNA and DNA Gels

Challenges: - 6 billion base pairs of DNA - 1000s of RNA and protein nucleotides ----------------------------- Difficult to see individual molecules (smear like). There are 2 ways to look for presence of specific RNA, DNA, or protein

PCR Uses

Changed how DNA was studied - could create unlimited amounts of sample DNA - can be used in a wide variety of settings > Research > Diagnistics > Forensics > Geneology > Archeology World's oldest genome sequenced from ~700,000 years old horse DNA

Lac Operon

Codes for proteins needed to break down lactose for energy. Provides a example of different ways in which gene expression can be transcriptionally regulated in prokaryotes, as well as ways gens cab be examined Lactose found in frequently in environment o compared to bacteria is usual food (glucose). To conserve energy and resources, the bacteria only want to express the lac operon when two conditions are met: 1. When lactose is present 2. Glucose is not present --------------------------------------------------------- Regulations of the lack operon is controlled by 2 other proteins 1. *Lac Repressor Protein* - active and binds to region of DNA near promoter called operator - binding of the repressor to operator reduces expression of lac operon - if it is present, lactose bind to repressor protein causing the repressor to become inactive and leave operator, which allows expression of the operon to increase 2. *CAP Activator Protein* - lac operon only way of expressing high levels when CAP activator protein binds to promoter and help recruit RNA pol. complex - can be inactviated by presence of glucose causing expression of operon to drop --------------------------------------------------------- Q) Which of regulation does the trp operon display> *1) Lac Repressor Protein present & active: Negative regulation* 2) CAP protein present & active: Positive regulation* 3) If lactose sugar is present, signal increase expression: inducible regulation 4) If glucose present, act as signal that decrease present: repressible regulation* Note: Lac Operon display positive, negative, inducile, and repressive all at once.

How is Information Organized in DNA

Crosses in fruit flies provide clues - traits inherited w/ specific chromosomes - recombination -> info is stored in discrete regions: "genes" What are genes like? *Bead Theory* (model) - genes are fundamental unit of structure and change and are indivisible: *nothing smaller than gene* Conflicted w/ new finding that DNA was composed of strings of nucleotide, so... -------------------------------------------------------------- *Seymour Benzer (1955-1959)* Bacteriophage (Phage) Experiment - Phage kill ("lyse") host cell and create plaques (holes) in a "lawn" of bacteria grown on a petri dish - Phage size depends on how quickly the phage lyse the bacteria (slower = smaller plaques) He identified 2 version of phage: 1) *wildtype (r+)*- made of small plaque 2) *Mutants (r#)* - made large plaques Experiment: infect bacteria w/ 2 diff. phage - creates bacteria that are partial (pseudo) *diploids* (contain 2 version of phage DNA) - What type of plaques do u get? Benzer identified 2 complementation groups rI and rII (2 diff genes) and examined >2.4k rII mutants and obtain results: - examined diploids, it had mutations for 2 rII gene, he obtained large plaque indicating the mutation affect the same gene - however, he also saw multiple small plaque?

How Do DNA binding protein find specific sequences

DNA binding protein scans exposed DNA region 1) Can bind loosely to 1 region of DNA and slide along the molecule until they find where they can stick more tightly 2) Can also move long distance along DNA by transferring 1 region to another and let go of DNA to move to other region of the open DNA by *diffusion*

How Gene Expression Controlled?

DNA-binding proteins are able to recognize and bind to specific sequences in DNA molecules. When they bind, these proteins can change the behavior of the DNA molecule in different ways depending on which protein has bound. --------------------------------------------------------- Ex. Prokaryotic Gene DNA---------Promoter------RNA-Coding region----- Coding 5'----TTGAGA--------------------------------3' Template 3'--AACTCT--------------------------------5' RNA polymerase core -------------- Sigma Subunit RNA pol holoenzyme ------------ Transcription start Sigma factor (DNA binding protein) help RNa pol complex recognize and bind to the promoters of genes Different sigma factors recognize different consensus sequences in different promoter Result: A single type sigma factor can control the expression of several different genes at once ----------------------- Promoter------------------------ Coarse control - additional mechanism needed to provide more precise level of regulation

LacI Footprinting

Data from a DNAse protection exit, SAM, the lac operon. - Lane 1 shows lacZ DNA without any protein added other than DNAse. -Lane 2 shows lacZ DNA with RNA polymerase added. Notice how a large section of DNA is protected by the binding of RNA polymerase enzyme. - Lane 3 shows lacZ DNA with lacI protein added. Notice how the region of DNA protected by lacI overlaps with the region recognized by RNA polymerase. This suggests that the lacI protein might physically get in the way of RNA polymerase, preventing RNA polymerase from binding to the DNA at the start of transcription.

