Is Transposition Really Random?
(Dwi Suryanto)
5
IS TRANSPOSITION REALLY RANDOM?
Dwi Suryanto
Departemen Biologi FMIPA Universitas Sumatera Utara
Jl. Bioteknologi No. 1 Kampus USU Medan 20155
Abstract
To characterize bacteria, transposon mutagenesis is still one of the most extensively utilized techniques available. These elements were believed to insert at random location. In this study, transposition was done by diparental mating technique to transfer pJFF350 carrying Omegon-Km to a Gram-negative Serratia marcescens DS-8. The result showed that diparental mating was successfully transfer pJFF350 into DS-8 cells. Interestingly, Southern hybridization analyses showed that transposon was inserted not randomly, but tended to insert into limited targets. It also indicated that duplication occurred on the target sequences upon insertion.
Keywords: Omegon-Km, Transposition Mutagenesis, Serratia Marcescens
INTRODUCTION
Transposition is a recombination process in which DNA sequences termed transposable elements move from an original site on a DNA molecule to a new site on the same or on different DNA molecule. In addition, transposable elements can cause, and are associated with, other types of genetic rearrangement such as deletions, inversions, and chromosome fusion (Reznikoff, 1993).
To characterize bacteria, transposon mutagenesis is still one of the most extensively utilized techniques available. This technique is especially useful for bacterial species with poorly described genetic systems or when existing molecular techniques are insufficient (Dennis and Zylstra, 1998). These elements have been extremely valuable as insertional mutagens because they were believed to insert at random locations (Scott, 1991).
In this study, Omegon-Km (pJJF350) were used to determine whether it insert randomly or tend to insert into specific sequences. Omegon-Km was designed to carry the artificial interposon Omegon-Km flanked by two synthetic inverted 28-bp repeats of IS1. The reason using these transposons is that inserted fragment could
be cloned easily and derived plasmids were stable (Fellay et al., 1989; Dennis and Zylstra, 1998; Civolani et al. 2000; Downing
et al., 2000).
MATERIALS AND METHOD
Strains and Plasmids
Escherichia coli S17-1 was used to promote a transfer of plasmid pJFF350 (Omegon-Km) to DS-8. Bacterial strain and plasmids are listed below.
Bacterial strains and plasmids used in this study.
Diparental Mating
Jurnal Sains Kimia
Vol. 11, No.1, 2007: 5-8
6
Bacterial strains or plasmid Relevant genotype/phenotype Strains
E. coli S17-1 recAthipro hdsR4 (rK- mK+) (RP4-2Tc-Mu-Km-Tn7) Tpr Smr
E. coli DH5α supE44 ΔlacU169 (Φ80 lacZΔM15) hsdR17 recA1 endA1
gyrA96 thi-1 relA1
Serratia marcescens DS-8 wildtype Ampr Plasmid
pJFF350 Kmr (Omegon-Km)
Transformation of Flanking DNA
Suspected colonies of transposition were grown in LB kanamycin and ampicillin broth overnight in 30°C at 200 rpm. Modified phenol-chloroform-isoamylalcohol treatment and ethanol precipitation were used to extract the genomic DNA as described previously. The DNA were digested with KpnI and transformed to DH5α using method as described by Sambrook et al. (1989).
A 1-ml overnight culture of DH5α was sub-cultured in LB broth for 3 h. The culture was harvested by centrifugation at 5000 rpm for 2 minutes at 4oC. The supernatant was discharge. Pellet was resuspended in 200 ml of ice-cold 50 mM CaCl2 + 50 mM Tris and
incubated on ice for 20 minutes. The cells were pelleted by centrifugation at 5000 rpm for 2 minutes at 4oC. The supernatant was discharged. Pellet was resuspended in 250 ml of ice-cold 0.1 M CaCl2 and reincubated on
ice for 10 minutes. KpnI-digested DNA was put into the microtube and gently mixed by swirling. The tube was heated at 42oC for 45-60 seconds. The tube was rapidly placed on ice to cool for 60 minutes. The cells were transferred into 2 ml of SOC broth. The culture was incubated for 45-60 minutes at 37oC to allow the cell to recover. A 50-100 ml of the transformation mix were plated onto LB-kanamycin agar and incubated overnight.
