DNA Pre-AP 2013
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    DNA Pre-AP 2013 DNA Pre-AP 2013 Presentation Transcript

    • Chapter 12: DNA and RNA Biology S.Rucker
    • DNA DNA- Deoxyribonucleic acid A large polymer used to carry the genetic code of all living organisms
    • DNA – Heredity & Structure What we know about DNA was not discovered overnight! Many different scientists contributed information. Because of the efforts of all these scientists, we now have a model of DNA that consistently fits the observations we make. It also allows us to make useful predictions!
    • DNA History Oswald Avery  (1944)  genes are composed of DNA Rosalind Franklin   (1952) studied the DNA molecule using a technique called X-ray diffraction HERSHY – CHASE – DNA & Viruses James Watson/Francis Crick (1953)  Developed the double helix model of DNA structure
    • Griffith‘s Experiment- 1928 Was trying to develop a vaccination for the pneumococcus bacteria.  Vaccine- a prepared substance from killed or weakened disease causing agents used to prevent future infections He was working with two strains of bacteria.   Rough - bacteria had a rough appearance in culture, non-virulent (doesn't kill) Smooth - bacteria had a smooth appearance in culture, virulent (kills) He discovered that something was being transferred between the ―dead‖ smooth bacteria and the living rough bacteria that caused them to undergo transformation.  Avery, MacLeod, McCarthy identified that DNA was being transferred killing the rats in 1944
    • Avery, MacLeod and McCarthy- 1944 1. Avery, MacLeod, McCarthy (1944)- proved that the transfer of DNA is what killed Griffith‘s rats 1. took extract (from heated smooth bacteria) and treated it with DNAase (destroys DNA) - then mixed with rough bacteria and injected into rats -> the rats lived 2. in other side of experiment, treated extract with protease (digests proteins) -then mixed with rough bacteria and injected into rats -> rat died This showed that DNA, not protein, has ability to transform cells
    • Heat-killed, disease-causing bacteria (smooth colonies) Disease-causing bacteria (smooth colonies) Harmless bacteria (rough colonies Dies of pneumonia Lives Heat-killed, diseasecausing bacteria (smooth colonies) Control (no growth) Lives Harmless bacteria (rough colonies) Dies of pneumonia Live, disease-causing bacteria (smooth colonies)
    • Erwin Chargaff- 1950 Base pairing rule is A-T and G-C Thymine is replaced by Uracil in RNA Bases are bonded to each other by Hydrogen bonds Discovered because of the relative percent of each base; (notice that A-T is similar and C-G are similar)
    • Chargaff‘s Data Source of DNA A T C G Streptococcus 29.8 31.6 20.5 18 Yeast 31.3 32.9 18.7 17.1 Herring 27.8 27.5 22.2 22.6 Human 30.9 29.4 19.9 19.8
    • Hershey and Chase- 1952 Hershey and Chase proved that the genetic material is DNA in 1952. Previously, scientists thought that proteins were the hereditary molecule Hershey and Chase used radioactively labeled bacteriophages (viruses) to determine that DNA was being injected by the viruses instead of proteins. This proved that DNA was the hereditary material of life.
    • Martha Chase (left) & Alfred Hershey (right)
    • Virus Structure DNA is located in the head. The outside and tail of the virus is made out of protein.
    • Virus ATTACKS!!
    • Bacteriophages ATTACK!!
    • Hershey – Chase Experiment – DNA in Viruses Bacteriophage with phosphorus-32 in DNA Bacteriophage with sulfur-35 in protein coat Phage infects bacterium Phage infects bacterium Radioactivity inside bacterium No radioactivity inside bacterium
    • Wilkins & Franklin- 1952 MHF Wilkins and Rosalind Franklin studied the structure of DNA crystals using X-rays. They found that the crystals contain regularly repeating subunits. The pattern generated by the diffraction of the x-rays suggested that the overall structure of DNA was a double helix.
    • Watson & Crick- 1953 James Watson and Francis Crick used Chargaff's base data and Franklin‘s X-ray diffraction data to construct a model of DNA. Their model showed that DNA is a double helix with sugar-phosphate backbones on the outside and the paired nucleotide bases on the inside, in a structure that fit the spacing estimates from the X-ray diffraction data. The paired bases can occur in any order, giving an overwhelming diversity of sequences.