How Do These Tools Help Us>

Detecting chromosomal abnormalities - Traditionally tools > Amniocenteses > Chorionic villus sampling > 0.5-1 risk of miscarriage Cell-Free fetal DNA testing - Fetal DNA in mother's blood stream = Sequence DNA from blood sample - Identify fetal (vs maternal) DNA - Examine for mutations and chromosome # > very low risk > can perform earlier > can test for more genetic disorder

*Nirenberg and Matthaei (1962)*

Developed an *in vitro* *cell free) system for making protein Examined synthesis using labeled (14C) amino acids --------------------------- In 1st experiment, they removed different molecules who blocked particular steps in their system - Adding antibiotic Puromycin blocks protein production - Adding DNA destroying enzyme still allows protein production - Adding RNA destroying enzyme stopped protein Result: *RNA affects protein production. More RNA = more protein --------------------------- In 2nd experiment, they added different RNA molecule 1) RNA from living cells added -> all diff. amino acids used to make protein 2) PolyU RNA molecule (Uracil only) added -> cell-free system made mostly amino acid phenylalaine Result: *Different RNA result in different protein means that RNA also direct what protein is made*

In Situ Hybridization

Different cells and tissue can express different gene, but this is how you know where gene is being expressed Detection of nucleotide sequence "n position" in the organism - sequence on chromosome - expression of RNA DNA 5' ATGATGCGA 3' ------3' TACTACGT 5' RNA 5" AUGAUGCA 3' 1) Tissue sample is treated with crosslinking agent to lock al large molecules in place 2) DNA is then destroyed, leaving RNA as the only nucleic acid left in tissue 3) Tissue is then incubated with a probe complimentary to target RNA and the excess probe is washed out 4) Location of the gene expressed is determined by where probe is stuck

mRNA in Eukaryotes

Differs in prokaryotes in many ways 1) Addition of 5' cap, which consist of a Guanine (G) nucleotide that is added to the RNA backwards, 5' to 3' 2) Long string of Adenine (A) to the 3' of the RNA called *poly-A-tails* -------------------------------- -5' capping (5 methyl guanosine) - added "backwards" 3' poly-A-tail: 20-200 adenine ribonucleotide added - Promote export from nucleus - Promote RNA stability and ribosome binding - Regulate RNA splicing (5' cap)

How are Different Cells and Tissues Created During Development

Do all cells in an organism have the same DNA? Tested by *John Gurdon 1958* whether different cells had the same DNA by removing the nucleus and DNA from frog oocytes. These cells have all the raw material to make new individuals, but no instruction 1) Adult frog 2) Skin cells in culture dish 3) McLees in pipette 4) Nucleus injected into egg 5) Nucleus destroyed by UV light 6) Unfertilized egg He transplanted a diploid nucleus from adult frog to frog egg - If embryo only formed skin cells -> Skin cells might only have DNA needed for skin cells - If embryo formed other tissues -> skin cells contained the DNA needed to make other type of cells as well Result: Gurdon found the modified eggs form a complete tadpoles, which suggest that adult tissues have all the same DNA *all tissues have the same DNA (Genomic Equivalence* Animal cloning used in mammals and primates (2018)

31 tRNA molecules and Charging Enzymes

Each tRNA also has a unique anticodon sequence with which to recognize codons in the mRNA during translation. However, though there are 61 different codons that code for amino acids, most cells have fewer than 61 tRNAs, with some having as few as 31 different tRNAs. How can 31 tRNAs be used to recognize 61 different codons? ----------------------------- Answer: The 3rd codon is less important than the first 2 and binds to the anticodon only weakly and imprecisely Result: a single tRNA can recognize more than 1 codon called *Third Base wobble*

