Unit+3+Molecular+Genetics

Unit 3 Molecular Genetics

Summary of Topics Covered in Class: - Structure of DNA (Chemical Composition)  - Edwin Chargaff's Data (Notion of Complementary Nitrogenous Bases) - Wilkins and Franklin (Do uble Helix was suggested through x-ray crystallography results, no link to nitrogenous bases, therefore analysis was incomplete) **//* Personal Notes had to be taken from pages 276-277// ** - Watson and Crick (Accepted theory of Double Helix) **__Notes: __** **//Structure of DNA //** - DNA structure: the monomer of DNA is the nucleotide - The phosphate and deoxyribose form the rails of the molecule; while these can be either a purine or pyrimidine nitrogenous base. (Prime 3 and 5 refers to carbons in deoxyribose) **//Edwin Chargaff's Data (Early 1950s) //** - Did not believe there were equal amounts of the 4 nitrogenous bases like everyone else. - He looked at purine vs. pyrimidine (nitrogenous base) composition in different samples of DNA - He measured molar ratios of the various nucleotides  <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">- Ratios of A:T and G:C were very similar in many species investigated  <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">- Thus suggesting the pairing of these bases with each other for some reason  <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">- This lead to the notion of complementary pairs! <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">A approx= T--> in % of base in DNA <span style="font-family: 'Times New Roman',serif; font-size: 12pt;">G approx = C--> in % of base in DNA **//<span style="font-family: 'Times New Roman',serif; font-size: 12pt;">Wilkins and Franklin: Another Piece of the Puzzle //** <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- New technology of their time: X-ray crystallography <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">X ray crystallography: X rays hitting a sample of a compound (solid crystal form); Atoms in crystal deflect the x-rays in a certain way making a pattern on a photographic plate. The pattern is analyzed to determine molecular structure of the original. <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- W & F used crystallography to study the shape of the DNA molecule. <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- Both worked independently, but came to similar conclusions. <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- W--> produced some preliminary crystallographs of DNA that suggested its helical structure <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- F--> prepared purer crystallized DNA samples and was able to produce clear crystallographs. Pattern was in the shape of an X. <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">--> Suggested the sugar phosphate backbones of DNA faced __the__ outside of the molecule <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- DNA had the diameter of 2nm and 1 turn of the helix was 3.4 nm in length <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- She couldn't explain how the nitrogenous bases were in the centre of the helix <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;"> <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">//**Structure of DNA- Watson and**// **Crick** <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- Drawing on their own research and that of others they suggested a structure for the DNA molecule <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- Consisting of a double helix <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- Purine, pyrimidine pairing (AT, GC) from one helix to another <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- Pairing was held together by two hydrogen bonds b/w A and T and three bonds b/w G and C <span style="display: block; font-family: 'Times New Roman',serif; font-size: 16px; text-align: justify;">- Helices were arranged anti- parallel to one another (one spiraled up to the right, the other down to the right) HOMEWORK: Notes on Wilkins and Franklin on pg 276-277 (My notes are written above: you're welcome!)
 * Shanukha Thivyanath**
 * Thursday November. 14/ 2013**

