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  • 1
    ISSN: 1436-2813
    Keywords: Key words Lung cancer ; Operation ; Hemophilia ; Recombinant DNA ; Coagulation factor VIII
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Hemophilia A is a sex-linked recessive hereditary disease that is relatively rare and the number of patients with this disorder who undergo major surgery is limited. Although replenishing coagulation factors can allow hemophiliac patients to undergo similar surgery to that performed for patients without hemophilia, there have been few reports on major surgery and none on the resection of lung cancer in patients with hemophilia A. We recently performed completion pneumonectomy of the left lung in a 70-year-old man with hemophilia A, for squamous cell carcinoma in the residual left lung. The administration of a recombinant DNA coagulation factor VIII preparation allowed this operation to be successfully carried out. This case serves to demonstrate that the recombinant DNA coagulation factor VIII preparation described may enable us to safely perform major surgery on hemophiliac patients, since there is no risk of viral infection or any other adverse effects, such as deterioration of immunocompetence or hemolysis, which are occasionally encountered with human plasma-derived preparations.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Urological research 27 (1999), S. 83-96 
    ISSN: 1434-0879
    Keywords: Key words Genitourinary malignancies ; Recombinant DNA ; Gene cloning ; DNA chip technology ; Diagnostics ; Gene therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Cancer, including genitourinary malignancy, is a consequence of accumulated genetic aberrations in genes involved in crucial regulatory pathways. The result is a deregulation of cellular behaviour, leading to neoplastic transformation, uncontrolled cell proliferation and acquisition of metastatic ability. The development and perfection of techniques in the field of recombinant DNA technology, gene cloning, (differential) analysis of gene expression, and sequencing of genes and proteins have provided a wealth of information about the genetic aberrations associated with cancer development. This “recombinant DNA and gene cloning” technology and the recently developed DNA chip technology may provide new molecular diagnostic tools. Furthermore, the technology of gene cloning in combination with the progress in in vivo gene delivery techniques offers new treatment modalities, like gene therapy, additional to conventional therapies. This review is intended to provide a general introduction to the fundamentals or strategies of recombinant DNA and gene cloning techniques as a basis for understanding the rapidly expanding range of new diagnostic and therapeutic opportunities. Some illustrative examples are provided, addressing basic and biomedical research and possible clinical applications in genitourinary oncology.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1617-4623
    Keywords: Key words Restriction endonuclease ; Methylase selection ; Gene expression ; DNA methylation ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.␣coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Theoretical medicine and bioethics 17 (1996), S. 279-314 
    ISSN: 1573-1200
    Keywords: Recombinant DNA ; transgenesis ; patents ; human life forms
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Philosophy
    Notes: Abstract The rationale of patents on transgenic organisms leads to the startling notion of the human qua infringement. The moral reasons by which we may tenably reject such notion are not conclusive as to human life forms outside the body. A close look at recombinant DNA experimentation reveals ingenious processes, but not entities that the body lacks. Except for artificial genes, the genes of biotechnology are found on chromosomes, albeit nonconsecutively, and their uninterrupted transcripts appear in messenger RNA. An enhanced form of protection for ingenious processes, the “human methods patent,” is proposed and defended as a replacement for product patents. The proposed patent would pertain to biotechnology manufacturing and genetic intervention in somatic and germ cells. A counterpart could govern nonhuman life forms. It is argued that compulsory licensing protections should be a condition of such patent. Contrary to the conservative assumption that statutory sobriquets suffice, the reckoning of what qualifies as a patentable ingenious process will continue to require systematic scientific guidance.
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  • 5
    ISSN: 1432-0983
    Keywords: Recombinant DNA ; Filamentous fungi ; 5-fluoro-orotic acid ; Homologous transformation system
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOAR), on the average 60% of the 5-FOAR colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5′ end of the pyrG gene with vectors containing a mutation near the 3′ end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.
    Type of Medium: Electronic Resource
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  • 6
    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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  • 7
    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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  • 8
    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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  • 9
    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.
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  • 10
    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.
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  • 11
    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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  • 12
    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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  • 13
    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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  • 14
    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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  • 15
    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.
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  • 16
    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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  • 17
    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.
