Plasmid vector for cloning, determination of nucleotide sequence and directed assembly of DNA fragments

A plasmid vector pNIMB has been constructed (starting) from the pUR222 plasmid as a result of substitution of the polylinker containing restriction sites: PstI, SalGI, AccI, HindII, BamHI EcoRI and by other synthetic linkers with additional sites for HindIII and HgaI. Plasmid pNIMB does not differ from the parent one phenotypically. Compared to pUR222 the vector contains an additional site for cloning HindIII fragments of DNA and allows to clone SalGI/BamHI- and PstI/SalGI-fragments. Cloning of DNA fragments in all seven unique sites of pNiMB gives the possibility for sequencing the fragments avoiding their isolation from the gel. Moreover, this vector may be useful for cloning and directed assembly of chemically synthesised DNA fragments when the endonuclease HgaI sites are used.     

Design of recombinant-stable plasmids of the pFH series

By cloning synthetic oligonucleotides into pUC18 plasmid, pFH123--pFH127 plasmids have been constructed. Their polylinker area, along with sites of widely used restriction endonucleases, contains two pairs each of FokI and HgaI sites in the opposite orientation to provide subfragment with unique predetermined 5'-ends. Comparative stability of the new plasmids and their derivatives has been studied and compared with that of the earlier constructed pMB plasmids.     

Comparison of the state of the area between B- and Z-segments of superhelical plasmids in vitro and in situ]

The structure of B-Z junction in a cloned plasmid pGC20 containing a (dG-dC)10 insert at the SmaI site has been studied in vitro and in situ by modifying the DNA with O-beta-diethylaminoethylhydroxylamine (OHA). The latter is an analog of hydroxylamine possessing specificity with respect to unpaired cytidine. Experiments in vitro showed a complicated pattern of inhibiting the restriction hydrolysis of the OHA-modified DNA within the polylinker region of the plasmid. As the duration of the DNA reaction with OHA grows, a gradual increase in the inhibition of restriction is observed at the BamHI site neighboring the Z-insert and at the HindIII site at a distance of about 30 bp from the insert, while an intact segment (containing the SalGI site) is retained in the intermediate region. On passing to the cell level, only the region immediately adjacent to the Z-insert appears to be modified. According to estimates, about 30 to 40% of pGC20 molecules have the (dG-dC)10 insert in the Z-form when modified in situ in 1M OHA, pH 5.0.     

FokI method of gene synthesis

An accurate, fast and simple method is presented for synthesis of a gene, or any DNA fragment with a defined sequence. The method is based on the observation that large (approx. 100 bp long) inserts can be cloned into a plasmid using a technique of oligodeoxynucleotide (oligo)-directed double-strand (ds) break repair. The procedure involves transformation of Escherichia coli with a denatured mixture of an insert-carrying oligo and linearized plasmid DNA [Mandecki, Proc. Natl. Acad. Sci. USA 83 (1986) 7177-7181]. The nucleotide (nt) sequences are inserted between two FokI restriction nuclease sites in one of four pUC-derived plasmids. Since FokI makes a staggered ds break at a DNA site 9 and 13 nt away from its recognition site, upon cleavage of the plasmid DNA with FokI, a restriction fragment is liberated that by design contains unique 4-nt-long 5'-protruding ends. The uniqueness of ends permits efficient and directed simultaneous ligation of several restriction fragments to form a gene. The method offers flexibility due to the modular-type assembly and does not require any restriction sites within the constructed gene. The sequence error rate is low: about one error per 4000 bp of DNA cloned. Synthetic DNA for only one DNA strand needs to be provided. The method was applied to the synthesis of a gene fragment encoding the N-terminal 143 amino acid residues of the human immunodeficiency virus transmembrane protein (p41).     

