Vectors containing infrequently cleaved restriction sites for use in BAL 31 nuclease-assisted and end-label-mediated analysis of cloned DNA fragments

Cloning vectors derived from plasmids pUC8 and pUC18 and phage M13mp10 were constructed so as to have multiple cloning sites (MCS) flanked by the recognition/cleavage sites for the Sfi I and Not I restriction nucleases. Cleavage of vectors containing cloned DNA fragments with either of the infrequently cleaving Sfi I or Not I endonucleases will usually yield linear DNAs cleaved only at the corresponding site in the MCS, so that the cloned insert can be degraded unidirectionally by the duplex exonuclease activity of the BAL 31 nucleases until an amount equal to the length of the vector has been degraded. The ends of the above constructs resulting from cleavage with Not I or Sfi I can readily be labeled, with labeling at only the terminus of the cloned DNA available for the Sfi I site. The BAL 31 nuclease-mediated procedures enhance a previous technique for mapping of restriction enzyme fragments, allow for localization of sequences in cloned segments for which a probe is available, and improve a method for sequencing cloned inserts through the production of sets of nested unidirectional deletions from either end of the parent cloned fragment. The advantages of end-label-mediated restriction site mapping using the above vectors over existing such procedures are also demonstrated.     

Cloning and characterization of the termination site tI for the gene int transcript in phage lambda

A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed. In one of these vectors a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM). The drug-resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with either various sticky ends or blunt ends. These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites. Insertion mutants are selected by their resistance to kanamycin. When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated. In addition, two new MCSs having single restriction sites have been formed by altering the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter. The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.     

Sse8647I, a new type II restriction endonuclease from a Streptomyces species cutting at 5'-AG/GWCCT-3'.

We isolated and characterized a new type II restriction endonuclease which recognizes the palindromic heptanucleotide sequence 5'-AGGWCCT-3' and cleaves double-stranded DNA after the first G in the sequence from a microorganism belonging to Streptomyces species. This enzyme cleaves adenovirus 2 DNA at eight sites, but does not cleave lambda phage, pBR322, pUC18 and 19, M13mp18 and 19, SV40, ColE1 and phi X174 DNAs.     

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.     

Construction of two stable bifunctional plasmids for Streptomyces spp. and Escherichia coli

Two bifunctional plasmid vectors pZG5 (7.45 kb) and pZG6 (6.95 kb), for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of the multicopy broad-host-range Streptomyces plasmid pIJ350 with E. coli plasmids Bluescribe M13- (pZG5) or pUC18 (pZG6). Both plasmids possess several unique restriction sites suitable for DNA cloning. Stable transformants of Streptomyces rimosus R6 and S. lividans 66 were obtained, harboring intact plasmids regardless of colony age or multiple subculturing. Moreover, pZG5 and pZG6 were successfully used to introduce several homologous transfer RNA genes into S. rimosus.     

Sse8387I, a new type-II restriction endonuclease that recognizes the octanucleotide sequence 5'-CCTGCAGG-3'.

A type II restriction endonuclease designated Sse8387I was partially purified from Streptomyces sp. 8387. This enzyme cleaved adenovirus 2 DNA at three sites, lambda phage DNA at five sites, and pUC18 and M13mp18 RF DNA at one site each, but did not cleave the DNAs from pBR322, SV40, or phi X174. Sse8387I recognized the octanucleotide sequence 5'-CCTGCA decreases GG-3', cleaving where shown by the arrow. Sse8387I is the first restriction endonuclease to be reported that recognizes an octanucleotide sequence consisting of all four nucleotides, G, A, T, and C. The frequency of occurrence of Sse8387I sites within sequenced regions of primate genomes was 2.4 times that of NotI sites.     

Construction of pRES18 and pRES19, Streptomyces-Escherichia coli shuttle vectors carrying multiple cloning sites

Abstract We developed two Streptomyces-Escherichia coli shuttle vectors. The plasmid pRES102, consisting of the essential region of pRES1 and the thiostrepton resistance gene ( tsr ) fragment of pIJ702, was combined with the E. coli plasmid vector pUC18 or pUC19. The resulting shuttle vectors, designated pRES18 and pRES19, respectively, have relatively compact size (6.25 kb), low copy number, multiple cloning sites reciprocally arranged in opposite directions, and selection markers for both Streptomyces ( tsr ) and E. coli (β-lactamase ( bla ) and β-galactosidase ( lacZ )). These shuttle vectors are capable of carrying DNA fragments as long as 10 kb, of being maintained in S. griseus, S. lavendulae and S. lividans , and are compatible with pIJ702.     

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.     

Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives.

