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The genetically engineered human manganese superoxide dismutase crystallizes in space group P2(1)2(1)2 with a = 75.51 A, b = 79.00 A, c = 67.95 A. At room temperature the crystals are not stable against radiation, so we cooled them to 90 K and collected a data set to 3 A resolution at this temperature.
Three new ascomycins produced by genetic engineering of Streptomyces hygroscopicus ATCC 14891 have been purified and characterized. Replacement of the 13-methoxyl group of ascomycin was accomplished by substitution of the corresponding acyltransferase domain of the polyketide synthase with a domain specific for either malonyl-CoA or methylmalonyl-CoA. The strain containing the methylmalonyl-specific acyltransferase domain produced a compound with properties consistent with those expected for 13-demethoxy-13-methylascomycin. NMR analysis revealed this material to be predominantly the cis amide rotamer, similar to ascomycin. The strain containing the malonyl-specific acyltransferase domain produced a mixture of two compounds, 13-demethoxyascomycin and the 9,14-hemiacetal isomer of 13-demethoxyascomycin, in nearly equal amounts. NMR analysis revealed both compounds to be predominantly the trans amide rotamers.
The technology needed to genetically engineer antibodies is evolving rapidly and the potential utility of these novel reagents is being explored with vigor. The process includes cloning of the antibody genes, their in vitro manipulation and mutagenesis, expression in a suitable host/vector system, and, for commercial production, scale-up, purification, and product evaluation. At each step, significant advances have been achieved recently. For example: at first, antibody genes were cloned from genomic libraries by using adjacent DNA probes; techniques for rapid sequencing by primer extension of total mRNA allowed more specific screening with synthesized oligomers; finally, antibody genes can now be created de novo by chemical synthesis. Moreover, such synthesis allows total control over the antibody sequence so that molecules of any configuration can be produced. New reagents created in this way include murine antibodies whose constant regions and variable-region frameworks have been replaced with human sequence to enhance immunocompatibility with patients, to switch immunoglobulin class, or both.
Present evidence on the use of genetic engineering methods in studying the molecular mechanism of radiation damage and repair of DNA, as well as radiation mutagenesis and carcinogenesis has been summarized. The new approach to radiobiological research has proved to be extremely fruitful. Some previously unknown types of structural disorders in DNA molecule have been discovered, some repair genes isolated and their primary structure established, some aspects of radiation mutagenesis elucidated, and research into deciphering the molecular bases of neoplastic transformations of exposed cells are being successfully investigated. The authors discuss the perspectives of using genetic engineering methods in radiobiology.
Two genetically engineered E. coli strains L+ and CAG+ possessing the ability to produce the enzyme Pro-urokinase and showing additionally ampicillin resistance, and wild-strains L- and CAG-, were characterized using 328 physiological tests. Their test profiles were compared with those of 30 clinical and nonclinical E. coli isolates. This biotyping made a differentiation and recognition of the genetically manipulated strains possible. It also allowed distinguishing them from the other tested isolates. The genetically engineered strains showed a narrower activity spectrum compared with their wild-strains. However, based on differentiating characteristics, all strains could be clearly biochemically identified as E. coli. Under different laboratory test conditions (organic load, pH, salt content, temperature), the E. coli strains showed no striking features or peculiarities with respect to their survival compared to data from literature. However, low pH (pH less than 5), high salt content (greater than 7%) as well as low (less than 8 degrees C) and high (greater than 37 degrees C) incubation temperatures clearly reduced their ability to survive. Apart from a few exceptions (e.g. survival of strain L+ at 44 degrees C and pH 7 with high cell densities), the survival of the genetically engineered strains corresponded to that of the control and wild-strains. Both CAG strains, especially the genetically manipulated strain CAG+, showed in many cases reduced viability compared with the other strains.
