Vectors for cloning promoters and terminators.
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Drug Resistance, Microbial↗
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A plasmid vector and quantitative techniques for the study of transcription termination in Escherichia coli using bacterial luciferase.
We have developed a plasmid expression vector for the study of transcription terminators in Escherichia coli that utilizes the lux genes coding for the enzyme luciferase of the bioluminescent marine bacterium, Vibrio harveyi. The pBR322-derived plasmid, called pHV100, contains the E. coli lac promoter, the polylinker regions from the plasmid vector pUC18, and the V. harveyi lux genes. Insertion of transcription termination sites into the polylinker region results in decreased luciferase expression. Because the bioluminescence genes are not indigenous to E. coli, their expression can be studied in virtually any host strain without the complications of background activity. This facilitates sensitive measurements of terminator efficiency in hosts containing termination factor mutations. Bioluminescence can be easily monitored with high sensitivity, using a rapid photographic technique or a more quantitative photometric assay.
Base Sequence↗
Sequence analysis, transcriptional organization, and insertional mutagenesis of the envA gene of Escherichia coli.
The Escherichia coli cell permeability-cell separation gene envA and the region immediately downstream were sequenced. The envA gene consisted of 305 codons which encoded a 34-kilodalton polypeptide that lacked a signal sequence and hydrophobic membrane-spanning regions. The envA1 mutation was determined to be a missense mutation in codon 19 resulting in a change in the amino acid sequence from histidine to tyrosine. Located 299 base pairs downstream of the envA gene was an unidentified open reading frame consisting of 147 codons. This open reading frame was followed by an additional open reading frame starting 59 base pairs further downstream and corresponded to the secA gene. A transcription terminator was located just downstream of envA on a fragment that contained a sequence corresponding to a typical rho-independent terminator. Transcription of envA and the upstream fts genes terminated at this terminator and was probably uncoupled from the downstream genes, including secA. Gene disruption experiments indicated that the envA gene was an essential gene.
Amino Acid Sequence↗
Construction of cloning, promoter-screening, and terminator-screening shuttle vectors for Bacillus subtilis and Streptococcus lactis.
Shuttle vectors have been constructed which are suitable both for the selection of regulatory sequences and for gene cloning in Bacillus subtilis and Streptococcus lactis. The promoter screening vectors contain a promoterless chloramphenicol acetyltransferase gene; the insertion of suitable DNA fragments upstream of the gene restored the enzyme activity. With a related set of vectors, transcription termination signals can be selected.
Acetyltransferases↗
The Xenopus laevis ribosomal gene terminator contains sequences that both enhance and repress ribosomal transcription.
A DNA segment approximately 200 base pairs upstream of the Xenopus laevis ribosomal promoter acts both as an upstream promoter element that augments transcription and as a transcription terminator. It is, however, unclear to what extent these two activities are related. A segment of the X. laevis ribosomal DNA, containing the terminator and the upstream promoter element, was subjected to point mutation, and the effects of the resulting mutations were investigated by oocyte microinjection. Analysis of 26 point mutants revealed not only sequences that augment 40S transcription but also those that repress it. The sequences that augmented transcription lay within the T3 homology box and also near the site of 3'-end formation. These sequences also played a role in termination. The sequences that repressed transcription lay within the G+C-rich DNA flanking the T3 box. It can be concluded that termination is probably essential but may not be sufficient for the activity of the upstream promoter element.
Animals↗
Specific transcription of cloned Methanobacterium thermoautotrophicum transcription units by homologous RNA polymerase in vitro.
Specific in vitro transcription by partially purified RNA polymerase from Methanobacterium thermoautotrophicum of DNA sequences cloned in between the promoter and terminator regions of the methyl CoM reductase transcription unit of the same organism is described. The 5'-terminus of the product has been mapped. Deletion analyses of the promoter region show the limits of the sequences essential for the promoter function.
Base Sequence↗
Structure and function of the trp operon control regions of Brevibacterium lactofermentum, a glutamic-acid-producing bacterium.
