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  • 1
    ISSN: 1432-0827
    Keywords: Amelogenin ; Expression ; Enamel ; Recombinant DNA ; Tooth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Physics
    Notes: Abstract A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20–50 mg of 95–99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4–8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Journal of cancer research and clinical oncology 120 (1994), S. 454-464 
    ISSN: 1432-1335
    Keywords: Xeroderma pigmentosum fibroblasts ; DNA repair ; Recombinant DNA ; cDNA libraries ; Phage λgt10 ; pBluescript vector ; In vitro transcripts ; Differential hybridization ; Mitochondrial neuromyopathies ; Genetic defects ; Uracil-DNA glycosylase ; Glyceraldehyde-3-phosphate dehydrogenase ; Lactate dehydrogenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Differential hybridization was used to detect repair defects in xeroderma pigmentosum (XP) that are not amenable to current analyses. cDNA libraries were constructed from cytoplasmic RNA of normal and XP fibroblast strains (complementation groups A and D) and analyzed for differential gene expression. More than 40000 λgt10 cDNA clones were differentially screened with in vitro transcripts made from cDNA in the pBluescript vector. Six differential clones were detected in the libraries of the XP group A and D strains which caused stronger or weaker signals when probed with transcripts from XP strains than with those from the normal strains. Two clones coded for mitochondrial genes: mitochondrial 16 S rRNA and ATPase 6L. Overexpression of mitochondrial genes in XP may indicate that functions of the ATP-generating system are impaired since such functions are intensified whenever they become insufficient, for example as a consequence of DNA damage. It is tempting to assume that abnormal mitochondria are one of the causes for the neurological malfunctions in XP. Furthermore, densitometric analysis of Northern blots revealed that mRNA of lactate dehydrogenase, chain M, was less abundant in four XP group A strains (extent of reduction: 70%) and in two XP group D strains (extent of reduction: 58%). Enzyme activity was also diminished. In addition, mRNA of the gene for glyceraldehyde-3-phosphate dehydrogenase was less expressed in the same XP group A and D fibroblast strains investigated (reduction in both complementation groups: 50%). Both glycolytic enzymes have nuclear functions apart from their role in sugar metabolism. Lactate dehydrogenase, chain M, is identical to a helix-destabilizing protein; it is closely associated with chromatin and unfolded DNA, suggesting a role in DNA synthesis and transcription. The 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase is involved in transcription and was shown to be identical to uracil-DNA glycosylase, a base-excision repair enzyme. We presume that the nuclear functions of these glycolytic enzymes may be thwarted in the XP strains investigated and may account for malfunctions in XP, particularly for neurological disturbances.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 243 (1994), S. 253-260 
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Saccharomyces cerevisiae ; Endo-β-glucanase ; Endo-xylanase ; Heterologous expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have developed a method for fast and efficient isolation of enzyme genes from filamentous fungi by combining the ability of Saccharomyces cerevisiae to express heterologous genes with the utilisation of sensitive and reliable enzyme assays. A cDNA library from the fungus Humicola insolens was constructed in a S. cerevisiae/Escherichia coli shuttle vector in E. coli. Sub-pools of the library were subsequently screened for enzyme activity in S. cerevisiae. More than 130 clones were identified as positive in either an endo-β-glucanase or an endo-xylanase assay. Based on a partial characterization of the DNA sequence of the individual clones, they could be grouped into five distinct types of endo-β-glucanases and three types of endo-xylanases. A representative cDNA from each type was sub-cloned in an Aspergillus vector and expressed in A. oryzae. The new cloning method may be an important alternative to traditional cloning methods based on amino acid sequence information.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 10 (1994), S. 659-662 
    ISSN: 0749-503X
    Keywords: Recombinant DNA ; purine salvage enzymes ; conserved sequences ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nucleotide sequence of a Saccharomyces cerevisiae gene encoding a potential adenine phosphoribosyltransferase (APRT) has been determined. The protein encoded by this gene shows a high degree of similarity with APRTs from a variety of other species. The S. cerevisiae gene, named APT2, has been mapped to chromosome IV. The sequence has been deposited in the GenBank data library under Accession Number L14434.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 23 (1993), S. 547-548 
