1. 1 Transposable Element (Transposon) Transposable elements in eukaryotes : Barbara McClintock (1902-1992) Cold Spring Harbor Laboratory, NY Nobel Prize in Physiology and Medicine 1983 “for her discovery of mobil genetic elements” Studied transposable elements in corn (Zea mays) 1940s-1950s (formerly identified as mutator genes by Marcus Rhoades 1930s)

2. Transposable element : genetic elements of a chromosome that have the capacity to mobilize and move from one location to another in the genome. Normal and ubiquitous components of prokaryote and eukaryote genomes. Nonhomologous recombination : transposable elements insert into DNA that has no sequence homology with the transposon. Transposable elements cause genetics changes and make important contributions to the evolution of genomes: Insert into genes. Insert into regulatory sequences; modify gene expression. Produce chromosomal mutations. Transposable Elements in Prokaryotes 1. Insertion sequence (IS) 2. Transposons (Tn) 3. Bacteriophage Mu 2

3. 3 Insertion sequence (IS) elements : 1. 2. 3. 4. 5. Simplest type of transposable element found in bacterial chromosomes and plasmids. Encode only genes for mobilization and insertion. Range in size from 768 bp to 5 kb. IS1 first identified in E. coli’s glactose operon is 768 bp long and is present with 4-19 copies in the E. coli chromosome. Ends of all known IS elements show inverted terminal repeats (ITRs). Transposition of insertion sequence (IS) elements : 1. 2. 3. 4. 5. 6. 7. Original copy remains in place; new copy inserts randomly. IS element uses host replication enzymes for replication. Transposition requires transposase, coded by the IS element. Transposition initiates when transposase recognizes ITRs. Site of integration = target site. Staggered cuts are made in DNA at target site, IS element inserts, DNA polymerase and ligase fill the gaps. Small direct repeats (~5 bp) flanking the target site are created.

4. 4 Fig. Integrated IS in the chromosomal DNA

5. 5 Transposons (Tn) : Similar to IS elements but are more complex structurally and carry additional genes 2 types of transposons: 1. 2. Composite transposons Noncomposite transposons Noncomposite transposons (Tn) : Carry genes (e.g., a gene for antibiotic resistance) but do not terminate with IS elements. Ends are non-IS element repeated sequences. Tn3 is 5 kb with 38-bp ITRs and includes 3 genes; bla (  -lactamase), tnpA (transposase), and tnpB (resolvase, which functions in recombination).

6. 6 Composite transposons (Tn) : Carry genes (e.g., a gene for antibiotic resistance) flanked on both sides by IS elements. Tn10 is 9.3 kb and includes 6.5 kb of central DNA (includes a gene for tetracycline resistance) and 1.4 kb inverted IS elements. IS elements supply transposase and ITR recognition signals. Fig. Structures of some composite transposons

7. 7 Models of transposon transposition : Similar to that of IS elements; duplication at target sites occurs. Cointegration = movement of a transposon from one genome (e.g., plasmid) to another (e.g., chromosome) integrates transposon to both genomes (duplication). May be replicative (duplication) or non-replicative (transposon lost from original site). Result in same types of mutations as IS elements: insertions, deletions, changes in gene expression, or duplication. Crossing-over occurs when donor DNA with transposable element fuses with recipient DNA.

8. 8 Advantage of Transposon mutagenesis: Easily selectable markers Strain construction Physical and genetic mapping Transposon Tn5 and Tn10 is often used as models system Transposon used for mutagenesis should have the following properties: High frequency of transposition should not very selective in its target seq Should carry selectable marker (Antibiotic Res). Broad host range for transposition

9. Figure Map of pUTmini-Tn5Km1 (A), and transposon Mini-Tn5Km1 (B) 9 Km1 tnp * X baI PstI SphI SfiI SalI PstI BglII PstI pUTmini-Tn5Km1 7.055 Kb o ri R6 K ApR PstI m o b RP4 SfiI EcoRIKpnI (A) IS Not I EcoN I Ssp I HindIII Xba I PstI Sph I Sfi I H indIII Xh oI S fiI EcoR I Kpn I ClaI Kan amycin R esistan ce G ene (Km 1) 1835 bp O end (B) I end

