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Chapter 21 DNA Biology and Technology Mader, Sylvia S. Human Biology. 13th Edition. McGraw-Hill, 2014. Points to Ponder • • • • • • • • • • • • • What are three functions of DNA? Review DNA and RNA structure. What are the 3 types of RNA and what are their functions? Compare and contrast the structure and function of DNA and RNA. How does DNA replicate? Describe transcription and translation in detail. Describe the genetic code. Review protein structure and function. What are the 4 levels of regulating gene expression. What did we learn from the human genome project and where are we going from here? What is ex vivo and in vivo gene therapy? Define biotechnology, transgenic organisms, genetic engineering and recombinant DNA. What are some uses of transgenic bacteria, plants and animals? 21.1 DNA and RNA structure and function What must DNA do? 1. Replicate to be passed on to the next generation 2. Store information 3. Undergo mutations to provide genetic diversity 21.1 DNA and RNA structure and function DNA structure: A review • • • • Double-stranded helix Composed of repeating nucleotides made of – a pentose sugar – phosphate – a nitrogenous base Sugar and phosphate make up the backbone while the bases make up the “rungs” of the ladder Bases have complementary pairing – cytosine (C) pairs with guanine (G) – adenine (A) pairs with thymine (T) The Bases Figure 2–22b, c 21.1 DNA and RNA structure and function DNA structure 21.1 DNA and RNA structure and function How does DNA replicate? 1. Two strands unwind by breaking the H bonds 2. Complementary nucleotides are added to each strand by DNA polymerase 3. Each new double-stranded helix is made of one new strand and one old strand (semiconservative replication) **The sequence of bases makes each individual unique DNA Replication • DNA helicases unwind the DNA and separates the strands • DNA polymerase bind to the DNA and synthesizes complementary antiparallel strands • DNA rewinds into double helix molecules – New molecules contains one strand of the original DNA and one newly synthesized strand 21.1 DNA and RNA structure and function DNA replication Mutations • Cell has repair enzymes that usually fix errors in DNA replication • A replication error that persists is a mutation – A permanent change in the sequence of bases that can cause a change in phenotype and introduce variability • Most non-infectious disease, conditions, and disorders are due to mutations in the DNA that change the amino acids in the protein • Point mutations – change in 1 base of DNA can be a silent mutation if the amino acids is not changed • Insertion mutation – addition of a base which changes the reading frame – whole protein after the mutation is wrong • Deletion Mutation – removal of a base, alter reading frame, wrong protein is made 21.1 DNA and RNA structure and function RNA structure and function • • • • • Single-stranded Composed of repeating nucleotides Sugar-phosphate backbone Bases are A, C, G and uracil (U) Three types of RNA – Ribosomal (rRNA): • joins with proteins to form ribosomes – Messenger (mRNA): • carries genetic information from DNA to the ribosomes – Transfer (tRNA): • transfers amino acids to a ribosome where they are added to a forming protein 21.1 DNA and RNA structure and function RNA structure 21.1 DNA and RNA structure and function Comparing DNA and RNA • Similarities: – Are nucleic acids – Are made of nucleotides – Have sugar-phosphate backbones – Are found in the nucleus • Differences: – DNA is double stranded while RNA is single stranded – DNA has T while RNA has U – DNA has a deoxyribose sugar while RNA has a ribose sugar – RNA is also found in the cytoplasm as well as the nucleus while DNA is not Gene Expression • DNA provides the cell with blueprints for synthesizing proteins • DNA resides in the nucleus and protein synthesis occurs in the cytoplasm 1. mRNA carries a copy of DNA’s blueprint into the cytoplasm 2. Other RNA molecules (rRNA and tRNA) are involved in protein synthesis 21.2 Gene expression Proteins: A review • Composed of subunits of amino acids • Sequence of amino acids determines the shape of the protein • Synthesized at the ribosomes • Important for diverse functions in the body including hormones, enzymes and transport • Can denature causing a loss of function Proteins: A review of structure 21.2 Gene expression 2 steps of gene expression 1. Transcription – DNA is read to make a mRNA in the nucleus of our cells 2. Translation – Reading the mRNA to make a protein in the cytoplasm Overview of transcription and translation Genetic code • • • Made of 4 bases Bases act as a code for amino acids in translation Every 3 bases on the mRNA is called a codon that codes for a particular amino acid in translation Step 1: Gene Activation • Uncoils DNA • Start and stop codes on DNA mark location of gene – Locates area for mRNA transcription Step 2: DNA to mRNA • Enzyme RNA polymerase transcribes DNA: – binds to promoter (start) sequence – reads DNA code for gene – binds nucleotides to form messenger RNA (mRNA) – mRNA duplicates DNA coding strand, uracil replaces thymine Step 3: RNA Processing • At stop signal, mRNA detaches from DNA molecule: – code is edited (RNA processing) – unnecessary codes (introns) removed – good codes (exons) spliced together – triplet of 3 nucleotides (codon) represents one amino acid 21.2 Gene expression 1. Transcription • • • • mRNA is made from a DNA template mRNA is processed before leaving the nucleus mRNA moves to the ribosomes to be read Every 3 bases on the mRNA is called a codon and codes for a particular amino acid in translation Processing of mRNA after transcription Modifications of mRNA: • One end of the RNA is capped • Introns removed • Poly-A tail is added – important for the nuclear export, translation and stability of mRNA Processing of mRNA after transcription • mRNA Splicing – Cells use only certain exons rather then all to form a mature RNA transcript – Result can be a different protein product in each cell – Alternate mRNA splicing may account for the ability of a single gene to result in