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  • 1
    Electronic Resource
    Electronic Resource
    The true meaning of ‘exotic species’ as a model for genetically engineered organisms (1993)
    Springer
    Cellular and molecular life sciences 49 (1993), S. 225-234 
    Details
    ISSN: 1420-9071
    Keywords: Ecological theory ; environmental safety ; exotic species ; genetic engineering ; introduced species ; recombinant DNA ; risk analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The exotic or non-indigenous species model for deliberately introduced genetically engineered organisms (GEOs) has often been misunderstood or misrepresented. Yet proper comparisons of ecologically competent GEOs to the patterns of adaptation of introduced species have been highly useful among scientists in attempting to determine how to apply biological theory to specific GEO risk issues, and in attempting to define the probabilities and scale of ecological risks with GEOs. In truth, the model predicts that most projects may be environmentallysafe, but a significant minority may be very risky. The model includes a history of institutional follies that also should remind workers of the danger of oversimplifying biological issues, and warn against repeating the sorts of professional misjudgments that have too often been made in introducing organisms to new settings. We once expected that the non-indigenous species model would be refined by more analysis of species eruptions, ecological genetics, and the biology of select GEOs themselves, as outlined. But there has been political resistance to the effective regulation of GEOs, and a bureaucratic tendency to focus research agendas on narrow data collection. Thus there has been too little promotion by responsible agencies of studies to provide the broad conceptual base for truly science-based regulation. In its presently unrefined state, the non-indigenous species comparison would overestimate the risks of GEOs if it were (mis) applied to genetically disrupted, ecologically crippled GEOs, but in some cases of wild-type organisms with novel engineered traits, it could greatly underestimate the risks. Further analysis is urgently needed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01923530
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  • 2
    Electronic Resource
    Electronic Resource
    Malaria vaccine (1991)
    Springer
    Cellular and molecular life sciences 47 (1991), S. 157-163 
    Details
    ISSN: 1420-9071
    Keywords: Malaria vaccine ; recombinant DNA ; surface proteins ; antigen polymorphism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Among infectious diseases caused by protozoa, malaria is still the greatest killer of children. Mortality in adults living in endemic areas is significantly lower because they frequently acquire partial or complete immunity to the major pathogen,Plasmodium falciparum. This natural protection indicates that vaccination may be possible, and the first candidate antigens were cloned with the use of human immune sera as probes. Genetic and biochemical analysis of the parasite proteins revealed that they are polymorphic, and frequently gene sequences were discovered which were specific for a particular parasite isolate, which eliminated most antigens for purposes of vaccine development. The most promising candidate antigens today are the major surface proteins of sporozoites and blood stage parasites. However, the immune response against those is not sufficient for complete protection, and additional, intensive research is necessary to identify new molecules to be included in a vaccine cocktail against malaria. The current spread of the disease due to increasing drug resistance of parasites and mosquito vectors emphasizes the urgent need for a vaccine.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01945417
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  • 3
    Electronic Resource
    Electronic Resource
    Transgenic regulation in laboratory animals (1991)
    Springer
    Cellular and molecular life sciences 47 (1991), S. 866-877 
    Details
    ISSN: 1420-9071
    Keywords: Transgenic mice ; microinjection ; recombinant DNA ; gene expression ; transcription factors ; chromatin ; homologous recombination ; episomal maintenance ; embryonic stem cells ; germ line ; position-effect ; mosaicism ; globin genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract This chapter is an attempt to summarize some commonly accepted and some more subjective opinions about the regulation of transgene expression in laboratory animals. After a short historical introduction, I present some general notions regarding gene structure/function. The spotlight shifts then to the description of the most popular techniques for gene transfer, including the targeted gene replacement. The different approaches are briefly discussed in terms of intrinsic advantages and limitations regarding gene expression patterns. Furthermore, the role of enhancers, promoters and othercis-acting elements such as silencers and dominant control regions as well as their involvement in the chromatin on-off state are discussed on the basis of a specific example studied in our laboratory. The review concludes by presenting recent results and the new perspectives opening in the field of ‘surrogate’ (also called ‘reversed’) genetics. Some problems which remain to be solved both at the technical as well as at the social-ethical level are also briefly presented.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01929876
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  • 4
    Electronic Resource
    Electronic Resource
    Nucleotide sequence and genomic structure analyses of the p70 subunit of the human Ku autoantigen: evidence for a family of genes encoding Ku (p70)-related polypeptides (1992)
    Springer
    Molecular biology reports 16 (1992), S. 91-97 
    Details
    ISSN: 1573-4978
    Keywords: autoantibodies ; DNA-binding protein ; gene ; Ku protein ; recombinant DNA ; systemic lupus erythematosus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract cDNA encoding the p70 polypeptide subunit of the human Ku autoantigen was isolated. In vitro expression analysis of the cDNA demonstrates that it encodes the entire open reading frame. Nucleotide sequence analysis and comparison to other previously described sequences indicate the existence of several single-nucleotide and amino acid polymorphisms. Southern blot analyses demonstrate the presence of multiple copies of homologous DNA sequences in the human genome. These data support the hypothesis that multiple genes encode a family of Ku(p70)-related polypeptides.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00419754
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  • 5
    Electronic Resource
    Electronic Resource
    Production of recombinant proteins in serum-free media (1991)
    Springer
    Cytotechnology 5 (1991), S. 47-55 
    Details
    ISSN: 1573-0778
    Keywords: recombinant DNA ; biopharmaceutical proteins ; perfusion culture ; fed-batch culture ; serum-free media
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Description / Table of Contents: Abstract The advantages of serum-free culture for the manufacture of recombinant biopharmaceuticals from mammalian cells are reviewed. The process favoured is fed-batch serum-free cell culture. This process is applicable to the majority of cell lines, is practical on the large scale, gives the lowest manufacturing cost, and can be carried out without the use of any serum.
    Notes: Abstract The advantages of serum-free culture for the manufacture of recombinant biopharmaceuticals from mammalian cells are reviewed. The process favoured is fed-batch serum-free cell culture. This process is applicable to the majority of cell lines, is practical on the large scale, gives the lowest manufacturing cost, and can b e carried out without the use of any serum.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365533
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  • 6
    Electronic Resource
    Electronic Resource
    The role of genetic factors in the etiology of the affective disorders (1990)
    Springer
    Behavior genetics 20 (1990), S. 235-250 
    Details
    ISSN: 1573-3297
    Keywords: genetic linkage ; affective disorders ; genotype ; pathogenesis ; recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Psychology
    Notes: Abstract Recent application of genetic linkage analysis to the affective disorders has suggested that there are at least three genotypic forms. This is an important step toward defining the genetic etiology involved, as it had previously been suggested that the complex nature of the clinical phenotype would preclude any attempt to apply such a technique. However, to date no clinical evidence exists to discriminate these genotypes at the phenotypic level. Molecular geneticists now face a formidable task of identifying the aberrant gene and relating the gene product, a protein, to the observed psychopathology. Current molecular genetic research in the affective disorders is discussed and similar work applied to the study of nonpsychiatric disorders such as cystic fibrosis and Duchenne muscular dystrophy is reviewed. The clinical value of genetic risk analysis for individuals with a family history of the affective disorders is also considered.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01067792
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  • 7
    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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  • 8
    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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  • 9
    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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  • 10
    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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  • 11
    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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  • 12
    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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