Characterization of a Chlamydomonas Transposon, Gulliver, Resembling Those in Higher Plants

While pursuing a chromosomal walk through the mt+ locus of linkage group VI of Chlamydomonas reinhardtii, I encountered a 12-kb sequence that was found to be present in approximately 12 copies in the nuclear genome. Comparison of various C. reinhardtii laboratory strains provided evidence that the sequence was mobile and therefore a transposon. One of two separate natural isolates interfertile with C. reinhardtii, C. smithii (CC-1373), contained the transposon, but at completely different locations in its nuclear genome than C. reinhardtii; and a second, CC-1952 (S1-C5), lacked the transposon altogether. Genetic analysis indicated that the transposon was found at dispersed sites throughout the genome, but had a conserved structure at each location. Sequence homology between the termini was limited to an imperfect 15-bp inverted repeat. An 8-bp target site duplication was created by insertion; transposon sequences were completely removed upon excision leaving behind both copies of the target site duplication, with minor base changes. The transposon contained an internal region of unique repetitive sequence responsible for restriction fragment length heterogeneity among the various copies of the transposon. In several cases it was possible to identify which of the dozen transposons in a given strain served as the donor when a transposition event occurred. The transposon often moved into a site genetically linked to the donor, and transposition appeared to be nonreplicative. Thus the mechanism of transposition and excision of the transposon, which I have named Gulliver, resembles that of certain higher plant transposons, like the Ac transposon of maize.     

转座子挽救法对转座子突变菌株中插入位点的定位分析

为寻找谷氨酸棒杆菌转座子插入突变菌株中的转座子插入位点,采用了转座子挽救法对转座子及其插入位点附近的序列进行分离,并测定插入位点相邻DNA序列,获得了三个转座子插入位点DNA序列,其中一个是柠檬酸合成酶基因,另两个为目前未知基因,暂命名为orfA和orfB。该方法简便易行,是分析转座子插入位点的理想方法。     

The construction and preliminary analysis of a Tn5 transposon based random mutant library of baculovirus

A transposon-based random mutation library of AcMNPV,the type species of baculovirus,was constructed using a Tn5 transposon.The green fluorescence protein gene under the control of the Drosophila hsp70 promoter was inserted into the transposon for easy tracking in insect cells.In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome.The transposed genome was then used to transfect Sf21 insect cells,and a library of mutant viruses capable of expressing green fluorescence protein was obtained.Two mutant viruses,B9F and Li6A were isolated,and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes,respectively.Both genes were determined to be nonessential in viral replication and infection.This technique will be very useful in the functional study of baculovirus genes.     

IS911 transposon circles give rise to linear forms that can undergo integration in vitro

High levels of expression of the transposase OrfAB of bacterial insertion sequence IS911 leads to the formation of excised transposon circles, in which the two abutted ends are separated by 3 bp. Initially, OrfAB catalyses only single-strand cleavage at one 3' transposon end and strand transfer of that end to the other. It is believed that this molecule, in which both transposon ends are held together in a single-strand bridge, is then converted to the circular form by the action of host factors. The transposon circles can be integrated efficiently into an appropriate target in vivo and in vitro in the presence of OrfAB and a second IS911 protein OrfA. In the results reported here, we have identified linear transposon forms in vivo from a transposon present in a plasmid, raising the possibility that IS911 can also transpose using a cut-and-paste mechanism. However, the linear species appeared not to be derived directly from the plasmid-based copy by direct double-strand cleavages at both ends, but from preformed excised transposon circles. This was confirmed further by the observation that OrfAB can cleave a cloned circle junction both in vivo and in vitro by two single-strand cleavages at the 3' transposon ends to generate a linear transposon form with a 3'-OH and a three-nucleotide 5' overhang at the ends. Moreover, while significantly less efficient than the transposon circle, a precleaved linear transposon underwent detectable levels of integration in vitro. The possible role of such molecules in the IS911 transposition pathway is discussed.     

Electron microscopic observation of new transposable elements inserted into P22 phage genome from R plasmids

By using phage P22spl, a deletion mutant of phage P22, the structures of two new transposons on P22 genomes were studied by the electron microscopic heteroduplex method. One of these was the Cm (chloramphenicol) transposon derived from an R plasmid, NR1, and the other the Km (kanamycin) transposon frin obr502. the heteroduplex between P22 phage DNAs with and without the Cm transposon revealed that the Cm transposon was similar in structure to the Tn9 element, a well-known Cm transposon derived from the R plasmid pMS14. On the other hand, the Km transposon of pNR502 was quite different in structure from other Km transposons reported previously. This transposon consists of a 6.8 kilobase (kb) segment of DNA, in which a short inverted repeat is contained. The heteroduplex experiments showed that a 4.5 kb segment of DNA was deleted from the P22 genome in the P22spl genome. Because of a shorter unit length of the genome, phage P22spl is considered to be useful of assaying various kinds of transposable elements.     