*Blotting*

Gel -> Filter -> DNA/RNA/Protein (through filter) - molecules separated by by gel electrophoresis are transferred onto a thin membrane or filter where molecules become attached - Transferring the molecules can be doing by using vacuum or changing the orientation of the electric field - Placing the molecules onto a filter exposes the molecules and enables them to interact with other molecules, which is not possible when they are embedded inside of the gel - Once your sample is attached to a filter, the sample can be examined for the presence of a specific molecule using a prob > For DNA and RNA, a probe is usually a complimentary DNA or RNA sequence that has a visible tag attached, such as a radioactive isotope or an enzyme that produces a colored product. > For PROTEINS, the probe is usually an antibody that specifically recognizes and attaches to the protein being sought. A secondary antibody that carries a visible tag is then used to identify the first antibody ----------------------------- Protein prones *Western Blot* - protein Nucleic Acid Prons *Southern* - DNA target Northern* - RNA target Possible to combine 2 to find more than 1 type at the same time To detect the presence of a molecule, the filter is incubated with a probe for a period of time and then unbound probe that hasn't bound to any target molecules is washed off. The filter is then examined for the presence of the visible tags on the probe, where it is attached to the molecule being looked for.

Genes (transcriptional units) have different regions (E. Coli)

Gene Promotor ------- Termination 5' --*P*---C-Region---*T*--3' 3'----X-----------------Y----5' X--> +1 transcription start Y--> trascription terminated *Promotor*: beginning of gene region where transcription should begin (*Upstream*) *Termination*: end of most but not all gene region where transcription stops (*Downstream*) *Template Strand*: 5'->3' strand of DNA ready to make RNA transcription *Complementary DNA strand*: coding strand b/c same nucleotide sequence as the RNA produced by transcription w/ exception of uracil (U) instead of thymine (T) The first nucleotide added into transcribed RNA and corresponding complementary nucleotide in the DNA template is the *Plus One Position* +1 Transcription begins after promotor at +1 and continues until terminator is reached

How is Gene Expression Controlled

Genes contain 2 types of info 1) *What* should be made - RNA sequence - Amino acid sequence 2) *How* should it be made - How much - in what cells - under what conditions In the past couple of decades, researchers have come to realize that the latter information, how a gene is expressed, is just as important as the product that the gene codes for Ex. Baking a cookies - can recipe could result in small or large impact - how you bake it could make a differences too ----------------------------------------------------------

Reporter Gene

How could mutations that don't even affect protein that are being made affect a phenotype? TERT promoter -> Luciferase enzyme -> Put into tissue culture cells Researchers attached the upstream region of the telomerase gene, the promoter, and other DNA nearby to the coding region of the luciferase enzyme. The luciferase enzyme comes from fireflies and the protein it codes for produces light in the presence of certain chemicals. Cells that produce the luciferase gene more strongly produce more light. The researchers attacked the telomerase promoters with and without the mutation to the luciferase coding regions and then examined gene expression in tissue culture cells. Result: - Telomerase helps cells replicate the ends of linear chromosome - Increase in telomerase enzyme levels could enable cells to continue dividing after they would normally have stopped Q) Could mutations be responsible for melanoma risk? Yes "Increasing the likelihood a person may develop cancer"

Rho-dependent

In Rho0-depedent termination, a protein called *Rho* binds to specific sequence found in the newly produced RNA and slides along RNS towards 3' end. When RNA reaches termination signal, the RNA poly complex forms stem-loop and paauses, but the complex does not disassemble Rho has helicase activity and when it reaches the complex, it separates the RNA and DNA molecules causing transcription to stop

How Does Transcription Stop?

In prokaryotes, 2 common type of termination signal *Rho-Indepedent (E. coli)* - Inverted sequence in RNA cause stem loop structure to form in the RNA - Stem loop causes RNA pol to stall - Creates an unstable structure Rho-depedent termination (E. coli) - Inverted repeated in RNA causes stem loop structure to form - Stem loop causes RNA pol to stall - rho protein unwinds RNA-DNA duplex -------------------------------- 1) DNA pol continues to open up the DNA strands as i moved down the DNA making complimentary RNA 2) He strands comes back together after the complex has passed 3) Transcription of most gene stops when pol. complex reaches termination signal. --------------------------------

Transcription and Translation History

Information in DNA used to make protein? Clues from labs - DNA in nucleus - Protein produed in cytoplasm on large particles (ribosome) - RNA is in cytoplasm and similar to DNA (nucleotide) - Protein and RNA are both turned over (replaced) quickly. DNA is not - Infect bacteria with phage --> spike in new RNA *Francis Crick (1957) proposed "Central Dogma"* - educated guess and no proof - proposed RNA can be used as template for more RNA and DNA copies (proved by telomerase) *Nirenberg and Matthaei (1962)* - developed an *in vitro* *cell free) system for making protein - examined synthesis using labaled (14C) amino acids

