Novel narrow-host-range vectors for direct cloning of foreign DNA inPseudomonas

Narrow-host-range vectors, based on an indigenous replicon and containing a multiple cloning site, have been constructed in aPseudomonas host capable of growth on unusual substrates. The new cloning vectors yield sufficient amounts of DNA for preparative purposes and belong to an incompatibility group different from that of the incP and incQ broad-host-range vectors. One of these vectors, named pDB47F, was used to clone, directly inPseudomonas, DNA fragments fromAgrobacterium, Pseudomonas, andRhizobium. A clone containingAgrobacterium and Km^R gene sequences was transformed with a higher efficiency than an RSF1010-derived vector (by as much as 1250-fold) in four out of fivePseudomonas strains tested. The considerable efficiency obtained with this system makes possible the direct cloning and phenotypic selection of foreign DNA inPseudomonas.     

Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter.

Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, have been constructed that carry four different single restriction sites down-stream from the phage lambda promoter pL. The promoting activity of pL is switched off at low temperature in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction. cIts was located either on the host chromosome, or on a second plasmid pRK248 that is compatible with the cloning vehicle, or on the vehicle itself. Three different restriction fragments, each carrying the gene trpA of Salmonella typhimurium or Shigella dysenteriae, have been inserted into the EcoRI, BamHI and SalI sites, respectively, of these plasmids and pL dependent expression of the inserted gene in Escherichia coli was determined by measuring the enzymatic activity of the trpA gene product. Heat induction resulted in a level of expression of trpA corresponding to 1 to 6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.     

Molecular cloning of DNA sequences coding for mouse embryonic globins

Yolk sac derived erythroid cells in mouse embryos synthesize four embryonic globins of which two are alpha-like and two are beta-like. Pure globin messenger RNAs from these cells were used as templates for two successive polymerizing reactions and a mixture of double stranded cDNAs coding for the four globins was obtained. These molecules were blunt-end ligated to an ECoR1 digested pBR322 plasmid and the recombinant plasmids were used to transform E. coli Hb101. Bacterial clones which proved positive upon hybridization with 32P-labelled embryonic globin cDNA were amplified and their plasmid DNA was isolated. Three different plasmids were studied, namely no. 2, 16 and 54. The restriction map of these plasmids showed that: 1) plasmid no. 2 and 54 had lost extensive DNA sequences comprising the genes responsible for tetracycline resistance; 2) the size of inserted sequences ranges from 427 base pairs of plasmid no. 16 to about 280 base pairs of plasmid no. 54; 3) plasmid no. 2 does not share any of the studied restriction sites with the other plasmids, while no. 2 and 54 have at least one site in common. The coding properties of inserted DNA were determined by positive hybrid translation showing that no. 2 codes for the alpha-like embryonic chain x, while no. 16 and 54 code for a beta-like embryonic chain, either y or z.     

PCR preparation of DNA inserts from lambda and plasmid vectors for RFLP mapping

A simplified method is described for preparing insert DNA for labelling reactions to be used in Southern hybridization. This method works with sequences cloned into both plasmid and lambda phage, and eliminates many of the steps leading to the labelling reaction. Small quantities of hostE. coli or lambda phage carrying a probe sequence are lysed and amplified via the polymerase chain reaction using standard sequencing primers. Unincorporated nucleotides are removed by ethanol precipitation or gel purification and insert DNA is ready for radio-labelling. This method reduces the time and expense associated with conventional insert preparation, and greatly simplifies the use of sequences cloned into lambda phage.     

GC level and expression of human coding sequences

Several groups have addressed the issue of the influence of GC on expression levels in mammalian genes. In general, GC-rich genes appeared to be more expressed than GC-poor ones. Recently, expression levels of GC₃-rich and GC₃-poor versions of genes (GC₃ is the third codon position GC), inserted in vector plasmids, were compared in order to eliminate differences associated with their genomic context. Transfection experiments showed that GC₃-rich genes were expressed more efficiently than their GC₃-poor counterparts, indicating that GC₃ dramatically and intrinsically boosts expression efficiency. Here we show that, while the protocols used eliminated the original genomic context, they replaced it with the plasmid contexts whose compositional properties affected the results.     