two different proteins in a cell 21.2 Gene expression 2. Translation 1. Initiation - mRNA binds to the small ribosomal subunit and causes 2 ribosomal units to associate 2. Elongation: polypeptide lengthens • • • tRNA picks up an amino acid tRNA has an anticodon that is complementary to the codon on the mRNA tRNA anticodon binds to the codon and drops off an amino acid to the growing polypeptide 3. Termination - a stop codon on the mRNA causes the ribosome to fall off the mRNA Overview of transcription and translation 21.2 Gene expression Regulation of gene expression 1. Transcriptional control (nucleus): – chromatin density must decondense to allow for activation – transcription factors DNA-bind proteins regulate activity of a gene 2. Posttranscriptional control (nucleus) – mRNA processing 3. Translational control (cytoplasm) – Differential ability of mRNA to bind ribosomes 4. Posttranslational control (cytoplasm) – changes to the protein to make it functional What did we learn from the human genome project (HGP)? • • • • Genes are a sequence of DNA bases that is transcribed into RNA molecules and may be translated into protein Humans consist of about 3 billion bases (A,T,G,C) and 25,000 genes Human genome sequenced in 2003 Many polymorphisms or small regions of DNA that vary among individuals were identified – – • Some have not physiological effects Others contribute to the diversity of human beings and possibly disease Genome size is not correlated with the number of genes or complexity of the organisms 21.3 Genomics What is the next step in the HGP? • Functional genomics • • • Understanding how the 25,000 genes function Understanding the function of gene deserts (82 regions that make up 3% of the genome lacking identifiable genes) Comparative genomics • • • Help understand how species have evolved Comparing genomes may help identify base sequences that cause human illness Help in our understanding of gene regulation 21.3 Genomics • New endeavors Proteomics – the study of the structure, function and interactions of cell proteins • Can be difficult to study because: • • protein concentrations differ greatly between cells protein location, concentration interactions differ from minute to minute – understanding proteins may lead to the discovery of better drugs • Bioinformatics – the application of computer technologies to study the genome – May allow scientists to find cause-and-effect relationships between genetic profiles and disorders caused by multifactorial genes How can we modify a person’s genome? • Gene therapy - insertion of genetic material into human cells to treat a disorder – Ex vivo therapy – cells are removed for a person altered and then returned to the patient – In vivo therapy – a gene is directly inserted into an individual through a vector (e.g. viruses) or directly injected to replace mutated genes or to restore normal controls over gene activity • Gene therapy has been most successful in treating cancer Ex vivo gene therapy Retrovirus -RNA virus that is replicated in a host cell via the enzyme reverse transcriptase to produce DNA from its RNA genome. - DNA is incorporated into the host's genome - Virus replicates as part of the host cell's DNA. 21.4 DNA technology DNA technology terms • Genetic engineering – altering DNA in bacteria, viruses, plants and animal cells through recombinant DNA technology • Recombinant DNA – contains DNA from 2 or more different sources • Transgenic organisms – organisms that have a foreign gene inserted into them • Biotechnology – using natural biological systems to create a product or to achieve an end desired by humans 21.4 DNA technology DNA technology 1. 2. 3. 4. Gene cloning through recombinant DNA Polymerase chain reaction (PCR) DNA fingerprinting Biotechnology products from bacteria, plants and animals 21.4 DNA technology • Recombinant DNA – contains DNA from 2 or more different sources that allows genes to be copies – – • 1. Gene cloning The gene of interest is inserted into a vector Vector is typically a plasmid, small accessory rings of DNA found in bacteria An example using bacteria to clone the human insulin gene: – Restriction enzyme • cut the vector (plasmid) and the human DNA with the insulin gene – DNA ligase • seals together the insulin gene and the plasmid – Bacterial cells uptake plasmid and the gene is copied and product can be made 21.4 DNA technology 2. Polymerase chain reaction (PCR) • • Used to clone small pieces of DNA Requires – – • DNA polymerase to carry out DNA replication Nucleotides (phosphate, deoxyribose, and base) Important for amplifying DNA for analysis such as in DNA fingerprinting 21.4 DNA technology 3. DNA fingerprinting • • • DNA Fragments are separated by their charge/size ratios Results in a distinctive pattern for each individual Often used for paternity or to identify an individual at a crime scene or unknown body remains 21.4 DNA technology 4. Biotechnology products: Transgenic bacteria • Important uses: – Insulin – Human growth hormone (HGH) – Clotting factor VIII – Tissue plasminogen activator (t-PA) – Hepatitis B vaccine – Bioremediation – cleaning up the environment such as oil degrading bacteria 21.4 DNA technology 4. Biotechnology products: Transgenic plants • Important uses: – Produce human proteins in their seeds such as hormones, clotting factors and antibodies – Plants resistant to herbicides – Plants resistant to insects – Plants resistant to frost • Corn, soybean and cotton plants are commonly genetically altered • In 2001: – 72 million acres of transgenic crops worldwide – 26% of US corn crops were transgenic crops 21.4 DNA technology 4. Biotechnology products: Transgenic plants 4. Biotechnology products: Transgenic animals • Gene is inserted into the egg that when fertilized will develop into a transgenic animal • Current uses: – Gene pharming: production of pharmaceuticals in the milk of farm animals – Larger animals: includes fish, cows, pigs, rabbits and sheep – Mouse models: the use of mice for various gene studies – Xenotransplantation: pigs can express human proteins on their organs making it easier to transplant them into humans