LacO

Isolated constituive mutants not in LacI Map near Lac Z and LacY: *LacO^c* Genotype/ Phenotype 1) lacO^c lacZ+ / Constitutive B-gal lacI+ lacO+ lacZ+ / 2) ------------------ / Constitutive B-gal lacO^c lacZ+ / lacI+ lacO+ lacZ+ / 3) -----------------/ Expressed if lactose is present lacO^c lacZ- / (inducible) Q) What does this say how lacO region functions? *Results indicated that the operator worked insists relative to the gene being regulated. In other words, the operator could only influence gene expression of or the expression of genes that were located nearby on the same chromosome*

What is an mRNA Vaccine

Katalin Kariko (1990) use mRNA to make human cells produce specific proteins General assumption: possible but too difficult and impractical Kariko had difficulty getting funding for her resrarch and demoted 2005: Kariko and her collaborators solved some 2006-Present: 2 pharmaceutical companies begin work develop mRNA-based therapy's Kariko is now senior vice president at BioNTech for RNA based gene therapy

Jacob and Manod Proposal on Lac I - Lac Z

LacI codes for a protein Protein binds DNA and turn of lacZ and LacY LacI- mutants: don't make protein - lacI protein must recognize and bind to specific DNA sequence in the lac operon? - If the model was correct, mutations in the DNA sequence recognized by the lacI protein should have the same effect as mutants in the protein itself Screened for additional constitutive mutants located outside of the lacI gene ---------------------------------------------------------

How Do we Know if the Regions Identified Do anything?

Mess with them and see what happens *Promotor Bashing* Modify DNA that codes for visible protein product (*reporter gene*) Conserved region being examined -> transcribed region -> expression of reporter protein Remove DNA near 5' -> no expression 1) Attach DNA sequences they want to study to another region of DNA codes for a protein can be detected (reporter genes_ 2) Put the modified DNA back into an organism to see what happens 3) What is expressed? How strongly? What happens if you modified conserved region? Ex. reporter gene is expressed with a conserved region attached but not expressed if the conserved region is missing or altered, this would tell the researchers that this region of DNA was needed for transcription Result: trimming and modifying a larger region of DNA, researchers can eventually identify the specific DNA sequences that are most important.

Polymerase Chain Reaction (PCR)

Mimics DNA replication Can unlimited copies of *specific* region od DNA Extremely sensitive - 1 molecule of DNA is enough template Key Differences: - 1 enzyme: heat stable DNA polymerase (Taq) - 2 ssDNA primers (18-30 bases) > enables specificity - only regions rexognzed by primer will be replicated > requires knowledge of the region's sequence - Repilcation repeated multiple time (20-30x)

RNA-Seq

Modern DNA sequencing to examine gene expression 1) mRNA is isolated from a cell culture or tissue and convert it into cDNA 2) CNA molecules are then sequenced using massive parallel sequencing techiques 3) When each sequence is read, its location on the genome is marked Note: - The height of the bar on the chart indicates how often sequence is observed, which means how many copies of mRNA are originally presented in the sample This approach tends to be more expensive, but it does have an advantage of letting the researcher examined the expression of all genes in the sample at the same time.

Benzer Experiment Cont.

Mutation on different gene: r1 wt/ wt r2 -> all small plaques (Wilde type: rII+) Mutation on same gene: wt r2 / wt r2 -> all big plaques (Mutant rII) How did you get mostly mutant and few wild type copes in the rII gene? Benzer proposed that the mutation were at different locations on the same genetic unit (gene) --> *recombination* Ex. rII (101) --x------ non functional product rII (104) -----x--- non functional product *If recombination took place b/w mutations* rII (101/104) --x----x-- non functional product wt ----------- intact gene - functional allows the production of small plaque ------------------------------------------------------------- Why did consistently get different percentage of small plaques in his results> If mutation are located on different parts of gene, the different percentage could correspond to different *recombination rates* between these mutations - reflects how far apart the mutations are in the gene - further mutation show higher recombination rates than those that were closer When Benzer mapped the relative locations of different mutations he observed onto a map of these genes, he was able to identify mutations that were literally just one nucleotide apart.

