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DNA in Detail

DNA Structure & Replication
How We Discovered Genetic Material: DNA vs. Proteins 1952: Alfred Hershey and Martha Chase set out to determine whether DNA or protein was the genetic material Virus Reproduction  Viruses are made of 2 parts:  Inner core made of nucleic acid  Outer coat, called the capsid, made of protein T2 (type 2) virus reproduces inside bacteria Hershey and Chase reasoned that if they could determine which part of the virus, DNA or protein, enters bacteria to produce more viruses, they would know if genes are made up of DNA or proteins Set up 2 experiments to test this out

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Hershey-Chase Experiments  Experiment 1:  Labeled DNA with radioactive 32P (red)  Viruses were allowed to attach to bacteria and inject genetic material  Agitated mixture in a kitchen blender to shake off whatever was left on the outside of the bacteria  Centrifuged (spin at super high speed) culture so bacteria collected in a “pellet” at bottom of centrifuge tube  Tested pellet: found most of 32P-labeled DNA in bacteria and not in the liquid medium  Conclusion: DNA entered bacteria Experiment 2:  Labeled protein capsid with radioactive 35S (red)  Viruses were allowed to attach to bacteria and inject genetic material  Agitated mixture to shake off whatever was on the outside of the bacteria  Centrifuged culture to form a pellet on bottom of centrifuge tube  No 35S found in pellet; most 35S found in liquid medium  Conclusion: Radioactive protein capsids remained on outside of bacteria and were shaken off by blender into the liquid medium

Hershey & Chase Conclusion   Experiments proved that the DNA of a virus, and not its protein, enters the host, where virus reproduction occurs Thus, DNA is genetic material: it transmits all genetic information needed to produce new viruses.

DNA Structure Review      DNA is a polynucleotide Composed of 3 subunits: nitrogenous base, phosphorus group, and pentose sugar 4 bases: 2 purines (Adenine, Guanine) with double ring; 2 pyrimidines (Cytosine, Thymine) with single ring Backbone made of sugar and phosphates Double helix structure of 2 twisting strands held together by hydrogen bonds between bases (A–T, C– G)

5’ and 3’     The 2 DNA strands are antiparallel; they run in opposite directions Sugar molecules are oriented differently (see diagram below) 5th Carbon is uppermost in one strand, 5’ (5 prime) 3rd Carbon (attached to phosphate) is uppermost in 2nd strand, 3’ (3 prime)

Read the Science Focus on “Finding the Structure of DNA” (page and chapter varies between textbooks)  Who discovered the structure of DNA?  James Watson, an American biologist, and Francis Crick, a British Physicist (early 1950s)  Used previous research (including Chargaff’s rules and the work of Franklin and Wilkins) to deduce that DNA has a twisted, ladder-like structure, with sides made of sugar-phosphate molecules and rungs made of bases  Determined that A is hydrogen bonded with T, and G is hydrogen bonded with C; this model suggests that complementary base pairing plays a role in DNA replication What is Chargaff’s rule?  Regardless of the species, DNA always contains an equal number of purines and pyrimidines  the frequency of A equals that of T  the frequency of G equals that of C How did Rosalind Franklin and Maurice Wilkins contribute to the research?  Prepared an X-ray diffraction photograph of DNA  Showed that DNA is a double helix of constant diameter, and that the bases are regularly stacked on top of one another

DNA Replication    Process of copying one DNA helix into 2 identical helices Each old strand serves as a template for the formation of a complementary new strand Semiconservative: each new helix has one old (conserved) strand and one new (not conserved) strand

Video: DNA Replication Process <http://www.youtube.com/watch?v=teV62zrm2P0> Parent-Daughter DNA replication 1. 2. 3. 4. 5. Strands H-bonded to each other DNA helicase (an enzyme) unwinds and unzips the double-stranded DNA (breaks up weak H-bonds) Complementary DNA nucleotides are joined to old strand by DNA polymerase (an enzyme) DNA ligase (an enzyme) seals any breaks in sugar-phosphate bonds The 2 new double helices are identical to the original strand

Cancer Treatment   Some chemotherapeutic drugs are analogs that have similar, but not the same, structure as the 4 nucleotides. When cancer cells mistakenly use these to synthesize DNA, replication stops and cancer cells die off

Homework 1. What do we mean when we say that 2 strands of DNA are antiparallel? 2. Label the 5 Carbons in ribose and attach a phosphate group and a base to the right Carbon; connect them to another nucleotide that has its Carbons labeled too. 3. Draw 3 sugar-phosphate backbones (using pentagons and circles and sticks); label the 5’ Carbon and 3’ Carbon in each one. 4. Using 2 different colours (1 for old strand, 1 for new strand), draw a simple ladder-like DNA double helix. In a cartoon, show how the helix unwinds, how new bases are added, and why the new strands are semiconservative.

