AP Biology Ch. 20 Slides for Test

DNA Cloning Plasmids -used to insert foreign DNA Recombinant plasmid is inserted into a bacteria Reproduction in bacteria cell results in cloning of the plasmid including foreign gene Useful for making copies of a gene and producing a protein product

Making recombinant DNA Use bacterial restriction enzymes Cuts DNA at specific restriction sites Cuts covalent bonds between sugar phosphate backbone Making restriction fragments Most useful restriction enzymes cut DNA in a staggered way forming “sticky ends” DNA ligase seals the bonds between restriction fragments. Cloning vector is original plasmid carrying foreign gene into the host cell.

Storing Cloned Genes in DNA Libraries A genomic library that is made using bacteria is the collection of recombinant vector clones produced by cloning DNA fragments from an entire genome. A genomic library that is made using bacteriophages is stored as a collection of phage clones. A bacterial artificial chromosome (BAC)is a large plasmid that can carry a large insert with many genes.

Storing Cloned Genes in DNA Libraries Complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell. cDNA library represents only part of the genome--only the subset of genes transcribed into mRNA in th original cells

Reverse transcriptase Poly-A tail mRNA Fig. 20-6-3 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA strand Primer Degraded mRNA Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene

Reverse transcriptase Poly-A tail mRNA Fig. 20-6-4 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA strand Primer Degraded mRNA Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene DNA polymerase

Reverse transcriptase Poly-A tail mRNA Fig. 20-6-5 DNA in nucleus mRNAs in cytoplasm Reverse transcriptase Poly-A tail mRNA DNA strand Primer Degraded mRNA Figure 20.6 Making complementary DNA (cDNA) for a eukaryotic gene DNA polymerase cDNA

Screening a Library for Clones Carrying a Gene of Interest A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene This process is called nucleic acid hybridization

For example, if the desired gene is A probe can be synthesized that is complementary to the gene of interest For example, if the desired gene is – Then we would synthesize this probe … … 5 G G C T A A C T T A G C 3 3 C C G A T T G A A T C G 5

The DNA probe can be used to screen a large number of clones simultaneously for the gene of interest Once identified, the clone carrying the gene of interest can be cultured

Radioactively labeled probe molecules Fig. 20-7 TECHNIQUE Radioactively labeled probe molecules Probe DNA Gene of interest Multiwell plates holding library clones Single-stranded DNA from cell Film • Figure 20.7 Detecting a specific DNA sequence by hybridizing with a nucleic acid probe Nylon membrane Nylon membrane Location of DNA with the complementary sequence

Detecting DNA sequence by hybridization with a nucleic acid probe Results The location of the black spot on the photographic film identifies the clone containing the gene of interest. By using probes with different nucleotide sequences, researchers can screen the collection of bacterial clones for different genes.

Expressing Cloned Eukaryotic Genes After gene has been cloned, its protein products can be produced in larger amounts for research. Cloned genes can be expressed as protein in either bacterial or eukaryotic cells.

Problems with expressing eukaryotic genes in bacterial host cells Scientists use an expression vector, a cloning vector that contains a highly active prokaryotic promoter This allows the bacteria to recognize the promoter and proceed to express the foreign gene. This allows synthesis of many eukaryotic proteins in bacteria cells. Another problem is the presence of introns in eukaryotic genes. Bacteria cells do not have RNA splicing machinery. This can be overcome by using cDNA which includes only the exons.

Eukaryotic cloning and Expression Systems The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems YACs behave normally in mitosis and can carry more DNA than a plasmid Eukaryotic hosts can provide the post-translational modifications that many proteins require

Amplifying DNA using Polymerase Chain reaction (PCR) PCR can produce many copies of a specific target segment of DNA Three-step cycle-heating--cooling--and replication Brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8 TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleo- tides Figure 20.8 The polymerase chain reaction (PCR) Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

TECHNIQUE 5 3 Target sequence Genomic DNA 3 5 Fig. 20-8a Figure 20.8 The polymerase chain reaction (PCR)

Cycle 1 yields 2 molecules Fig. 20-8b 1 Denaturation 5 3 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension Figure 20.8 The polymerase chain reaction (PCR) New nucleo- tides

Cycle 2 yields 4 molecules Fig. 20-8c Cycle 2 yields 4 molecules Figure 20.8 The polymerase chain reaction (PCR)

molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8d Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Figure 20.8 The polymerase chain reaction (PCR)

Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene DNA cloning allows researchers to Compare genes and alleles between individuals Locate gene expression in a body Determine the role of a gene in an organism Several techniques are used to analyze the DNA of genes

Gel Electrophoresis and Southern Blotting Gel electrophoresis uses a gel as a molecular sieve to separate nucleic acids by size A current is applied to the gel that causes the charged molecules to move DNA is negatively charged and it moves towards a positive pole. Molecules are sorted into “bands” by their size

Mixture of DNA mol- ecules of different sizes Fig. 20-9a TECHNIQUE Power source Mixture of DNA mol- ecules of different sizes – Cathode Anode + Gel 1 Power source Figure 20.9 Gel electrophoresis – + Longer molecules 2 Shorter molecules

Fig. 20-9b RESULTS Figure 20.9 Gel electrophoresis

In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene The procedure is also used to prepare pure samples of individual fragments

Fig. 20-10 Normal -globin allele Normal allele Sickle-cell allele 175 bp 201 bp Large fragment DdeI DdeI DdeI DdeI Large fragment Sickle-cell mutant -globin allele 376 bp 201 bp 175 bp 376 bp Large fragment DdeI Figure 20.10 Using restriction fragment analysis to distinguish the normal and sickle-cell alleles of the β-globin gene DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