两种pUC18高效T载体的构建

T载体是用于直接克隆PCR产物的线性载体.在此之前,克隆PCR片段时一般先用Klenow片段酶或T4DNA聚合酶削平PCR产物两端,克隆过程中又大都不能使用碱性磷酸酶为载体片段脱磷,因为绝大多数PCR引物5’端未磷酸化,T载体的诞生使分子生物学工作者摆脱了这一窘境,而且,T载体的3’端突出的T碱基与PCR产物3’端由于Taq酶非模板依赖的末端转移酶活性而添加的A碱基[1]互补,使载体与PCR产物的连接效率大大提高.由于具有上述优点,T载体从一产生就引起人们极大的兴趣,很多公司也相继推出了各自的T载体系统,并运用该技术改造了很多传统载体.本…     

The SalGI restriction endonuclease. Enzyme specificity.

We have analysed the kinetics of DNA cleavage in the reaction between the SalGI restriction endonuclease and plasmid pMB9. This reaction is subject to competitive inhibition by DNA sequences outside the SalGI recognition site; we have determined the Km and Vmax. for the reaction of this enzyme at its recognition site and the KI for its interaction at other DNA sequences. We conclude that the specificity of DNA cleavage by the enzyme is only partly determined by the discrimination it shows for binding at its recognition sequence compared with binding to other DNA sequences.     

Convenient vectors for cloning and sequencing EcoRI and HindIII fragments

The polylinker regions of plasmid pUC and bacteriophage M13mp vectors have been specifically modified to provide alternative positions for cloning and reexcising EcoRI and HindIII fragments; the EcoRI and HindIII sites have been moved internal to BamHI and Bg/II sites. The location of EcoRI and HindIII sites in these HinEco vectors allows either selective linearization or excision of the cloned fragments at unique flanking sites.     

The SalGI restriction endonuclease. Mechanism of DNA cleavage.

The cleavage of supercoiled DNA of plasmid pMB9 by restriction endonuclease SalGI has been studied. Under the optimal conditions for this reaction, the only product is the linear form of the DNA, in which both strands of the duplex have been cleaved at the SalGI recognition site. DNA molecules cleaved in one strand at this site were found to be poor substrates for the SalGI enzyme. Thus, both strands of the DNA appear to be cleaved in a concerted reaction. However, under other conditions, the enzyme cleaves either one or both strands of the DNA; the supercoiled substrate is then converted to either open-circle or linear forms, the two being produced simultaneously rather than consecutively. We propose a mechanism for the SalGI restriction endonuclease which accounts for the reactions of this enzyme under both optimal and other conditions. These reactions were unaffected by the tertiary structure of the DNA.     

Construction and properties of hybrid plasmids carrying the E. coli gal operon.

A family of hybrid plasmids carrying the entire gal operon of E. coli and designated pgal was constructed in vitro. In the case of pgal 1 (mol. wt. 16.4 Md), a fragment cut by Bam HI endonuclease from lambda gal phage DNA (lambda D-J-gal-att-int) was joined to pMB9 and cloned in the gal-strain of E. coli, which was grown on selective media with galactose as a sole source of carbon. Plasmid pgal2 was derived from pgal 1 by elimination of the 1.1 Md fragment located between the two EcoRI sites and carrying the lambda att-int region and part of pMB9. To obtain pgal3, the 10.7 Md fragment of lambda DNA located between the two SmaI sites (lambda D-J and part of pMB9) in pgal2 was cut out and the resulting flush-end fragments were sealed by the T4DNA ligase. The mol. wt. of pgal3 containing one SmaI site amounted to 4.6 Md, while several pgal3 variants that had lost their SmaI site were still smaller. Plasmid pgal1 inhibited the growth of the gal- host cells, which effect could be overcome by the accompanying helper pMB9. The presence of pgal2 and pgal3 supported the growth and multiplication of gal- cells on selective media even without the helper plasmid. The total amount of pgal plasmid DNA per cell was constant and equalled 60--70 Md (4 copies of pgal1 or 15--16 copies of pgal3, ColE1 or pMB9). This might explain why the co-presence of pMB9 helper does alleviate the "harmful" effects of the plasmid pgal1 (which carries att-int genes), by reducing the copy number of the latter from four to one.     

