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  • 1
    Electronic Resource
    Electronic Resource
    pH responsive microdomain formation in a De Novo polypeptide (1997)
    New York : Wiley-Blackwell
    Biopolymers 41 (1997), S. 521-532 
    Details
    ISSN: 0006-3525
    Keywords: recombinant DNA ; pH responsive hydrophobic microdomains ; dn31 gene ; gel filtration chromatography ; CD ; fluorescence probe analysis ; Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Recombinant DNA technology has been employed to produce a polypeptide capable of forming pH responsive hydrophobic microdomains. The design of this peptide is based upon an idealized conceptual model in which electrostatic, hydrophobic, and hydration forces are responsible for the association of amphipathic α-helical elements. Reduction in solution pH is responsible for reducing electrostatic repulsions between similarly charged residues, promoting the hydrophobic collapse of helical elements. A polymerizable synthetic element (dn31) has been synthesized and inserted into an appropriate expression vector. A clone containing a single copy of the dn31 gene (designated dn31x1) was isolated and the corresponding gene product DN3Lx1 isolated. The physical properties of DN3Lx1 were examined in solution by gel filtration chromatography, CD, and fluorescence probe analysis. It was determined that DN3Lx1 self-associates in solution with the degree of aggregation dependent on pH and ionic strength. An initial objective of this work was to examine domain organization in higher molecular weight species containing ten or more repetitive sequences. However, attempts to express multiple repeats of DN3Lxn from concatemers were unsuccessful. © 1997 John Wiley & Sons, Inc. Biopoly 41: 521-532, 1997.
    Additional Material: 8 Ill.
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  • 2
    Electronic Resource
    Electronic Resource
    Quantification of cell culture factors affecting recombinant protein yields in baculovirus-infected insect cells (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 614-618 
    Details
    ISSN: 0006-3592
    Keywords: baculovirus ; aeration ; insect cell ; medium ; recombinant DNA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An experimental study was undertaken to quantify the effects of infection cell density, medium condition, and surface aeration on recombinant protein yields in insect cells. In the absence of surface aeration and fresh medium, insect cells generated higher product yields (on a per cell basis) when infected with recombinant baculovirus at low cell densities, LCD (3 × 105-4 × 105 cells/mL), than at high cell densities, HCD (〉0.9 × 106 cells/mL), for two distinct baculovirus types. Surface aeration of a HCD culture infected in spent medium improved β-glactosidase yields 5-fold over the nonaerated case. Surface aeration and medium replenishment improved β-galactosidase yields of a HCD culture by 20-fold (compared to a 1.6-fold improvement for a LCD culture), resulting in cultures with productivties that were independent of the cell density at infection.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260390605
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  • 3
    Electronic Resource
    Electronic Resource
    Effects of glucose on the production of recombinant protein C in mammalian cell culture (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 39 (1992), S. 953-959 
    Details
    ISSN: 0006-3592
    Keywords: recombinant DNA ; protein C ; glucose ; Chinese hamster ovary cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Effects of glucose on a cultured Chinese hamster ovary cell line producing recombinant human protein C were investigated. After the recombinant cells reached confluency, they were maintained in the medium containing 10% serum and different levels of glucose in either batch or daily-exchange mode. High concentrations of glucose to the cultures yielded higher cell densities. Daily exchanges of media produced higher cell densities than the corresponding batch culture. Total protein C production per cell decreased with time in batch culture, in accordance with the declined glucose metabolism. Supplementation of the media with high levels of glucose diminished both the expression and γ-carboxylation activities of the recombinant cells. Production of protein C persisted in daily-exchange culture, resulting in a constant production rate of protein C. In this case again, glucose reduced the specific productivity of recombinant protein C. An apparent glucose inhibition constant was determined to be 0.11 mg/mL by Dixon plots. The ability to γ-carboxylate recombinant protein C was also impaired at the highest level of glucose. From these results, a strategy to maximize recombinant protein C productivity is discussed.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260390910
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  • 4
    Electronic Resource
    Electronic Resource
    Different environmental stresses can activate the expression of a heat shock gene in rabbit blastocysts (1984)
    New York, NY : Wiley-Blackwell
    Gamete Research 10 (1984), S. 45-56 
    Details
    ISSN: 0148-7280
