Power Lecture Chapter 22 DNA Genes and Biotechnology

Power. Lecture: Chapter 22 DNA, Genes, and Biotechnology

Learning Objectives Ø Ø Understand how the instructions for producing heritable traits are encoded in DNA. Know the parts of a nucleotide, and know how nucleotides are linked together to make DNA. Understand how DNA is replicated and what materials are needed for replication. Know how the structure and behavior of DNA determine the structure and behavior of the forms of RNA during transcription.

Learning Objectives (cont’d) Know how the structure and behavior of the three forms of RNA determine the primary structure of polypeptide chains during translation. Ø Know the various ways that gene activity (replication and transcription) are turned on (activated) and off (inactivated). Ø Understand what plasmids are and how they may be used to insert new genes into recombinant DNA molecules. Ø

Learning Objectives (cont’d) Know how DNA can be cleaved, spliced, cloned, and sequenced. Ø Be aware of several limits and possibilities for future research in genetic engineering. Ø

Impacts/Issues Ricin and Your Ribosomes

Ricin and Your Ribosomes Ricin could be used as a biochemical weapon. Ø l l Ø Ricin was identified as a biochemical weapon as long ago as the 1880 s and was considered for use during WWII by both the US and England. It is a poisonous byproduct formed during production of castor oil from the castor bean. Ricin inactivates ribosomes by damaging the part of the ribosome where amino acids are joined together; protein synthesis stops and the person dies because there is no antidote.

How Would You Vote? To conduct an instant in class survey using a classroom response system, access “Join. In Clicker Content” from the Power. Lecture main menu. Ø Given the threat of biochemical warfare, would you be willing to be vaccinated – or does the threat seem too remote? l l a. Yes, serious bioterror threats can be lethal in small doses and easy to manufacture. b. No, we must be selective in what diseases to vaccinate against.

Section 1 DNA: A Double Helix

DNA: A Double Helix DNA is built of four kinds of nucleotides. Ø l Each nucleotide consists of a five carbon sugar (deoxyribose), a phosphate group, and one of four bases—adenine (A), guanine (G), thymine (T), cytosine (C). Figure 22. 1

adenine A base with a double-ring structure thymine T base with a single-ring structure sugar (deoxyribose) guanine G base with a double-ring structure cytosine C base with a single-ring structure Fig 22. 1 b, p. 406

DNA: A Double Helix l Watson and Crick were the first to discover the structure of DNA. • • DNA consists of two strands of nucleotides twisted into a double helix. Nucleotides are joined along the molecule’s length by covalent bonds and run in opposite directions; the two strands are held together with weaker hydrogen bonds. Figure 22. 2

The pattern of base pairing (A only with T and G only with C) is consistent with the known composition of DNA (A=T and G=C Fig 22. 2, p. 407

DNA: A Double Helix Chemical “rules” determine which nucleotide bases in DNA can pair up. Ø l l l Base pairs are formed by the hydrogen bonding of A with T, and G with C. In a DNA molecule, the amount of adenine always equals thymine, and G = C. The base pairs can occur in any sequence along the length of the DNA molecule.

DNA: A Double Helix A gene is a sequence of nucleotides. Ø l l A gene is a sequence of nucleotides in a DNA molecule. The nucleotide sequence of each gene codes for a given polypeptide chain. Figure 22. 3

Section 2 Passing on Genetic Instructions

Passing on Genetic Instructions How is a DNA molecule duplicated? Ø l DNA replication is the process that duplicates DNA before a cell divides. • • l First, the two strands of DNA unwind and expose their bases to serve as a template. Then, unattached nucleotides are linked by hydrogen bonds to exposed bases according to base pairing rules. Replication results in DNA molecules each consisting of one “old” strand one “new” strand—semiconservative replication.

A. Parent DNA molecule, two complimentary strands of base paired nucleotides. B. Replication begin; the two strands unwind and separate from each other at specific sites along the length of the DNA molecule. C. Each “old” strand serves as a structural pattern (a template) for the addition of bases according to the base paring rule. D. Bases positioned on each old strand are joined together into a “new” strand. Each half old, half new DNA molecule is just like the parent molecule Fig 22. 4, p. 408

Passing on Genetic Instructions Mistakes and damage in DNA can be repaired. Ø l DNA polymerases and other enzymes are involved in DNA repair mechanisms. • • These enzymes detect and correct the sequence of bases if it becomes altered; they do so by reading the complementary sequence on the other strand restoring it. If an error is not fixed, a mutation results.