Martha G. Monday November 18, 2013 DNA Replication
 * The Discovery of DNA Replication
 * Meselon and Stahl
 * Investigated whether DNA replication is conservative (one strand is 100% the old DNA and the second strand is 100% new DNA) or semi-conservative (both strands of DNA are 50% old DNA and 50% new DNA)
 * They used E. coil and grew it in heavy nitrogen, 15N, medium (for example a petri dish) so all nitrogenous bases of the DNA would be labelled
 * They then transferred the bacteria to a 14N medium
 * They compared the density of the bacteria in the 15N and 14N mediums which reflected the semi-conservative process
 * [[image:pierce_12_3_FULL.jpg width="540" align="left"]]
 * DNA polymers consist of nucleotides linked by 3’ – 5’ phosphodiester bonds
 * Chemically, the concentration of purine equals the concentration of pyrimidine in DNA
 * [A] = [T] and [G] = [C]
 * Heating DNA slightly changes its physical properties without breaking the 3’ – 5’ phosphodiester bonds
 * The DNA would uncoil and unzip under heat
 * DNA molecules have a double helical structure
 * The DNA double helix is unzipped with the use of enzymes
 * DNA polymerase adds new nucleotides to the now unzipped portions
 * New nucleotides are attached to the 3’ end of the newly synthesized strand by a DNA polymerase
 * Bacteria have 3 types of DNA polymerase while eukaryotes have 5 types
 * The steps of DNA Replication:
 * 1) DNA gyrase unwinds the DNA from its helical shape
 * 2) DNA helicase unzips the DNA by breaking the hydrogen bonds that hold it together
 * 3) Single stranded binding proteins (SSBs) are bound to the single stranded DNA section to keep them from pairing up or re-annealing again
 * 4) Primase puts down RNA primers that will indicate to DNA polymerase III to start replication


 * DNA cannot fully unwind due to its large size so replication occurs immediately when areas of the DNA are exposed as a single strand*
 * Replication forks and within bacteria, replication bubbles form

5. DNA polymerase III (5’ – 3’) adds the complementary nucleotides to the 3’ end of the new strand (called the leading strand) 6. DNA polymerase I cuts out the RNA primers and replaces them with appropriate DNA nucleotides 7. DNA ligase joins the gaps between the lagging strand by creating phosphodiester bonds 8. DNA polymerase I and III proofread the complementary strands 9. DNA polymerase II fixes errors in the DNA

HOMEWORK! à Read 6.4

Resources: [] [] []

Nejaha Abdulmalik November 19th 2013 Today was a study period for the Unit test Make sure to review everything from the beginning of chapter 3 to chapter 5

Topics Covered in Class: - Recall of DNA Replication (10 steps) - Easy to Understand Diagrams of the process
 * Shanukha Thivyanath**
 * November 19, 2013**

__Recall:__ DNA in prokaryotes= circular, DNA in eukaryotes = linear 1. Hydrogen bonds hold the two strands of DNA together and it is twisted in the helical shape. 2.DNA gyrase unwinds DNA. 3. DNA helicase unzips DNA by breaking those hydrogen bonds. 4. Now you have two single stranded sections BUT they have a natural tendency to re-anneal (pair up again. 5.To keep them seperate, single stranded binding proteins (SSBs) are bound. 6. Enzyme primase puts down RNA primers that will indicate to DNA polymerase III as starting point of replication. 7. DNA polymerase III (5' To 3') adds the complementary nucleotides to the 3' end of the new strand- this is called leading strand vs lagging strand (Okazaki fragments), that are built discontinuously from the replication fork. 8. DNA polymerase I excises (cuts out) the RNA primers and replaces them with appropriate deoxyribonucleotides (DNA nucleotides) 9. DNA ligase joins the gaps between the Okazaki fragments by creating phosphodiester bonds. 10. Finally, DNA polymerase I and III proof read the complementary strands.
 * Process of DNA Replication:**


 * Diagrams of the Process (Enlarge the photos in edit to see them clearly!)**
 * Homework: Pg. 294 Q- 1, 3, 4, 6, 8 + Pg. 298 Q- 1, 3**