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Genetically engineered mice with a specific deletion of targeted genes provide a novel and useful tool to study the endogenous mechanisms underlying aberrant behaviour. In this review we take the stress hormone (hypothalamic-pituitary-adrenocortical) system as an example to demonstrate how refined molecular technologies have allowed to target individual genes involved in stress hormone regulation. We describe different gene targeting methods: the generation of "conventional" knock-out mice enables us to delete a gene of interest in every cell of the body. Equally important for the studies of gene function in the mouse is the use of tissue-specific regulatory systems that allow gene inactivation to be restricted to specific tissues and, in some cases, to specific time points during development, such as the "conditional" knock-out, or the application of antisense techniques. Importantly, deletion of individual genes is not providing animal models for certain psychiatric disorders as these are caused by a manifold of minor changes in a series of so-called susceptibility genes. However, these gene targeting methods have become valuable tools to dissect the functions of individual components of complex biological systems in behavioural neuroscience: genetically engineered animals help to unravel the complex interactions and correlations between individual genes, hormonal regulation and behaviour, the most complex form of biological organization.
Earlier studies have shown that genetically engineered herpes simplex viruses (e.g., HSV-1) are effective in killing malignant tumor cells both in vitro and in various murine tumor models. This report focuses on a panel of five genetically engineered viral mutants of the gamma(1)34.5 gene, which was shown previously to cause reduction in viral replication and associated neurovirulence of HSV. These include R3616, which has both copies of gamma(1)34.5 deleted, R4009, which has a stop codon inserted after codon 28 in both copies of the gamma(1)34.5 gene, R849, which contains a lacZ gene inserted in place of the gamma(1)34.5, R908, which lacks 41 codons in frame after codon 72 of the gamma(1)34.5, and R939, which carries a stop codon precluding the translation of the COOH-terminal domain of the gamma(1)34.5 gene. We report the following: (a) all five mutant HSVs were avirulent in experimental animals but were cytotoxic for human tumor cells in vitro and in vivo; (b) the gamma(1)34.5- HSV replicated in human glioma cells almost as efficiently as wild-type HSV-1(F) based on replication assays, in situ hybridization for viral DNA, and expression of infected cell protein 27; (c) capacity of mutant HSVs to kill human cells derived from glioblastoma multiforme (CH-235MG, D-37MG, D-54MG, D-65MG, U-251MG, U-373MG, and SK-MG-1), anaplastic astrocytoma (Hs-683), anaplastic glioma (U-87MG and U-138MG), gliosarcoma (D-32GS), or normal human astrocytes demonstrated that glioma cells varied in their susceptibility to HSV-mediated cytotoxicity and that cultured astrocytes were two to three orders of magnitude less susceptible to killing than were malignant glia; and (d) scid mice, which received 0.5 or 5 x 10(6) plaque-forming units of R4009, either were coinoculated at the time of intracranial transplantation with 106 U251MG or D-54MG human glioma cells or received the cells intratumorally 5 days after tumor induction and experienced significant increases in median survivals, with no histopathological indication of an infectious encephalitic process. Genetically engineered gamma(1)34.5- HSV mutants appear to be a potentially safe biotherapeutic agent for experimental treatment of uniformly fatal malignant brain tumors.
Intrinsic value and animal integrity are two key concepts in the debate on the ethics of the genetic engineering of laboratory animals. These concepts have, on the one hand, a theoretical origin and are, on the other hand, based on the moral beliefs of people not directly involved in the genetic modification of animals. This 'external' origin raises the question whether these concepts need to be adjusted or extended when confronted with the moral experiences and opinions of people directly involved in the creation or use of transgenic laboratory animals. To answer this question, 35 persons from the practice of biomedical research who are directly involved in genetic engineering (scientists, biotechnicians, animal caretakers and laboratory animal scientists) were interviewed. They were asked to give their moral opinion on different aspects of the genetic engineering of animals and to react to statements about the concepts of intrinsic value and animal integrity. Analysis of the interviews showed that, contrary to what is often assumed, the respondents embraced these concepts, even those senses that (more) specifically apply to genetic engineering. And although the respondents raised some objections that go beyond issues of animal welfare, these objections could quite well be expressed in terms of the concepts of intrinsic value and animal integrity. In short, the results of the present study strongly suggest that these concepts do not have to be adjusted or extended in the light of the moral experiences and opinions from practice.
One goal of plant genetic engineering is the manipulation of protein levels within crop plants. New insights into the ubiquitin-dependent proteolytic pathway provide potential novel ways of enhancing levels of desired proteins by synthesizing them as ubiquitin fusions, and reducing levels of undesired proteins by selective protein degradation. As a result, the ubiquitin pathway should become a useful tool for many aspects of plant biotechnology.
