Pitt BIOSC 1850 - Exam 3 Study Guide (13 pages)

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Exam 3 Study Guide



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Exam 3 Study Guide

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Pages:
13
Type:
Study Guide
School:
University of Pittsburgh
Course:
Biosc 1850 - Microbiology
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BIOSC 1850 1st Edition Exam 3 Study Guide Textbook Chapters 10 11 13 14 15 Chapter 10 Microbial Genomics 10 1 Genome Sequencing Sanger Dideoxy Sequencing cloning of the fragment to be sequenced DNA synthesis gel electrophoresis o Primer is added complementary to flanking vector sequences o Bases are attached to a tri phosphate group for marking and are added to the mix These altered bases stop further replication so we re left with a lot of shorter bases o Gel electrophoresis is run for each of the four bases to determine sequence Primer Walking consecutive runs of sequencing and primer for longer sequences Next Generation Sequencing Methods not reliant on gel electrophoresis o Pyrosequencing the four altered bases are added one at a time If it matches it produces light due to the release of PPi which gets converted into ATP that drives a light producing reaction o 454 DNA Sequencing DNA fragments don t have to be added into vectors Shotgun Sequencing shearing DNA into short pieces sequencing those fragments Computer programs are used to map the overlapping sequences to link together individual sequences 10 2 Genomic Analysis of Gene Expression Genomic library a collection of cloned DNA fragments that comprise the entire genome of that organism cDNA Library genomic library of the mRNA sequences only o Varies under different conditions Proteome collection of proteins present in a cell under certain conditions Transcriptome set of transcripts encoded by each gene within a genome o Transcription patters o Northern Blotting Binding a probe to a known sequences of mRNA to detect its presence and the level of transcription o Microarray Simultaneous examination of the transcriptional activity of all genes in the genome Relative abundance of each transcript determined by the color output 10 3 Comparative Genomics Determining evolutionary relationships Paralogs genes that arise from duplication events Orthologs genes with the same function in different organisms that have evolved from the same ancestor Horizontal gene transfer sharing of genetic information by microbes Genomes vary in the ratio of G C pairs and A T pairs o G C 10 4 Metagenomics Process in which DNA is extracted directly from microbial communities and sequenced or cloned into vectors to make libraries Chapter 11 Regulation of Gene Expression 11 1 Differential Gene Expression Constitutive expression genes that are always expressed Inducible expression genes that are expressed only under certain conditions Allosteric inhibition the inhibitory product binds to the enzyme making it so the substrate cannot bind Covalent modification phosphorylation methylation etc Transcription can be turned off 11 2 The Operon Operon transcriptional unit consisting of o Series of structural genes coding for polypeptides o Regulatory elements that affect their transcription Operator DNA sequence to which regulatory components can bind Common promoter for initiation of transcription Common in bacteria since they re polycistronic Negative control involves a repressor to block transcription o Repressor binds to operator to form a dimer and inhibits binding of RNA polymerase o Effectors intermediate molecules of the metabolic process involved Interact with repressor to modulate their ability to bind to the operator Can be inducers or co repressors o Lac operon inducible catabolic pathway involving negative control of transcription Absence of lactose repressor binds Presence of lactose allolactose acts as an effector Binds to repressor to prevent it from binding to the operator Positive control regulatory molecules lead to an increased transcription o Activator molecule increases the affinity of RNA polymerase to promoters that otherwise to not bind strongly Bind to activator binding site ABS o Removal of inducer shuts down the pathway o The lac operon also exhibits positive control For transcription to occur activator protein CRP binds to the ABS to initiate transcription Also require a co activator cAMP Low levels in presence of glucose but high levels when glucose levels are low Enzymes still aren t produced since there s no allolactose need both in order for transcription to occur 11 3 Global Gene Regulation Regulons collection of genes regulated in a global manner Catabolite repression a substance shuts down operons that produce enzymes utilizing a number of other nutrients o Preferred nutrient SOS response to repair damaged DNA o Experiment shining UV light on cells and measure amount of phage infection After phage exposure to UV light the phage particles had a reduced rate of infection of E coli no increase in mutation UV light damaged the phage DNA unable to replicate After E coli exposure to UV light rate of phage infection was high rate of DNA mutation was also high UV treatment of the host E coli enhances their ability to repair the damage of the phage DNA allowing it to replicate Repair mechanism is error prone resulting in lots of mutations DNA repair required protein synthesis experiment adding in an antibiotic that blocks protein synthesis o Reporter gene promoter less gene not expressed unless it inserts within an actively transcribed gene o RecA recombination and regulation of SOS response binds single stranded DNA Cells detect a molecule that results from DNA damage o Single stranded DNA o SOS repair system uses alternative DNA polymerases that can fill in missing DNA strands Lacks the ability to proofread high rate of mutation Mutations either turned on the reporter gene constitutive induction of the SOS genes or turned off the reporter gene abolishing the inducibility of SOS genes Mutations were in recA and lexA o RecA controls activity of LexA o LexA repressor of SOS regulons Alternative sigma factors allow for another means of global gene regulation o Generally are active only under specific conditions and are not required for cell viability 11 4 Post Initiation Control of Gene Expression sRNA small non coding sequences 50 400 nucleotides with a regulatory function o Typically affects gene expression by interacting with existing mRNA Antisense RNA single stranded RNA that interacts with specific mRNA through complementary base pairing Attenuation regulatory mechanism that occurs after the initiation of transcription but before translation of the operon is complete decrease in virulence Riboswitches sequences of mRNA that binds an effector molecule that regulates transcription of the mRNA molecule 11 5 Quorum Sensing Chemical


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