Showing papers in "Gene in 1988"

Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.

Abstract: Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum. In the majority of cases, fusion proteins are soluble in aqueous solutions and can be purified from crude bacterial lysates under non-denaturing conditions by affinity chromatography on immobilised glutathione. Using batch wash procedures several fusion proteins can be purified in parallel in under 2 h with yields of up to 15 micrograms protein/ml of culture. The vectors have been engineered so that the GST carrier can be cleaved from fusion proteins by digestion with site-specific proteases such as thrombin or blood coagulation factor Xa, following which, the carrier and any uncleaved fusion protein can be removed by absorption on glutathione-agarose. This system has been used successfully for the expression and purification of more than 30 different eukaryotic polypeptides.

CLUSTAL: A package for performing multiple sequence alignment on a microcomputer

Abstract: An approach for performing multiple alignments of large numbers of amino acid or nucleotide sequences is described. The method is based on first deriving a phylogenetic tree from a matrix of all pairwise sequence similarity scores, obtained using a fast pairwise alignment algorithm. Then the multiple alignment is achieved from a series of pairwise alignments of clusters of sequences, following the order of branching in the tree. The method is sufficiently fast and economical with memory to be easily implemented on a microcomputer, and yet the results obtained are comparable to those from packages requiring mainframe computer facilities.

New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites.

Abstract: We describe the production of new alleles of the LEU2, URA3 and TRP1 genes of Saccharomyces cerevisiae by in vitro mutagenesis. Each new allele, which lacks restriction enzyme recognition sequences found in the pUC19 multicloning site, was used to construct a unique series of yeast-Escherichia coli shuttle vectors derived from the plasmid pUC19. For each gene a 2 mu vector (YEplac), an ARS1 CEN4 vector (YCplac) and an integrative vector (YIplac) was constructed. The features of these vectors include (i) small size; (ii) unique recognition site for each restriction enzyme found in the pUC19 multicloning site; (iii) screening for plasmids containing inserts by color assay; (iv) high plasmid yield; (v) efficient transformation of S. cerevisiae. These vectors should allow greater flexibility with regard to DNA restriction fragment manipulation and subcloning.

The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions.

Abstract: Polymerase chain reaction conditions were established for the in vitro amplification of eukaryotic small subunit ribosomal (16S-like) rRNA genes. Coding regions from algae, fungi, and protozoa were amplified from nanogram quantities of genomic DNA or recombinant plasmids containing rDNA genes. Oligodeoxynucleotides that are complementary to conserved regions at the 5' and 3' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis. The fidelity of synthesis for the amplification products was evaluated by comparisons with sequences of previously reported rRNA genes or with primer extension analyses of rRNAs. Fewer than one error per 2000 positions were observed in the amplified rRNA coding region sequences. The primary structure of the 16S-like rRNA from the marine diatom, Skeletonema costatum, was inferred from the sequence of its in vitro amplified coding region.

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

Abstract: 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.

Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli.

Abstract: A series of new plasmid expression vectors (the pTrc series) has been constructed for the regulated expression of genes in Escherichia coli. Based on pKK233-2 [Amann and Brosius, Gene 40 (1985) 183-190], the vectors carry a strong hybrid trp/lac promoter, the lacZ ribosome-binding site (RBS), the multiple cloning site of pUC18 and the rrnB transcription terminators. With the aid of synthetic oligodeoxynucleotides, the multiple cloning site has been inserted behind an NcoI site in three reading frames. Thus, the vectors are equally useful for the expression of proteins in their authentic, non-fused form (by using the NcoI site) and for the expression of fusion proteins (by choosing any of the cloning sites in the correct translational frame). To ensure complete repression of the hybrid trp/lac promoter during construction and growth in any host strain, the lacIq allele of the lac repressor gene was added to some of the vectors. The complete vector nucleotide sequence and examples of heterologous gene expression (human coagulation factor XIIIa and human placental anticoagulant protein PP4) with the new vectors are presented.

Antibody-selectable filamentous fd phage vectors: affinity purification of target genes.

Abstract: Foreign DNA fragments can be inserted into a minor coat protein gene of filamentous phage, creating a fusion protein that is incorporated into the virion; we call these particles “fusion phage”. The foreign amino acids are displayed on the surface, allowing fusion phage bearing antigenic determinants from a target gene to be purified in infectious form by affinity to antibody directed against the gene product. Here we introduce fusion-phage vectors that accept foreign DNA inserts with little effect on phage function; and describe affinity purification of virions bearing a target determinant from a 108-fold excess of phage not bearing the determinant, using minute amounts of antibody. These “antibody-selectable” vectors are a promising alternative to conventional expression systems for using antibodies to clone genes, though the ability to isolate rare clones from actual libraries remains to be demonstrated.

