Biol 118 1st Edition Lecture 19 Outline of Last Lecture I. Differential Gene ExpressionII. Mechanisms of Gene Regulation- An OverviewIII. Three Additional Levels Unique to EukaryotesIV. What is Chromatin’s Basic StructureV. Chromatin Structure is Altered in Active GenesVI. Negative Control of Eukaryotic GenesVII. How is Chromatin Altered?VIII. Chromatin Modifications Can Be InheritedIX. How Is Transcription Occurring in Eukaryotic Cells, And How Might It Be Regulated?X. Characteristics of EnhancersXI. Enhancers & SilencersXII. What Role Do Regulatory Proteins Play?XIII. Types of Transcription FactorsXIV. The Mediator ComplexXV. Transcription Initiation in Eukaryotic CellsXVI. Post-Transcriptional ControlXVII. Post-Translational ControlXVIII. Comparing Gene Expression in Bacteria & EukaryotesOutline of Current Lecture I. Chapter 20a. Introduction to Genetic Engineeringb. Engineering a Safe Supply of Growth Hormonec. Using Reverse Transcriptase to Produce cDNAsd. Using Plasmids in Cloninge. Cutting & Pasting DNAf. The Importance of the Creation of Sticky Endsg. Transformationh. Producing a cDNA Library i. Screening a DNA Libraryj. Mass-Producing Growth Hormonek. Ethical Concerns Over Recombinant Growth Hormonel. The Polymerase Chain Reactionm. Requirements of PCRn. The Steps of Polymerase Chain Reactiono. Dideoxy DNA SequencingThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.p. The Potential of Gene Therapyq. Introducing Novel Alleles Into Human Cells r. Ethical Concerns Over Gene TherapyII. Chapter 21a. Genomeb. Whole-Genome Sequencingc. How Are Complete Genomes Sequenced?d. Shotgun Sequencing Processe. The Role of ‘Next-Generation Sequencing’ Strategiesf. The Natural History of Prokaryotic Genomesg. Lateral Gene Transferh. Evidence for Lateral Gene Transferi. How Does Lateral Gene Transfer Occur?j. Eukaryotic Genomesk. How Do Transposable Elements Work?l. Repeated Sequencesm. DNA FingerprintingCurrent LectureChapter 20Introduction to Genetic Engineering- Genetic Engineering is the manipulation of DNA sequences in organisms- Techniques used to engineer genes is called recombinant DNA technologyEngineering a Safe Supply of Growth Hormone- The recombinant DNA strategy for producing human growth hormone involved o Cloning the human geneo Introducing the gene into bacteriao Having the recombinant bacterial cells produce the hormoneUsing Reverse Transcriptase to Produce cDNAs- The enzyme reverse transcriptase can synthesize DNA from an RNA template- Researchers used reverse transcriptase to make complementary DNA (cDNA) from mRNA isolated in pituitary cells- cDNA: any DNA made from an RNA template- Used DNA cloning (producing many identical copies of a gene) to copy the cDNAs for analysiso Determine which encoded the growth hormone proteinUsing Plasmids in Cloning- Plasmid: small, circular DNA molecules often found in bacteriao Replicate independently of the chromosomeo Plasmids can serve as vector (vehicle for transferring recombinant genes to new host)- If a recombinant plasmid can be inserted into a bacterial or yeast cell foreign DNA will be copied & is then transmitted to new cells as the host cell grows & divides o Produces millions of identical copies of specific genes Cutting & Pasting DNA- Restriction endonucleases: bacterial enzymes that cut DNA at specific base sequences called recognition siteso Often make staggered cuts in the DNA, resulting in sticky ends (complementary single stranded ends)- The first step in cloning genes into plasmids is to cut the plasmid and the cDNA with the same restriction endonuclease- The sticky ends of the plasmids and cDNAs will bind together by complementary base pairing - DNA ligase seals the recombinant pieces of DNA together forms the phosphodiester bond linkageThe Importance of the Creation of Sticky Ends- If restriction sites in different DNA sequences are cut with the same restriction endonucleaseo Presence of the same sticky ends in both samples of DNA allows the resulting fragments to be spliced together by complementary base pairingThe Essence of Recombinant DNA Technology - The ability to create novel combinations of DNA sequences by cutting specific sequences& pasting in new locationsTransformation- If a recombinant plasmid can be inserted into a bacterial or yeast cell, the foreign DNA will be copiedo Transmitted to new cells as the host cell grows and divideso Produces millions of identical copies of specific genes - Plasmid vectors can be introduced into bacteria by transformation: The process of takingup DNA from the environment & incorporating it into the genomeProducing a cDNA Library- DNA library: a collection of transformed bacterial cells, each containing a vector with an inserted gene- cDNA Library: a collection of bacterial cells each containing a vector with one cDNA - Genomic Library: made up of cloned DNA fragments representing an entire genomeo Important because they give researchers a way to store information from a particular cell type or genome in an accessible formScreening a DNA Library- DNA probe: single stranded fragment of a known gene that binds a complementary sequence in the DNA being analyzedo Must be labeled in some fashion (i.e. radioisotopes) so that it can be found after it has bound the target sequence- The growth hormone researchers inferred the approximate sequence for the GH1 gene from their knowledge of the amino acid sequence of human growth hormoneo From this knowledge, they constructed a probe and radioactively labeled ito Then used this probe to screen a cDNA library for the plasmid containing the GH1cDNAMass-Producing Growth Hormone- Once the human growth hormone cDNA was isolated, it was cloned into a plasmid in such a way that is was under the control of a bacterial promoter (so bacteria would start transcribing)- The plasmid was inserted into E. coli bacteria- The transformed E. coli cells produced human growth hormone- The hormone could be isolated & purified in large quantitiesEthical Concerns Over Recombinant Growth Hormone- The increased supply of growth hormone led to its use to treat children who were short, but did not suffer from pituitary dwarfismo Also for athletic or non-medical purpose- The U.S. Food & Drug Administration has now approved use of the hormone only for children projected to reach heights of less than 5’3” for males & 4’11” for
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