Lecture 10 - Chapter 8: Transposition & Transposon Mutagenesis PDF

MICR 321: Advanced Microbiology Lecture 10: Chapter 8: Transposition & Transposon Mutagenesis Transposition Transposons – “Jumping genes” - DNA elements that can hop or transpose from one place in the DNA to another Discovered by Barbara McClintock in corn in the early...

MICR 321: Advanced Microbiology Lecture 10: Chapter 8: Transposition & Transposon Mutagenesis Transposition Transposons – “Jumping genes” - DNA elements that can hop or transpose from one place in the DNA to another Discovered by Barbara McClintock in corn in the early 1950s Exist in every organism on Earth (half of our genome may be transposons!) Transposition – the movement of transposons Transposases - the enzymes that promote transposition and are encoded within the transposon Transposition offers a way of introducing genes from one bacterium into the chromosome of another to which it has little DNA sequence homology Homologous recombination accounts for the majority of recombination in a cell and results from the breaking and rejoining of two DNA molecules that have similar or identical sequences – the benefit of using “homology arms” in cloning Non-homologous recombination does not require two DNAs to have the same sequence and depends on enzymes that recognize specific regions in the DNA that may/may not have similar sequences  Fig 8.1 Overview of Transposition Result of transposition - transposon appears in a different site in the genome from where it was originally DNA is cut out of one DNA strand → may/may not be copied → inserted into another location in the genome Transposase cuts the donor DNA at ends of transposon and inserts the transposon into target DNA Donor DNA - the DNA strand that the transposon originated from Recipient DNA – the DNA strand that the transposon hops into Transposition must be tightly regulated and occur only rarely. Why?  Fig 8.2 Transposon Structure Smaller transposons (~1000 bp) encode only the gene for the transposase that will promote movement Larger transposons often encode additional genes for regulation of movement or factors beneficial for the host, ie. antibiotic resistance Two common features of transposons: Bacterial transposons contain inverted repeats at their ends – recognized by transposases that bind to form synapse for excision  Fig 8.3 Transposon Structure Smaller transposons (~1000 bp) encode only the gene for the transposase that will promote movement Larger transposons often encode additional genes for regulation of movement or factors beneficial for the host, ie. antibiotic resistance Two common features of transposons: Bacterial transposons contain inverted repeats at their ends – recognized by transposases that bind to form synapse for excision After integration, direct repeats form in target DNA that bracket the transposon  Fig 8.4 Types of Transposons Insertion sequence elements (IS elements) – smallest bacterial transposons Only about 750 to 2000 bp, carry no selectable genes Generally inactivate genes they hop into, resembling deletion mutations but they can revert Can cause polar effects. Why? Composite transposons – two copies of the same IS elements can combine to form a large transposon that will transfer everything in between – often contain selectable genes  Fig 8.5 IS Element V. Composite Transposon Using transposons in bacterial genetics Reverse genetics: Creating a targeted mutation and then studying the resulting phenotype Gene locus isolated → introduce mutation → test for phenotype/function Forward genetics: Discover the gene responsible for phenotypes Phenotype/function → locus Transposon Mutagenesis Qualities of effective mutagenic transposons Transpose at high frequency Not very selective target sequence Carry an easily selectable marker (AbxR) Broad host range for transposition Once selected these transposons can be cloned into a vector that cannot replicate inside the recipient host cell → the transposon can move from the plasmid into the genome and interrupting a gene(s) A high-throughput method of creating mutations and can be done in organisms without a complete or high-quality gDNA sequence. Newer techniques can integrate transposon mutagenesis with next- generation sequencing technologies Transposon Mutagenesis Insertions in the coding regions of genes, within the first half of the gene are most likely to result in loss of function alleles. Why? Where have you seen this before?? Ideally, you want ~ one insertion per gene. Coverage – You want insertions in as many genes as possible. Can you recover every gene in the genome? What did you learn from the Keio collection? In traditional transposon mutagenesis, mutations were mapped by cloning or arbitrary PCR after screening TnSeq - Now with next generation sequencing, we can map mutations prior to screening, screen all the mutants at once, and sequence after screening to see what is left or what drops out. New and improved barcoded-transposon mutant libraries – Wetmore, et al. 2015. Constructing a transposon mutant library Saturated krmit-Tn library Modified mariner transposon, inserts at TA 68,857 unique insertion sites insertion every ~ 35 nt You have mutants – Now you can pool them OR you can index/array the library Pros and Cons What can we do if the library is arrayed? When have you seen this before?? Image courtesy of Dr. Jenny Heppert What can we do if the library is pooled? How do we analyze these data?? Day3 GBS 6 Reads are checked for quality and trimmed 5 -Log10 p value Mapped to bacterial reference 4 genome 3 Verified leading ‘TA’ transposon 2 insertion site, all reads without ‘TA’ are omitted going forward 1 Differential analyses are 0 performed, significance cutoff of -40 -20 0 20 40 p