The trp genes of Brevibacterium lactofermentum lie within a 7.72-kb HapII-BamHI fragment whose sequence has been determined (Matsui et al., 1986). The 5'- and the 3'-flanking regions of this gene cluster were subcloned as a 1.8-kb PstI-PstI and a 0.6-kb XhoI-BamHI fragment, respectively. The 5'-flanking region encodes two open reading frames (ORFs); one corresponds to trpL, while the other corresponds to the N-terminal half of the trpE gene. Within the 17 amino acid residues of the predicted leader peptide encoded by trpL are found three contiguous tryptophan residues. By subcloning parts of the 1.8-kb PstI-PstI fragment into promoter probe vectors, a promoter situated 32 bp upstream from the presumptive trpL gene was identified. The -35 and the -10 region of this promoter, TACACA and AATAAT, respectively, are very similar to the Escherichia coli trp promoter. The trp promoter of B. lactofermentum functions in E. coli. A 14-bp imperfect palindrome that overlaps the -10 region apparently functions in B. lactofermentum but not in E. coli as a trp operator. Upstream of trpE, attenuator-like sequences are found that resemble the corresponding E. coli sequences. The 0.6-kb XhoI-BamHI 3'-flanking fragment contains one ORF that encodes the C-terminal part of trpA. Downstream from trpA lies a Rho-independent terminator that resembles E. coli sequences that are situated downstream from the E. coli trp operon. Thus the trp control regions of the Gram-positive B. lactofermentum are more closely related in structure to the corresponding regions of the Gram-negative E. coli than to those of the Gram-positive Bacillus subtilis.
Base Sequence↗
Lorist2, a cosmid with transcriptional terminators insulating vector genes from interference by promoters within the insert: effect on DNA yield and cloned insert frequency.
Transcription terminators have been included in a phage-lambda-replicon-based cosmid vector, Lorist2, to insulate vector genes against transcriptional interference from cloned insert DNA. DNA yields of recombinant clones containing Escherichia coli genomic DNA inserts are more even for Lorist2 than with its progenitor LoristB. However, the terminators provide only a partial reduction in the over-representation of r X DNA-containing clones generally observed in cosmid libraries of Caenorhabditis elegans DNA, suggesting that causes other than transcriptional readthrough into the vector contribute to this problem.
Animals↗
Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants.
The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.
Aspartate Ammonia-Lyase↗
Surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12. Determination of the nucleotide sequence and promoter and terminator activities.
The DNA encoding the surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12 has been sequenced and the biological activity of the various terminators and promoters determined. The data show that traS encodes a 16,861 Mr protein with no apparent signal sequence, as expected for its cytoplasmic membrane location. The protein is extremely hydrophobic. traS has its own promoter and a weak terminator region follows the gene. After the traS termination loop there is a small intergenic region before the traT promoter. The traT gene encodes a 25,932 Mr precursor for the 23,709 Mr mature protein. The amino-terminal signal peptide is 21 amino acid residues, consistent with it being an outer membrane lipoprotein. A very strong termination loop follows the gene and adjacent to this a further loop can be predicted from the sequence. These secondary structures would be expected to enhance the stability of the mRNA in the presence of 3' specific ribonucleases accounting for the apparent long half-life of the messenger. The amino acid sequence of the mature product of traT of F differs from that of R100 by only a single amino acid substitution (Gly for Ala at position 119), whereas that of pED208 (Folac) differs at 40 positions. traT lies in a region of heteroduplex homology between F and R100, and the nucleotide sequence confirms this and demonstrates that this homology breaks down immediately preceding and following the coding region. Sequence analysis shows that this is also so for pED208. Thus the entire traS of F, R100 and pED208 are very different at the DNA level. An open reading frame, preceded by a typical promoter sequence and a weak and poorly located Shine-Dalgarno sequence, follows traT and corresponds to the start of traD. Alone, this promoter appears to be inactive.
Amino Acid Sequence↗
IS10 transposition is regulated by DNA adenine methylation.
We show that dam- mutants are a major class of E. coli mutants with increased IS10 activity. IS10 has two dam methylation sites, one within the transposase promoter and one within the inner terminus where transposase presumably binds. Absence of methylation results in increased activity of both promoter and terminus, and completely accounts for increased transposition in dam- strains. Transposition of Tn903 and Tn5 are also increased in dam- strains, probably for analogous reasons. Transposition is also increased when IS10 is hemimethylated. One hemimethylated species is much more active than the other and is estimated to be at least 1000 times more active than a fully methylated element. Evidence is presented that the promoter and inner terminus of IS10 are coordinately activated in a dam-dependent fashion, presumably because they are hemimethylated at the same time. Thus, in dam+ strains, IS10 will transpose preferentially when DNA is hemimethylated. We suggest specifically that IS10 transposition may preferentially occur immediately after passage of a chromosomal replication fork.
Adenine↗
Effect of the promoter structure on the nutL transcription antitermination function.