Passing on Genetic Instructions l l DNA is vulnerable to damage from chemicals and UV light, which can form thymine dimers that increase replication errors. Thymine dimers can lead to the genetic disorder xeroderma pigmentosum, which further increases an individual’s chance of developing lethal skin cancers. Figure 22. 5

Passing on Genetic Instructions A mutation is a change in the sequence of a gene’s nucleotides. Ø l Gene mutations are small scale changes in the nucleotide sequence of genes. • Base-pair substitutions can result in the substitution of one amino acid for another in a protein, as in sickle cell anemia. Figure 22. 6 a b

Passing on Genetic Instructions • A deletion occurs when a base has been lost. • In an expansion mutation, a nucleotide sequence is repeated many times, as in Huntington disease and fragile X syndrome. Figures 22. 6 a, c, and 22. 7 a

Passing on Genetic Instructions l l Neurofibromatosis is caused by segments of DNA called transposable elements (in this case, a specific element called Alu); such elements can move from location to location in the chromosomes. Mutations are only inherited if they occur in the germ cells that form the gametes. Figure 22. 7 b

Section 3 DNA into RNA—The First Step in Making Proteins

DNA into RNA— The First Step in Making Proteins Genes become proteins through the processes of transcription and translation. Ø l RNA is involved in both processes; RNA is single stranded, contains the sugar ribose, and substitutes the base uracil for the thymine of DNA. • • In transcription, molecules of RNA are produced on the DNA templates in the nucleus. In translation, RNA molecules are shipped from the nucleus to the cytoplasm to be used in polypeptide assembly.

DNA into RNA— The First Step in Making Proteins l Genes are transcribed into three kinds of RNA: • • • Ribosomal RNA (r. RNA) combines with proteins to form ribosomes upon which polypep tides are assembled. Messenger RNA (m. RNA) carries the protein “code” to the ribosome. Transfer RNA (t. RNA) brings the correct amino acid to the ribosome and pairs up with an m. RNA nucleotide code for that amino acid.

DNA into RNA— The First Step in Making Proteins In transcription, DNA is decoded into RNA. Ø l Transcription differs from replication in three ways: • • • Only one region of one DNA strand is used as a template. RNA polymerase is used instead of DNA polymerase. The result of transcription is a single stranded RNA.

DNA RNA DNA © 2007 Thomson Higher Education base-pairing in DNA replication base-pairing in

DNA into RNA— The First Step in Making Proteins l Transcription begins when RNA polymerase binds to a promoter region (a base sequence at the start of a gene) and then moves along to the end of a gene. • • The result is a RNA transcript, which will have a 5 cap and a 3 tail. The RNA is also modified: introns (noncoding portions of the RNA) are removed, and exons (those portions that will be translated) are stitched together before the finished transcript leaves the nucleus.

Gene Transcription [Step art] Figure 22. 8

DNA into RNA— The First Step in Making Proteins Gene transcription can be turned on or off. Ø l l Most of the cells of the human body carry the same genes, but only certain genes are expressed in any given cell at any given time. Genes are turned on and off by regulatory proteins that speed up or halt transcription.

Section 4 Reading the Genetic Code

Reading the Genetic Code Codons are m. RNA “words” for building proteins. Ø l l l Three base triplets, a codon, specify each amino acid to be included into a growing polypeptide chain. The genetic code consists of a total of 64 triplet codons: most specify amino acids, one is a start codon (AUG) and three are stop codons (UAA, UAG, UGA). Most amino acids can be specified by more than one codon.

DNA m. RNA a m. RNA codons amino acids b threonine proline glutamate lysine Fig 22. 9, p. 412

The Genetic Code Figure 22. 10

Reading the Genetic Code t. RNA translates the genetic code. Ø l Each kind of t. RNA has an anticodon that is complementary to an m. RNA codon; each t. RNA also carries one specific amino acid. Figure 22. 11

Reading the Genetic Code l l After the m. RNA arrives in the cytoplasm, a specific anticodon on a t. RNA bonds to the codon on the m. RNA by complementary base pairing, and so a correct amino acid is brought into place. There are fewer t. RNAs than the number of possible codons because third position in the codon anticodon pairing is loose; the wobble effect allows some t. RNAs to match multiple amino acids to the right codon.