Phage lambda and plasmid expression vectors with multiple cloning sites and lacZ alpha-complementation

Two new lambda vectors were constructed which permit cloning of genes that are potentially lethal if cloned in analogous plasmid vectors. lambda DL10 and lambda DL11 contain the alpha-complementing fragment of lacZ and multiple cloning sites found in the polylinker region of M13mp10 and M13mp11, respectively. DNA cloned into the unique cloning sites of these vectors can be detected by inactivation of alpha-complementation. These lambda vectors provide a lac promoter for expression of foreign genes as well as the ability to make fusion proteins. Two plasmid expression vectors, pPR110 and pPR111, were constructed from lambda DL10 and lambda DL11 respectively, and pCQV2. These plasmids, which express lacZ alpha-complementing activity from the lambda PR promoter, contain multiple cloning sites immediately downstream of the PR promoter. They allow cloning of genes under the control of the PR promoter and the plasmid-encoded thermosensitive (cI857) repressor, and allow easy detection of inserted fragments by inactivation of alpha-complementation.     

Phasmids. Properties and the use in genetic engineering. I. Construction of the phasmid vector

A phasmid vector molecule designated pMYF11 has been constructed. The vector combines some useful features of plasmid and phage vector molecules. lambda pMYF11 is a hybrid of lambda 47.1 vector and pBR322 plasmid. CI- marker of pMYF11 is replaced with cI+ marker by recombination between the plasmid and prophage 434. The phasmid molecule can be used as a replacement vector for BamHI, HindIII, SalGI endonucleases. The maximum size of fragments to be cloned is 21 kilobase pairs. Positive selection for hybrid molecules is possible because of the Spi phenotype expression after replacement of the central HindIII or BamHI DNA fragment with foreign DNA. A library of Escherichia coli genes is constructed with the help of lambda pMYF11 as a vector molecule. A hybrid phage harboring genes of the proline operon is detected by means of complementation.     

Cloning and expression of the Clostridium thermocellum gene in cyanobacteria Anacystis nidulans R2 cells]

The XhoI-SalGI fragment of the plasmid pCI DNA was inserted into the SalGI site of the cyanobacterium Anacystis nidulans R2 integrative vector plasmid pIAH4. The fragment incorporates the endoglucanase gene of Clostridium thermocellum cloned earlier within the 6.7 kb DNA sequence. The recombinant plasmid DNA was transformed into Anacystis nidulans R2 cells. The cloned endoglucanase gene was shown to express in the cyanobacterium cells. The enzyme synthesized is accumulated within the cytoplasm of Anacystis nidulans cells and is not secreted into the periplasm.     

Construction of plasmid vectors that facilitate subcloning and recovery of yeast and Escherichia coli DNA fragments

We have constructed a plasmid vector (pNF2) which is a derivative of the multicopy yeast cloning vehicle YEp24. This derivative contains a single BamHI site flanked immediately on each side by SalI sites. The latter site was selected because it appears to be infrequent in yeast nuclear DNA. Thus, DNA fragments produced by partial digestion with enzymes (such as Sau3A) that cut at frequent intervals and leave single-stranded ends that have sequence homology with BamHI sites, can be conveniently subcloned into this site. Such fragments can then be excised intact by digestion with SalI enzyme. Plasmid pNF2 also contains the kanamycin-resistance (kanR) gene derived from Tn903 and confers resistance in yeast to the antibiotic G418. pNF2 was converted into an integrating vector (pNF3) by deleting a 2.2-kb EcoRI fragment containing a sequence that determines autonomous replication in yeast. Further deletion of a HindIII fragment containing the yeast URA3 gene converts the plasmid into one containing only pBR322 sequences plus the kanR gene (pNF4).     

The mutH gene regulates the replication and methylation of the pMB1 origin.

