High efficiency vectors for cosmid microcloning and genomic analysis

We describe the construction and use of cosmid vectors designed for microcloning, gene isolation and genomic mapping starting from submicrogram amounts of eukaryotic DNA. These vectors contain (1) multiple cos sites to allow for simple and efficient cloning using non size-selected DNA; (2) bacteriophage T3 and T7 promoter sequences flanking the cloning site to allow for the synthesis of end-specific probes for chromosome walking; (3) a selectable gene for immediate gene transfer of cosmid DNA into mammalian cells; (4) recognition sequences for specific oligodeoxyribonucleotides to allow rapid restriction mapping; (5) unique NotI, SacII or SfiI sites flanking the cloning site to allow for removal of the cloned DNA insert from the vector. These cosmid vectors allow the construction of high quality genomic libraries in situations where the quantity of purified DNA is extremely limited, such as when using DNA prepared from purified mammalian chromosomes isolated by fluorescence-activated cell sorting.     

Functional cloning vectors for use in directional cDNA cloning using cohesive ends produced with T4 DNA polymerase.

This paper describes the construction of 'Prime' cloning vectors, which include phage lambda and plasmid vectors useful for functional cloning in oocytes, yeast, and mammalian cells, and their use in a 'Prime' cloning system. The system takes advantage of the very active and precise 3' exonuclease activity of T4 DNA polymerase to produce single-stranded (ss) ends (cut-back) of vector and insert DNA. This results in the highly efficient directional cloning of cDNA and PCR-amplified DNA. The system obviates the need to digest insert DNA with a restriction endonuclease to unveil cloning sites, and thus eliminates the chance of internal digestion of the insert DNA. The cloning of PCR-amplified DNA, which is sometimes difficult, is made routine with this system. The 'Prime' sequence is included in vector cloning sites and cDNA and PCR primers. The 'Prime' sequence was chosen so that the ss sticky ends are nonpalindromic and will hybridize only to the appropriate partners. This makes cloning with the 'Prime' system very efficient, because neither the vector nor insert DNA is lost to unproductive self-hybridization.     

A phagemid vector library for cloning DNA with four-nucleotide 5' or 3' overhangs

A phagemid vector library for cloning DNA with four nucleotide 5' or 3' overhangs has been constructed. This library is based on the pT7T3 vector (Pharmacia) which is a modification of the phagemid pTZ18U vector. We have chosen pT7T3 as the parent vector because it can be used for Sanger's dideoxy sequencing and for the generation of RNA probes with either the T7 or T3 promoter. Each member of the cloning vector series pBM has recognition sites for both of the restriction enzymes BspM1 and BstX1 in addition to the basic multiple cloning sites. BspM1 recognizes the sequence 5'...ACCTGC NNNN/NNNN...3' whereas BstX1 recognizes the sequence 5'...CCAN NNNN/NTGG...3'. Thus these two sites can be overlapped, so that only 256 vectors (instead of 512 vectors) need be constructed to cover all the theoretical possible combinations of sites which give complementary cohesive ends for cloning DNA with four nucleotide 5' or 3' overhangs. This vector library can be used for amplification cloning of DNA in a tandem array by choosing appropriate vectors which have nonpalindromic sequences. We have obtained approximately 200 members of the 256 possible clones and have organized the vectors using a MacIntosh HyperCard program for easy retrieval.     

3' RACE walking along a large cDNA employing tiered suppression PCR

Large genes present particular cloning difficulties, especially when expressed at relatively low levels. We describe a novel method, termed 3' rapid amplification of cDNA ends (RACE) walking, for the rapid determination of unknown 3' flanking sequence of a large cDNA. The technique is a derivative of the anchored PCR 5' RACE procedure but includes a specific and limited second-strand cDNA synthesis and a tiered "panhandle" suppression of nonspecific products. The method generated 900 bp of new sequence for the large tammar wallaby ATRY gene in two easy steps, in which standard 3' RACE and PCR-based cDNA library walking proved unsuccessful. This robust approach represents a new tool for isolating unknown sequence under challenging cloning scenarios such as poor library representation, long coding regions, long 3' untranslated regions, and difficult template regions.     