Reading the Genetic Code r. RNAs are ribosome building blocks. Ø l l Translation occurs on the surface of ribosomes where the t. RNAs and m. RNA interact. Ribosomal subunits are synthesized from r. RNA and proteins in the nucleus, then shipped to the cytoplasm where they are combined into ribosomes during translation. Figure 22. 12

Section 5 Translating the Genetic Code into Protein

Translating the Genetic Code into Protein Translation has three stages. Ø l l In initiation, a complex forms in this sequence: initiator t. RNA + small ribosomal subunit + m. RNA (specifically, the AUG start codon) + large ribosomal subunit. In elongation, the m. RNA passes through the ribosome attracting a series of t. RNAs that deliver amino acids in sequence by codon anticodon matching; a peptide bond joins each amino acid to the next in the sequence.

Elongation intact ribosome INITIATION m. RNA transcript © 2007 Thomson Higher Education Fig. 22.

binding site for m. RNA (first binding site for t. RNA) © 2007 Thomson

© 2007 Thomson Higher Education Fig. 22. 13 f-i, p. 415

Translating the Genetic Code into Protein l With termination, a stop codon is reached that has no corresponding t. RNA; release factors cause the polypeptide chain and the m. RNA to be released.

© 2007 Thomson Higher Education Fig 22. 13 j-l, p. 415

Translating the Genetic Code into Protein Cells use newly formed proteins in various ways. Ø l l To increase the efficiency of the translation process, several ribosomes can be aligned on one m. RNA (polysome), allowing synthesis of more than one polypeptide at a time. After new polypeptide chains are complete, they may join the pool of proteins in the cytoplasm or may enter the ER for modification.

Transcription Different gene regions of DNA: Transcript processing: m. RNA mature m. RNA Translation At ribosome, a polypeptide chain is synthesized at the binding sites for m. RNA and t. RNAs r. RNA protein subunits ribosomal subunits RNAs converge t. RNA mature t. RNA amino acids, ribosome subunits, and t. RNAs in the cytoplasm FINAL PROTEIN For use in cell or for export © 2007 Thomson Higher Education Fig 22. 26, p. 424

Section 6 Tools for “Engineering” Genes

Tools for “Engineering” Genes Recombinant DNA technology encompasses a range of techniques that allow for the specific creation of genetic changes in DNA. Ø l l DNA from different species can be cut, spliced together, and inserted into bacteria, which then multiply the recombinant DNA molecules. Genetic engineering involves the isolation, modification, and reinsertion of DNA back into an organism.

Tools for “Engineering” Genes Enzymes and plasmids from bacteria are basic tools. Ø l l Many bacteria possess plasmids, circular DNA molecules that carry only a few genes and which can replicate independently of the single “main” chromosome. Restriction enzymes are used by bacteria to cut apart DNA; this capability makes them useful to researchers as tools for doing genetic recombination in the laboratory.

Tools for “Engineering” Genes • • l Restriction enzymes produce DNA fragments with staggered cuts resulting in sticky ends; some fragments may be thousands of bases long, allowing the study of the genome (all of the DNA in a haploid set of chromosomes). The sticky ends of the fragments can be spliced together by other enzymes to create a recombinant DNA molecule. Using these tools, it is possible to insert foreign DNA into bacterial plasmids, creating DNA clones; DNA clones are sometimes called cloning vectors.

