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Today’s Plan: 2/16/11 Bellwork: Talk about yesterday and the test (30 mins) Casting gels for tomorrow (30 mins) DNA Tech Notes (the rest of class) Today’s Plan: 2/18/2010 Bellwork: Test discussion (15 mins) Transformation Notes, if time Today’s Plan: 2/19/2010 Bellwork: Flies and Test Corrections (15 mins) AP Lab 6 Lab Bench Intro to the Molecular Biology Lab (30 mins) Continue notes (the rest of class) Pack/Wrap-up (last few mins of class) Today’s Plan: 2/22/10 Bellwork: Cast Gels (30 mins) Count flies and look at bacteria (while gel is dissolving Practice and run gels (45 mins) Continue with notes (the rest of the period) Today’s Plan: 2/23/10 Bellwork: Read Gels/answer questions/fly counts (30 mins) Set up for Lunch demos (30 mins) Continue notes (the rest of class) Today’s Plan: 2/24/10 Bellwork: Flies/Test Q&A (20 mins) DNA Tech test (the rest of class) Today’s Plan: 9/15/09 Bellwork: Intstructions (5 mins) Research for Discussion (30 mins) Bioethics discussion (the rest of class) Today’s Plan: 9/16/09 Bellwork: Last Presentation (10 mins) Senses Stations (50 mins) Continue with DNA Tech notes (the rest of class) Today’s Plan: 9/17/09 Bellwork: Class Optional Taste Demo (10 mins) Finish Senses Stations (50 mins) Finish DNA Tech notes (the rest of class) Today’s Plan: 9/18/09 Bellwork: Test Q&A (15 mins) DNA Tech Test (as needed) If you finish your test early, work on the senses questions. Regulating Gene Expression Prokaryotes (Bacteria) Often live in erratic environments Need to turn on and off genes in response to the environment Use Operons to regulate genes. These are DNA sections that are regulated by repressors which can turn off the promoter site, and keep transcription from happening. Operons contain several related genes, each with its own start and stop codon The convenience of the operon is that there only has to be 1 on/off switch for all of the genes Regulating Operons Operons contain a promoter region (consisting of an attachment point for RNA polymerase and an operator for the repressor to attach to when not needed), and the genes for a specific job Repressors come from a regulatory gene at a point away from the operon Repressible operons are always on unless a repressor is bound (ex: trp operon-repressor is inactive until bound to a tryptophan molecule) Inducible operons are off unless an inducer is present to inhibit the repressor’s hold on the DNA (ex: lac operon-repressor is active unless bound to allolactose) Figure 17-4 E. coli Galactoside permease -Galactosidase Glucose Galactose Lactose Plasma membrane Figure 17-8 lac operon DNA lacl promoter lacl Promoter Operator of lac operon lacZ lacY lacA Figure 17-7 Repressor present, lactose absent: Repressor binds to DNA. No transcription occurs. The repressor blocks transcription Repressor synthesized DNA Normal lacl gene Repressor present, lactose present: Lactose binds to repressor, causing it to release from DNA. Transcription occurs Repressor (lactose acts as inducer). synthesized Normal lacl gene lacZ lacl+ RNA polymerase bound to promoter (blue DNA) TRANSCRIPTION BEGINS -Galactosidase Permease mRNA lacZ lacl+ RNA polymerase bound to promoter (blue DNA) No repressor present, lactose present or absent: Transcription occurs. No functional repressor synthesized Mutant lacl gene lacY lacY Lactose-repressor complex TRANSCRIPTION BEGINS mRNA -Galactosidase Permease Lacl – lacZ RNA polymerase bound to promoter (blue DNA) lacY Figure 17-9 When tryptophan is present, transcription is blocked. Repressor Tryptophan No transcription Operator RNA polymerase bound to promoter When tryptophan is absent, transcription occurs. TRANSCRIPTION 5 genes coding for enzymes involved in tryptophan synthesis RNA polymerase bound to promoter Figure 17-10 lac operon trp operon Catabolism (breakdown of lactose) Anabolism (synthesis of tryptophan) Repressor Lactose Repressor Tryptophan Tryptophan binds to repressor Lactose binds to repressor Lactoserepressor complex releases from operator Operator Tryptophanrepressor complex binds to operator Operator Transcription of lac operon TRANSCRIPTION No more transcription of trp operon Positive Gene Regulation Bacteria needs to sense whether or not glucose as well as lactose are present in its environment. Bacteria prefer to use glucose for glycolysis, and therefore only use lactose when there isn’t glucose available Cyclic AMP (cAMP) accumulates when glucose is scarce. cAMP binds to a regulatory protein, catabolite activator protein (CAP) and the complex becomes an activator that binds to the DNA just upstream of the promoter. The activator makes it more likely that RNA polymerase will attach to the operon and transcribe Figure 17-14 Glucose inhibits the activity of the enzyme adenylyl cyclase, which catalyzes production of cAMP from