Increased synthesis of human parathyroid hormone in Escherichia coli through alterations of the 5′ untranslated region

The expression of human parathyroid hormone (hPTH) in Escherichia coli was optimized by variations of the spacing sequence between the ribosome-binding site (RBS) and the beginning of the gene (ATG) and by increasing the complementarity of the RBS to the 16 S rRNA. The expression level of 3 μg/liter increased more than 100-fold to 475 μg/liter as a direct consequence of modifications in the region 5′ of the gene.     

The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli

A gene coding for bovine pancreatic DNase I has been constructed from synthetic oligonucleotides. This gene has been cloned into a plasmid vector pDOC55 designed to allow very tight control of expression of potentially lethal proteins. Induction of protein synthesis from the gene yielded a peptide of molecular weight of approximately 31,000, consistent with DNase I. The yield of this protein from the pDOC55 construct (pAW5) was approximately 150 micrograms/liter of cell culture. Attempts to clone the gene into a less tightly controlled expression vector based on the tac-promoter (pKK223-3) were unsuccessful, presumably due to the expected lethality of the product. Mutagenesis of the gene to replace the active site histidine (His-134) in the protein with glutamine yielded a gene readily clonable into both expression systems. Yields of the mutagenized protein were approximately 6 micrograms/liter from a pDOC55 system and 20 mg/liter from a pKK223-3 system. The activity of the proteins were assayed using the Kunitz procedure and their cleavage selectivities by digestion of the Escherichia coli tyr T promoter. The recombinant native enzyme had both the same specific activity and DNA cleavage selectivity as the protein isolated from bovine pancreas using these two assays. The H134Q mutant had a specific activity of about 0.001% of the native protein but had an unaltered DNA cleavage selectivity.     

Establishment and characterization of Roberts syndrome and SC phocomelia model medaka (Oryzias latipes)

Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.     

High-level expression of porcine liver cytochrome P-450 reductase catalytic domain in Escherichia coli by modulating the predicted local secondary structure of mRNA

A direct expression system for the solubilized catalytic domains of NADPH-cytochrome P-450 reductase (sCPR) from rat (RsCPR) and porcine (PsCPR) in Escherichia coli cells was constructed using the expression plasmid pCWori(+). PsCPR was minimally expressed, whereas RsCPR was highly expressed. Replacement of the nucleotides encoding Thr(60)Ser(61)Ser(62) in PsCPR with those for Ala(60)Pro(61)Pro(62) in RsCPR markedly increased the expression level of the protein. The local secondary structures of the mRNAs, which were predicted with the prediction program GeneBee (http://www.genebee.msu.su), suggested that the intramolecular double strand between the ribosome binding site (RBS) and the Thr(60)Ser(61)Ser(62) codons in PsCPR, and/or the base-pairing at the initiation codon of the mRNAs significantly affected protein expression. Silent mutations were systematically introduced into the codons for Thr(58) and Thr(60)Ser(61) in PsCPR to modulate the local secondary structure of the mRNA. The expression level of the silently mutated PsCPR suggests that the expression level of PsCPR depends on the stability of the local structure at the RBS in the mRNA. A high-level expression system for wild-type PsCPR was constructed by introducing silent mutations at the codons for Thr(60)Ser(61) in PsCPR. The purified PsCPR showed the characteristic absorption spectral changes of sCPR after reduction with NADPH. The yield of purified PsCPR from 1 liter of culture fluid was 45.8 mg. These results substantiate that the introduction of silent mutations in the section of the gene encoding the N-terminal region of the protein based on the predicted local secondary structure of the mRNA at the RBS is a useful approach to control and increase the expression level of heterologous proteins in E. coli cells.     

Use of a portable ribosome-binding site for maximizing expression of a eukaryotic gene in Escherichia coli

To maximize expression of a eukaryotic gene in Escherichia coli, a series of plasmids were constructed containing various synthetic ribosome-binding sites (RBS). These sites consist of a Shine-Dalgarno (SD) region (with translation stop codons in all three reading frames) positioned at distances 5-9 nucleotides (nt) from the AUG initiator codon of the gene coding for human T-cell growth factor (TCGF or IL-2). The region encompassing the RBS through the TCGF structural gene from each of these plasmids was inserted as a 'cassette' into seven different E. coli expression vectors, and TCGF production was measured. Our results demonstrate a greater than 2000-fold range of TCGF synthesis dependent upon the promoter and the synthetic RBS used. The translational efficiency of the TCGF gene was found to be influenced by the quality of the RBS, which is in part determined by the external sequence context of this site. The synthetic RBS, containing the necessary information for the translation initiation process, readily accessible by restriction sites, should be of general usefulness in obtaining maximum expression of eukaryotic genes in E. coli.     

