Vectors for cloning promoters and terminators.
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Drug Resistance, Microbial↗
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Cloning with tandem gene systems for high level gene expression.
A method has been devised for increasing the copy number of a gene (or genes) cloned into a plasmid while minimizing the size of the plasmid. If n copies of a transcriptional unit are cloned, including the promoter, coding region and terminator, the size of the plasmid will increase by n times the total size of the unit. However, if we borrow the concept of polycistronic operon and sandwich n structural genes, each with its own ribosome binding-site, between a promoter and a transcription terminator, there will be a space saving equivalent to n-1 promoters and n-1 transcription terminators. We have constructed plasmids in which an E. coli lipoprotein promoter is followed by 1 to 4 human leukocyte interferon genes and a transcription terminator. The applications of this method in genetic engineering are discussed.
Amino Acid Sequence↗
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↗
A T7 promoter vector with a transcriptional terminator for stringent expression of foreign genes.
An expression vector, pKK233-2 [Amann and Brosius, Gene 40 (1985) 183-190], was modified by replacing the trc promoter with the T7 phi 10 promoter and introducing a strong transcriptional terminator upstream from this promoter. This modification successfully suppressed background transcription and resulted in strict dependence upon induction.
Bacteriophage T7↗
A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase.
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Bacteriophage lambda↗
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↗
Biosensors as new analytical tool for detection of Genetically Modified Organisms (GMOs).
Three different biosensors for detection of Genetically Modified Organisms (GMOs) are presented. The sensing principle is based on the affinity interaction between nucleic acids: the probe is immobilised on the sensor surface and the target analyte is free in solution. The immobilised probes are specific for most inserted sequences in GMOs: the promoter P35S and the terminator TNOS. Electrochemical methods with screen-printed electrodes, piezoelectric and optical (SPR) transduction principles were applied.
Biosensing Techniques↗
In vitro analysis of transcription initiation and termination from the Lhcb1 gene family in Nicotiana sylvestris: detection of transcription termination sites.
Genes encoding chlorophyll a/b-binding proteins of photosystem II (Lhcb) constitute a multigene family. Nine Lhcb1 genes have previously been isolated from the tobacco species, Nicotiana sylvestris, and the transcription initiation sites in vivo have been mapped. Reaction conditions from a previously developed in vitro transcription system from tobacco cultured cells were optimized for the Lhcb1 genes. Transcription initiation sites in vitro predominantly coincided with those found in vivo and were typically cytidines, a system unique to N. sylvestris Lhcb1 genes. CTC*A (C* for initiation site) was a consensus motif for the initiation region in vitro, as reported in vivo. Mutation analysis defined functionally that the TATA box is essential for transcription initiation and that the CTCA motif is a determinant of transcription initiation sites but not transcript levels. Polyadenylation sites were determined from in vivo transcripts, located 12-21 nt downstream from likely poly(A) signals. Four major 3'-ends of in vitro transcripts from Lhcb1*6 were detected, 40-300 nt downstream of the poly(A) site, suggestive of multiple, discrete transcription termination sites. These 3'-ends are mapped in or nearby T-rich sequences.
3' Untranslated Regions↗
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↗
Neomycin resistance as a selectable marker in Methanococcus maripaludis.
We cloned the aminoglycoside phosphotransferase genes APH3'I and APH3'II between the Methanococcus voltae methyl reductase promoter and terminator in a plasmid containing a fragment of Methanococcus maripaludis chromosomal DNA. The resulting plasmids encoding neomycin resistance transformed M. maripaludis at frequencies similar to those observed for pKAS102 encoding puromycin resistance. The antibiotic geneticin was not inhibitory to M. maripaludis.
Anti-Bacterial Agents↗
Regulation of the Escherichia coli K5 capsule gene cluster by transcription antitermination.
The expression of the Escherichia coli K5 (group II) capsular polysaccharide requires the rfaH gene. By reverse transcriptase-polymerase chain reaction (RT-PCR) it was possible to demonstrate that RfaH increases the transcription of region 2 genes by readthrough transcription from the region 3 promoter. A mutation in the rfaH gene reduced this readthrough transcription from the region 3 promoter by 10-fold as measured by quantitative RT-PCR. The region 3 promoter was mapped to 741 bp 5' of the initiation codon of the kpsM gene. Deletion and insertion mutagenesis of the JUMPstart sequence, which is 28 bp 5' of kpsM and is conserved upstream of RfaH-regulated operons and other polysaccharide biosynthesis genes, confirmed that this sequence was required for expression of the K5 antigen and for the antitermination activity of RfaH. The JUMPstart sequence could cause RfaH-dependent antitermination at other Rho-dependent terminators, suggesting that the JUMPstart sequence may function in a manner analogous to a lambda nut site. On the basis of these results we propose a model by which RfaH regulates expression of E. coli group II capsule gene clusters by allowing readthrough transcription to proceed from region 3 into region 2 and that sequences within the JUMPstart sequence are essential for this process.
Antigens, Bacterial↗
Antitermination of early transcription in phage HK022. Absence of a phage-encoded antitermination factor.
In phage lambda and its relatives most early phage genes are located downstream from transcription termination sites, and full gene expression requires suppression of termination (or antitermination). Phage HK022, a lambda relative, also antiterminates early transcription, but, unlike its relatives, does so in the absence of any active phage gene product. We found no functional equivalent of the lambda N antitermination protein in HK022. In addition, nus mutations, which alter host proteins required for lambda antitermination, have no apparent effect on HK022 early gene expression. We have shown that terminators located several thousand base-pairs from the start point of transcription are suppressed, and that in the left operon suppression requires a short, promoter-proximal segment. A 40 bp region within this segment is repeated in the right operon. The chromosomal locations of these repeated segments resemble those of the nut antitermination sites of other lambdoid phages, but the HK022 sites lack the conserved sequence elements of the nut sites. It appears that HK022 antiterminates early transcription in a novel way.
Bacteriophage lambda↗
The light-responsive promoter of cyanobacterial ORF76 gene overlaps with the htpG terminator.
A Synechococcus PCC7942 gene, encoding 76 amino acids of an unknown protein (designated ORF76), is located at the same orientation and downstream of the htpG gene. The processed site of ORF76 transcripts is located at position +39 with respect to the true transcription initiation site. The non-E. colisigma70-like basal promoter of ORF76 (-51 to -9) is controlled by three cis-acting elements: positively acting element (-160 to -86), negative regulatory element (-86 to -51) and light-responsive element (-51 to +63) that, together, respond to high light induction at transcriptional level. The ORF76 gene is expressed as monocistronic transcript. The promoter elements of ORF76 gene overlap with the coding sequence and 3' end formation signal of htpG gene.
Amino Acid Sequence↗
An improved plasmid DNA expression vector for direct injection into skeletal muscle.
In previous work, the direct injection of 50 micrograms of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences. Expression of the modified vectors was then compared to VR1205 using the intramuscular injection assay. In general, modifications to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect. However, modifications to the polyadenylation and transcriptional termination sequences, plasmid backbone elements, and the luciferase gene itself each increased luciferase expression levels. The best-expressing vector, designated VR1255, contained a combination of these incrementally beneficial changes. A single intramuscular injection of 50 micrograms of VR1255 produced 300 ng of luciferase at 7 days postinjection, an expression level 46-fold higher than the VR1205 vector (or 22-fold higher, excluding modifications to the luciferase gene) and 154-fold higher than a commercially available luciferase expression vector. Thus, VR1255 represents an improved plasmid DNA vector that may be useful for gene therapy applications.
Animals↗