With more than 30 genetic variants human cytochrome P450 2D6 (CYP2D6) presents the most extensive variation among all cytochromes P450. At the same time, roughly 30% of all drugs are metabolised by CYP2D6. Therefore, V79 Chinese hamster cells were genetically engineered for the genetic variants *1, *2, *9, *10, and *17 encoding active enzymes. These cells are to be used to understand and to predict variant-dependent metabolism of drugs and drug candidates. The V79-derived cell lines were extensively characterised for stable expression of mRNA, for enzyme activity using bufuralol hydroxylation, for CYP content by CO difference spectra, and for protein distribution and cellular location by in situ immunofluorescence. Based on these results, CYP-mediated metabolism of tamoxifen was investigated.
Discussions of the environmental risks and benefits of adopting genetically engineered organisms are highly polarized between pro- and anti-biotechnology groups, but the current state of our knowledge is frequently overlooked in this debate. A review of existing scientific literature reveals that key experiments on both the environmental risks and benefits are lacking. The complexity of ecological systems presents considerable challenges for experiments to assess the risks and benefits and inevitable uncertainties of genetically engineered plants. Collectively, existing studies emphasize that these can vary spatially, temporally, and according to the trait and cultivar modified.
Heavy metal wastewater poses a serious threat to the environment. In comparison to the existing methods of chemical precipitation, ion exchange and carbon adsorption, biosorption is an attractive alternative for the recovery of heavy metals from industrial effluents. However, nickel ion, different from other heavy metal ions, is a more recalcitrant pollutant and has low affinity to many metal tolerant microorganisms. In this study, Escherichia coli JM109 was genetically engineered to simultaneously express a Ni2+ transport system (the product of nixA gene) andoverexpress metallothionein (MT). NixA protein has a high affinity for Ni2+, and metallothioneins (MTs) are capable of binding a variety of heavy metals including Ni2+ . The Ni2+ bioaccumulation performance of the genetically engineered E. coli JM109 was evaluated. Time-course test showed that the bioaccumulation rate was rapid, and 95% of the accumulation was achieved within the first 10 minutes. The maximum Ni2+ bioaccumulation by genetically engineered E. coli cells was dramatically increased from 1.54 mg/g to 10.11mg/g, a more than five-fold increase than that of the original E. coli strain. The isotherm was of Langmuir type. Within the tested pH range (pH 4-10), the engineered cells displayed more resistance to pH variation, retaining up to 80% of the Ni2+ binding capacity at pH 4, while the original E. coli host cells lost 80% of Ni2+ binding capacity at pH 4. The presence of Na+ and Ca2+ affected Ni2+ bioaccumulation, but the effects were not serious, as 71% and 66% of the Ni2+ binding capacities were retained respectively at the concentrations of 1000 mg/L Na+ and 1000 mg/L Ca2+ . However, Mg2+ exerted a severe adverse effect on Ni2+ bioaccumulation, 83% of Ni2+ accumulating capacity was lost when Mg2+ concentration reached 200 mg/L. The effects of different kinds of heavy metals on Ni2+ accumulating were different. The genetically engineered E. coli cell lost less than 45% of its Ni2+ bioaccumulation activity in the presence of 50 mg/L lead or cadmium, 66% in the presence of 25mg/L mercury and 84% in the presence of 40 mg/L copper. The presence of glucose did not improve Ni2+ uptake. Our study suggests that the genetically engineered E. coli JM109 has potential application for effective and efficient recovery of nickel from aqueous solutions.
For many years, ex vivo gene transfer has been used for genetic manipulation of various organs. In the kidney, ex vivo gene transfer was reported using mesangial cells and macrophages. In rats, cultured cells injected into the renal artery are accumulated selectively in the glomerulus. With this approach, it is possible to transfer genetically engineered cells to normal and diseased glomeruli. The transfer of genetically engineered cells to glomeruli can be used for several purposes. With the use of resident glomerular cells engineered in vitro, it is possible to examine how the cells that overexpress certain genes behave differently in normal and diseased glomeruli. Both gain-of-function and loss-of-function strategies are useful for this purpose. For the latter, stable expression of antisense cDNA, ribosomes, or dominant-negative mutants is available. By transfer of engineered cells producing secretory, recombinant proteins, it is possible to modify glomerular microenvironment in vivo. Transfer of genes encoding therapeutically relevant molecules could be useful for therapeutic intervention. Transfer of engineered leukocytes to the glomerulus also allows investigation of cross talk between leukocytes and resident cells. Transfer of stimulated leukocytes is useful for investigation of the pathologic actions of infiltrating cells on glomerular structure and function. Leukocytes in which certain gene functions are selectively reinforced or deleted would be useful for elucidation of the exact functions of leukocyte-associated genes in glomerular diseases. This article summarizes current experience with the adoptive transfer of engineered cells to the glomerulus for investigation of and therapy for glomerular diseases.