Martha G. November 25, 2013 //**<span style="font-family: 'Times New Roman',Times,serif; font-size: 170%;">Protein Synthesis **//
 * Beadle and Tatum
 * Came up with the one gene one enzyme hypothesis which states each gene is unique and codes for the synthesis of a single enzyme
 * Experiment:
 * Neurospora (bread mould) can generally be grown on a medium of sugar, inorganic acids and salts, ammonium compounds and biotin
 * The reasoned that Neurospora used these chemicals to make the complex chemicals needed for growth and reproduction
 * They used x-rays to cause mutations in many samples of the mould cells then took the mutated cells and saw that some did not grow in the original medium anymore
 * The radiation produced mutant Neurospora which required additional specific nutrient supplements in order to grow in the medium
 * Their hypothesis:
 * A mutation has blocked a metabolic step leading to the synthesis of a specific compound
 * Process of Protein Synthesis
 * DNA doesn't replicate, it encodes to be decoded
 * DNA is synthesized during replication but it's transcribed during protein synthesis:
 * DNA acts as an original tape, preserving information while temporary transcripts make use of the information
 * The transcripts are made in the nucleus and consists of RNA molecules called mRNA
 * The mRNA is then translated into polypeptides in the cytoplasm which eventually becomes active proteins
 * There are three major classes of RNA:
 * mRNA - The information that is transcribed into the final protein
 * tRNA - Transfers appropriate amino acids to the ribosomes determined by mRNA
 * rRNA - Structural components of the ribosomes site of protein synthesis
 * STAGE ONE: Transcription
 * Information is transcribed onto another medium (from DNA to RNA)
 * RNA polymerase transcribes DNA and read 3' to 5'
 * The synthesized single strand of RNA is complementary to the one strand of DNA which essentially is mRNA
 * mRNA contains coding information for a specific protein
 * Post-transcriptional Changes:
 * Addition of 5' cap consisting of 7 G's which protects the mRNA from digestion of enzymes
 * Removal of introns (non-coding regions and useless) using spliceosomes
 * Addition of a poly-Adenine tail (20 to 30 long) which extends the life of the mRNA molecule
 * Overall Process of Transcription
 * RNA polymerase does not require a primer
 * RNA polymerase begins unzipping the DNA at an initiation sequence called a promoter region (TAC)
 * RNA polymerase reads 3' to 5' and complements the DNA strand
 * The first bit of "writing" is the 5' UTR (untranslated region) followed by the exons and introns then ends with 3' UTR which is essentially mRNA
 * It proceeds until it reaches the termination sequence signal in RNA is released and processed
 * Transcription ---> making mRNA and post-transcriptional changes

HOMEWORK ---> 7.2 Questions #1 - 6 ---> If the diagrams in class is not in your possession, then copy it off of a friend!!!

November 26, 2013
 * Annie Vimalanathan**

__** 7.3 Translation **__
 * At the ribosome
 * We read the mRNA in the 5' to 3' direction
 * Also read mRNA in TRIPLETS aka CODONS
 * Begin at start codon [ AUG ] found at the beginning of an exon
 * There are 3 stop codes/ codons [UGA,UAA,UAG]

1) What is involved
 * AMINOACYL SITE a.k.a the A site is for READING the mRNA and retrieving the correct tRNA
 * PEPTIDYL SITE a.k.a the P site is for HOLDING onto the amino acid and forming peptide bonds
 * EXIT SITE is a.k.a the E site is where the empty tRNA is moved, then released.
 * As the ribosome moves along the mRNA, it will read it and each codon on the mRNA will go into each site [ A site --> P site --> E site]

2) Transfer RNA
 * tRNAs are the smallest types of RNA
 * On the bottom (opposite of the 3' end) you will find the ANTICODON - a three nucleotide segment that pairs with a codon in an mRNA
 * tRNAs are active or non active, which is determined by whether it has an amino acid attached or not
 * This process of adding an amino acid to a tRNA is called aminoacylation
 * The a.a is added to the 3' end of the tRNA via an ester bond
 * It is done by an enzyme called synthetase
 * The final product after is called an aminoacyl-tRNA

__ Step by Step: __

//__ INITIATION: __//
 * 1) Small ribosomal subunit binds to mRNA and scans it until it finds the start codon [AUG]
 * 2) [AUG] is sitting in the P site
 * 3) Active tRNA enters the P site and matches its ANTICODON to the CODON
 * 4) This matching signals for the large ribosomal subunit to bind