A family of cloning vectors derived from plasmid pACYC184 and, therefore, compatible with pBR322 and its derivatives (especially the pUC family of vectors), is described. They all contain a multiple cloning site (MCS) and the lacZ alpha reporter gene for easy cloning. They have been grouped in three sets: (i) six of the vectors contain a chloramphenicol-resistance (CmR)-encoding gene and each a different MCS with 16 unique restriction sites overall; (ii) another six vectors contain a kanamycin-resistance (KmR)-encoding gene and the same six MCS; and (iii) two CmR vectors that contain the SP6 and T7 promoters flanking the MCS and lacZ alpha reporter gene of pUC18/19.     

Insertional restoration of beta-galactosidase alpha-complementation (white-to-blue colony screening) facilitates assembly of synthetic genes

The assembly of synthetic genes from oligodeoxynucleotides can be an inefficient process. Upon ligation of a synthetic assembly into a plasmid vector and transformation of an Escherichia coli host, it is often found that only a minor fraction of the putative recombinant plasmids contains synthetic sequences. Moreover, the synthetic sequences cloned are often altered versions of those originally designed. We have designed a biological test to detect those plasmids that contain synthetic sequences of the proper length, termini and reading frame. The test is the reversal of the beta-galactosidase alpha-complementation (blue-to-white) test used to detect the insertion of DNA segments into the polylinker sequences of the phage M13 mp, plasmids pUC, and related vectors. We begin with a modified vector defective in alpha-complementation and use insertion of the synthetic DNA segment to restore alpha-complementation. The alpha-complementation activity of the original vector (e.g., pUC18) was first abolished by a frameshift or DNA insertion within the polylinker sequence of the lacZ' gene segment. The alpha-complementation was then restored by insertion of the synthetic DNA sequence between the cohesive ends generated by digestion of two polylinker restriction sites. Formation of blue colonies requires the insertion of a DNA segment of appropriate length and termini to reconstruct the lacZ' open reading frame and thus is much more selective than the usual insertional inactivation strategy. We show that this 'insertional restoration' screening method markedly enhances the proper assembly of synthetic genes and describe manipulations to readily and reliably frameshift various polylinker sequences.     

New versatile cloning and sequencing vectors based on bacteriophage M13

A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed. These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors [cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276], unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI. A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors.     

A vector for directional cloning and expression of polymerase chain reaction products in Escherichia coli

This paper describes the construction of a modified vector for the cloning and expression of protein-encoding genes in Escherichia coli. The vector, pfXblue, is derived from the system originally developed by Nagai and Th?gerson [Nature 309 (1984) 810-812], but contains a modified multiple cloning site (MCS) from M13mp18 to allow directional insertion of foreign coding sequences. The MCS is located within the M13mp18 lacZ' gene and thus allows blue/white screening of colonies for inserts. The inserted gene is expressed as a fusion protein, which, when cleaved by the coagulation factor Xa protease, yields the mature product. This vector was successfully used for the production of a mitochondrial [2Fe-2S]ferredoxin using polymerase chain reaction products generated from a chick kidney cDNA library.     

基于XcmⅠ酶切的高效TA克隆载体的构建

目的：制备基于XcmⅠ酶切的高效TA克隆载体,并检测其克隆PCR产物的效果。方法：设计一对互补配对的寡核苷酸,经过变性及退火后插入质粒pUC19的多克隆位点,从而在该多克隆位点中引入2个XcmⅠ酶切位点,用XcmⅠ酶切后即获得含有3’突出T碱基的T载体;为了提高该T载体的克隆效率,优化了2个XcmⅠ酶切位点之间的碱基数目,排除了载体自连产生白色克隆的可能性,使假阳性大大减少;此外,为了便于完全酶切与未完全酶切载体的分离,在2个XcmⅠ之间插入了一段无关DNA片段。结果：改进得到的T载体可以有效克隆PCR产物,其阳性克隆率可达95％。结论：构建了基于XcmⅠ酶切的TA克隆载体,经过改进的T载体具有很高的克隆效率。     

A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments

The strategy of shotgun cloning with M13 is based on obtaining random fragments used for the rapid accumulation of sequence data. A strategy, however, is sometimes needed for obtaining subcloned sequences preferentially out of a mixture of fragments. Shotgun sequencing experiments have shown that not all DNA fragments are obtained with the same frequency and that the redundant information increases during the last third of a sequencing project. In addition, experiments have shown that particular fragments are obtained more frequently in one orientation, allowing the use of only one of the two DNA strands as a template for M13 shotgun sequencing. Two new M13 vectors, M13mp8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations. Consequently, each of the two strands becomes a (+) strand in a pair of vectors. The fragments to be cloned are cleaved with two restriction enzymes to produce a fragment with two different ends. The insertion of such a fragment into the vector can occur only in one orientation. Since M13mp8 and M13mp9 have their array of cloning sites in an antiparallel order, either orientation for inserting a double-digest fragment can be selected by the choice of the vector.     