    ISSN: 1432-0983
    Keywords: Autonomously replicating sequence ; Recombinant DNA ; Nested deletions ; Mutagenesis ; Schizosaccharomyces pombe
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Plasmids pSP1 and pSP2 are two new Schizosaccharomyces pombe ars1 multicopy vectors with the Saccharomyces cerevisiae LEU2 and URA3 genes as selectable markers. They are derivatives of S. cerevisiae integrative plasmids. These plasmids allow classical molecular genetic techniques, such as mutagenesis, nested deletions and sequencing, to be performed directly.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 23 (1993), S. 435-442 
    ISSN: 1432-0983
    Keywords: Heat shock ; Recombinant DNA ; Membrane protein ; Nutritional limitation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using differential hybridization, a gene preferentially expressed during entry into stationary phase has been isolated. Subsequent analysis indicated that this gene corresponds to a heat-shock gene. The nucleotide sequence has been determined. It revealed a 332 aminoacid protein. No similarities to any previously known protein have been noted. The protein is very hydrophobic and is predicted to have a membraneous localisation. In agreement with this hypothesis, the analysis of membrane proteins from stationary-phase cells showed that a strain carrying this gene on a multicopy vector overproduces a protein of 30 kDa. This protein was recognized by antibodies directed against the N-terminal portion of the gene product. Considering its induction in response to heat shock and the apparent molecular weight of its product, this gene was designated HSP30.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Journal of cancer research and clinical oncology 119 (1993), S. 675-684 
    ISSN: 1432-1335
    Keywords: Xeroderma pigmentosum fibroblasts ; DNA repair ; Recombinant DNA ; cDNA libraries ; Phage λgt10 ; pBluescript vector in vitro transcripts ; Differential hybridization ; Mitochondrial neuromyopathies ; Genetic defects
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract cDNA libraries constructed from cytoplasmic RNA of normal and xeroderma pigmentosum (XP) fibroblast strains were screened for differential gene expression. XP fibroblast strains included one representative of the complementation groups A, C, D, and one XP variant strain. The XP λgt10 cDNA libraries were differentially screened with in vitro transcripts made from cDNA in the pBluescript vector using both the same XP strain and the normal fibroblast strain. Eight differential clones were detected in the libraries of the XP group A, D, and C strains, which caused stronger signals when probed with transcripts from XP strains than with those from the normal strain. The cDNA clones were sequenced. Seven of the eight clones detected coded for three mitochondrial genes: subunit I of cytochromec oxidase (complex IV of the respiratory chain), apocytochromeb (subunit of complex III), and 16-S rRNA. Two clones representing essentially (a) subunit I of cytochromec oxidase and (b) 16-S rRNA diverged from the sequence of the human mitochondrial genome present in the data-base libraries. Clone a exhibited a transition mutation, clone b reflected a transcript of a mitochondrial genome rearranged in the 16-S rRNA gene, including four nucleotides of the adjacent tRNALeu gene. The apparently enhanced expression of mitochondrial genes in XP cells, together with the changes in DNA sequence, seem to indicate that functions of the ATP-generating system were impaired. This defect may have originated from mutations due to lack of DNA repair. The data can be interpreted in the light of mitochondrial changes that cause human neuromyopathies to occur. In analogy to these diseases the neurological symptoms in XP might be explained by abnormal mitochondria.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Journal of industrial microbiology and biotechnology 10 (1992), S. 185-190 
    ISSN: 1476-5535
    Keywords: β-Glucosidase ; C. flavigena ; Cellobiase ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Plasmid-coded β-glucosidase produced byEscherichia coli was characterized and compared to the enzyme produced byCellulomonas flavigena. Cell-free extracts, non-denaturing PAGE and 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-glu) as substrate were used to compare both enzymes. The β-glucosidase was assayed for cellobiose andp-nitrophenyl-glucopyranoside (PNPG). Cellobiose hydrolysis was performed at 50°C for the enzyme fromC. flavigena and at 37°C for that fromE. coli pJS3, both with an optimal pH of 6.5. For PNPG hydrolysis, the optimal conditions were pH 5.5 and 37°C for both cell extracts. Most of the β-glucosidase activity was intracellular. When cultures ofC. flavigena were grown with cellobiose or carboxymethylcellulose (CMC) as inducers, the expression of β-glucosidase was increased considerably.E. coli pJS3 produces a cellobiase which hydrolyzes cellobiose and PNPG. TheK m values for cellobiose and PNPG indicated that the β-glucosidase activity ofC. flavigena had a higher affinity for cellobiose as substrate, whereas the β-glucosidase fromE. coli pJS3 showed higher affinity for PNPG.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 232 (1992), S. 498-504 
