High-level expression vectors to synthesize unfused proteins in Escherichia coli

A new class of plasmid vectors (pANK-12, pANH-1, and pPL2) for synthesizing unfused proteins was constructed by inserting synthetic linkers at the NdeI site (CATATG) of plasmid pJL6, which contains the lambda cII gene initiator codon. These expression vectors contain the lambda pL promoter, the cII ribosome-binding site, cII start codon and unique restriction sites (KpnI, Asp718, HpaI, BamHI) downstream from the initiator ATG for expression of unfused proteins. The main advantage of these vectors is that any DNA fragment with an open reading frame that does not possess a start and/or a stop codon can be directed to overproduce protein in an unfused form.     

Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering

An efficient method for introducing two (or four) codons into a cloned gene has been developed. Single-stranded (ss) hexameric linkers are inserted into a plasmid linearized at cohesive-end restriction sites. The resultant 6 (or 12)-bp insertion creates a new 6-bp restriction site. Plasmids containing linker insertions are enriched by using biochemical selection, or selected by using a kanamycin-resistance (KmR) cassette (biological selection). A total of 57 new linkers have been designed, and compatible KmR cassettes flanked by eleven different restriction sites have been constructed. Two-codon insertions into the tetracycline-resistance (TcR) gene of pBR322 yielded a series of new plasmid vectors. Moreover, proteins with internally duplicated domains have been constructed from beta-lactamase (ApR) insertions into the ApR gene of pBR322. Some of the resulting "gemini" proteins retained the beta-lactamase activity.     

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.     

Precise nucleotide sequence modifications with bidirectionally cleaving class-IIS excision linkers

Bidirectionally cleaving blunt-ended DNA linkers have been constructed to generate defined nucleotide sequence modifications. The oligodeoxynucleotides (termed 'excision linkers'), contain two back-to-back recognition sites for class-IIS restriction endonucleases and provide a new instrument for modifying DNA primary structure. Following insertion of these linkers into host DNA, digestion with the cognate class-IIS enzyme results in a cleavage upstream and downstream from the adjoining enzyme recognition sites. Bidirectional cleavage efficiency can be improved by including spacer nucleotides between the two recognition sites. The number of nucleotides removed from or added to the host DNA depends upon the cleavage shift characteristic of the class-IIS enzyme, the design of the linker (including lateral spacer nucleotides to set the cleavage position), and the method used to make blunt ends from staggered ends following excision of the linker. BspMI linkers constructed in this study have been used to generate defined deletions in the ApR and TcR genes of pBR322. BsmI excision linkers are also described.     

Psoralen photo-cross-linking by triplex-forming oligonucleotides at multiple sites in the human rhodopsin gene.

Targeting DNA damage by triplex-forming oligonucleotides (TFOs) represents a way of modifying gene expression and structure and a possible approach to gene therapy. We have determined that this approach can deliver damage with great specificity to sites in the human gene for the G-protein-linked receptor rhodopsin, mutations of which can lead to the genetic disorder autosomal dominant retinitis pigmentosa. We have introduced DNA monoadducts and interstrand cross-links at multiple target sites within the gene using TFOs with a photoactivatable psoralen group at the 5'-end. The extent of formation of photoadducts (i.e., monoadducts and cross-links) was measured at target sites with a 5'-ApT sequence at the triplex-duplex junction and at a target site with 5'-ApT and 5'-TpA sequences located four and seven nucleotides away, respectively. To improve psoralen reactivity at more distant sites, psoralen moieties were attached to TFOs with nucleotide "linkers" from two to nine nucleotides in length. High-affinity binding was maintained with linkers of up to 10 nucleotides, but affinities tended to decrease somewhat with increasing linker length due to faster dissociation kinetics. DNase I footprinting indicated little, if any, interaction between linkers and the duplex. Psoralen-TFO conjugates formed DNA cross-links with high efficiency (56-65%) at 5'-ApT sequences located at triplex junctions. At a 5'-ApT site four nucleotides away, the efficiency varied with linker length; a four-nucleotide linker gave the highest efficiency. Duplexes with 5'-TpA and 5'-ApT sites two nucleotides away, in otherwise identical sequences, were cross-linked with efficiencies of 56 and 38%, respectively. These results indicate that TFO-linker-psoralen conjugates allow simultaneous, efficient targeting of multiple sites in the human rhodopsin gene.     

Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus.

The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine.     

Formation of MboII vectors and cassettes using asymmetric MboII linkers

Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.     

The complete nucleotide sequence of the gene coding for diphtheria toxin in the corynephage omega (tox+) genome.