A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins

Recombinant immunotoxins (rITs) are highly specific anti-tumor agents composed of monoclonal antibody fragments or other specific carriers coupled to plant or bacterial toxins. A major problem in the purification of rITs is the low periplasmic yield in currently available expression systems. Thus, the aim of this study was the development of a new bacterial expression system for high-level production of rITs. We constructed a series of pET-based vectors for pelB-directed periplasmic secretion or cytoplasmic production under the control of the T7lac promoter. Expression in Escherichia coli BL21(DE3)pLysS allowed a tightly regulated isopropyl beta-d-thiogalactopyranoside (IPTG) induction of protein synthesis. An enterokinase-cleavable poly-histidine cluster was introduced into this setup for purification by affinity chromatography. A major modification resulted from the insertion of a specifically designed multiple cloning site. It contains only rare restriction enzyme recognition sites used for cloning of immunoglobulin variable region genes, as well as unique SfiI and NotI restriction sites for directed insertion of single-chain variable fragments (scFv) available from established bacteriophage systems. For this purpose, we deleted two naturally occurring internal SfiI consensus sites in a deletion mutant of Pseudomonas aeruginosa exotoxin A (ETA'). Each single structural element of the new vector (promoter, leader sequence, purification tag, scFv sequence, selectable marker, and toxin gene) was flanked by unique restriction sites allowing simple directional substitution. The fidelity of IPTG induction and high-level expression were demonstrated using an anti-CD30 scFv (Ki-4) fused to ETA'. These data confirm a bacterial vector system especially designed for efficient periplasmic expression of ETA'-based fusion toxins.     

LoxP-directed cloning: use of Cre recombinase as a universal restriction enzyme.

We have developed a novel way to use the Cre/loxP system for in vitro manipulation of DNA and a technique to clone DNA into circular episomes. The method is fast, reliable, and allowsflexible cloning of DNA fragments into episomes containing a loxP site. We show that a loxP site can serve as a universal target site to clone a DNA fragment digested with any restriction enzyme(s). This technique abolishes the need for compatible restriction sites in cloning vectors and targets by generating custom-designed 5' 3', or blunt ends in the desired orientation and reading frame in the vector Therefore, this method eliminates the limitations encountered when DNA fragments are cloned into vectors with a confined number of cloning sites. The 34-bp loxP sequence assures uniqueness, even when large episomes are manipulated. We present three examples, including the manipulation of a bacterial artificial chromosome. Because DNA manipulation takes place at a loxP site, we refer to this technique as loxP-directed cloning.     

Mulcos: a vector for amplification and simultaneous expression of two foreign genes in mammalian cells

A method was developed for amplification and expression of foreign genes in mammalian cells. This procedure exploits the fact that an SfiI cleavage site, GGCCGCCT/CGGCC (the recognition sequences are underlined), is present at the SV40 replication origin and the cleaved ends, CCT-3' and AGG-3', are not rotationally equivalent. Thus DNA fragments flanked by the SfiI sites can be ligated in head-to-tail tandem arrays and cloned in cosmids; the resulting construct is called a mulcos. The cosmid vector we have used, pCHD2L, contains the single SfiI site as well as HmBR and dhfr genes, selectable markers in mammalian cells. Cassette plasmid pmoRH contains two expression units, each of which consists of SV40 early promoter, EcoRI or HindIII cloning site, small T splicing region, and poly(A) signal, and the two units as a whole are flanked by the SfiI sites. A set of alpha- and beta-chain cDNAs of a human major histocompatibility class-II antigen were inserted into the EcoRI and HindIII sites, respectively. The purified SfiI fragment, containing both expression units, was then ligated with SfiI-linearized cosmid vector pCHD2L at a molar ratio of 20:1. A mulcos containing eight pairs of the alpha- and beta-chain expression units was isolated by in vitro packaging in phage lambda heads and subsequent transfection into Escherichia coli. Drug-selected cells transfected with the mulcos contained significantly higher copy numbers of the expression units and higher expression levels than those obtained using conventional plasmids. More than 85% of these cells expressed class-II antigen on their cell surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)     