    ISSN: 1617-4623
    Keywords: luminescence ; Recombinant DNA ; Grampositive organisms ; Shuttle vectors ; luc and lux genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A group of vectors for luciferase expression in Bacillus subtilis was constructed. So far, only bacterial luciferases have been expressed in Bacillus, but in this study we wanted also to express genes encoding eukaryotic luciferases to perform direct comparisons of the light levels produced by the two different systems in B. subtilis. The vectors constructed can replicate both in Escherichia coli and B. subtilis, and the luciferase expression is strictly regulated due to the dual plasmid system used. Nearly a 100-fold increase in light production compared to previous results was achieved when genes encoding bacterial luciferase were inserted into the constructs and transformed into B. subtilis. An additional tenfold increase in light production was obtained when luciferase genes from the North American firefly (Photinus pyralis) or a click beetle (Pyrophorus plagiophtalamus) were introduced in a similar fashion into B. subtilis. Measurement of the light emission was performed without disruption of bacterial cells in a real-time manner, which is a common feature when working with all of these constructions. Structures of the shuttle vector constructs and results from light emission measurements are presented.
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  • 10
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Shotgun cloning ; Filamentous fungi ; Promoter sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A transformation system for the thermophilic cellulolytic fungus Talaromyces sp. CL240 has been developed, using the phleomycin resistance gene from Streptoalloteichus hindustanus (Sh ble) as a dominant selectable marker. The plasmids (pAN8-1 and pUT720) carrying the Sh ble gene under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter, allowed selection of phleomycin-resistant transformants. A new promoter sequence cloned from chromosomal DNA of Trichoderma reesei (pUT737) was also able to drive efficient expression of the Sh ble gene in Talaromyces sp. CL240, resulting in the selection of transformants that were highly resistant to phleomycin.
    Type of Medium: Electronic Resource
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  • 11
    ISSN: 1617-4623
    Keywords: Secretion ; Recombinant DNA ; Hemolysin ; HlyB/H1yD complementation ; OmpT protease
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A fusion gene (ces-hlyA s) was constructed by ligating the genetic information for the C-terminal 60 amino acids (hlyA s) ofEscherichia coli hemolysin (H1yA) to the ces gene for a cholesterol esterase/lipase (CE) from aPseudomonas species. Part (about 30 %) of the expressed fusion protein CE-H1yAs was secreted inE. coli carryinghlyB andhlyD genes. Following the insertion between the reporter gene andhlyA s of a linker sequence that contains the information for potential cleavage sites for the outer membrane protease OmpT, two different fusion proteins (PhoA-H1yAs and CE-HlyAs) were shown to be cleaved by OmpT between the two parts during H1yB/H1yD-mediated secretion. Processed PhoA and CE accumulated in the supernatant. The efficiency of cleavage by OmpT was considerably improved by increasedompT gene dose. It was further shown that OmpT preferentially recognizes potential cleavage sites within the linker sequence.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 31 (1992), S. 161-169 
    ISSN: 1040-452X
    Keywords: Sperm cell ; Recombinant DNA ; Fertilization ; Genetic transformation ; Transgenic animals ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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  • 13
    Electronic Resource
    Electronic Resource
    Springer
    European archives of psychiatry and clinical neuroscience 240 (1991), S. 197-203 
    ISSN: 1433-8491
    Keywords: Schizophrenia ; Recombinant DNA ; Genetic markers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Interest in genetic marker studies of schizophrenia has been considerably enhanced by the advent of recombinant DNA technology, which has dramatically increased the number of available markers. In the present paper, we review studies that have been carried out using classical markers as well as the more recent molecular studies. The problems that arise when schizophrenia is studied in this way are discussed and attempts are made to account for some of the conflicting findings in this area.
