today’s plan: 2/16/11 bellwork: talk about yesterday and the test (30 mins) casting gels for...

Todays 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)

Todays Plan: 2/18/2010 Bellwork: Test discussion (15 mins) Transformation Notes, if time

Todays 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)

Todays 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)

Todays 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)

Todays Plan: 2/24/10 Bellwork: Flies/Test Q&A (20 mins) DNA Tech test (the rest of class)

Todays Plan: 9/15/09 Bellwork: Intstructions (5 mins) Research for Discussion (30 mins) Bioethics discussion (the rest of class)

Todays Plan: 9/16/09 Bellwork: Last Presentation (10 mins) Senses Stations (50 mins) Continue with DNA Tech notes (the rest of class)

Todays Plan: 9/17/09 Bellwork: Class Optional Taste Demo (10 mins) Finish Senses Stations (50 mins) Finish DNA Tech notes (the rest of class)

Todays 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 repressors hold on the DNA (ex: lac operon-repressor is active unless bound to allolactose)

Figure 17-4 E. coli -Galactosidase Galactoside permease Lactose Plasma membrane Glucose Galactose

Figure 17-8 lacl promoter DNA Promoter of lac operon Operator lacZlacYlacA lac operon

Figure 17-7 Repressor present, lactose absent: Repressor present, lactose present: No repressor present, lactose present or absent: Transcription occurs. Repressor synthesized DNA lacl + RNA polymerase bound to promoter (blue DNA) lacZ lacY TRANSCRIPTION BEGINS -Galactosidase Permease mRNA lacZ lacY RNA polymerase bound to promoter (blue DNA) Lactose-repressor complex Repressor synthesized No functional repressor synthesized mRNA TRANSCRIPTION BEGINS -Galactosidase Permease lacZ lacY RNA polymerase bound to promoter (blue DNA) Lacl Repressor binds to DNA. No transcription occurs. Lactose binds to repressor, causing it to release from DNA. Transcription occurs (lactose acts as inducer). Normal lacl gene Normal lacl gene lacl + Mutant lacl gene The repressor blocks transcription

Figure 17-9 When tryptophan is present, transcription is blocked. When tryptophan is absent, transcription occurs. Repressor Tryptophan No transcription Operator RNA polymerase bound to promoter RNA polymerase bound to promoter TRANSCRIPTION 5 genes coding for enzymes involved in tryptophan synthesis

Figure 17-10 lac operontrp operon Catabolism Anabolism (breakdown of lactose) (synthesis of tryptophan) Repressor Lactose Tryptophan Lactose binds to repressor Tryptophan binds to repressor Lactose- repressor complex releases from operator Operator Tryptophan- repressor complex binds to operator No more transcription of trp operon Transcription of lac operon TRANSCRIPTION

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 isnt 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. The amount of cAMP and the rate of transcription of the lac operon are inversely related to the concentration of glucose. ATP Adenylyl cyclase Glucose inhibits this enzyme cAM P Two phosphate groups Infrequent transcription of lac operon (Cell continues to use glucose as energy source.) CAP does not bind to DNA CAP LOW cAMP INACTIVE adenylyl cyclase HIGH glucose concentration LOW glucose concentration ACTIVE adenylyl cyclase HIGH cAMP CAP CAP-cAMP complex binds to DNA Frequent transcription of lac operon (Cell uses lactose if lactose is present.)