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  • 18
    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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  • 19
    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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  • 20
    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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  • 21
    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.
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  • 22
    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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  • 23
    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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  • 24
    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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  • 25
    Electronic Resource
    Electronic Resource
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    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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  • 26
    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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  • 27
    ISSN: 1617-4623
    Keywords: metC ; Cystathionine-β-lyase ; Nucleotide sequence ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The DNA sequence of the Salmonella typhimurium metC gene and its flanking regions was determined. The metC gene contains an open reading frame of 1185 nucleotides encoding a polypeptide of 395 amino acids with a predicted molecular weight of 42874 daltons. S1 nuclease mapping experiments located the transcription start site of the metC gene. The nucleotide sequence and the deduced amino acid sequence for the metC genes of S. typhimurium and Escherichia coli were compared. Although there are 279 nucleotide replacements, most do not change the amino acid sequence. Nucleotide sequence analysis of the flanking regions of the S. typhimurium metC gene shows that there is an open reading frame upstream and an open reading frame downstream of the gene. The existence of the divergently transcribed upstream open reading frame (designated ORF1) was confirmed by the construction of an ORF1-lacZ fusion. The transcription start site of ORF1 was determined by S1 nuclease mapping.
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  • 28
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Small subunit ; Ribosomal DNA ; Sequence comparison ; Lycopersicon esculentum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The gene of a cytoplasmic 18 S ribosomal RNA (18 S rDNA) of the dicotyledonous plant tomato (ycopersicon esculentum) cv. Rentita has been cloned, and its complete primary structure has been determined. The tomato 18 S rDNA is 1805 by long with a G+C content of 49.6%. Its sequence exhibits 94%–96% positional identity when it is colinearly aligned with the previously reported sequences of the 17–18 S rDNAs of the dicot soybean and the monocots maize and rice. A model of the secondary structure of the 18 S rRNA of angiosperms is presented and its genera-specific structural features are compared with a current eukaryotic 18 S rRNA consensus model.
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  • 29
    Electronic Resource
    Electronic Resource
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 4 (1989), S. 551-554 
    ISSN: 0884-3996
    Keywords: Recombinant DNA ; luminescence ; luciferase gene ; Vibrio harveyi ; toxic substances ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We have studied possibilities for constructing Escherichia coli strains capable of producing stable light. Light production in E. coli is achieved by cloning the genes encoding bacterial luciferase from Vibrio harveyi. To gain the advantage of sensitive detection of light we transferred the genes under the control of a strong, regulatable promoter system.Stabilization of light produced by E. coli clones was accomplished by finding the optimal plasmid construction and growth conditions as well as suitable measuring buffers. The adjustment of the luciferase synthesis for bioluminescence measurements to a high but not harmful level gives healthy cells and stable luciferase. Cultivation at 30 °C in an uninduced state was found to be the most important factor in getting stable-light production. The overall cell metabolism being unstressed gives us the possibility of monitoring cell physiology and factors affecting it via bioluminescence reactions in vivo. To make the results easy to interpret the light emission has to be stable during a measurement period of one to several hours. In the case of the original light-producing bacteria, Vibrio and Photobacterium strains it has not thus far been possible to find conditions where light emission would be stable for several hours. Based on our findings an automated biosensor system can be developed to monitor the effects of biologically active compounds against stable-light-producing bacteria.
    Additional Material: 1 Ill.
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  • 30
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    Journal of molecular medicine 66 (1988), S. 241-245 
    ISSN: 1432-1440
    Keywords: Erythropoietin ; Stability ; Radioimmunoassay ; Polycythemic mouse bioassay ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Radioimmunoassays for erythropoietin are limited so far to a few specialized laboratories and this requires transport and storage of samples. We therefore tested the stability of immunoreactive erythropoietin in serum and plasma samples obtained from a uremic and a nonuremic anemic patient. No significant change in the concentration of immunoreactive erythropoietin was found in either serum or plasma samples for up to 14 days of storage. This type of stability was observed no matter whether the samples were stored at room temperature, 4° C, or −20° C. There was no difference between the estimates of erythropoietin in serum and heparinized plasma. Validity of the radioimmunoassay used in this study was demonstrated by parallelism of dilution curves of test specimens and the 2nd International Reference Preparation for erythropoietin and by a close correlation between the immunoreactivity and the bioactivity of the hormone, as assessed in the same samples by the exhypoxic polycythemic mouse bioassay. In conclusion the data obtained clearly indicate that the necessity of storage and transport of clinical samples does not limit the practicability of the radioimmunoassay for erythropoietin.