A segment of corynephage omega (tox+) DNA, containing the gene for diphtheria toxin (tox) was fragmented with restriction enzymes and the fragments cloned into M13 vectors for nucleotide sequence determination. A long open reading frame was shown to encode the tox gene by comparing the predicted amino acid sequence with that of peptides derived from the mature toxin molecule. Analysis of the nucleotide sequence shows RNA polymerase and ribosome binding signals preceding a GTG codon in the open reading frame: if this is the correct starting signal for translation, then a 25 amino acid signal peptide can be predicted for the toxin molecule.     

The nucleotide sequence and transcript map of the herpes simplex virus thymidine kinase gene

This paper presents the nucleotide sequence of the Herpes Simplex Virus thymidine kinase (tk) gene. The position on the DNA sequence corresponding to the 5' and 3' termini of tk messenger RNA have been mapped. The mRNA termini are separated by slightly more than 1,300 nucleotides. The same 2,300 nucleotide segment of tk coding strand DNA is fully protected from S1 nuclease digestion when hybridized to tk mRNA. The location and size of the mRNA-coding segment corresponds to a region of the viral DNA that is essential for tk gene expression in microinjected frog oocytes. The nucleotide sequence of the HSV tk gene exhibits an open translational reading frame of 376 codons that extends from the methionine codon most proximal to the 5' terminus of tk mRNA to a UGA stop codon approximately 70 nucleotides from the poly-A addition site. The results of these experiments indicate that the tk gene is not interrupted by intervening DNA sequences, and that certain oligonucleotide sequences adjacent to the termini of the tk gene are homologous to similarly positioned sequences common to structural genes of eukaryotic cells.     

Characteristics of the structural organization of the DIP1 gene in Drosophila melanogaster strains mutant for the flamenco gene

Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flamSS (SS) and flamMS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flampy + (P) carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco+. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flamSS, flamMS, flampy +(P), and flamenco+ considerably differ from one another in the structure of DIP1. Strains flamss and flamMS have no Dral restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The Dral gene of strainsflamSS andflamMS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tcl/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIPI and, probably, neighboring genes. In strains flamenco+ and flampy + (P), the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either directly or through interaction with other elements of the genome.     

Characterization of the ksgA gene of Escherichia coli determining kasugamycin sensitivity

In the plasmid pUC8ksgA7, the coding region of the ksgA gene is preceded by the lac promoter (Plac) and a small open reading frame (ORF). This ORF of 15 codons is composed of nucleotides derived from the lacZ gene, a multiple cloning site and the ksgA gene itself. The reading frame begins with the ATG initiation codon of lacZ and ends a few nucleotides beyond the ATG start codon of ksgA. The ksgA gene is not preceded by a Shine-Dalgarno (SD) signal. Cells transformed with pUC8ksgA7 produce active methylase, the product of the ksgA gene. Introduction of an in-phase TAA stop codon in the small ORF abolishes methylase production in transformed cells. On the plasmid pUC8ksgA5, which contains the entire ksgA region, the promoter of the ksgA gene was found to reside in a 380 base pair Bgl1-Pvu2 restriction fragment, partly overlapping the ksgA gene, by two independent methods. Cloning of this fragment in front of the galK gene in plasmid pKO1 stimulates galactokinase activity in transformants and its insertion into the expression vector pKL203 makes beta-galactosidase synthesis independent of the presence of Plac. The sequence of the Bgl1-Pvu2 fragment was determined and a putative promoter sequence identified. An SD signal could not be distinguished at a proper distance upstream from the ksgA start codon. Instead, an ORF of 13 codons starting with ATG in tandem with an SD signal and ending 4 codons ahead of the ksgA gene was identified. This suggests that translation of the ORF is required for expression of the ksgA gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

The cloning and nucleotide sequence of the serine protease gene (aspA) of Aeromonas salmonicida ssp. salmonicida

The gene for Aeromonas salmonicida serine protease has been cloned into phagemid pTZ18R in two restriction fragments, 2.0-kb PstI and 2.3-kb KpnI, of genomic DNA. The nucleotide sequences of the two fragments have been determined, in both directions, after subcloning, by double-stranded sequencing of nested deletions. An open reading frame of 1863 bp translated into a sequence of 621 amino acids, a 24-amino acid signal peptide and a 597-amino acid mature enzyme of molecular mass 64,173 Da. The consensus sequence, NGTS, of a serine protease substrate primary binding site was identified and a putative ribosome-binding site GGAG occurred 6 bp upstream of the ATG initiation codon.