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Nucleic acids Genetic Information Genetic Information { { { { August2010 Genetic information is stored in DNA in a linear sequence of nucleotides, packaged into structures called chromosomes Most bacteria and viruses have a single chromosome while eukaryotes usually have many A single chromosome contains thousands of segments of DNA which are referred to as genes The sum of all the genes on all chromosomes of a cell is referred to as the organism’s genome HBC108/2010 UON EGK 2 Size of DNA molecules Prokaryotes { Sizes of DNA molecules differ in both prokaryotes (viruses and bacteria) and eukaryotes { Genetic information in viruses is small and it can either be DNA or RNA { DNA viruses can have either single or double stranded DNA { DNA in viruses can be circular in shape August2010 HBC108/2010 UON EGK 3 Bacterial DNA { { { { August2010 Bacteria have much more DNA than viruses (e.g E. coli DNA is 200 times more than bacteriophage λ virus E. coli has a single double stranded circular molecule of DNA Many species of bacteria contain one or more small circular DNA molecules that are free in the cytosol called plasmids Plasmids also carry important genetic information HBC108/2010 UON EGK 4 Eukaryotic cell DNA { { { August2010 Yeast, one of the simplest eukaryotes has DNA 4 times as E.coli (human cell has as many as 600 times) Nuclear DNA molecules of eukaryotic cells have linear, double stranded DNA The length of DNA in a human cell is about 2m compared with 1.7 mm for E.coli. HBC108/2010 UON EGK 5 Structure of chromosomes { { August2010 Bacteria have one chromosomes per cell and in most cases containing only one copy of any given gene Eukaryotic cells contain many chromosomes in a cell and many genes exist as multicopy genes HBC108/2010 UON EGK 6 Chromatin Structure { { { { August2010 DNA exists within cells as chromatin The structure of chromatin is determined and stabilized through the interaction of the DNA with nucleoproteins Examples of nucleoproteins include DNAbinding proteins in the nucleus There are 2 classes of DNA-binding proteins HBC108/2010 UON EGK 7 DNA binding proteins { { { August2010 The histones are the major class of DNAbinding proteins involved in maintaining the compacted structure of chromatin. There are 5 different histone proteins identified as H1, H2A, H2B, H3 and H4 Histones package and order DNA into structural units called nucleosomes HBC108/2010 UON EGK 8 Non-histone proteins { { { August2010 The other class of DNA-binding proteins is a diverse group of proteins called simply, nonhistone proteins. This class of proteins includes the various transcription factors, polymerases, hormone receptors and other nuclear enzymes. In any given cell there are greater than 1000 different types of non-histone proteins bound to the DNA. HBC108/2010 UON EGK 9 Nucleosome { { { August2010 The binding of DNA by the histones generates a structure called the nucleosome. The nucleosome core is a protein structure consisting of 2 subunits each of H2A, H2B, H3 and H4. These nucleosomal structures would appear as beads on a string if the DNA were pulled into a linear structure HBC108/2010 UON EGK 10 Nucleosomes beads August2010 HBC108/2010 UON EGK 11 Summary of genetic flow of information Nucleic acids Manipulation of purified DNA DNA manipulations { DNA manipulative techniques including:1. 2. 3. 4. 5. 6. August2010 Cutting Joining Shortening Lengthening Copying DNA into RNA or into new DNA molecules Modifying DNA by addition or removal of specific chemical groups HBC108/2010 UON EGK 14 DNA manipulations { { Almost all the DNA manipulative techniques make use of purified enzymes DNA manipulative enzymes are grouped into 1. 2. 3. 4. 5. August2010 Nucleases Ligases Polymerases Modifying enzymes Topoisomerases HBC108/2010 UON EGK 15 Nucleases { These degrade DNA molecules by breaking the phosphodiester bonds There are two types 1. 