Arabidopsis thaliana small subunit leader and transit peptide enhance the expression ofBacillus thuringiensis proteins in transgenic plants

The expression of the modified gene for a truncated form of thecryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein fromBacillus thuringiensis var.kurstaki (B.t.k.) HD73, under control of theArabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunitats1A promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants. Examination of leaf tissue revealed that theats1A promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10-fold to 20-fold increase incryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-En35S) was used to express the samecryIA(c) gene. Transient expression assays in tobacco protoplasts and the whole plant results support the conclusion that the transit peptide plus untranslated sequences upstream of that region are both required for the increase in expression of the CryIA(c) protein. Furthermore, the CaMV-En35S promoter can be used with theArabidopsis ats1A untranslated leader and transit peptide to increase expression of this protein. While subcellular fractionation revealed that the truncated CryIA(c) protein fused to theats1A transit peptide is located in the chloroplast, the increase in gene expression is independent of targeting of the CryIA(c) protein to the chloroplast. The results reported here provide new insight into the role of 5 untranslated leader sequences and translational fusions to increase heterologous gene expression, and they demonstrate the utility of this approach in the development of insect-resistant crops.     

The cya gene region of Erwinia chrysanthemi B374: organisation and gene products

Summary A DNA fragment containing the cya gene region of Erwinia chrysanthemi, B374 was cloned in vivo and transferred into cells of E. coli using a plasmid pULB113 derived from RP4 followed by subcloning in vitro into the vector pBR322. The cya gene encodes a 95 kDal protein that complemented E. coli cya mutants. Apparently, cya genes truncated at the 3 end could still produce proteins complementing cya-defective strains, thus showing that adenylate cyclase truncated at its carboxy-terminal end could synthesise cAMP. A protein of unknown function (40 kDal) is encoded by a gene that is transcribed divergently from the control region of the adenylate cyclase gene.     

A DNA sequence from Dictyostelium discoideum complements ura3 and ura5 mutations of Saccharomyces cerevisiae

Summary A 3.7 kilobase fragment of Dictyostelium discoideum genomic DNA has been cloned by its ability to complement a yeast ura5 mutation affecting the activity of orotidine-5-phosphate carboxy-lyase (EC 4.1.1.23). This fragment also complements a yeast ura5 mutation that leads to a defect in orotate phosphoribosyl transferase (EC 2.4.2.10). The orotidine-5-phosphate carboxy-lyase and the orotate phosphoribosyl transferase activities that result from Dictyostelium gene expression in yeast have been detected. The size of the DNA required for both complementations has been localised to a segment of less than 2 kb. A unique Dictyostelium RNA species of 1,600 base pairs hybridises to this fragment. In vitro deletions in this fragment lead to the simultaneous loss of the two activities. The two enzymatic activities coelute as a protein of 120.000 daltons during gel filtration of a Dictyostelium extract. These results favour the existence, on the cloned Dictyostelium DNA fragment, of a unique structural gene which codes for a bifunctional enzyme carrying the two activities, orotidine-5-phosphate carboxy-lyase and orotate phosphoribosyl transferase.Abbreviations bp basepair - kb kilobasepair - MOPS Morpholino propane sulfonic acid     

A DNA Region That Complements on Escherichia coli cysG Mutation in Thiobacillus Ferrooxidans

Thiobacillus ferrooxidans AP19-3 has a novel NADH-dependent sulfite reductase in the periplasmic space. The gene responsible for the appearance of NADH-dependent sulfite reductase activity was cloned into a vector plasmid pBR322 to give a 5.7-kb hybrid plasmid, pTHS1, which contains a 1.3-kb DNA fragment of T. ferrooxidans AP19-3. When pTHS1 was used to transform sulfite reductase deficient E. coli mutants, strain AT2455 (cysG), JM246 (cysl), and AT2427 (cysJ), it complemented only the E. coli cysG mutation. Since cysG codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase, the enzyme involved in siroheme synthesis, the results indicate that the DNA region that codes for S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase is present in a T. ferrooxidans 1.3 kb DNA fragment on pTHS1.     

