共查询到20条相似文献,搜索用时 15 毫秒
1.
Tetsurou Muroi Takaaki Kokuzawa Yoshihiko Kihara Ryuichi Kobayashi Nobutaka Hirano Hideo Takahashi Mitsuru Haruki 《Applied microbiology and biotechnology》2013,97(9):4039-4048
Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ~2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ~10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ~10-kbp DNA into the genome with an efficiency of ~104 transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli “centromere” analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC–migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species. 相似文献
2.
Kentaro Morita Tomoyuki Yamamoto Naoki Fusada Mamoru Komatsu Haruo Ikeda Nobutaka Hirano Hideo Takahashi 《Molecular genetics and genomics : MGG》2009,282(6):607-616
We have previously shown that, in vivo, the integration system based on the gene encoding the TG1 integrase and the corresponding
attB
TG1
and attP
TG1
sites works well not only in Streptomyces strains, but also in Escherichia coli. Furthermore, the attachment sites for TG1 integrase are distinct from those of ϕC31 integrase. In this report, we expressed
TG1 integrase as a GST-TG1 integrase fusion protein and then used affinity separation and specific cleavage to release purified
integrase. Conditions for in vitro recombination were established using the purified TG1 integrase and its cognate attP
TG1
and attB
TG1
sites. TG1 integrase efficiently catalyzed a site-specific recombination between attB
TG1
and attP
TG1
sites irrespective of their substrate topology. The minimal sequences of attP
TG1
and attB
TG1
sites required for the substrates of TG1 integrase were demonstrated to be 43 and 39-bp, respectively. These results provide
the basic features of the TG1 integrase system to be used as biotechnological tools, as well as to unravel the mechanism of
the serine integrase. 相似文献
3.
Morita K Morimura K Fusada N Komatsu M Ikeda H Hirano N Takahashi H 《Applied microbiology and biotechnology》2012,93(1):295-304
The serine-type phage integrase is an enzyme that catalyzes site-specific recombination between two attachment sites of phage
and host bacterial genomes (attP and attB, respectively) having relatively short but distinct sequences without host auxiliary factor(s). Previously, we have established
in vivo and in vitro site-specific recombination systems based on the serine-type integrase produced by actinophage TG1 and
determined the minimal sizes of attP
TG1
and attB
TG1
sites required for the in vitro TG1 integrase reaction as 43- and 39-bp, respectively. Here, DNA databases were surveyed
by FASTA program with the authentic attB
TG1
sequence of Streptomyces avermitilis as a query. As a result, possible attB
TG1
sequences were extracted from genomes of bacterial strains belonging to Class Alphaproteobacteria in addition to those of
Class Actinobacteria. Those sequences extracted with a high similarity score and high sequence identity (we took arbitrarily
more than 80% identity) turned out to be located within a conserved region of dapC or related genes encoding aminotransferases and proved to be actually recognized as the cognate substrate of attP
TG1
site by the in vitro TG1 integrase assay. Furthermore, the possible attB
TG1
site of Rhodospirillum rubrum revealed to be used actually as a native (endogenous) attachment site for the in vivo TG1-based integration system. These
features are distinct from other serine-type phage integrases and advantageous for a tool of genome technology in varied industrially
important bacteria belonging to Class Alphaproteobacteria. 相似文献
4.
Herrmann S Siegl T Luzhetska M Petzke L Jilg C Welle E Erb A Leadlay PF Bechthold A Luzhetskyy A 《Applied and environmental microbiology》2012,78(6):1804-1812
The feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the genera Streptomyces, Micromonospora, and Saccharothrix. Two different systems based on Cre/loxP and Dre/rox have been utilized for numerous applications. The activity of the Cre recombinase on the heterospecific loxLE and loxRE sites was similar to its activity on wild-type loxP sites. Moreover, an apramycin resistance marker flanked by the loxLERE sites was eliminated from the Streptomyces coelicolor M145 genome at a surprisingly high frequency (80%) compared to other bacteria. A synthetic gene encoding the Dre recombinase was constructed and successfully expressed in actinomycetes. We developed a marker-free expression method based on the combination of phage integration systems and site-specific recombinases. The Cre recombinase has been used in the deletion of huge genomic regions, including the phenalinolactone, monensin, and lipomycin biosynthetic gene clusters from Streptomyces sp. strain Tü6071, Streptomyces cinnamonensis A519, and Streptomyces aureofaciens Tü117, respectively. Finally, we also demonstrated the site-specific integration of plasmid and cosmid DNA into the chromosome of actinomycetes catalyzed by the Cre recombinase. We anticipate that the strategies presented here will be used extensively to study the genetics of actinomycetes. 相似文献
5.