2. August2010 Exonucleases which remove nucleotides one at a time from either end of a DNA molecule Endonucleases which are able to break internal phosphodiester bonds within a DNA molecule HBC108/2010 UON EGK 16 Restriction enzymes/endonucleases { { { { August2010 Restriction enzymes are DNA-cutting enzymes found in bacteria Because they cut within the DNA molecule, they are often called restriction endonucleases A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIII that cuts DNA wherever it encounters the sequence 5'GGCC3‘ 3'CCGG5' HBC108/2010 UON EGK 17 DNA Ligase DNA ligase is used to repair single stranded breaks in the cell { DNA ligases will also join together two individual fragments of double-stranded DNA { They form covalent bonds along the backbone of each strand { August2010 HBC108/2010 UON EGK 19 Polymerases These are enzymes that synthesize a new strand of DNA complimentary to an existing DNA or RNA template { DNA polymerase I is used to synthesize a completely new strand of DNA { Reverse transcriptase is an enzyme that can synthesize DNA from an RNA template is essential for cDNA cloning { August2010 HBC108/2010 UON EGK 20 DNA modifying enzymes { Numerous enzymes modify DNA molecules by addition or removal of specific chemical groups 1. 2. 3. August2010 Alkaline phosphatase will remove phosphate gps present at the 5`-terminus of a DNA molecule Polynucleotide kinase will add a phosphate gp on a free 5`-terminus of a DNA molecule Terminal deoxynucleotidyl transferase adds one or more deoxyribonucleotides on the 3`-terminus of a DNA molecule HBC108/2010 UON EGK 21 Topoisomerases These are able to change the conformation of covalently closed-circular DNA e.g. plasmids by introducing or removing supercoils { This process is necessary during DNA replication { August2010 HBC108/2010 UON EGK 22 DNA manipulations 1. 2. 3. August2010 Genetic engineering Recombinant DNA technology Genetic modification/manipulation (GM) and gene splicing are terms that are applied to the manipulation of genes HBC108/2010 UON EGK 23 Genetic manipulations It involves the isolation, manipulation and reintroduction of DNA into cells or model organisms, usually to express a protein { The aim is to introduce new characteristics or attributes physiologically or physically { z August2010 such as making a crop resistant to a herbicide, introducing a novel trait, enhancing existing ones, or producing a new protein or enzyme HBC108/2010 UON EGK 24 Recombinant DNA { August2010 Recombinant DNA is a form of artificial DNA which is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences which would not normally occur together. HBC108/2010 UON EGK 25 Summary of recombinant DNA technology August2010 HBC108/2010 UON EGK 26 Example { { August2010 GloFish are a type of zebrafish with recombinant DNA. Genes for fluorescent proteins have been inserted into their genome to produce their fluorescent colors HBC108/2010 UON EGK 27 Polymerase chain reaction (PCR) The (PCR) is a technique for isolating and exponentially amplifying a DNA fragment or sequence of interest via enzymatic replication, without using a living organism { PCR is an in vitro technique, it is performed in the lab { It can be extensively modified to perform a wide array of genetic manipulations { August2010 HBC108/2010 UON EGK 28 Applications of rDNA technology { { { { August2010 The first genetically engineered drug was human insulin, Another early application was to create human growth hormone In 1986 the first genetically engineered vaccine for humans, for hepatitis B was approved in the US. One of the best known applications of genetic engineering is the creation of genetically modified organisms (GMOs). HBC108/2010 UON EGK 30 GMOs { { { August2010 Golden rice is a genetically modified rice crop that produces beta-carotene, which can be metabolized into Vitamin A within the body. Scientists hope that golden rice will eventually be a cheap source of beta-carotene in malnourished countries, reducing the number of children worldwide that go blind from Vitamin A deficiency. Bt maize is a genetically modified corn crop that produces a toxin that kills stalk borers feeding on the maize HBC108/2010 UON EGK 31 DNA fingerprinting { { { { August2010 Genetic fingerprinting, DNA testing, DNA typing, and DNA profiling are DNA techniques used to distinguish between individuals Genetic fingerprinting exploits highly variable repeating DNA sequences called minisatellites. Minisatellites patterns are unique for each person Genetic fingerprinting is used in forensic science, to match suspects to samples of blood, hair, saliva or semen HBC108/2010 UON EGK 32 Somatic cell nuclear transfer Somatic cell nuclear transfer (SCNT) is a laboratory technique for creating an ovum with a donor nucleus. { It can also be used as the first step in the process of reproductive cloning { August2010 HBC108/2010 UON EGK 33 August2010 HBC108/2010 UON EGK 34