人CD46启动子真核表达载体的构建

为了构建人CD46（hCD46）启动子指导目的基因表达的真核表达载体,提取HeLa细胞基因组DNA,用PCR扩增出hCD46基因的启动子区域,序列分析结果表明其与GenBank中hCD46基因5’端某片段的同源性为99.9％。用此启动子替换pcDNA3EGFP中的CMV启动子,并在hCD46启动子和EGFP基因之间插入兔β-球蛋白基因第二内含子（RGI）,得到的重组表达载体转染CHO和SP2/0两种鼠源细胞,流式细胞术检测表明CHO细胞EGFP的表达量高于SP2/0细胞,表达特性与人体CD46相似;RGI可以增强EGFP的表达量,但不改变其表达的组织特异性,提示克隆的hCD46启动子可以用于研制模拟人体CD46基因表达特性的转基因小鼠。     

Construction of direct selection vectors for use inBacillus sphaericus

Genetic manipulation in the mosquito larvicidal organismBacillus sphaericus has been hampered by a lack of effective molecular biological techniques and tools. Currently available vectors forBacillus subtilis are inappropriate as they are selected with antibiotics to whichB. sphaericus is naturally resistant. Construction of chimeras between theStaphylococcus aureus plasmid pE194 and theB. subtilis direct selection vector pBD214 permitted selection of plasmid transformants with erythromycin. As with pBD214, direct selection of recombinants is accomplished by insertional inactivation of the plasmid-borne thymidylate synthetase gene and subsequent selection with trimethoprim.     

Construction of cloning vectors for Bacillus thuringiensis.

The replication region of the Bacillus thuringiensis plasmid, pHT1030, was treated with hydroxylamine. Various copy-number mutants were selected and subsequently used to construct shuttle vectors with multiple cloning sites. These recombinant plasmids are very stable and allowed the cloning of a delta-endotoxin-encoding gene in B. thuringiensis. Comparison between gene expression level and vector copy-number indicated that a plateau in delta-endotoxin production is reached with a copy-number of about fifteen per equivalent chromosome.     

New vectors for high level expression of recombinant proteins in bacteria.

A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.     

Construction of shuttle cloning vectors for Bacteroides fragilis and use in assaying foreign tetracycline resistance gene expression

Shuttle vectors capable of replication in both Escherichia coli and Bacteroides fragilis have been developed. Conjugal transfer of these plasmids from E. coli to B. fragilis is facilitated by inclusion of the origin of transfer of the IncP plasmid RK2. The vectors pDK1 and pDK2 provide unique sites for cloning selectable markers in Bacteroides. pOA10 is a cosmid vector containing the replication region of pCP1 necessary for maintenance in Bacteroides. pDK3, pDK4.1, and pDK4.2 contain the Bacteroides clindamycin resistance gene allowing selection and maintenance in B. fragilis of plasmids containing inserted DNA fragments. pDK3 was used to test the expression in B. fragilis of five foreign tetracycline resistance (TcR) genes. The tetA, -B, and -C markers from facultative gram-negative bacteria, as well as a TcR determinant from Clostridium perfringens, did not express TcR in B. fragilis. The tetM gene, originally described in streptococci, encoded a small but reproducible increase of TcR in Bacteroides. These studies demonstrate the utility of shuttle vectors for introducing cloned genes into Bacteroides and underscore the differences in gene expression in these anaerobes.     

Construction of broad-host-range vectors for general cloning and promoter selection in Pseudomonas and Escherichia coli

We have constructed two promoter-selection vectors based upon the broad-host-range plasmid pRO1614. pQF40 (6 kb) contains a promoterless tetA gene downstream from a large multiple cloning site while pQF26 (5.4 kb) possesses a promoterless cat cartridge. The latter vector displayed a copy number of 13 in Pseudomonas aeruginosa and 39 in Escherichia coli. When promoter sequences derived from the Pseudomonas phage phi PLS27 were cloned into pQF26, high levels of chloramphenicol-acetyltransferase were detected in P. aeruginosa. In E. coli the activity was approximately one-third that in P. aeruginosa when corrections were made for the plasmid copy number.