BIOSC 1850 1st EditionExam 3 Study GuideTextbook Chapters 10, 11, 13, 14, 15Chapter 10: Microbial Genomics10.1: 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 baseso 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 reactiono 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 onlyo 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 genomeo Transcription patterso 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 sameancestor - Horizontal gene transfer: sharing of genetic information by microbes- Genomes vary in the ratio of G:C pairs and A:T pairso % G+C10.4: Metagenomics - Process in which DNA is extracted directly from microbial communities and sequenced or clonedinto vectors to make librariesChapter 11: Regulation of Gene Expression11.1: 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 off11.2: The Operon- Operon: transcriptional unit consisting of:o Series of structural genes coding for polypeptideso 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 pathwayo 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 numberof other nutrientso Preferred nutrient- SOS response to repair damaged DNAo 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 geneo 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 lexAo 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 functiono 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