__// ELONGATION: //__
 * The ribosome will read the mRNA codon by codon. This is also known as the reading formula
 * 1) A site "reads" the second CODON. The correct aminoacyl - tRNA binds in the A site
 * 2) Peptidyl transferase breaks the ester bond of the 1st tRNA and creates the peptide bond that connects the 1st amino acid to the 2nd amino acid (that is still connected to the 2nd tRNA)
 * 3) Ribosome SHIFTS the reading frame
 * non-active tRNA enters into the E site, then exits the ribosome
 * 2nd tRNA enters into the P site
 * 3rd CODON enters into A site ... (repeat step 5-7) until a stop codon enters

__// TERMINATION: //__


 * 1) Stop codon enters into the A site
 * 2) Instead of an aminoacyl- tRNA entering, a protein release factor binds to the site
 * 3) Polypeptide is released from the ribosome. Ribosome subunits separate and protein release factor and mRNA are released

For more information visit: [] [] []

Homework: Page 331 #1,2,6,8 > Initiation step of Translation was drawn out. Copy this from a friend if you do not have it.

Annie Vimalanathan November 27, 2013

Chapter 6 quiz took place. Homework: Bring in Field Trip Forms and Money

=**Saad Sheikh**= =**November 28, 2013**=

**Overall Process of Trancription**

 * RNA polymerase does not require a primer
 * RNA polymerase begins unzipping the DNA at an INITIATION SEQUENCE called a promoter region (TAC)
 * RNA polymerase reads in the 3' - 5' direction
 * RNA polymerase then complements the single DNA strand
 * The first bit of "writing" is the 5' UTR (untranslated region) followed by the exons and introns and ends with the 3' UTR
 * This newly transcribed RNA is the mRNA
 * it proceeds until it reaches the termination sequence signal in RNA is released and processed

Control of Gene Expression

 * Gene expression is controlled at various levels**
 * There are four types of control in Eukaryotic Cells**
 * 1) **Transcriptional**
 * regulates which genes are transcribed
 * controls the RATE of transcription
 * 1) **Post - transcriptional**
 * removes introns
 * 5' cap and 3' poly A tail added
 * 1) **Translational**
 * how often and how fast mRNA is translated
 * length of time for mRNA to be active
 * Poly A tail: shorter tail --> shorter lifespan
 * 1) **Post - translational**
 * Modifications to proteins in Golgi: addition of functional groups or cleave of parts of the Protein


 * Example 1: lac Operon (negative feedback control)**


 * found in E. coli: breaks down lactose into glucose and galactose as its source of energy
 * it uses the enzyme B-galactosidase
 * a negative control system is used to block the production of B-galactosidase until it is needed in the bacteria
 * the gene for the enzymes are normally turned off
 * an activator needs to be present
 * once lactose is broken down, the [lactose] decreases and the inhibitor is free to find to the operator again
 * this system helps the bacteria's survival by limiting the energy used for the manufacturing of enzymes


 * Operon -** a cluster of genes under the control of one promoter and operator
 * Operator** - regulatory sequence where the repressor protein will bind


 * In this specific example:**
 * lac operon has 3 genes that code for proteins
 * lac Z - b-galactosidase
 * lac Y - b-galactosidase permease
 * lac A - unknowon functioning protein
 * repressor protein = lac I binds to the operator
 * Inducer = lactose

> pg. 339 #1-6
 * Homework**
 * build project

[] []
 * References**

November 29, 2013
 * Rajavi Kanagaiyah**

We had a supply in class and used the time to: -Read page 340-345 and make notes -Do questions 1-8 on page 345 -Work on our "build" project

Martha G. December 02, 2013

CONTINUATION OF LESSON ON NOVEMBER 28TH!


 * Example 2: trp Operon
 * Found in E.coli
 * It needs tryptophan to produce proteins
 * Is a positive control system:
 * The gene of the enzymes are usually "on"
 * When tryptophan is present, trp binds to the repressor
 * This creates a trp - trp repressor complex that binds to the operator region and prevents transcription
 * This system helps the bacteria survive via limiting energy used to manufacture enzyme only when needed (i.e. when trp is not present)
 * In this example:
 * Trp operon has five genes that code for five polypeptides that make three enzymes used to synthesize trp
 * Repressor protein = inactive trp repressor and tryptophan



Under Normal Conditions:
 * The cell needs trp.
 * The trp repressor proteins is inactive
 * RNA will transcribe
 * The concentration of trp will increase

When trp is present:
 * Trp will bind to the inactive repressor forming a trp - trp repressor complex which binds to the operator
 * RNA polymerase cannot transcribe
 * The concentration of trp will decrease

HOMEWORK: Build Project !!!!