Gene Expression
 Gene: segment of DNA that specifies a specific amino acid sequence of a protein (in the 21st century, we also know that portions of a gene may help in its expression/regulation) RNA    contains the bases A, U, G, and C Single stranded, not double Does not form a double helix

3 Major Types:  mRNA: messenger RNA  Takes “message” from DNA and brings it to ribosomes (DNA stays in nucleus; ribosomes are in cytoplasm) tRNA: transfer RNA  Transfers amino acids to the ribosomes rRNA: ribosomal RNA  Along with proteins makes up the ribosome, where new proteins are made (synthesized) Transcription and Translation  Gene expression (production of a protein) requires 2 processes:  Step 1) transcription  Step 2) translation

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Transcription (in brief)    Genetic information on DNA in nucleus is transferred (transcribed) to RNA Polynucleotide is transcribed letter by letter from DNA template (master copy) to mRNA copy (actually a mirror copy, as in A  U, T  A, C  G, and G  C) The mirror copy is later used to retranslate the original message

Translation (in brief)  The RNA transcript (message fragment produced during Step 1: transcription) directs the sequence of amino acids that are added to a polypeptide

Nucleotide vs. Amino Acid Sequence   Nucleotide sequence is very different from amino acid sequence The nucleotide sequence coded by mRNA helps direct the addition of amino acids by the ribosomes Step 1: Transcription in Detail    RNA polymerase: an enzyme that binds to a promoter region and opens up the double helix (unzips it) so that complementary base pairing can occur just in this region Promoter region: region that contains special sequence of DNA that signals, “Start here!” RNA polymerase also joins the complementary base pairs to form an RNA sequence, which results in an mRNA molecule

Video: Transcription <http://www.youtube.com/watch?v=WsofH466lqk> mRNA Maturation    Before entering the cytoplasm, the mRNA must be processed. The original DNA and the transcribed mRNA contain intron and exon regions. Introns are genetic areas that do not code for proteins and must be removed before the mRNA leaves for the cytoplasm. They are called introns because they are intragene segments.  Exons are portions of the gene that are ultimately expressed.  Only the exons result in a protein product.  Modifications to mRNA: 1) A cap and tail are added to the primary RNA  Cap is composed of an altered guanine nucleotide  Tail is composed of many adenosine nucleotides. It is often referred to as a poly-A tail because “poly” means many and A stands for Adenosine. 2) Introns are removed and exons rejoined together  Splicing done by a complex of both RNA and protein (RNA is the enzyme here, not protein, and we call it a ribozyme).  Capped, tailed, spliced (so there are exons only), mRNA is now a mature mRNA fragment. All or None? Or Some?    Processing normally brings together all the exons of a gene; however, in some cases, some exons are kept while others are removed or not expressed. This results in a different protein for each different combination. We suspect white blood cell recognition of specific antibodies for each antigen encountered is a result of this. Step 2: Translation in Detail   2nd step of gene expression that leads to protein synthesis. Requires several enzymes: mRNA, tRNA, rRNA

Genetic Code

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Just 4 nucleotides for 20 amino acids! How does the code work?  If 1 base = 1 amino acid: 4 possibilities  If 2 bases = 1 amino acid: 16 possibilities  If 3 bases = 1 amino acid: 64 possibilities (refer to table on right) DNA is based on a triplet code. Codon: a 3 letter (nucleotide) unit of mRNA that codes for an amino acid. 61/64 code for amino acids 3/64 code for “stop codon”: signals polypeptide termination Methionine codon (AUG) is also the “start codon”: signals polypeptide initiation Why repetition? Why do some amino acids have many codons? (ie. several have 4 codons, and one has 6 codons)  Allows for some protection from harmful mutations (while also promoting variation)

tRNA (transfer RNA)    brings amino acids to ribosomes is single-stranded “boot-shaped” nucleic acid that loops back on itself and carries a specific amino acid to the forming polypeptide chain based on its anticodon, the CBP for a specific codon of mRNA

Codon-Anticodon  The anticodon of a tRNA-amino acid complex pairs with the mRNA codon  The amino acid is added to the forming polypeptide chain  Once the amino acid is taken from the tRNA, that tRNA leaves the area

The mRNA codon sequence determines the order that tRNA-amino acids arrive at the ribosome