    Keywords: blastocyst ; messenger RNA ; heat shock ; recombinant DNA ; actin ; transcription ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have utilized the rabbit 6-day blastocyst as a model system in which to examine the effect of environmental stress on embryonic gene expression. Elevation of the incubation temperature from 37 to 43 °C, exposure to 50 μM sodium arsenite or mechanical injury (resulting in the structural collapse of the 6-day rabbit blastocyst) was found to depress total protein synthesis as well as enhance the synthesis of a 70,000-dalton stress-induced protein. The molecular mass of this stress protein is similar to a heat shock protein (HSP) found in other eukaryotic systems. A recombinant DNA probe consisting of the 5′ end of a mouse gene for a 70,000-dalton HSP hybridized to RNA isolated from heat shocked, sodium arsenite-treated, and mechanically injured blastocysts but not to RNA isolated from control embryos. These results as well as in vitro translation data suggest that the expression of the 70 K HSP is controlled at the transcriptional level. The levels of actin mRNA, as detected by means of a recombinant DNA probe encoding a Drosophila actin gene, did not undergo a major alteration following these different stresses. The relevance of these observations to embryonic cellular homeostatis is discussed.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1120100106
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  • 5
    Electronic Resource
    Electronic Resource
    The transforming sequences of avian myelocytomatosis virus (MC29) (1981)
    New York, NY : Wiley-Blackwell
    Journal of Supramolecular Structure and Cellular Biochemistry 16 (1981), S. 193-207 
    Details
    ISSN: 0275-3723
    Keywords: recombinant DNA ; acute leukemia virus ; bacleriophage λ ; R-looping ; Chemistry ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo, and transforms fibroblasts and hematopoietic target cells in vitro. We have used recombinant DNA technology to isolate and characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R-loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2 kb cloned DNA insert contains approximately 4 kb of viral sequences and 5.2 kb of quail cellular sequences. The viral sequences contain all of the MC29-specific sequences and 5′ helper related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of trans-formed cells with high efficiency.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jsscb.1981.380160209
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  • 6
    Electronic Resource
    Electronic Resource
    Factors affecting homologus overexpression of the Sacchromyces cerevisiae Lanosterol 14α-demethylase gene (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 519-533 
    Details
    ISSN: 0749-503X
    Keywords: Cytochrome P450 14DM ; recombinant DNA ; plasmid copy number ; regulated gene expression ; galactose induction ; mRNA and protein levels ; chemostat cultivation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Saccharomyces cerevisiae Lanosterol 14α-demethylase (14DM) gene was overxpressed in S. cerevisiae using Promoter sequences of the highly expressed S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase TDH3 gene. To investigate factors affecting 14DM overproduction, the levels of 14DM-specific specific RNAs, apoprotein, and heme protein, repectively, were determined and the 14DM-specific RNA levels compared with the RNA levels originating from the enodogenous TDH gene(s). The quantitative measurements revealed that the 14DM steady-state RNA levels reached were some three-to five-fold below the theoretically expected values. With a View towards futrher improving expression of the 14DM gene, the specing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment. Furthermore, the effects of the gene copy number on 14DM overproduction were investigated. From the analysis of the improved expression constructs five conclusions could be reached: (1) experssion from the native 14DM gene is comparable to the expression driven by the TDH3 promoter-14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter-14DM construct on single-copy vectors is nearly as effcient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter-14DM constructs; (4) the steady-state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production. The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320080704
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  • 7
    Electronic Resource
    Electronic Resource
    Effect of leader primary structure on the translational efficiency of phosphoglycerate kinase mRNA in yeast (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 6 (1990), S. 473-482 
    Details
    ISSN: 0749-503X
    Keywords: Leader ; phosphoglycerate kinase ; recombinant DNA ; Saccharomyces cerevisiae ; trailer ; translation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In order to determine the effect of nucleotide composition of the 5′-untranslated (leader) region on the translational efficiency of mRNA in yeast, we replaced a large part of the leader region of the phosphoglycerate kinase (PGK) gene by various deoxyoligonucleotides of defined sequence. All mutations left the context of the transcription initiations site and AUG start codon intact. The mutant genes were introduced into yeast cells on a multicopy vector and the ratio of the steady-state levels of PGKmRNA and protein were determined.We found the translational efficiency to be unaffected by the presence of either an 18 nucleotides (nt) long poly A or poly C tract or by sequences consisting of mixtures of A and C residues in any proportion. In contrast, a polyU tract, as well as mixtures of U and C residues, reduced translational efficiency by a factor of two to three, presumably by long-rang base -pairing between the leader and sequences elsewhere in the coding or 3′-non-coding regions of the messenger. In agreement with this hypothesis, a five-fold reduction in translational efficiency was found for an mRNA carrying a polyC tract in the leader as well as a polyG tract in the trailer, neither of which had any effect on translational efficiency by itself. Therefore, we conclude that the leader and trailer regions (including the polyA tail) of PGK mRNA are sufficiently close to base-pair when containing complementary sequences. The resulting secondary structure evidently constitutes a barrier for incoming 40S subunits on their way to the AUG start codon.The presence of an 18 nt long polyG tract in the leader completely abolished translation of the PGK mRNA in accordance with earlier observations. However, we found the leaders containing up to 40% G residues interspersed with either A or U, still allow highly efficient translation. This value is about four times as high as the average G content of leader sequence in naturally occurring yeast mRNAS.Finally, neither deletion of about 40% of the trailer sequence of PGK mRNA, not replacement of this sequence by homopolymer tracts had any effect on translational efficiency.