Topics Covered in Class: - Notes on Plasmid Construction - Notes on Plasmid Mapping - Examples of Plasmid Mapping Questions
 * Shanukha Thivyanath**
 * December 4, 2013**

__Plasmid Construction: How Plasmids are made__ - A Plasmid contains numerous sites for restriction enzymes to act - Are single super-coiled circular pieces of DNA inside bacteria - Not the genomic DNA of the bacteria a specific characteristic (gives them an advantage) i.e ampicillin -Plasmids can be designed as vehicles to transfer genetic information into a cell. These are called vectors. i.e used in insulin production - A cell that can take up foreign DNA and express the products of the gene are called competent.

__Plasmid Mapping__ - A restriction map is a diagram showing the restriction enzyme sites on a plasmid - It includes the distances (measured in base pairs) between the sites - This allows molecular biologists to determine which plasmids are best suited to create a given recombinant DNA To create a map. scientists first cut the plasmid with the different restriction enzymes (alone and in combination) and determine the lengths of the fragments formed by cleaving (using gel electrophoresis) -They then use the fragment length data to create their map vectors are created by using restriction enzymes to insert the gene of interest.

__Things to Consider when using Plasmids:__ -Plasmids may have many restriction sites, but the one you use must occur only once (and have a sticky end if possible). - The point of insertion should be downstream of an existing control sequence like lac or trp operons -This allows for control of eventual gene product - The gene of interest must have no introns or control sequences - Genes must insert in the correct direction

__Examples with Diagrams__

Homework: Finish Plasmid Mapping Worksheet, front and back. **Try Level 3 Questions! Quiz is coming soon! = = = = =**Saad Sheikh**= =**December 5, 2013**=

**Gel Electrophoresis**

 * **Gel electrophoresis** is a technique used to separate pieces of DNA based on their size (kb) kilobases
 * samples are inserted into each well
 * one sample is the control: it has fragments of known molecular sizes aka "the standard" - aka standard ladder
 * set up the voltage standard - positive at the bottom, negative at the


 * Recall: DNA is negatively charged due to the phosphate groups
 * when the current is turned on, the negatively charged fragments will travel towards the positive electrode
 * the shorter the fragment, the faster it will travel through the gale (made of agarose)
 * to see the DNA fragments, we use a loading dye
 * it is made up of a dye (Ethidium bromide) + glycerol (heavy molecule)
 * gel electrophoresis can be used for __protein separation__ as well
 * it would use gels made up of a polyacrylamide (smaller pores)


 * samples are applied to each of the wells, with an "X" sample containing fragments of known molecular mass (standard)
 * Apply the voltage across the gel with the positive at the bottom and the negative at the top
 * gel electrophoresis involves the separation of charged molecules on the basis of size/mass
 * the medium used is some kind of gel, that acts as filter screen or sieve
 * DNA is negatively charged due to the phosphates, and may be combined with SDS (a type of detergent)
 * Once loaded into the gel, a charge will attract the DNA towards the positive end with the smallest molecules moving the most quickly
 * the gel can be thought of as a forest, and the DNA samples like people running through
 * if there are more people holding hands together (10 or 20) it will be harder (slower) to run through the woods
 * the people that are running alone or only in pairs will be able to run faster through the woods