How Do We Determine How Gene is regulated

Mutations can be used to investigate regulation of gene expression The MET operon codes for enzyme needed to use methane for energy. You examine mutations in second gene (metB) that you believe regulates the met operon. Based on the result below, how does this gene appears to regulated? *Regulated by positive inducible regulation* The way to solve these types of questions is to look at one factor at a time, either the effect of the presence, or absence of the signal, or the effect of the presence of a functional regulatory protein indicated by a plus symbol, or the absence of this regulatory protein indicated by a minus symbol. If we look at how the signal affects expression in the absence of any mutations, as is shown in Row 1, we can see that the operon is expressed in the presence of the signal methane and is not expressed if methane is absent. This indicates that expression of the operon requires the presence of the signal, which suggests that this operon is inducibly expressed. If we look at the effect of the mutation alone, the first column, we would see that the operon can be expressed if a functional regulatory protein is available metB plus, but is never expressed if the regulatory gene is mutated. This suggests that the regulatory protein is needed to increase expression of the operon, which further suggests that regulatory protein produced is an activator. Together, this data suggests that the met operon is regulated by positive inducible regulation."T

What is A Code?

Nirenberg & Matthaei: In Vitro Translation System Poly U ->(UU..) -> Phe -> UUU Poly C ->(CC..) -> Pro -> CCC Poly A ->(AA..) -> Lys -> AAA Poly G ->(GG..) ->Gly -> GGG *Identify 4 code for amnio acids* -------------------------------- Gobind Khorana Mae repeating di-, tri-, and tetranucleotide mRNAs - E.g. the repeat (UC)n makes the mRNA 5' UCUCUC.. 3' - 2 possible codon (UCU and CUC) - Polypeptides w/ alternating amino acids (Ser-Leu) *Figured most/rest of codon. However some codon didnt produced protein* - The sequence prob have stop codon to prevent production of long protein

The Last Codon

Nirenberg, Leder, and Khorana Mixed mini-RNAs (3 nucleotides) w/ ribosome and tRNA amino acids complexes 1) mixed mini RNAs with correct tRNA amino acids common molecule to form large structure 2) catches by the filters 3) all the other tRNA amino molecule pass through Results identified the genetic code

Model for Lac Operon Induction (No glucose)

No lactose - repressor binds to operator physically preventing transcription Lactose (Inducer) present - repressor cannot bind to operator - transcription proceeds

Genetic Code

Non-overlapping Codons are 3 nucleotide long More codon (64) than amino acids (20) with most (61 or 63) coding for amino acid - code is degenerate (some amino acids have more than 1 codon) - 1st 2 nucleotide most important Codon is (almost) universal)

How Does RNA Polymerase Recognize the Promoter

Often recognize "specific" sequences in DNA - sequence can be very small (<10 bp) -------------------------------- 1) RNA Pol recognize the promoter w/ the help of DNA binding protein 2) DNA binding protein recognize specific DNA sequence by forming H-bond w/ nucleotide 3) Allows protein to stick tightly to those region than others -------------------------------- In bacteria, the DNA binding protein helps RNA pol identify and bind to promotor called *Sigma Factor*

Elongation

Peptidly transferase transfer Met to second amino acid creating peptide bond Ribosome moves down mRNA by 1 codon - 2nd tRNA attached to di-peptide moves from A to P site - empty tRNA in E site exits the ribosome Cycle repeats until release factor binds to stop codon Note: - Different tRNAs in a cell have different sequences recognized by tRNA charging enzymes that attached the correct amino acid to the correct tRNA

Quantitative Real TIme PCR (qRT-PCR)

Precise way to determine how strong a gene is expressed (thick, stronger band) Isolated mRna and reverse transcribe -> *copy DNA (cDNA)* 5' GUUAU ... ACUUAAA 3' *<-------------TGAATTT 5'* - Poly T probe used to recognzied Poly A tail of mRNA and serve as a primer for reverse transcriptase to make DNA copy of RNA - Use cDNa as template for PCR - rate of amplification determined by the staring mRNA concentration Note: Dont need gels - can meausre copies as they are made

Translation

Process by which mRNA is decoded and a protein is produced Note: - The nucleotide at the end of the mRNA does ot contain info that is translated to protein. These RNA ends are called 3' and 5' untranslated regions, which help faciliate and regulate translation ----------------------------- Production of protein begins multiple nucleotide downstreams from 5' end of the RNA at the *start codon AUG* and stops several nucleotide before the 3' end of mRNA at 1 of 3 possible start codon

Trp Operon

Promoter-Operator-Enzyme for Tryptophan synthesis - trpEDCBA ----> Co-repressor Tryptophan & TrpR (inactive repressor) ----> Active repressor ---> Operator - Tryptophan binding causes allosteric change in TrpR repressor - Allows TrpR to bind to operator Q) What type of regulation does the trp operon display? - Negative regulation and repressible expression

