An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

Baculovirus is a rod-shaped virus containing a large circular dsDNA genome with the size of 80—180 kb[1]. Baculoviruses have been used as insecticides for biological control of forest and agricultural pests[2]. In addition, baculovirus is of great interest as it can be used as efficient eukaryotic expression vector[3], surface display vector[4], and gene therapy vector[5]. Till April 2002, the complete genome sequences of 13 baculoviruses have been reported. The functional genomics has now…     

利用Red系统快速敲除家蚕核型多角体病毒orf60基因

用Red重组系统和最近构建的家蚕核型多角体病毒(BmNPV)bacmid在大肠杆菌BW25113中快速地敲除BmNPVorf60基因。从大肠杆菌BmDH10Bac中提取BmNPVbacmid,将其电转化到含有质粒pKD46(能表达Red重组酶)的大肠杆菌菌株BW25113中,获得了可用于BmNPV基因打靶的菌株BW25113-Bac。设计一对长63bp的引物(5′端为orf60基因的左右同源臂,长45bp;3′端长18bp,为氯霉素抗性基因(cat)的首尾序列),以pKD3质粒(含cat)为模板,PCR扩增携带orf60左右同源臂的cat,即打靶线性化片段。将该线性化片段电转入BW25113-Bac菌株,在Red重组酶的作用下,线性化片段与BmNPVbacmid中的orf60基因发生同源重组。设计3对特异引物,用PCR方法证明cat成功地替换了BmNPVorf60基因。重组bacmid DNA转染BmN细胞后,Western blot分析未检测到orf60基因的表达。     

Red重组技术研究进展

主要从Red系统组成元件、作用机理、重组策略以及先进性和发展前景四个方面综述了利用Red 重组系统敲除或替换细菌染色体目的基因的方法。首先简要介绍了传统的细菌染色体重组技术,指出了其中的缺陷。然后提出了Red重组技术的定义：利用噬菌体Red系统介导来实现外源线性DNA片断与细菌染色体的靶基因进行同源重组的方法,外源线性DNA通常是PCR产物、寡核苷酸片断等,在它们的两翼各含有与染色体靶基因两翼同源的序列40~60bp。这种Red重组技术省去了体外DNA酶切和连接等步骤,使细菌染色体靶基因的敲除与替换操作相对简单,逐渐成为基因功能探索以及新菌株构建的有力手段。     

An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments

This paper describes a rapid method of constructing homologous recombinant baculovirus inE. coli with PCR-amplified fragments. By using this method, the traditional steps of constructing transfer vector are omitted. The method is based on phage λ red system which can promote the recombination between the homologous fragments with the length above 36 bp. Taking HaSNPV as an example, this paper describes the rapid recombination process by using chloramphenicol resistance gene (Cm ^R ) to replaceorf135 in HaSNPV genome. A pair of primers with length of 60 bp was synthesized, in which 40 bp was homologous to the each end sequence oforf135, and the rest 20 bp was homologous to the each end sequence ofCm ^R . By using these primers, a linear fragment containing the completeCm ^R gene between 40 bp of homologous arms oforf135 was generated by PCR with the plasmid pKD3 which containsCm ^R as the template. By transforming the linear fragment into theE. coli containing the bacterial artificial chromosome of HaSNPV and with the help of a plasmid expressing λ recombinase, the recombinants on which the homologue replacement had taken place were selected by chloramphenicol resistance. This method greatly shortens the process of constructing recombinant baculovirus since the process was performed inE. coli and does not need to construct transfer vectors. It can be further used for gene replacement and gene deletion of other large viral genomes.     

Scarless and sequential gene modification in <Emphasis Type="Italic">Pseudomonas</Emphasis> using PCR product flanked by short homology regions

Background  

The lambda Red recombination system has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment containing antibiotic cassette flanked by homology regions to the target locus into a strain that can express the lambda Red proteins (Gam, Bet, Exo).     

