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PubMed1998

Autocrine TGFalpha expression in the regulation of initiation of human colon carcinoma growth.

Previously, we reported that unaggressive, growth factor-dependent FET human colon carcinoma cells downregulated their transforming growth factor alpha (TGFalpha) expression in a quiescent state (G0/G1) induced by growth factor and nutrient deprivation (Mulder, 1991, Cancer Res., 51:2256-2262). In contrast, highly aggressive, growth factor-independent HCT116 human colon carcinoma cells aberrantly upregulated this autocrine activity in the quiescent state (Mulder, 1991, Cancer Res., 51:2256-2262; Howell et al., 1998, Mol. Cell. Biol., 18:303-313). In this report, the role of autocrine TGFalpha and the mechanism of its regulation of expression during reentry into the cell cycle from a noncycling growth state were determined in FET cells. Optimal induction of DNA synthesis from a quiescent state in FET cells is dependent upon autocrine TGFalpha as well as exogenous transferrin and insulin. Reentry into the cell cycle resulting from treatment with exogenous transferrin and insulin resulted in approximately 3-fold induction of TGFalpha expression within 1 hr. TGFalpha induction was controlled at the transcription level, and the cis-controlling element was localized to the region between bp -370 - -201 relative to the translation start codon within the TGFalpha promoter. Thus neutralization of autocrine TGFalpha protein revealed that the induced TGFalpha autocrine activity was necessary for DNA synthesis and acted only in the early G1 phase of the cell cycle. Blockade of autocrine TGFalpha expression early in the cell cycle resulted in the reduction of DNA synthesis, whereas treatment with neutralization antibody at later times had no effect. This suggested that autocrine TGFalpha functions to initiate cell growth from noncycling states. This was further confirmed by the dependence of FET cells upon autocrine TGFalpha for colony formation in experiments where the plating density was sufficiently low to generate a lag phase in tissue culture. In contrast, TGFalpha autocrine activity was not required for exponential phase cells, as evidenced by the failure of TGFalpha neutralizing antibody to inhibit proliferation in this growth state. Taken together, these results suggest that autocrine TGFalpha acts primarily in the process of growth initiation by moving cells from a noncycling state back into the cell cycle, rather than supporting cell growth already initiated.

Autocrine Communication↗
PubMed2024

A Proteogenomic Pipeline for the Analysis of Protein Biosynthesis Errors in the Human Pathogen Candida albicans.

Candida albicans is a diploid pathogen known for its ability to live as a commensal fungus in healthy individuals but causing both superficial infections and disseminated candidiasis in immunocompromised patients where it is associated with high morbidity and mortality. Its success in colonizing the human host is attributed to a wide range of virulence traits that modulate interactions between the host and the pathogen, such as optimal growth rate at 37 °C, the ability to switch between yeast and hyphal forms, and a remarkable genomic and phenotypic plasticity. A fascinating aspect of its biology is a prominent heterogeneous proteome that arises from frequent genomic rearrangements, high allelic variation, and high levels of amino acid misincorporations in proteins. This leads to increased morphological and physiological phenotypic diversity of high adaptive potential, but the scope of such protein mistranslation is poorly understood due to technical difficulties in detecting and quantifying amino acid misincorporation events in complex protein samples. We have developed and optimized mass spectrometry and bioinformatics pipelines capable of identifying rare amino acid misincorporation events at the proteome level. We have also analyzed the proteomic profile of an engineered C. albicans strain that exhibits high level of leucine misincorporation at protein CUG sites and employed an in vivo quantitative gain-of-function fluorescence reporter system to validate our LC-MS/MS data. C. albicans misincorporates amino acids above the background level at protein sites of diverse codons, particularly at CUG, confirming our previous data on the quantification of leucine incorporation at single CUG sites of recombinant reporter proteins, but increasing misincorporation of Leucine at these sites does not alter the translational fidelity of the other codons. These findings indicate that the C. albicans statistical proteome exceeds prior estimates, suggesting that its highly plastic phenome may also be modulated by environmental factors due to translational ambiguity.

Candida albicans↗
PubMed1992

Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991.

Amino Acid Sequence↗
PubMed2002

Characterization of mycoplasma arginine deiminase expressed in E. coli and its inhibitory regulation of nitric oxide synthesis.