10. IS (I) R D E. coli S17-1 λ pir (pUTmini-Tn5Km1) M Km 50µ g/ml Km1 tnp * PstI X baI SphI SfiI EcoR I K pnI SfiI SalI PstI B glII pUTmini-Tn5Km1 7.055 Kb ori R6K Ap PstI pi r Tra Philus MSGM agar plate MSGM agar + Magnetospirillum magneticum AMB-1 mini-Tn5Km1 EcoNI Ssp I Hind II Eco RV HindI II XhoI Figure Molecular mechanism of transposon mutagenesis primer2 DNA amplified IS (O) EcoRV digest DNA genome of mutant Ligation (Circularization) IS (O) IS (I) primer1 mini-Tn 5Km1 IPCR Amplification primer1 EcoRV primer2 Fig. The strategy for inverse PCR 10 EcoRV Primer 1 : 5'-ACACTGATGAATGTTCCGTTG-3' Primer 2 : 5'-ACCTGCAGGCATGCAAGCTTC-3'

11. Number of non-magnetic mutant Position of Mini-Tn5 (bp) NMA35; 1014919; 911 NMA25; 1034763; 928 NMA9; 1142283; 1035 NMA59; 1376420; 1239 NMA29; 1443282; 1300 3265; 3645630; NMA42 3208; 3592045; NMA40 3149; 35294558; NMA8 3097; 3477510; NMA58 2998; 3377665; NMA20 NMA61; 1812678; 1661 2838; 3212025; NMA21 2575; 2880230; NMA32 2488; 2796870; NMA17 -; 2772204; NMA6 2462; 2757024; NMA51 NMA1; 1818420; 1665 NMA47; 1885655; 1725 NMA41; 2185779; - NMA10; 2211399; 2018 NMA60; 2220262; 2026 NMA49; 2294609; 2085 3696; 4066333; NMA45 NMA5; 803094; 713 3712; 4095710; NMA4 NMA19; 823416; 731 4442; 4900925; NMA44 4357; 4810423; NMA56 4272; 4709356; NMA38 M. magneticum AMB-1 4,967,148 bp ORF index NMA14; 545355; 474 NMA48; 557992; 492 NMA11; 558163; 492 NMA36; 635112; 557 -; 4491265; NMA34 -; 4491256; NMA33 4027; 4456954; NMA15 3992; 4422128; NMA50 3973; 4399224; NMA53 4967148/0 2301; 2544096; NMA13 NMA26; 2534508; 2293 Fig. Position of Mini-Tn5 insertion sites on the genome map of M. magneticum AMB-1 Inverse PCR of Tn10 interrupted genes Sequence align- ment against whole genome sequence Figure. Position of Tn10 insertion on the genome map of Escherichia coli 11

12. 12 Temperate bacteriophage Mu (Mu = mutator) : 37 kb linear DNA with central phage DNA and unequal lengths of host DNA at each end. On infection, Mu integrates by non-replicative transpostion and replicates when E. coli replicates. During the lytic cycle, Mu remains integrated in E. coli chromosome, and shifts to replicative transposition. McClintock’s discovery of transposons in corn : Purple spots in white corn kernels are results of transposons. c/c = white kernels and C/- = purple kernels Kernel color alleles/traits are “unstable”. If reversion of c to C occurs in a cell, cell will produce purple pigment and a spot. Earlier in development reversion occurs, the larger the spot. McClintock concluded “c” allele results from a non-autonomous transposon called “Ds” inserted into the “C” gene (Ds = dissassociation). Autonomous transposon “Ac” controls “Ds” transposon (Ac = activator).

13. 13 General properties of plant transposons : Possess ITR sequences and generate short repeats at target sites. May activate or repress target genes, cause chromosome mutations, disrupt genes… Two types: Autonomous elements transpose themselves; possess transposition gene. Nonautonomous elements do not transpose themselves; lack transposition gene and reply on presence of another Tn McClintock demonstrated purple spots in otherwise white corn (Zea mays) kernels are results of transposons. Fig. 20.11, Transposon effects on corn kernel color.

14. McClintock’s discovery of transposons in corn (cont.) : Ac element is autonomous/Ds element in nonautonomous. Ac is 4,563 bp with 11 bp ITRs and 1 transcription unit encoding an 807 amino acid transposase. Ac activates Ds; Ds varies in length and sequence, but possesses same ITRs as Ac. Many Ds elements are deleted or rearranged version of Ac; Ds element derived from Ac. Ac/Ds are developmentally regulated; Ac/Ds transpose only during chromosome replication and do not leave copies behind. Structure of Ac autonomous and Ds non-autonomous transposable elements in corn. 14

15. Ac transposition mechanism during chromosome replication. Ty elements in yeast : Similar to bacterial transposons; terminal repeated sequences, integrate at non-homologous sites, target site duplication… Ty elements share properties with retroviruses, retrotransposons : Synthesize RNA copy and make DNA using reverse transcriptase. cDNA integrates at a new chromosomal site. Fig. 20.14 15