Several boxA-less 74-bp nutL antiterminator fragments do not function as antiterminators of plac-promoted transcription, but are active with the pp and p'R promoters at 30 degrees C. At elevated temperatures (42 degrees C), these defective 74-bp nutL modules retain 50% of their activity when controlled by the p'R promoter; with the pp promoter the antitermination activity is 5-10 times less efficient. Similarly, deletions of 1-3 bp in the spacer region between the boxA and boxB subunits of nutL (which abolish antitermination promoted by plac), allow rather efficient but thermosensitive antitermination when controlled by the pp or p'R promoter. These results point to possible conditional interactions between the promoter and antiterminator elements and functions. Also noted were unexpectedly slow mobilities of the p'R-nutL-bearing fragments, suggesting some unusual secondary structures.
Bacteriophage lambda↗
Nonlinear waves in DNA and regulation of transcription.
The transmission of conformational changes along the DNA double helix is interpreted as a propagation of nonlinear solitary waves. Experimental and theoretical data in favour of this approach are presented. The possible role of nonlinear waves in regulation of transcription is discussed.
DNA↗
A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure.
The neo (neomycin-resistance) gene of transposon Tn5 encodes the enzyme neomycin phosphotransferase II (EC 2.7.1.95), which confers resistance to various aminoglycoside antibiotics, including kanamycin and G418. The gene is widely used as a selectable marker in the transformation of organisms as diverse as bacteria, yeast, plants, and animals. We found a mutation that involves a glutamic to aspartic acid conversion at residue 182 in the protein encoded by the chimeric neomycin phosphotransferase II genes of several commonly used transformation vectors. The mutation substantially reduces phosphotransferase activity but does not appear to affect the stability of the neomycin phosphotransferase II mRNA or protein. Plants and bacteria transformed with the mutant gene are less resistant to antibiotics than those transformed with the normal gene. A simple restriction endonuclease digestion distinguishes between the mutant and the normal gene.
Anti-Bacterial Agents↗
Molecular evolution of the Escherichia coli chromosome. I. Analysis of structure and natural variation in a previously uncharacterized region between trp and tonB.
We present the sequence of a 3500-bp region of the Escherichia coli strain K12 chromosome lying between the tryptophan operon and the tonB gene. Analysis of the sequence yields six open reading frames that have properties characteristic of genes for proteins. The reading frames are closely spaced, and putative transcription units and control sites compose over 95% of the DNA. The sequences of several wild strains of E. coli have been determined for a large segment of the region described. Comparison of these sequences reveals the effects of base substitutions, DNA rearrangements, and recombination. In the regions presumably expressed as polypeptides, most of the natural variation results from synonymous substitutions. However, the DNA rearrangements identified have end points within the open reading frames and disrupt them in a variety of ways. The effects of genetic recombination between strains, recently found to be significant on a large scale in E. coli, are also apparent in the region between trp and tonB.
Base Sequence↗
Direct selection vectors, promoter-active adaptors and their use for gene assembly by a single-stranded cloning method.
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Cloning, Molecular↗
Bacteriophage 82 gene Q and Q protein. Sequence, overproduction, and activity as a transcription antiterminator in vitro.
Phage 82 gene Q encodes a phage-specific positive regulator of late gene expression, thought, by analogy to the corresponding gene of phage lambda, to be a transcription antiterminator. We have cloned and sequenced the phage 82 gene Q and have overproduced and purified the 82 Q protein. We also have identified and sequenced DNA containing the phage 82 late gene promoter and terminator. We show that purified 82 Q protein is active and specific for DNA containing the 82 late gene promoter in a well defined in vitro transcription reaction: RNA polymerase initiating at the phage 82 late gene promoter and modified by 82 Q protein reads through a downstream transcriptional terminator. We used T1 RNase mapping to confirm that the putative readthrough RNA made in the presence of 82 Q protein is in fact an elongation product of the shorter RNA.
Amino Acid Sequence↗
The mouse ribosomal DNA promoter has more stringent requirements in vivo than in vitro.
Using mouse ribosomal DNA templates bearing polymerase I terminators to prevent transcriptional interference (S. L. Henderson, K. Ryan, and B. Sollner-Webb, Genes Dev. 3:212-223, 1989) and facilitate promoter analysis in intact cells, we demonstrate that a -140 promoter domain (as well as the core region) is essential for appreciable levels of initiation in vivo. This in vivo polymerase I promoter can also be detected in vitro but only under very stringent conditions.
Animals↗