We previously reported that a cytostatic protein that is found in ASC-17D Sertoli cell-conditioned media was Mycoplasma arginine deiminase (ADI), which hydrolyzes L-arginine into L-citrulline and ammonia. Here, we report the over-expression of recombinant ADI (rADI) in E. coli and the down-regulation of lipopolysaccharide (LPS) induced-nitric oxide (NO) production by rADI treatment. We cloned the ADI gene from Mycoplasma arginini genomic DNA by a polymerase chain reaction, and changed five TGA tryptophan codons (stop codon in E. coli) to TGG codons in the coding region by site-directed mutagenesis in order to express in E. coli. The rADI was purified to apparent homogeneity by DEAE-Sepharose and arginine-affinity chromatography. The rADI expressed in E. coli was identified as 45 kDa on SDS-PAGE and 90 kDa on native PAGE, implying that it exists as a dimer like ADI of M. arginini. The Km for arginine of rADI was approximately 370+/-50 microM. Its optimal temperature and pH were 41 degrees C and pH 6.4, respectively, and enzyme activity remained > or = 50% for 5 d at physiological temperature and pH. Treatment of purified rADI suppressed NO production in macrophage-like RAW 264.7 and primary glial cells that were exposed to LPS. Furthermore, an intraperitoneal injection of rADI significantly suppressed the rise of blood nitrite/nitrate levels that were induced by the systemic administration of bacterial endotoxin LPS to mice, resulting in an improvement in their survival rate. These results suggest that the depletion of blood arginine with an arginine-metabolizing enzyme, such as ADI, could suppress excessive production of NO that is caused by inducible NOS (iNOS) during the endotoxemia. Also, rADI may be used as a new approach to control NO-related diseases, such as sepsis.

Animals↗
PubMed2001

Application of genetic semihomology algorithm to theoretical studies on various protein families.

Several protein families of different nature were studied for genetic relationship, correct alignment at non-homologous fragments, optimal sequence consensus construction, and confirmation of their actual relevance. A comparison of the genetic semihomology approach with statistical approaches indicates a high accuracy and cognition significance of the former. This is particularly pronounced in the study of related proteins that show a low degree of homology. The sequence multiple alignments were verified and corrected with respect to the questionable, non-homologous fragments. The verified alignments were the basis for consensus sequence formation. The frequency of six-codon amino acids occurrence versus position variability was studied and their possible role in amino acid mutational exchange at variable positions is discussed.

Algorithms↗
PubMed2005

Detection of TP53 mutation using a portable surface plasmon resonance DNA-based biosensor.

A DNA-based surface plasmon resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETA SPR-EVM-BT, from Texas Instruments. A direct immobilisation procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridisation reactions between the immobilised probe and a short sequence (26 mer) complementary, non-complementary and one-point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples, DNA extracted from both normal, wild-type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base; and hence, it appears to be a strong candidate technique for the detection of gene mutation.

Biosensing Techniques↗
PubMed2004

Optimal designing of beta-conglycinin to genetically incorporate RPLKPW, a potent anti-hypertensive peptide.

Previously, we introduced the RPLKPW sequence, a highly potent hypotensive peptide designed based on ovokinin (2-7), into three homologous sites in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The modified protein expressed in Escherichia coli reduced blood pressure of spontaneously hypertensive rats (SHRs) after oral administration at a dose of 10 mg/kg, which suggested about 30% of the introduced peptide was released in vivo. In this study amino acid residues around the RPLKPW sequence were optimized with a use of synthetic peptides to facilitate release of RPLKPW by gastrointestinal proteases. Then, fourth RPLKPW was also introduced into the extension domain of the protein. The newly modified protein, which was produced in E. coli, significantly lowered blood pressure in SHRs at a dose of 2.5 mg/kg 4 h after oral administration. Furthermore, we produced an extension domain that corresponds to residues 1-143 of the modified alpha' subunit containing four RPLKPW sequences by introducing a termination codon. The minimum effective dose of the modified extension domain was 1.0 mg/kg, which is 1/2000 that of ovalbumin.

Administration, Oral↗
PubMed1994

Biochemical characterization of human PC-1, an enzyme possessing alkaline phosphodiesterase I and nucleotide pyrophosphatase activities.