    • Watson & Crick with their model of DNA
    • DNA Structure
    • There are 3 main components of a strand of DNA 1. DNA is a large polymer (macromolecule) Made up of monomers called nucleotides. Nucleotides A nucleotide is made of 3 parts: 1. 2. 3. phosphate functional group Nitrogen base (A, T, G, C) Deoxyribose sugar (in DNA)
    • Nucleotide Hydrogen bonds Phosphate Nitrogen base Sugar-phosphate backbone Key Adenine (A) Thymine (T) Cytosine (C) Guanine (G)
    • DNA Structure Dna twists into a double helix due to the attraction between the negatively charged phosphates and net positive charge of the hydrogen bonds between the Bases - DNA has an overall negative charge due to the phosphates of the sugar/phosphate backbone • ―Rails‖ of the ladder are made of alternating sugar and phosphates The nitrogen base (A, T, G, C) are always attached to the deoxyribose sugar
    • Nitrogenous Bases Two types:   Purines (two rings) Pyrimidines (one ring) Purines  Adenine and Guanine Pyrimidines  Thymine and Cytosine
    • Nucleotide Hydrogen bonds Phosphate Nitrogen base Sugar-phosphate backbone Key Adenine (A) Thymine (T) Cytosine (C) Guanine (G)
    • Practice Pairing… TEMPLATE STRAND A T C G G C G C T A A T
    • Bonding TEMPLATE STRAND A T C G G C G C T A A T Weak HYDROGEN bonds form between the Nitrogen Base Pairs.
    • The backbone of it all… TEMPLATE STRAND A T C G G C G C T A A T The backbone is made of alternating sugars and phosphates. - Remember: Sugar ALWAYS attaches to the Nitrogen base
    • Chromosome Coils Supercoils Histones
    • DNA Replication Part 2
    • DNA & RNA continued! 1. Before mitosis (during S phase of interphase) , a complete copy of a cell‘s DNA is made through a process called DNA replication. 2. When a cell divides, each daughter cell gets one complete copy of the DNA. 1. Similar to photocopying a document – the end result is two identical documents that contain the same information.
    • Step 1 1) DNA must unwind and break the hydrogen bonds. DNA Helicase ―unzips‖ the strands. 1. DNA Helicase- the enzyme that unzips DNA like a zipper so it can be copied. The area where the DNA is split is called the replication fork.
    • Step 2 2. Each strand of original DNA is used as a template (blueprint). DNA Primase ―flags‖ or ―marks‖ the spot for it to begin  DNA Primase- Begins DNA replication by attaching a short fragment of RNA called a primer to the place where replication will begin. This primer ―tells‖ DNA polymerase where to start copying DNA replication Created by DNA Primase Replication Fork
    • Step 3 1. DNA Polymerase- makes the new strand of DNA like a copying machine. It can only read in one direction 3‘ to 5‘ just like how we read a page from left to right. As a result, the new strand it makes is made in the 5‘ to 3‘ direction. 1. 2. Leading strand- the continuous strand that DNA polymerase makes in the 5‘3‘ direction. It never stops once it starts until it reads the entire strand of DNA. DNA replication 5' to 3‗ Lagging strand- DNA polymerase can only read in the 3‘ to 5‘ direction, it must make the new strand in small chunks. These small chunks are called Okazaki fragments. They are normally between Direction of replication 100-200 base pairs long. 2. DNA Ligase- connects okazaki fragments together on the lagging strand to make a complete strand
    • 1. Because of Chargaff‘s rule, only the correct, complementary bases will fit, so chances are good that the DNA polymerase will make a perfect copy. 2. What would happen if DNA polymerase made a mistake? How long do you think these animals will survive?
    • Protein Synthesis Transcription
    • DNA‘s Purpose DNA has genes that code for the synthesis (creation) of specific PROTEINS Here‘s the problem…  Where is DNA located? Nucleus  Where does Protein Synthesis occur? At ribosomes in the cytoplasm  Can DNA ever leave the nucleus? No.
    • RNA Ribonucleic acid Single-stranded Sugar is ribose Thymine is replaced by URACIL 3 types of RNA 1) Messenger RNA (mRNA) o carries information from DNA to ribosome 2) Transfer RNA (tRNA) o Carries amino acids 3) Ribosomal RNA (rRNA) o Makes up ribosomes
    • RNA can be Messenger RNA also called Ribosomal RNA which functions to mRNA also called rRNA Carry instructions which functions to Combine with proteins from to to make up DNA Ribosome Ribosomes Transfer RNA also called tRNA Bring amino acids to ribosome
    • Differences between DNA and RNA RNA Structure: Single stranded  Sugar:  Ribose Bases: Adenine Guanine Cytosine Uracil DNA Structure:  Double stranded Sugar:  Deoxyribose Bases: Adenine Guanine Cytosine Thymine
    • Transcription1. Transcription- creating a strand of mRNA from an original strand of DNA 1. occurs in the nucleus!!!