Prokaryote vs. Eukaryote Genomes Smaller Genome Fewer Genes Higher gene density (more genes in a smaller segment of DNA Relatively few noncoding regions and protein genes are continuous 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 Chromatin (DNA-protein complex) 1. Chromatin remodeling 2. Transcription Open DNA (Some DNA not closely bound to proteins) 3. RNA processing Primary transcript (pre-mRNA) Cap Tail Mature mRNA Cytoplasm 4. mRNA stability 5. Translation Degraded mRNA (mRNA lifespan varies) mRNA Polypeptide Active protein 6. Post-translational modification (folding, transport, activation, degradation of protein)

DNA Regulation Chromatin is DNA that is packaged with proteins, called histones. The basic unit of chromatin is called the nucleosome 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 Its also been shown that methyl groups are also added to the histone tails, which can promote condenstion of the chromatin DNA Methylation 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 Group of 8 histone proteins Nucleosome 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 dont code but bind proteins

Figure 18-7 Start site Exon Intron EnhancerPromoterEnhancer Intron Exon Promoter- proximal element 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 its the combination of control elements (proteins, activators, etc) that is unique to each gene

Figure 18-10 THE ELEMENTS OF TRANSCRIPTIONAL CONTROL: A MODEL Regulatory transcription factor Chromatin remodeling complex (or HATs) 1. Regulatory transcription factors recruit chromatin- remodeling complex, or HATs. Chromatin decondenses. Exposed DNA Promoter-proximal element Promoter Exon Intron Exon Promoter Transcribed portion of gene for muscle-specific protein Co-activators Regulatory transcription factors Promoter-proximal element Basal transcription complex TRANSCRIPTION RNA polymerase II Basal transcription complex 2. When chromatin decondenses, a region of DNA is exposed, including the promoter. 3. Regulatory transcription factors recruit proteins of the basal transcription complex to promoter. Note looping DNA. 4. RNA polymerase II completes the basal transcription complex; transcription begins. Enhancer

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 Cytoplasm Signaling molecule Cell-surface receptor Inactive STAT protein (two single polypeptide chains) Activated STAT protein (dimer of two polypeptide chains) Nuclear envelope Enhancer TRANSCRIPTION Transcription activated Nucleus

Post-transcriptional Gene Regulation RNA Processing-Alternative RNA splicing The same transcript may result in different mature RNA depending on which segments are treated as introns mRNA Degradation In Prokaryotes, mRNA is degraded within a few minutes, but EukaryotesmRNA can last for days or weeks Initiation of Translation 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 Processed mRNAs Skeletal muscle Smooth muscle 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, doesnt

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 theyre complimentary at some part), and can degrade it (if theyre complimentary along the length) Another type of RNA, small interfering RNA (siRNA) can also interfere with mRNAs 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 1. Transcription of a microRNA gene. DNA RNA polymerase Precursor miRNA Cytoplasm Enzyme Mature miRNA Single-stranded miRNA RISC protein complex Target mRNA 2. Initial transcript is processed into a precursor micro RNA (miRNA). 3. Enzyme in cytoplasm cuts out hairpin loop, forming a mature miRNA. 4. miRNA becomes single-stranded and binds to RISC protein complex. 5. miRNA, held by RISC, binds to complementary sequence on 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 embryos 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 single- stranded DNA or RNA probe, complementary in sequence to target mRNA. 4. Treat preserved cells or tissues to make them permeable to probe. Add many copies of probe. 3. Preserve the specimen (in this case, a Drosophila embryo). 2. Add label to probe (a radioactive atom or an enzyme that catalyzes a color- producing reaction). 5. Probe binds to target mRNA. Labeled probe that does not bind to target mRNA is excess, and is washed away. 6. In this case, target mRNAs are concen- trated in the anterior end of the embryo. The label shows up as black in this image. Posterior Anterior Target mRNA DNA probe DNA probe Embryo DNA probe Label

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 genes- when there is a mutant in the mother, there are mutations in the offspring, regardless of the offsprings 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 embryoA bicoid mutant Abdominal segments Abdominal segments Abdominal segments Thoracic segments Head segments

Figure 21-13 Head The location of Hox genes on the mouse chromosome correlates with their pattern of expression in mouse embryos. Hox genes The location of Hox genes on the fly chromosome correlates with their pattern of expression in fly embryos. Fly embryo Mouse embryo Hox genes Thorax Abdomen