Molecular cloning and characterisation of the gua regulatory region of Escherichia coli K12

Summary The regulatory region of the gua operon of Escherichia coli is contained within a 2.1 kb EcoR1 restriction fragment isolated from a pgua transducing phage. This DNA fragment was inserted into pPV33-H, a promoter-cloning vector, where it activated the gene(s) for tetracycline resistance. The level of tetracycline resistance conferred by the hybrid plasmid was reduced by the addition of guanine and increased by adenine, indicating the presence of the gua promoter. The cloned fragment codes for a polypeptide that complements in vivo the defective enzymes present in certain guaB mutants. This polypeptide was characterised using minicells and immunoprecipitation, and is presumed to correspond to the N-terminal region of IMP dehydrogenase.     

Isolation of specialized transducing bacteriophages carrying deletions of the regulatory region of the Escherichia coli K-12 tryptophan operon.

A lysogen of a wild-type strain of Escherichia coli K-12 carrying a heat-inducible lambda-phi80 hybrid prophage was induced to yield transducing phages carrying all of the structural genes of the tryptophan operon. The presence or absence of elements of the trp regulatory region was determined by examining the effects of lambda genes N and cI on trp gene expression. The phages were further characterized by transduction studies and by examining anthranilate synthetase (EC 4.1.3.27) (TRYPE+D) synthesis in the presence of the lambda cI product. A number of phages deleted for the trp promoter were found. Recombination studies between trpOc bacteria and the transducing phages have yielded information that can be used to order the trp end points of some phages and to provide an estimate of the size of the trp promoter region.     

Production of an Expression System for a Synaptobrevin Fragment to Monitor Cleavage by Botulinum Neurotoxin B

Recombinant DNA techniques were used to develop an expression system for a 51-amino acid peptide fragment that encompasses residues 44–94 of human synaptobrevin 2. This protein is associated with secretory vesicles of nerve terminals and is a substrate for four of the seven serotypes of botulinum neurotoxin (BoNT). The DNA for the recombinant peptide was amplified by the polymerase chain reaction and cloned into the pTrxFus vector. The resulting synaptobrevin peptide was expressed as a thioredoxin fusion protein in E. coli and released into the medium by osmotic lysis. The 18.7-kDa thioredoxin-synaptobrevin protein, designated as TSB-51, is intended for use in a cell-free assay to test potential inhibitors of BoNT/B-mediated proteolysis of synaptobrevin with the ultimate aim of developing clinically effective therapeutic agents to counteract botulism. Incubation of TSB-51 with the purified light chain of BoNT/B resulted in proteolysis which was evident within 30 min and increased with time until completion (4 hr). Cleavage of TSB-51 appeared to be at the appropriate BoNT/B cleavage site as indicated by a reduced intensity of the 18.7-kDa band and the appearance of a band at 16.4 kDa on Tris-tricene polyacrylamide gradient gels. The concentration of free Zn²⁺ had a significant effect on the cleavage rate; low Zn²⁺ concentrations stimulated substrate cleavage, whereas high concentrations were inhibitory. Cleavage was not significantly depressed by the naturally occurring metalloprotease inhibitor phosphoramidon when tested at concentrations up to 5 mM. TSB-51 appears to be a useful substrate for studying BoNT/B and is expected to aid in the discovery of effective BoNT inhibitors.     

Molecular cloning and characterization of the alkB gene of Escherichia coli

Summary Using methods of in vitro recombination we constructed hybrid plasmids that can suppress the increased methylmethane sulfonate sensitivity caused by alkB mutation. Since the cloned DNA fragment was mapped at 47 min on the Escherichia coli K12 genetic map, an area where the alkB gene is located, we concluded that the cloned DNA fragment contains the alkB gene itself but not other genes that suppress alkB mutation. Specific labeling of plasmid-encoded proteins by the maxicell method revealed that the alkB codes for a polypeptide with a molecular weight of about 27,000. Introduction of a small deletion into the alkB region of the bacterial chromosome resulted in inactivation of both the alkB and ada genes, thereby suggesting that the two genes are adjacent on the E. coli chromosome.Abbreviations Ap ampicillin - Cm chloramphenicol - HPLC high performance liquid chromatography - kb kilobases - kd kilodaltons - MMS methylmethane sulfonate - MNU methylnitrosourea - MNNG N-methyl-N-nitro-N-nitrosoguanidine - Tc tetracycline - SDS sodium dodecyl sulfate     