Sigma Factor

Protein that recognize specific DNA sequence in the promoter, which helps rest RNA poly to recognize promoter to start transcription - Different Sigma factor that recognize diff. promoter and allow the bacteria to regulate the expression of large # of genes that are involved in similar practice *Using diff Sigma Factor allows coordinated expression of several genes* Examples: *Sigma Factor 32*: express gens that code for protein used by bacteria to respond to stressful situtations such as high temp. *Sigma factor 54*: recognize promoters for genes needed for bacteria to meet metabolize nitrogen Sigma factors and other DNA binding protein of diff gene are not identical in each of these genes. Instead the sequence is recognized by Sigma Factor similar to each other but a few differences. The differences in sequence could cause DNA binding proteins to bind more or less strongly to promoter and influence 1) how much gene is expressed, 2) how many copies of RNA is produced or how quickly. *COnsensus Sequence*: is the avg sequence recognized by a particular DNA binding protein. Shown in weblogo

RNA can be Single or Double stranded (RNA-RNA or RNA-DNA)

RNA is a single molecule, but it can fold and form complex base pairs with itself Allows RNA to form complex structure that are important for the different functions role played by RNA like tRNA

Example: "C" sequencing reaction

Reactions contain all dNTPS and ddCTP - 1 ddCTP for every -200dCTP *Incorporation of dCTP allows the cina to continue growing, but incorporation of ddCTP terminates chain elongation* Random insertion of ddCTP creates fragments of different sizes Mulitple replication being produced but some stops, but others continue until the next cytosine is added. Small % receives ddCTP to stop and the rest of the strands receive dCTP to continue producing Result: Different size fragment whose length correspond to the location of cytosine in the DNA sequences

Single Molecule Sequencing

Reads a single molecule of DNA (no PCR needed) Makes longer sequences (fewer fragments to align) Potential of whole human genome sequence - in a few min - < $100

Regulation of Gene Expression

Refers to production or presence of a functional product In theory, could occur at any level V - chromatin structure DNA->1->RNA->2->Protein *Translation* - Initiation - Elongation - Termination - Splicing *RNA* - stability - activity - location *Translation* - initiation '- elongation - termination - modification *Protein* - activity - location - stability

WebLogo

Represent the information content in DNA. High of the column indicates how strongly conserved a DNA sequence is at a particular location Size of the letter indicates how often a parrticular nucleotide is found at the location

DNA Sequencing

Researcher can now read DNA sequences *Sanget Didoexy sequencing* - requires ssDNA template - DNA polymerase' - ssDNA primer - All 4 dNTPs in each reaction (dATP, dTTP, dCTP, dGTP) - 4 diff. reaction tubes each w/ *didoexy nucleotide (ddNTP)* > no new nucleotide cannot be added b/c of no hydroxyl group (no O)

Terminology

Response to transcription factor addon - *Positive regulation*: protein binding INCREASES expression - *Negative regulation*: protein binding DECREASES expression Response to signal (internal or environment) - *Inducible*: signal present -> INCREASE in expression - *Repressible*: signal present -> DECREASE in expression Transcription factor proteins that - increase expression are called *activator protein* - decrease expression are called *repressor protein* Signals that - increase expression are called *inducers* - decrease expressions are called *repressors (or-repressor* when working with a repressor protein

Antibody Staining of Tissues

Similar approach can be used to identify protein locations with the antibodies that recognize protein of interest called *Immunofluoescene staining* Similar to Western blot but w/ cell/tissue as target - easier than in situ - cellular/tissue locationlization of protein produced.

Centra Dogma of Molecular Biology

Simple in theory, complicated in practice. "DNA can serve as a template for making a complementary RNA molecule through the process of transcription. The RNA molecule can move into the cytoplasm where the information stored in the nucleotide sequences is read three nucleotides at a time to join amino acids together in the correct order to make a protein." Complicated b/c requires actions of many different enzyme and other molecule to perform steps for many challenges --------------------------------Challenges: 1) *How to identify individual genes* - chromosome contain a single very large DNA molecule - problem for human's 23 diff. chromosome w/ 6 billion base pair that stores info for 20,000 gens 2) *How do we figure out where different genes begin or end* - uses the RNA molecule that cells produce by transcribing genes - compare sequence of diff. RNA molecule - Look near DNA used to make the RNA (*RNA nucleotide sequence should match region of DNA used to transcribe this RNA*) > Similar DNA and RNA found in diff. molecule are conserved