Figure 21-12 Homeotic mutant Normal fruit fly Homeotic mutant Legs in place of antennae Wings in place of halteres Haltere 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 DNA 7. New copy of LINE is integrated into genome. New copy Cytoplasm LINE protein Nuclear envelope Ribosome Original copy cDNA mRNA Reverse transcriptase Integrase Reverse transcriptase Original location of LINE (15 kb) RNA polymerase LINE mRNA LINE mRNA and LINE proteins Gene for reverse transcriptase Gene for integrase 6. Integrase cuts chromosomal DNA and inserts LINE cDNA. 5. Reverse transcriptase makes LINE cDNA from mRNA, then makes cDNA double stranded. 4. LINE mRNA and proteins enter nucleus. 3. LINE mRNA exits nucleus and is translated. 2. RNA polymerase transcribes LINE, producing LINE mRNA. 1. A long interspersed nuclear element (LINE) exists in DNA.

Figure 20-8 GENE DUPLICATION BY UNEQUAL CROSSOVER Homologous chromosomes 1 1 1 1 1 2 2 2 2 2 3 3 3 3 4 4 4 4 4 5 5 5 5 5 6 6 6 6 6 Gene deletion Gene duplication 1. Start with two homologous chromosomes containing the same genes (numbered 16). 2. The genes misalign during meiosis I. Crossing over and recombination occur. 3. Gene 3 has been deleted from one chromosome and duplicated in the other chromosome. 12334 56

The Molecular Biology of Cancer As we learned before, cancers are unregulated cells. Scientists think that that oncogenes-are the cancer- causing 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 cells 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 theyre specific to their hosts-the capsid must fit into a receptor on the host cell in order to infect the cell, and theres 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 Genome (in this case, RNA) Capsid (protein) Envelope (phospholipid bilayer) Host protein Viral protein

Figure 35-17

Figure 35-6 Tobacco mosaic virus Adenovirus Influenza virus Bacteriophage T4

Figure 35-21 Leaf infected with virus Healthy leaf

Figure 35-1-1 Heart coxsackie Trachea and lungs parainfluenza RSV influenza adenovirus Lymphatic and immune systems Epstein-Barr HIV paramyxovirus (e.g., measles) Brain and CNS encephalitis rabies polio herpes zoster yellow fever Ebola dengue West Nile

Figure 35-1-2 Digestive track and liver hepatitis A, B, C, D, E rotavirus Blood vessels and blood cells erythrovirus Ebola hantavirus Skeletal muscles coxsackie Skin rubella variola papillomavirus herpes 1 molluscum contagiosum Reproductive organs herpes 2 papillomavirus 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 Lytic Viruses Also called virulent phages, because these infect, degrade the hosts DNA, reproduce, and kill the host cell right away (rhinovirus, influenza, T4 bacteriophage) Lysogenic Viruses These are called temperate phages because these inject their genome (prophage), and integrate it within the hosts DNA, so they can hide inside of the host until theyre 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 hosts chromosomes (ex: HIV)

Figure 35-8a LYTIC REPLICATION RESULTS IN A NEW GENERATION OF VIRUS PARTICLES AND THE DEATH OF THE HOST CELL. Virus particle ProteinmRNA DNA Host-cell genome Protein DNA 1. Viral genome enters host cell. 2. Viral genome is transcribed; viral proteins are produced. 3. Viral genome is replicated. 5. Particles exit to exterior. 4. Particles assemble inside host. 6. Free particles in tissue or environment are transmitted to new host.

Figure 35-8b LYSOGENIC REPLICATION RESULTS IN VIRUS GENES BEING TRANSMITTED TO DAUGHTER CELLS OF THE HOST. 1. Viral genome enters host cell. 2. Viral genome integrates into host- cell genome. 3. Host-cell DNA polymerase copies chromosome. 4. Cell divides. Virus is transmitted to daughter cells.