ATP. Glucose inhibits this enzyme ATP Adenylyl cyclase cAM P Two phosphate groups The amount of cAMP and the rate of transcription of the lac operon are inversely related to the concentration of glucose. CAP HIGH glucose concentration INACTIVE adenylyl cyclase LOW cAMP CAP does not bind to DNA Infrequent transcription of lac operon (Cell continues to use glucose as energy source.) cAMP CAP LOW glucose concentration ACTIVE adenylyl cyclase HIGH cAMP CAP-cAMP complex binds to DNA Frequent transcription of lac operon (Cell uses lactose if lactose is present.) Prokaryote Genomes vs. Smaller Genome Fewer Genes Higher gene density (more genes in a smaller segment of DNA Relatively few noncoding regions and protein genes are continuous Eukaryote Large Genome Many more genes Lower gene density Many noncoding regions (introns) and protein genes are not continuous Eukaryotic Gene Regulation Recall that in complex organisms, the complete genome is in all cells, but only the genes necessary for the function of the individual cell are turned on in that individual cell In stead of regulating just transcription, as bacteria do, eukaryotic cells can regulate gene expression at any step from DNA to protein Figure 18-1 Nucleus 1. Chromatin remodeling 2. Transcription Chromatin (DNA-protein complex) “Open” DNA (Some DNA not closely bound to proteins) Primary transcript (pre-mRNA) 3. RNA processing Cap Tail Mature mRNA Cytoplasm 4. mRNA stability Degraded mRNA (mRNA lifespan varies) 5. Translation mRNA Polypeptide 6. Post-translational modification (folding, transport, activation, degradation of protein) Active protein DNA Regulation Chromatin is DNA that is packaged with proteins, called histones. The basic unit of chromatin is called the nucleosome DNA Methylation Under normal conditions, the lysine tails of histones extend out from the nucleosome and are attracted to other nucleosomes Histone acetylation attaches acetyl groups to these tails, making them no longer attracted to other histones, which loosens up the chromatin to make transcription easier It’s also been shown that methyl groups are also added to the histone tails, which can promote condenstion of the chromatin Methyl groups can be attached to cytosine, again causing condensation of the chromatin Some research shows that heavily methylated areas recruit deacetylation enzymes, which dually promotes condensation This appears to be an important regulatory step from embryo to mature organism. In cases where a template strand is methylated, the cell matches the methylation in the daughter strand after replication so that the cell stays specialized Epigenetic Inheritance Inheritance of traits not directly involved with the DNA sequence, such as alteration of methylation patterns Figure 18-2 Nucleosomes in chromatin Nucleosomes DNA Nucleosome structure Linker DNA H1 protein attached to linker DNA and nucleosome DNA Nucleosome Group of 8 histone proteins In some cases, nucleosomes may be grouped into 30-nanometer fibers. 30 nm Figure 18-4 Condensed chromatin Decondensed chromatin Acetyl group on histone The Eukaryotic Gene Recall that even Eukaryotic genes contain a promoter site, on which the transcription initiation complex assembles There are introns and exons within the gene and control elements that don’t code but bind proteins Figure 18-7 Enhancer Promoter Start site Enhancer PromoterExon Intron Exon proximal element Intron Exon Enhancer Regulating Transcription Transcription factors bind to the DNA and make it easier for RNA polymerase to bind These can be general transcription factors, if they are necessary for all protein-coding genes, and if they result in a low rate of transcription Specific transcription factors are proteins that attach to only certain genes and generally produced a high rate of transcription in cells needing particular genes Enhancers are generally found thousands of nucleotides upstream from the gene and are called Distal control elements Activators and repressors can bind to these elemets to regulate the initiation of transcription by interacting with mediator proteins The DNA can also be bent so that these form a transcription initiatinon complex The actual number of activators is small, but it’s the combination of control elements (proteins, activators, etc) that is unique to each gene Figure 18-10 THE ELEMENTS OF TRANSCRIPTIONAL CONTROL: A MODEL Chromatin remodeling complex (or HATs) Regulatory transcription factor 1. Regulatory transcription factors recruit chromatinremodeling complex, or HATs. Chromatin decondenses. Exposed DNA Promoter-proximal element Promoter Intron Exon Intron Exon 2. When chromatin decondenses, a region of DNA is exposed, including the promoter. Transcribed portion of gene for muscle-specific protein 3. Regulatory transcription Regulatory transcription factors Promoter-proximal element