含外源类蜗牛毒素基因的AcMNPV的虫体感染性研究

类蜗牛毒素基因(conotoxinlike,ctl)是在一些杆状病毒基因组中存在的与蜗牛毒素类似的一类基因,其功能尚不清楚。本文利用苜蓿银纹夜蛾核多角病毒(AcMNPV)bacmid表达系统构建了含油桐尺蠖核多角体病毒(BusuNPV)ctl基因的重组病毒AcBac-ph-ctl。在细胞水平上对ctl基因的RT-PCR分析表明,该基因转录出mRNA。在甜菜夜蛾体内进行了生物活性测定,结果表明AcBac-ph-ctl与对照野生型AcMNPV的LC50,ST50无显著性差异,表明在此系统中,外源的CTL无杀虫增效性能。     

The role of DNA recombination in herpes simplex virus DNA replication

In many organisms the processes of DNA replication and recombination are closely linked. For instance, in bacterial and eukaryotic systems, replication forks can become stalled or damaged, in many cases leading to the formation of double stranded breaks. Replication restart is an essential mechanism in which the recombination and repair machinery can be used to continue replication after such a catastrophic event. DNA viruses of bacteria such as lambda and T4 also rely heavily on DNA recombination to replicate their genomes and both viruses encode specialized gene products which are required for recombination-dependent replication. In this review, we examine the linkage between replication and recombination in the eukaryotic pathogen, Herpes Simplex Virus Type 1 (HSV-1). The evidence that recombination plays an intrinsic role in HSV-1 DNA replication and the infection process will be reviewed. We have recently demonstrated that HSV-1 encodes two proteins which may be analogous to the lambda phage recombination system, Red(alpha) and beta. The HSV-1 alkaline nuclease, a 5' to 3' exonuclease, and ICP8, a single stranded DNA binding protein, can carry out strand annealing reactions similar to those carried out by the lambda Red system. In addition, evidence suggesting that host recombination proteins may also be important for HSV-1 replication will be reviewed. In summary, it is likely that HSV-1 infection will require both viral and cellular proteins which participate in various pathways of recombination and that recombination-dependent replication is essential for the efficient replication of viral genomes.     

A strategy to construct vector-free amylolytic strains through nondisruptive homologous recombination: application to Enterococcus faecalis.

In order to direct chromosomal integration of the alpha-amylase-encoding gene from Bacillus licheniformis (amyL) under the control of expression and secretion signals from Enterococcus faecalis, the chromosomal fragment (named AB) from the pGIP3124 plasmid [Hols et al., Gene 118 (1992) 21-30] was chosen and split into two fragments (A and B). A translation fusion between the A fragment and 'amyL, deleted of its expression and secretion signals, was made and this fusion was flanked with the AB fragment at its right end. The A::'amyL:AB integration module was cloned into a thermosensitive pE194 replicon (chloramphenicol resistant; CmR) and electro-transformed into E. faecalis OG1X. After an overnight culture in selective liquid medium, the offspring from the amylolytic transformants obtained was shown to yield CmR colonies with two distinct halo sizes on iodine-stained starch plates. Southern analysis clearly showed that the smaller halos corresponded to descendants in which the plasmid had integrated into the chromosome through homologous recombination. One such Amy+ integrant in the AB site was further cultured under nonselective conditions at 42 degrees C for about 20 generations, and the offspring was screened for Amy+/CmS clones. Such revertants were indeed found, and Southern analysis clearly showed that the vector matrix had been excised through homologous recombination between the redundant A sites, leaving the integrated amy gene intact.     

中国棉铃虫单粒包埋核多角体病毒（HaSNPV）HindⅢ-Ⅰ片段的序列分析与基因排列研究

对中国棉铃虫单粒包埋核多角体病毒（HaSNPV)基因组中的HindⅢ-Ⅰ片段的序列进行分析,该片段全长7501nt,包括10个开放阅读框：AcMNPV ORF111的同源基因（Ac 111),晚期表达因子2（lef-2）基因,病毒核壳结构蛋白p24基因、gp16基因、多角体包膜蛋白（polyhedron envelope protein,pep)基因、AcMNPV ORF63的同源基因（Ac63),38.7kD基因,晚期表达因子1（lef-1) 基因,及两个特有基因HaSNPV orf591和HasSNPV orf435。基因组的排列比较分析发现与AcMNPV有较大区别。     

Restoration of gene function by homologous recombination: from PCR to gene expression in one step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdaPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage lambda Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding beta-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.     