Video: Protein Synthesis: Translation Process <http://www.youtube.com/watch?v=B6O6uRb1D38> Ribosomes        Small structural bodies found in cytoplasm and endoplasmic reticulum where translation occurs Composed of many proteins and several ribosomal RNA (rRNA) rRNA produced in nucleolus joins with proteins formed in cytoplasm to form 2 ribosomal subunits (the large and small subunits), which leave and join together in cytoplasm for protein synthesis 1 binding site for mRNA 2 binding sites for 2 tRNA at a time Ribosome moves down mRNA; new tRNAs then arrive and add amino acids to the growing polypeptide chain Ribosomes dissociate once protein is fully formed

Polyribosomes    Often, as one ribosome moves along an mRNA molecule, another ribosome will attach to the beginning of the same mRNA Allows many proteins to be formed at the same time from one mRNA strand The entire complex (ie. mRNA + ribosomes) is called a polyribosome (much more efficient than making a single protein from one mRNA strand)

Video: Polyribosome <http://www.youtube.com/watch?v=bJmml5KLJO8> Translation in 3 Steps:  1) 2) 3) Translation must be very orderly so that the amino acid sequence in a polypeptide is correct Chain Initiation: steps necessary to begin translation Chain Elongation: steps necessary to effect polypeptide synthesis Chain Termination: steps necessary to end the process of translation

Video: mRNA Splicing <http://www.youtube.com/watch?v=FVuAwBGw_pQ> Video: Translation <http://www.youtube.com/watch?v=5bLEDd-PSTQ> Chain Initiation    Step 1: small ribosomal subunit attaches to mRNA around start codon (AUG) Step 2: anticodon of the initiator tRNA-methionine complex binds with this codon Step 3: large ribosomal subunit joins with small ribosomal subunit in order to begin elongation

Chain Elongation  a ribosome has 2 binding sites for tRNA – each newly-arrived tRNA at site 2 receives a peptide from a tRNA at site 1

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tRNA at site 1 leaves, and tRNA at site 2 moves to site 1, making room for another tRNA to now arrive at site 2 this movement of ribosomes along the mRNA during protein synthesis is called translocation Ribozyme: an RNA enzyme that is part of the larger ribosomal subunit  Attaches peptide to newly arrived amino acid (formation of a peptide bond)  Occurs over and over as protein is synthesized  tRNAs that leave are now free to pick up a new amino acid and return entire cycle of CBP to new tRNA, transfer of peptide, and translocation is rapid (15 times/s in E. coli)

Chain Termination    occurs when protein synthesis comes to an end at a “stop codon” peptide is enzymatically cleaved from the last tRNA by a release factor tRNA and polypeptide leave, and ribosome dissociates into its 2 subunits

In short,   Step 1: Transcription – DNA base sequence is copied into mRNA Step 2: Translation – mRNA serves as a template for tRNA to bring amino acids that form a protein Genes and Gene Mutations   Gene: a sequence of DNA bases that code for a product, which is usually a protein Gene Mutation: a change in sequence of bases within a gene  May lead to malfunctioning proteins in cells (or it may not).

Causes of Mutation   3 general types:  Errors in replication  Mutagens If it occurs  in gametes, then offspring may be affected  in cells (somatic), then cancer may result  Transposons

Errors in Replication   Mistakes in DNA replication occur, but are rare DNA polymerase proofreads new strand against old strand and corrects mismatched pairs: only 1 mistake out of 1 000 000 000 replicated nucleotides occur

Mutagens  Mutagens: environmental influences that cause mutations in cells  eg. radiation (radioactive elements and isotopes, X-rays, UV light, gamma rays), certain organic chemicals (some chemicals found in pesticides or in cigarette smoke)  Mutation rate ends up being low because of repair rate and efficiency of DNA repair enzymes

Transposons  Transposons: segments of DNA that have ability to move within and between chromosomes  Movement affects neighbouring genes by increasing their expression (up-regulating) or decreasing it (down-regulating)  Jumping genes: genes that have been up- or down- regulated due to transposons; responsible for certain genetic conditions (Charcot-Marie-Tooth disease) in humans and plants (white corn kernels)

There are 2 major types of mutation: Frameshift Mutations  Frameshift Mutation: one or more nucleotides are inserted or deleted from a sequence, resulting in an incorrect reading of mRNA that leads to an incorrect amino acid sequence in the polypeptide  eg. CCC GGG AAA TTT add a T in the 2nd position  PRO, GLY, LYS, PHE CTC CGG GAA ATT T (frame has shifted 1 over)  LEU, ARG, GLU, ILE