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  • 14
    Electronic Resource
    Electronic Resource
    Springer
    European journal of epidemiology 7 (1991), S. 213-221 
    ISSN: 1573-7284
    Keywords: Rickettsiae ; Rickettsial genetics ; Genes ; Recombinant DNA ; Cloning ; Electroporation ; Transformation Rickettsia ; Coxiella ; Rochalimaea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Classical genetic approaches useful with free-living bacteria are difficult to apply to the rickettsiae. Although rickettsial mutants have been isolated over the years, the genetic basis of these mutants is unknown, limiting their usefulness. The application of molecular biological techniques to rickettsial studies has provided the opportunity to isolate and study specific genes. Genes encoding metabolic enzymes from rickettsiae were cloned in Escherichia coli and shown to retain their regulatory properties, suggesting that recombinant DNA technology may be useful for studies of rickettsial enzymes and regulatory mechanisms. The potential use of rickettsial surface components, or virulence factors as possible antigens for protective subunit vaccines, has led to the cloning and expression in E. coli, of rickettsial chromosomal and plasmid genes encoding outer membrane proteins. The number of genes characterized in recent years has increased dramatically giving rise to an increasing source of information on rickettsial gene structure. Plasmids have only been identified in C. burnetii and possibly Rochalimaea quintana. The plasmid sequences present in C. burnetii are highly conserved suggesting that they are important to the growth and virulence of this organism. To understand the role of genes in the rickettsia-host relationship, it is critical that a genetic exchange system be developed. The recent description of transformation of R. quintana by electroporation is an important first step in this direction. The ability to introduce genetic material is necessary to address questions that cannot be resolved by studying rickettsial gene expression in E. coli.
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  • 15
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 227 (1991), S. 28-32 
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Antibiotic production ; Genetic organization ; Directed mutant screen ; Prodigiosin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Fifteen mutants ofStreptomyces coelicolor A3(2) blocked in both the bipyrrole branch (redA) and a second site specific to the undecylprodigiosin pathway were characterized. Some of the mutants were ordered biosynthetically based on cosynthesis experiments. Complementation of each of the mutants with wild-type DNA cloned in low- and high-copy number plasmid vectors allowed the mutants to be separated into 12 new classes which are physically clustered within approximately 37 kb on theS. coelicolor genome. Early-step biosynthetic genes are centrally located and are flanked by later-step and regulatory genes.
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  • 16
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Gene cloning ; Ubiquitin fusion gene ; Ubiquitin extension protein ; Expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The genome of Tetrahymena pyriformis has been shown to contain a ubiquitin multigene family consisting of several polyubiquitin genes and at least one ubiquitin fusion gene. We report here the isolation and characterization of one genomic clone (pTUl1), that encodes a ubiquitin extension protein. A comparison of the predicted amino acid sequence of the ubiquitin extension protein gene of T. pyriformis with those from other organisms indicated a high degree of homology. However, the Tetrahymena ubiquitin extension protein contains 53 and not 52 amino acids. This feature is different from all ubiquitin 52-amino-acid extension protein genes thus far sequenced. Furthermore, we found an array of four cysteine residues similar to those found in nucleic acid binding proteins. Also, the C-terminal sequence possesses a conserved motif which may represent a nuclear translocation signal. The ubiquitin 53-amino-acid extension protein gene encodes the smallest class of ubiquitin mRNAs in T. pyriformis.
    Type of Medium: Electronic Resource
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  • 17
    Electronic Resource
    Electronic Resource
    Springer
    Current genetics 17 (1990), S. 223-227 
    ISSN: 1432-0983
    Keywords: Orotidine-5′-phosphate decarboxylase ; Cephalosporium acremonium ; Recombinant DNA ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have cloned the Cephalosporium acremonium pyr4 gene by cross-hybridization with the equivalent gene from Neurospora crassa, the closest relative from which this gene is available. The C. acremonium pyr4 gene complements an E. coli pyrF mutant lacking orotidine-5′-phosphate decarboxylase (OMPdecase), and most probably does not contain introns. Maxicell analysis in E. coli shows that it encodes a 46 kDa polypeptide. The C. acremonium OMPdecase contains a highly conserved pentadecapeptide characteristic for this category of enzyme. Extensive sequence comparison suggests an important role of this region in enzymatic activity.