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  • 31
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    European archives of psychiatry and clinical neuroscience 238 (1988), S. 110-113 
    ISSN: 1433-8491
    Keywords: Schizophrenia ; Cytomegaloviruses ; Virus diseases ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Using highly sensitive nucleic acids hybridization techniques, which allow the detection of 0.1–0.5 single copy gene equivalents per cell, DNA from the temporal cortex of seven definite schizophrenics, five persons with schizophrenia-like psychoses, three patients with Huntington's chorea and nine mentally normal individuals were probed with human cytomegalovirus (HCMV) DNA. A clear hybridization signal was obtained with DNA from the temporal lobe of a young schizophrenic patient, whereas DNA from the temporal cortex of controls did not hybridize to the HCMV probe. This finding is in agreement with the cytomegalovirus hypothesis of schizophrenia and hints at the possibility that viral infection of the temporal cortex may in some sporadic cases be a contributing factor to the development of schizophrenic psychoses. There is no indication, however, that infection of the central nervous system with HCMV is an aetiological factor in the great majority of schizophrenic disorders. Clearly further studies, preferably in situ hybridizations of whole brains, are needed to prove or disprove the cytomegalovirus hypothesis of schizophrenia.
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  • 32
    ISSN: 1617-4623
    Keywords: metF ; 5,10-methylenetetrahydrofolate reductase ; Recombinant DNA ; Nucleotide sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The Salmonella typhimurium LT2 metF gene, encoding 5,10-methylenetetrahydrofolate reductase, has been cloned. Strains with multicopy plasmids carrying the metF gene overproduce the enzyme 44-fold. The nucleotide sequence of the metF gene was determined, and an open reading frame of 888 nucleotides was identified. The polypeptide deduced from the DNA sequence contains 296 amino acids and has a molecular weight of 33 135 daltons. Mung bean nuclease mapping experiments located the transcription start point and possible transcription termination region for the gene. There is a 25bp nucleotide sequence between the translation termination site and the possible transcription termination region. This region possesses a GC-rich sequence that could form a stable stem and loop structure once transcribed (ΔG=-9 kcal/mol), followed by an AT-rich sequence, both of which are characteristic of rho-independent transcription terminators. The nucleotide and deduced amino acid sequences of the S. typhimurium metF gene are compared with the corresponding sequences of the Escherichia coli metF gene. The nucleotide sequences show 85% homology. Most of the nucleotide differences found do not alter the amino acid sequences, which show 95% homology. The results also show that a change has occurred in the metF region of the S. typhimurium chromosome as compared to the E. coli chromosome.
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  • 33
    ISSN: 1617-4623
    Keywords: Autolysin ; Hydrolase ; In vitro transcription and translation ; Recombinant DNA ; Transcriptional and translational signals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Several hundred bacterial isolates were screened for bacteriolytic activity by growing them on agar medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells as the substrate. A Bacillus sp. producing the largest lytic zone was selected. A genomic bank of this selected bacterium was constructed in the multi-functional vector pTZ18R, with partial SauIIIA DNA fragments inserted at the SalI restriction site. Screening of 800 colonies of this bank for cell lysis gave 5 recombinants exhibiting lytic activity, as detected by analysis of extracts of sonicated Escherichia coli cells on denaturing polyacrylamide gels containing autoclaved, lyophilized M. lysodeikticus cells as the substrate. One clone (pBH2500), expressed inE. coli strain NM522, was found to code for a lytic enzyme corresponding, in molecular weight, to the 27 kDa Bacillus sp hydrolase. This clone with an insertion of 2.5 kb was then subcloned as a 929 bp EcoRI-SauIIIA fragment in pTZ18R (pBH929) and showed higher cell lytic activity. A unique open reading frame for a protein of 251 amino acids, followed by a putative terminator sequence, was found after a consensus ribosome binding site. A putative leader sequence was identified in the first 37 amino acids. One truncated subclone (pBH703), corresponding to 196 out of 251 residues from the protein N-terminal end, still possessed lytic activity.