PC-1 is an ecto-enzyme possessing alkaline phosphodiesterase I and nucleotide pyrophosphatase activities. In this paper, we demonstrate the expression, biochemical characterization and biosynthesis of human PC-1. Previously, there has been uncertainty concerning which of two methionine residues is the initiator. It is now shown that expression of PC-1 is much greater if the first methionine residue is present, and that the sequence between the two methionine residues is translated in both human and mouse, in both transfected cells and cells naturally expressing PC-1. The first methionine residue is therefore the initiator. Human PC-1 is capable of autophosphorylation, and conditions are described in which PC-1 is the only labelled phosphoprotein on the plasma membranes of intact cells, allowing the demonstration that the mature membrane form of human PC-1 is approximately 10 kDa larger than that of the mouse form. Pulse-chase biosynthetic studies and treatment with two different endoglycosidases show that most of this difference is due to N-linked oligosaccharides. The polypeptide backbone of human PC-1 is 20 amino acids longer than that of the mouse PC-1, with most of the difference in polypeptide chain length being in the cytoplasmic domain. The revised cytoplasmic domain of human PC-1 has 76 amino acids, while the mouse cytoplasmic domain has 58 amino acids. Optimal alignment of mouse and human cytoplasmic domains reveals areas of sequence conservation in which the third bases vary. It is suggested that these regions of conservation may point to functionally important sequences in the cytoplasmic domain.

Amidohydrolases↗
PubMed1993

Heterogeneous mutations in the gene encoding the alpha-subunit of the stimulatory G protein of adenylyl cyclase in Albright hereditary osteodystrophy.

Albright hereditary osteodystrophy (AHO) is an inherited disorder associated with deficient activity of the alpha-subunit of the guanine nucleotide-binding regulatory protein (Gs alpha) that couples receptors to adenylyl cyclase. To identify mutations that lead to Gs alpha deficiency, we isolated genomic DNA from patients with AHO and used the polymerase chain reaction to amplify exons of the Gs alpha genes. DNA was amplified using intron-specific oligonucleotide primers flanking exons of the Gs alpha gene. To optimize our ability to detect mutations, one oligonucleotide from each primer pair was synthesized with a 5' GC-clamp. Amplified Gs alpha gene fragments were analyzed by denaturing gradient gel electrophoresis in order to detect mutations that alter the melting point of the double-stranded DNA fragment. Using this technique, we have identified and characterized three mutations and one neutral polymorphism. The polymorphism, located in exon 5, consisted of a T-->C substitution that conserves the isoleucine residue at codon 131 (ATT-->ATC). Two mutations were missense mutations, which in one family consisted of a nucleotide substitution (T-->C) in exon 4 that results in replacement of Leu by Pro at codon 99 of the Gs alpha molecule. Affected subjects in a second family had a single base (C-->T) mutation in exon 6 that resulted in replacement of Arg by Cys at codon 165. A 4-base pair deletion (GTGG) in exon 8 at position +214 was identified in one Gs alpha allele from each affected subject in the third family. This mutation causes a frameshift after the codon for Gln213 that results in a premature stop codon 81 base pair after the deletion. Immunoblot analysis of plasma membranes prepared from cultured fibroblasts or erythrocytes indicated that levels of immunoactive Gs alpha protein were decreased in all affected subjects. We conclude that heterogeneous mutations in the gene encoding Gs alpha, including deletions and single amino acid substitutions, are responsible for Gs alpha deficiency in AHO.

Adenylyl Cyclases↗
PubMed2002

Expression of enzymatically-active phospholipase Cgamma2 in E. coli.

Phospholipase C-gamma-2 (PLCgamma2) activation is a key signaling event for many cell functions. In order to delineate the pathways that lead to PLCgamma2 activation, we devised a quick method for obtaining sufficient PLCgamma2. We obtained the full-length cDNA for human PLCgamma2 and expressed it in E. coli using the expression vector pT5T. To enhance the protein expression, tandem AGG-AGG arginine codons at the amino acid positions 1204-1205 were replaced by CGG-CGG arginine codons. The protein expression was detected in a Western blot analysis by both anti-PLCgamma2 antibodies and the antibodies that are raised against the tripeptide epitope (Glu-Glu-Phe) tag that are genetically-engineered to its carboxyl terminal. Crude lysates that were prepared from bacteria that express PLCgamma2 were found to catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate. Similar to previous reports on PLCgamma2 that is isolated from mammalian tissue, the recombinant enzyme was Ca2+ dependent with optimal activity at 1-10 microM Ca2+.

Calcium↗
PubMed1996

A phosphoglucomutase-like gene essential for the optimal expression of methicillin resistance in Staphylococcus aureus: molecular cloning and DNA sequencing.

