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PubMed1992

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↗
PubMed1987

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↗
PubMed1993

Transactivation of cellular promoters by an integrated hepatitis B virus DNA.

A new Hepatitis B virus(HBV) DNA integrant clone DA2-6, isolated from a human hepatocellular carcinoma(HCC) genomic library, was tested for its ability to transactivate expression of other genes. DA2-6 consists of 3.7 kb flanking cellular sequences and an integrated 2.8 kb HBV DNA which covers the region of preS, S, and the 3' truncated X. Using a chloramphenicol acetyltransferase (CAT) assay, a number of cellular and viral promoters were transactivated by DA2-6, and the spectrum of transactivational effect was the same as that by the wild type X gene of the virus. Deletion mutant analyses indicated that the transactivation function of DA2-6 is expressed by the region that encodes a truncated X-cell fusion product.

Amino Acid Sequence↗
PubMed2005

Plasmid vaccines and therapeutics: from design to applications.

In the late 1980s, Vical and collaborators discovered that the injection into tissues of unformulated plasmid encoding various proteins resulted in the uptake of the plasmid by cells and expression of the encoded proteins. After this discovery, a period of technological improvements in plasmid delivery and expression and in pharmaceutical and manufacturing development was quickly followed by a plethora of human clinical trials testing the ability of injected plasmid to provide therapeutic benefits. In this chapter, we summarize in detail the technologies used in the most recent company-sponsored clinical trials and discuss the potential for future improvements in plasmid design, manufacturing, delivery, formulation and administration. A generic path for the clinical development of plasmid-based products is outlined and then exemplified using a case study on the development of a plasmid vaccine from concept to clinical trial.

Anthrax Vaccines↗
PubMed1993

Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei.

A cDNA encoding for the glucoamylase P enzyme (GAMP) of the fungus Hormoconis resinae was introduced into the cellulolytic filamentous fungus Trichoderma reesei under the control of the promoter of the major cellulase gene (cbh1) of Trichoderma. The transforming vector plasmid used was found to be integrated into the genome of T. reesei at various locations and in multiple copies. The size of the GAMP secreted by Trichoderma varied because of different glycosylation patterns. The best transformant strains secreted about 700 mg/l of active GAMP, which is 20-fold more than obtained with H. resinae.

Blotting, Northern↗
PubMed1985

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↗
PubMed1986

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↗
PubMed1988

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↗
PubMed1990

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↗
PubMed1988

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↗
PubMed1998

A vector for systematic gene inactivation in Bacillus subtilis.

To study the functions of the uncharacterized open reading frames identified in the Bacillus subtilis genome, several vectors were constructed to perform insertional mutagenesis in the chromosome. All the pMUTIN plasmids carry a lacZ reporter gene and an inducible Pspac promoter, which is tightly regulated and can be induced about 1000-fold. The integration of a pMUTIN vector into the target gene has three consequences: (1) the target gene is inactivated; (2) lacZ becomes transcriptionally fused to the gene, allowing its expression pattern to be monitored; (3) the Pspac promoter controls the transcription of downstream genes in an IPTG-dependent fashion. This last feature is important because B. subtilis genes are often organized in operons. The potential polar effects generated by the integration of the vectors can be alleviated by addition of IPTG. Also, conditional mutants of essential genes can be obtained by integrating pMUTIN vectors upstream of the target gene. The vectors are currently being used for systematic inactivation of genes without known function within the B. subtilis European consortium. pMUTIN characteristics and the inactivation of eight genes in the resA-serA region of the chromosome are presented.

Bacillus subtilis↗
PubMed2005

Topological insulators inhibit diffusion of transcription-induced positive supercoils in the chromosome of Escherichia coli.

The double helical nature of DNA implies that progression of transcription machinery that cannot rotate easily around the DNA axis creates waves of positive supercoils ahead of it and negative supercoils behind it. Using topological reporters that detect local variations in DNA supercoiling, we have characterized the diffusion of transcription-induced (TI) positive supercoils in plasmids or in the chromosome of wild type Escherichia coli cells. Transcription-induced positive supercoils were able to diffuse and affect local supercoiling several kilobases away from the site of origin. By testing the effect of various DNA sequences, these reporters enabled us to identify elements that impede supercoil diffusion, i.e. behave as topological insulators. All the elements tested correspond to DNA gyrase catalytic targets. These results correlate the ability of a DNA sequence to be cleaved by DNA gyrase with topological insulator activity. Implications of the asymmetry in supercoil diffusion for the control of DNA topology are discussed.

Chromosomes, Bacterial↗
PubMed1987

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↗
PubMed1990

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↗
PubMed1987

Regulation of the Escherichia coli uvrD gene in vivo.

The roles of two putative promoter sequences, P1 and P2, and a potential antiterminator sequence found in the uvrD control region were examined in vivo. Constitutive and SOS-induced levels of uvrD mRNA were determined by S1 mapping, and it was shown that the majority of uvrD transcripts are from P1, while P2 plays only a minor role. A series of increasing deletions from the 5' end of the uvrD gene was used to assay transcription in the promoterless vector pKO-1. Loss of just the -35 region of P1 was sufficient to switch off detectable transcription from both P1 and P2. Disruption of the antiterminator by site-specific mutagenesis had no effect on constitutive levels of transcription, but led to a significant increase over wild-type levels following SOS induction. This suggests that the attenuator comes into play following DNA damage to moderate the increase in UvrD protein synthesis.

Adenosine Triphosphatases↗