Exon Co-activators Promoter Intron factors recruit proteins of the basal transcription complex to promoter. Note looping DNA. Exon Basal transcription complex 4. RNA polymerase II Intron Exon RNA polymerase II Basal transcription complex completes the basal transcription complex; transcription begins. Do Eukaryotes have Operons? While there are co-expressed genes in Eukaryotes, each gene has its own promoters Some co-expressed genes are clustered, while others are on different chromosomes Coordinate control of co-expressed genes seems to be regulated by the genes having the same combination of control elements at the same time, usually in response to a signal outside of the cell Figure 18-14 Signaling molecule Cell-surface receptor Inactive STAT protein (two single polypeptide chains) Cytoplasm Activated STAT protein (dimer of two polypeptide chains) Enhancer TRANSCRIPTION Transcription activated Nucleus Post-transcriptional Gene Regulation RNA Processing-Alternative RNA splicing mRNA Degradation In Prokaryotes, mRNA is degraded within a few minutes, but Eukaryotes’mRNA can last for days or weeks Initiation of Translation The same transcript may result in different mature RNA depending on which segments are treated as introns Proteins can block the 5’ end of an mRNA, preventing attachment by the ribosome In other cases, the poly-A tail is not synthesized long enough until the organism is ready for the protein Protein processing and Degradation Many protiens require post-translation modifications, such as reversible phorphorylation Some proteins are tagged with ubiquitin, which alerts the proteasomes to their presence and degrades them Figure 18-12 Tropomyosin gene Intron Exon Intron Exon Processed mRNAs Skeletal muscle Smooth muscle Intron Exon Exon Some exons are specific to tropomyosin in skeletal or smooth muscle; some exons are common to both muscle types Noncoding RNAs These are other molecules, like tRNA and rRNA Many more RNAs are discovered frequently and have a variety of functions within the cell Apparently, not all DNA is supposed to code for proteins, and in fact, doesn’t MicroRNAs These are small pieces of RNA that are complimentary to mRNA Called miRNA Formed from a large primary transcript that bends into one or more hairpin turns An enzyme, called a “dicer” cuts these away, forming double-stranded mRNA One strand degrades, while the other forms a complex with a protein These complexes can bond with and interfere with mRNA (if they’re complimentary at some part), and can degrade it (if they’re complimentary along the length) Another type of RNA, small interfering RNA (siRNA) can also interfere with mRNA’s function. These are formed from larger, double-stranded precursor RNA molecules Collectively, this is called RNAi (RNA interference) Figure 18-13 miRNAs TARGET CERTAIN mRNAS FOR DESTRUCTION RNA hairpin DNA 1. Transcription of a microRNA gene. RNA polymerase 2. Initial transcript is Precursor miRNA processed into a precursor micro RNA (miRNA). Cytoplasm 3. Enzyme in cytoplasm Enzyme cuts out hairpin loop, forming a mature miRNA. Mature miRNA Single-stranded miRNA RISC protein complex 4. miRNA becomes single-stranded and binds to RISC protein complex. 5. miRNA, held by RISC, binds to complementary sequence on target mRNA. Target mRNA 6. Enzyme inside RISC cuts mRNA. Gene Expression and Embryonic Development The genome (including the cytoplasmic genome) contains a program for cell differentiation in the embryo Cytoplasmic determinants (RNA and DNA in the cytoplasm-matrolineal) get divided unevenly, which may contribute to cellular differentiation The embryo’s own cells may also induce changes in the other embryonic cells Sequential Regulation of Gene Expression during Differentiation Once a cell begins the process of differentiation, it is irreversible-even if the cell is moved to another part of the embryo Each cell type produces its own tissue-specific proteins from transcribed mRNA in genes that are turned on Figure 21-7 VISUALIZING mRNAs BY IN SITU HYBRIDIZATION 1. Start with a singleDNA probe Label stranded DNA or RNA probe, complementary in sequence to target mRNA. 2. Add label to probe Embryo (a radioactive atom or an enzyme that catalyzes a colorproducing reaction). 3. Preserve the DNA probe specimen (in this case, a Drosophila embryo). 4. Treat preserved DNA probe Target mRNA cells or tissues to make them permeable to probe. Add many copies of probe. 