A simple system of cloning in phage M13 and DNA sequencing with terminators

A compilation of techniques for DNA cloning in filamentous phage M13 based vectors for a novice in cloning is presented. It does not require either specialized microbiological facilities, or any specific knowledge in Escherichia coli genetics. The cloning strategy uses only blunt-end ligation into a vector that has been prepared once for several hundred experiments. The first part describes the isolation, preparation and checking of a blunt-ended M13 vector (with M13 mp series vectors as an example), and also the isolation of clonable fragments, transformation of competent cells and preliminary analysis of recombinants. The second part describes procedures and equipment, which enable to sequence recombinant M13 clones by the chain termination procedure of Sanger et al. It includes simplified procedures for the preparation of sequencing gels, and the rules of interpretation of the sequencing ladders. Reference material is added, which includes trouble-shooting guide, E. coli K12 strain list and polylinker sequences for use of mp-series vectors as well as a fully documented cloning and sequencing experiment.     

The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing

A series of nic- cloning vectors have been constructed analogous to the pUC plasmids but which are smaller in size and carry more extensive polylinker regions within the lacZ' gene. The vectors pMTL20 and pMTL21 carry six additional sites (AatII, MluI, NcoI, BglII, XhoI and StuI) to those present in pUC18 and pUC19, while pMTL22 and -23 possess eleven new cloning sites (ActII, MluI, NcoI, BglII, XhoI, StuI, NaeI, EcoRV, ClaI, NdeI and NruI). More importantly, the relative order of the restriction sites within the polylinker of these latter vectors has been totally rearranged, relative to pUC18 and pUC19, to facilitate the conversion of DNA fragments with incompatible ends to fragments with compatible termini. The availability of such DNA fragments is a crucial requirement when M13 templates are generated for dideoxy sequencing by the sonication procedure. Derivatives of these vectors have also been constructed which demonstrate improved segregational stability by incorporation of the pSC101 par locus. During the construction of these new vectors data were obtained which demonstrated that the pUC and pMTL plasmids contain a previously unreported single base pair difference within the RNA I/RNA II region (compared to pBR322) responsible for a three-fold increase in plasmid copy number. The pUC and pMTL plasmids were also shown to be functionally nic-, thus affording the lowest categorisation in genetic manipulation experiments.     

Construction of mobilizable vectors derived from plasmids RP4, pUC18 and pUC19

Mobilizable narrow-host-range plasmids were constructed from pUC18 and pUC19 by addition of a segment of pSUP2021 bearing the basis of mobilization (bom) site and origin of transfer (oriT) of RP4. One pair of expression vectors, pARO180 and pARO190, retains the beta-lactamase (bla) gene and twelve of the 13 restriction enzyme multiple cloning sites (MCS) of pUC18/19. Another pair was created by replacing the bla gene with the gene encoding kanamycin resistance (kan) from Tn5. The molecules replicate to high copy number in Escherichia coli and Enterobacter aerogenes. They can be transferred efficiently to other Gram- bacteria from the mobilizing strain, E. coli S17-1. In non-enteric strains, the new plasmids can be used as suicide vectors in site-specific insertional mutagenesis.     

An improved method for the detection of Dcm methylation in DNA molecules

The intrinsic insensitivity of EcoRII recognition sites in RF DNAs of phage M13 and vector M13mp18 towards this restriction endonuclease can be overcome by adding site-specific oligodeoxyribonucleotide duplexes to the restriction sample. Since Dcm- DNA but not Dcm(+)-methylated DNA becomes susceptible under these conditions, this procedure constitutes an improvement of the Dcm methylation assay.     

Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria

Improved broad-host-range plasmid vectors were constructed based on existing plasmids RSF1010 and RK404. The new plasmids pDSK509, pDSK519, and pRK415, have several additional cloning sites and improved antibiotic-resistance genes which facilitate subcloning and mobilization into various Gram-negative bacteria. Several new polylinker sites were added to the Escherichia coli plasmids pUC118 and pUC119, resulting in the new plasmids, pUC128 and pUC129. These plasmids facilitate the transfer of cloned DNA fragments to the broad-host-range vectors. Finally, the broad-host-range cosmid cloning vector pLAFR3 was improved by the addition of a double cos casette to generate the new plasmid, pLAFR5. This latter cosmid simplifies vector preparation and has permitted the rapid cloning of genomic DNA fragments generated with Sau3A. The resulting clones may be introduced into other Gram-negative bacteria by conjugation.