pUC12-STOP: An expression vector with portable translation stop signals

Summary A DNA linker with TAA translational stop codons in all three reading frames was inserted into the polylinker region of pUC12. The new plasmid pUC12-STOP is useful for the expression of DNA in cases where defined translational stops are desired. The STOP linker is flanked by unique restriction sites and thus can be excised as portable STOP linker fragments. The STOP linker was used to express in Escherichia coli a truncated form of the Herpes simplex virus type 1 glycoprotein D antigen.     

Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the expression of fully functional eukaryotic proteins inE. coli. Essential to the success of these procedures is the transport of such proteins across the inner membrane to the periplasmic space, allowing proper folding and the establishment of disulfide bonding. Subsequently, fully functional proteins can be exposed on the surface of filamentous (bacterio)phages, provided a system is employed that consists of a cloning vector (e.g. the phagemid pComb3, Barbas et al., 1991) that generates phage particles in the presence of a helper phage. The main advantage of surface display of recombinant proteins is to facilitate the screening of very large numbers of different molecules by simple selection methods (panning). In addition, periplasmic expression yields relatively large quantities (e.g. 1 mg l^–1 of culture) soluble protein. In this review, the principle aspects of this novel expression system based on the phagemid pComb3 will be discussed. Two examples for functional periplasmic expression of human proteins inE. coli will be presented, namely i) the antigen-binding moiety (Fab fragment) of human immunoglobulins (IgGs) and ii) the human plasminogen activator inhibitor 1, an essential regulator of the plasminogen activation system. Finally, perspectives for the application of this system to express mutant proteins, fragments of proteins and peptides are indicated.Abbreviations Ap^R ampicillin resistance - cfu colony forming unit(s) - cpIII gene III-encoded coat protein of M13 - cpVIII gene VIII-encoded coat protein of M13 - ER endoplasmic reticulum - Fab fragment of Ig containing light chain, variable region and first constant region of heavy chain - Fd variable region and first constant region of the heavy chain - Fv fragment containing variable regions of heavy and light chain - Ig immunoglobulin - Km^R kanamycin resistance - kb kilobase or 1000 basepairs - PAI-1 plasminogen activator inhibitor 1 - t-PA tissue-type plasminogen activator - u-PA urokinase-type plasminogen activator     

Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase

Summary The Bacillus subtilis cdd gene encoding cytidine/2-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order rpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis 43 and 37 RNA polymerase holoenzymes during growth and stationary phase.Abbreviations SDS sodium dodecyl sulphate - Ap ampicillin resistance - Tet^r tetracycline resistance - Km^r kanamycin resistance     

Overproduction of E. coli xylose isomerase

Summary A promoterles DNA fragment containing theE. coli xylose isomerase gene and its ribosome binding site was ligated into a plasmid downstream from the strong P_L promoter. The plasmid was then used to transformE. coli strains containing a temperature-sensitive repressor (cI857). The transformants overproduced xylose isomerase when the repressor was thermally inactivated.     

Cis regulatory elements directing tuber-specific and sucrose-inducible expression of a chimeric class I patatin promoter/GUS-gene fusion

Summary The 5-upstream region of the class I patatin gene B33 directs strong expression of the -glucuronidase (GUS) reporter gene in potato tubers and in leaves treated with sucrose. Cis-acting elements affecting specificity and level of expression were identified by deletion analysis in transgenic potato plants. A putative tuber-specific element is located downstream from position –195. Nuclear proteins present in leaf and tuber extracts bind specifically to a conserved AT rich motif within this region. A DNA fragment between –183 and –143, including the binding site is, however, not able to enhance the expression of a truncated 35S promoter from cauliflower mosaic virus. Independent positive elements contributing to a 100-fold increase relative to the basic tuber-specific element are located between –228 and –195; –736 and –509, –930 and –736 and –1512 and –951. Sucrose inducibility is controlled by sequences downstream of position –228, indicating that the tuber-specific and sucrose-inducible elements are in close proximity.     

Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose.

Results

The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the P_R promoter and kanamycin resistance gene (P_R-kan ^R ) at 30 °C, but have no effect on P_R-kan ^R gene at 37 °C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~10⁷ cfu per µg of genomic DNA, with no empty vectors detected.

Conclusions

Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.

     

Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum

Immunoglobulin light and heavy chains are synthesized in mammalian cells as precursors containing a signal peptide. Processing and assembling result in formation of active antibodies. Chimeric genes have been made containing the coding sequence of the barley -amylase signal peptide which has been fused to cDNAs coding for either the mature light or the mature heavy chain of a monoclonal antibody. A plasmid was constructed linking both chimeric genes under the control of plant active promoters in an expression cassette. This DNA fragment was stably integrated into the genome of Nicotiana tabacum by Agrobacterium tumefaciens mediated gene transfer. Synthesis of light and heavy chains and assembly to antibodies was detected in transgenic tobacco tissue using specific secondary antibodies. By electron microscopic immunogold labeling, the presence of assembled antibody could be detected within the endoplasmic reticulum. Affinity chromatography indicated biological activity of the assembled immunoglobulin produced in plant cells. Unexpectedly, a significant amount of assembled antibodies was found within chloroplasts.     

Physical mapping of the immunity region of phage phi 80

Mapping of the sites of cleavage of five restriction endonucleases (BglI, BglII, EcoRV, KpnI and PvuII) in the immunity region of bacteriophage phi 80 DNA is described. The order of the 27 restriction sites was established. This helped to localize the phi 80 cI gene within the 640 bp fragment of the immunity region. Recombinant plasmids carrying phi 80 "kil" function were constructed. The function is suppressed by the cI-coded repressor. The deletion AB43 which is present in the phi 80 vir mutant is located within the phi 80 DNA fragment carrying the cI gene.     

Nucleotide sequence coding for the insecticidal fragment of the Bacillus thuringiensis crystal protein

The insecticidal crystal protein (ICP) gene, icp, from a 68-kb plasmid derived from Bacillus thuringiensis subsp. sotto was cloned in Escherichia coli. The icp expression in E. coli cells was confirmed by both immunological and insect-toxicity assays of the cell extract. The entire icp gene resides in the 6.6-kb PstI fragment, which codes for a 144-kDal peptide identical to the intact ICP, as determined by its size and reaction with anti-ICP antibody. Deletion analysis further revealed that the 2.8-kb region within the 6.6-kb PstI fragment codes for ICP. Analysis of the nucleotide sequence indicated that a peptide of 934 amino acid residues truncated at the C-terminal end is encoded by this 2.8-kb fragment. A unique feature of this truncated ICP is the abundance of cysteine and lysine residues within its C-terminal region.     

Glutathione production by Escherichia coli cells with hybrid plasmid containing tandemly polymerized genes for glutathione synthetase

Summary A DNA fragment containing the structural and promoter regions of glutathione synthetase (GSH II) gene (gsh II) from Escherichia coli B were polymerized. The dimeric and trimeric DNA fragments obtained were inserted into Bam HI site of vector plasmid pBR325 and the resulting hybrid plasmids were designated pGS401-02 and pGS401-03, respectively. The GSH II activity of E. coli cells with these hybrid plasmids increased depending on the number of the genes (gsh II) contained. To construct hybrid plasmids useful for glutathione production, another DNA fragment with a gene (gsh I) for -glutamylcysteine synthetase (GSH I) from E. coli B was inserted into Pst I sites of pGS401-02 and pGS401-03 and the hybrid plasmids obtained (pGS501-12 and pGS501-13, respectively) were introduced into E. coli B cells. Although the glutathione-producing activities of the cells with these plasmids were little improved as compared with that of cells with the hybrid plasmid (pGS501-11) containing both gsh I and gsh II because of the low activity of GSH I, our method has brought to light a new type of gene amplification.