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  • 18
    ISSN: 1476-5535
    Keywords: Fermentation ; Recombinant DNA ; Phage λp L promoter ; Expression vector ; α1-Antitrypsin ; Malaria vaccine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The major leftward early promoter of phage λp L, has frequently been used to drive expression of heterologous genes inEscherichia coli.p L is typically maintained fully repressed by the lambda cl protein. When induction of heterologous protein synthesis is desired, one of several potential mechanisms of destroying cl function is employed and the expression of the foreign gene commences. One method of derepressingp L involves exposing cells to nalidixic acid, which results in the “activation” of RecA protein and the subsequent RecA-mediated proteolytic cleavage of cl. Activated RecA also mediates the cleavage of theE. coli LexA protein, resulting in induction of the SOS regulon (at least 15E. coli genes, includingrec A). We have examined the effect of two chromosomal mutations on the productivity of nalidixic acid inductions. One of the tested mutations (recA o) increased the intracellular concentration of RecA prior to induction; the other (lexAind−) resulted in a mutated lexA protein insensitive to RecA-mediated cleavage. These mutations were introduced into a strain carrying acl+ defective lysogen. Synthesis of two heterologous proteins, human α1-antitrypsin and a fusion protein partially derived from thePlasmodium falciparum circumsporozooite surface antigen, was examined in the wild-type and mutant strains. The maximum α-1 antitrypsin concentration achieved was improved by 50% when therecA o strain was used rather than the wild type; however; only smaller changes (20% or less) in the maximum concentration of the malaria fusion protein wer observed. Use of thelexAind− strain resulted in a decrease in the maximum concentration attained for both heterologous products.
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  • 19
    ISSN: 1617-4623
    Keywords: Monooxygenase ; Recombinant DNA ; Overexpression ; Upstream reading frames ; Complementation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The gene coding for benzoate-para-hydroxylase (bphA) of Aspergillus niger was cloned using differential hybridisation techniques and complementation of mutants deficient in this enzyme activity. The nucleotide sequence of the gene was determined, the presence of two introns was shown and the transcription start and termination sites were determined. The structure of the mRNA upstream from the long open reading frame (ORF) is unusual. It contains two small, overlapping ORFs whose function is unknown. Comparison of the deduced amino acid sequence of the protein with the sequences present in the databanks, indicated a significant similarity of BPH to the superfamily of cytochrome P450 enzymes. Further analysis revealed that this protein is a member of a new P450 gene family designated P450LIII. The gene is designated CYP53. To increase the BPH activity of A. niger, multiple copies of the bphA gene were introduced into the genome of a recipient strain by transformation. Although increased intracellular levels of the BPH protein could be detected, the BPH enzyme activity was decreased, suggesting titration of another essential component.
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  • 20
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 222 (1990), S. 249-256 
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Plant virus ; T7 promoter ; In vitro transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Full-length cDNA copies of cucumber mosaic virus (CMV) RNAs 1 and 2 of the Fny strain were constructed from partial cDNA clones and were cloned downstream of bacteriophage T7 promoters. In one pair of clones, transcription proceeded from an unaltered T7 promoter such that in vitro transcripts representing RNAs 1 and 2 contained an additional 17 nucleotides at their 5′ termini. In a second pair of clones, the T7 promoter/cDNA junction was altered by oligonucleotide-directed mutagenesis such that the in vitro transcripts contained only an additional G residue at their 5′ ends. In addition, a full-length cDNA copy of Fny-CMV RNA 3 was constructed from two overlapping cDNA clones and was cloned downstream of an altered T7 promoter such that the resultant in vitro transcripts also contained only an additional G residue at their 5′ ends. In vitro transcripts derived from all clones contained an additional C residue at their 3′ ends. In vitro transcripts representing RNAs 1, 2 and 3 which contained an additional residue at each terminus were shown to be infectious together in several hosts of CMV.
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  • 21
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Vaccine development ; Carrier protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Hypervariable regions (HRs) of the major subunit of F11 fimbriae were exploited for insertion of foreign epitopes. Two insertion vectors were created that contain a unique cloning site in HR1 or HR4 respectively. Several oligonucleotides, coding for antigenic determinants derived from different pathogens, were cloned in both insertion vectors. Hybrid fimbrial subunits were generally shown to be assembled in fimbriae when the length of the inserted peptide did not exceed 14 amino acids. The inserted peptides appeared to be exposed in the fimbrial filament. One hybrid fimbrial protein induced detectable levels of antibodies against the inserted epitope if injected into mice.
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