EXAM 3 Regulation of Gene Expression Microorganisms are able to control how much of each building block it makes When bacterial cell are growing on simple defined medium they needs When same cells are growing on complex medium they can get all the to make all the amino acids amino acids from the medium Since biosynthesis of building blocks involve a series of enzymes reactions the cells either turn on or turn off enzyme synthesis or enzyme activity feedback regulation Control of protein synthesis Regulation of enzyme synthesis Example Lactose Operon Proposed by Jacob and Monod An example of negative transcription regulation Lactose Operon contains DNA that encodes for Three enzyme LacZ b galactosidase LacY permease LacA transacetylase And a regulatory protein LacI Repressor protein Lactose Operon Transcription is controlled by a regulatory protein that binds to DNA Lac I Repressor protein Operator DNA site bound by LacI Off state No Lactose present for the cell to use 2 Repressor bound to DNA at operator site Results Very little transcription of lacZ lacY lacA genes ON State 3 Lactose present High lactose results in accumulating allolactose Inducer side reaction of b galactosidase allolactose interacts with repressor and alters the repressor s ability to bind to the operator DNA sequence Thus RNA polymerases can interact with promoter DNA Results high level of transcription of lacZ lacY lacA genes Bacterial Genetics Mutations Recombination B Bacterial Mutations Spontaneous mutations Induced mutations DNA repair mechanism Transposable genetic elements Ames test for mutagens C Bacterial Recombination Transformation Conjugation Transduction B Mutations and Mutagenesis A mutation is a change in the DNA sequence of a chromosome note a mutation involves a change in a nucleotide pair compared to wild type Can arise spontaneously without an identifiable cause Can arise by mutagens UV radiation chemicals that modify bases nitrous acid deaminated cytosine and adenine A mutant is a organism that has a mutation Induced Mutations Ultraviolet light UV induces adjacent thymine molecules to link together Nitrous acid converts DNA adenine to hypoxanthine Hypoxanthine complemented by C Give AT to GC base pair change Base analogs substances bearing a chemical resemblance to nitrogenous bases Example 5 bromouracil Base analogs can inhibit DNA replication Acyclovir a drug against Herpes viruses is a base analog Types of mutations 1 Base pair change 2 Deletion loss of DNA 3 Insertion gain of DNA Insertions deletions can be cause by agent like benzopyrene smoke and aflatoxin made by fungi Insertions deletions cause Frameshift mutations DNA Repair Enzymes DNA repair enzymes locate and repair alteration mismatched bases and distortions thymine dimers of the DNA Mismatch repair first proof reading by DNA polymerase as it is synthesizing Damage excision repair nucleases that cut out of damaged DNA which is repaired by DNA polymerases Transposable genetic elements Insertion sequences Transposons jumping genes Insertion sequences small segments of DNA about 1000 base pairs That forms a copy of itself and Can move trans locate elsewhere on the chromosome Cause of some spontaneous mutations Transposons Larger than insertion sequences and often carry drug resistant genes Transposons move from Plasmid to plasmid Plasmid to chromosome Chromosome to plasmid End of transposons have inverted repeat sequences Fig 7 18 Replica plating Positive selection Ames Mutagen Test Purpose to look for revertant mutants of a histidine requiring strain on media without histidine Bacterial Recombination Chapter 8 Recombination involves genetic DNA exchange between two organisms Donor cell contributes chromosomal DNA or plasmid DNA Recipient cell recombines with chromosome or now harbors the plasmid DNA Prokaryotes have three methods for bacterial recombination Transformation Conjugation Transduction Transformation Uptake of naked DNA from the environment Active transferred of DNA from one cell into another mediated by a cell cell contact It often involves plasmids Cells obtain chromosomal DNA by means of a phage virus infection Some species of prokaryotes can take up pieces of DNA from their environment and recombine that DNA into their chromosomes Cells that can be transformed are called competent Example strains Haemophilus Streptococcus Bacillus Neisseria Competence is often expressed when cells are in a high cell density late log phase There is increased expression of a competence factor Natural Competence A Classic transformation experiment was first performed by Griffith and later performed by Avery et al in 1944 who demonstrated that DNA was the transforming factor They transformed a avirulent Streptococcus pneumonia strain to virulent with heat hilled virulent cells Griffith s Experiment interpretation Type R Rough live cells avirulent took up the DNA of the S Smooth dead cells which were virulent Recombination of S donor DNA with R recipient chromosome converted R cells into S virulent cells Conjugation Mechanism of gene transfer Observed first in Gram negative E coli in 1946 by Lederberg and Tatum Involves single stranded DNA transfer directly from one cell to another by a rolling circle mechanism of DNA replication See next slide Involves a conjugative plasmid F in E coli that carries the genes necessary for the DNA transfer The F plasmid Also called the F factor or fertility factor Cells that have an F plasmid are know as F cells male Cells that do no have the F plasmid are know as F cells female F cells make sex pili External protein filament that allows donor F cells to form a contact with recipient F cells Transfer of F plasmid F donor cell synthesizes a sex pili Sex pili contacts the recipient F cell Sex pili retracts pulls the donor and recipient cell together By means of rolling circle DNA replication F plasmid is transferred as a single DNA synthesis results in F in both the original donor and original recipient stranded DNA molecule which is now become F Relevance of Conjugation Conjugation can occur between various Gram negative bacteria Escherichia and Shigella Salmonella and Serratia Escherichia and Salmonella This intergenic transfer is significance in a transfer of antibiotic resistance genes carried on plasmids b introduction of drug resistant transposons between Genera Definition Transduction head and then transfers this DNA by phage particle infection into a recipient strain When a phage packages donor