DNA methylation is known to regulate several prokaryotic replication origins. In particular, the Escherichia coli chromosomal origin oriC and the pMB1 plasmid origin (which is homologous to the ColE1 origin) replicate poorly when hemimethylated at dam (GATC) sites. Because the mismatch repair protein MutH is known to recognize hemimethylated dam sites, its role in the replication of these origins was investigated. The results presented here show that the mutH gene product is partially responsible for the poor replication of the pMB1 origin when hemimethylated but has no effect on the replication of oriC. Methylation levels at individual dam sites suggest that the MutH protein binds to an inverted repeat in the pMB1 replication primer promoter. These findings suggest a mechanism for the coordinated control of DNA repair and replication.     

Specific-purpose broad-host-range vectors

Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 were constructed and characterized. Vector pAYC30 was constructed by insertion in vivo into the genome of RSF1010 the Hgr transposon Tn501, originating from the plasmid pVS1 of Pseudomonas aeruginosa. Plasmids with inserts of PstI or SacI fragments may be selected by inactivation of genes sul and aph, respectively. The cloning at unique site SalGI leads to the appearance of HgCl2--sensitive transformants. Versatile cloning vector pAYC1 consists of two replicons, RSF1010 and plasmid pMZ7, a derivative of R6K. The constructed plasmid is 16.9 kb in length and determines resistance to five drugs. Two promoter-probe broad-host-range vectors, pAYC36 and pAYC37, were obtained by replacing a small segment from the DNA sequence of the aph gene promoter of previously described plasmid pAYC32 with the polylinker from plasmid pUC19. Therefore, vector plasmids retained the intact gene aph (Smr); however, they have Sms phenotype because of the insertional inactivation of the promoter. The genetic structure of promoter-probe vectors allows one to select clones, containing hybrid plasmids with an active promoter for gene aph expression.     

Shuttle cloning vectors for the cyanobacterium Anacystis nidulans.

Hybrid plasmids capable of acting as shuttle cloning vectors in Escherichia coli and the cyanobacterium Anacystis nidulans R2 were constructed by in vitro ligation. DNA from the small endogenous plasmid of A. nidulans was combined with two E. coli vectors, pBR325 and pDPL13, to create vectors containing either two selectable antibiotic resistance markers or a single marker linked to a flexible multisite polylinker. Nonessential DNA was deleted from the polylinker containing plasmid pPLAN B2 to produce a small shuttle vector carrying part of the polylinker (pCB4). The two polylinker-containing shuttle vectors, pPLAN B2 and pCB4, transform both E. coli and A. nidulans efficiently and provide seven and five unique restriction enzyme sites, respectively, for the insertion of a variety of DNA fragments. The hybrid plasmid derived from pBR325 (pECAN1) also transforms both E. coli and A. nidulans, although at a lower frequency, and contains two unique restriction enzyme sites.     

The HgaI restriction-modification system contains two cytosine methylase genes responsible for modification of different DNA strands.

A DNA fragment of about 3.4 kilobase pairs that expressed the HgaI modification activity was cloned from the chromosomal DNA of Haemophilus gallinarum, and its nucleotide sequence was determined. Two open reading frames (ORF) which could code for structurally similar proteins were identified in the upstream and middle regions and a truncated ORF in the downstream region in the same orientation. When the respective ORFs were separately cloned, the clones carrying the upstream and middle ORFs both expressed the modification activity, indicating that the two genes are involved in modification of the HgaI restriction-modification system. In order to determine the sites of modification precisely, the respective genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the HgaI recognition site was treated with each of these enzymes, and, after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to HgaI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by each gene product. Analysis of the species and positions of modified bases by the Maxam-Gilbert method further demonstrated that the gene products from the upstream and middle ORFs participated in methylation of the internal cytosine residues of the strands carrying 3'-CTGCG-5' and 5'-GACGC-3', respectively. We concluded that the HgaI modification system consisted of two cytosine methylase genes responsible for modification of different strands in the target DNA.