mRNA Splicing

Slicing apparatus recognize sequence at splice sites Strongly regulated of sequence inside and outside of the introns -------------------------------- Due to the process of mRNA splicing, which removes some sections of the transcribed RNA that are not used for translation *Introns*: section of transcribed RNA not use for translation and removed *Exons*: section of mRNA that are kept Before the processing of product of transcription is called *pre-mRNA* After processing, a mature RNA is produced that can be sent out of the nucleus and into the cytoplasm for translation ------------------------------ Splicing is performed by the enzyme called a *splicer zone* w/ the help of small nuclear RNA (snRNA) The splices zone recognizes the introns and exons by the presence of consensus sequences located at the ends of these regions in the RNA. The process of splicing is regulated by RNA sequences found in both the introns and exons called *splice enhancers.* Transcription and translation in prokaryotes does not involve RNA splicing. *RNA splicing can be very useful for eukaryotes because it allows a gene to be used to make more than one protein*

Differential Gene Expression

Some genes encodes product that are always needed: -> Constitutively expressed (housekeeping) Some genes are encode products only needed at certain times or in certain circumstances/environment -> Regulated expression Ex. bacteria may need to produce one set of molecules when a predator is present, and a different set when food is present. Expressing genes only when they're needed is also less wasteful of an organism's resources and energy In some cases, genes aren't expressed at the same time b/c each could conflict with each other Ex. Protein insulin signals the body to take up glucose from the blood, lowering blood sugar levels. In contrast, the protein glucagon has the opposite effect and signals the body to transport glucose into the blood, raising blood sugar levels. As a result, levels of both of these proteins must be carefully controlled to ensure that the level of sugar in the blood remains at an optimal level ---------------------------------------------------------- Ability to control expression of gene is important in multicellular organism b/c all their tissues are derived from the same original cells and contain the same genetic info However, different tissue and organs may need to respond differently to the same environment conditions Ex. Hormone Adrenaline (fight or flight) prodiced in response to emergency. - Affects blood vessels to the heart, brain, lungs, and muscles expands to increase blood floe and transport of oxygen and nutrient to these organs to work at maximum efficiency - At the same time, other blood vessels to digestive tract respond to adrenaline signal by contracting, reducing the flow of blood to these organ because the digesting a breakfast food is not a priority

How DO You Know if Someone Has Been Infected With COVID-19?

Symptoms are similar to other illness Up to 30% show no symptoms ---------------------------------------------------------- COVID Testing - Nasal Swab - Saliva - Blood PCR Testing -Look for presence of virus and very accurate 1) Nasopharngeal (NP) or Oropharyngeal (OP) swab - cotton swab inserted into nostril to absorb secretion 2) Collected specimen - stored at 2-8 C for 72 hours or proceed to RNA extraction 3) RNA extraction - purified RNA is extracted from deactivated virus 4) RT-qPCR - purified RNA is reverse transcribed to cDNA and amplified by qPCR 5) Test reports - passes threshold = positive for Covid - Slow and labor intensive New modified PCR (LAMB) is faster and cheaper

Complementation Test

Test to determine if 2 recessive mutations affect the same or different genes Note: - If mutations affect *different* gene, the DNA from 1 mutation provide a function copy of 1 gene and the so would the other mutant DNA --> small plaques - If 2 mutations affect the *same* gene, there would be no functional copies of that gene --> large plaque

Alternative Splicing

The Same RNA can be spliced different way to produce different proteins Pre mRNA molecule can be sliced in different ways to retain different combination of exons When the mature mRNA are translated, the different exons are retained can be used to make different protein w/ different functions Ex. Calcitonin gene contains six exons. In thyroid cells, exons 5 and 6 are removed and the finished mRNA is translated to produce a hormone called calcitonin, which helps to regulate calcium levels in the body. In neuronal cells, the pre mRNA is spliced differently. Exon 4 is removed and exons 5 and 6 are kept. This mature mRNA is translated to produce a different hormone, CGRP, which helps to regulation dilation of the blood vessels, appetite, and connect as neurotransmitter Note: - >90% of mammalain genes alternativelt spliced - 1/3 of diease associated mutations associated w/ splicing - roughly 20,000 genes identified in human can poteitnally produce far more than 20,000 different protein

What DNA sequence does the repressor bind?