A. A selected restriction enzyme cuts wherever a specific base sequence occurs in a molecule of chromosomal DNA or c. DNA. C. DNA or c. DNA fragments with sticky ends. E. The foreign DNA, the plasmid DNA, and modification enzymes are mixed together. F. A collection of recombinant plasmids containing foreign DNA. B. The same enzyme cuts the same sequence in plasmid DNA. D. Plasmid DNA with sticky ends G. Host cells able to divide rapidly take up recombinant plasmids. © 2007 Thomson Higher Education Fig 22. 14, p. 416

Tools for “Engineering” Genes The polymerase chain reaction (PCR) is a faster way to copy DNA. Ø l The reactions are done in test tubes, starting with primers. • • Primers are man made, short nucleotide sequences that will base pair with sequences of DNA that are to be amplified. A heat stable DNA polymerase is also needed. Figure 22. 15

Tools for “Engineering” Genes l The steps are relatively simple: • • • Researchers mix primers, polymerase, DNA of choice, and nucleotides. The mixture is exposed to precise temperature cycles in a dedicated machine. Starting with tiny quantities of DNA, the procedure doubles the DNA molecules in each round.

a. PCR starts with a fragment of doublestranded DNA b. The DNA is heated to 90º 94ºC to unwind it. The single strands will be templates. c. Primers designed to basepair with ends of the DNA strands will be mixed with the DNA. d. The mixture is cooled. The lower temperature promotes Base pairing between the primers and the ends of the DNA strands. e. DNA polymerases recognize the primers as start tags. They assemble complimentary sequences on the strands. This doubles the number of identical DNA fragments. © 2007 Thomson Higher Education Fig. 22. 15, p. 417

f. The mixture is heated again. The higher temperature make all of the double-stranded DNA fragments unwind. g. The mixture is cooled. The lower temperature promotes Base pairing between more primers added to the mixture and the single strands h. DNA polymerase action again doubles the number of identical DNA fragments. © 2007 Thomson Higher Education Fig. 22. 15, p. 417

Section 7 “Sequencing” DNA

“Sequencing” DNA Automated DNA sequencing can reveal the sequence of nucleotides in DNA in a few hours. Ø l l The machines are loaded with four standard nucleotides (A, T, G, and C) and four modified versions of the nucleotides, which fluoresce a different color. The DNA molecule to be sequenced, primer, and polymerase are also added.

“Sequencing” DNA Ø A series of segments tagged with fluorescing molecules are separated into sets of fragments, which are analyzed by the machine to reveal the original DNA’s nucleotide sequence. printout of DNA sequence: T C C A T G G A C C A Figure 22. 16

“Sequencing” DNA Ø To identify a particular gene among many in a gene library (say, inside bacteria), researchers use a radioactive probe that will match up with the DNA nucleotide sequence of interest.

Section 8 Mapping the Human Genome

Mapping the Human Genome Ø Results of the Human Genome Project indicate that the human genome is composed of roughly 2. 9 billion nucleotide bases subdivided into about 21, 500 genes. Figure 22. 17

Mapping the Human Genome mapping provides basic biological information. Ø l l Exons comprise only 1. 5% of our DNA; the remainder is non coding DNA but should not be labeled “junk. ” Our DNA is sprinkled with SNPs (single nucleotide polymorphisms), each of which has a change in one nucleotide in sequence; these account for the slightly different versions of the genes that make us all different.

Mapping the Human Genome DNA chips help identify mutations and diagnose diseases. Ø l Each chip is a microarray of thousands of DNA sequences stamped onto a small glass plate. • • l When a sample of body tissue is placed on the plate, the reactions can pinpoint which genes are silent and which are being expressed. Some chips are being used to design better drug therapies for disease. As new genes are identified, it may be possible to derive a complete genetic profile from a small sample of a person’s blood. Figure 22. 18 a

Mapping the Human Genome Chromosome mapping shows where genes are located. Ø l Sequencing of the genome can identify where specific genes are located on chromosomes. • • We know that chromosome 21 carries genes for early onset Alzheimer’s, epilepsy, and amyotrophic lateral sclerosis (ALS). More than 60 disorders have been mapped to chromosome 14.

P 1 12 11 Amyloidosis, cerebroarterial, Dutch-type Alzheimer’s disease, one form Schizophrenia, chronic, one form Amyotrophic lateral sclerosis, one form q 2 21 22 Down syndrome (critical region) Epilepsy, progressive myoclonus Hemolytic anemia due to phosphofructokinase deficiency Homocystinuria, B 6 responsive and B 6 unresponsive Leukemia, acute myeloid Leukocyte adhesion deficiency Fig 22. 19, p. 419

Mapping the Human Genome l But there may be a down side to all of this progress if genetic profiling leads to discrimination in employment or insurance coverage. Figure 22. 18 b

Section 9 Some Applications of Biotechnology

Some Applications of Biotechnology Researchers are exploring gene therapy. Ø l l There are 15, 500 known genetic disorders in humans. Gene therapy attempts to replace mutated genes with normal ones, or to insert genes that restore normal controls over gene activity. Genes can be inserted two ways. Ø l Transformation involves exposing cells cultured in the laboratory to DNA that contains the gene of interest; some small portion of the DNA will be taken up by the cells and integrated into the host’s genome.