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  • 34
    ISSN: 1617-4623
    Keywords: Competence ; Autolysins ; Choline ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have worked out conditions for the study of competence development and genetic transformation in Streptococcus oralis NCTC 11427 (type strain), a species that contains choline in the cell wall. The peak of competence was found at the early exponential phase of growth and the optimal conditions for transformation were achieved with shuttle plasmids prepared from S. pneumoniae or from Escherichia coli serving as donor DNA. Transformation with dye-bouyant density gradient purified plasmid preparations followed first-order kinetics. The pneumococcal amidase can be expressed in S. oralis harbouring a plasmid carrying the lytA gene. This enzyme lysed the cell wall of the transformed cell in the presence of detergents.
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  • 35
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    Molecular genetics and genomics 213 (1988), S. 269-277 
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Helix-turn-helix motifs ; In vitro transcription-translation ; Phage immunity ; Exonuclease III deletions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The nucleotide sequence of the 3.4 kb SphI-G fragment that contained the repressor gene (c) of the temperate Streptomyces phage ϕc31 was determined. Analysis of this sequence revealed a large open reading frame with protein coding character and sequence changes in c gene point and deletion mutants identified this as the coding region of the repressor. Two of the mutants studied had undergone deletions of 1.1 kb and 1.4 kb that had occurred across short direct repeats of 6 bp and 11 bp, respectively. Coupled in vitro transcription-translation experiments using the cloned SphI-G fragment and Streptomyces lividans cell free extracts identified a protein product of approximately 72 kDa, in close agreement with that predicted from the nucleotide sequence. A strongly predicted helix-turn-helix motif that may be involved in DNA binding occurred towards the carboxy-terminus of the amino acid sequence. Initial attempts to clone the SphI-G fragment in Streptomyces failed; using information gained from the sequence analysis a smaller segment of this DNA fragment was cloned in S. lividans and conferred immunity to a clear plaque mutant (c1) of ϕc31.
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  • 36
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    Molecular genetics and genomics 215 (1988), S. 19-25 
    ISSN: 1617-4623
    Keywords: IGF-1 ; Escherichia coli secretion/export ; LamB leader peptide ; Heterologous gene expression ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The processing of LamB-IGF-1 fusion protein and the export of processed IGF-1 (insulin-like growth-factor-1) into the growth medium was examined in the Escherichia coli host strain, JM101. Several strain or plasmid modifications were tried to increase export of periplasmic (Processed) IGF-1 into the growth medium of JM101. These included: (1) use of a lon null mutant strain to increase accumulation levels of unprocessed LamB-IGF-1 fusion protein; (2) use of an alternative drug resistance marker on the expression plasmid rather than beta-lactamase, thereby reducing any competition for processing of LamB-IGF-1 by signal peptidase; (3) examination of whether phage M13 gene III protein expression caused more periplasmic IGF-1 to be exported into the growth medium due to increased outer membrane permeability; and (4) examination of the effect of E. coli or yeast optimized IGF-1 codons. None of these strain or plasmid modifications caused any significant increase in export of IGF-1 into the growth medium of JM101. Solubility studies of LamB-IGF-1 and processed IGF-1 showed that virtually all of the LamB-IGF-1 and IGF-1 remaining within the cell after a 2 h induction period was insoluble. This implied that only soluble LamB-IGF-1 was processed to IGF-1 and that only soluble IGF-1 was exported into the growth medium. Taken together, the results indicated that LamB-IGF-1 and IGF-1 solubility were the limiting factors in secretion of IGF-1 into the periplasm and export of IGF-1 into the growth medium.