A simple phase-extraction assay for chloramphenicol acyltransferase activity

Abstract: A simple and convenient phase extraction assay for chloramphenicol (Cm) acetyltransferase (CAT) activity has been developed, based on the enzymatic butyrylation of radiolabelled Cm. The assay is linear over two to three orders of magnitude of enzyme concentration, is highly sensitive, and substantially less expensive than all presently available alternatives. Methods for convenient CAT assay, adapted for mammalian cells, plant protoplasts, and mammalian cell culture supernatants, are described. These methods should also simplify measurement of CAT activity in other organisms, such as yeast and bacteria. In addition, a simple pre-extraction procedure is presented for purifying radiolabelled Cm which allows a 25-fold increase in sensitivity using tritiated substrates.

Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cells.

Abstract: This paper describes a novel eukaryotic reporter gene, secreted alkaline phosphatase (SEAP). In transient expression experiments using transfected mammalian cells, we demonstrate that SEAP yields results that are qualitatively and quantitatively similar, at both the mRNA and protein levels, to parallel results obtained using established reporter genes. However, SEAP offers significant advantages in terms of ease of assay and assay expense, and also has the potential for quantitative assay at levels as low as 0.2 pg/ml of culture medium. These attributes suggest that SEAP may have general utility in experiments which rely on the accurate measurement of reporter gene expression levels.

Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein.

Abstract: Vectors were constructed that allow foreign peptides to be expressed in Escherichia coli as fusion proteins. The peptides are fused to the C terminus of maltose-binding protein (MBP), which allows them to be purified by the MBP's affinity to cross-linked amylose (starch). The fusion protein can be directed to the periplasm by including the leader sequence from the phoA gene on the vector.

An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein.

Abstract: A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.

Vectors for Drosophila P-element-mediated transformation and tissue culture transfection.

Abstract: We describe nine P-element vectors that can be used to study gene regulation and function in Drosophila. These vectors were designed for use in germline transformation and cell culture transfection assays. One set consists of five P elements that can be used to study transcriptional regulatory sequences. These vectors contain several unique restriction sites for insertion of a foreign promoter upstream from either a cat or lacZ reporter gene. Two of the beta-galactosidase-coding vectors also require the insertion of a start codon for translation of the reporter enzyme and thus can be used to study translational regulatory sequences. The second set of P elements consists of four vectors that contain the Drosophila cytoplasmic actin 5C promoter and polyadenylation signals. Upon insertion of a foreign DNA segment, these vectors direct constitutive expression of the encoded RNA and protein.

A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers

Abstract: Several families of positive-selection cloning vectors were constructed, based on the principle of palindrome nonviability first used by Hagan and Warren [Gene 19 (1982) 147-151]. Each vector, derived from either pBR322 or RSF1010 (a broad-host-range plasmid), contains a long inverted repeat (2 x 366 to 2 x 1008 bp) ending in a symmetrical polylinker. Plasmids with long palindromes are not viable in most strains of Escherichia coli and in at least one Gram-positive bacterium. These palindrome-containing vectors therefore transform such strains at a very low frequency unless a DNA fragment is cloned within the polylinker at the center of the palindrome. Transformation by plasmids lacking an insert is reduced by two to four orders of magnitude. Such vectors can be propagated in a palindrome-tolerant strain; however, long symmetrical deletions then occur within the palindrome. To suppress the resulting deletion derivatives, vectors have been constructed so that an extensive deletion would remove the selectable marker. Alternatively, the vectors can be propagated in any strain of E. coli so long as the palindrome is interrupted by a nonpalindromic DNA fragment. We also present several symmetrical polylinkers and drug-resistance cassettes within the vectors. These components can be interchanged to make new positive-selection vectors as needed, and the cassettes are useful in insertional mutagenesis as well. A general method is described to convert virtually any small or medium-sized plasmid into a positive-selection vector.