Some Applications of Biotechnology l In transfection, DNA segments are inserted into viruses (often retroviruses) and then the modified viruses are allowed to infect the target host cell; infection generally leads to insertion of the DNA into the host genome.

Normal gene Clone normal gene into retrovirus vector Infect patient’s white blood cells with virus In some cells viral DNA Inserts into chromosome Inject cells into patient © 2007 Thomson Higher Education Fig 22. 20, p. 420

Some Applications of Biotechnology Results of gene therapy have been mixed. Ø l l Severe combined immune deficiency (SCID-X 1) was one of the first successfully treated diseases using gene therapy; however, several of the initial children treated for the disease went on to develop cancer. Cystic fibrosis therapy trials have attempted to deliver the corrective gene into the body using a viral vector in a nasal spray; results have been disappointing. Figure 22. 21

Some Applications of Biotechnology l One of the most successful gene therapy efforts is the treatment of some cancers such as malignant melanoma, leukemia, and lung cancer. • • Viruses have been used to introduce interleukin encoding genes to tumor cells; the interleukins serve as a “suicide tag, ” encouraging destruction of the tumor by T cells. “Lipoplexes” composed of plasmid wrapped in lipid have also been used to deliver markers to tumors to stimulate T cell destruction.

Some Applications of Biotechnology Genetic analysis also is used to read DNA fingerprints. Ø l Each of us has a unique set of DNA fragments inherited from our parents in a Mendelian pattern—a DNA fingerprint. • • Fingerprints form from short repeated segments called tandem repeats. Tandem repeats can be separated and visualized by gel electrophoresis.

Some Applications of Biotechnology l Variation can also be detected using restriction fragment length polymorphisms (RFLPs); in RFLP analysis, DNA is cut into fragments using restriction enzymes and then separated by electrophoresis. Figure 22. 28

Section 10 Issues for a Biotechnological Society

Issues for a Biotechnological Society Some important concerns brought up for discussion in recent years include: Ø l l l The possibility that transgenic bacteria or viruses could mutate, possibly becoming new pathogens. Bioengineered plants could escape from test plots and become “superweeds” resistant to herbicidal control measures. Crop plants with added insect resistance could bring forth new, even more formidable, insect pests.

Issues for a Biotechnological Society l Transgenic species, such as fish, could feed voraciously and displace natural species. Genetically modified plants, especially those used for food, are particularly controversial. Ø l Critics allege that these “Frankenfoods” may be toxic, less nutritious, and could promote antibiotic resistance. Figure 22. 23

Issues for a Biotechnological Society l On the other hand, advocates for such foods envision a Green Revolution where these plants may help feed the world’s hungry people or be used to clean up pollution in a process called bioremediation. Figures 22. 23 and 22. 24

Section 11 Engineering Bacteria, Animals, and Plants

Engineering Bacteria, Animals, and Plants Bacteria were the first bioengineered organisms; today they help produce many important human medicines such as human growth hormone, insulin, and interferons. Ø Animals have been used in bioengineering experiments, and transgenic barnyard animals may become the sources for pharmaceuticals: the blood clot dissolver t. PA, CFTR protein for cystic fibrosis, and blood clotting factor VIII. Ø

Engineering Bacteria, Animals, and Plants Ø Animals have been used in bioengineering experiments, and transgenic barnyard animals may become the sources for pharmaceuticals: the blood clot dissolver t. PA, CFTR protein for cystic fibrosis, and blood clotting factor VIII. Figures 22. 23 a and 22. 27

Engineering Bacteria, Animals, and Plants Ø Plants can be conferred with desirable traits in the laboratory, such as resistance to pathogens or herbicides, and then grown in the field. Figure 22. 25 b c