The mutations isolated by Jacob and Manod indicated that a repressor protein recognize DNA sequences located in the operon. (Indirect evidence) DNA from lac+ operon w/ & w/o lac repressor protein - treat with DNase-I > randomly cuts DNA > *DNA protein assay (foot print analysis) - Use Gel electrophoresis and autoradiograph afterwards W/o repressor: -- --- ------ -------- W/ repressior -- --- g a p -------- ------------- -------------------------------------------------------- Other researchers (direct evidence) used DNAs protection assays, also known as a footprint analysis. DNA is an enzyme that randomly cuts DNA to create smaller DNA fragments. If another protein is attached to a DNA molecule, this can protect the DNAse from DNAse and prevent DNAse from cutting where this other protein is bound. In a DNAse protection act. Say one end of a DNA molecule is labeled with an easily detectable tag. The DNA molecule isn't allowed to incubate with the protein being examined that you think sticks to the DNA, and is then treated with small amounts of DNA's enzyme so that each DNA molecule is cut by the enzyme just once in a random location. The resulting DNA fragments are then separated using gel electrophoresis and the sizes of the different fragments are examined. Regions that DNA that are not protected will be randomly cut, producing different sized fragments. Regions that are protected can't be cut where the protein is binding to the DNA. These regions will show up as a gap in the sizes of different size fragments of DNA appearing on the gel.

Eukaryotes and Prokaryotes Transcription

Transcription in eukaryotes follows the same principle as in prokaryotes, but some differences 1) Promoter sequence are more complex with more consensus sequence 2) Transcription can be influenced by how DNA is packaged 3) Diff. RNA pol. enzymes that transcribe different genes --------------------------------

Regulation of Transcription

Usually involves transcription factors (DNA binding proteins) that influence RNA polymerase activity The activity of transcription factors themselves is often influenced by environment or cellular conditions (signals) -

REVIEW 3/11/21

When bacteriophage (phage) infect bacteria, the bacteria make new phage particles and are killed. As a phage infection spreads on layer of bacteria (a "lawn") growing on the surface of an agar plate, it causes the formation of a clear spot called a plaque. Wild-type (wt) phage cause the production of small plaques. Some mutations in phage DNA can cause a phage infection to spread faster and results in the production of large plaques. 1) If bacteria are infected with both wt and mutant phage, small plaques are produced. What does this indicate? a) Mutant phage are unable to infect bacteria b) Mutant phage phenotype is dominant to wt phage *c) Mutant phage phenotype is recessive to wt phage* 2) If bacteria are infected with two different phage that have different mutations (mut#1 and mut #1), small plaques are produced. What does this indicate a) Mut #1 is dominant to mut #2 b) Mut #1 is recessive to mut #2 c) Mut #1 and mut #2 both affect the same gene * d)Mut #1 and mut #2 affect different gene* 3) How many complementation groups (genes) can you identify? Gene 1: r47, r105, r106 Gene 2: r102, r51, *B 2* ii) What kind of plaqus would you expect a combination or r102 and r106 to make? *Small plaques b/c both on different gene* 4) Combination / % small plaques 1 and 2 / 1.7 1 and 3 / 3.1 1 and 4 /2.1 2 and 3 / 4.8 2 and 4 / 3.8 3 and 4 / 1.0 You infect bacteria with different combinations of mutations that you believe affect the same gene. Most of the plaques produced are large, but you consistently see a small percentage of small plaques as shown with your data here. Which of these maps best reflects the relative locations of these mutations? *D 3-4-1-2* 5) Which region probably contains the promotor? *VII, promotor starting point but not transcribed * - Promoter reads 3' to 5' and make 5' to 3' 6) Which is the template strand for transcription? *A) Top b/c DNA is the template b/c read 3' and 5' - bottom (5' -> 3') is the coding strand b/c same sequence of preMRNA (T instead of U) 7) Which regions correspond to introns? C) III and V b/c it isn't in mRNA* 8) Where is the start codon most likely located? *E Read mRNA 5'-3', so the start codon is in VI* - 5' untranslated 9) Which region would not be found in the DNA? *VIII b/c it's in mRNA, but not DNA* 10) 5' GCUUGGCUAAUAACUAC *AUG AAU CCA GGU CCU UCU UAG* CCU AGC UGU UCA 3" - *Start codon is AUG*, the ones before are 5' UTR Met-Asn-Pro-Gly-Pro-Set - *Stop codon is UAG and UGA* 11) How would the following mutations affect the protein coded for by this gene? *G _ A at position 19 b/c the first nucleotide is changed, which makes GGG (Gly) -> AGG (Arg)* * Deletion of U at position 13 would also affect the protean coded*