Biol 118 1st Edition Lecture 19 Outline of Last Lecture I Differential Gene Expression II Mechanisms of Gene Regulation An Overview III Three Additional Levels Unique to Eukaryotes IV What is Chromatin s Basic Structure V Chromatin Structure is Altered in Active Genes VI Negative Control of Eukaryotic Genes VII How is Chromatin Altered VIII Chromatin Modifications Can Be Inherited IX How Is Transcription Occurring in Eukaryotic Cells And How Might It Be Regulated X Characteristics of Enhancers XI Enhancers Silencers XII What Role Do Regulatory Proteins Play XIII Types of Transcription Factors XIV The Mediator Complex XV Transcription Initiation in Eukaryotic Cells XVI Post Transcriptional Control XVII Post Translational Control XVIII Comparing Gene Expression in Bacteria Eukaryotes Outline of Current Lecture I Chapter 20 a Introduction to Genetic Engineering b Engineering a Safe Supply of Growth Hormone c Using Reverse Transcriptase to Produce cDNAs d Using Plasmids in Cloning e Cutting Pasting DNA f The Importance of the Creation of Sticky Ends g Transformation h Producing a cDNA Library i Screening a DNA Library j Mass Producing Growth Hormone k Ethical Concerns Over Recombinant Growth Hormone l The Polymerase Chain Reaction m Requirements of PCR n The Steps of Polymerase Chain Reaction o Dideoxy DNA Sequencing These 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 II p The Potential of Gene Therapy q Introducing Novel Alleles Into Human Cells r Ethical Concerns Over Gene Therapy Chapter 21 a Genome b Whole Genome Sequencing c How Are Complete Genomes Sequenced d Shotgun Sequencing Process e The Role of Next Generation Sequencing Strategies f The Natural History of Prokaryotic Genomes g Lateral Gene Transfer h Evidence for Lateral Gene Transfer i How Does Lateral Gene Transfer Occur j Eukaryotic Genomes k How Do Transposable Elements Work l Repeated Sequences m DNA Fingerprinting Current Lecture Chapter 20 Introduction to Genetic Engineering Genetic Engineering is the manipulation of DNA sequences in organisms Techniques used to engineer genes is called recombinant DNA technology Engineering a Safe Supply of Growth Hormone The recombinant DNA strategy for producing human growth hormone involved o Cloning the human gene o Introducing the gene into bacteria o Having the recombinant bacterial cells produce the hormone Using 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 analysis o Determine which encoded the growth hormone protein Using Plasmids in Cloning Plasmid small circular DNA molecules often found in bacteria o Replicate independently of the chromosome o 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 sites o 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 linkage The Importance of the Creation of Sticky Ends If restriction sites in different DNA sequences are cut with the same restriction endonuclease o Presence of the same sticky ends in both samples of DNA allows the resulting fragments to be spliced together by complementary base pairing The Essence of Recombinant DNA Technology The ability to create novel combinations of DNA sequences by cutting specific sequences pasting in new locations Transformation If a recombinant plasmid can be inserted into a bacterial or yeast cell the foreign DNA will be copied o Transmitted to new cells as the host cell grows and divides o Produces millions of identical copies of specific genes Plasmid vectors can be introduced into bacteria by transformation The process of taking up DNA from the environment incorporating it into the genome Producing 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 genome o Important because they give researchers a way to store information from a particular cell type or genome in an accessible form Screening a DNA Library DNA probe single stranded fragment of a known gene that binds a complementary sequence in the DNA being analyzed o 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 hormone o From this knowledge they constructed a probe and radioactively labeled it o Then used this probe to screen a cDNA library for the plasmid containing the GH1 cDNA Mass 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 quantities Ethical 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 dwarfism o 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 females The Polymerase Chain Reaction Polymerase Chain Reaction An in vitro DNA synthesis reaction o
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