We describe here the cloning and sequencing of a new auxiliary gene identified by Tn551 insertional mutagenesis of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL. The insertionally inactivated mutant RUSA315 had intact mecA and normal amounts of PBP2A, but drastically reduced antibiotic resistance (drop in methicillin MIC from 1600 to 1.5 micrograms ml-1), a unique heterogeneous phenotype, and a compositional change in the cell wall characterized by the complete disappearance of the unsubstituted disaccharide pentapeptide from the peptidoglycan. Cloning in E. coli followed by sequencing located the Tn551 insert omega 720 in an open reading frame of 451 codons, provisionally called femR315, defining a polypeptide with a deduced amino acid sequence that showed over 26% sequence identity and 57% overall sequence similarity with the phosphoglucomutase (PGM) gene of E. coli. The Tn551 insertion site of a previously described mutant 12F (femD) also lies in the same gene as femR315. The wild-type form of femR315 subcloned in a shuttle vector fully restored expression of high level (parental) methicillin resistance in mutant RUSA315. The exact biochemical function of femR315 is not known. However, enzymes similar to PGM catalyze the isomerization of hexose and hexosamine phosphates leading to the formation of glucosamine-1-P, which is an obligate precursor in the biosynthesis of UDP-N-acetylglucosamine (UDP-NAGA). We propose that the suppression of methicillin resistance in RUSA315 is related to some functional or quantitative abnormality of UDP-NAGA metabolism.

Amino Acid Sequence↗
PubMed1997

Phylogenetic relationships of the liverworts (Hepaticae), a basal embryophyte lineage, inferred from nucleotide sequence data of the chloroplast gene rbcL.

Sequence data from the chloroplast-encoded gene rbcL were obtained for 24 liverworts, a basal group of embryophytes. Maximum likelihood and parsimony analyses of these data, along with data from other major green plant lineages, confirm hypotheses based on morphological data, such as the paraphyly of bryophytes, and the basal position of liverworts. Molecular data corroborate the deep separation between the complex thalloid and leafy/simple thalloid liverworts implied by morphological data, but the monophyly of liverworts could not be rejected. The effects of accounting for site-to-site rate heterogeneity in these data were examined using maximum likelihood methods. Comparison of trees obtained with and without rate heterogeneity showed that simply allowing for heterogeneity had a greater improvement on likelihood score than optimization of transition/transversion bias. Incorporation of site-to-site rate heterogeneity in the larger analysis, however, did not necessarily change which topology was favored. Properties of rbcL sequences from the two liverwort groups were compared. Significantly different substitution rates were found between leafy/simple thalloid and complex thalloid liverwort taxa, with rates of rbcL sequence evolution in leafy/simple thalloid taxa being higher and more indicative of those of vascular plants, and with those of complex thalloid taxa (such as Marchantia) being slower. Codon usage in rbcL in complex thalloid liverworts was biased toward NNU and NNA, compared to the leafy/simple thalloid liverworts. Although base composition and relative substitution rates differed between the two groups, no significant differences were detected within each of the two groups of liverworts. The signal present in first and second codon sites versus third codon sites was compared. While the third codon positions in rbcL across this taxon sampling are highly variable (with only 15 constant sites of 439), the trees obtained were in general agreement with trees from the entire data set and with trees obtained from independent sources of data. The presence of signal in third codon positions across greater than 400 MY of plant evolution means that definitions of saturation based on pair-wise comparisons of sequences inadequately assess phylogenetic signal.

Chloroplasts↗
PubMed1986

Isolation of a cDNA coding for human galactosyltransferase.

Human milk galactosyltransferase (EC 2.4.1.22) was purified to homogeneity using affinity chromatography. Edman degradation was used to determine the amino acid sequences of eight peptide fragments isolated from the purified enzyme. A 60-mer "optimal" oligonucleotide probe that corresponded to the amino acid sequence of one of the galactosyltransferase peptide fragments was constructed and used to screen a lambda gt10 cDNA library. Two hybridization-positive recombinant phages, each with a 1.7 Kbp insert, were detected among 3 X 10(6) recombinant lambda gt10 phages. Sequencing of one of the cDNA inserts revealed a 783 bp galactosyltransferase coding sequence. The remainder of the sequence corresponded to the 3'-region of the mRNA downstream from the termination codon.

Amino Acid Sequence↗
PubMed1996

Mutational analysis of a transmembrane segment in a bacterial chemoreceptor.

Trg is a member of a family of receptors that mediates chemotaxis by Escherichia coli. Its transmembrane domain is a loose four-helix bundle consisting of two helices from each of the two identical subunits. This domain mediates transmembrane signaling through a conformational change in which the second transmembrane segment (TM2) is thought to move relative to TM1, but mutational analysis of TM2 by cysteine scanning had identified only a few positions at which substitutions perturbed function or induced signaling. Thus, we performed mutational analysis by random mutagenesis and screening. Among 42 single-residue substitutions in TM2 that detectably altered function, 16 had drastic effects on receptor activity. These substitutions defined a helical face of TM2. This functionally important surface was directed into the protein interior of the transmembrane domain, where TM2 faces the helices or the other subunit. The functionally perturbing substitutions did not appear to cause general disruption of receptor structure but rather had more specific effects, altering aspects of transmembrane signaling. An in vivo assay of signaling identified some substitutions that reduced and others that induced signaling. These two classes were distributed along adjacent helical faces in a pattern that strongly supports the notion that conformational signaling involves movement between TM2 and TM1 and that signaling is optimal when stable interactions are maintained across the interface between the homologous helices in the transmembrane domain. Our mutational analysis also revealed a striking tolerance of the chemoreceptor for substitutions, including charged residues, usually considered to be disruptive of transmembrane segments.