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  • 37
    ISSN: 1617-4623
    Keywords: Complementary chromatic adaptation ; Gene expression ; Photoregulation ; Phycobilisome ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In cyanobacteria, light is harvested by phycobilisomes which are essentially made up of chromophoric proteins called phycobiliproteins. We have characterized two gene clusters (cpcB1, cpcA1 and cpcB3, cpcA3) each encoding the two subunits of phycocyanin (βPC and αPC, respectively), one of the major phycobiliproteins in Calothrix 7601. Downstream from the gene encoding the PCα subunit in cluster 1, an open reading frame was found, cpcE1. These genes are organized in two transcriptional units, namely: cpcB3 A3 and cpcB1 A1 E1. All these genes are transcribed whatever the chromatic light received during cell growth. Consequently, although only one type of “constitutive” PC has been biochemically characterized, we have demonstrated that there are two cpc operons “constitutively” transcribed in this strain. With the previously described red light “inducible” cpcB2 A2 operon, there are three copies of the PC encoding genes in Calothrix 7601. The significance of this newly described multigene family in cyanobacteria is discussed. We have also mapped the 5′ and 3′ termini of the major transcript from the cpc1 operon. Analysis of the 5′ untranslated region of this transcript has revealed alternative secondary structures which are proposed to play a role in the regulation of the expression of this operon.
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  • 38
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Chloramphenicol resistance ; cat gene ; Plasmids pC194 and pUB110 ; Inducible gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two non-homologous chloramphenicol (Cm) acetyltransferase (CAT) genes, designated catA and catB, were cloned from Clostridium butyricum type strains and characterized by restriction mapping. Both genes are efficiently expressed in Escherichia coli and Bacillus subtilis. In contrast to analogous genes from staphylococci and bacilli, gene expression is not dependent on induction by Cm. The genes are considered as chromosomal, since no association with endogenous plasmids was detectable. Southern hybridization revealed a homology between catA and the staphylococcal Cm resistance plasmid, pC194. The subunit size of the clostridial CAT enzymes expressed in E. coli was determined as 22.5 kDa (catA) and 24 kDa (catB), respectively. The C. butyricum cat genes provide potentially useful selection markers for the construction of cloning vectors from cryptic clostridial plasmids.
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  • 39
    ISSN: 1432-069X
    Keywords: Collagen ; Fibroblasts ; Scleroderma ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Fibroblast cultures were started from affected and unaffacted skin areas of six patients with localized scleroderma in an active stage. The cell lines were studied for synthesis of procollagens and fibronectin by metabolic labeling with 3H-proline and for their contents of mRNAs for pro α1(I) and pro α2(I) collagen. For this purpose a cDNA clone for human pro α1(I) collagen mRNA was constructed. The clone was identified by restriction site mapping and hybridization to the specific mRNAs. All the scleroderma fibroblast lines produced increased amounts of type I and type III collagens and fibronectin during the early passages. The cell lines gradually reduced their elevated synthesis of collagen and fibronectin to normal or near normal levels by the tenth passage. The ratios of α1(I) and α2(I) chains and of type I and type III collagens, and the extent of type I procollagen processing, remained relatively unchanged in all the cultures. The cellular levels of type I procollagen mRNAs were increased in all the cells exhibiting an increased synthesis of collagen. The results suggest that in localized scleroderma the fibroblasts have undergone a coordinated activation of collagen synthesis at transcriptional level.
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  • 40
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    Journal of industrial microbiology and biotechnology 1 (1987), S. 277-282 
    ISSN: 1476-5535
    Keywords: Recombinant DNA ; Gene expression ; Genetic engineering ; Biotechnology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary A variety of factors affect the expression of foreign proteins inEscherichia coli. These include: promoter strength, efficiency of ribosome binding, stability of the foreign protein inE. coli, location of the foreign protein inE. coli, the codons used to encode the foreign protein, the metabolic state of the cell, and the location, stability and copy number of the foreign gene. This paper contains a critical review of these factors with the idea that a detailed understanding of them is the key to the development of strategies for the efficient large-scale production of foreign proteins inE. coli.
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  • 41
    ISSN: 1432-0983
    Keywords: Schizophyllum commune ; Transformation ; Gene isolation ; Basidiomycetes ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune. Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation. The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ural, encoding orotidine-5′-phosphate decarboxylase; OMPdecase), tryptophan (rpl, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pab1). In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S. commune mutant. Total DNA from transformants was restricted, religated, and used to transform E. coli. Ampicillin resistant plasmids were recovered from E. coli and tested for their ability to transform the corresponding mutant of S. commune. Plasmids complementing the ade2, adeS, pabl, trpl, and ural mutations were recovered.