 * Homework**
 * work on plasmid mapping questions
 * build project


 * Reminder**
 * plasmid mapping quiz on Monday

[] []
 * Helpful Links**

=**Saad Sheikh**= =**December 6, 2013**=


 * Polymerase Chain Reaction**

Purpose: to amplify a gene fragment (like one for insulin)
 * 1) Given a short segment of DNA, heat it to 95 degrees Celsius (replaces gyrase and helicase) until it dissociates into single stranded (ssDNA)
 * 2) Cool it to about 57 degrees Celsius. Incubate the single stranded DNA with a forward and a reverse DNA primers (5' - 3'). This allows the primers to anneal (stick to) the ssDNA
 * 3) Heat it to 72 degrees Celsius, then add Taq polymerase (heat insensitive) and allow new complementary strands to form. (Why can't you use DNA polymerase III? Because the temperature is too high)
 * 4) Heat the strands up to 95 degrees Celsius to dissociate the new pieces and the templates
 * 5) Cool it to 57 degrees Celsius again and allow the primers to anneal, and heat to 72 degrees Celsius to allow Taq to replicate the DNA (Repeat the heating/cooling, synthesizing)
 * 6) After the second cycle, the new pieces have gone from primer to primer exactly. They are shorter than either the original DNA of the first copy.
 * 7) Continued cycling causes an exponential increase in the number of short but "consistent"/ constant length pieces. Eventually the proportion of strands that are "odd"/ variable lengths will be so small as to be insignificant

==
 * Applications of PCR:**
 * forensic criminal investigations
 * DNA in a hair follicle or saliva is VERY LITTLE
 * Use PCR to amplify the DNA so that various tests can be used on it (ex. paternity tests)
 * genetic testing

**Restriction Fragment Length Polymorphism (RFLP)**


 * a technique in which organisms may be differentiated by analysis at patterns derived from digestion of their DNA
 * if two organisms differ in the distance between sites of digestion of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme
 * patterns generated can be used to differentiate species (and even strains) from one another


 * VNTRs (**variable number of tandem repeats) aka microsatelites
 * a noncoding region of DNA
 * often used in RFLP
 * a sequence of base pairs that repeat over and over (eg. TAGTAGTAGTAGTAG)
 * between individuals, VNTRs can vary in length and position within the genome


 * DNA Sequencing**


 * a sequence of DNA is obtained and divided into four test tubes
 * Each tube contains everything necessary for DNA replication
 * also each tube also has a small percentage of nucleotides made with ddDNA (deoxyribonucleic acid)
 * Each tube has different ddDNA nucleotide
 * As the DNA is synthesized, occasionally a ddDNA is used
 * this stops the synthesis at that point due to the absence of an -OH and Carbon #3 of the deoxyribose
 * Each tube has various sized pieces of DNA each stopped at the same base
 * By running each sample in parallel in gel, each piece can be separated in gel

**Homework** page 385, 1-7 Build project Plasmid mapping worksheets

**Helpful Links** []

=**December 9,2013**=
 * Annie Vimalanathan**


 * Plasmid Mapping Quiz took place**

__**Transformation**__
 * This is the introduction of DNA from another source into a bacterial cell**

__**The Process**__

1. Bacteria are put into a solution of Calcium Chloride 2. The solution is set at 0 degrees 3. Now foreign DNA is introduced 4. The solution is subjected to a sudden heat treatment of 42 degrees for about 90 seconds 5. A nutrient broth is added and the solution incubated at 37 degrees 6.The solution is placed into agar that contains an antibiotic.
 * This creates pores in the membrane and the calcium ions "neutralize" the negative charges on both the cell membrane and the plasmid
 * This makes the cell membrane less fluid
 * This DNA is also neutralized by Calcium ions
 * This creates a draft that sweeps in the DNA into the bacterial cell
 * This helps the bacteria recover and begin to grow

=**December 10, 2013**=
 * Irteza Junaid**
 * ==== It was an ISP today. ====
 * Good Luck for tomorrow's Test everyone!

=December 13th 2013= Tahrik Rodriques

Today we performed the pGLO Lab, in hopes that our plasmid colonies glow green under UV light.

Homework: Finish the DNA "build' project that is due on Monday

Have a great weekend, and good job to all those that performed or helped in the musical!