6.
Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model. 相似文献
7.
Site-specific recombination systems for the genetic manipulation of eukaryotic genomes 总被引:4,自引:0,他引:4
Site-specific recombination systems, such as the bacteriophage Cre-lox and yeast FLP-FRT systems, have become valuable tools for the rearrangement of DNA in higher eukaryotes. As a first step to expanding the repertoire of recombination tools, we screened recombination systems derived from the resolvase/invertase family for site-specific recombinase activity in the fission yeast Schizosaccharomyces pombe. Here, we report that seven recombination systems, four from the small serine resolvase subfamily (CinH, ParA, Tn1721, and Tn5053) and three from the large serine resolvase subfamily (Bxb1, TP901-1, and U153), can catalyze site-specific deletion in S. pombe. Those from the large serine resolvase subfamily were also capable of site-specific integration and inversion. In all cases, the recombination events were precise. Functional operation of these recombination systems in the fission yeast holds promise that they may be further developed as recombination tools for the site-specific rearrangement of plant and animal genomes. 相似文献
8.
Site-specific integration of the actinophage R4 genome into the chromosome of Streptomyces parvulus upon lysogenization. 总被引:3,自引:1,他引:3
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The lysogenization of Streptomyces parvulus by actinophage R4 occurs by site-specific integration of the phage genome into the chromosome. The DNA fragments containing the attachment sites on the host chromosome, the phage genome, and the two junctions created by insertion of the phage genome were cloned and sequenced. The attachment sites were found to share a common core of 12 bp. This common core sequence was not detected in chromosomal DNAs of S. coelicolor and S. lividans. 相似文献
9.
The Streptomyces phage BT1 encodes a site-specific integrase of the large serine recombinase subfamily. In this report, the enzymatic activity of the BT1 integrase was characterized in vitro. We showed that this integrase has efficient integration activity with substrate DNAs containing attB and attP sites, independent of DNA supercoiling or cofactors. Both intra- and intermolecular recombinations proceed with rapid kinetics. The recombination is highly specific, and no reactions are observed between pairs of sites including attB and attL, attB and attR, attP and attL, or attP and attR or between two identical att sequences; however, a low but significant frequency of excision recombination between attL and attR is observed in the presence of the BT1 integrase alone. In addition, for efficient integration, the minimal sizes of attB and attP are 36 bp and 48 bp, respectively. This site-specific recombination system is efficient and simple to use; thus, it could have applications for the manipulation of DNA in vitro. 相似文献
10.
Xiao-Xing Wei Zhen-Yu Shi Zheng-Jun Li Lei Cai Qiong Wu Guo-Qiang Chen 《Applied microbiology and biotechnology》2010,87(4):1533-1541
A method for construction of bacterial strains with multiple DNA inserted into their chromosomes has been developed based
on the mini-Mu transposon and FLP/FRT recombination. Exogenous DNA can be integrated by Mu transposition with an FRT cassette
containing selection marker and conditional replicative origin (R6Kγori). Subsequently, with the introduction of a helper plasmid bearing gene of FLP recombinase, drug-resistant selection marker
is excised from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of transposition and excision
of selection marker. Each cycle can add further foreign gene(s) to the chromosome. As an example, resistance genes of chloramphenicol,
tetracycline, and gentamicin were successively integrated into the chromosome of Escherichia coli BW25113 by three cycles of insertion and excision as described above. This method proved to be simple and time-saving, which
could be applicable to a variety of microorganisms. 相似文献
11.