    • Steps of transcription 1. Just as DNA polymerase copies DNA, a similar enzyme called RNA polymerase makes new RNA from the DNA strand. 2. RNA polymerase temporarily separates the strands of a small section of the DNA molecule which exposes some of the bases of the DNA molecule. 3. Along one strand of the DNA, the RNA polymerase binds complementary RNA nucleotides to the exposed DNA bases and makes a strand of mRNA. 1. 2. It is called messenger RNA because it carries DNA‘s message out of the nucleus and into the cytoplasm. mRNA is SINGLE STRANDED! A=U T=A C=G G=C
    • 5. When the RNA polymerase is done reading the gene in the DNA, it seperates from the DNA 6. The separated DNA strands reconnect, ready to be read again when necessary. 7. mRNA moves out of the nucleus and finds a ribosome RNA polymerase mRNA DNA
    • Translation Translation- (also known as protein synthesis) making a protein from the instructions found on mRNA. These instructions are originally found in genes. 1. A gene is a region of DNA that contains the instructions for making proteins. This is why we refer to DNA as the ―blueprints‖ Protein
    • Where does this happen? Where is the DNA located? Where are proteins made in the cell?
    • Genetic Code Genetic code – the language of mRNA instructions (blueprints)  Read in three bases (codon) at a time by a ribosome Codon found on mRNA; consists of three bases (one right after the other)   There are 64 different codons that code for 20 amino acids Each codon ―codes‖ for a specific amino acid • Ex: Consider the following RNA sequence: UCGCACGGU • The sequence would be read three base pairs at a time: UCG – CAC – GGU • The codons represent the amino acids: Serine – Histidine - Glycine
    • Special codons- Start and Stop AUG – start codon which codes for the amino acid Methionine. All protein chains begin with this UAA, UAG, UGA – These three codons are ―stop‖ codons. When a ribosome reaches these codons it tells the ribosome to end the protein chain.
    • Ribosomes- the protein ―factory‖ 1. Ribosomes are organelles in the cell designed to make proteins by reading mRNA made during transcription 2. Ribosomes are found in two main locations in a cell1. 2. Rough ER Freely floating in the cytoplasm 3. Ribosomes are made of rRNA 4. Ribosomes have two main parts or ―subunits‖ that attach to mRNA to ―read‖ it. 5. A ribosome can fit two ―codons‖ inside of it at a time
    • tRNA (transfer RNA) tRNA carries (or transfers) the correct amino acid to the codon on the mRNA. One end of the tRNA has an ANTICODON that is paired with the codon on the mRNA strand There are 1000‘s of tRNA‘s floating around in the cytoplasm to be used for translation
    • Step 1 of Translation (protein synthesis) 1. mRNA is made during transcription. It then leaves the nucleus and combines with a ribosome. The ribosome then reads the mRNA to make a protein
    • Translation (don‘t copy) mRNA GUA UCU GUU ACC GUA •Codon: a sequence of 3 nitrogen bases on mRNA that code for 1 amino acid •It‘s a TRIPLET code •Example: This strand of mRNA has 5 codons, so it would code for 5 amino acids.
    • Translation (don‘t copy) mRNA GUA UCU GUU ACC GUA Ribosome •The mRNA molecule travels to the ribosomes where the mRNA codes are ―read‖ by the ribosomes •Ribosomes hold the mRNA so another type of RNA, transfer RNA (tRNA) can attach to the mRNA
    • Step 2 of translation mRNA GUA UCU GUU ACC GUA CA U A G A Ribosome Covalent bond VAL SER 1. As the ribosome reads down the mRNA strand, it will pair each mRNA codon with the correct tRNA anticodon. 2. Remember, only 2 tRNA‘s can fit in a ribosome at a time 3. After it has been paired, a covalent bond will form between the amino acids creating a chain of amino acids also known as a protein
    • Translation mRNA GUA UCU GUU ACC GUA CA U A G A CA A
    • Translation- step 3 The ribosome will read through the entire strand of mRNA making a protein in the process until it reaches a ―stop‖ codon. Once it reaches a stop codon, the ribosome releases the mRNA and the protein is completed. Protein Synthesis Video
    •  As the ribosome reads the mRNA strand, amino acids linked together to form a protein. The new protein could become cell part, an enzyme, a hormone etc.