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  • 42
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    Journal of industrial microbiology and biotechnology 2 (1987), S. 117-121 
    ISSN: 1476-5535
    Keywords: Fermentation ; Recombinant DNA ; Hepatitis B surface antigen
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Fermentations were performed to determine parameters affecting the expression of hepatitis B surface antigen (HBsAg) in the yeastSaccharomyces cerevisiae containing the HBsAg gene. These studies emphasized inereasing both the relative abundance (HBsAg: cell mass) and total production of HBsAg. Specific activity was increased 70-fold when cells were grown in shake flasks containing nonselective rather than selective medium. The addition of adenine, ammonium sulfate or glucose to the complex medium reduced the production of antigen. Results similar to those achieved in shake flasks were obtained when the growth was performed in fermenters. A nutrient addition system was employed to increase the production of cells and HBsAg. The addition of glucose to the culture medium increased cell mass 6-fold but decreased the production of antigen. This imbalance was corrected by supplementing the glucose with complex nutrients.
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  • 43
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    Plant and soil 99 (1987), S. 185-196 
    ISSN: 1573-5036
    Keywords: Antibody ; H+-ATPase ; Membrane ; Recombinant DNA ; Transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary Mineral transport across the plasma membrane of plant cells is controlled by an electrochemical gradient of protons. This gradient is generated by an ATP-consuming enzyme in the membrane known as a proton pump, or H+-ATPase. The protein has a catalytic subunit of Mr=100,000 and is a prominent band when plasma membrane proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We generated specific rabbit polyclonal antibody against the Mr=100,000 H+-ATPase and used the antibody to screen λgtll expression vector libraries of plant DNA. Several phage clones producing immunoreactive protein, and presumably containing DNA sequences for the ATPase structural gene, were isolated and purified from a carrot cDNA library and a Arabidopsis genomic DNA library. These studies represent our first efforts at cloning the structural gene for a plant plasma membrane transport protein. Applicability of the technique to other transport protein genes and the potential for use of recombinant DNA technology in plant mineral transport research are discussed.
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  • 44
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Methionine metabolism ; Negative control ; Regulatory regions
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In Saccharomyces cerevisiae, the expression of several genes implicated in methionine biosynthesis is coregulated by a specific negative control. To elucidate the molecular basis of this regulation, we have cloned two of these genes, MET3 and MET25. The sequence of MET25 has already been determined (Kerjan et al. 1986). Here, we report the nucleotide sequence of the MET3 gene along with its 5′ and 3′ flanking regions. Plasmids bearing different deletions upstream of the transcribed region of MET3 were constructed. They were introduced into yeast cells and tested for their ability to complement met3 mutations and to respond to regulation by exogenous methionine. The regulatory region was located within a 100 bp region. The sequence of this regulatory region was compared with that of MET25. A short common sequence which occurs 250–280 bp upstream of the translation initiation codon of the gene was found. This sequence is a good candidate for the cis-acting regulatory element.
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  • 45
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    Molecular genetics and genomics 209 (1987), S. 570-574 
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Methylase ; Plasmid vector ; Gel electrophoresis ; Clearage map
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The modification genes of Flavobacterium okeanokoites and Haemophilus galinarum have been cloned into the vector pBR322 and expressed in Escherichia coli cells. FokI methylase gene is contained on a 3.80 kb piece of F. okeanokoites DNA. Plasmid constructs carrying this fragment of DNA are resistant to digestion by FokI restriction endonuclease but are sensitive to cleavage by HindIII, EcoRI and PstI. Unmodified λ DNA molecules, exposed in vitro to cell extracts prepared from cells habouring this plasmid, became resistant to digestion by FokI. The smallest HgaI methylase clone carries the pBR322 plasmid containing a 3.50 kb piece of H. galinarum DNA. This plasmid is resistant to digestion by HgaI. Neither the FokI nor the HgaI restriction endonuclease was detected in either clone. This is the first report of cloning modification genes whose protein products recognise asymmetric nucleotide sequences.