5. Probe binds to target mRNA. Labeled probe that does not bind to target mRNA is excess, and is washed away. Target mRNA 6. In this case, target Anterior mRNAs are concentrated in the anterior end of the embryo. The label shows up as Posterior black in this image. Setting up the body plan Pattern formation is the organization of the body and is regulated by cytoplasmic determinants and inductive signals from neighboring cells Early on, positional information, such as where the “head” and “tail” are to be, is established In Drosophila, a series of homeotic (hox) genes control the segmentation of the body and position of body parts Body Axis is determined by maternal effect geneswhen there is a mutant in the mother, there are mutations in the offspring, regardless of the offspring’s genotype Bicoids are two-tailed mutants come from mutations in these maternal effect genes These genes produce Morphogens, that concentrate in certain segments and determine what that segment will become Figure 21-6 A normal fruit-fly embryo Head Thoracic segments segments Abdominal segments A bicoid mutant Abdominal segments Abdominal segments Figure 21-13 The location of Hox genes on the fly chromosome correlates with their pattern of expression in fly embryos. Hox genes Fly embryo Head Thorax Abdomen The location of Hox genes on the mouse chromosome correlates with their pattern of expression in mouse embryos. Hox genes Mouse embryo Figure 21-12 Homeotic mutant Normal fruit fly Homeotic mutant Antennae Haltere Wings in place of halteres Legs in place of antennae Wrapping up Eukaryotic Genomes Eukaryotic Genomes consist of many noncoding and repetitive sequences that scientists now suspect actually serve important purposes within the cells Transposable Elements-”Jumping Genes” Transposons are genes that move via a DNA intermediate Retrotransposons are what most transposable elements are and they move via an RNA intermediate Figure 20-5 HOW LINE TRANSPOSABLE ELEMENTS SPREAD Gene for reverse transcriptase Gene for integrase 1. A long interspersed nuclear element (LINE) exists in DNA. DNA Cytoplasm Original location of LINE (1–5 kb) 2. RNA polymerase LINE protein transcribes LINE, producing LINE mRNA. LINE mRNA 3. LINE mRNA exits RNA polymerase Ribosome LINE mRNA and LINE proteins nucleus and is translated. Reverse transcriptase 4. LINE mRNA and Integrase proteins enter nucleus. cDNA 5. Reverse mRNA transcriptase makes LINE cDNA from mRNA, then makes cDNA double stranded. Reverse transcriptase Integrase 6. Integrase cuts chromosomal DNA and inserts LINE cDNA. 7. New copy of LINE is Original copy New copy integrated into genome. Figure 20-8 GENE DUPLICATION BY UNEQUAL CROSSOVER 1 2 3 4 5 1. Start with two homologous 6 chromosomes containing the same genes (numbered 1–6). Homologous chromosomes 1 2 3 4 1 2 3 4 1 3 2 5 5 6 2. The genes misalign during 6 meiosis I. Crossing over and recombination occur. 4 5 6 Gene deletion 1 2 4 5 6 Gene duplication 1 2 3 3 4 5 6 3. Gene 3 has been deleted from one chromosome and duplicated in the other chromosome. The Molecular Biology of Cancer As we learned before, cancers are unregulated cells. Scientists think that that oncogenes-are the cancercausing genes as well as random mutation from DNA damage Proto-oncogenes are the normal genes that code for proteins that regulate the cell cycle Tumor supressor genes inhibit cell division There are generally 3 ways that protooncogenes become oncogenes DNA movement w/in a genome Point mutations Amplification of a proto-oncogene The development of Cancer Cancer requires multiple mutations and at least 1 oncogene Cancers can begin as benign polyps, tumors, etc, but the longer that these exist, the longer there is for the necessary mutations to accumulate Viruses also play a role in the development of some cancers Retroviruses have oncogenes that can be donated to the host cell The viral DNA may also be inserted in such a way that it disrupts a tumor-supressing gene. What about Genetic Predisposition? It makes sense, that if oncogenes are partially responsible for cancer, that certain cancers should run in families Examples of cancers with a strongly-heritable component are colorectal cancer and breast cancer In Breast cancer, mutations in the BRCA1 or BRCA2 genes appear to be responsible for many breast cancers These genes play a role in the cell’s DNA damage repair proteins It makes sense, then, that avoiding mutagens would lower the risk of cancer, even if one has the mutations in his/her genome Viruses At their simplest, these are a piece of genetic material with a protein coat (called the capsid) These are considered non-living b/c they have no metabolism, homeostasis, growth, and require a host cell to carry out their functions Are extraordinarily small, since they are active inside of cells. They can contain traditional, double-stranded DNA, single-stranded DNA, or even RNA Recall that they’re specific to their hosts-the capsid must fit into a receptor on the host cell in order to infect the cell, and there’s a lot of variety in the capsid A few, like influenza, have a viral envelope, derived from the membranes of their host cell Figure 35-7 Nonenveloped virus Genome (in this case, DNA) Capsid (protein) Enveloped virus Viral