Effects of GUG and AUG initiation codons on the expression of lacZ in Escherichia coli

We have replaced the ribosomal binding site (RBS) of the lacZ gene of E. coli by those of the maturation (A) gene of phage MS2 and that of the tufA gene. Both RBSs contain a GUG initiation codon. The expression with the tufA RBS is at least 25-fold higher than with the phage RBS. Changing the GUG into AUG results in a 3-fold increase in expression in both cases. In general, higher expression is accompanied by an increase of lac-specific mRNA. It is argued that this is a consequence of the more efficient translation of the mRNA.     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dissolved organic carbon and second substrates on the biodegradation of organic compounds at low concentrations.

Pseudomonas acidovorans and Pseudomonas sp. strain ANL but not Salmonella typhimurium grew in an inorganic salts solution. The growth of P. acidovorans in this solution was not enhanced by the addition of 2.0 micrograms of phenol per liter, but the phenol was mineralized. Mineralization of 2.0 micrograms of phenol per liter by P. acidovorans was delayed 16 h by 70 micrograms of acetate per liter, and the delay was lengthened by increasing acetate concentrations, whereas phenol and acetate were utilized simultaneously at concentrations of 2.0 and 13 micrograms/liter, respectively. Growth of Pseudomonas sp. in the inorganic salts solution was not affected by the addition of 3.0 micrograms each of glucose and aniline per liter, nor was mineralization of the two compounds detected during the initial period of growth. However, mineralization of both substrates by this organism occurred simultaneously during the latter phases of growth and after growth had ended at the expense of the uncharacterized dissolved organic compounds in the salts solution. In contrast, when Pseudomonas sp. was grown in the salts solution supplemented with 300 micrograms each of glucose and aniline, the sugar was mineralized first, and aniline was mineralized only after much of the glucose carbon was converted to CO2. S. typhimurium failed to multiply in the salts solution with 1.0 micrograms of glucose per liter. It grew slightly but mineralized little of the sugar at 5.0 micrograms/liter, but its population density rose at 10 micrograms of glucose per liter or higher. The hexose could be mineralized at 0.5 micrograms/liter, however, if the solution contained 5.0 mg of arabinose per liter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of premature translational termination in the regulation of expression of the phi X174 lysis gene

Expression of the phi X174 lysis (E) gene, a member of an overlapping gene pair, appears to depend on a frameshift-induced chain termination by ribosomes translating the upstream D gene. A -1 reading frameshift, possibly induced by misreading of an alanine codon as a doublet, causes ribosomes to terminate translation at two different sites, suggesting two modes of regulating expression of the E gene. One frameshift can cause translational termination at a stop codon(s) near the E gene ribosome binding site (RBS), resulting in reinitiation by ribosomes at the E gene RBS. Termination at a second site some 70 bases upstream from the E gene RBS, while too far away to allow ribosomal re-initiation at the E gene RBS, probably results in an unmasking of the message, allowing entry of a new ribosome at the E gene RBS.     

Efficacy of copper and silver ions and reduced levels of free chlorine in inactivation of Legionella pneumophila.

Water disinfection systems utilizing electrolytically generated copper and silver ions (200 and 20, 400 and 40, or 800 and 80 micrograms/liter) and low levels of free chlorine (0.1 to 0.4 mg/liter) were evaluated at room (21 to 23 degrees C) and elevated (39 to 40 degrees C) temperatures in filtered well water (pH 7.3) for their efficacy in inactivating Legionella pneumophila (ATCC 33155). At room temperature, a contact time of at least 24 h was necessary for copper and silver (400 and 40 micrograms/liter) to achieve a 3-log10 reduction in bacterial numbers. As the copper and silver concentration increased to 800 and 80 micrograms/liter, the inactivation rate significantly (P less than or equal to 0.05) increased from K = 2.87 x 10(-3) to K = 7.50 x 10(-3) (log10 reduction per minute). In water systems with and without copper and silver (400 and 40 micrograms/liter), the inactivation rates significantly increased as the free chlorine concentration increased from 0.1 mg/liter (K = 0.397 log10 reduction per min) to 0.4 mg/liter (K = 1.047 log10 reduction per min). Compared to room temperature, no significant differences were observed when 0.2 mg of free chlorine per liter with and without 400 and 40 micrograms of copper and silver per liter was tested at 39 to 40 degrees C. All disinfection systems, regardless of temperature or free chlorine concentration, showed increase inactivation rates when 400 and 40 micrograms of copper and silver per liter was added; however, this trend was significant only at 0.4 mg of free chlorine per liter.     