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  • 46
    ISSN: 1432-0983
    Keywords: Recombinant DNA ; Cross hybridization ; 2D-electrophoresis ; Hybrid selection translation ; Transformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Using the structural gene for the ribosomal protein L3 from Saccharomyces cerevisiae as a probe, we isolated a homologous fragment from genomic DNA of Schizosaccharomyces pombe. Analysis of the plasmid carrying this fragment by hybridization selection and 2D-electrophoresis revealed a 31 kDa ribosomal protein. Transformation of the vector pDB248x containing this fragment into Schizosaccharomyces pombe leads to an increased level of mRNA suggesting that we have cloned the entire and actively transcribed gene.
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  • 47
    ISSN: 1432-0983
    Keywords: Mitochondrial DNA ; Population heterogeneity ; Molecular evolution ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Physical characterization of the mitochondrial genome derived from the obligate mosquito parasite, Romanomermis culicivorax has generated some surprising physical properties regarding the molecular structure of nematode mitochondrial DNA (mtDNA). Restriction enzyme analysis of this mtDNA has revealed a mitochondrial genome size of approximately 26 kb, the largest metazoan mtDNA reported to date. Isofemale lineages are monomorphic for one of three size variants, differing by 500-1,000 base pairs, present in our original field population. Cloned hybridization probes derived from a single region exhibiting a 600 by size polymorphism share strong homology with several spatially separated sites distributed about the mtDNA. This suggests that the homology is a result of repeated DNA sequence elements contained within this mitochondrial genome that contribute to mtDNA size polymorphism.
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  • 48
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    Springer
    Molecular genetics and genomics 202 (1986), S. 271-275 
    ISSN: 1617-4623
    Keywords: Aspergillus ; Isocitrate lyase ; Cloning ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary An Aspergillus nidulans gene library was constructed in a high-frequency transformation vector, pDJB3, based on the Neurospora crassa pyr4 gene. This gene library was used to isolate the structural gene for isocitrate lyase (acuD) by complementation of a deficiency mutation following transformation of A. nidulans. Plasmids rescued in Escherichia coli were able to transform five different A. nidulans acuD mutants. Transformation using plasmids containing the cloned fragment resulted in integration at the acuD locus in six of nine transformants.
    Type of Medium: Electronic Resource
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  • 49
    ISSN: 1617-4623
    Keywords: Gene transfer ; Plant cell transformation ; Plant tissue culture ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary DNA from a bacterial plasmid containing the T-DNA border sequences of Agrobacterium tumefaciens was transferred into the nucleus or the cytoplasm of tobacco mesophyll protoplasts by microinjection. Following culture in hanging drops, some of these protoplasts produced calli containing the foreign DNA sequences. Evidence for the presence of the injected plasmid DNA in these calli was provided by Southern hybridization analysis. The results demonstrated that random portions of the bacterial plasmid were linked to plant DNA and that integration did not occur at the T-DNA borders present on the injected plasmid. The average number of integrated copies ranged from less than one to 1–2 per tobacco genome. The frequency of integration averaged 14% with intranuclear injections compared to 6% with cytoplasmic injections. With further refinement, the use of microinjection may allow the introduction of many different types of genetic elements into plants.
    Type of Medium: Electronic Resource
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  • 50
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    Springer
    Molecular genetics and genomics 205 (1986), S. 546-549 
    ISSN: 1617-4623
    Keywords: Transposition ; Genomic libraries ; Recombinant DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A general in vivo procedure for cloning Escherichia coli genes into cosmids has been developed. The method we describe here uses a deleted Mu phage (a mini-Mu) to transpose E. coli genes into cosmids during mini-Mu replication. The resulting cosmids clones are packaged in-vivo into λ phage particles. Plasmids carrying a particular DNA sequence can be selectively recovered after infection of a new host with the in vivo constructed genomic cosmid library. This system was used succesfully to clone several E. coli genes.
