BIOLOGY 207 1st Edition Lecture 8Outline of Previous Lecture I. Basic aspects of microbial growthII. Central metabolism in biosynthesisIII. Evolution and synthetic biologyOutline of Current Lecture I. Mutations and mutagenesisII. Gene transfer in bacteriaIII. CRISPR interferenceCurrent LectureGenetics of bacteria and archaeaI. Mutations and mutagenesisa. Mutations: heritable changes in the base sequence of a genomei. Natural strains are known as ‘wild-type’ gene strains; the strain that straysfrom the wild-type is a ‘mutant’ii. The difference between the wild-type parent strain comes in regards to the genotypeb. Naming conventions for a single genei. Notice the differences in this example of the italics and capitalizations; these are trends for most genes1. Genotype: hisC2. Protein: HisC3. 1st isolated mutant: hisC14. Phenotype: His+ vs. His-c. Nutritional auxotrophs can be identified by replica platingi. Auxotrophs: strains that have a nutritional supplement requirement (eg: cell requires oxygen)ii. Start with the master plate- stamp onto other plates of the growth medium and examine the growth of the cells vs. those that experienced mutations These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.d. Types of mutationsi. Induced mutations1. Environmental agents2. Can be made deliberately by humansa. Tool in laboratories to study different types of traitsii. Spontaneous mutations1. Without any external intervention2. Mostly occurring through errors during DNA replication3. Spontaneous mutations happen at a rate of about one in a millionfor any gene per round of replicationa. Remember that a typical gene has about 1000 base pairsb. 10-6 per 1000 base pairsiii. Base-pair substitutions1. Silent mutationa. Mutation does not affect phenotypeb. Based on redundancy of genetic codec. The amino acid created produces the same results in the protein as the parent strain2. Missense mutationa. Results in the change in the amino acid sequence of the polypeptide 3. Nonsense mutation a. Changes in the sense codon to a nonsense codonb. Results in a failed proteinsc. Affects the phenotype, often harmfullyiv. Insertions and deletions 1. Frameshift mutationsa. One or more bases are inserted or deleted in the sequenceb. This creates a completely different amino acid than it is meant to during translationc. Can result in serious complications and consequences for the cellv. Reversions 1. Same site reversionsa. Mutations in the same place as the original mutation- restores activity of translated polypeptideb. If the original sequences are restored, the mutation is referred to as a true revertant2. Second site revertantsa. Mutation occurs at a separate DNA siteb. Can compensate for the effect for the original mutation on the phenotypec. This is referred to as a suppressor mutatione. Mutagensi. Agents that cause mutationsii. Allows us to do study on mutations and their effectsiii. Chemical mutagens1. Nucleotide base analogs resemble DNA bases but don’t pair correctly2. Alkylating agents can cause chemical modificationsa. Cell stops replicating3. Intercalating agents (acridines) insert between two DNA base pairsa. Causes frameshift mutationsiv. Radiation1. Nonionizing (UV) vs ionizinga. Can cause really extensive cell damageb. Lesions in DNA resulting in failures during replication and other complications2. Results in pyrimidine dimmersa. Purines: AGb. Pyrimidines: CUTf. DNA damage repair i. Large-scale damage is repaired with the SOS response system1. Cells have multiple mechanisms to protect and repair DNA damage2. In some cases, the damage is too extensive for replication to occur3. SOS response is activated in these cases4. Allow for replication to proceed via random incorporation of nucleotides5. Two proteins that regularly regulate the response:a. LexA – normally prevents expression of SOS systemi. LexA is a repressor; that prevents transcription of SOS genes; cleaves itself (no longer binds to operator)ii. Partial repressionb. RecA – normally prevents functions in genetic recombinationi. It is activated by extreme DNA damage; interacts with LexA6. Generates a lot of mutations as a last ditch effort to survivea. Genes involved can now be transcribed, allowing for replicationII. Gene transfer in bacteriaa. Transferred DNA in bacteria can go down three different pathsi. Degraded by restriction enzymes1. Note: even though we use restriction enzymes as a tool in the lab, they are naturally a defense mechanism against foreign nucleic acids for the bacteria ii. Replicates itself 1. If it has its own origin of replicationiii. Recombination with host chromosomeb. Homologous recombinationi. Genetic recombination between similar DNA sequences ii. This process generates diversity during meiosisiii. Takes place in all three domains of life iv. Plays a major role in DNA repair for bacteriav. Allows bacteria to recombine foreign DNA into the genomec. DNA transfers in 3 different waysi. Transformation1. Occurs when the cell takes free DNA upa. Often corresponds with about 10 genesb. Cells that are able to take up DNA are called competenti. Inducing competence is possible in labs to make cells take up foreign DNA c. Bacteria than can transform increase diversity and fitness in their communityii. Transduction1. Occurs when DNA is transferred by a bacteriophage/phage2. Generalized transductiona. Donor cannot replicate independentlyb. Donor genes are lost unless they recombine with recipient chromosome3. Specialized transductiona. Bacteria’s DNA is part of temperate phage genome b. Can be integrated into host during lysogeny4. Not all phages can undergo transduction/not all bacteria are transducible5. Ex: antibiotic resistance genes and genes encoding photosynthetic proteinsiii. Conjugation1. Requires cell contact2. Plasmid-encoded mechanism3. Fertility plasmid (F) contains:a. Genes regulating DNA replicationb. Transposable elementsc. Transfer functions4. Sex pilus allows pairing to take place between two cells a. Cells drawn togetherb. Remain in contact by binding proteinsc. DNA transferred to recipient cell through junctionIII. CRISPR interference (CRISPRi)a. “Clustered Regularly Interspaced Short Palindromic Repeats”b. RNA-based defense program against external DNA that tries to infiltrate the cellc. Made of “spacers” of foreign DNA from previous invadersi. These spacers are broken up