Figure 35-14 Double-stranded DNA First, reverse transcriptase synthesizes cDNA from RNA Then, reverse transcriptase synthesizes double-stranded DNA from cDNA cDNA template cDNA RNA template

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 1. Viral antigens (in red) are intro- duced into the body. 2. Antigens bind to receptors on certain immune system cells. 3. These cells stimulate other immune system cells to produce antibodies (in green) to the virus. 4. Later, if the host organism is exposed to actual virus particles, the antibody-producing cells are activated. The virus particles become coated with antibodies. 5. Viruses that are coated with antibodies are destroyed by immune system cells. The antigens are usually protein components of a virus capsid or envelope The cells that produce specific antibodies remain active for a long timeyears or decades Virus

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 another- especially 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 plants 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 abnormally- folded 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 bacterias 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 5 3 3 Plasmid 5 5 3 3 Recognition site Restriction endonuclease (EcoR1) Plasmid Recombinant plasmid 1. Plasmid DNA contains a recognition site for a restriction endonuclease. 2. Attach the same recognition site to the gene that will be inserted into the plasmid. 3. A restriction endonuclease makes staggered cuts at each of the recognition sites, creating sticky ends. 4. Sticky ends on plasmid and on gene to be inserted bind by complementary base pairing. 5. Use DNA ligase to catalyze a phosphodiester bond at points marked by green arrows, sealing the inserted gene. Sticky end

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 sticky- ends 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 mRNA Single- stranded cDNA Reverse transcriptase Double- stranded cDNA 3. Make the cDNA double- stranded. 2. Use reverse transcriptase to synthesize a cDNA from each mRNA. 1. Isolate mRNAs from cells in pituitary gland.

Screening a Library for a Gene This involves creating a nucleic acid probe that has a complimentary sequence to the DNA were looking for We can then see where this probe hybridizes to find the gene

Figure 19-4 Labeled probe USING A DNA PROBE TO FIND A TARGET SEQUENCE IN A COLLECTION OF MANY DNA SEQUENCES 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 DNAand 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 heat- stable DNA polymerase molecule

Figure 19-6 PCR primers must be located on either side of the target sequence, on opposite strands. 3 When target DNA is single stranded, primers bind and allow DNA polymerase to work. 5 3 3 3 3 3 5 5 5 5 5 Primer Region of DNA to be amplified by PCR

Figure 19-7 THE POLYMERASE CHAIN REACTION IS A WAY TO PRODUCE MANY IDENTICAL COPIES OF A SPECIFIC GENE 1. Start with a solution containing template DNA, synthesized primers, and an abundant supply of the four dNTPs. dNTPs Primers 5 5 3 3 3 3 5 5 3 3 5 5 5 One cycle 5 5 5 5 5 3 3 3 3 2. Denaturation Heating leads to denaturation of the double-stranded DNA. 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 (24) again, doubling the copies of DNA. 6. Repeat cycle again, up to 2030 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 Lane sources: X: An unrelated individual M: A mother B: A boy the mother claims is her own U: Undisputed children of the mother A gel showing minisatellite seqences from unrelated and related individuals X M B U U U

Figure 19-8l Location of restriction endonuclease cuts Sample 1 Samples from four individuals Double- stranded DNA Double-stranded DNA SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A LARGE COLLECTION OF DNA FRAGMENTS 1. Restriction endonucleases cut DNA sample into fragments of various lengths. Each type of restriction endonuclease cuts a specific sequence of DNA. 2. A sample consists of all the DNA fragments of various lengths. The sample is loaded into a gel for electrophoresis. 3. During electrophoresis, a voltage gel separates DNA fragments by size. Small fragments run faster. Power supply 1 2 3 4