Conserved sequences and structures of group I introns: building an active site for RNA catalysis — a review

Abstract: Group I introns fold to form an active site to mediate their own RNA splicing. Sequence elements conserved among the available set of 66 group I introns are compiled. Comparative sequence analysis leads to the prediction of some conserved structural features that have not been widely appreciated. The possible significance of conserved nucleotides within base-paired duplexes is discussed; they might be involved in base triplets or alternate pairing interactions.

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

Abstract: 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.

EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme

Abstract: A novel endoglucanase from Trichoderma reesei, EGIII, has been purified and its catalytic properties have been studied. The gene for that enzyme (egl3) and cDNA have been cloned and sequenced. The deduced EGIII protein shows clear sequence homology to a Schizophyllum commune enzyme (M. Yaguchi, personal communication), but is very different from the three other T. reesei cellulases with known structure. Nevertheless, all the four T. reesei cellulases share two common, adjacent sequence domains, which apparently can be removed by proteolysis. These homologous sequences reside at the N termini of EGIII and the cellobiohydrolase CBHII, but at the C termini of EGI and CBHI. Comparison of the fungal cellulase structures has led to re-evaluation of hypotheses concerning the localization of the active sites.

pACYC184-derived cloning vectors containing the multiple cloning site and lacZα reporter gene of pUC8/9 and pUC18/19 plasmids

Abstract: A new series of vectors, pSU2716, pSU2717, pSU2718, and pSU2719, has been constructed. The plasmids contain (i) the P15A replicon, (ii) the chloramphenicol acetyl transferase (CAT)-coding gene from Tn 9 , and (iii) the Hae II fragment which carries the multiple cloning site and the lacZ α reporter gene of pUC8, pUC9, pUC18 and pUC19, respectively. These vectors allow rapid and simple transfer of inserts from pUC plasmids, have an intermediate copy number (which allows regulated expression from the lac promoter), and are compatible with ColE1-derived vectors (and, therefore, can be used in studies requiring the joint expression of two genes, for example, in genetic complementation analysis). Furthermore, the accumulation of CAT instead of β-lactamase, allows an easy visualization in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of proteins of 28–35 kDa, which can otherwise be obscured by the β-lactamase.

A variation in the structure of the protein-coding region of the human p53 gene

Abstract: An extensive analysis of genomic DNA preparations from a number of normal and malignant tissues revealed Bgl II site polymorphism of the human p53 gene. Approximately 10% of p53 gene alleles were found to contain an additional Bgl II site localized in a region of intron I. This allelic form of p53 gene was also responsible for p53 protein having altered electrophoretic mobility. Molecular cloning and sequencing of both the alleles of p53 gene revealed a base-pair change in codon 72 causing arginine → proline substitution in the allele with the additional Bgl II site. Both variants of the p53 gene may occur in homozygous state and are therefore functional.

Mechanisms of mRNA decay in bacteria: a perspective

Abstract: Messenger RNA decay plays an important role in prokaryotic gene expression. The disparate stabilities of bacterial messages in vivo are a consequence of their differential susceptibility to degradation by cellular endoribonucleases and 3'-exoribonucleases, which in turn results from differences in mRNA sequence and structure. RNase II and polynucleotide phosphorylase, the major bacterial exonucleases involved in mRNA turnover, rapidly degrade single-stranded RNA from the 3' end, but are impeded by 3' stem-loop structures. At present, the identity and substrate specificity of the endonucleases that control mRNA decay rates are relatively poorly defined. Ribosomes and antisense RNA also can influence the stability of transcripts with which they associate. Differences in mRNA stability can contribute to differential expression of genes within polycistronic operons and to modulation of gene expression in response to changes in bacterial growth conditions.

Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum.

Abstract: The phosphinothricin (Pt) N-acetyltransferase gene (pat) of Streptomyces viridochromogenes Tu494 is located on a 0.8-kb BglII fragment [Strauch et al., Gene 63 (1988) 65-74]. By sequencing a 1.3-kb BglII-SstII fragment, an open reading frame representing the pat gene was found. It encodes a polypeptide of 183 amino acids with an Mr of 20,621. The base composition of the pat gene is typical for Streptomyces [70.1 mol% (G + C) in total and 93.5 mol% (G + C) in the third position]. Translation of pat is initiated by a GTG codon which was identified by frameshift mutations in Escherichia coli as well as in Streptomyces lividans. Significant homology of the pat gene was found to the bialaphos-resistance gene (bar) of Streptomyces hygroscopicus [Thompson et al., EMBO J. 9 (1987) 2519-2523]. However, variations were detected in the 5'-noncoding region of the two resistance genes which may reflect differences in regulation. Since Pt is a potent herbicide, the pat gene was modified and introduced into Nicotiana tabacum by Agrobacterium-mediated leaf-disc transformation. The GTG start codon of pat was replaced by ATG. Subsequently the modified pat-coding region was fused to the 35S promoter of the cauliflower mosaic virus. Transgenic plants could directly be selected on Pt-containing medium.