A technique called Southern blotting combines gel electrophoresis of DNA fragments with nucleic acid hybridization Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

Restriction fragments DNA + restriction enzyme I II III Fig. 20-11a TECHNIQUE Heavy weight Restriction fragments DNA + restriction enzyme I II III Nitrocellulose membrane (blot) Gel Sponge I Normal -globin allele II Sickle-cell allele III Heterozygote Paper towels Alkaline solution Figure 20.11 Southern blotting of DNA fragments 1 Preparation of restriction fragments 2 Gel electrophoresis 3 DNA transfer (blotting)

Radioactively labeled probe for -globin gene Fig. 20-11b Radioactively labeled probe for -globin gene Probe base-pairs with fragments I II III I II III Fragment from sickle-cell -globin allele Film over blot Figure 20.11 Southern blotting of DNA fragments Fragment from normal -globin allele Nitrocellulose blot 4 Hybridization with radioactive probe 5 Probe detection

Studying the Expression of Interacting Groups of Genes Automation has allowed scientists to measure expression of thousands of genes at one time using DNA microarray assays DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions

Labeled cDNA molecules (single strands) Fig. 20-15 TECHNIQUE Tissue sample 1 Isolate mRNA. 2 Make cDNA by reverse transcription, using fluorescently labeled nucleotides. mRNA molecules Labeled cDNA molecules (single strands) 3 Apply the cDNA mixture to a microarray, a different gene in each spot. The cDNA hybridizes with any complementary DNA on the microarray. DNA fragments representing specific genes Figure 20.15 DNA microarray assay of gene expression levels DNA microarray DNA microarray with 2,400 human genes 4 Rinse off excess cDNA; scan microarray for fluorescence. Each fluorescent spot represents a gene expressed in the tissue sample.

Concept 20.3: Cloning organisms may lead to production of stem cells for research and other applications Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell

Cloning Plants: Single-Cell Cultures One experimental approach for testing genomic equivalence is to see whether a differentiated cell can generate a whole organism A totipotent cell is one that can generate a complete new organism

EXPERIMENT RESULTS Transverse section of carrot root 2-mg fragments Fig. 20-16 EXPERIMENT RESULTS Transverse section of carrot root 2-mg fragments Figure 20.16 Can a differentiated plant cell develop into a whole plant? Fragments were cultured in nu- trient medium; stirring caused single cells to shear off into the liquid. Single cells free in suspension began to divide. Embryonic plant developed from a cultured single cell. Plantlet was cultured on agar medium. Later it was planted in soil. A single somatic carrot cell developed into a mature carrot plant.

Cloning Animals: Nuclear Transplantation In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg However, the older the donor nucleus, the lower the percentage of normally developing tadpoles

EXPERIMENT RESULTS Frog embryo Frog egg cell Frog tadpole UV Fig. 20-17 Frog embryo Frog egg cell Frog tadpole EXPERIMENT UV Fully differ- entiated (intestinal) cell Less differ- entiated cell Donor nucleus trans- planted Donor nucleus trans- planted Enucleated egg cell Egg with donor nucleus activated to begin development RESULTS Figure 20.17 Can the nucleus from a differentiated animal cell direct development of an organism? Most develop into tadpoles Most stop developing before tadpole stage

Reproductive Cloning of Mammals In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus

TECHNIQUE RESULTS Mammary cell donor Egg cell donor Fig. 20-18 TECHNIQUE Mammary cell donor Egg cell donor 1 2 Egg cell from ovary Nucleus removed Cultured mammary cells 3 Cells fused 3 Nucleus from mammary cell 4 Grown in culture Early embryo Figure 20.18 Reproductive cloning of a mammal by nuclear transplantation For the Discovery Video Cloning, go to Animation and Video Files. 5 Implanted in uterus of a third sheep Surrogate mother 6 Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor RESULTS

Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”

Fig. 20-19 Figure 20.19 CC, the first cloned cat, and her single parent

Problems Associated with Animal Cloning In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development

Stem Cells of Animals A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem cells; these are able to differentiate into all cell types The adult body also has stem cells, which replace nonreproducing specialized cells

From bone marrow in this example Fig. 20-20 Embryonic stem cells Adult stem cells Early human embryo at blastocyst stage (mammalian equiva- lent of blastula) From bone marrow in this example Cells generating all embryonic cell types Cells generating some cell types Cultured stem cells Different culture conditions Figure 20.20 Working with stem cells Different types of differentiated cells Liver cells Nerve cells Blood cells

The aim of stem cell research is to supply cells for the repair of damaged or diseased organs

Many fields benefit from DNA technology and genetic engineering Concept 20.4: The practical applications of DNA technology affect our lives in many ways Many fields benefit from DNA technology and genetic engineering One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

Human Gene Therapy Gene therapy is the alteration of an afflicted individual’s genes Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of genes into specific types of cells, for example bone marrow Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

Insert RNA version of normal allele into retrovirus. Fig. 20-22 Cloned gene 1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Figure 20.22 Gene therapy using a retroviral vector Bone marrow 4 Inject engineered cells into patient.

Pharmaceutical Products Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases

Protein Production in Cell Cultures Host cells in culture can be engineered to secrete a protein as it is made This is useful for the production of insulin, human growth hormones, and vaccines

Environmental Cleanup Genetic engineering can be used to modify the metabolism of microorganisms Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels

Agricultural Applications DNA technology is being used to improve agricultural productivity and food quality

Animal Husbandry Protein Production by “Pharm” Animals and Plants Genetic engineering of transgenic animals speeds up the selective breeding process Transgenic animals are made by introducing genes from one species into the genome of another animal Beneficial genes can be transferred between varieties or species Protein Production by “Pharm” Animals and Plants Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use “Pharm” plants are also being developed to make human proteins for medical use