protein Genome (in this case, RNA) Host protein Capsid (protein) Envelope (phospholipid bilayer) Figure 35-17 Figure 35-6 Bacteriophage T4 Tobacco mosaic virus Adenovirus Influenza virus Figure 35-21 Healthy leaf Leaf infected with virus Figure 35-1-1 Brain and CNS encephalitis rabies polio herpes zoster yellow fever Ebola dengue West Nile Lymphatic and immune systems Epstein-Barr HIV paramyxovirus (e.g., measles) Trachea and lungs parainfluenza RSV influenza adenovirus Heart coxsackie Figure 35-1-2 Digestive track and liver hepatitis A, B, C, D, E rotavirus Blood vessels and blood cells erythrovirus Ebola hantavirus Reproductive organs herpes 2 papillomavirus Skin rubella variola papillomavirus herpes 1 molluscum contagiosum Skeletal muscles coxsackie Peripheral nerves rabies Figure 35-15-Table 35-2 Viral Cycles All cycles begin with the virus binding to the host cell Some are taken in by endocytosis Others inject their genome Also called virulent phages, because these infect, degrade the host’s DNA, reproduce, and kill the host cell right away (rhinovirus, influenza, T4 bacteriophage) Lytic Viruses Lysogenic Viruses These are called temperate phages because these inject their genome (prophage), and integrate it within the host’s DNA, so they can “hide” inside of the host until they’re triggered (ex: HSV, HPV, lambda phage) Retroviruses These are special lysogenic viruses whose prophage is made of RNA, so they must inject reverse transcriptase (rt) as well as the prophage in order to integrate with the host’s chromosomes (ex: HIV) Figure 35-8a LYTIC REPLICATION RESULTS IN A NEW GENERATION OF VIRUS PARTICLES AND THE DEATH OF THE HOST CELL. Host-cell genome Virus particle DNA mRNA Protein 1. Viral genome enters 2. Viral genome host cell. is transcribed; viral proteins are produced. DNA Protein 3. Viral genome is replicated. 6. Free particles in tissue or environment are transmitted to new host. 5. Particles exit 4. Particles assemble to exterior. inside host. Figure 35-8b LYSOGENIC REPLICATION RESULTS IN VIRUS GENES BEING TRANSMITTED TO DAUGHTER CELLS OF THE HOST. 1. Viral genome 2. Viral genome 3. Host-cell DNA 4. Cell divides. Virus is transmitted enters host cell. integrates into hostcell genome. polymerase copies chromosome. to daughter cells. Figure 35-14 cDNA RNA template First, reverse transcriptase synthesizes cDNA from RNA Double-stranded DNA cDNA template Then, reverse transcriptase synthesizes double-stranded DNA from cDNA Preventing Viruses Some cells have evolved defenses against these viruses in the form of restriction enzymes that can destroy the viral DNA Vaccination helps animals avoid viral infection Being infected allows the immune system to learn to detect and fight existing strains of viruses Figure 35-9 HOW VACCINATION WORKS The antigens are usually protein components of a virus capsid or envelope The cells that produce specific antibodies remain active for a long time—years or decades Virus 1. Viral antigens 2. Antigens bind 3. These cells stimulate 4. Later, if the host organism 5. Viruses that are (in red) are introduced into the body. to receptors on certain immune system cells. other immune system cells to produce antibodies (in green) to the virus. is exposed to actual virus particles, the antibody-producing cells are activated. The virus particles become coated with antibodies. coated with antibodies are destroyed by immune system cells. Emerging Viruses New Viruses occur because of 3 main causes: Mutation of existing viruses-especially RNA viruses, which mutate faster Viruses coming from a small, isolated human population Viruses jumping from one species to anotherespecially in closely-related species Epidemic=emergence of a new strain of an existing virus Pandemic=global epidemic Plants and viruses Yes, plants get viruses too Transmission occurs in 1 of 2 ways: Horizontal transmission-plant is infected by an external source of virus, especially if the epidermis of the plant is damaged (herbivore damage is especially bad b/c herbivores can act as horizontal transmitters) Vertical transmission-plant inherits the virus from the parent. The virus can spread through the plasmodesmata Viroids and Prions Viroids=circular pieces of RNA that infect plants These reproduce inside of the plant’s cells and cause errors in the regulation of growth Infected plants typically exhibit stunted growth Prions=infectious proteins (ex: BSE=mad cow disease) These develop slowly (up to 10 year incubation period) These are indestructible Scientists believe that these are abnormallyfolded proteins, that, when they enter a cell that has the normal proteins, corrupt these DNA Technology Involves a number of techniques for identifying, copying, cutting, and modifying DNA These are all part of the field of biotechnology Genetic engineering-directly manipulating the DNA of an