负链不分节段RNA病毒反向遗传系统中通用型真核表达载体的构建

本研究选择pVAX1作为供体载体,通过分子克隆方法,分别用人工合成的锤头型核酶、丁型肝炎核酶序列(用于获得转录后病毒基因组RNA的精确末端)和含有9个常用限制性酶切位点的linker序列(用于病毒基因组插入的多克隆酶切位点)取代真核表达载体pVAX1的多克隆酶切位点,将其改造成负链不分节段RNA病毒反向遗传系统的通用型表达载体。通过酶切鉴定和序列测定表明,pVAX1载体中插入的核酶及linker序列正确无误,并将CTN株狂犬病病毒全长基因组cDNA插入pVAX-R中,通过与辅助质粒的共转染成功拯救CTN株狂犬病毒,证明通用型真核表达载体构建成功,为快速建立负链不分节段RNA病毒反向遗传系统奠定基础。     

Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.

Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.     

Cloning and characterization of the O-methyltransferase I gene (dmtA) from Aspergillus parasiticus associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin in aflatoxin biosynthesis.

O-Methyltransferase I catalyzes both the conversion of demethylsterigmatocystin to sterigmatocystin and the conversion of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis. In this study, both genomic cloning and cDNA cloning of the gene encoding O-methyltransferase I were accomplished by using PCR strategies, such as conventional PCR based on the N-terminal amino acid sequence of the purified enzyme, 5' and 3' rapid amplification of cDNA ends PCR, and thermal asymmetric interlaced PCR (TAIL-PCR), and genes were sequenced by using Aspergillus parasiticus NIAH-26. A comparison of the genomic sequences with the cDNA of the dmtA region revealed that the coding region is interrupted by three short introns. The cDNA of the dmtA gene is 1,373 bp long and encodes a 386-amino-acid protein with a deduced molecular weight of 43,023, which is consistent with the molecular weight of the protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal half of the deduced protein exhibits 76.3% identity with the coding region of the Aspergillus nidulans StcP protein, whereas the N-terminal half of dmtA exhibits 73.0% identity with the 5' flanking region of the stcP gene, suggesting that translation of the stcP gene may start at a site upstream from methionine that is different from the site that has been suggested previously. Also, an examination of the 5' and 3' flanking regions of the dmtA gene in which TAIL-PCR was used demonstrated that the dmtA gene is located in the aflatoxin biosynthesis cluster between (and in the same orientation as) the omtA and ord-2 genes. Northern blotting revealed that expression of the dmtA gene is influenced by both medium composition and culture temperature and that the pattern correlates with the patterns observed for other genes in the aflatoxin gene cluster. Furthermore, Southern blotting and PCR analyses of the dmtA gene showed that a dmtA homolog is present in Aspergillus oryzae SYS-2.     

Restriction digestion monitors facilitate plasmid construction and PCR cloning

Plasmid construction by "forced" or "directional" ligation of fragments digested with two different restriction enzymes is highly efficient, except when inhibited digestion of one site favors vector recircularization. Such failures often result because incomplete double digestion is undetected in vector polylinkers or at terminal cloning sites on a PCR fragment. To test cleavage efficiency indirectly, a "monitor" plasmid is added to the digest. In a suitable monitor, the two test sites are separated by enough DNA (approximately 20% of full length) to distinguish the double digest from the failed single digest. To make this applicable to combinations of 32 popular cloning enzymes, we constructed a set of 4 monitors (pDM1, pDM2, pDM3, and pDM4). Each contains three polylinkers separated by stuffer segments of approximately 1 kb. The 32 sites are distributed in the polylinkers such that at least one plasmid in the set is diagnostic for each enzyme pair. The set is designed to be extended to up to 81 sites. A linearized version of the monitor allows for the determination of which of the two enzymes has failed in an incomplete double digest and is also useful when the target DNA is close to the size of the pDM backbone. The plasmids also serve as versatile self-monitoring cloning vectors for any site combination.