Plasmid Preparation
In general, DNA plasmid minipreparation was done with Quantum
PrepTM Plasmid Miniprep Kit (Bio-Rad, Hercules, CA). The preparation was done as specified by the manufacturer.
Southern Hybridization
Total bacterial DNA was extracted as previously described (Sambrook et al. 1989). After digested with KpnI, DNA was fractionated on 1.5% agarose gel in 1x TAE buffer. The gel was stained with EtBr and photographed under UV illumination. DNA was denatured by soaking the gel into denaturing solution (1.5 N NaCl and 0.5 N NaOH) for 30 minutes at room temperature with constant, gentle agitation and then rinsed briefly in deionized water. Neutralization was done by soaking the gel for 15 minutes 2 times into the neutralization solution pH 7.5 (1 M Tris and 1.5 N NaCl) at room temperature with constant, gentle agitation.
Is Transposition Really Random?
(Dwi Suryanto)
7 RESULTS AND DISCUSSION
Diparental mating was successfully transfer pJFF350 into S. marcescens DS-8 cells. Mating of S. marcescens DS-8 with E. coli (pJFF350) was obtained at a frequency of 5x10-7 to 2x10-6. Downing et al. (2000) and Fellay et al. (1989) reported that the mutations caused by this transposable element were random. The data in this study showed that the artificial interposon Omegon-Km has specific site preferences. It also showed duplication on the target sequence upon insertion. Berg et al. (1983), Scott (1991), and Wall et al. (1996) showed that many transposons have specific sites of transposition either in Gram-negative or Gram-positive bacteria.
Southern-blot analysis of total cellular DNA DS-8 and its Omegon-Km mutants digested with KpnI. The DNA of lane 1 was marker, lanes 2-5 were mutants, lane 6 was pJFF350 digested with EcoRI, and lane 7 was DS-8.
Site preferences were reported in Tn5
transposition in tet genes of pBR322(Berg et al., 1983), Tn7 in Desulfovibrio desulfuricans (Wall et al., 1996), and in B. subtilis (Scott, 1991). Furthermore, the DNA
sequence terminations indicated that GC base pairs occupied the first and ninth positions in some target sequence duplication at each of the five Tn5 insertion hotspots suggested
GC-cutting preference during Tn5
transposition (Berg et al., 1983). The GC-cutting preference was proposed earlier to guide IS1 and Tn9 insertion (Galas et al., 1980).
DAFTAR PUSTAKA
Berg, D.E., M.A. Schmandt, and J.B. Lowe. 1983.
Specificity of Transposon Tn5 Insertion. Genetics 105: 813-828.
Civolani, C., P. Barghini, A.R. Roncetti, M. Ruzzi, and A. Schiesser. 2000. Bioconversion of Ferulic Acid Into Vannilic Acid by Means of a Vannilate-Negative Mutant of Pseudomonas Fluorescens Strain BFB. Appl. Environ. Microbiol. 66: 2311-2317.
Dennis, J.J. and G.J. Zylstra. 1998. Plasposons: Modular Self-Cloning Minitransposon Derivative for Rapid Genetic Analysis of Gram-Negative Bacterial Genomes. Appl. Environ. Microbiol. 64: 2710-2715.
Downing, K.J., G. Leslie, and J.A. Thomson. 2000.
Biocontrol of the Sugarcane Borer Elsana Saccharina by Expression of the Bacillus Thuringiensis Cryac7 and Serratia Marcescens Chia Genes in Sugarcane-Associated Bacteria.
Appl. Environ. Microbiol. 66:2804-2810.
Fellay, R., H.M. Krisch, P. Prentki, and J. Fey. 1989. Omegon-Km:a Transposable Element Designed for in Vivo Insertional Mutagenesis and Cloning of Genes in Gram-Negative Bacteria.
Gene 76: 215-226.
Galas, D.J., M.P. Calos, and J.H. Miller. 1980.
Sequence Analysis of Tn9 Insertions in the LacZ Gene. J. Mol. Biol. 144: 19-41.
Reinkoff, W.S. 1993. The Tn5 Transposon. Annu. Rev. Microbiol. 47: 945-963.
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989.
Molecular Cloning. Cold Spring HarborLaboratory Press. Cold Spring Harbor. New York.
Jurnal Sains Kimia
Vol. 11, No.1, 2007: 5-8
8
