LS-MCRB 121 1st EditionExam # 2 Study Guide Lectures: 4-7Lecture 1 (February 19)Microbial GeneticsTerms- Genetics: The study of what genes are, how they carry information, how information is expressed, and how genes are replicated- Gene: A segment of DNA that encodes a functional product, usually a protein- Chromosome: Structure containing DNA that physically carries hereditary information; contain the genes- Genome: all genetic information in a cell- Genomics: the molecular study of genomes- Genotype: The genes of an organismo Blueprint of a cell- Phenotype: Expression of the geneso How the “blueprint” is expressed, how the finished product appears- DNA Gyrase: relaxes supercoiling ahead of the replication fork- DNA ligase: makes covalent bonds to join DNA strands; joins Okazaki fragments and new segments in excision repair- DNA Polymerase: Synthesizes DNA; proofreads and repairs DNA- Endonucleases: Cut DNA backbone in a strand of DNA; facilitate repair and insertionso Cleave in the middle- Exonucleases: Cut DNA from an exposed end of DNA; facilitate repairo Cleave on the ends- Helicase: Unwinds double-stranded DNA- Methylase: Adds methyl group to selected bases in newly made DNA- Photolyase: Uses visible light energy to separate UV-induced pyrimidine dimers- Primase: Makes RNA primers from a DNA template- Ribozyme: RNA enzyme that removes introns and splices exons together- RNA Polymerase: Copies RNA from a DNA template- snRNP: RNA-protein complex that removes introns and splices exons together- Topoisomerase: relaxes supercoiling ahead of the replication fork; separates DNA circles atthe end of DNA replication- Transposase: Cuts DNA backbone leaving single-stranded “sticky ends”E. coli- Single chromosome- 4.6 million bases or 4.6 Mb (mega bases)- 1 mm in length- The cell is 1 um- Circular DNADNA Structure- Polymer of nucelotides: Adenine, thymine, cytosine, and guanine- Double helix associated with proteins- “Backbone” is deoxyribose-phosphate- Strands are held together by hydrogen bonds between AT and CGo Mild base can separate hydrogen bonds or something will bind to them to lose energy and separate- Strands are antiparallelo Critical because after separation, process goes off polarityDNA Synthesis- DNA is copied by DNA polymerase in 5’ 3’ direction- DNA is bidirectional- Initiated by an RNA primer- Leading strand is synthesized continuously- Lagging strand is synthesized discontinuously- Okazaki fragments- RNA primers are removed and Okazaki fragments joined by a DNA polymerase and DNA ligaseo Okazaki fragments are on both strands- Process has high fidelity- Error rate contributes to spontaneous mutation frequency- Primingo DNA polymerases cannot synthesize DNA strand de novoo Requires a free 3’ –OHo RNA polymerases can synthesize RNA polymer de novoo Necessitates the involvement of RNA polymerase laying down RNA primerTranscription- DNA is transcribed to make RNA (mRNA, tRNA, and rRNA)- Begins when RNA polymerase binds to the promoter sequence- Proceeds in the 5’ 3’ direction- Stops when it reaches the terminator sequenceTranslation- mRNA is translated in codons (three nucleotides)- Begins at start codon: AUG- Ends at nonsense codons: UAA, UAG, UGAThe Genetic Code- 64 sense codons on mRNA encode the 20 amino acids- Genetic code is degenerate because there’s more sense codons than amino acidso There are amino acids that are encoded by more than one sense codon- tRNA carries the complementary anticodonWhat is the substance of inheritance?- Nucleic acids and DNAWhat is the genome of an organism?- The nucleus in animal cellsGene Regulation- Constitutive genes: expressed at a fixed rate- Repressible genes: transcribed until they are turned off; usually in response to the abundance of an end-product (end product inhibition)- Inducible genes: transcribed only in presence of inducer- Catabolite repression: regulation of carbon source utilization based on environmental and/or metabolic signalsIn the presence of glucose, cAMP levels are low; as glucose is consumed, cAMP levels rise.Mutation- A change in genetic material- May be neutral, beneficial, or harmful- Mutagen: agent that causes mutations- Spontaneous mutations: occur in the absence of a mutagen- Base substitutiono Point mutationo May be silent (degeneracy of code) Doesn’t change the amino acid coded- Missense mutationo Change in one baseo Result in change in amino acid A change in one base will change the amino acid (conservative change)- Nonsense mutationo Results in a nonsense codon- Frameshift mutationo Insertion or deletion in one or more nucleotide pairso Happens often from faulty replication, replication influenced by a mutageno Protein is messed up from the point of the mutation onFrequency of Mutation- Spontaneous mutation rate = (10^-9) base pairs or (10^-6) replicated genes- Population Frequency = Given a mutation frequency of 10^-7 a population of 10^8 bacterial cells will likely harbor ~10 mutations in 1 gene- Mutagens: Increase spontaneous mutation frequencyo 10^-5 or 10^-3 per replicated geneSelection of Mutations- Positive (direct) selection: detects mutant cells because they grow or appear different- Negative (indirect) selection: detects mutant cells because they don’t growo Replica plating: method for physically isolating mutant cells (strains)o Ames TestGenetic Recombination- Vertical gene transfer: occurs during reproduction between generations of cells- Horizontal gene transfer: The transfer of genes between cells of the same generation- Exchange of genes between two DNA moleculeso Crossing over occurs when two chromosomes break and rejoin- Transformation: first form of recombinationo Requires “naked” DNAo Must be homologouso Select groups of bacteriao Doesn’t require cell to cell contacto Process sensitive to DNase in the surrounding medium- Bacterial Conjugation: 2nd form of recombinationo Sex pilus: ones that have this property are able to pass a trait to another cell that don’t have the specific traito Mating through conjugation requires a mating bridge, subject to mechanical interruptiono Conjugation in E. coli Requires the presence of Fertility factor (F-plasmid) in donor and absenceof F factor in recipient (F-) Requires cell to cell contact Insensitive to DNase in surrounding medium Transfer of chromosomal genes- Hfr formation only occurs with certain conjugative (F-like) plasmids- Once formed conjugation is extremely efficient-
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