Point Mutations: involve the substitution of one nucleotide for another

Silent Mutation: Consider CTT: what does it code for?  Substitute an A in the 3rd position: what does it code for?  Substitute a C in the 3rd position: what does it code for?  Substitute a G in the 3rd position: what does it code for?  Answer: they all code for leucine (LEU) Nonsense Mutation: Consider TGT (CYS):  Substitute an A in the 3rd position: what does it code for? Stop  Substitute a C in the 3rd position: what does it code for? CYS  Substitute a G in the 3rd position: what does it code for? TRP  Which one do you think is a nonsense mutation?  Answer: TGA is a nonsense mutation because it results in a premature stop codon. Missense Mutation: Consider the above cases. Which one(s) do you think are missense mutations? How would you define missense mutation?  Answer: TGG is the missense mutation. Substitution of an incorrect amino acid results in a change in the protein which may affect its function.

Questions  How did researchers crack the code for what each codon represents?  DNA is a triplet code because this provides enough possibilities for 4 bases to code for 20 different amino acids.  Artificial RNA was added to a medium containing bacterial ribosomes and a mixture of amino acids. By comparing the bases in the RNA with the polypeptide produced, investigators were able to decipher the code. Thinking question: at which codon position (2 or 3) do you think the most missense mutations occur? What about silent mutations? Explain your answers.  I think the most missense mutations occur at the 2nd codon position, while the most silent mutations occur at the 3rd codon position.  Looking at a chart relating base sequence and amino acid sequence, one can see that some amino acids have more than one codon. With the exceptions of leucine and arginine, these codons only differ by the base in the 3rd codon position, so changing this might still yield the same amino acid.

On the contrary, changing the base in the 2nd position often results in a different amino acid than the one intended, potentially affecting the function of the protein. Discuss with a friend the causes and types of mutations.  Errors in replication only occur in 1 out of 1 000 000 000 replicated nucleotides, since DNA proofreads the new strand against the old strand and corrects mismatched pairs.  Mutagens include environmental influences such as radiation from radioactive elements and isotopes, X-rays, UV light, and gamma rays; as well as certain organic chemicals found in pesticides or cigarette smoke. DNA repair enzymes have such a great repair rate and efficiency that the mutation rate ends up being low.  Transposons are segments of DNA that have the ability to move within and between chromosomes. Their movement affects neighbouring genes by increasing their expression (upregulating) or decreasing it (down-regulating). Jumping genes which have been up- or downregulated due to transposons are responsible for certain genetic conditions such as Charcot-MarieTooth disease in humans, and white corn kernels in plants.  Frameshift mutations occur when one or more nucleotides are inserted or deleted from a sequence. This results in an incorrect mRNA reading, leading to an incorrect amino acid sequence in the polypeptide.  Point mutations involve the substitution of one nucleotide for another. Silent Mutations do not cause a change in the amino acid. Nonsense Mutations result in a premature stop codon and a shortened polypeptide. Missense Mutations produce a different amino acid, which may or may not affect the function of the final protein. Explain again in detail transcription and translation  Transcription: The nucleotide base sequence of a DNA template is copied letter by letter into mRNA. It begins when RNA polymerase binds to the promoter region, a region containing a special sequence of DNA that signals the start of transcription. RNA polymerase unzips the helical DNA to allow CBP in just one specific region, and it also joins the complementary base pairs to form an RNA sequence and subsequently, an mRNA molecule.  Before entering the cytoplasm, primary mRNA must undergo processing to remove any introns. Splicing is done by a complex of RNA and protein, called a spliceosome.  Translation in 3 steps:  Chain Initiation: A small ribosomal subunit composed of rRNA and proteins attaches to a mature mRNA strand around the start codon, AUG. An initiator tRNA-methionine complex with the complementary anticodon, UAC, binds to the mRNA codon. A large ribosomal subunit then joins with the small ribosomal subunit to being elongation.  Chain Elongation: Ribosomes have 2 binding sites for tRNA. New tRNA arrive at Site 2 and receive a peptide from the tRNA at Site 1, which then leaves. In a process known as translocation, the ribosome moves down the mRNA molecule, so the tRNA at Site 2 is now at Site 1, making room for another tRNA at Site 2. Ribozymes attach the growing polypeptide chain to the newly arrived amino acid at Site 2 by forming a peptide bond. This occurs over and over again as protein is synthesized.  Chain Termination: Protein synthesis ends when the ribosome reaches a “stop codon.” The new polypeptide is cleaved enzymatically from the last tRNA by a release factor. tRNA and the polypeptide leave, while the ribosome dissociates into its 2 subunits.