Amino Acid Sequence↗
PubMed2000

Mutational analysis of the GB virus B internal ribosome entry site.

GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5' nontranslated GBV-B RNA (5'NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5'NTR (M. Honda, et al. RNA 2:955-968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5' end by structural domain II, a location analogous to the 5' limit of the IRES in both the HCV and pestivirus 5'NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3' limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.

5' Untranslated Regions↗
PubMed2000

Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems.

Monocotyledonous crop plants are usually more resistant to herbicides than grass weeds and most dicots. Their resistance to herbicides is mediated in many cases by P450 oxygenases. Monocots thus constitute an appealing source of P450 enzymes for manipulating herbicide resistance and recombinant forms of the major xenobiotic metabolizing mooxygenases are potential tools for the optimization of new active molecules. We report here the isolation and functional characterization of the first P450 and P450 reductase coding sequences from wheat. The first attempts at expressing these cDNAs in yeast and tobacco led to levels of protein, which were extremely low, often not even detectable. The wheat P450 cDNAs were efficiently transcribed, but no protein or activity was found. Wheat coding sequences, like those of other monocots, are characterized by a high GC content and by a related strong bias of codon usage, different from that observed in yeast or dicots. Complete recoding of genes being costly, the reengineering their 5'-end using a single PCR megaprimer designed to comply with codon usage of the host was attempted. It was sufficient to relieve translation inhibition and to obtain good levels of protein expression. The same strategy also resulted in a dramatic increase in protein expression in tobacco. A basis for the success of such a partial recoding strategy, much easier and cheaper than complete recoding of the cDNA, is proposed.

Amino Acid Sequence↗
PubMed1996

Extensive regions of pol are required for efficient human immunodeficiency virus polyprotein processing and particle maturation.

Human immunodeficiency type 1 particle maturation is dependent upon proteolytic cleavage of the gag and gag-pol precursors by the pol-encoded viral protease. We have investigated the importance of domains of pol other than the protease for particle maturation and gag proteolytic processing. Truncations of the gag-pol polyprotein precursor of HIV-1 were created by deleting segments of the reverse transcriptase coding region or by introducing stop codons in the integrase region of an HIV-1 infectious molecular clone. In these mutants, the protease coding sequence was left intact. Particles produced by all of the mutants displayed abnormal morphologies and impaired proteolytic processing of gag. The severity of particle morphology abnormalities and of gag polyprotein processing impairment appeared to be affected both by the size and by the position of the deletions in pol, suggesting that the integrity of several pol domains within the gag-pol precursor is required for optimal protease activation and particle maturation. Additionally, cotransfection of a deletion mutant with wild-type provirus led to a marked reduction in the titer of infectious virus, suggesting that truncated gag-pol precursors can interfere with wild-type virus assembly and maturation.

Cloning, Molecular↗
PubMed1994

A copy-number-controlled expression vector for the fission yeast Schizosaccharomyces pombe.

A novel expression vector for the fission yeast Schizosaccharomyces pombe carries the neomycin-resistance-encoding gene regulated by the SV40 early promoter, and its copy number is controlled by the level of Geneticin (G418). Foreign gene expression is driven by the human cytomegalovirus (hCMV) promoter which is transcriptionally active in S. pombe. Moreover, the vector expresses foreign genes at high levels, due to the 5'-untranslated region (5'-UTR) containing an A + T-rich sequence of about 50 nucleotides located between the TATA box of the hCMV promoter and the start codon. Recombinant human lipocortin I was produced at levels of up to 50% of the total soluble protein in the presence of 100-200 micrograms/ml of G418 in the media. Southern and Northern blotting showed that this high level of expression was due to an increase in copy number induced by G418, the high transcriptional activity of the hCMV promoter and the high translational efficiency of the 5'-UTR. We modified the vector into an 'ATG vector', named pTL2M, that maintains the 5'-UTR optimized for gene expression and into which any foreign gene, whose exact sequence is known, can be easily inserted.

Annexin A1↗