Ribosomal Binding Site Switching: An Effective Strategy for High-Throughput Cloning Constructions

Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is essential for gene expression. Change from A/G to T/C in the RBS blocks its activity and thereby abolishes gene expression. Based on this property, we introduced an inactive RBS upstream of a selectable marker gene, and designed a fragment insertion site within this inactive RBS. Forward and reverse insertions of specifically tailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverse insertions will be eliminated due to the non-expression of the marker gene. The effectiveness of our strategy for TA cloning and blunt end ligation are confirmed. Application of this strategy to gene over-expression, a bacterial two-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure, together with its low cost and high efficiency, makes our strategy extremely useful in HTP cloning constructions.     

Assessment of bacterial growth and total organic carbon removal on granular activated carbon contactors.

The overall growth rate of bacteria on granular activated carbon (GAC) contactors at the Philadelphia Torresdale Water Treatment Pilot Plant facility was found to decrease until steady state was reached. The growth rate was found to fluctuate between 6.94 X 10(-3) and 8.68 X 10(-4) doublings per h. The microbiological removal of total organic carbon (TOC) was calculated by considering the GAC contactors as semiclosed continuous culture systems and using growth yield factors determined in laboratory experiments. After ozonation, the average TOC entering the contactors was 1,488 micrograms/liter, and the average effluent TOC was 497 micrograms/liter. Microbiological TOC removal was found to average 240 micrograms/liter on GAC contactors, which was not significantly different from microbiological TOC (220 micrograms/liter) removal across a parallel sand contactor where no adsorption took place. Thus, GAC did not appear to enhance biological TOC removal. Bacterial growth and maintenance was responsible for approximately 24% of the TOC removal on GAC under the conditions of this study.     

Roles of the sister chromatid cohesion apparatus in gene expression,development, and human syndromes

The sister chromatid cohesion apparatus mediates physical pairing of duplicated chromosomes. This pairing is essential for appropriate distribution of chromosomes into the daughter cells upon cell division. Recent evidence shows that the cohesion apparatus, which is a significant structural component of chromosomes during interphase, also affects gene expression and development. The Cornelia de Lange (CdLS) and Roberts/SC phocomelia (RBS/SC) genetic syndromes in humans are caused by mutations affecting components of the cohesion apparatus. Studies in Drosophila suggest that effects on gene expression are most likely responsible for developmental alterations in CdLS. Effects on chromatid cohesion are apparent in RBS/SC syndrome, but data from yeast and Drosophila point to the likelihood that changes in expression of genes located in heterochromatin could contribute to the developmental deficits.     

The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.     

‘ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli

A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage A or a large portion of the coding region of the Herpes simplex virus type l glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.     

Efficient translation in chloroplasts requires element(s) upstream of the putative ribosome binding site from atpI

Thousands of proteins make up a chloroplast, but fewer than 100 are encoded by the chloroplast genome. Despite this low number, expression of chloroplast-encoded genes is essential for plant survival. Every chloroplast has its own gene expression system with a major regulatory point at the initiation of protein synthesis (translation). In chloroplasts, most protein-encoding genes contain elements resembling the ribosome binding sites (RBS) found in prokaryotes. In vitro, these putative chloroplast ribosome binding sequences vary in their ability to support translation. Here we report results from an investigation into effects of the predicted RBS for the tobacco chloroplast atpI gene on translation in vivo. Two reporter constructs, differing only in their 5'-untranslated regions (5'UTRs) were stably incorporated into tobacco chloroplast genomes and their expression analyzed. One 5'UTR was derived from the wild-type (WT) atpI gene. The second, Holo-substitution (Holo-sub), had nonchloroplast sequence replacing all wild-type nucleotides, except for the putative RBS. The abundance of reporter RNA was the same for both 5'UTRs. However, translation controlled by Holo-sub was less than 4% that controlled by WT. These in vivo experiments support the idea that translation initiation in land plant chloroplasts depends on 5'UTR elements outside the putative RBS.     

Overexpression of the phage lambda lysozyme cloned in Escherichia coli: use of a degenerative mixture of synthetic ribosome binding sites and increase of the protein stability in vivo.

The R gene of the phage lambda coding for a lysozyme expressed at the end of an infection cycle in Escherichia coli has been cloned in a series of vector plasmids. Two methods for improving the efficiency of translation have been tested. First, the use of a bicistronic construction in which the ribosome binding site (RBS) of the first cistron is that of a highly expressed gene or the use of a degenerate mixture of synthetic oligonucleotides for the optimization of a RBS. The second strategy is more efficient: the analysis of a number of clones reveals that the LaL expression levels are increased by a factor between 3 and 6 times compared with the clone using the natural RBS. The expression levels are described by an approximately Gaussian histogram. The translation promoter that was found to afford the best expression (PL) is under the control of a thermolabile repressor. Under the expression conditions, the protein is partially proteolysed. The proteolysis is significantly decreased by adding salt to the growth medium. After optimization, an increase in expression by a factor of 40 is obtained compared with the initial conditions. An efficient purification protocol is described.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water.

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.