Figure 19-8r Single- stranded DNA Stack of blotting paper Labeled probe DNA Filter Gel Sponge in alkaline solution SOUTHERN BLOTTING: ISOLATING AND FINDING A TARGET DNA IN A LARGE COLLECTION OF DNA FRAGMENTS 4. The DNA fragments are treated to make them single stranded. 5. Blotting. An alkaline solution wicks up through the gel into blotting paper. DNA fragments from the gel are carried to the filter, where they are permanently bound. 6. Hybridization with labeled probe. The filter is put into a solution containing labeled probe DNA. The probe binds to DNA fragments containing complementary sequences. 7. Visualize fragments bound by probe. Fluorescence or autoradiography (see BioSkills 7) is used to find label. 1 2 3 4

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 3 3 5 5 Normal dNTP (extends DNA strand) ddNTP (terminates synthesis) ddGTPs Template DNA 3 No OH 3 5 Labeled primer Non-template DNA ddCTPs ddATPs ddTTPs 5 end3 end Smaller fragments Larger fragments Non-template DNA 5 5 3 3 Template DNA 1. Incubate a large number of normal dNTPs with a small number of ddNTPs (in this case starting with ddGTPs), template DNA, a primer for the target sequence, and DNA polymerase. 2. Collect DNA strands that are produced. Each strand will end with a ddGTP (corresponding to a C on the template strand). 3. Repeat process three more times using ddCTPs, ddATPs, and ddTTPs, which will terminate synthesis where Gs, Ts, and As occur on the template strand, respectively. 5 4. Line up different-length strands by size using gel electrophoresis to determine DNA sequence. DNA sequence

Figure 19-10 FLUORESCENT MARKERS IMPROVE SEQUENCING EFFICIENCY. DNA polymerase Template DNA Long fragments Short fragments Capillary tube Output 1. Do one sequencing reaction instead of four. Reaction mix contains ddATP, ddTTP, ddGTP, ddCTP with distinct fluorescent markers. (With radioactive labels, four reactions are neededone labeled ddNTP at a time.) 2. Fragments of newly synthesized DNA that result have distinctive labels. 3. Separate fragments via electrophoresis in mass- produced, 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 Genomic DNA BAC library 1-kb fragments Shotgun clones Shotgun sequences BAC Main bacterial chromosome 1. Cut DNA into fragments of 160 kb, using sonication. 2. Insert fragments into bacterial artificial chromosomes; grow in E. coli cells to obtain large numbers of each fragment. 3. Purify each 160-kb fragment, then cut each into a set of 1-kb fragments, using sonication, so that 1-kb fragments overlap. 4. Insert 1-kb fragments into plasmids; grow in E. coli cells. Obtain many copies of each fragment. 5. Sequence each fragment. Find regions where different fragments overlap. Draft sequence 6. Assemble all the 1-kb fragments from each original 160-kb fragment by matching overlapping ends. 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 were looking for mRNA in cells at different stages of development to see if the protein were 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 were 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 Exon 286 Exon 287 Exon 288 Microarray slide Each spot on the slide contains many single- stranded copies of a different exon

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. 2. React with labeled nucleotides 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. 4. Shine laser light to induce fluorescence. Analyze the pattern of hybridization between the two cDNAs and the DNA on the microarray. cDNA mRNA cDNA probes Microarray Microarray computer output: 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, theyre 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 already- differentiated 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 werent as vigorous as the original organism.

Figure 21-3 Surrogate mother Cloned sheep Dolly Early embryo Fused cell Mammary cellsEgg cell Mammary-cell donor sheep Egg-cell donor sheep CLONING A SHEEP 1. Start with two female sheep. Each will donate one cell. 5. Grow in culture. Embryo begins development. 6. Implant early embryo in uterus of third sheep. 7. Embryo develops 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. 2. Culture mammary- gland cells. Remove nucleus from egg cell. 3. Fuse the mammary-gland cell to enucleated egg cell. 4. Egg cell now contains nucleus from mammary- gland cell.

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 pluripotent- can 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 arent as vigorous Obtaining Stem Cells Where to draw the line with research?

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