Sequence of mdr3 cDNA encoding a human P-glycoprotein.

Abstract: We have determined the sequence of the human mdr3 gene using cDNA derived from liver RNA. The mdr3 gene codes for a member of a family of membrane proteins, the P-glycoproteins, overproduced in many multi-drug-resistant (MDR) cell lines. Like its relatives, the protein encoded by mdr3 has a deduced Mr of 140,000, which is presumably increased by glycosylation after synthesis. The sequence consists of two similar halves, each with a series of six hydrophobic segments that may form a membrane channel. The halves also possess nucleotide-binding consensus sequences, which presumably act as ATPases and drive drug transport. The presumed ATPase domains are all but identical to those of the human mdr1 gene product [Chen et al., Cell 47 (1986) 381-389]. We attribute this high level of sequence conservation to the repeated gene conversion that is evident from segments in which mdr1 and mdr3 differ only in a few silent mutations. Divergence between P-glycoprotein family members is greatest at the N terminus and in the 60 amino acid linker connecting the two halves. In the putative trans-membrane domains approx. 80% of the amino acids are conserved between the products of mdr1 and mdr3. Although the function of mdr3 is not yet known, its high homology with mdr1 suggests that it also encodes an efflux pump with broad specificity.

A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants

Abstract: We describe an in vitro selection procedure for oligodeoxynucleotide-directed mutagenesis, which produces mutants at frequencies of greater than 90%, facilitating the identification of mutants directly by nucleotide sequencing. The method is based on the selective methylation of the mutant strand by the incorporation of 5-methyl-dCTP. Restriction endonuclease digestion of the resulting hemimethylated DNA with MspI results in the nicking of only the nonmethylated-parental strand. The parental strand is removed by treatment with exonuclease III. The mutants are recovered by transformation of a mcrAB strain of Escherichia coli with the nascent strand.

Antisense RNA: its functions and applications in gene regulation--a review.

Abstract: All known antisense RNAs existing in nature are described. Of 11 natural antisense RNAs, nine function at the level of transcription and two at the level of DNA replication. On the basis of their inhibitory mechanisms they can be separated into three classes. From what can be found in the naturally occurring antisense RNAs, strategies in designing artificial antisense RNAs for gene expression are proposed, and applications and potential problems discussed.

Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene.

Abstract: An industrial Penicillium chrysogenum strain was transformed using two dominant selection markers, namely the bacterial gene for phieomycin resistance (ble) fused to a fungal promoter, and the acetamidase (amdS) gene from Aspergillus nidulans. Transformation frequencies of up to 20 transformants per μg of DNA were obtained with the ble system. With the amdS marker the frequency was up to 120 transformants. Cotransfonnation was very efficient when using amdS as a selection marker. The introduction ofpAN5-41B, a plasmid carrying the Escherichia coli lacZ gene fused to the strong glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter from A. nidulans, resulted in the formation of blue colonies on XGal plates indicating expression of the lacZ fusion gene in P. chrysogenum. A more detailed analysis of expression levels in several transformants showed that up to 6% of the total amount of soluble protein consists of the β-galactosidase fusion protein.

Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a γ-actin

Abstract: The single actin gene from the filamentous fungus Aspergillus nidulans has been isolated and characterized. The only other organism reported to contain just one actin gene is another Ascomycete, the budding yeast Saccharomyces. The nucleotide sequence of the A. nidulans actin gene predicts a polypeptide containing the N-terminal sequence identifying the gamma-actin isotype. Until now this characteristic N terminus has only been reported to occur in vertebrate actin sequences. A monospecific anti-gamma-actin antiserum recognizes a single 42-kDa band in immunoblots of total Aspergillus protein. None of the six introns in the A. nidulans actin gene sequence aligns precisely with those found in other actin genes. One, unlike other known actin introns, is located in the 3'-untranslated region of the gene. The 5' and 3' ends of the gene have been characterized. The Aspergillus actin gene has a heterogeneous transcript size due to the presence of several different 3' termini. Of four characterized polyadenylated transcripts, only the longest contains a typical AATAAA polyadenylation signal near its 3' terminus. Using an integrative plasmid containing Aspergillus actin sequences and the pyr4 gene from Neurospora, the A. nidulans actin gene has been mapped to the first chromosome.

Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae

Abstract: Two different cellobiohydrolases, CBHI and CBHII, of the filamentous fungus Trichoderma reesei both hydrolyse highly crystalline cellulose. Cellulolytic strains of the yeast Saccharomyces cerevisiae were constructed by transferring cDNAs coding for these enzymes into yeast on an expression plasmid. These cellulolytic yeasts were able to secrete efficiently the large, heterologous proteins to the culture medium. The recombinant cellulases were observed to be heterogeneous in M r due, at least partly, to variable N -glycosylation. Recombinant CBHII was able to bind to crystalline cellulose, although slightly less efficiently than the native enzyme. Both of the two recombinant cellulases were able to degrade amorphous cellulose. In a fermenter cultivation, around 100 μg/ml of CBHII was secreted into the yeast growth medium.

A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae.

Abstract: A simple assay called the sectoring shuffle was developed to monitor the mutational state of essential genes cloned into yeast centromeric plasmids. The essence of this assay is the creation of a conditional phenotype, colony color sectoring, for an essential gene in the absence of conditional thermosensitive or cold-sensitive alleles of that gene. This allows the quick determination of the mutational state of a cloned essential gene by observing its effect on the sectoring phenotype of the tester strain. During the course of this work we developed a family of 20 Escherichia coli-yeast shuttle vectors, pUN plasmids, containing ARS1 CEN4 and a variety of selectable markers as well as the SUP11 gene which can act as a color marker in the proper background. These vectors are compact and have been very useful for the sectoring-shuffle assay and for gene analysis in general. This paper describes these vectors, the sectoring shuffle and several applications of sectoring phenotypes.

Nucleotide sequence, transcription and deduced function of a gene involved in polyketide antibiotic synthesis in Streptomyces coelicolor.

Abstract: The Bam HI fragment containing the act III gene, from the actinorhodin (Act) biosynthetic gene cluster of Streptomyces coelicolor A3(2), was sequenced. The derived amino acid sequence for the act III gene shows homology to known oxidoreductases and the act III product is believed to be responsible for catalysing a β-keto reductive step during assembly of the Act polyketide chain. High resolution transcript mapping identified the transcription start point at 33 nucleotides upstream of the putative translation start codon. The transcript ends in a large invertedly repeated sequence. In vivo promoter-probe studies suggest that efficient transcription of the act III gene requires the product of the act II gene.

The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli.

Abstract: Expression of foreign genes in Escherichia coli requires the juxtaposition of prokaryotic transcription and translation elements with a coding region for the foreign gene. Commonly, this results in only modest expression of the foreign gene product. Here we describe a novel ribosome-binding site (RBS; phage T7 'gene 10 leader') which is able to drive the translation of several foreign genes. This RBS dramatically enhanced the translation efficiency of all the genes we have tested to date, and was particularly effective for foreign genes. The enhanced expression was often more than 40-fold greater than that obtained using a 'consensus' RBS. A general plasmid vector has been constructed, incorporating the T7 gene 10 leader sequence, which allows the facile expression of important gene products. In this report we demonstrate the application of this system for the high-level expression of plant, mammalian and bacterial proteins in E. coli.

Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis

Abstract: The nucleotide sequence of the protective antigen (PA) gene from Bacillus anthracis and the 5′ and 3' flanking sequences were determined. PA is one of three proteins comprising anthrax toxin; and its nucleotide sequence is the first to be reported from B. anthracis . The open reading frame (ORF) is 2319 bp long, of which 2205 bp encode the 735 amino acids of the secreted protein. This region is preceded by 29 codons, which appear to encode a signal peptide having characteristics in common with those of other secreted proteins. A consensus TATAAT sequence was located at the putative −10 promoter site. A Shine-Dalgarno site similar to that found in genes of other Bacillus sp. was located 7 bp upstream from the ATG start codon. The codon usage for the PA gene reflected its high A + T (69%) base composition and differed from those of genes for bacterial proteins from most other sequences examined. The TAA translation stop codon was followed by an inverted repeat forming a potential termination signal. In addition, a 192-codon ORF of unknown significance, theoretically encoding a 21.6-kDa protein, preceded the 5′ end of the PA gene.