The literatures on potential of genetic engineering in sugarcane, transformation method and its efficiency, effects of promoter and selectable marker on gene expression and on identification of transformant, and achievement of genetic engineering in sugarcane were reviewed. Directions of further research were also suggested.
Bollworm, Helicoverpa zea (Boddie), larvae are commonly observed feeding in genetically engineered Bollgard cotton. Although no information is currently available characterizing the levels of injury bollworms cause, aproximately 25% of the Bollgard acreage in the United States receives at least one insecticide application annually targeting bollworm populations. Studies were conducted to determine the levels of fruiting form injury that can occur from bollworm larvae feeding on white flowers of two types of genetically engineered cotton. The two types of genetically engineered cotton included the original Bollgard that produces one protein (Cry1Ac) from Bacillus thuringiensis variety kurstaki Berliner and Bollgard II that produces two proteins (Cry1Ac + Cry2Ab) from B. thuringiensis kurstaki. In one study, individual larvae (24 +/- 6 h old) were placed in first position white flowers of Deltapine 5415 (non-Bollgard) and Deltapine NuCOTN 33B (Bollgard). Larval infestations were made on 50 plants for each of 5 d during 2000 and 2001. Each plant was visually examined at 3 d and every 2 d thereafter, until larvae were no longer recovered. Larvae injured a total of 46.6 fruiting forms per 50 plants on non-Bollgard cotton, compared with only 18.9 fruiting forms per 50 plants on Bollgard cotton. Mean larval injury per insect was 4.3 fruiting forms on non-Bollgard cotton compared with 2.7 fruiting forms on Bollgard cotton. In a second study, individual larvae (24 +/- 6 h old) were placed in first position white flowers of Deltapine 50 (non-Bollgard), Deltapine 50B (Bollgard), and an experimental Bollgard II line. Larval infestations were made on 10 plants per day for each of six consecutive days during 2001. Larvae injured a total of 25.0 fruiting forms per 10 plants on non-Bollgard, 11.5 on Bollgard, and 6.4 on Bollgard II cottons. Mean larval injury per insect was 6.6 fruiting forms on non-Bollgard, 3.5 on Bollgard, and 0.8 on Bollgard II cottons. These data indicate that supplemental insecticide applications may be necessary to prevent yield losses on Bollgard cotton. In contrast, injury to Bollgard II cotton was minimal and may not require additional insecticide applications for bollworms.
Genetically engineered kanamycin-resistant Escherichia coli HB101 containing the mutant chimeric plasmid pAW6194-T17 specifying biphenyl dioxygenase and dihydrodiol dehydrogenase and lacking the ability to produce active 3-phenylcatechol dioxygenase was used to produce 1,2-dihydroxy-4'-chlorobiphenyl (DHCB) from 4-chlorobiphenyl. Resting-cell suspensions of genetically engineered E. coli in mineral salts medium (pH 7.0) containing 880 microM 4-chlorobiphenyl produced 110 microM DHCB. The Km for 4-chlorobiphenyl was 3.3 mM. Biotransformation of DHCB from 4-chlorobiphenyl was maximum when cells (2.5 mg of protein per ml) were incubated with shaking (150 rpm) at pH 7.0 and 30 degrees C for 6 h. The enzymatically produced DHCB was a suitable substrate for assaying 3-phenylcatechol dioxygenase activity. Biologically produced DHCB showed UV and mass spectra similar to those of chemically synthesized DHCB. The bioconversion rate of ortho-substituted chlorobiphenyl was slower than that of the para- or meta-substituted chlorobiphenyl.