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McMaster University
Lovaye Kajiura

1 S2013 BIOLOGY 1A03: CELLULAR & MOLECULAR BIOLOGY DR. KAJIURA’S UPDATED LECTURE OUTLINES TH TH WEEK 6 : MONDAY, JUNE 10 & WEDNESDAY, JUNE 12 2013 IMPORTANT The posted Biology 1A03 AVENUE Lecture Outlines are NOT detailed lecture notes. Students are expected to supplement the posted AVENUE Lecture Outlines with their own written notes. Since Supplementary Lecture Topics and Examples will be discussed EXCLUSIVELY in lectures (they are NOT found in the textbook), it is very important that students attend ALL Biology 1A03 Lectures. Note that Chapter 2 in the Biology 1A03 textbook is considered assumed knowledge that should have been previously obtained in high school biology 12U. CHAPTER 17 CONTROL OF GENE EXPRESSION IN BACTERIA Recall the steps of information flow from DNA to proteins DNA → mRNA → protein → activated protein Fig. 17.1 Gene Expression can be Regulated at Three Levels - transcriptional control - tranlational control - post-translational control Fig. 17.2 Glucose Affects the Regulation of the B-Galactosidase Gene - experiments supported that the presence of lactose, without glucose, stimulates the gene expression of the gene for beta-galactosidase Fig. 17.3 Replica Plating is a Technique for Identifying Mutant Cells Table 17.1 Three Distinct Types of Mutants in Lactose Metabolism of E. coli Fig. 17.6 Genes are Regulated by Negative Control, Positive Control, or Both 2 Repressible Operons (Fig. 17.9 The trp Operon is under Negative Control) - genes are switched “ON” until a specific metabolite activates the repressor - operons have their transription inhibited - repressible enzymes catalyze the anabolic pathway, e.g. synthesis of tryptophan - accumulation of tryptophan represses the production of the enzymes that catalyzes its synthesis pathway end-products switches “OFF” its production by repressing synthesis of enzymes When tryptophan is present, transcription does not occur : Tryptophan PRESENT  repressor protein ACTIVE conformation  BINDS with operator  trp operon switched “OFF” (transcription is blocked) When tryptophan is absent, transcription takes place : Tryptophan ABSENT  repressor protein INACTIVE conformation  trp operon switched “ON” (transcription occurs) Fig. 17.13 Positive Control of the lac Operon Inducible Operons - genes are switched “OFF” until a specific metabolite inactivates the repressor - have their transcription stimulated - associated with catabolic pathways - enzyme synthesis is switched “ON” by the nutrient utilized by the pathway allolactose binds to the repressor  inactivated repressor loses its affinity for the lac operon  operon is transcribed  enzymes for metabolism of lactose are produced 3 Positive control: cAMP receptor protein lac operon with dual regulation: negative control by repressor determines whether the operon will or will not transcribe structural genes positive control by CRP (cAMP receptor protein) determines the transcription rate Fig. 17.14 Cyclic AMP (cAMP) is Synthesized when Glucose Levels Are Low Fig. 17.15 An Overview of Lactose use Glucose concentration LOW  cAMP concentration increases  cAMP binds to CRP  cAMP-CRP complex binds to lac promoter  transcription (efficient) of lac operon large amounts of lac mRNA synthesized Glucose concentration HIGH  cAMP concentration decreases  CRP releases its cAMP  CRP separates from lac promoter  transcription (slow) of lac operon small amounts lac mRNA synthesized USEFUL TERMINOLOGY gene expression transcriptional control translational control lactose repressor inducer operator operon negative control positive control lac operon trp operon tryptophan master plate replica plate indicator plates Beta-galactosidase 4 CHAPTER 19 ANALYZING AND ENGINEERING GENES & CHAPTER 20 GENOMICS DNA TECHNOLOGY Overview of how bacterial plasmids are used to clone genes for biotechnology Recombinant DNA Technology - provides tools for examining the structure & function of the eukaryotic genome. What are some of the important components of DNA technology? 1. Biochemical tools 2. Method 3. Vectors 4. Techniques Fig. 19.1 Reverse Transciptase Catalyzes the Synthesis of DNA from RNA Fig. 19.2 Genes Can be Cloned into Plasmids Using a restriction enzyme and DNA ligase to make recombinant DNA Restriction - protection involves foreign DNA being cut into small segments Restriction enzymes - valuable tool for recombinant DNA technology (1960’s 1 discovered) - recognizes short, specific nucleotide “recognition sequences” or “restriction sites” - cut only at specific sites within those sequences Bacteria naturally protect their own DNA from restriction by a modification, which involves the methylation of DNA (methyl groups are added to nucleotides within the recognition sequences). - restriction enzymes cut the phosphodiester bonds of both strands in a staggered manner, which results in single-stranded ends called “sticky ends”. Sticky Ends of restriction fragments - are utilized to join DNA pieces from different sources (cells or different organisms) - unions are temporary since they are held by a few H bonds, but can be made permanent
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