organism, is also part of biotechnology DNA Cloning Involves copying DNA-useful for studying specific genes, since you can keep a library of cloned genes, rather than search an entire genome for them Most cloning is done with bacterial plasmids-circular pieces of DNA in a bacteria that contain only a few genes and are separate from the bacteria’s main chromosome (these are called cloning vectors) In recombinant DNA, a plasmid is removed from the bacteria and spliced with a new piece of DNA. This can be re-inserted into the bacteria, which will both express the gene and copy it every time the cell divides The gene we inserted is called the donor gene The process of putting the gene back into the bacteria is called transformation Figure 19-2 GENES CAN BE CLONED BY INSERTING THEM INTO PLASMIDS Recognition site 5 3 3 5 Recognition site 5 3 Restriction endonuclease (EcoR1) 3 5 Plasmid Plasmid Recombinant plasmid Sticky end 1. Plasmid DNA 2. Attach the same 3. A restriction endonuclease 4. Sticky ends on 5. Use DNA ligase to contains a recognition site for a restriction endonuclease. recognition site to the gene that will be inserted into the plasmid. makes staggered cuts at each of the recognition sites, creating “sticky ends.” plasmid and on gene to be inserted bind by complementary base pairing. catalyze a phosphodiester bond at points marked by green arrows, “sealing” the inserted gene. Restriction Enzymes These are enzymes that cut DNA at specific recognition sequences (usually palindromic) Useful for many biotechnology applications because we know their recognition sequences Each resulting restriction fragment (DNA cut with a restriction enzyme), has stickyends so that it is easy to splice Storing Cloned Genes Genomic Library=cell clones containing the recombinant plasmid Sometimes, phages are used as genomic libraries b/c they can carry bigger inserts Scientists have also found mRNA extracts useful in producing libraries b/c of the poly-A tail The tail is a useful primer for reverse transcriptase, and can be used to make cDNA (complimentary DNA) The cDNA can then be inserted into the cloning vector Bacterial Artificial Chromosomes (BAC) can also act as libraries This is simply a large plasmid which contains the inserts and genes necessary for replication Figure 19-3-1 CREATING A cDNA LIBRARY THAT CONTAINS THE HUMAN GROWTH HORMONE GENE Singlestranded cDNA mRNA Doublestranded cDNA mRNA Reverse transcriptase 1. Isolate mRNAs from cells in pituitary 2. Use reverse transcriptase to 3. Make the cDNA double- gland. synthesize a cDNA from each mRNA. stranded. Screening a Library for a Gene This involves creating a nucleic acid probe that has a complimentary sequence to the DNA we’re looking for We can then see where this probe hybridizes to find the gene Figure 19-4 USING A DNA PROBE TO FIND A TARGET SEQUENCE IN A COLLECTION OF MANY DNA SEQUENCES Labeled probe 1. Single-stranded DNA probe has a label that can be visualized. 2. Expose probe to collection of single-stranded DNA sequences. 3. Probe binds to complementary sequences in target DNA—and only to that DNA. Target DNA is now labeled and can be isolated. Expressing Eukaryotic cloned DNA Eukaryotic expression in bacteria is sometimes difficult b/c the promoters and control sequences are often different Scientists use an expression vector, a vector that has a very active promoter region upstream from the donor gene Scientists also occasionally need to use cDNA donor genes b/c of the presence of introns in the eukaryotic genes, making them unwieldy Yeasts can be used as cloning vectors to completely bypass this problem Yeast Artificial Chromosomes (YACs) combine the necessary origin for DNA replication, centromeres, and telomeres, with the donor genes Sometimes, you need to use a eukaryotic vector b/c only it is capable of the post-translational protein modifications necessary for the protein to function PCR Polymerase Chain Reaction allows the scientist to amplify a sample of DNA Produces results within hours, rather than days Involves thermal cycling to denature (unzip) the DNA molecule with heat, then cooling to promote annealing (hydrogen bonding), and uses a heatstable DNA polymerase molecule Figure 19-6 PCR primers must be located on either side of the target sequence, on opposite strands. 5 3 Primer 3 5 Primer Region of DNA to be amplified by PCR When target DNA is single stranded, primers bind and allow DNA polymerase to work. 5 3 3 5 3 Primer Primer 5 3 5 Figure 19-7 THE POLYMERASE CHAIN REACTION IS A WAY TO PRODUCE MANY IDENTICAL COPIES OF A SPECIFIC GENE 3 dNTPs 5 3 5 1. Start with a solution containing template DNA, synthesized primers, and an abundant supply of 3 Primers the four dNTPs. 5 2. Denaturation Heating leads to denaturation of the double-stranded DNA. 5 3 5 5 5 5 3 5 3 5 3 5 5 3 3 3 3. Primer annealing At cooler temperatures, the primers bind to the template DNA by complementary base pairing. 