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320060604
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  • 8
    Electronic Resource
    Electronic Resource
    Constitutive expression of the Saccharomyces cerevisiae CUP1 gene in Kluyveromyces lactis (1991)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 7 (1991), S. 127-135 
    Details
    ISSN: 0749-503X
    Keywords: Metallothionein ; resistance to metal ions ; expression vectors ; CUP1 ; β-galactosidase ; upstream activation sequences ; recombinant DNA ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Shuttle plasmids, pE1.CUP1B and pE1.CUP1E of 10·6 kb, have been constructed between the metallothioneinencoding CUP1 gene of Saccharomyces cerevisiae and a vector capable of replication in Kluyveromyces lactis. Introduction of these plasmids into K. lactis confers resistance to copper as well as to cadmium and silver. Resistance to these latter metal ions, in the absence of induction by copper, suggested that the CUP1 gene is constitutively expressed in the foreign background. Introduction of the lacZ reporter gene from Escherichia coli into a cloning site downstream from the CUP1 promoter showed that expression of this gene is constitutive in K. lactis but in S. cerevisiae induction by copper is necessary. Sequences upstream from the CUP1 promoter are involved in the constitutive expression since deletion of 91 nucleotides from this region abolishes metal resistance. It is suggested that a K. lactis protein, normally involved in activating transcription of the resident CUP1 gene in the presence of copper, can promote transcription in the absence of metal ion by binding to the upstream activation sequence of the introduced CUP1 gene.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320070206
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  • 9
    Electronic Resource
    Electronic Resource
    Genetically-modified brewing yeasts for the 21st century. Progress to date (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 1613-1627 
    Details
    ISSN: 0749-503X
    Keywords: yeast ; brewing ; Saccharomyces ; genetic-modification ; recombinant DNA ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Academic studies and traditional breeding of yeasts depend upon their sporulation lifestyle. The strains used have been specially selected to sporulate readily and to mate producing new yeast types. Unfortunately brewing yeast strains do not behave in this way. They sporulate poorly, any spores which are formed are usually non-viable and any haploid strains produced are invariably non-maters. Only in recent years, with the development of recombinant-DNA techniques, has the specific breeding of new brewing yeast strains become widespread. Strains have been produced with the ability to ferment a wider range of carbohydrates, with altered flocculation properties and which produce beers with modified flavours. Many have been tested on the pilot scale and one, an amylolytic brewing yeast, has received approval for commercial use.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320111606
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  • 10
    Electronic Resource
    Electronic Resource
    Detection of specific RNA sequences in yeast by in situ colony hybridization (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 6 (1990), S. 31-34 
    Details
    ISSN: 0749-503X
    Keywords: Yeast ; transcription ; recombinant DNA ; in situ hybridization ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Recently a convenient method for detection of specific RNA sequences in bacteria has been developed (Ivanov and Gigova, 1986) but the original protocol was inapplicable to microorganisms with a rigid cell wall. Here we report a modification of the RNA colony hybridization for use with yeast. The modified method includes the following consecutive procedures: (a) treatment of the yeast colonies on the membrane filter with 10% SDS at 65°C for 30 min; (b) treatment of the same filter with 3 × SSC, 10% formaldehyde at 65°C for 30 min; (c) hybridization with 32 P-labelled oligonucleotide or (DNA) specific for the RNA sequence of interest. The intensity of the radioactive signals thus obtained is comparable with that of the E. colsi colonies.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320060103
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  • 11
    Electronic Resource
    Electronic Resource
    Cloning, sequencing and disruption of the ARG8 gene encoding acetylornithine aminotransferase in the petite-negative yeast Kluyveromyces lactis (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 281-285 
    Details
    ISSN: 0749-503X
    Keywords: recombinant DNA ; K. lactis genomic library ; pCXJ22 ; arginine biosynthesis ; KlARG8 ; mitochondrial transformation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.