Takamasa Miura Akito Nishizawa Tomoyasu Nishizawa Munehiko Asayama Hideo Takahashi Makoto Shirai 《Molecular genetics and genomics : MGG》2014,289(4):615-623
The integrase of actinophage R4, which belongs to the large serine-recombinase family, catalyzes site-specific recombination between two distinct attachment site sequences of the phage (attP) and actinomycete Streptomyces parvulus 2297 chromosome (attB). We previously reported that R4 integrase (Sre) catalyzed site-specific recombination both in vivo and in vitro. In the present study, a Sre-based system was developed for the stepwise site-specific integration of multiple genes into the chromosome of cyanobacterium Synechocystis sp. PCC 6803 (hereafter PCC 6803). A transgene-integrated plasmid with two attP sites and a non-replicative sre-containing plasmid were co-introduced into attB-inserted PCC 6803 cells. The transiently expressed Sre catalyzed highly efficient site-specific integration between one of the two attP sites on the integration plasmid and the attB site on the chromosome of PCC 6803. A second transgene-integrated plasmid with an attB site was integrated into the residual attP site on the chromosome by repeating site-specific recombination. The transformation frequencies (%) of the first and second integrations were approximately 5.1 × 10?5 and 8.2 × 10?5, respectively. Furthermore, the expression of two transgenes was detected. This study is the first to apply the multiple gene site-specific integration system based on R4 integrase to cyanobacteria. 相似文献
12.
13.
Targeted integration of foreign genes into plant genomes is a much sought-after technology for engineering precise integration
structures. Homologous recombination-mediated targeted integration into native genomic sites remained somewhat elusive until
made possible by zinc finger nuclease-mediated double-stranded breaks. In the meantime, an alternative approach based on the
use of site-specific recombination systems has been developed which enables integration into previously engineered genomic
sites (site-specific integration). Follow-up studies have validated the efficacy of the site-specific integration technology
in generating transgenic events with a predictable range and stability of expression through successive generations, which
are critical features of reliable and practically useful transgenic lines. Any DNA delivery methods can be used for site-specific
integration; however, best efficiency is mostly obtained with direct DNA delivery methods such as particle bombardment. Although
site-specific integration approach provides unique advantages for producing transgenic plants, it is still not a commonly
used method. The present article discusses barriers and solutions for making it readily available to both academic research
and applicative use. 相似文献
14.
E. J. Nücken R. B. Henschke F. R. J. Schmidt 《Molecular & general genetics : MGG》1991,229(1):137-146
Summary Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA
– background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5 CTAAAACAAAGTTA 3 (hs2). The double-site-specific recombination occurred with a frequency of 10–5–10–6. The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a trans-acting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations. 相似文献
15.
Site-specific recombinases are the enzymes that catalyze site-specific recombination between two specific DNA sequences to
mediate DNA integration, excision, resolution, or inversion and that play a pivotal role in the life cycles of many microorganisms
including bacteria and bacteriophages. These enzymes are classified as tyrosine-type or serine-type recombinases based on
whether a tyrosine or serine residue mediates catalysis. All known tyrosine-type recombinases catalyze the formation of a
Holliday junction intermediate, whereas the catalytic mechanism of all known serine-type recombinases includes the 180° rotation
and rejoining of cleaved substrate DNAs. Both recombinase families are further subdivided into two families; the tyrosine-type
recombinases are subdivided by the recombination directionality, and the serine-type recombinases are subdivided by the protein
size. Over more than two decades, many different site-specific recombinases have been applied to in vivo genome engineering,
and some of them have been used successfully to mediate integration, deletion, or inversion in a wide variety of heterologous
genomes, including those from bacteria to higher eukaryotes. Here, we review the recombination mechanisms of the best characterized
recombinases in each site-specific recombinase family and recent advances in the application of these recombinases to genomic
manipulation, especially manipulations involving site-specific gene integration into heterologous genomes. 相似文献
16.
17.