4. Extension During incubation, Taq polymerase uses dNTPs to synthesize complementary DNA strand, starting at the primer. 5. Repeat cycle of three steps (2–4) again, doubling the copies of DNA. 6. Repeat cycle again, up to 20–30 times, to produce millions of copies of template DNA. DNA Sequences Gel Electrophoresis Uses charge and size to pull fragments of DNA across a Gel Useful for generating characteristic banding patterns, but also for looking at differences in sequences, as the DNA fragments are cut with restriction enzymes Southern Blotting Is a combination of gel electrophoresis and DNA hybridization Probe is radioactive Figure 20-7b A gel showing minisatellite seqences from unrelated and related individuals X M B U U U Lane sources: X: An unrelated individual M: A mother B: A boy the mother claims is her own U: Undisputed children of the mother Figure 19-8l SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A LARGE COLLECTION OF DNA FRAGMENTS Location of restriction endonuclease cuts Samples from four individuals Sample 1 1 2 3 4 Doublestranded DNA Power supply Double-stranded DNA 1. Restriction endonucleases cut 2. A sample consists of 3. During electrophoresis, DNA sample into fragments of various lengths. Each type of restriction endonuclease cuts a specific sequence of DNA. all the DNA fragments of various lengths. The sample is loaded into a gel for electrophoresis. a voltage gel separates DNA fragments by size. Small fragments run faster. Figure 19-8r SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A LARGE COLLECTION OF DNA FRAGMENTS 1 2 3 4 Singlestranded DNA Stack of blotting paper Labeled probe DNA Filter Gel Sponge in alkaline solution 4. The DNA 5. Blotting. An alkaline 6. Hybridization with labeled 7. Visualize fragments are treated to make them single stranded. solution wicks up through the gel into blotting paper. DNA fragments from the gel are carried to the filter, where they are permanently bound. probe. The filter is put into a solution containing labeled probe DNA. The probe binds to DNA fragments containing complementary sequences. fragments bound by probe. Fluorescence or autoradiography (see BioSkills 7) is used to find label. DNA Sequencing This is when the sequence of bases on the molecule is determined Mostly, this is automated now. Dideoxy Chain Method of Sequencing is one of these methods, using fluorescent dyes and can sequence a segment up to about 800 bps Figure 19-9 DIDEOXY SEQUENCING Smaller fragments 5 end Template DNA 3 5 5 Normal dNTP (extends 3 DNA strand) Larger fragments 3 end 5 ddCTP’s ddNTP (terminates synthesis) 3 ddATP’s No OH ddTTP’s 5 3 Labeled primer Non-template DNA Non-template DNA 5 3 Template DNA 3 5 ddGTP’s 1. Incubate a large number of normal dNTP’s with a small number of ddNTP’s (in this case starting with ddGTP’s), template DNA, a primer for the target sequence, and DNA polymerase. 2. Collect DNA strands that are produced. Each 3. Repeat process three more 4. Line up different-length strands by size using gel strand will end with a ddGTP (corresponding to a C on the template strand). times using ddCTPs, ddATPs, and ddTTPs, which will terminate synthesis where G’s, T’s, and A’s occur on the template strand, respectively. electrophoresis to determine DNA sequence. DNA sequence Figure 19-10 FLUORESCENT MARKERS IMPROVE SEQUENCING EFFICIENCY. Long fragments Template DNA DNA polymerase 1. Do one sequencing reaction 2. Fragments of newly instead of four. Reaction mix contains ddATP, ddTTP, ddGTP, ddCTP with distinct fluorescent markers. (With radioactive labels, four reactions are needed—one labeled ddNTP at a time.) synthesized DNA that result have distinctive labels. Short fragments Capillary Output tube 3. Separate fragments via electrophoresis in massproduced, gel-filled capillary tubes. Automated sequencing machine reads output. Sequencing Whole Genomes HGP was set up to create chromosome maps of the human genome This was done with a 3-step approach The first step was to create a linkage map (like we did with Sordaria Next, a physical map was constructed, using linkage mapping data Finally, the genes were sequenced (dideoxy sequencing) Shotgun sequencing Uses cut-ups of human DNA, inserted into bacteria for cloning, then analysis of the small sequences and reconstruction Figure 20-2 SHOTGUN SEQUENCING A GENOME 160 kb fragments 1. Cut DNA into fragments of 160 kb, using sonication. Genomic DNA 2. Insert fragments into bacterial artificial BAC library BAC Main bacterial chromosome chromosomes; grow in E. coli cells to obtain large numbers of each fragment. 3. Purify each 160-kb fragment, then cut 1-kb fragments each into a set of 1-kb fragments, using sonication, so that 1-kb fragments overlap. 