    Type of Medium: Electronic Resource
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  • 51
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    Archives of microbiology 140 (1984), S. 218-224 
    ISSN: 1432-072X
    Keywords: Recombinant DNA ; Cloning ; Staphylococcus carnosus and S. hyicus subsp. hyicus ; Ribose degradation ; Ribokinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A gene library with DNA of Staphylococcus hyicus subsp. hyicus was established in S. carnosus by using the plasmid vector pCT20. Two clones of S. carnosus were isolated which were able to ferment d-ribose. The two hybrid plasmids (pRib 1) and (pRib 2) were isolated and characterized. They contained inserted DNA fragments of S. hyicus subsp. hyicus with sizes of 10.2 and 8.2 kb, respectively. d-Ribose uptake and enzyme activities were studied. All strains tested [S. hyicus subsp. hyicus, S. carnosus (wild type) and the two S. carnosus clones] possessed an inducible uptake system for d-ribose. S. hyicus subsp. hyicus possessed in addition enzyme activities of d-ribokinase and d-ribose-5-P isomerase. None of these enzyme activities could be detected in S. carnosus (wildtype). Only in the S. carnosus clones containing (pRib 1) or (pRib 2) could a d-ribokinase activity be demonstrated, indicating that the gene for d-ribokinase of S. hyicus subsp. hyicus was cloned in S. carnosus.
    Type of Medium: Electronic Resource
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  • 52
    ISSN: 1432-0983
    Keywords: Aspergillus ; 5S rRNA ; Recombinant DNA ; Restriction Analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Genes coding for 5S rRNA were found to be dispersed in the Aspergillus nidulans genome. Three different recombinant plasmids hybridizing to 5S rRNA were isolated and their restriction enzyme maps were established.
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  • 53
    ISSN: 1432-0983
    Keywords: Recombinant DNA ; 2 µm DNA ; Heterologous gene expression ; Eukaryotic host
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The expression of the lacZ gene of E. coli in S. cerevisiae has been studied. The enzymatic activity coded by the lacZ gene in E. coli, β-galactosidase, is detectable in yeast cells harboring a chimeric plasmid carrying the gene. On the basis of size and immunological criteria, no difference was detected between the Coli-in-yeast β-galactosidase and the E. coli enzyme.
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  • 54
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    Amsterdam : Elsevier
    Gene 102 (1991), S. 249-254 
    ISSN: 0378-1119
    Keywords: Recombinant DNA ; deduced amino acid sequence ; evolutionary conservation ; primer extension
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 55
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    Amsterdam : Elsevier
    Gene 102 (1991), S. 255-259 
    ISSN: 0378-1119
    Keywords: Ca^2^+ binding ; EF-hand ; Recombinant DNA ; S-100 proteins ; cDNA cloning ; nucleotide sequence
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 56
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    Amsterdam : Elsevier
    Gene 102 (1991), S. 261-264 
    ISSN: 0378-1119
    Keywords: J gene segments ; LOUVAIN rat ; Recombinant DNA ; nucleotide sequence ; polymerase chain reaction ; pseudogene
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 57
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    Amsterdam : Elsevier
    Gene 150 (1994), S. 371-373 
    ISSN: 0378-1119
    Keywords: murine and human genes ; PCR ; Recombinant DNA ; cytokine ; homology to porcine ; pathology
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 58
    ISSN: 0378-1119
    Keywords: AIDS ; Recombinant DNA ; murine, rat, and human cells ; phage λlibrary ; transcription factor
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 59
    ISSN: 0378-1119
    Keywords: Recombinant DNA ; animal virus vector ; homologous recombination ; lacZ ; live vaccine ; replacement insertion
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 60
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    Amsterdam : Elsevier
    Gene 101 (1991), S. 223-229 
    ISSN: 0378-1119
    Keywords: Recombinant DNA ; avian keratin ; cDNA ; feather ; gene expression ; gene family ; scale ; β-keratin
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 61
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    Amsterdam : Elsevier
    Gene 101 (1991), S. 247-250 
    ISSN: 0378-1119
    Keywords: Recombinant DNA ; internal duplication ; inverted repeats ; sequence analysis ; sugar beet ; tandem arrangement
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 62
    ISSN: 0378-1119
    Keywords: Recombinant DNA ; Southern hybridization ; maxicells ; promoter ; sequence homology
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 63
    ISSN: 0378-1119
    Keywords: Recombinant DNA ; fusion proteins ;