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Slides%20for%20lectures%202-11%20F09

Slides%20for%20lectures%202-11%20F09 - Basic Molecular...

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Basic Molecular Biology
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QuickTimeª and a TIFF (Uncompressed) decompressor are needed to see this picture. Structure of dsDNA G:C pairs have 3 hydrogen bonds A:T pairs have 2 hydrogen bonds 5’ phosphate, 3’ hydroxyl
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“coding” strand 5’ ACGTGCTACCTTACCTAAG 3’ “non-coding” strand 3’ TGCACGATGGAATGGATTC 5’ 5’ ACGUGCUACCUUACCUAAG 3’ RNA Important: 1. Coding DNA strand is usually written above non-coding strand if both are shown. 2. Coding DNA strand sequence is the same as the RNA 3. Coding DNA strand has the same polarity as the RNA transcript 4. Only non-coding DNA strand is involved in the process of transcription (as template for RNA polymerase) 5. When only one strand of transcribed DNA is shown it is usually the coding strand unless explicitly indicated otherwise. dsDNA Summary of terms regarding DNA strands:
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template strand refers to the sequence of DNA that is copied during the synthesis of mRNA •the opposite strand (the strand with a base sequence directly corresponding to the mRNA sequence = the “mRNA-like” strand) is called the coding strand because the sequence corresponds to the codons that are translated into protein http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/chroms-genes-prots/temp-strand.html
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Although RNA polymerase must recognize sequences on the template strand, by convention we draw the DNA sequence and regulatory signals on the "mRNA-like" strand. This makes it simpler to directly determine the sequence of the resulting RNA. http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/chroms-genes-prots/temp-strand.html RNA polymerase transcribes DNA in a 5’ to 3’ direction (only) RNA polymerase does NOT need a primer to start transcription
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Figure 13.3 The three most important signals for gene expression in E. coli . Gene Cloning and DNA Analysis by T.A. Brown. © 2006 T.A. Brown.
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QuickTimeª and a TIFF (Uncompressed) decompressor are needed to see this picture. Transcription = synthesis of mRNA Translation = synthesis of protein Transcription / Translation are coupled in a bacterial cell
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Lux System
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lux operon and luxR -luxR is constitutively expressed -Expression of lux operon is induced (auto-induction) NOTE: This figure is incorrect. It is the LuxR-autoinducer complex that binds to the lux operon to Promoter to stimulate transcription; LuxR without autoinducer does not bind
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QuickTimeª and a TIFF (LZW) decompressor are needed to see this picture. Lux Region - elements involved in gene expression
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Genome maps of V. fischeri Chromosome 1: 2,900 kb Chromosome 2: 1,300 kb Lux Sal1 fragment: ~ 9 kb 100 kb
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Cloning - Cloning Lux Region
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Desirable characteristics of plasmids used for cloning Need to be able to replicate in host – ori Have a genetic marker that allows for selection of host cells that have taken up vector - amp R Choice of restriction sites for cloning - mcs Minimal size Selection marker for cells with plasmids with inserts versus cells with plasmids only – lacZ α Promoter for expression of protein?
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pGEM mcs is 69 bp
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Lux region can be inserted in either direction in pGEM: = Sal1 site
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