4. Insert 1-kb fragments into plasmids; grow “Shotgun clones” in E. coli cells. Obtain many copies of each fragment. 5. Sequence each fragment. Find regions Shotgun sequences where different fragments overlap. 6. Assemble all the 1-kb fragments from each original 160-kb fragment by matching overlapping ends. Draft sequence 7. Assemble sequences from different BACs (160-kb fragments) by matching overlapping ends. Analyzing Gene Expression Northern Blotting Same basic procedure as Southern Blotting, but we’re looking for mRNA in cells at different stages of development to see if the protein we’re studying is needed at these steps Reverse-transcriptase PCR Will accomplish the same thing as Northern Blotting, but uses rt to make cDNA from the mRNA, which is then put through PCR and run on a gel The gene we’re observing only occurs in samples that contained the mRNA with that gene DNA Microarray Assays Hybridization of cDNA with a pre-fixed slide of mRNA This helps scientists to see which genes may be turned on at the same time and thus working together Figure 20-11 Microarray slide Exon 286 Each spot on the slide contains many singlestranded copies of a different exon Exon 287 Exon 288 Figure 20-12 PROTOCOL FOR A MICROARRAY EXPERIMENT Normal temperature High temperature 1. Use reverse transcriptase to prepare single-stranded cDNA from mRNA of control cells and treatment cells. cDNA mRNA Microarray computer output: 2. React with labeled nucleotides cDNA probes to add fluorescent green label to control cDNA and fluorescent red label to treatment cDNA. 3. Probe a microarray with the labeled cDNAs. Probe cDNA will bind and label spots containing complementary sequences. Microarray 4. Shine laser light to induce fluorescence. Analyze the pattern of hybridization between the two cDNAs and the DNA on the microarray. Green = genes transcribed in control cells Yellow = genes transcribed equally in both cells Dark = low gene expression Red = genes transcribed in treatment cells Determining Gene Function Usually, scientists disable a gene which has been identified by DNA tech, then observe the consequences in the cell This is called in vitro mutagenesis Cloning Organisms Plants can be cloned using single-cell cultures Differentiated cells from the root can be grown in culture and become entire organisms, genetically identical to the parent When mature cells are capable of dedifferentiating and redifferentiating, they’re called totipotent Recall that through propogation, plants are cloned as well! Animals can be cloned via nuclear transfer Originally, an unfertilized egg was used, which worked, except that the ability of the new nucleus to control the resultant clone decreased with donor nucleus age Dolly was different because she was made from an alreadydifferentiated mammary cell. Dedifferentiation was accomplished by culturing the cell in a nutrient poor medium Dolly died at age 6, when she was euthanized after suffering from a lung disease that usually effects much older sheep, leading scientists to speculate that clones weren’t as vigorous as the original organism. Figure 21-3 CLONING A SHEEP Mammary-cell Egg-cell donor sheep donor sheep 1. Start with two female sheep. Each will donate one cell. 2. Culture mammarygland cells. Remove nucleus from egg cell. Mammary cells Egg cell 3. Fuse the mammary-gland cell to enucleated egg cell. Fused cell Early embryo Surrogate mother 4. Egg cell now contains nucleus from mammarygland cell. 5. Grow in culture. Embryo begins development. 6. Implant early embryo in uterus of third sheep. 7. Embryo develops Cloned sheep “Dolly” normally, resulting in lamb that is genetically identical to mammary-cell donor. This result supports the hypothesis that mature cells contain all the genes in the genome. Problems with Organismal Cloning Cloning is inefficient-only a small percent of cloned embryos develop normally, and there are often defects (like pneumonia, obesity, liver failure, and premature death) Scientists are working to improve the efficiency of cloning by studying systematic changes to the chromatin as the nucleus matures Stem Cells These are unspecialized cells Ultimately, this is what scientists would like to achieve through cloning for the treatment of disease The most common place to find these is in embryos (these are pluripotentcan develop into a wide variety of cell types), although, there are some less flexible stem cells in adults Applications of Biotechnology Medical Applications Diagnosis of disease Gene Therapy Pharmaceuticals Forensic Evidence Environmental Clean-up Ag Apps “old school”=selective breeding Ethics Issues with Biotechnology Safety questions about GMOs Problems with the technologies leading to “super bugs” and maldeveloped mutants Creating organisms with medical issues since clones aren’t as vigorous Obtaining Stem Cells Where to “draw the line” with research?