Bacteriophage SPP1 Chu is an alkaline exonuclease in the SynExo family of viral two-component recombinases

Many DNA viruses concatemerize their genomes as a prerequisite to packaging into capsids. Concatemerization arises from either replication or homologous recombination. Replication is already the target of many antiviral drugs, and viral recombinases are an attractive target for drug design, particularly for combination therapy with replication inhibitors, due to their important supporting role in viral growth. To dissect the molecular mechanisms of viral recombination, we and others previously identified a family of viral nucleases that comprise one component of a conserved, two-component viral recombination system. The nuclease component is related to the exonuclease of phage lambda and is common to viruses with linear double-stranded DNA genomes. To test the idea that these viruses have a common strategy for recombination and genome concatemerization, we isolated the previously uncharacterized 34.1 gene from Bacillus subtilis phage SPP1, expressed it in Escherichia coli, purified the protein, and determined its enzymatic properties. Like lambda exonuclease, Chu (the product of 34.1) forms an oligomer, is a processive alkaline exonuclease that digests linear double-stranded DNA in a Mg(2+)-dependent reaction, and shows a preference for 5'-phosphorylated DNA ends. A model for viral recombination, based on the phage lambda Red recombination system, is proposed.     

一种新颖的棉铃虫单粒包埋核多角体病毒表达系统

将含有低拷贝数的mini Freplicon、一个卡那霉素抗性基因和一个lacZα基因 8.6kb的DNA片段经同源重组置换到棉铃虫核型多角体病毒基因组中的多角体蛋白基因内 ,构建了既能在E .coli内复制又可在昆虫细胞内复制形成完整的病毒粒子棉铃虫核型多角体病毒Bacmid(HaBacmid HZ8)。另外将HaSNPV的多角体蛋白基因和P10启动子序列取代 pFastBacDual质粒上的AcMNPV的多角体启动子序列和P10启动子序列 ,构建插入HaSNPV多角体蛋白基因和P10启动子序列的HapFastBacPhP10供体质粒。利用HapFastBacPhP10供体质粒将eGFP基因转位至HZ8的Tn7附着位点上 ,随后将含有eGFP基因的重组HaBacmidDNA转染至HZAm1细胞内。转染 5d后 ,细胞核内能形成典型的多角体 ,在萤光显微镜下观察到细胞内显示出强烈的绿色萤光。结果证明我们构建的HaBactoBcac表达系统能有效的表达外源基因     

利用Red同源重组系统进行牛β酪蛋白基因敲除

利用EL350基因工程菌进行同源重组, 成功进行基因敲除已有报道, 但利用该系统进行乳腺生物反应器质粒构建的研究却未见报道。实验采用含有完整的牛b 酪蛋白基因的CSN2质粒作为基因打靶的载体, 设计不同的同源臂, 成功地敲除了b 酪蛋白基因的编码区。并且同时利用同源重组技术对敲除不同大小的DNA片段的效率进行了研究。为进一步利用CSN2质粒两端的调控序列, 插入新的基因, 研究其表达功能, 或者进行乳腺生物反应器的研究奠定了基础。     

Cloning and sequence analysis of a plasmid-encoded chloramphenicol acetyltransferase gene from Staphylococcus intermedius.

The chloramphenicol acetyltransferase gene (cat) of a 3.9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCS1, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194.     

Recombination-promoting activity of the bacteriophage lambda Rap protein in Escherichia coli K-12

The rap gene of bacteriophage lambda was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the lambda red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recGDelta red(+) rap(+) strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicol-resistant bacterial progeny. The results of these experiments indicated that the lambda rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.     

A rearranged DNA sequence possibly related to the translocation of immunoglobulin gene segments.