DNA Biotechnology
Cloning of a Gene    Cloning is the production of identical copies of an organism. This occurs naturally in new plant shoots, bacterial colonies, and identical human twins. Gene cloning is the production of identical copies of a gene.

Video: How to Clone Animals <http://www.youtube.com/watch?v=dV2OxSGhwjY>. Using Recombinant DNA Technology  Recombinant DNA (rDNA – do NOT get this mixed up with rRNA) contains DNA from two or more different sources. A vector plasmid (a small accessory ring of DNA in bacteria) or virus is necessary to insert foreign DNA into a host cell. Restriction enzymes cleave the vector DNA and the source DNA at a specific sequence, leaving “sticky” ends that allow a portion of source DNA to be inserted into the vector DNA. DNA ligase then seals the openings and recombinant DNA is formed.

Cloning    After recombinant DNA enters a host cell, it may be copied. If successful, some particular gene(s) have been cloned and can be recovered. Bacterial cells take up recombinant plasmids and clone the new DNA.

Reverse Transcription    When a viral vector is used, the cloned DNA is inside newly formed bacteriophages. To express a human gene in a bacterium, it must not have introns. Reverse transcriptase can be used to make a DNA copy of mRNA; this complementary DNA does not contain introns. (see picture on the next page)

Using the Polymerase Chain Reaction     The Polymerase Chain Reaction (PCR) produces many copies of a single gene or piece of DNA. PCR requires DNA polymerase and a supply of nucleotides for the new DNA strands. Artificially cycles through a) unzipping DNA; b) copying DNA; and c) rezipping DNA. Cycle repeats 30-36 times so that with 1 starting copy, up to 235 new copies may be made.

DNA Fingerprinting  The entire genome of an individual can be cut by restriction enzymes to yield variable fragment lengths.  Gel electrophoresis separates fragment lengths.  Use of probes results in a pattern unique to the individual (sometimes called a DNA fingerprint). CSI  You can identify a person who has committed a crime, or tell who is related to whom by doing a DNA fingerprint.  Since PCR can amplify the smallest amount of DNA, a single sperm, or one cell on a cigarette butt, provides enough DNA to be identified by comparison with sample DNA. Video: How does DNA fingerprinting work? <http://www.youtube.com/watch?v=ZxWXCT9wVoI>. Biotechnology   Using natural biological systems to create a product or to achieve an end desired by human beings. Transgenic organisms have had a foreign gene inserted into them.

Video: Fincasters Episode 8 GloFish <http://www.youtube.com/watch?v=SA9PEBPnhWU>.  Transgenic bacteria are used to produce biotechnology products such as insulin, human growth hormone, t-PA, and hepatitis B vaccine. They also add insecticidal toxins to plants, reduce frost damage, degrade wastes, produce chemicals, and help mine metals. Transgenic plants: Foreign genes are added to protoplasts, plant cells that lack a cell wall, using an electric current. Foreign genes in cotton, corn, and potatoes have given them pest resistance; soybeans are made resistant to herbicide for no-till farming. Transgenic plants produce human hormones, clotting factors, and antibodies in their seed. One weed has even been engineered to produce plastic granules.

Video: What is Genetically Modified Food? <http://www.youtube.com/watch?v=jAP6ZtfP9ZQ>.  Transgenic animals: Foreign genes can be inserted into animal eggs by hand or by vortex mixing. Gene pharming uses transgenic farm animals to produce pharmaceuticals in their milk. There are plans to use animals to produce drugs for the treatment of cystic fibrosis, cancer, blood diseases, etc. In vitro fertilization is necessary.

Cloning of Animals

Cloning of mammals was once considered impossible, but has now been accomplished with sheep, calves, and goats. A 2N nucleus from a bioengineered animal is inserted into enucleated (original nucleus removed) eggs from a donor. A surrogate mother gives birth to the cloned animals.

Animal Organs as Biotechnology Products  Scientists are genetically engineering pigs to serve as organ donors for humans who need transplants. Transplants of organs across species is xenotransplantation.

Video: BioBytes Genetically Engineered Animals < http://www.youtube.com/watch?v=_6Q5oM94bOQ>  Xenotransplant concerns: Researchers are trying to make organs less antigenic to humans. One concern is whether pig organs might carry animal viruses into humans. HIV is a virus that jumped from monkeys into humans. Tissue engineering is an alternative method of securing transplant material from culturing human tissue from a mixture of cells and synthetic polymers.