    • Protein synthesis in prokaryotes vs eukaryotes Prokaryote vs eukaryote protein synthesis Prokaryotes lack a nucleus. While RNA polymerase begins making the strand of mRNA from the template DNA, the ribosome floating around in the cytoplasm can simultaneously read the mRNA strand that‘s being made and translate it into a protein In Eukaryotes, the mRNA strand must first exit the nucleus through a nuclear pore before it can be translated into a protein
    • Mutations
    • Point Mutations- Substitutions  Point mutations – mutations involving changes in one or a few nucleotides in a DNA sequence. Point mutations come from a substitution in the copied DNA strand  Substitutions – one base is changed to another  ATGC  AAGC  3 types of point mutations:    Silent mutation- No change in the protein Missense mutation- changes one amino acid (missense)  Sickle-cell anemia is caused by this Nonsense mutation- Inserts a pre-mature STOP codon
    • Frameshift Mutations  A frameshift mutation occurs when the “reading” frame of the ribosome is changed.  How frameshift mutations can affect the protein:  This may change every amino acid that follows the point of the mutation.  Can alter a protein so much that it cannot perform its function.  Frameshift mutations can come from 2 different changes to the DNA sequence  Insertion – a extra base is inserted into the original strand of DNA  Deletion – a base is removed from the original strand of DNA Frameshift due to insertion Frameshift due to deletion
    • Guess the mutation… Deletion Substitution Insertion
    • Significance of mutations  Mutations can be neutral, beneficial, or harmful  Neutral mutations  Generally have little or no effect on an organism.  Beneficial mutations  May produce proteins with new or altered activities  Useful to organisms in different or changing environments  Plant an animal breeders take advantage of these  Polyploidy often results in larger, stronger plants.  Bananas and other citrus fruits have been made polyploid.
    •  Harmful mutations  Can cause dramatic change in protein structure or gene activity  Defective proteins can disrupt normal biological activities  May result in genetic disorders Normal Fruit fly face Antennae replaced by legs
    • Mutations & Inheritance  Mutations in somatic (body) cells affect only that organism, but the effects can be dramatic.  Harmful mutations cause many forms of cancer.  Mutations in gametes (sperm & egg) are passed along to offspring.  These mutations become the basis for new genetic variation within a species, which is important to understand evolution.
    • Chromosomal Mutations Mutations can also occur when a chromosome is changed. A chromosomal mutation is a change in the number or structure (genes) of chromosomes. 4 main types of chromosomal mutations:     Deletion Duplication Inversion Translocation
    • Deletion Duplication Inversion Translocation
    • Part 3- Genetic Techniques
    • What is Genetic Engineering? • Genetic Engineering- Making changes in the DNA code of living organisms in an effort to achieve a more desirable trait
    • Techniques in Genetic Engineering DNA extraction  Removal of DNA from a cell Cutting DNA  Small sections are cut from the DNA using Restriction enzymes Separating DNA   DNA is separated in a technique called Gel Electrophoresis (separates according to size) CSI- crime scene investigation- DNA is often used to link criminals to crime scenes by matching DNA fingerprints of a suspect with DNA found at the crime scene. Making Copies  Many copies of DNA can be made in a technique known as Polymerase Chain Reaction (PCR)
    • Figure Section 13-2 13-6 Gel Electrophoresis DNA plus restriction enzyme Power source Longer fragments Shorter fragments Gel Mixture of DNA fragments DNA fingerprinting
    • Recognition sequence Section 13-2 Restriction Enzymes DNA sequence Restriction enzyme EcoRI cuts the DNA into fragments. Sticky end
    • Recombinant DNA DNA from different species that is cut and recombined; usually human DNA is cut and combined with bacterial DNA
    • Applications of Genetic Engineering Transgenic – organisms that contain genes from other species (recombinant DNA)
    • Transgenic Microorganisms Reproduce rapidly Easy to grow Produces a host of important useful substances such as human forms of proteins such as insulin, growth hormone, and clotting factor
    • Figure 13-9 Making Recombinant DNA Recombinant DNA Section 13-3 Gene for human growth hormone Gene for human growth hormone Human Cell Sticky ends DNA recombination DNA insertion Bacterial Cell Bacterial chromosome Plasmid Bacterial cell for containing gene for human growth hormone
    • Transgenic Animals Used to study genes and improve food supply Mice have been produced with human genes that make immune system act similar to human
    • Transgenic Plant Genetically modified Many contain genes that produce natural insecticide Others resist weed-killing chemicals Eventually produce human antibodies
    • Cloning Member of a population of genetically identical cells produced from a single cell Cloned sheep – DOLLY Ethical and moral issues
    • Figure 13-13 Cloning of the First Mammal A donor cell is taken from a sheep‘s udder. Donor Nucleus These two cells are fused using an electric shock. Fused Cell Egg Cell The nucleus of the egg cell is An egg cell is taken removed. from an adult female sheep. Cloned Lamb The fused cell begins dividing normally. Embryo The embryo develops normally into a lamb—Dolly Foster Mother The embryo is placed in the uterus of a foster mother.