[目的]研究构建稳定表达外源基因、无抗性标记基因的苏云金杆菌(Bacillus thuringiensis简称Bt)工程菌的方法.在构建Bt工程菌时,高拷贝外源质粒的转入导致Bt芽孢数量减少,芽孢形成期延滞,影响Bt菌株的杀虫活力.而且,外源质粒在Bt中的稳定性较差,外源基因容易丢失.将基因整合人染色体是一种构建遗传性状稳定、杀虫活力高的Bt工程菌的有效方法.[方法]本研究采用PCR技术,分两段扩增定位于Bt无晶体突变株XBU001染色体上的trigger factor基因片段作为同源臂,克隆入温度敏感型载体pKSV7,构建了定点整合载体pKTF12.并利用pKTF12质粒将crylAc基因定点整合入XBU001染色体上.[结果]利用载体pKTF12将crylAc定点插入triggerfactor位点,对宿主菌XBU001的正常生长没有影响.重组菌株KCTF12中的crylAc基因能够稳定遗传、表达并形成菱形晶体.与携带高拷贝外源质粒的Bt菌株HTX42相比较,KCTF12具有芽孢数量增多、芽孢形成期提前的优势.[结论]定点整合法是一种构建稳定表达外源基因、无抗性标记基因Bt工程菌的有效方法. 相似文献
18.
In this report, we describe an optimized method for generation of ephA8 BAC transgenic mice expressing the lacZ reporter gene under ephA8 regulatory sequences. First, we constructed a targeting vector that carries a 1.2 kb ephA8 DNA upstream of its first exon, a lacZ expression cassette, a kanamycin cassette, and a 0.7 kb ephA8 DNA downstream of its first exon. Second, the targeting vector was electroporated into cells containing the ephA8 BAC and pKOBEGA, in which recombinases induce a homologous recombination between the ephA8 BAC DNA and the targeting vector. Third, the FLP plasmid expressing the Flipase was electroporated into these bacteria to eliminate a kanamycin cassette from the recombinant BAC DNA. The appropriate structures of the modified ephA8 BAC DNA were confirmed by Southern analysis. Finally, BAC transgenic mouse embryos were generated by pronuclear injection of the recombinant BAC DNA. Whole mount X-gal staining revealed that the lacZ reporter expression is restricted to the anterior region of the developing midbrain in each transgenic embryo. These results indicate that the ephA8 BAC DNA contains most, if not all, regulatory sequences to direct temporal and spatial expression of the lacZ gene in vivo. 相似文献
19.
Site-specific integration of an adeno-associated virus vector plasmid mediated by regulated expression of rep based on Cre-loxP recombination 总被引:1,自引:0,他引:1
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Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the rep gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-loxP recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the rep coding sequence; in this configuration, rep expression is silent. However, Cre circularizes the rep expression unit, directly joining the p5 promoter to the 5' end of the rep78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV. 相似文献
20.
NBU1 is a 10.3 kbp Bacteroides mobilizable transposon. A previous study had identified a 2.7 kbp segment of the excised circular intermediate that was sufficient to mediate integration of the element after transfer. This segment contained an integrase gene, intN1, and a region spanning the ends of the circular form within which integration occurred (attN1). The integrase protein, IntN1, appeared to be a member of the tyrosine recombinase family because it contains the canonical C-terminal RKHRHY [RK(H/K)R(H/W)Y] motif that characterizes members of that family. In this study, we describe an Escherichia coli-based integration assay system that has allowed us to characterize attN1 in detail. We first localized attN1 to a 250 bp region. We then used site-directed mutations to identify directly repeated sequences within attN1 that were required for site-specific integration. The locus of NBU1 site-specific integration in the Bacteroides thetaiotaomicron chromosome, attBT1-1, contains a 14 bp sequence that is identical to a 14 bp sequence that spans the joined ends of the NBU1 attN1 site (common core sequences). The effects of mutations in the common core were different from the expected results if NBU1 integration was similar to lambda integration. In particular single base changes near one end of the common core region, which introduced heterology, actually increased the frequency of integration. By contrast, compensating changes that restored homology in the common core region reduced the integration frequency. The recombination mechanism also differs from the one used by conjugative transposons that have coupling sequences between the sites of strand cleavage and exchange. These results indicate that although NBU1 integrase is considered to be a member of the tyrosine recombinase family, it catalyses an integrative recombination reaction that occurs by a different crossover mechanism. 相似文献