Use of Transposons in Cloning Poorly Selectable Genes of Escherichia coli: Cloning of uvrA and Adjacent Genes

A transposon was introduced close to a poorly selectable gene. This gene could be cloned by using selection for the antibiotic resistance marker of the transposon.     

Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome

Genetic analysis of Bacteroides fragilis (BF) is hindered because of the lack of efficient transposon mutagenesis methods. Here, we describe a simple method for transposon mutagenesis using EZ::TN5, a commercially available system that we optimized for use in BF638R. The modified EZ::TN5 transposon contains an Escherichia coli conditional origin of replication, a kanamycin resistance gene for E. coli, an erythromycin resistance gene for BF , and 19 basepair transposase recognition sequences on either ends. Electroporation of the transposome (transposon-transposase complex) into BF638R yielded 3.2 ± 0.35 × 10(3) CFU μg(-1) of transposon DNA. Modification of the transposon by the BF638R restriction/modification system increased transposition efficiency sixfold. Electroporation of the EZ::TN5 transposome results in a single-copy insertion of the transposon evenly distributed across the genome of BF638R and can be used to construct a BF638R transposon library. The transposon was also effective in mutating a BF clinical isolate and a strain of the related species, Bacteroides thetaiotaomicron. The EZ::TN5-based mutagenesis described here is more efficient than other transposon mutagenesis approaches previously reported for BF.     

Sleeping Beauty transposition: from biology to applications

Sleeping Beauty (SB) is the first synthetic DNA transposon that was shown to be active in a wide variety of species. Here, we review studies from the last two decades addressing both basic biology and applications of this transposon. We discuss how host–transposon interaction modulates transposition at different steps of the transposition reaction. We also discuss how the transposon was translated for gene delivery and gene discovery purposes. We critically review the system in clinical, pre-clinical and non-clinical settings as a non-viral gene delivery tool in comparison with viral technologies. We also discuss emerging SB-based hybrid vectors aimed at combining the attractive safety features of the transposon with effective viral delivery. The success of the SB-based technology can be fundamentally attributed to being able to insert fairly randomly into genomic regions that allow stable long-term expression of the delivered transgene cassette. SB has emerged as an efficient and economical toolkit for safe and efficient gene delivery for medical applications.     

Germline mutagenesis mediated by <Emphasis Type="Italic">Sleeping Beauty</Emphasis>transposon system in mice

Following the descovery of its transposition activity in mammalian culture systems, the Sleeping Beauty (SB) transposon has since been applied to achieve germline mutagenesis in mice. Initially, the transposition efficiency was found to be low in cultured systems, but its activity in germ cells was unexpectedly high. This difference in transposition efficiency was found to be largely dependent on chromosomal status of the host genomic DNA and transposon vector design. The SB transposon system has been found to be suitable for comprehensive mutagenesis in mice. Therefore, it is an effective tool as a forward genetics screen for tagged insertional mutagenesis in mice.     

Transposon Tn5 mutagenesis of Pseudomonas fluorescens to isolate mutants deficient in antibacterial activity

Abstract Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn 5 -GM into the chromosome of P. fluorescens was achieved by heat ttreatment of the transformed cells at 42°C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens . One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis . These results introduce Tn 5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens .     

Physical map of the genome of Acholeplasma oculi ISM1499 and construction of a Tn4001 derivative for macrorestriction chromosomal mapping.

A physical chromosomal map of Acholeplasma oculi ISM1499 was constructed by using field inversion gel electrophoresis. To assist in the ordering of the chromosomal fragments, a modified transposon, Tn4001.1064, was constructed. It was also used to rescue mycoplasmal chromosomal sequences adjacent to transposon insertion sites in a one-step cloning procedure. The total size of the A. oculi ISM1499 genome was estimated to be 1,633 kb. The restriction enzyme sites for ApaI, BssHII, EagI, and SmaI were positioned on the map along with several transposon insertion sites.     

Template secondary structure promotes polymerase jumping during PCR amplification.

Pairs of primers flanking known miniTn10 transposon insertion sites were used to confirm the presence of the transposon in DNA isolated from Legionella pneumophila mutants. It was expected that the polymerase chain reaction products derived from the mutant template would be larger than those from the wild-type (WT) template due to the presence of the 1.8-kb transposon. Instead, it was observed that the mutant template yielded a product of almost the same size as that yielded by WT template. We present evidence to indicate that the aberrant product from the mutant template is a direct result of secondary structure of the template resulting from an inverted repeat sequence present in the miniTn10 transposon.     