A 5.3 kb EcoRI fragment (T3, abbreviations in ref. 2) has been cloned from DNA of a kappa light chain producing mouse myeloma. The fragment hybridizes to the k' flanking sequences of the J1 gene segment but not to C gene sequences of kappa light chain DNA. Restriction nuclease mapping and partial nucleotide sequencing showed that the fragment consists of sequences from the 5' side of the J1 and form the 3' side of a V gene segment, which apparently had been linked in a genomic rearrangement process. These rearranged flanking sequences are not the flanking sequences of the V and J gene segments which had been joined to form the two kappa light chain genes of the myeloma. Fragments with the hybridization properties of T3 have been found also in two other kappa and one lambda chain producing myelomas. The linking of flanking sequences in the myeloma genome is discussed with respect to the mechanism of recombination between V and J gene segments.     

Endpoint distribution for deletions into imm lambda region forming p lambda CM replicons: phage lambda gene rex affects plasmid establishment

For the p lambda CM family of lambda-derived self-encapsidating plasmids, the rexB gene product facilitates plasmid establishment following injection into a new host cell. Temperature-stable chloramphenicol resistance (CmR at 40 degrees C) conferred by low-multiplicity infection with lambda::Tn9 cI857 lysates (Tn9 sites tested: 22.60 or 24.08 kb, in the b region, or 28.41 kb, in int) is usually due to a lambda::Tn9 plasmid (p lambda CM) formed by a deletion penetrating the lambda immunity region. These grow either as plasmids in the absence of, or lytic phages in the presence of N function supplied by a host such as lambda cI857 delta H1 lysogen MS1449. The 'groplaque' (plaque-shaped growth spot) assay, which selects for CmR growth in an MS1449 lawn at 32 degrees C after an initial plaquing period at 37 degrees C, reveals two distinguishable classes of p lambda CM isolates. All variants whose deletions extend into or beyond rexB give rise to visible CmR growth only after the temperature shift to 32 degrees C, and thus produce a hollow-centered 'donut' type of groplaque. In contrast, 16 out of 17 variants whose deletions fall short of rexB produce 'solid' groplaques which appear before the temperature shift. Tests of T4rII phage exclusion show the exceptional 17th variant to be Rex-, confirming the identification of rex as the lambda component whose loss results in the 'donut' groplaque morphology. More specific physiological tests showed that in the absence of Rex the establishment of a newly injected p lambda CM plasmid becomes temperature-sensitive (ts), while plasmid maintenance remains unaffected. This indicates that the role of Rex in plasmid survival is confined to the early stages of transduction, where it might either assist plasmid replication or retard host replication, to help the plasmid replicon achieve a copy number sufficient for stable transmission.     

Rapid construction of adenoviral vectors by lambda phage genetics

Continued improvements of adenoviral vectors require the investigation of novel genome configurations. Since adenovirus can be generated directly by transfecting packaging cell lines with viral genomes isolated from plasmid DNA, it is possible to separate genome construction from virus production. In this way failure to generate a virus is not associated with an inability to generate the desired genome. We have developed a novel lambda-based system that allows rapid modification of the viral genome by double homologous recombination in Escherichia coli. The recombination reaction and newly generated genome may reside in a recombination-deficient bacterial host for enhanced plasmid stability. Furthermore, the process is independent of any restriction endonucleases. The strategy relies on four main steps: (i) homologous recombination between an adenovirus cosmid and a donor plasmid (the donor plasmid carries the desired modification[s] and flanking regions of homology to direct its recombination into the viral genome); (ii) in vivo packaging of the recombinant adenoviral cosmids during a productive lambda infection; (iii) transducing a recombination-deficient E. coli lambda lysogen with the generated lysate (the lysogen inhibits the helper phage used to package the recombinant andenoviral cosmid from productively infecting and destroying the host bacteria); (iv) effectively selecting for the desired double-recombinant cosmid. Approximately 10,000 double-recombinant cosmids are recovered per reaction with essentially all of them being the correct double-recombinant molecule. This system was used to generate quickly and efficiently adenoviral genomes deficient in the E1/E3 and E1/E3/E4 regions. The basis of this technology allows any region of the viral genome to be readily modified for investigation of novel configurations.