    Additional Material: 3 Ill.
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  • 12
    Electronic Resource
    Electronic Resource
    Recombinant DNA analysis indicates that the multiple chromosomes of maize mitochondria contain different sequences (1979)
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 1 (1979), S. 363-378 
    Details
    ISSN: 0192-253X
    Keywords: maize ; mitochondrial DNA ; recombinant DNA ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Twenty-eight Bam H 1 restriction fragments were isolated from normal mitochondrial DNA of maize by recombinant DNA techniques to investigate the organization of the mitochondrial genome. Each cloned fragment was tested by molecular hybridization against a Bam digest of total mitochondrial DNA. Using Southern transfers, we identified the normal fragment of origin for d each clone. Twenty-three of the tested clones hybridized only to the fragment from which the clone was derived. In five cases, labeling of an additional band indicated some sequence repetition in the mitochondrial genome. Four clones from normal mitochondrial DNA were found which share sequences with the plasmid-like DNAs, S-1 and S-2, found in S male sterile cytoplasm. The total sequence complexity of the clones tested is 121×106 d (daltons), which approximates two thirds of the total mitochondrial genome (estimated at 183×106 d).Most fragments do not share homology with other fragments, and the total length of unique fragments exceeds that of the largest circular molecules observed. Therefore, the different size classes of circular molecules most likely represent genetically discrete chromosomes in a complex organelle genome. The variable abundance of different mitochondrial chromosomes is of special interest because it represents an unusual mechanism for the control of gene expression by regulation of gene copy number. This mechanism may play an important role in metabolism or biogenesis of mitochondria in the development of higher plants.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/dvg.1020010409
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  • 13
    Electronic Resource
    Electronic Resource
    Organization of mitochondrial DNA in normal and texas male sterile cytoplasms of maize (1981)
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 2 (1981), S. 319-336 
    Details
    ISSN: 0192-253X
    Keywords: maize ; mitochondrial DNA ; recombinant DNA ; cms-T ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Recombinant DNA and hybridization techniques have been used to compare the organization of mitochondrial DNA (mtDNA) from normal (N) and Texas male sterile (T) cytoplasms of maize. Bam H1 restriction fragments of normal mtDNA were cloned and used in molecular hybridizations against Southern blots of Bam H1 digested N and T mtDNA. Fifteen of the 35 fragments were conserved in both N and T as indicated by hybridization to comigrating bands in their restriction patterns. Only three fragments produced autoradiographs whose differences could reasonably be attributed to single changes in the cleavage site of the enzyme while approximately half (17/35) of the clones resulted in more complicated differences between N and T. The autoradiographs produced by these 17 clones indicated multiple cleavage site changes and/or sequence rearrangements of the mtDNA. Patterns of six of these 17 clones indicated partial duplication of the sequence and two showed variation in the intensity of hybridization between N and T, which may be related to the molecular heterogeneity phenomenon found in maize mitochondrial genomes. The large proportion of changes observed between N and T mtDNA indicates that rearrangements may have played an important role in the evolution of the maize mitochondrial genome.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/dvg.1020020402
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