Specific binding of integrase to the origin of transfer (oriT) of the conjugative transposon Tn916

Purified integrase protein (Int) of the conjugative transposon Tn916 was shown, using nuclease protection experiments, to bind specifically to a site within the origin of conjugal transfer of the transposon, oriT. A sequence similar to the ends of the transposon that are bound by the C-terminal DNA-binding domain of Int was present in the protected region. However, Int binding to oriT required both the N- and C-terminal DNA-binding domains of Int, and the pattern of nuclease protection differed from that observed when Int binds to the transposon ends and flanking DNA. Binding of Int to oriT may be part of a mechanism to prevent premature conjugal transfer of Tn916 prior to excision from the donor DNA.     

新型二合一PiggyBac转座系统的构建及功能验证*

目的:PiggyBac(PB)转座子是一种可移动的遗传元件,采用“剪切和粘贴”机制在载体和染色体之间进行转座;通过将转座子元件和转座酶表达框整合到一个表达载体中,构建简便易用的二合一PB转座系统。方法:通过聚合酶链式反应(polymerase chain reaction,PCR)获取PiggyBac转座系统所需转座子元件和转座酶表达框,利用T4 DNA连接酶将转座酶表达框插入到pUC18载体上,再利用Gibson同源重组技术将转座子元件与重组载体结合构建二合一PB转座系统;使用该系统携带的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)以及功能性损伤抑制蛋白(damage-suppressing protein,DSUP)检测其有效性及可靠性。结果:在所有筛选获得的嘌呤霉素抗性细胞中,EGFP都是明亮可见;利用此二合一PB转座系统成功获得了可高效表达功能性损伤抑制蛋白的稳定细胞系,证明外源基因可被有效整合到基因组DNA中并表达。结论:成功构建了新型二合一PB转座系统,使稳定表达细胞系的建立更加经济简便。     

A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens

A mini-Tn5 transposon derivative, mini-Tn5gfp-km, has been constructed which contained a promoter-less artificial operon consisting of two open reading frames, green fluorescent protein (GFP) and neomycin phosphotransferase II (NptII). When this transposon was used to mutagenize Agrobacterium tumefaciens, all the mutants selected in the presence of kanamycin exhibited GFP expression, which could be conveniently monitored by a fluorometer. The transposon appeared to be bifunctional and could provide both selection and reporter functions. Even the mutants showing minimal levels of GFP expression were still resistant to kanamycin. This suggests that this transposon can be used to select for insertions downstream of both weak and strong promoters, as long as the insertions themselves are non-lethal. This system was used to identify A. tumefaciens genes that were upregulated in response to acidic pH. Screening only 20 colonies led to identification of two promoters that were specifically induced by low pH and one promoter that was specifically induced by acetosyringone in a minimal medium of pH 5.5.     

Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene

A 1.3-kb restriction fragment carrying a cat gene derived from Staphylococcus aureus was inserted by ligation in both possible orientations into a HpaI restriction site located less than 300 bp from one end of Tn917. The resulting transposon derivatives were unimpaired in their ability to make and resolve transpositions into the chromosome of Bacillus subtilis and they displayed no detectable defect in expression of the inducible erm gene carried by the transposon. This demonstrates that the HpaI site itself, and perhaps the entire 250- to 300-bp region between the HpaI site and the nearest transposon terminal inverted repeat consists of nonessential DNA, and is there fore available to be modified or used as a cloning site with the expectation that the resulting transposon derivatives should be capable of normal transposition activity. To facilitate such manipulations, the HpaI site was "replaced" by a 24-bp DNA segment which contains a BamHI site flanked on either side by SmaI sites; these BamHI and SmaI sites are unique to the transposon. Several of the plasmid constructions undertaken in the course of this work illustrate ways in which homologous recombination may be used in conjunction with ligation in B. subtilis (and other bacteria, such as Streptococcus pneumoniae, which have similar mechanisms for DNA uptake during competence) to facilitate significantly the recovery of certain kinds of recombinant molecules.     

GENETIC SCREENING FOR IDENTIFICATION OF SALMONELLA TYPHIMURIUM TN5 MUTANTS WITH POTENTIAL HYPERSENSITIVITY TO SHORT-CHAIN FATTY ACIDS

This study was conducted to test if transposon footprinting could be used to identify transposon mutants of Salmonella typhimurium with growth defects in a media containing short-chain fatty acids (SCFA) as the test selective condition. High concentrations of SCFA are one of the characteristic conditions in the animal intestine that has been suggested to play a role in inhibiting colonization by nonindigenous bacterial pathogens. When the mutant pools containing 25 Tn5 mutants/pool were analyzed for transposon footprints before and after selection, a polymerase chain reaction (PCR) product could be identified that was present in an input pool, but not in a corresponding output pool. The results indicate that transposon footprinting can be used for negative screening of genes sensitive to SCFA in the S. typhimurium bacterial genome.     

A system of transposon mutagenesis for bacteriophage T4

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.